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Tutorial Review Capillary electrochromatography Maria G. Cikaloa, Keith D. Bartle*a, Mark M. Robsona, Peter Myersa and Melvin R. Euerbyb a School of Chemistry, University of Leeds, Leeds, UK LS2 9JT b Pharmaceutical and Analytical R and D, Astra Charnwood, Bakewell Road, Loughborough, Leicestershire,UK LE11 5RH Capillary electrochromatography (CEC) has seen a resurgence of interest during the 1990s, despite having origins in the 1970s. The technique combines the desirable features of both high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE): the separation process is based on differential interactions between the stationary and mobile phases, whilst the electroosmotic flow transports the mobile phase through the capillary.Thus, it has demonstrated advantages over both HPLC and CE, which are yet to be fully exploited over a wide field of application; already the popularity of CEC is on the increase, as reflected in the number of scientific publications and seminars held. The aim of this tutorial review is to increase awareness and understanding of both theoretical and practical aspects of CEC.Whilst it does not provide an in-depth account of CEC, it does attempt to cover the more important, relevant work available in the open literature: only major advancements associated with CEC applications are highlighted. Material presented in the review was typically obtained by literature searches involving Analytical Abstracts, Chemical Abstracts and ‘BIDS’ (for academic use only).Keywords: Review; capillary electrochromatography; microseparations Introduction Capillary electrochromatography (CEC) is a recently developed (Table 1) variant of high-performance liquid chromatography (HPLC) in which the flow of mobile phase is driven through the column by an electric field, rather than by applied pressure. This electroosmotic flow (EOF) is generated by applying a large voltage across the column; positive ions of the added electrolyte accumulate in the electrical double layer of particles of column packing, move towards the cathode and drag the liquid mobile phase with them.As in capillary electrophoresis (CE) and micellar electrokinetic chromatography (MEKC), small internal diameter (50–100 mm) columns with favourable surface area-tovolume ratios are employed to minimise thermal gradients from ohmic heating, which can have an adverse effect on bandwidths. CEC differs crucially from CE and MEKC, however, in that the separating principle is partition between the liquid and solid phases (Table 2).Avoiding the use of pressure results in a number of important advantages for CEC over conventional HPLC. First, the pressure driven flow rate through a packed bed depends (Table 3) directly on the square of the particle diameter and inversely on column length; for practical pressures, generally used particle diameters are seldom less than 3 mm, with column lengths restricted to approximately 25 cm.By contrast (Table 3), the electrically driven flow rate is independent of particle diameter and column length so that, in principle, smaller particles and longer columns can be used. It follows that considerably higher efficiencies can be generated in CEC than in HPLC. A second consequence of employing electrodrive is that the flow–velocity profile in EOF reduces dispersion of the band of solute as it passes through the column, further increasing column efficiency. The combined effect of reduced particle diameter, increased column length and plug flow leads to CEC efficiencies of typically 200 000 plates per metre and substantially improved resolution.Thus the two tipredane Maria Cikalo graduated from Kingston Polytechnic where she studied for a GRSC part time whilst working in formulation development in the pharmaceutical industry. Following a year travelling abroad, in which she obtained experience of working in quality control, she briefly returned to the same field before embarking on an MSc in Analytical Science at the University of Hull.Moving slightly north, she obtained her DPhil from the University of York where her research was chiefly concerned with the use of indirect detection in capillary electrophoresis. Since then, she has maintained her interest in electroseparation science and is currently undertaking research in the field of capillary electrochromatography at the University of Leeds.Table 1 Landmarks in CEC First report of use of EOF in chromatography Strain 1939 Separation of polysaccharides using EOF through a colloidal membrane Mould and Synge 1954 Use of EOF in column chromatography Pretorius 1974 Electroosmosis in capillaries Jorgenson and Lukacs 1981 CEC in open-tubular columns Tsuda 1986 Theory of CEC and technique development Knox and Grant 1987, 1991 Analysis of pharmaceutical compounds by CEC Smith and Evans 1994 Analyst, July 1998, Vol. 123 (87R–102R) 87Rdiastereoisomers, which were very difficult to separate by conventional HPLC, were readily resolved by CEC (see Applications). Voltages up to 30 kV are supplied to generate the electric field usually for solutions of 1–50 mm buffers in aqueous reversed-phase mobile phases; non-aqueous CEC has also been carried out with ammonium acetate buffer.1 The dependence of EOF flow rate on solvent dielectric constant has been confirmed, but the electrical potential (the zeta potential) of the boundary between the fixed and diffuse layers (the double layer) of positive ions at the stationary phase wall (Fig. 1) is less well understood. The conclusion of a theoretical study by Rice and Whitehead which suggested that flat EOF profiles in a capillary of diameter d would result if d were considerably greater than the double layer thickness, d, has been confirmed by experiment; for channels between particles, however, the influence of d is less clear.Current indications are that it should be possible to use monodisperse particles with diameters down to 0.5 mm. Pores sizes of commonly used HPLC particles are too small to give rise to EOF, but larger pore packings show promise. Although CEC has been demonstrated for stationary phases bonded to the walls of open tubes, and in sol–gel derived phases, most work has been carried out on columns packed with HPLC stationary phases; a new generation of packings custom synthesised for CEC is, however, now beginning to make an impact.Principles of electroosmotic flow Electroosmosis is best described as the movement of liquid relative to a stationary charged surface under an applied electric field.2 Substances tend to acquire a surface charge as a result of ionization of the surface and/or by interaction with ionic species. In a fused silica capillary, the ionization of silanol groups gives rise to a negatively charged surface, which affects the distribution of nearby ions in solution.Ions of opposite charge (counter-ions) are attracted to the surface to maintain the charge balance whilst ions of like charge (co-ions) are repelled. The double layer of electric charge thus formed (see Fig. 1) is generally explained by a revised version of the Gouy–Chapman model, which is covered extensively in the literature.2–4 Essentially the counter-ions are arranged in two layers, fixed and diffuse, with a surface of shear at just beyond the interface.The voltage drop between the wall and this surface of shear is known as the zeta potential, z. In the diffuse layer, the potential falls exponentially to zero, and the distance over which it falls by e21 is known as the double layer thickness, d. When the voltage is applied, the solvated cations in the diffuse layer migrate towards the cathode, dragging the solvent molecules along with them. The linear velocity of the EOF, ueo, is best described by the equation shown in Table 3, which shows how the EOF is governed by changes in the dielectric constant and viscosity of the electrolyte and the zeta potential; z, itself, depends on the charge density and d, which is inversely related to the ionic strength of the electrolyte.The flow profile is assumed to be near-plug-like as essentially it originates from the capillary wall, but in reality it depends on the capillary internal diameter, d, and d.Theoretical studies by Rice and Whitehead5 proposed that ueo is only independent of the capillary diameter when d 9 d. As d approaches d, double layer overlap occurs with a simultaneous reduction in flow velocity, until finally a parabolic flow profile is obtained when d and d are similar. It has been proposed6 that the EOF velocity is acceptable when d � 10d. In CE, however, double layer overlap is unlikely to be a problem: for a salt concentration of 1 mm in water, the double layer thickness is calculated to be 10 nm.6 The use of microscope optics to image flow profiles in narrow capillaries has produced conflicting results.Taylor and Yeung7 have observed the plug Table 2 Comparison of electrically driven separation methods CE MEKC CEC Separation principle Different mobilities of ions in electric field Partition between bulk solution and micelle moving in opposite direction to analyte Partition between solid stationary phase and mobile phase Column diameter/mm 50–100 50–100 50–100 Stationary phase None None Silica or cellulose particles with bonded groups; bonded or imprinted polymeric matrices Mobile phase Electrolyte solution Electrolyte solution Electrolyte solution Sample type Charged species Neutrals Neutrals and charged species Table 3 Equations of note in microchromatography Pressure drive: u d P L p = 2D fh Electrodrive: u E eo o r = e e z h Resolution: R N k k s = - Ê Ë Á � � � + Ê Ë Á � � � 1 2 4 1 1 a a where dp = particle diameter, DP = pressure drop across column, f = column resistance factor, h = mobile phase viscosity, L = column length, eo = permittivity of a vaccum, er = mobile phase permittivity, z = zeta potential, E = electric field strength, N = number of theoretical plates, a = selectivity, k = retention factor Fig. 1 Double-layer structure at a silica wall. Reprinted with permission from ref. 15. 88R Analyst, July 1998, Vol. 123flow profile predicted from theory,6,8 whereas Tsuda et al.9 have not: they found the EOF at the capillary wall to be greater than that at the centre of the capillary.The importance of the EOF profile in CEC necessitates further research in this area. The EOF in CEC The current CEC development has much to owe to the theoretical and experimental papers published by Knox and coworkers6,10 –12 over the last 10 years. More recently, CEC has been reviewed by Colón and co-workers,13,14 Robson et al.15 and Kowalczyk.16 Crego et al.17 focused on the fundamental principles of CEC, whilst Dittman et al.18 gave an overview of the theory and practice of CEC and Rathore and Horváth19 compared HPLC, CE and CEC.With the exception of Ståhlberg,20 who considered the migration of charged species in CEC, theoretical treatments have mainly focused on neutral species. In packed CEC, both the capillary wall and column packing carry surface charges that are capable of supporting EOF. To date, most of the work carried out suggests that the column packing is responsible for the generation of EOF;21,22 there is a greater number of free silanol groups present since the solid packing has a far larger surface area compared with that of the internal silica wall.If the column is assumed to consist of a closely packed array of non-porous spherical particles, then the EOF arises from the channels between the particles. The average interparticle channel is estimated to be one-quarter to one-fifth the particle diameter.6,23 Knox and Grant6 subsequently suggested that, on the basis of the Rice and Whitehead treatment,5 the particle diameter should be no less than 40d if double layer overlap and subsequent loss of plug flow are to be avoided.With the ionic strengths typically used in CEC, namely 1–10 mm, this means that particle sizes as small as 0.4 mm can be utilised with little loss of EOF velocity. Since the particle sizes routinely used in CEC are typically 3 mm, there is considerable scope for the use of smaller particle diameters before double layer overlap becomes a problem.However, this is not the case for porous materials, where EOF generation can occur within the pores. Li and Remcho24 have studied the role of pore size in CEC using materials with pores ranging from 6 to 400 nm. Packing materials of large channel diameter ( > 200 nm) were found to be capable of supporting through-particle (perfusive) EOF. In addition, a significant increase in efficiency was observed.The EOF velocity in a CEC column is most likely to be reduced compared with that in an open tube, on account of the tortuosity and porosity of the packed bed. Although there does not appear to be any adverse effect as a result of packing irregularities,25 further investigations are now being made on packing structure using electrical conductivity measurements.26 The results have been promising in that the electrical conductivities obtained for open and packed capillaries can give an indication of the flow permeability of the column.However, a factor often overlooked in CEC is the contribution of the open capillary present: most packed capillaries have an open section for detection purposes. Choudhary and Horváth27 discussed both theoretical and practical aspects of having open and packed sections of capillary of differing conductivities, across which the voltage gradients, and hence electric field strengths, will vary.Their study found that having identical charge on both the capillary wall and packing material always resulted in a reduced EOF, which could not be explained readily. In conclusion, they suggested that the characterization of the individual column segments is necessary if a better understanding of the EOF in CEC is to be obtained. The use of open tubular columns in CEC has several advantages: the approach to understanding and generating the EOF is less complex, their fabrication is easier and they are far more robust.Columns have been prepared for reverse, normal and chiral separations, by applying the general procedures used in open tubular liquid chromatography (OTLC).28–30 Several groups have studied the role of surface modifications in open tubular electrochromatography (OTEC), in terms of both the EOF generated and the separations obtained. The EOF has been found to vary dramatically between capillaries ranging from untreated to those etched and coated with octadecylsilanol groups.30 Since the EOF arises from both the surface coating and residual silica, it is likely to be reduced compared with untreated capillaries as a consequence of effective shielding of the silanol groups.31 Although an enhanced EOF and improved peak shape could be obtained in polymer coated capillaries when using surfactants in the buffer,32 Francotte and Jung33 reported that these parameters were most likely dependent on the coating thickness.In contrast, Tan and Remcho34 demonstrated that the flow velocity does not exhibit an obvious trend with monomer and/or cross-linker concentration. However, they showed that the selectivity could be controlled by careful adjustment of the monomer and cross-linker concentrations, and by incorporation of other functional groups into the polymer matrix. A recent comparison of open tubular liquid chromatography and open tubular electrochromatography for chiral separations35 indicated that although OTEC exhibits greater efficiency and resolution due to the plug flow, OTLC remains the faster technique.How does CEC improve on HPLC? If existing HPLC analyses are to be replaced by CEC methods, the practising analyst must perceive substantial advantages along with at least equivalent performance in quantitative analysis. The first question likely to be asked is whether the undoubted increased efficiency, actual and potential, discussed above is relevant to a given analysis.For comparatively simple mixtures, increased theoretical plate numbers, N, may not always be required; many HPLC separations are achieved on the basis of selectivity, a, which along with N is the major factor influencing resolution, Rs (Table 3). Probably more relevant is the peak capacity, the number of peatogram between realistic retention factor limits. Clearly, CEC will offer substantial advantages here, and for very complex mixtures of, for example, biological compounds separation by CEC may become the method of choice.It has to be said, of course, that CE now separates many such mixtures with high resolution. Nonetheless, there is probably an analogy here with the progress of gas chromatography, where the advent of fused silica column technology offered the resolution necessary to make routine the analysis of complex fuel and environmental mixtures. Pressure drop across an HPLC column restricts the mobile phase flow rate, but in any case the well known rising graph of plate height, h, (i.e., decreasing N) against mobile phase velocity, u, is a considerable disincentive to shortening analysis times in this way.On the other hand, plug flow in CEC means that the plate height increases much less with increasing ueo, so that in principle shorter analysis times are possible without loss of resolution. It may be, however, that higher applied voltages than are currently commonly used may be necessary to achieve very high flow rates, and Choudhary and Horváth27 pioneered experiments with voltages up to 60 kV.An influential factor in CEC development is that charged analytes may be subject to separation by both electrophoresis and chromatography. The miniaturisation of HPLC has been driven by the necessity of analysing very small amounts (picomoles) of substance available, for example, in small volumes of body fluids or in the products of single-bead combinatorial chemistry. If small amounts are to be analysed, micro-HPLC (mHPLC) is carried out on packed capillary columns with diameter of 300 mm or less.It is comparisons of CEC with mHPLC that are Analyst, July 1998, Vol. 123 89R0 2 4 6 8 10 12 14 16 18 20 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Geometric diameter/mm d N/d d probably most meaningful, and the necessary development for mHPLC of robust, easily installed columns with reliable injection procedures and available gradient elution methods parallels the practical requirements of CEC for future routine use.The great test of CEC will be whether regulatory authorities will specify its use in analysis, especially of pharmaceuticals. Here, precision, accuracy, trace analysis and repeatability are vital, and promising results have already been obtained. Figures of merit for repeat analyses of a mixture of test compounds that are not dissimilar from those observed in HPLC analysis have been reported.Robson et al.36 showed that with both unpressurised and pressurised systems, highly repeatable separations can be obtained; for a series of injections of a test mixture, relative standard deviations were less than 1% for retention time and, typically, 1–3% for peak height and corrected peak area. In addition, retention time, column efficiency and retention factor have been demonstrated to remain essentially constant for at least 200 repeat injections on the same column.18,36,37 Still lacking, however, are convincing demonstrations of trace analysis for, say, impurities at the 0.1% level. Longer light-path flow cells are becoming available, and it may be that current experiments with low (or even zero!) electrolyte concentrations, and hence reduced ohmic heating, will permit larger column diameters to be utilised.Packing materials used in CEC Working from the proposition that the support material must have a large zeta potential, the materials used have been, and are mostly, HPLC supports that are not ‘end capped’.Traditionally, this has meant that the bonded phase is reacted in nonstoichiometric quantities on to the support; thus, in the case of a silica support, there are unreacted silanol groups left on the surface (Fig. 2) which are capable of generating EOF. An example of this type of material is the octadecylsilane ODS1 class of bonded phases that were developed in the late 1970s as the first HPLC phases.End capping is a process performed after the phase has been attached to the surface, to minimise the number of these residual silanol groups. For CEC, however, although the particle size is normally 3 mm, the silica itself, in terms of its physical characteristics and particle size distribution, is still the same as that developed for HPLC. The pore size of the supports is commonly of the order of 8–10 nm. This means that EOF flow will only occur on the outside of the particles as double layer overlap will occur in the pores.11 A variety of results have been reported from these HPLC derived phases: Table 4 outlines published results on the separation of PAHs using isocratic CEC.The differences shown, although not normalised in any way, are far greater than one would expect from HPLC comparisons and may result from the packing of these materials into narrow-bore(50–200 mm id) capillaries. Although they may be listed as 3 mm material, all the packings will have unique particle size distributions.In addition, in a manner analogous to the molecular size distribution of polymers, the distribution will vary according to how it is measured; currently there are three ways of characterising particle size distribution, namely number, area and volume. Manufacturers typically do not stipulate which method was used to characterise a particular stationary phase, and thus a nominal 3 mm material may vary from company to company.However, extremely noticeable in all of the number distributions is the presence of fine material below 2 mm (see Fig. 3), which is thought to impede the packing process. This material is very difficult to remove via the normal air classification used by manufacturers to produce different particle sizes. However, work by some manufacturers has led to new particle size distributions that are optimised for the packing procedures used in the packing of 50–200 mm fused silica capillaries.Monodisperse solid silicas are now available for use in CEC,42 but as yet there are insufficient data to compare these with porous silicas. The first specially manufactured phases for CEC were prepared by Myers and reported in papers by Smith and Evans.43 These were based on a new 3 mm particle size distribution silica and bonded with propylsulfonic acid. Efficiencies from this phase have been reported in terms of millions Fig. 2 Molecular model of the surface of a silica support that is not ‘end capped’. Key: yellow, silicon; white, hydrogen; red, oxygen; green, carbon. Table 4 Efficiencies obtained for isocratic CEC of PAHs using HPLC stationary phases Range of efficiencies Stationary phase (plates per metre) Ref. 3 mm Spherisorb ODS1 200 000–240 000 38 3 mm Nucleosil 100 C18 91 000–147 000 39 3 mm Spherisorb C18 PAH Up to 260 000* 36 3 mm Synchrom 102 000–138 000 40 3 mm Vydac C18 > 160 000 41 3 mm CEC Hypersil 240 000–280 000 21 * Calculated for a 50 mm id column of length 280 mm, dp 3 mm and a minimum reduced plate height of 1.3. Fig. 3 Number distribution of a 5 mm CEC silica. 90R Analyst, July 1998, Vol. 123of plates for the analysis of basic drugs.43,44 However, the reproducibility of these very high efficiencies is extremely poor and, because of the low carbon load, very short retention times are obtained for neutral compounds. At present, we are attempting to increase the hydrophobic nature of the support by bonding octadecyl groups on to the silica with the sulfonic acid (Fig. 4).These materials have yet to show the same high efficiencies on basic compounds, but promising results have been obtained for neutrals. New phases are also being developed, by adjusting the carbon chain length and the ratio of the alkane chain to the sulfonic acid, in an attempt to obtain a silica based phase that provides a good EOF over a wide pH range and provides good selectivity for neutrals.Other work on the effect of the particle size and the bonding of the stationary phase is being undertaken in the hope that the focusing effect can be understood and controlled, thus permitting the production of purpose made supports for CEC. Work has been reported on the use of wide pore material with pore sizes up to 400 nm.24 In these systems it has been shown that above 200 nm the materials are capable of supporting through-particle electroosmosis, which in turn results in a significant increase in efficiency.New CEC columns have been manufactured by polymerisation of either silica or polymers inside the column to produce a monolithic bead, which is then derivatised with the stationary phase.45 This technique removes the problem of the inlet and outlet frits that are required for producing particle columns, and is a more readily available technique to researchers who may not have access to the small particle size silicas produced by speciality manufacturers.Other monolithic capillary columns have been produced in a single step copolymerisation process,46 which allows fine control of the porous properties of the final column. The EOF through the column is dependent on the monolith pore size and the proportion of charged groups on the surface. The use of macroporous polyacrylamide–poly(ethylene glycol) gels in CEC has also been reported;47 in these gels the EOF is generated by a sulfonic acid group as opposed to the silanol on silica.Practical variables in CEC Since CEC is essentially a hybrid of CE and HPLC, there appear to be a large number of variables to consider before attempting any separations; the selection of the most appropriate conditions for an application is not for the fainthearted. It helps to go back to the basics of analytical chemistry and define the analytical problem, i.e., the nature of the sample, the end use of the results, the species to be separated and what information is required.Established HPLC or CE methods can provide a good starting point, but may not be ideal for a particular requirement. In addition, it is a good idea to have available such sample information as pKa values and solubility data, since these are often overlooked and may have significant implications for the analysis. The general theory of HPLC and CE can be found described in a number of texts,48–51 so only more practical considerations will be covered here. In HPLC, chromatographic separation is the result of specific interactions between sample molecules with the stationary and mobile phases.Hence it follows that these are the most important source of variables in HPLC, with mobile phase flow rate and column temperature playing a lesser role. At the heart of the separation is essentially the chromatographic column, which can be varied in both the physical dimensions (length, internal diameter) and the characteristics of the packing material (nature and quality of the stationary phase, particle size and porosity).The various components of the mobile phase (water, organic solvent, buffer, etc.) are adjusted to control such factors as solvent strength and viscosity. For CE, however, where separation is primarily based on mobility in an electric field, factors that affect the charge and effective size of the analyte and the magnitude of the EOF play the dominant role.In particular, the electrolyte pH is of primary importance since it affects the degree of ionisation of both the analyte and the silanol groups on the capillary wall. The physical dimensions of the capillary have typically taken a secondary role; capillaries of larger inner diameter tend to be used in cases where increased detection is required, and smaller capillaries when ohmic heating, which can adversely affect resolution and efficiency, needs to be minimised. In CEC, the fundamental driving force is the EOF, which is mainly influenced by parameters affecting the surface charge of the capillary column and the double layer thickness, i.e., the stationary phase properties and the mobile phase composition. In practice, EOF control is achieved most readily by selecting the required stationary phase type, e.g., chiral, ion-exchange, then varying mobile phase characteristics such as pH, concentration of electrolyte and proportion of organic solvent to give the EOF and separation required.Recent investigations into the influence of these parameters have been carried out by Li and Lloyd,52 Lelièvre et al.53 Dittman and Rozing,22 Euerby et al.,44 Kitagawa and Tsuda,54 Wan,55 Seifar et al.56 and Wright et al.57 For simplicity, an overview of their findings follows. The majority of CEC analyses have typically used capillaries (50–100 mm id) packed with 3 mm HPLC phases; although open tubular CEC has also been reported,30–35 it will not be discussed here.In CEC, the relationships which describe the variation of EOF are far less well defined than in CE. In part this can be attributed to the experimental conditions chosen; in many cases, there has been little attempt to keep all variables, except the one under investigation, constant. Subsequent results may be misleading. pH is a typical example of this: low pH buffers are often prepared by adjusting a higher pH buffer with acid, hence the decrease in EOF observed on going from high to low pH could be due to both an increased ionic strength and a decreased surface charge.In addition, the incorporation of organic solvents into the electrolyte can alter the ionisation equilibrium and analyte solvation. Adding organic solvents to the electrolyte generally shifts the pKa values of the surface silanol groups to higher values; this has been demonstrated in CE by Schwer and Kenndler58 and in CEC by Kitagawa and Tsuda.54 As expected, at high pH values ( > 9), where all the surface silanols should be dissociated, the EOF exhibits very little change.At low pH, however, substantial EOF has been demonstrated despite surface silanol groups being predominantly non-ionised.21,44 The pH dependence of the solute must not be ignored; CEC Fig. 4 Molecular model of sulfonic acid on a silica support. Key: yellow, silicon; white, hydrogen; red, oxygen; green, carbon; pink, sulfur. Analyst, July 1998, Vol. 123 91Rpermits the separation of both charged and uncharged species. Often it is beneficial to work with the analytes in their nonionised forms (subsequently referred to as the ion-suppressed mode), for example to minimise ionic interactions with the packing, or in the case where the analytes are negatively charged and would migrate away from the detector. Most of the work reported in CEC has employed low concentration buffer solutions in order to avoid ohmic heating effects; typical concentrations are 1–10 mm for inorganic salts such as phosphate and borate.For applied voltages in the range 5–30 kV, we have observed a virtually linear relationship between EOF velocity and electric field strength, implying that the heat generated is negligible at low electrolyte concentration. However, poor migration time reproducibility and ion depletion may occur as a result of the low buffering capability.59 The use of higher concentrations of low-conductivity zwitterionic buffers such as 2-morpholinoethanesulfonic acid (MES) and TRIS, is therefore to be recommended.In accordance with theory, the EOF in open capillaries was found to decrease as the buffer concentration was raised from 0.04 to 1 mm:55 the double layer thickness, and subsequently z, are reduced with increasing ionic strength. In contrast, the EOF in packed columns remained relatively constant as the electrolyte concentration was decreased, a factor attributed to double layer overlap.The EOF velocity was, however, seen to be dependent on the concentration of a salt (sodium chloride) added to the buffer to increase the ionic strength.60 Although the effect of changing the buffer and salt concentrations was different, this was hardly surprising since the concentration of sodium chloride added ranged from 0.01 to 0.45 m, concentrations rarely used in CE, let alone CEC. As we have observed in our work, the nature of the anion or cation influences the EOF rate; in this case the phosphate exerted a greater influence on EOF than did the chloride.In addition, tetrabutylammonium bromide had a significant effect on EOF whilst sodium dodecyl sulfate (SDS) did not. SDS is a surfactant which, although typically used to form micelles, can also be used as a dynamic surface modifier like tetrabutylammonium bromide. In CEC, SDS has been found to be effective in not only controlling but also stabilising EOF.56 The EOF was found to increase with increasing concentration of SDS, and was attributed to changes in zeta potential due to adsorbed SDS molecules.This behaviour can be explained more simply by considering the structure of the SDS molecule: the tails of the SDS molecules will interact with the stationary phase, thus leaving the negatively charged heads to impart more negative character to the surface, hence increasing EOF. The reversal of EOF in CE by the use of triethylammonium acetate (TEA) has also been demonstrated in CEC.52 Studies in open capillaries have shown that increasing the organic component in a buffer solution up to around 80% results in a decrease in the EOF58 for a variety of solvents; the overall reduction was found to be least for acetonitrile, and greatest for ethanol and propan-2-ol. A number of papers have demonstrated similar findings in CEC for mobile phases containing an added electrolyte,22,61 but only Wright et al.57 have reported behaviour in solvents without supporting electrolyte.They confirmed these results but showed that as the organic content approaches 100%, the EOF is further increased; the EOF typically passed through a minimum corresponding to an organic proportion of around 70–80%. Although the variation of the e/h ratio with solvent composition follows a similar pattern, the change in EOF velocity cannot be attributed to this alone. The extremely high EOF observed for CE separations in 100% acetonitrile, namely 17 31028 m2 V21 s21, is partially explained by solvatochromatic solvent polarity studies, which take into consideration hydrogen bond donor ability and polarizability.For practical purposes, these results illustrate that substantial EOFs can be generated in the absence of an electrolyte and in non-aqueous media. Euerby et al.62 have demonstrated two important HPLC concepts in CEC: (i) linearity between the logarithm of the retention factor (ln k) and the percentage of acetonitrile in a mobile phase containing TRIS buffer (50 mm, pH 7.8) over the range 50–80% acetonitrile; and (ii) isoeluotropic strength.From these findings it is evident that well established theories used in HPLC method development are directly applicable to CEC, as are HPLC optimization programmes. CEC Equipment The equipment required for CEC is very simple. Essentially a capillary electrochromatograph (Fig. 5) can be broken down into four main components: (a) a system for either electrokinetic or pressure driven injection; (b) a column in which EOF and chromatographic processes take place; (c) a detector; and (d) a high voltage power supply. Most CEC is performed on laboratory-built or modified CE equipment which has the option to pressurise one or both ends of the capillary column; at present, only Hewlett-Packard have produced a commercial instrument for CEC which allows the column to be pressurised.Although it has been found that with proper degassing of the mobile phase (using helium sparging) column pressurisation is not necessarily required, it is extremely useful for conditioning columns on the instrument and for method development.For long automated runs, where mobile phases are likely to need further degassing, it is essential. In addition, under these circumstances there may be appreciable solvcnt evaporation from the sample and mobile phase vials; to minimise the effect of solvent losses, the vial compartment should ideally be cooled.However, owing to solubility constraints of buffers in the mobile phase some compromise may be necessary. Performing reproducible CEC requires stringent control of parameters such as temperature, voltage and pressure if used. The use of commercial CE equipment that permits automatic control of these parameters has led to significant improvements in reproducibility. The majority of CEC analyses reported to date have used aqueous isocratic mobile phases with equipment similar to that described previously. Examples of non-aqueous isocratic CEC are far less documented despite their potential.This has been Fig. 5 Schematic diagram of a CEC instrument. 92R Analyst, July 1998, Vol. 123realised in a separation of PAHs and fullerenes using a mobile phase consisting of acetonitrile, methanol and tetrahydrofuran but no buffers.41 The elution of coronene and C70 in approximately 11 and 170 min, respectively, demonstrated the applicability of non-aqueous CEC to a whole range of petrochemicals not normally separated by reversd phase LC.In situations where an increased flow of mobile phase is desirable, the use of pneumatic pressure, applied at the injection end of the capillary, should be considered. The idea was developed initially by Tsuda,63 and later by Dekkers et al.,64 who combined it with electrospray mass spectrometry where an increased flow was required. This approach decreased retention times without compromising resolution, and appeared to reduce bubble formation.The use of continuous gradient CEC techniques is increasing, despite the current lack of commercial instrumentation. Generally the systems used are of two types. In the first, a conventional gradient LC pump supplies a changing mobile phase across the CEC column inlet and the EOF drives it through the column.65 In the second, two high-voltage power supplies are used to control the EOF in two different mobile phases which then mix and enter the column.66 Although the use of the LC pump gives control of the mobile phase composition, it wastes mobile phase and may impose a small pressure driven flow, which will distort the plug flow profile. However, whilst no mobile phase is wasted in the EOF flow controlled system, the exact composition of the mobile phase as it enters the column is unknown.Results on both systems have been extremely encouraging.An alternative approach to continuous gradient, which can be performed on normal instrumentation, is that of the step gradient. This is achieved by changing the inlet vial during the chromatographic run with a new buffer vial containing a different mobile phase. Euerby et al.67 applied this technique to the analysis of a mixture of six diuretics of widely differing lipophilicity; the rapid separation they obtained demonstrated the advantage of step gradient over isocratic conditions.Column packing techniques Fused silica capillary columns are mainly packed using either supercritical CO2 36 or an organic solvent11,59 at pressures of up to 600 bar. One end of the fused silica capillary is connected to a packing reservoir containing packing material (approximately 200 mg) and the other is connected to a 1/16 in union containing a sintered metal frit. Alternatively, a retaining frit made at one end of the capillary may be substituted for the union when packing with organic solvents.The reservoir is then connected to a high-pressure pump by means of a high-pressure valve that allows the introduction of the solvent into the packing reservoir, thus transporting the packing material into the column. After the column has been packed to the required length, as seen under a microscope, the column is disconnected from the reservoir and flushed with distilled water. Two other methods that have been used to pack capillary columns are centrifugal and electrokinetic packing, but they have not gained widespread use.It must be mentioned, however, that there are several commercial suppliers of packed capillaries for CEC. These offer a variety of column dimensions to suit different instrument configurations, and a wide selection of packing materials and particle sizes. In addition, many companies offer a service in which they will instal the capillary column into the instrument cartridge sent to them, albeit at a cost.Frit manufacture and bubble formation There are three principal methods which have been used to form frits: the reaction of sodium silicate solution with formamide to form a porous silica plug41,68 and the use of either an electrical heating element27,59 or micro torch27,40 to fuse the stationary phase. The electrical heating element is the preferred method owing to its ease of use and the reproducibility of the frits formed. The heating element is made from a few turns of resistance wire mounted on a thermocouple plug, and can be powered by simply a battery or a more sophisticated power supply, with time and current control.This technique relies on the stationary phase having a high sodium content, e.g., Spherisorb material which contains approximately 1500 ppm Na, which may not be found in the newer types of silica manufactured from tetraethoxysilicate. The frits are made by heating the silica stationary phase to a temperature sufficient to form a porous sodium silicate plug.Columns produced using this sintering process for the frits are generally based on variations of the following procedure. The capillary column is connected to a HPLC pump (Fig. 6) and flushed with water at approximately 100 bar for about 60 min. With the HPLC pump still on, a frit is formed near the column outlet. The second frit is formed at a distance back from the first frit according to the dimensions set by the CEC instrument and required detection mode, and the pump switched off.Detection can be made either through the frit, through a packed section of capillary or just below the outlet frit on an unpacked part of capillary; in the last case any excess packing material must be removed by flushing. Prior to use the column is conditioned with the required CEC mobile phase (degassed with helium or by vacuum) for at least 60 min and until no bubbles are observed leaving the column. The problem of bubble formation in a CEC column is undesirable since it may lead to the breakdown of current and subsequent loss of EOF.In addition, it may cause the column to dry out, although this can be rectified by reflushing the column with mobile phase at high pressure. Bubbles are thought to arise within the packed section of the capillary or the frits as a result of either ohmic heating in the column, or a change in the EOF velocity on moving from the packed bed through the frit into the open capillary.Bubble formation is typically observed when using non-pressurised systems but can be minimised by proper degassing of mobile phases (helium sparge) and careful selection of buffers. The use of low conductivity buffers, such as TRIS and MES, is to be recommended since they are zwitterionic in nature and thus can be utilised at higher concentrations without contributing significantly to ohmic heating. It has also been suggested that a higher proportion of organic solvent in the mobile phase could reduce self-heating and bubble formation.53 Column coupling Conventional CEC capillary columns are relatively fragile and prone to breakage owing to having a frit close to the detection window.A technique that overcomes these limitations is to use a separate detection cell from the chromatographic column. This consists of a conventional chromatographic column that is sealed with frits at each end, but is coupled to the detector cell Fig. 6 Schematic representation of apparatus used for conditioning capillaries. Analyst, July 1998, Vol. 123 93Rcapillary by a PTFE connector. The connector is manufactured from 1/16 in PTFE tubing 10–15 mm long with an interference fit hole for the fused silica capillary. With both the packed capillary and detector cell each pressed half way into the PTFE sleeve, it is possible to operate the PTFE connector at pressures up to 6 bar. Alternatively, 340 mm id Teflon tubing is commercially available. CEC detectors Detection in CEC is principally UV/VIS detection through the unpacked part of the capillary where the polyimide coating has been removed.This has several limitations: the small pathlength of the cell, the fragility imposed by the removal of the polyimide coating and the fact that detection takes place after the frit. Extended light path cells, such as the ‘bubble’ and ‘Z’ type cells developed by Hewlett-Packard for capillary electrophoresis, are available and do give increased sensitivity; their use in CEC is illustrated in the Applications section.Detection through the packed column is possible but we have found that baselines are typically more noisy owing to scattered light. One cannot, however, ignore the data collection system; with the high efficiencies observed, e.g., for bases on SCX stationary phases, it is imperative to use the correct sampling and detector data capture rates if the loss of valuable peak information is to be avoided.Euerby et al.69 have found the peak efficiency values to be highly dependent on the detector rise time employed. The use of fluorescence detection for PAH determinations has been reported by Rebscher and Pyell39,70 for capillaries ranging from 50 to 150 mm id and by Yan et al.40 In the latter case, laser-induced fluorescence was used to evaluate both onand off-column detection methods. Limits of detection were1029 –10210 m, with efficiencies of up to 400 000 and 150 000 plates per metre being obtained for on- and off-column detection, respectively.Since the first demonstrations of CEC–mass spectrometry (CEC–MS),71 the technique has developed rapidly into a powerful analytical tool. The extremely low flow rates ( < 1 ml min21) encountered in CEC can be utilised readily since an electrospray source typically requires a liquid make-up flow of 0.75–500 ml min21, and have allowed the direct coupling of electrospray (ESI) and atmospheric pressure ionisation (API) MS sources.This approach has been used successfully for the determination of sulfonamides,64 fluticasone propionate and cefuroxime axetil72,73 and non-ionic disperse textile dyes.74 Mixtures of benzodiazepines, corticosteroids and thiazide diuretics have been separated by gradient CEC with UV absorbance and ESI-MS detection.75 Gordon et al.76 have reported on the use of a FAB probe for the separation of steroids; the detection of [M + Na]+ peaks did not represent a problem even when a sodium-containing buffer was used.However, a loss of chromatographic resolution was observed on account of dispersion in the unpacked section of capillary used for coupling to the mass spectrometer. A novel solution to this problem has been designed and evaluated by Lane et al.73 Their CEC–ESI-MS interface incorporated a working CEC within the interface probe, thus allowing short columns and high electric field strengths to be employed.A significant improvement on previous CEC–MS systems has also been demonstrated by Schmeer et al.42 In their system, which uses a Sciex API mass spectrometer, the CEC column is placed directly in the MS source, thus eliminating the need for extra make-up flows or connecting capillary. CEC application review Since the re-emergence of CEC in the early 1990s, workers have mainly concentrated on establishing reliable column packing technologies36,43,59,77,78 and investigating the theory and mechanism underpinning CEC.6,11,18,21,38 Only in the last 3 years have we seen a rapid increase in the number of presentations and publications relating to CEC.Despite there being numerous separation techniques which can be classified under the general term of electrochromatography, this review of applications will be restricted to those involving EOF driven separations in packed capillaries only. The review will not cover pseudoelectrochromatography71,79 in which the hydrodynamic flow is augmented by the application of positive or negative voltage along the column, or open tubular CEC, in which a thin film is coated on the internal surface of the capillary.80 In addition, this review will only highlight the major advances associated with CEC applications and will not report on conference presentations, details of which are often not available in the open literature.After a brief introduction to the current scope and applicability of analysing various chemical functionalities, the review will be divided into chemical classes/application fields and, finally, cover the more specialised areas of chiral analyses and the analysis of compounds from various matrices.Scope and applicability of CEC Neutrals To date, most of the reported applications of CEC have been devoted to the analysis of neutral species of widely differing structures. Neutral species are particularly amenable to CEC because they can be chromatographed over the pH range 2–9. The pH of the mobile phase has been typically > 7 in order to promote a high EOF due to increased silanol ionisation.Older type HPLC stationary phases have generally been employed as they are ‘unendcapped’ and possess a large number of acidic silanol groups. However, certain manufacturers have now begun to market various reversed phases specifically for CEC use. Acids Acidic analytes which are separated in their ionised form tend to migrate towards the anode, i.e., against the EOF, and are either not loaded on the column during electrokinetic injection or are not swept towards the detector.Hence they may not be detected. In order to chromatograph acids successfully by CEC, a mobile phase pH must be employed which will allow the separation of the acids in their ion suppressed mode, i.e., as neutral species. As a consequence of using acidic mobile phases, the EOF and hence linear velocities are reduced, e.g., linear velocities obtained are typically in the region of 0.75 mm s21 compared with 1.5 mm s21 at pH 7.8.However, successful and rapid separations of acidic diuretics (anti-inflammatory arylpropionic acids) are still possible.44 Recently we have demonstrated the rapid separation of acids in their ion suppressed mode at acidic pH by the use of mixed mode phases which possess a C-alkyl and a sulfonic acid group bound to the same silica particle. The presence of the sulfonic acid group, which is ionised at all workable pH values, generates a good EOF over a wide pH range, thus enabling extremely rapid analyses to be performed without sacrificing the partitioning capacity of the phase.69 Bases The separation of basic analytes by CEC is problematic since, in order to generate a good EOF, a silica which is acidic in nature is required.This, however, causes severe peak tailing of basic analytes due to strong secondary interactions of the base with the ionised silanol groups.43 We have recently reported that, in an analogous manner to HPLC, these interactions can be minimised by the incorporation of triethylamine (which competes with the analyte for the silanol groups) in the mobile phase 94R Analyst, July 1998, Vol. 123(see Fig. 7 and 8). In addition, we have shown that certain basic drugs can be successfully chromatographed on a C-phenyl phase (see Fig. 7) when an unacceptable peak shape is obtained with a C18 bonded phase under similar conditions.It is suspected that the way in which the phenyl and C18 are bonded to the silica (proprietary information) restricts access of certain bases to the surface silanols in the case of the phenyl phase; this may shed new light on the way in which phases should be designed. Another alternative, depending on the pKa of the base, is to run them in their ion suppressed mode. Recently we have had reasonable success with analysing a basic drug candidate with a pKa of 8 at a pH of 9.3 on a C18 type bonded phase developed for CEC.Bases, not surprisingly, have been found to behave in a similar fashion on both the mixed mode phases and traditional C18 materials. Smith and Evans43 reported a possible solution to the analysis of basic drugs by using a strong cation-exchange stationary phase. An ‘on-column focusing’ phenomenon of the bases produced efficiencies of up to 8 3 106 plates per metre for the separation of tricyclic antidepressants, whereas concomitant application to neutral species only produced efficiencies comparable to those seen in reversed-phase CEC.These staggering efficiencies have also been obtained by other workers for a range of structurally diverse bases. However, all workers have experienced severe irreproducibility of the phase in that severe tailing and fronting have been unexpectedly observed in the middle of successful runs.44 Maruska and Pyell81 recently described a new cellulose based stationary phase (C18 Granocel-14Sh) which gave a good peak shape for the basic analyte pyridine.In comparison, under identical conditions using a silica based C18 phase, pyridine exhibited severe peak tailing due to the interaction with residual silanol groups. This indicated that the cellulose phase may be useful for the separation of bases, although in comparison with silica based phases low peak efficiencies were observed. Applications Environmental Polyaromatic hydrocarbons (PAHs) were one of the first types of compound class to be separated by CEC and as a consequence there are many published examples.40,82The separation of these PAHs by CEC is typified by efficiencies 75% higher than with HPLC.40 Recently it has been performed using non-aqueous CEC without the incorporation of electrolytes. 57, 83 The separation of 16 PAHs was attempted using a C18 type stationary phase and an acetonitrile (MeCN)–water composition of 80 + 20 (v/v): the addition of water was necessary to provide sufficient partitioning into the C18 phase.Although linear velocities comparable to those seen with conventional CEC were obtained, two pairs of co-eluting peaks still remained. CEC has also been shown to be beneficial for the separation of a range of triazine herbicides. Using a range of stationary phase materials, the new C6/SCX mixed mode phase appeared to exhibit the best selectivity for a given mobile phase composition of 1 + 1 (v/v) MeCN–25 mm sodium acetate (pH 8).22 In addition, polychlorinated benzene derivatives84 and phthalates38 have been separated.Pharmaceutical The pharmaceutical industry has been one of the driving forces for the development of CEC as the technique offers the potential for a separation mechanism orthogonal to the ubiquitously used HPLC. This, combined with highly efficient and rapid separations of complex mixtures, makes CEC an attractive adjunct to the conventional chromatographic methods employed. To date, most of the separations reported have not utilised the combined separation mechanisms of electromobility and partitioning; instead, workers have focused their attention on using the ion suppressed mode, thus utilising the partitioning mechanism only.This is partly due to the lack of suitable stationary phases that enable analytes to be run under conditions of high EOF whilst maintaining a satisfactory peak shape. Given these limitations, CEC has found a strong hold in the analysis of drug substances and intermediates such as cephalosporin antibiotics, 85,72 barbiturates,69 prostaglandins,85 diuretics,43,44,53 steroids, 43,44,62,73,76,85 macrocyclic lactones,73 C- and N-protected peptides,42,44 nucleosides and purine bases.44 Phthalates38 and parabens21 have also been successfully chromatographed by CEC using the standard conditions of a reversed phase column possessing a high proportion of acidic silanol groups and a mobile phase of pH !6.CEC appears to be of particular use in the early stages of drug discovery where rapid method development is essential and the demands of validation are less stringent. In nearly all cases for neutral and acidic compounds, using the ion suppressed mode where we were able to separate the components more efficiently and quickly, the development time was dramatically reduced. Two C- and N-protected tetrapeptides which differ in only the methylation of one amide function were found to be separated in less than 4 min using the standard CEC test chromatographic conditions which are employed to check the performance of our Fig. 7 CEC of basic drug candidate (I) using (a) 60 + 20 + 20 acetonitrile– TRIS (50 mm, pH 7.8)–water, 30 kV, CEC Hypersil C18 column (250 mm 3 100 mm id); (b) 60 + 20 + 20 acetonitrile–NaH2PO4 (50 mm, pH 2.3)–water, 30 kV, Hypersil C-Phenyl column (250 mm 3 100 mm id). Fig. 8 CEC of basic analytes using 60 + 20 + 20 acetonitrile–NaH2PO4 (50 mm, pH 2.3)–water with 0.1% v/v triethylamine, 30 kV: (a) basic drug candidate (I), (b) benzylamine. Analyst, July 1998, Vol. 123 95Rcapillaries. In comparison, the HPLC method previously used involved a 30 min gradient. Since the quantitative results from both techniques were comparable, the CEC method was therefore the obvious choice.44 We have shown that method development in CEC can be easily automated on the current commercially available CEC instrumentation.In addition, the chromatographic theory, central to computer optimisation in HPLC, holds in CEC for analytes using the ion suppressed mode.44 This enables method development to progress at a rapid pace. Miyawa et al.86 extended this work to show the usefulness of a modified central composite design to optimise the CEC separation of the antibacterial 3-[4-(methylsulfinyl)phenyl]-5S-acetamidomethyl- 2-oxazolidinone from its three related S-oxidation products.The variables included in the investigation were applied potential, volume fraction of MeCN and buffer (TRIS) concentration. The end result was the development of a rugged CEC method for the separation of the antibacterial from its thioether, sulfone and sulfoxide diastereoisomer on a 3 mm C18 bonded phase in 9 min. Steroids Steroids seem to be particularly amenable to separation by CEC; possibly the best example is that of the separation of the corticosteroid tipredane from its diastereoisomer and five related substances.44 As can be seen in Fig. 9, baseline separation of tipredane (14) from its diastereoisomer (15) was achieved using standard CEC conditions with no method development; in contrast, HPLC failed to achieve the baseline separation of the diastereoisomers despite extensive stationary and mobile phase optimisation. Boughtflower and Smith’s groups have also demonstrated the effectiveness of CEC, in this case for the separation of the synthetic corticosteroid fluticasone. 73,85 Subsequently, many reports have augmented these findings that steroids of widely differing structure and from differing sources can be successfully separated with higher efficiencies than by HPLC, using the traditional silica reversedphase materials with MeCN and a mobile phase pH > 6. Lord et al.87 highlighted the use of CEC in the separation of bufadienolide (bufalin, cinobufagin and cinobufatakin) and cardenolide (digoxigenin, gitoxigenin and digitoxigenin) steroids containing sugar residues and subsequently went on to couple this method with MS.There have been several reports on the separation of endogenous steroids such as testosterone, 17-a-methyltestosterone and progesterone56,88 and many synthetic corticosteroids such as triamcinolone, hydrocortisone, cortisone, methylprednisolone, betamethasone, dexamethasone, adrenosterone, fluocortolone, triamcinolone and triamcinolone acetonide by CEC.75 We have recently reported the use of a short-end injection technique with reverse polarity to achieve rapid analysis of extremely lipophilic steroids.89 This technique facilitated the separation of budesonide and related steroids in approximately 1 min.The attractiveness of this approach resides in the fact that most of the voltage drop occurs over the short packed capillary rather than over the entire capillary, and therefore higher EOF values are obtainable. Diuretics Euerby et al.44 and Taylor and Teale75 have both reported good chromatography of thiazide diuretics using CEC.However, we have found that the CEC must be performed at a pH of 2.5 to ensure that the acids are in their ion suppressed mode.44 As can be seen in Fig. 10(a), six thiazide diuretics could be successfully separated. In view of the reduced EOF it would beneficial to Fig. 9 Separation of tipredane (14) from its diastereoisomer (15) and structural analogues (11–13, 16). Electrochromatography was performed on an unpressurised HP3D CE system using a 3 mm Spherisorb ODS1 column (250 mm 3 50 mm id), 80 + 20 acetonitrile–TRIS (50 mm, pH 7.8) buffer, 15 kV, capillary temperature 15 °C.Reprinted with permission from ref. 44. Fig. 10 CEC separation of the diuretics chlorothiazide (1), hydrochlorothiazide (2), chlorthalidone (3), hydroflumethiazide (4), bendroflumethiazide (5) and bumetanide (6). (a) Isocratic separation, CEC Hypersil C18 column (250 mm 3 50 mm id), 40 + 20 + 40 acetonitrile– Na2HPO4 (50 mm, pH 2.5)–water.(b) Step gradient, column as for (a): 0–6.50 min, 40 + 20 + 40 acetonitrile–Na2HPO4 (50 mm, pH 2.5)–water; 6.50–17.25 min, 60 + 20 + 20; 17.25–25.00 min, 40 + 20 + 40. (c) Continuous gradient, Spherisorb ODS1 column (250 mm 3 100 mm id): mobile phase A, phosphate buffer (5 mm, pH 2.3); mobile phase B, phosphate buffer (5 mm, pH 2.3)–acetonitrile (20 + 80). (a) and (b) reprinted with permission from ref. 67. 96R Analyst, July 1998, Vol. 123investigate the use of a mixed mode phase. In addition, it is apparent from Fig. 10(a) that the six compounds possess widely differing octanol–water partition coefficients (log P values) and that a gradient CEC would be preferential. Although commercial CEC systems will allow automated step gradients67 to be performed [Fig. 10(b)], from a pharmaceutical viewpoint it would be advantageous to perform continuous gradient CEC. There have been several reports of laboratory-built gradient CEC systems in which the CEC capillary takes the changing mobile phase composition on demand;66,75,88 since there is no pressure flow down the CEC, capillary plug flow should be maintained.Fig. 10(c) illustrates the separation of diuretics using a prototype of a commercial gradient CEC system. Bases Owing to the severe problem of peak tailing associated with the separation of bases using traditional reversed-phase silica materials, only limited examples have been published. Taylor and Teale,75 however, reported the separation of two benzodiazepines (diazepam and nitrazepam) using a C18 type phase and an ammonium acetate–MeCN mobile phase.Under isocratic conditions the resultant peaks were broad and exhibited tailing, whereas under gradient conditions the peaks were more gaussian in appearance owing to the gradient effect on the tail of the peak. Other examples of successful separations of bases, notably those with highly efficient peaks, have been dealt with in a previous section.Biomolecules Amino acids Huber et al.88 have shown that reversed-phase gradient elution CEC is particularly suited to the separation of phenylthiohydantoin( PTH)-amino acids from the classical Edman degradation of peptides with phenyl isothiocyanate. Twelve PTH-amino acids were separated on a 3.5 mm C18 type packed capillary using a non-optimised gradient of 30–60% (v/v) MeCN with 5 mm phosphate, buffer (pH 7.55) (see Fig. 11). PTH-arginine, being positively charged, exhibited slight peak tailing due to interactions with the charged silanol groups, whereas the negatively charged PTH-aspartate and PTHglutamate did not elute. Peptides and oligosaccharides Horváth et al. have shown the worth of CEC for the separation of various peptides (tetra[Trp–Met–Asp–Phe] and pentapeptides [Trp-Gly–Gly–Phe–Met]) by the use of a 8 mm gigaporous (1000 Å) PLSCX (strong cation exchanger on highly crosslinked styrene-divinylbenzene particles) material with an MeCN–25 mm phosphate mobile phase.27 Palm and Novotny47 have shown that monolithic stationary phases based on macroporous polyacrylamide and poly(ethylene glycol) are suitable for the separation of various enkephalin derivatives and 2-aminobenzamide derivatised maltose oligosaccharides (Glu 1 to Glu 7) using an MeCN–TRIS–borate buffer (pH 8.2) mobile phase (see Fig. 12). Endogenous steroids As seen in the previous section, steroids of endogenous origin are particularly amenable to separation by CEC.Separation of analytes from various matrices by CEC Bioanalysis Until recently, the effectiveness of CEC in the analysis of biological samples has not been fully exploited. One of the major challenges for CEC is in the field of bioanalysis, where concentrations are usually low and the sample may contain varying types and amounts of endogenous interferences. Given the fact that the surface area of the packing material is small, then the likelihood of fouling the column increases.However, various workers have been able to show that CEC can be successfully used to separate a range of compounds from various biological matrices, e.g., urine and plasma from differing species. Taylor et al.90 have shown that corticosteroids (adrenosterone, hydrocortisone, dexamethasone, fluocortolone) in extracted horse urine and plasma can be separated by CEC using a laboratory-built gradient elution CEC system.The separation is performed on a 50 mm capillary packed with 3 mm C18 bonded material, using an MeCN–5 mm ammonium acetate gradient varying from 9 to 80% MeCN. In order to prevent early deterioration of the packed capillary, the urine samples were purified by a C8 followed by an SAX solid-phase extraction stage; in contrast, the plasma samples were purified by dialysis. The capillary was shown to be perfectly serviceable and efficient after over 200 injections of horse urine extract.A major advantage of CEC over HPLC was that the interferent, which eluted near the peaks of interest in HPLC, eluted near the EOF and well clear of the steroids in CEC, indicating that the interferences may have an amine functionality. The determination of hydrocortisone in equine urine by CEC with UV detection, after administration of tetracosactrin acetate, was shown to compare favorably with an in-house validated LC–MS method. The metabolite of hydrocortisone (20b-dihydrocortisone) was also detected (see Fig. 13). The detection levels achievable were well below that required by the regulatory bodies, and the reproducibility of the method was acceptable in terms of the precision obtained on automated runs; RSD values were typically below 2 and 7% for retention time and peak area, respectively. Paterson et al.91 elegantly illustrated the combined power of CEC and MS for the determination of a potential drug candidate Fig. 11 Capillary electrochromatography of PTH-amino acids with gradient elution. 3.5 mm Zorbax ODS column (207/127 mm 3 50 mm id): mobile phase A, phosphate buffer (5 mm, pH 7.55)–acetonitrile (70 + 30); mobile phase B, phosphate buffer (5 mm, pH 7.55)–acetonitrile (40 + 60); 0–100% B in 20 min. Reprinted with permission from ref. 88. Analyst, July 1998, Vol. 123 97Rin extracted plasma. Using a mixed mode C18–SCX phase and a mobile phase of MeCN–25 mm ammonium acetate (75 + 25) (pH 3.5), 13 structurally related compounds were separated from the parent drug candidate in 8 min.The plasma samples were purified by C2 solid-phase extraction prior to CEC–MS and an internal standard was employed. This resulted in an RSD of 1.7% over the whole concentration range, which was excellent considering the manual injection method employed. The power of CEC–MS was illustrated by the fact that severe co-elution would have occurred if only UV detection had been employed.A detection level of 1 ng ml21 could be routinely measured owing to the use of a peak stacking technique, which injected as much as a third of the column interstitial volume. This preconcentration technique in conjunction with increased pathlength detection cells should facilitate even lower detection limits (see Fig. 14). CEC has been successfully used for the separation of complex mixtures of neutral isomeric compounds derived from the in vitro reaction of carcinogenic hydrocarbon (benzo[g]- chrysene and 5,6-dimethylchrysene) dihydrodiol epoxides with calf thymus deoxyribonucleic acid (DNA).92 CEC demonstrated higher resolution, greater speed and lower analyte consumption than conventional HPLC.The use of a manual three-step gradient on a 3 mm C18, 75 mm id capillary using various proportions of MeOH, MeCN, THF and 6 mm ammonium acetate further improved the speed of analysis. This work was further extended by coupling the method with MS for the determination of two DNA adducts of acetylaminofluorene deoxyguanosine (AAF-dG) and G4 DNA.In order to achieve the detection level required, a 7 min injection was used with concomitant peak focusing. The use of such a high loading illustrated the potential advantage of using nanospray MS coupled with CEC.93 These examples indicate the potential of CEC for high speed, high sensitivity multi-component analyses on very small sample volumes. Plant origin The best example in this area is that of the difficult separation of triglycerides from various sources by CEC.1 By the use of a 3 mm C18 type material and the novel use of MeCN–propan-2-ol– hexane (57 + 38 + 5) plus 50 mm ammonium acetate as the Fig. 12 (A) Isocratic CEC of maltooligosaccharides using a capillary filled with a macroporous polyacrylamide–poly(ethylene glycol) matrix, derivatized with a C4 ligand and containing vinylsulfonic acid (effective column length 250 mm). Mobile phase 10 mm TRIS–15 mm boric acid (pH 8.2), acetonitrile content not stipulated.(B) Same analysis as (A), including the peak of the derivatization agent (14–16 min). Reprinted with permission from ref. 47. Fig. 13 Gradient CEC of equine urine samples after administration of tetracosactrin acetate and extracted by SPE. (A) 2 h post-administration; (B) 12 h post-administration. DH = 20b-dihydrocortisone; H = hydrocortisone, and A = adrenosterone (internal standard). Reprinted with permission from ref. 90. Fig. 14 Comparison of standard through capillary detection using (a) a 100 mm id fused silica capillary and (b) an extended pathlength flow cell. CEC separation of standard test mixture (thiourea, benzamide, anisole, benzophenone, and biphenyl) using a Hypersil C-Phenyl phase and standard test conditions [mobile phase composition acetonitrile–TRIS (50 mm, pH 7.8) (80 + 20)]. 98R Analyst, July 1998, Vol. 123isocratic mobile phase, the separation of triglycerides from over 30 samples of vegetable oils, foods, soya lecithin extracts and pharmaceuticals was successfully achieved.In contrast to reversed-phase HPLC, which does not separate the triglyceride isomeric forms of OLL and LLL, CEC yielded near baseline separation. As can be seen from Fig. 15, the separation of evening primrose oil by CEC resulted in better resolution in a shorter analysis time than using HPLC. In addition, separation of testosterone esters in a formulation based on peanut oil was demonstrated.Miscellaneous applications Li et al.94 have reported the interesting use of ion-exchange CEC for separating iodide, iodate and perhenate ions from the Hanford nuclear site environment. A 5 mm strong anion exchanger was used in conjunction with a 5 mm phosphate buffer (pH 2.6), and since the analytes were anionic, the polarity was reversed in order to sweep them past the detector. The analytes were easily separated but eluted in a different order to that observed in CE; this change in elution was easily rationalised on the basis of ion chromatography theory.Efficiencies were shown to be much better than with either HPLC or CE and detection was 20 times better than with the latter. It is believed that the anions experience a focusing effect as a decrease in efficiency was observed for higher mass loads. Chiral The technique of using CEC for chiral analyses has attracted much interest; it was expected that chiral selectivity would not be so important since the high efficiency associated with CEC would compensate for any short fall in selectivity.As in HPLC, investigations into the applicability of using chiral stationary phases and chiral mobile phase additives have been pursued. Mobile phase additives The feasibility of this approach has been established using hydroxypropyl-b-cyclodextrin as the mobile phase additive in the chiral separation of chlorthalidone and mianserin by CEC.53 Baseline separation was achieved but excessive analysis times were required.Protein phases CEC has been used with an immobilised a1-acid glycoprotein (AGP) on a silica based stationary phase for the enantiomeric separatation of racemic hexobarbital, pentobarbital, benzoin and cyclophosphamide.61 The separation efficiencies were slightly higher than those obtainable with chiral HPLC, but did not approach those seen with achiral CEC. The benzodiazepines temazepam and oxapam have been reported to be resolved using human serum albumin immobilised on a 7 mm silica.However, efficiencies were found to be very low and the EOF was lower than that of the AGP column.95 Cyclodextrin phases Hydroxypropyl-b-cyclodextrin as a chiral stationary phase has been shown to result in the baseline separation of chlorthalidone and the partial separation of mianserin.53 Li and Lloyd52 successfully used the standard b-cyclodextrin chiral stationary phase to separate a range of racemic 2,4-dinitrophenylamino acid derivatives, benzoin, dansylthreonine and hexobarbital.In order to separate the anionic analytes, triethylamine was incorporated into the mobile phase and the polarity of the applied voltage was reversed. Once again the expected high efficiencies were not observed. Pirkle type phases Wolf et al.96 have reported on the success of chiral CEC using (S)-naproxen derived and (3R,4S)-Whelk-O chiral stationary phases which were immobilised on 3 mm silica supports and packed into 100 mm id fused silica capillaries.Once again simple mobile phase compositions of MES (pH 6) buffer– MeCN were used to obtain efficiencies in the region of 200 000 plates per metre. Excellent enantiomeric selectivity with all of the 10 structurally diverse neutral analytes was achieved on these columns in run times of less than 10 min (see Figs. 16 and 17). Surprisingly, TRIS buffer failed to give satisfactory baseline stability. Molecular imprinting techniques Recently, molecular imprinting techniques have been used to produce chiral separation media used for CEC.These ap- Fig. 15 Triglyceride analysis of primrose oil by (A) micro-LC and (B) CEC. (A) Column, 50 cm 3 320 mm id FSOT, BioSil C-18 HL, 5 mm; mobile phase, acetonitrile–propan-2-ol–hexane (57 + 38 + 5). (B) Column, 25 cm 3100 mm id FSOT, Hypersil ODS, 3 mm; mobile phase, acetonitrile– propan-2-ol–hexane 57 + 38 + 5)–50 mm ammonium acetate. Reprinted with permission from ref. 1. Analyst, July 1998, Vol. 123 99Rproaches represent an interesting complementary alternative to conventional chiral selectors. Nilsson et al.97 have described the in situ preparation of a monolithic phase in fused silica capillaries based on (R)- propanolol molecular imprinted polymers to separate several badrenergic antagonists into their enantiomers (see Fig. 18). As expected, the phase exhibited the best chiral selectivity for propanolol itself. However, reasonable enantiomeric separation of rac-prenalterol, rac-atenolol and rac-pindolol was also achieved.Efficiencies in the region of 35 000–70 000 and 5000–20 000 plates per metre for the first and last enantiomers, respectively, were obtained. In a recent communication the same group has reported chiral separation of propanolol in less than 120 s by using one of these types of phases.98 Lin’s group have reported numerous examples of using molecular imprinted polymer stationary phases, either monolithic in nature or the conventional packed type, for the enantioseparation of a range of derivatised and underivatised amino acids.The peaks obtained by CEC were much sharper than those obtained by HPLC, and thus should improve detection limits.99–102 Future trends A number of applications of CEC have been described, which demonstrate the wide applicability of this relatively new technique. Progress over the last two years has been rapid; whilst much of the literature then dealt with separations of model compounds such as simple mixtures of hydrocarbons and other neutrals, it has now started to include samples of more complex nature and diversity.In the longer term, the extra peak capacity available in CEC may considerably extend its range. Already, the economic and environmental advantages of having low expenditure of solvents and stationary phases make it attractive. However, much work will still be necessary if CEC is to be recognised as an analytical technique and a viable alternative to CE and HPLC.In particular, the future of CEC is likely to depend greatly on the nature of the column. At present, it is far from ideal; columns tend to be fragile in their present format. In addition, we have found that prolonged use of acetonitrile removes the protective polyimide coating at the capillary ends, thus rendering the frits even more susceptible to breakage. New column materials, which can provide more control over EOF and selectivity, are expected to make an appearance on the market, as are monolithic type columns.With miniaturisation being increasingly popular, a new generation of small CEC instruments on a chip with ‘disposable’ columns may be the way forward. References 1 Sandra, P., Dermaux, A., Ferraz, V., Dittmann, M. M., and Rozing, G., J. Microcol Sep., 1997, 9, 409. Fig. 16 CEC separation of the enantiomers of compound 7 on (3R, 4S)- Whelko-O CSP, using MES (25 mm, pH 6.0)–acetonitrile (1 + 3.5).Reprinted with permission from ref. 96. Fig. 17 CEC separation of the enantiomers of compound 3 on (S)- naproxen derived CSP, using MES (25 mm, pH 6.0)–acetonitrile (1 + 3.5). Reprinted with permission from ref. 96. Fig. 18 Separation of non-racemic mixtures of propranolol on a capillary column containing imprints of (R)-propranolol, using acetonitrile–4 m acetate (pH 3.0) (80 + 20) at 60 °C. 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M., and Rozing, G. P., J. Chromatogr. A, 1997, 782, 175. 97 Nilsson, S., Schweitz, L., and Petersson, M., Electrophoresis, 1997, 18, 884. 98 Schweitz, L., Andersson, L. I., and Nilsson, S., Anal. Chem., 1997, 69, 1179. 99 Lin, J.-M., Nakagama, T., Uchiyama, K., and Hobo, T., J. Liq. Chromatogr., 1997, 20, 1489. 100 Lin, J.-M., Nakagama, T., Uchiyama, K., and Hobo, T., Biomed. Chromatogr., 1997, 11, 298. 101 Lin, J.-M., Nakagama, T., Uchiyama, K., and Hobo, T., J. Pharm. Biomed. Anal., 1997, 15, 1351. 102 Lin, J.-M., Nakagama, T., Wu, X. Z., Uchiyama, K., and Hobo, T., Fresenius’ J. Anal. Chem., 1997, 357, 130. Paper 8/01148F Received February 9, 1998 Accepted April 2, 1998 102R Analyst, July 1998, Vol. 123

ISSN:0003-2654    

DOI:10.1039/a801148f

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Critical Review Microwave digestion procedures for environmental matrices Kathryn J. Lamble and Steve J. Hill* Department of Environmental Sciences, University of Plymouth, Drake Circus, Plymouth, UK PL4 8AA Summary of contents Introduction Open or closed digestion systems? Closed microwave digestion techniques Open digestion techniques On-line digestion techniques Chemometrics Universal digestion procedures Biological samples Geological samples Water samples Conclusions References Keywords: Microwave digestion; environmental matrices; review Steve Hill is Professor of Analytical Chemistry at the University of Plymouth where he is currently Head of the Department of Environmental Sciences.Until recently he was Chariman of the Atomic Spectroscopy Group of the RSC and currently serves on several of the Society’s committees including Analytical Division Council. In addition he is on the Editorial Boards of The Analyst, JAAS, and Analytical Communications and is General Editor of Atomic Spectrometry Updates. His research interests are varied and to date he has over 120 scientific publications based on analytical chemistry and has made over 150 conference presentations.Introduction Quantitative analytical techniques are required to determine accurately and rapidly a variety of trace metals with sufficient sensitivity for application to a range of environmental samples. This requirement has led to major technological advances in the development of analytical instrumentation, enabling vast amounts of elemental information to be obtained in a shorter time and with increased sensitivity than was ever possible before. This is especially true of analytical techniques such as inductively coupled plasma atomic emission spectrometry (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS), which can obtain multi-element information in a fraction of the time needed to prepare the samples.However, prior to analysis by most analytical techniques there is an intrinsic requirement for the sample to be converted into a liquid form.For solid samples this can be achieved by undertaking some form of digestion procedure. To be effective, sample digestion methods must efficiently decompose the sample matrix so that the analytes of interest are completely released and solubilised and are in a form compatible with the analytical method of choice. Effective methods of sample digestion are therefore a crucial prerequisite to accurate analysis.However, technological advances in this area have been slow in comparison with the developments in analytical instrumentation. Until relatively recently, sample digestion methods were largely limited to the conventional techniques of wet digestion, dry ashing and fusion techniques. Such methods are often time consuming, may be the source of contamination and losses of analyte and generally require a great deal of operator attention, skill and experience in order to gain accurate and precise results.As a consequence, sample preparation is often regarded as the weak link in sample analysis, and an area which provides much scope for improvement. Significant improvements in sample preparation techniques have been made since 1975, however, when Abu-Samra1 first reported the use of microwaves as a heat source for wet digestion methods. Since then the microwave digestion technique has gradually gained widespread acceptance as an effective method of sample preparation.Using this technique, not only have digestion times been dramatically reduced (by a factor of 2–5) but also other benefits such as a reduction in contamination, less reagent and sample usage, a reduction in the loss of volatile species and improved safety have been reported.2 The advantages of the microwave digestion technique have led to its application as an effective sample preparation method for a wide range of sample matrices.Each year more and more laboratories replace the conventional methods with the new technology, as is reflected in the ever increasing amount of material published on the subject. A number of review articles and books have been published2–9 detailing the use of the technique for elemental analysis. Kuss5 listed the applications of microwave digestion techniques for elemental analyses cited in the literature before 1992. Included were references for the digestion of biological, geological, environmental and metallic materials.A publication by Zlotorzynski3 discusses the fundamental principles of microwave field interaction with the sample matrix, whereas de la Guardia and Morales-Rubio4 discuss the modern strategies available for the rapid determination of metals in sewage sludges. A review on the use of microwave assisted sample preparation in analytical chemistry has been undertaken by Smith and Arsenault9 and specifically for analysis by electrothermal atomic absorption spectrometry by Chakraborty et al.6 This paper reviews the application of microwave energy for the digestion of environmental samples (biological, geological and water) reported since 1992.Where it was felt that the Analyst, July 1998, Vol. 123 (103R–133R) 103Rmethodological approach would benefit the reader, matrices which are not strictly environmental are also included, e.g., bovine liver, foodstuffs. Details of the open and closed microwave digestion methods used to digest these samples, including the advantages and disadvantages of each technique, are discussed.The review also attempts to highlight any trends in research and to identify universal digestion procedures for particular matrices or elements. Tables 1, 2 and 3 summarise the different microwave digestion procedures employed during the review period for biological, geological and water samples, respectively. Each table characterises the matrix digested, elements determined, microwave system used, digestion method, i.e., specific reagents and heating time, and analytical technique used, and finally comments on the effectiveness of the method. In many cases certified reference materials have been used for validation of digestion procedures.It was observed that results are often classed as ‘good’ when in fact they lie outside the uncertainty limits of the certified value. For clarification, in this review, results described as ‘good’ indicate that they lie within the uncertainty limits of the certified value.In most cases this is defined as twice the standard deviation of the mean of the certified value. Other results are classified as ‘low’ or ‘high’ accordingly. A key to the abbreviations used in Tables 1–3 is given in Table 4. Open or closed digestion systems? During the review period, most studies have concentrated on the development of closed digestion methods.Most commonly these are carried out in multimode ovens, in which the microwaves are dispersed into a large cavity, in a similar manner as for domestic microwave ovens. Multimode ovens have also been used for open digestions; however, the majority of open vessel applications utilise monomode (focused) microwave ovens. In the latter case, microwave energy is directly applied to the sample by placing it within the waveguide. Hence, it may be better to describe microwave ovens in terms of the type of applicator used, i.e., ‘cavity’ for multimode and ‘waveguide’ for monomode ovens.A commercial closed monomode microwave digestion system is also available.27 Each microwave digestion system has certain advantages and disadvantages, so it is not possible to suggest either as being the most suitable for all applications. Closed microwave digestion techniques The closed digestion technique involves placing the sample in a vial (or bomb), usually constructed of a fluorinated polymer, such as polytetrafluoroethylene (PTFE) or perfluoroalkoxy (PFA).After adding the digestion reagents, the bomb is tightly sealed and placed in the microwave oven for irradiation by microwave energy. Initial closed vessel research was undertaken in domestic multimode microwave ovens. Digestion vessels were often placed inside evacuated desiccators or large plastic jars to contain the evolved acid vapours and improve safety in the event of overheating.In order to prevent damage to the magnetron from reflected microwaves unabsorbed by small samples, an additional load (usually water) was commonly placed in the microwave oven. However, as these auxiliary loads reduce the amount of microwave energy reaching the sample, a constant and reproducible supply cannot be guaranteed. A further disadvantage of the use of domestic microwave ovens is that the power output of the magnetron is static, the output being controlled by cycling the magnetron off and on to obtain an average power level.Domestic ovens typically have a high time base, generally between 10 and 30 s. Hence, to obtain 50% power, the magnetron will only be on for half of the time base. This approach may prove undesirable for analytical work as significant heat losses can occur during the periods of zero power output. As a result of the unsuitability of domestic ovens for use in analytical chemistry, a number of commercial systems have been specially developed to overcome the problems of acid fume damage, sample power reflection, field inhomogeneity, long time bases and safety.29,63,75,79,168,169 The major advantage of the closed microwave digestion technique is the high heating efficiency which can be obtained.Heating causes an increase in pressure, due to the evaporation of digestion acids and the gases evolved during the decomposition of the sample matrix. This is beneficial by increasing the boiling-point of the reagents, which aids the breakdown of the sample matrix.However, the excessive build-up of pressure, especially during the digestion of samples with a high organic content, can lead to the rupture of sealed vessels. For this reason, most digestion bombs are fitted with pressure relief valves, designed to open when the pressure becomes too great, and thus maintain safety. If venting does occur, sample losses are likely and owing to the reduction in acid vapours a less active digestion may result.Considerable research has therefore been undertaken to find ways of controlling or reducing pressure build-up during the digestion process. 31,34,42,47,92,101,112,120,123,128,187 One method of avoiding excess pressure is to pre-digest the sample, and thus enable the gases evolved from the decomposition of easily oxidised organic matter to escape before commencing the closed digestion procedure. This may be carried out by leaving the samples to pre-digest at room temperature, often overnight.31,45,47,49,75,80,92,112,123,128 However, if high sample throughput is required this extra step must be taken into account since a large number of digestion vessels will be required.Pre-digestion can also be carried out by microwave heating in an unsealed vessel, prior to the capping and digestion of the sample in the usual manner. Rhoades49 predigested samples by heating for 1 h with a heat lamp. However, an important consideration is that the pre-digestion step should not be too lengthy since it may counteract the benefit of rapid digestion using microwave systems.Also, excessive evaporation of digestion reagents and volatile elements must be avoided. Reid et al.47 overcame these problems by employing an open vessel heptane-cooled reflux pre-digestion step during which oxidation products could escape whilst retaining the analytes and digestion reagents for the ensuing closed digestion. Heltai and Peresich101 investigated the idea of controlling the vapour pressure by means of a water cooled spiral inserted into the closed space of the digestion bomb. During the digestion, the acid and water vapours evolved are condensed on the spiral, producing a reflux action which continuously renews the liquid phase over the sample for effective digestion.A different approach involves leaving the closed digestion to continue spontaneously after initial heating to induce the reaction.Using this method temperatures greater than 150 °C and pressures in excess of 150 lb in22 were achieved, sufficient, for example, to digest NIST Bovine Liver.120 Another technique reported in order to control pressure buildup is to monitor the pressure or temperature throughout the course of the reaction and subsequently apply microwave power only when the readings are below the required level. In this way the pressure can be controlled, thus minimising venting of the digestion vessel.One commercial company has achieved this by placing a pressure transducer inside one of the digestion vessels to monitor the pressure continuously.34 Other workers have taken temperature measurements using a non-invasive infrared probe attached to the bottom of the microwave oven.42 In this case the output from the probe is fed to a computer which switches the magnetron on and off to achieve a pre-set temperature–time programme. The technology for monitoring the temperature or pressure of the digestion offers the potential to produce far more reproducible and controllable procedures. 104R Analyst, July 1998, Vol. 123Table 1 Microwave digestion procedures for biological samples Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref. Biological tissues Se Prolabo (Paris, France) Microdigest 301 (200 W) Open focused HNO3–H2O2 digestion for 20 min (n = 1) FI–CSV Good recoveries were obtained for Se in BCR Lyophilised Pig Kidney 10 Marine biological tissues As Prolabo A320 (200 W) Solubilisation: HNO3 open focused digestion for 10 min.Mineralisation: HNO3–H2SO4– HNO3–H2O2 digestion for 70 min ICP-MS and HPLC–ICPMS Good results were obtained for total arsenic in BCR Cod Muscle after both solubilisation and mineralisation procedures 11 Biological tissues and botanical samples Se Prolabo Microdigest A301 (200 W) Open focused HNO3 digestion for 20 min, followed by addition of H2O2 and further heating for 25 min (n = 1) SeVI is reduced to SeIV prior to analysis by GC–MS Good agreement with certified values for NIST Bovine Liver and Mixed Diet (Finland), although for NIST Total Diet results were slightly low.Procedure was quicker than a conventional method without needing HClO4 12 Fat-rich foods Cu, Fe, Ni, Zn Prolabo Microdigest M300 (200 W) and domestic oven (700 W) Closed: HNO3–H2O2 PTFE bomb digestion for 20 min. Open focused: HNO3–H2SO4– HNO3 digestion for 25 min (n = 1) FAAS and ETAAS (for Ni) For the closed digestion, reasonable agreement was obtained with results from a wet pressure autoclave digestion method (for soybean flour and linseed samples).However, results were generally low for the open microwave method 13 Foods N Prolabo Maxidigest MX- 350 (350 W) Open focused H2SO4–H2O2 Kjeldahl nitrogen digestion for 20–45 min depending on food type (n = 1) NH3 titration Substantial time savings over conventional methods were demonstrated, without the need for a catalyst.However, for new matrices each step of the procedure must be re-optimised separately 14 Cereals and cereal products Cd, Cu, Pb, Se Prolabo Microdigest A301 and CEM (Buckingham, UK) MDS-2000 (630 W) (a) Open focused HNO3–H2O2 digestion for 17 min (n = 1). (b) Closed HNO3 digestion for 60 min (n = 12) ETAAS Generally good results were obtained for the open and closed methods for BCR Brown Bread, NRCCRM Wheat and Rice Flour, CGC Whole Wheat and NIES Rice Flour, Corn Bran, Durum Wheat Flour, Hard Red Spring Wheat Flour, Soft Winter Wheat Flour, Rice Flour and Wheat Flour 15 Marine biological tissues As, Cd, Cu, Mg, Mn, Ni, Sr, Zn Prolabo Microdigest A301 Open focused HNO3 digestion for 17 min, followed by 20 min cooling and further heating with H2O2 for 5 min (n = 1) ICP-MS Generally good results were obtained for NRCC LUTS-1, DORM-1 Dogfish Muscle, DOLT-2 Dogfish Liver and TORT-1 Lobster Hepatopancreas, except for high As and Ni 16 Marine biological tissues and botanical samples Cd Prolabo Microdigest A301 Open focused HNO3–H2O2 digestion for 42 min ETAAS Good results were obtained for NIST Wheat Flour and Bovine Liver, although for IAEA Fish Flesh Homogenate results were slightly low.Acceptable agreement with results from a conventional wet digestion was obtained for bovine muscle, bovine liver, oyster, barley straw, cabbage, carnations, oak leaves, pine needles, apple-fruit and grass meal 17 Biological tissues Bi, Cd, Co, Cs, Cu, Fe, Hg, Mn, Mo, Pb, Rb, Sb, Sn, Sr, Tl, Zn Prolabo Microdigest A301 and Milestone (Sorisole, Italy) MEGA 1200 (1200 W) HNO3–H2O2 digestion.Closed vessel: 26.5 min. Open focused: 45 min ICP-MS Open focused: good results were obtained for NIST Bovine Liver, except for low Cu, Sr and Zn. Closed vessel: Good results were obtained, except for low Cd, Pb and Sr 18 Biological tissues As, Ba, Ca, Cd, Cu, Fe, K, Mg, Mn, Na, P, Pb, Sr, Zn Prolabo Microdigest M300 and Floyd (Lake Wylie, SC, USA) RMS- 150 (600 W) Open focused vessel: HNO3–H2SO4–H2O2– NH4EDTA digestion for 30 min (n = 1). Closed vessel: HNO3 digestion for 32 min ICP-AES and residual carbon content analysis.Generally good results were obtained for the open digestion of NIST Bovine Liver and Oyster Tissue, IAEA Horse Kidney and NIES Mussel Tissue, although Na recoveries were slightly low. The residual carbon content of digests following the open method were far superior to those from the closed system 19 Tea leaves Al, Ba, Ca, Cu, K, Mg, Mn, Zn Prolabo Microdigest 301 Open focused HNO3–HClO4 digestion for 35 min (n = 1) ICP-AES Good agreement with the certified values for NIES Tea Leaves was obtained 20 Analyst, July 1998, Vol. 123 105RTable 1 Continued Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref. Marine biological tissues As Prolabo Microdigest 301 On-line system incorporating HPLC separation; potassium persulfate–NaOH oxidation and l-cysteine pre-reduction of As species in fish samples following enzymatic extraction On-line analysis by HG– AAS.Total As can be determined by removal of the HPLC column from the system The AsBet, DMA, MMA, AsV and total arsenic content of NRCC TORT-1 Lobster Hepatopancreas and DORM-1 Dogfish Muscle were determined by the on-line technique. Results for total arsenic were in good agreement with those obtained by ICP-MS analysis 21 Marine biological samples As Prolabo Microdigest 301 Open focused HNO3–H2O2 digestion for 15 min (n = 1) ICP-MS Good results were obtained for As in NRCC DORM-1 Dogfish Muscle and TORT-1 Lobster Hepatopancreas 21 Marine biological tissues Hg Prolabo Microdigest 301 On-line digestion of 0.15% slurries (in 50% HCl), including Br2–BrO32 oxidation of organomercury species On-line analysis by CVAFS A recovery of 97% was obtained for a standard solution of methylmercury chloride.Results for Hg in NRCC DORM-2 Dogfish Muscle were in good agreement with the certified value 22 Marine biological tissues Hg Prolabo Microdigest 301 Open focused HNO3–H2SO4– H2O2 digestion for 25 min (n = 1) CV-AAS Good results were obtained for Hg in NRCC DORM-2 22 Marine biological tissues As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Se, Sr, Zn Prolabo Maxidigest M401 (300 W) Open HNO3 or HNO3– H2SO4 digestion for 20–100 min (depending on sample size) followed by evaporation to a volume of 1 ml ETAAS Samples of up to 8 g were successfully digested.Generally good results were obtained for NRCC TORT-1 Lobster Hepatopancreas and LUTS-1 23 Sewage sludge Hg Prolabo Microdigest 301 On-line digestion of slurries prepared in nitric acid On-line FI–CV-AFS Good results were obtained for BCR Sewage Sludge (Domestic); however, results for BCR Sewage Sludge Amended Soil were slightly low 24 Marine biological tissues and botanical samples Al, As, Cd, Co, Cr, Cu, Hg, Mg, Mo, Ni, Pb, Zn Prolabo Microdigest A300 (200 W) Open focused digestion with (a) HNO3–H2O2, (b) HCl–HNO3–H2O2, (c) HNO3–H2SO4–H2O2 ICP-AES and ICP-MS Good results were obtained for procedure (a) for BCR Spruce Needles (except low Al, Mg), White Clover, Cod Muscle (except low Hg) and Plankton (except high Hg and low Mn).Procedure (b) gave high As and low Mn, whereas procedure (c) gave low Hg results for BCR Cod Muscle. Results by (b) were generally low for BCR Plankton, except for Cd, Zn 25 Biological tissues SbIII and SbV Prolabo Microdigest 301 Open focused digestion with (a) 1 m acetic acid (for SbIII), (b) H2SO4–KI (for total Sb) HG–AAS Good results were obtained for total Sb in spiked calve liver samples.SbV was calculated as the difference between total Sb and SbIII 26 Biological tissues Hg Prolabo Microdigest A301 and Superdigest (300 W) Open: HNO3–H2SO4–HNO3– H2O2 digestion for 20 min (n = 1). Closed: HNO3 digestion CV-AAS Good results were obtained for Hg in BCR Pig Kidney and IAEA Fish Tissue following both methods.For the open method, digestion with just HNO3 and with HNO3–H2SO4-HNO3 resulted in low recoveries 27 Marine biological tissues Hg Prolabo Microdigest A301 Open focused microwave assisted extraction for 2 min (n = 1) with (a) 25% TMAH, (b) methanolic KOH HG–CT–GC–ETAAS Good results were obtained for total Hg and methylmercury in NRCC DORM-1 Dogfish Muscle and TORT-1 Lobster Hepatopancreas and in BCR CRM 463 Tuna Fish Muscle 28 Marine biological tissues Cd, Cu, Zn CEM MDS-81D (600 W) HNO3 low volume Teflon bomb digestion for 49 min (n = 24) FAAS and ETAAS (for Cd) Good results were obtained for Cu, Zn and Cd in NIST Oyster Tissue, NRCC DOLT-1 Dogfish Liver and TORT-1 Lobster Hepatopancreas, for Cu and Zn in NRCC DORM-1 and for Zn in NIST Albacore Tuna 29 Botanical samples Ca, Cu, Fe, K, Mg, Mn, Na, P, Zn CEM MDS-2100 (950 W) HNO3–HCl Teflon bomb digestion for 74 min (n = 12) ICP-AES Good results were obtained for K, Na, P and Pb in NIST SRM 1572 Citrus Leaves; however Ca, Cu, Mg and Zn results were slightly low and Fe was very low. Results for corn samples were compared with the package label claims of the supplier 30 106R Analyst, July 1998, Vol. 123Table 1 Continued Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref. Botanical samples B, Se CEM MDS-2000 (630 W) Se: HNO3–H2O2–H2O PTFE bomb digestion for 30 min following pre-digestion for 4 h.B: HNO3–H2O2 PTFE bomb digestion for 45 min following pre-digestion for 4 h (n = 12) FAAS (for Se) and ICPAES (for B) Se recoveries for NIST Wheat Flour were: 23% with HNO3, 30% with HNO3–H2O2, 57% with HNO3–H2O2 and 80% with HNO3–H2O2–H2O. B recoveries for NIST Apple Leaves were: 60% with HNO3 and 66–96% with HNO3–H2O2. Longer digestion times or adding HCl (for Se) did not increase recoveries 31 Botanical and sludge samples Pb CEM SpectroPrep (550 W) On-line microwave digestion of slurried samples (prepared in 3 m HNO3 for botanical and HNO3–HClO4–HF for sludge samples), 10 min per sample Off-line analysis by ID–ICP-MS Good results were obtained for Pb in NIST SRM 1547 Peach Leaves and SRM 2781 Domestic Sludge 32 Cocoa Cu, Fe CEM MDS-81 HNO3 bomb digestion for 30 min (n = 7) FAAS The microwave digestion procedure was four times quicker than a conventional hot-plate method.Results from the two techniques were in good agreement 33 Biological tissues Ca, Fe, Mg, Zn CEM MDS-81 HNO3 bomb digestion for 30 min (n = 7) FAAS Good results were obtained for Fe and Mg in IAEA Horse Kidney, however, Zn results were slightly high and Ca low 34 Biological tissues and botanical samples Ca, Cd, Fe, Mg, Zn CEM MDS-81 On-line stopped-flow digestion (for 5 min) of slurries (prepared in Triton X-100 and HNO3) Off-line analysis by AAS Good Zn results were obtained in IAEA Horse Kidney, but those for Fe and Cd were low and Mg was high. The system was unsuitable for Ca determinations.Results for batch microwave digestion, on-line microwave digestion and hot-plate digestion of cocoa powder were in good agreement. Attempts at slurrying NIST Pine Needles, Oyster Tissue and Bovine Liver at concentrations needed for FAAS detection were unsuccessful 35 Biological tissues and botanical samples Ca, Fe, Mg, Zn CEM MDS-81 On-line digestion of HNO3 slurries On-line analysis by FAAS Good recoveries and precision was obtained for the results of NIES Chlorella, Sargasso and Pepperbush and NIST Bovine Liver, although for NIES Mussel, precision was slightly high. At the maximum stable slurry concentration, Zn was not detectable by FAAS in Chlorella and Sargasso, nor was Ca in Bovine Liver 36 Marine biological tissues Se CEM MDS-81 HNO3–H2O2 bomb digestion for 24 min followed by evaporation to near dryness (45 min) ETAAS Results for Se in lyophilised fish samples were in good agreement with those obtained by a slurry technique 37 Botanical samples As, Se CEM MDS-2000 H2O–HNO3–H2O2 PTFE bomb digestion for 25 min HG–AAS Good results were obtained for BCR FD8 Maize Leaves.Spike recoveries of 95 and 105% were obtained for As and Se, respectively 38 Botanical samples Al, Ca, Cu, Cr, Fe, K, Mg, Mn, Si, Ti, Zn CEM MDS-81D Teflon PFA bomb digestion with (a) HNO3–HCl, (b) HNO3–HF–H2O2 FAAS and DCP-AES (for Cu) (a) Good Ca, K, Mg, Mn and Zn results were obtained for NIST Citrus Leaves, Pine Needles and IAEA Mixed Diet, but those for Al, Cu and Fe were low.(b) Good results were obtained for Al, Fe and Mg in Citrus Leaves, but Cu results were slightly low 39 Bio-monitors Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Ni, Pb, Zn CEM MDS-2000 HNO3–H2O2 PFA bomb digestion for 15 min (moss and rye grass) and 17 min (humus and hay) (n = 10) FAAS and ETAAS Good results were obtained for BCR 281 Rye Grass and IAEA V-10 Hay, except for low Fe recoveries.Moss and humus samples were also successfully digested 40 Botanical samples Ca, Cu, Fe, K, Mn, Mg, Na, P, Zn CEM MDS-81D PTFE bomb digestion for 60 min (n = 12) with (a) HNO3 and (b) HNO3–H2O2 AAS and AES (for Na and K) No significant difference was observed between the results of the two microwave procedures, a conventional dry ashing and a wet ashing technique for lucerne leaves 41 Botanical samples Ba, Ca, Cu, Mg, Mn, Zn CEM MDS-81 with IR probe HNO3–HF PTFE bomb digestion for 20 min (n = 6) ICP spectrometry Good results were obtained for NBS Citrus Leaves, except for slightly low Cu 42 Analyst, July 1998, Vol. 123 107RTable 1 Continued Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref. Food samples Na CEM MDS-2000 PTFE bomb HNO3–H2O2 digestion for 1.5 h (n = 12) AAS, CIE and IC analysis No CRMs were analysed 43 Sewage sludge Ag, Al, Ba, Be, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, Pb, Sn, Ti, V, Zn CEM MDS-81D Closed vessel HNO3–50% HCl digestion for 20 min ICP-AES Results were presented for the acid leachable metals in NIST SRM 2781 Domestic Sludge and used to derive reference values for the sample.Generally results compared well with those of an open vessel hot-plate digestion method. Spike recoveries of 89–120% were obtained for the microwave method 44 Biological tissues and botanical samples B CEM MDS-81D H2O2–HNO3 Teflon bomb digestion for 10 min, following having left samples to predigest at room temperature for 10 min ICP-MS Generally good results were obtained for B in NIST SRM 1515 Apple Leaves, SRM 1547 Peach Leaves, RM 8433 Corn Bran and RM 8414 Bovine Muscle.Spike recoveries of 90–109% were obtained 45 Botanical samples As, Ba, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Tl, V, Zn CEM MDS-81D HNO3 Teflon bomb digestion (18 min) followed by 21 min heating with HF. H2O2 is then added and heating continued in a water-bath (15 min) before adding H3BO3 for a further 5–10 min heating (n = 12) AAS and ICP-AES Reasonable agreement with the certified values for NIST SRM 1547 Peach Leaves was obtained, except for low Ba.The method was also used to digest corn leaves 46 Botanical samples Mn CEM MDS-81D HNO3 Teflon PFA bomb digestion (a) without predigestion; (b) with predigestion at room temperature (18 h); (c) with microwave predigestion and reflux (7 min); (d) with microwave predigestion without reflux FAAS No significant differences were observed between the results obtained for sweet bay powder by each of procedures (a) to (d).Procedure (c) permits a fast, safe digestion without large evaporation of acids in the pre-digestion step 47 Food samples Decomposition products CEM MDS-81D Bomb digestion with HNO3, HNO3–H2O2 or HNO3 followed by H2O2–HClO4 treatment on a hot-plate. Carbon content analysis, IR, TLC Results illustrated the necessity of employing different sample decomposition methods according to the sample matrix and analytical technique of choice 48 Botanical samples As, B, Ba, Be, Bi, Ca, Cd, Co, Cu, Cr, K, La, Mg, Mn, Mo, Na, Ni, P, Pb, S, Se, Sr, Te, Zn CEM MDS-2100 After adding HNO3 samples are allowed to stand for 35–40 min, heated at 80 °C with a heat lamp (1 h), cooled and capped.Microwave energy is applied in three steps (total 3.5 h, n = 5) with cooling and venting between steps. After cooling samples are evaporated at 80 °C under N2 purge for 4 h ICP-AES Generally good results were obtained for NIST SRM 1547 Peach Leaves, 1571 Orchard Leaves and 1572 Citrus Leaves 49 Biological tissues Ca, Cu, Fe, K, Mg, Mn, PS, Zn CEM MDS-81D After initial closed heating with HNO3–H2O2 the digestion is allowed to continue spontaneously without further irradiation ICP-AES and FAAS Good recoveries were obtained for Ca, Fe, K, Mg, Mn and S in NIST Bovine Liver.P, Zn and Cu results were just outside the certified range.The heating time required depended on the sample size and amount of H2O2 used 50 108R Analyst, July 1998, Vol. 123Table 1 Continued Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref. Sludge samples As, Se CEM MDS-81D PFA bomb digestion for 1–2 h with (a) HNO3 –HCl, (b) H2O2–HCl–H2SO4, (c) H2O2–H2SO4, (d) HNO3–H2SO4 FI–HG–AAS Method (d) gave the best recoveries (validated using NIST San Joaquin Soil, see Table 2). Good results were obtained for As, but Se recoveries were slightly high.For NIST Domestic Sewage Sludge results agreed well with those of a conventional reflux method, but the microwave method was faster and HClO4 was not required 51 Marine biological tissues As, Cd, Pb CEM MDS-2000 HNO3 Teflon PFA bomb digestion for up to 2 h depending on the sample (n = 12) ICP-AES and ICP-MS Good results were obtained for NRCC TORT-1 Lobster Hepatopancreas and DORM-1 Dogfish Muscle, except for high As in the former. Spike recoveries were in the range 75–117%. 52 Botanical samples Ca, Fe, K, Mg, Mn, P, S CEM MDS-2000 Closed digestion with (a) HNO3–HClO4 for 1 h 23 min, (b) HNO3–H2O2 ICP spectrometry Generally good results were obtained for NIES Citrus Leaves, Pine Needles and Corn Leaves 53 Marine biological tissues Ag, As, Cd, Cr, Cu, Ni, Pb, Zn CEM SpectroPrep system and Floyd RMS-150 On-line: 0.5% m/v slurries were digested in 20% HNO3–3% H2O2. Batch: Teflon bomb HNO3–HF digestion for 70 min with cooling step half way through ID–ICP-MS (standard additions) and ETAAS (for As and Cr) On-line system: good results were obtained for NRCC LUTS-1 for Cd, Cu, Ni and Pb (Ag and Zn were low and Cr very high). For lobster hepatopancreas, results were in agreement with the closed vessel technique.Slurries of Pacific oyster tissue were not amenable to direct uptake by the SpectroPrep system unless prior digestion was undertaken 54 Botanical samples Al, B, Ba, Ca, Cr, Cu, Fe, K, Mg, Mn, Na, P, S, Sr, Zn CEM MDS-81D HNO3–HF closed digestion for 37 min (including time for cooling steps) followed by open heating with H2O2 and SiO2 for 30 min ICP-AES Generally good results were obtained for NIST SRM 1515 Apple Leaves, 1547 Peach Leaves, 1573a Tomato Leaves and 1575 Pine Needles 55 Biological tissues Al CEM MDS-81D HNO3–HF digestion for 30 min followed by cooling, addition of H2O2 and H3BO3 and evaporation to dryness for 30 min (n = 12) ICP-AES Good results were obtained for NIST SRM 1566a Oyster Tissue and NIES 1577b Bovine Liver.However following digestion with HNO3–HClO4 and HNO3–H2O2 low results were obtained in the former. Spike recoveries of 95–96% were obtained in crab and shrimp meat samples 56 Biological tissues B CEM MDS-81D HNO3 closed digetion for 35 min followed by cooling, addition of H2O2 and evaporation for 30 min ICP-AES Spike recoveries of 95–100% were obtained in five meat samples and in NIST Oyster Tissue and Bovine Liver (CRMs not certified for B) 57 Marine biological tissues Cd, Pb CEM MDS-2000 HNO3 digestion for 20–25 min (n = 12) ETAAS Good results were obtained for Cd and Pb in NRCC DORM-1 Dogfish Muscle 58 Marine biological tissues Hg CEM MDS-2000 HNO3 PTFE bomb digestion for 70 s CV-AAS Results were in good agreement with the certified value for NIST RM 50 Albacore Tuna and spike recoveries of 99–102% were obtained 59 Marine biological tissues As, Cd, Co, Cr, Cu, Hg, Pb, Zn CEM MDS-81D HNO3 Teflon bomb digestion for 2 min followed by preconcentration on a Chelex- 100 column NAA and ETAAS Good results were obtained for NRCC DORM-1 Dogfish Muscle 60 Biological tissues and botanical samples Al, Ca, Cu, Fe, K, Mg, Mn, Na, Ni, Zn CEM MDS-2000 Closed TMAH–EDTA ammoniacal leaching procedure for 30 min with prior stirring step (10 min) FAAS and ETAAS Variable results were obtained for NIST SRM 1577b Bovine Liver, NIST SRM 1515 Apple Leaves and NIES CRM No. 1 Pepperbush, No.3 Chlorella, No. 6 Mussel and No. 7 Tea Leaves 61 Marine biological tissues Hg Milestone MLS-1200 (1200 W) HNO3–H2O2 bomb digestion for 6 min FI–CV-AAS Good results were obtained for Hg in NRCC DORM-1 Dogfish Muscle 62 Analyst, July 1998, Vol. 123 109RTable 1 Continued Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref. Botanical samples Lanthanides and actinides Milestone MLS-1200 HNO3–H2O2 PTFE bomb digestion for 14 or 26 min depending on sample size ICP-MS Good results were obtained for Ce, Eu, Sm, Tb and 238U in NIST Apple Leaves, but those for 232Th were low.For NIST Orchard Leaves, results were low for 232Th and slightly low for 238U 63 Sewage sludge samples COD Milestone MLS-1200 On-line K2Cr2O7–H2SO4 oxidation (3 min) FI spectrophotometric detection Results were in good agreement with those of the standard COD method for sewage samples 64 Marine biological tissues As Milestone MLS-1200 HNO3–H2O2 Teflon bomb digestion for 12.5 min FI–HG–AAS Recoveries of 13 ± 10% and 2 ± 1% were obtained for As in BCR 278 Mussel Tissue and BCR 422 Cod Muscle due to incomplete oxidation of organoarsenicals.Results were compared with those obtained from high-pressure ashing and dry ashing procedures 65 Marine biological tissues Hg Milestone MEGA-1200 HNO3–H2O2 closed digestion procedure FI–CV-AFS Good agreement with the certified value was obtained for Hg in NRCC DORM-1 Dogfish Muscle and DOLT-1 Dogfish Liver 66 Biological tissues Ru Milestone MLS-1200 HNO3–H2O2 PTFE bomb digestion of homogenised samples (in water) for 30 min ETAAS Spike recoveries of 95–101% were obtained for liver and kidney samples, but no CRMs were analysed 67 Duck eggs Cu, Cd, Pb Milestone MLS-1200 HNO3–H2O2 Teflon bomb digestion for 10 min FAAS Recoveries of 82–101% were obtained in egg albumen and yolk samples. 68 Biological tissues and botanical samples B, Cd, Cu, Fe, Mn, P, Pb, Zn Milestone MLS-1200 MEGA and domestic oven (665W), Teflon bomb digestion with (a) HNO3, (b) HNO3–HF–hotplate evaporation to dryness– HNO3, (c) HNO3–H2O2, (d) HNO3–HClO4 ICP-AES, FAAS, ETAAS (a) Poor precision.Generally high P and low Fe results were obtained for Comit�e Inter-Instituts botanical reference materials. (b) Acceptable precision. Fe results were improved but most still outside certified range. (c) and (d) mixed Fe, Cu, Mn, Zn results for NIST Total Diet, NIES Mussel, Pepperbush and BCR Wholemeal Flour (many results too high) 69 Biological tissues As, Cd, Co, Cu, Fe, Hg, Mg, Mn, Mo, Pb, Se, V, Zn Milestone MLS-1200 MEGA HNO3 digestion (38 min) in dual PTFE containers for sample sizes of 35–45 mg (n = 20) ICP-MS Good results were obtained for all elements studied in NIST Bovine Liver.Good results were also obtained for As in IAEA MA-A-2 Fish Flesh, however Cd, Cu, Hg, Se and Zn were slightly low 70 Rice flour Cd, Cr, Fe, Pb Milestone MLS-1200 MEGA Closed digestion with (a) HNO3 for 8 min, (b) HNO3 for 8 min.Then cool, add HF and HClO4 and heat to dryness at 100 °C. HNO3 is then added and the sample heated to near dryness ID–ICP-MS following removal of the Ca matrix by passage through a microcolumn (a) For the rice flour reference materials NIST SRM 1568, NIES 10a and KRISS A and B, good Cd results were obtained. However, Fe and Cr were low. (b) Good results were obtained for Cr and Fe in the above samples. 71 Biological tissues and botanical samples Ca, Cu, Fe, K, Mg, Mn, Na, P, Zn Milestone MLS-1200 HNO3–H2O2 PTFE bomb digestion ICP-AES and AAS For NBS Orchard Leaves, Bovine Liver and Spinach low Fe but good Cu, Mn, Na, P and Zn results were obtained. Some Ca, K and Mg recoveries were slightly low 72 Almond kernels Ca, Cu, Fe, K, Mg, Mn, Na, P, S, Zn Milestone MLS-1200 MEGA HNO3–H2O2 Teflon bomb digestion for 22 min (n = 6) ICP-AES The elemental composition of the kernels of 19 almond cultivars from different origins was determined to investigate the influence of the cultivar on the mineral composition of the sample 73 Marine biological tissues and botanical samples Al Milestone MLS-1200 Teflon bomb HNO3–H2O2 digestion for 23 min (n = 3) ETAAS Good results were obtained for NIST Wheat Flour, but those for Rice Flour were slightly high.Reasonable agreement with the informational values for Total Diet and IAEA Fish Tissue were obtained. The addition of HF to the digestion did not increase recoveries 74 110R Analyst, July 1998, Vol. 123Table 1 Continued Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref. Marine biological samples Si Floyd RMS 150 (850 W) HNO3–H2O2–HF Teflon bomb digestion for 30 min, after having left samples to predigest overnight. HF is neutralised with H3BO3 A tertiary amine mixture is added before ICPAES analysis Good results were obtained for NIST 1566 Oyster Tissue. Results were also in acceptable agreement with those obtained by a LiBO2 fusion procedure for a range of food samples 75 Marine biological tissues and botanical samples As Floyd RMS 150 HNO3–H2O2 bomb digestion for 32 min (n = 6) ICP-MS Good results were obtained for NIST Oyster Tissue and Orchard Leaves 76 Marine biological tissues Ag, Al, As, Cd, Cr, Co, Cu, Fe, Hg, Mn, Ni, Pb, Se, Sn, Th, Zn Floyd HNO3–HF digestion for 42 min, followed by cooling, re-heating for 42 min, evaporation to dryness on a hot-plate (with/ without H2O2) and redissolution in HNO3/H2O ID–ICP-MS and ICP-MS Good results were obtained for Ag, Al, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, Sn, Th and Zn in NRCC DORM-2 Dogfish Muscle and DOLT-2 Dogfish Liver 77 Marine biological tissues and botanical samples Ca, Cu, Fe, Zn Floyd RMS-150 21 different digestion procedures using HCl–HNO3–HF in different ratios DCP-AES The most suitable procedure was chosen by fractional factorial design.For this procedure, good results were obtained for NRCC TORT-1 Lobster Hepatopancreas and NIST Pine Needles (Ca and Fe only). Spike recoveries of 96–105% were obtained. A detrimental effect of aqua regia was reported (HNO3 and HCl most effective in equal quantities) 78 Biological tissues and botanical samples As, Cd, Co, Cu, Ni, Pb PMD, Paar (Graz, Austria) Quartz tube closed HNO3–HClO4 digestion (procedure is dependent on the sample) DP-ASV and HG–AAS (for As) Mixed results were obtained for Cd, Cu and Pb in BCR Bovine and Cod Muscle, Bovine Liver, Mussel Tissue and Brown Bread. Results for Co in NRCC TORT-1 Lobster Hepatopancreas were good but were slightly low for Ni.For the determination of As in fish and cooking oil (by HG–AAS) addition of H2SO4 was needed. Good As results were obtained for BCR Cod Muscle, Mussel Tissue, NIST Orchard Leaves and for NRCC TORT-1 Lobster Hepatopancreas 79 Botanical samples Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, Ni, Pb, Sb, Se, Sr, Ti, Tl, Th, U, V, Zn Questron (Mercerville, NJ, USA) Q-Wave 1000 HNO3 PFA bomb digestion for 30 m, after leaving samples to pre-digest overnight (n = 12) ICP-MS Reasonable agreement with the certified values was obtained for NIST SRM 1515 Apple Leaves and 1547 Peach Leaves, although some results were too high and some too low 80 Biological tissues and botanical samples Ni Domestic oven (700 W) HNO3–HCl PTFE bomb digestion for 14 min (n = 3) ICP-AES analysis after extraction of Ni complex formed with DPTH into butan-1-ol Generally good agreement with certified values for BCR Olive Europea, Lagarosiphon Major, Platihpnidium Ripariodides; NRCC DORM-1 Dogfish Muscle, DOLT-1 Dogfish Liver, TORT-1 Lobster Hepatopancreas; NIST Citrus Leaves; BCR Pig Kidney and Bovine Muscle 81 Marine biological tissues and botanical samples Ni Domestic oven (700 W) HNO3–HCl PTFE bomb digestion for 14 min (n = 3) ICP-AES analysis after extraction of Ni complex formed with BPTH into IBMK Good agreement with the certified values was obtained for NIST Citrus Leaves, NRCC DORM-1 Dogfish Muscle and NRCC TORT-1 Lobster Hepatopancreas 82 Marine biological tissues Al Domestic oven (800 W) On-line HNO3 digestion of slurries (in 0.2% HNO3) Off-line analysis by ETAAS 90% recovery for Al in NIST SRM 1566a Oyster Tissue was obtained.Five fresh shellfish samples were also analysed 83 Analyst, July 1998, Vol. 123 111RTable 1 Continued Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref. Marine biological tissues Se Domestic oven (800 W) On-line digestion of slurries (in 0.2% HNO3) for 4 min (stopped flow) Off-line analysis by ETAAS Good results were obtained for NIST SRM 1566a Oyster Tissue. No significant differences were observed between the results obtained for lyophilised and unlyophilised samples 84 Botanical samples Hg Domestic oven (800 W) HNO3 PTFE bomb digestion for 3 min at 800 W (n = 5) FANES after reduction with SnCl2 and in situ preconcentration Good agreement with certified values for NIST Citrus Leaves and Pine Needles was obtained 85 Sewage sludge Cd, Cr, Cu, Ni, Pb, Zn Domestic oven (662 W) On-line digestion of 0.2–0.75% m/v slurries prepared in 1.5 m HNO3.On-line ICP-AES analysis Generally good results were obtained for BCR Sewage Sludge Industrial 86 Biological tissues Cu, Fe, Zn Domestic oven (700 W) HNO3–H2O2 open digestion for 14 min (n = 100) FI–AAS Good results were obtained for Cu, Fe and Zn in NBS Bovine Liver and for Zn in BCR Bovine Muscle, although results for Fe and Cu were just outside the certified range 87 Biological tissues and botanical samples Pb Domestic oven (700 W) On-line HCl–HNO3 digestion of samples (dispersed in Triton X- 100 solution) On-line analysis by FI– ETAAAS Good results were obtained for BCR Bovine Muscle, Pig Kidney and NIST Bovine Liver.For NIST Pines Needles and BCR Olea Europea results were slightly low 88 Fruit slurries and juices Pb Domestic oven (650 W) On-line HNO3 digestion of liquid and slurried samples (dispersed in Triton X-100) On-line analysis by FI– HG–AAS No significant differences were observed for the results obtained by FI–MW–HG–AAS and a conventional Al heating block digestion (ETAAS analysis) 89 Food and feed crops Pb Domestic oven (600 W) HNO3 PTFE bomb digestion with V2O5 catalyst for 90 s ETAAS Good results were obtained for NIST Citrus Leaves 90 Fruit slurries Cd, Cu, Fe, Pb, Se Domestic oven (600 W) HNO3 PTFE bomb digestion with V2O5 catalyst for 90 s ETAAS Results were in good agreement with those obtained by a slurry procedure 91 Marine biological tissues Hg Domestic oven (700 W) HNO3 PTFE bomb digestion for 20 min after leaving samples overnight to partially digest (n = 10) CV-AFS, ICP-MS and ID–ICP-MS Results obtained after analysis by CV-AFS, ICP-MS (standard additions) and ID–ICP-MS (spike added prior to overnight digestion) were in good agreement with the certified values for BCR Cod Muscle 92 Botanical and sewage sludge samples Cu, Mn Domestic oven (650 W) On-line digestion of HNO3–H2O2 slurries for 2 min (botanical) and 4 min (sewage sludge) On-line analysis by FAAS Good results were obtained for Mn in NIST Tomato Leaves. For CBR Sewage Sludge–Domestic and Industrial results were generally in good agreement with the certified values 93 Botanical samples Co, Cr, Ni Domestic oven (800 W) On-line microwave digestion of slurries prepared in 5% HNO3 Off-line analysis by ETAAS Good results were obtained for Cr and Ni in NIST Citrus Leaves.Results for Co, Cr and Ni in vegetable samples were compared with those obtained by direct slurry analysis and by a dry ashing method 94 Botanical and sewage sludge samples Cr Domestic oven (650 W) Aqua regia–H2O2 PTFE bomb digestion ETAAS Good results were obtained for Cr in NIST Tomato Leaves and Citrus Leaves.Results were also in agreement with the informational value for BCR Sewage Sludge. Spike recoveries of 98–103% were obtained 95 Biological tissues and sewage sludge samples Cd Domestic oven (650 W) Biological: closed vessel HNO3 (14 m)–H2O2 digestion (12.5 min) Sludge: closed vessel aqua regia–HF–H2O2 digestion followed by H3BO3 treatment ETAAS Good results were obtained for BCR Pig Kidney, BCR Sewage Sludge Domestic and for MA-M-2/TM Mussel Tissue (IAEA) 96 Marine biological, botanical and sewage sludge samples Ni Domestic oven (1100 W) Aqua regia–HF–H2O2 PTFE bomb digestion for 12 min (4 steps) with cooling between each step.After adding H3BO3 the sample is heated in a boiling water bath for 10 min ETAAS Good results were obtained for Ni in NIES No.10c Rice Flour (unpolished), IAEA No. 325 Mussel Tissue, BCR CRM 143R Sewage Sludge Amended Soil and in CRM 144 Domestic Sewage Sludge 97 112R Analyst, July 1998, Vol. 123Table 1 Continued Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref. Botanical and sewage sludge samples Cu, Mn, Pb, Zn Domestic oven (650 W) Slurries (prepared in HNO3) are merged on-line with H2O2 and digested at 100% power On-line FAAS Results were in good agreement with the certified value for Mn in NIST Tomato Leaves, for Cu, Mn and Pb in CBR Sewage Sludge–Industrial and for Pb in Sewage Sludge–Domestic. Results were poor for Cu and Mn in Sewage Sludge–Domestic and for Zn in both sewage CRMs 98 Biological tissues and botanical samples B Domestic oven HNO3–H2O2 PTFE bomb digestion for 1 h (n = 2) Photometry, fluorimetry, ICP-MS and ICP-AES Good results were obtained for NBS Tomato Leaves and Pine Needles, although recoveries for Bovine Liver were low 99 Marine biological tissues Cd Domestic oven Closed digestion for 135 s (n = 6) with (a) HNO3, (b) H2SO4 and (c) HNO3–H2SO4. After cooling H2O2 is added, but no heating is applied FAAS Spike recoveries of (a) 95–107%, (b) 60–71% and (c) 73–89% were obtained.Digestion (a) was chosen for further study and used to digest NIST SRM 1566a Oyster Tissue. Good agreement with the certified value for Cd was obtained 100 Botanical samples Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Ni, Pb, Zn Domestic oven (550W) HNO3–H2O2 Teflon bomb digestion with water cooled spiral for 6 min (n = 1) FAAS and FAES Good results were obtained for Co and Cr in ISS/MMM certified Green Algae.Pb and Ni results were just outside the certified range. Acceptable results were obtained for the ‘in-house’ reference material Lucerne, except for Fe and Zn 101 Octocorals Cd, Cu, Ni Pb, Zn Domestic oven (600 W) Microwave pre-drying (20–50 min) followed by HNO3 digestion in pyrex tubes for 1 min (4–6 times) with cooling between steps ETAAS and FAAS (for Zn) Good recoveries were obtained for a synthetic CRM prepared from a mixture of 61% NIES Mussel and 39% CaCO3.Eight octocoral species were also analysed 102 Biological tissues and botanical samples Ca, Cu, Fe, Mg, Mn, Zn Domestic oven (500 W) HNO3–HClO4–HCl–HF PTFE bomb digestion (with polypropylene jacket) for 14 min (n = 6) ‘One-drop’ FAAS Results were in good agreement with the certified values for NIST Bovine Liver, NIES Pepperbush and Mussel samples. For NIES Tea Leaves, Fe results were high and Ca low. Ca results were also low for NIES Sargasso 103 Botanical samples Cd Domestic oven HNO3–HClO4–HCl–HF PTFE bomb digestion (with polypropylene jacket) for 9 min (n = 6), followed by hotplate evaporation to dryness and dissolution in HClO4 Fe was removed with HIPT in benzene and the Cd complex formed with APDC was extracted into chloroform for ‘onedrop’ FAAS analysis Results were in good agreement with the certified values for NIES Pepperbush and Rice Flour (low and medium) and for NIST Pine Needles, Orchard and Citrus Leaves 104 Marine biological tissues Se Domestic oven (600 W) HNO3–H2SO4–H2O2 PTFE bomb digestion SeVI is reduced to SeIV.The complex Se(O)SO3 22 is formed and analysed by DPP Results were in good agreement with the certified value for Se in NIES No. 6 Mussel sample 105 Marine biological tissues Se Domestic oven (600 W) PTFE bomb digestion with (a) HNO3–H2O2, (b) HNO3– H2SO4–H2O2, (c) HNO3– H3PO4–H2O2 , (d) HNO3– K2S2O8–H2O2 SeVI is reduced to SeIV for analysis by HG–AAS Good results were obtained for NIES Mussel following digestion by procedure (b).Procedures (a), (c) and (d) gave low recoveries 106 Marine biological tissues As Domestic oven (700 W) On-line potassium persulfate– NaOH oxidation, following HPLC separation of As species in sample extracts On-line analysis by HG–AAS AsV, MMA, DMA, arsenocholine and arsenobetaine levels were determined in a synthetic fish extract, although no CRMs were analysed. Spike recoveries of 96–110% were obtained 107 Marine biological tissues As Domestic oven (750 W) Sample was heated with HCl–KI in a PTFE bomb for 8 min (n = 1).The distilled AsCl3 was collected in hydroxylamine hydrochloride HG–AAS Good spike recoveries were obtained for inorganic arsenic in a mussel sample. However, organoarsenic compounds were not decomposed 108 Analyst, July 1998, Vol. 123 113RTable 1 Continued Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref. Botanical samples Ca, K, Mg, P, S Domestic oven (750 W) HNO3–HClO4 open digestion for 15–30 min depending on the sample (n = 25) ICP-AES analysis Good results were obtained for P in NBS Pine Needles and Citrus Leaves, although results for Ca and K in the former were slightly outside the certified range 109 Biological tissues As Domestic oven (650 W) HNO3–H2SO4–H2O2 PTFE bomb digestion HG–AAS with a modified electrical heating system Good results for As in NIST Bovine Liver and spike recoveries of 91–108% were obtained 110 Food samples Bromide ions Domestic oven NaOH (0.5 m) bomb digestion for 4 min.After cooling, H2O2 is added and heating continued for a further 2 min (n = 2) Ion-exchange chromatography (UV spectrometric detection), following cation exchange removal of Na Bromide ions were determined in total diet samples. Spike recoveries of 87–119% were obtained in four different food samples 111 Marine biological tissues Hg Domestic oven (750 W) HNO3–H2O2 PTFE bomb digestion for 5 min, after leaving the sample to predigest overnight CV-AAS Good results were obtained for total Hg in NRCC DORM-1 Dogfish Muscle.Tuna fish samples were also analysed and spike recoveries of 91–93% were obtained 112 Marine biological tissues As Domestic oven (600 W) HNO3 PTFE bomb digestion with catalyst of V2O5 for 90 s HG–AAS Results were in good agreement with the certified value for BCR Mussel Tissue and spike recoveries of 93–101% were obtained 113 Marine biological tissues Hg Domestic oven (600 W) HNO3 bomb digestion of samples and standards for 90 s CV-AAS Good results for BCR Mussel Tissue and spike recoveries of 95–106% were obtained 114 Botanical samples As Domestic oven (600 W) HNO3 bomb digestion with catalyst of V2O5 for 90 s HG–AAS Results were in good agreement with the certified values for NBS Citrus Leaves 115 Total diet samples Al, Ca, Cu, Fe, K, Mg, Na, P, Zn Domestic oven (750 W) Digestion (quartz vessel) with (a) HNO3, (b) HNO3–H2O2, (c) HNO3–H2SO4, (d) HNO3–HCl ICP-AES Good results were obtained for NIST Total Diet except for (a) slightly low K and Zn, (b) slightly low K, (c) slightly high P and slightly low K, (d) low Al, K and Zn 116 Marine biological tissues As Domestic oven HNO3 Teflon bomb digestion for 90 s ETAAS Good results were obtained for NIST Oyster Tissue 117 Biological tissues Se Domestic oven (650 W) HNO3 Teflon bomb digestion (3 min) followed by evaporation to dryness with HClO4 (twice) and re-dissolution in H2O SW-CSV following reduction of SeVI to SeIV Results were in good agreement with the certified values for NIST Bovine Liver (sample amount 5 mg) 118 Biological tissues and botanical samples Ca, Fe, Mg, Zn Domestic oven (800 W) HNO3 closed digestion for 7 min (n = 2) ICP-AES Good results were obtained for NIST 1577 Bovine Liver (except slightly high Mg and Zn) and for 1570 Spinach (except slightly low Ca and Fe) 119 Food samples Al Domestic oven HNO3 PTFE bomb digestion (32 min) ICP-AES Generally low results were obtained for total diet samples.Higher recoveries were obtained by a HNO3–HF–HNO3–HClO4 digestion in a drying oven 120 Botanical samples Cd, Cu, Pb, Zn Domestic oven (850 W) HNO3–HClO4 digestion in quartz crucible placed inside Teflon bomb for 29 min (n = 4) followed by hot-plate evaporation to dryness DP-ASV Good results were obtained for NIST Citrus Leaves, Lucerne P-alfalfa (Slovakia) and CL-1 Cabbage leaves (Poland) (except low Cu).Cu, Pb and Zn results were low for NIST Apple Leaves 121 Biological tissues and botanical samples Cd, Cu, Pb, Zn Domestic oven HNO3–HClO4 digestion in quartz crucible placed inside Teflon bomb for 11 min (n = 10) DP-ASV Generally good results were obtained for CL-1 Cabbage Leaves (Poland), P-alfalfa Lucerne (Slovakia), BCR Rye Grass, SRM Apple Leaves, BCR and SRM 1577b Bovine Liver 122 114R Analyst, July 1998, Vol. 123Table 1 Continued Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref.Sewage sludge Cd, Cr, Pb Domestic oven HCl–HNO3 Teflon bomb digestion (20 min) following pre-digestion overnight ETAAS Good results were obtained for Pb and Cr in BCR Sewage Sludge (CRM 145R) but Cd results were slightly low 123 Biological tissues Po Domestic oven (650 W) HNO3 PFA bomb digestion (1h for plants and 2 h for animal tissue, n = 4), followed by evaporation to dryness and re-dissolution in HCl Alpha spectrometry after plating on silver discs A good level of precision for results was achieved with no loss of Po during the digestion 124 Marine biological tissues and botanical samples Cd, Co, Cu, Ni, Pb Domestic oven HNO3–HCl–HClO4–HF PTFE bomb digestion ICP-AES Good Cu and Pb, but low Cd, Co and Ni results were obtained for NIES Pepperbush.Results were also good for Cd, Cu and Pb in Chlorella. For NIES Mussel, Cd, Co and Cu results were good, however Ni and Pb were low.Generally low results were obtained for NIES Tea Leaves 125 Marine biological tissues Cd, Cu, Pb, Zn Domestic oven HNO3 PTFE bomb digestion for 6.5 min (n = 4) FAAS and ETAAS Generally good results were obtained in NRC-D Dogfish (except low Cu), NRC-E Scallop (except low Cd) and NRC-F Swordfish (except low Cu) 126 Botanical samples Co Domestic oven HNO3–HCl bomb digestion for 10 min followed by hotplate evaporation to dryness FI–spectrophotometric determination following complexation with PSAA Results were in good agreement with the certified values for NIES Pepperbush 127 Biological tissues and botanical samples Cd, Cu, Fe, Mn, Pb — Open HNO3–H2O2-digestion, following having left the sample to pre-digest overnight ETAAS Generally good results were obtained for BCR Bovine Mussel, Olea Europea (except high Cu), Lagarosiphoumajur (except low Cu), Pig Kidney (except low Cu, Fe, Zn) and for NBS Citrus Leaves (except high Cu), Pine Needles (except low Pb) and Wheat Flour 128 Marine biological tissues As — H2SO4–HNO3–H2O2 closed digestion for 30 min ICP-AES A selection of fish and shellfish samples were analysed; however, no CRMS were included 129 Botanical samples Al, Fe, Si — HNO3–H2O2–HF digestion for 13 min ICP spectrometry UCD 155 (Avocado); 176 (Citrus); 124 (Barley Hulls) and 190 (Rice Straw) and NIST 1547 (Peach) samples were analysed 130 Food samples Amino acids — Hydrolysis performed by heating with 6 m HCl in a closed vessel — The amino acid sequence obtained from microwave digestion and a conventional method were compared in bovine serum albumin and Durum wheat samples 131 Botanical samples Phenolic acids — Teflon bomb NaOH digestion at 700 W for 90 s HPLC The liberation of b-ether bound phenolic acids from plant cell walls of maize, wheat, oilseed rape stems and barley was an order of magnitude more effective than with a dioxane—HCl procedure and as effective, but far quicker than high-temperature alkaline digestions 132 Botanical samples P — HNO3–H2O2–HCl digestion ICP-AES Results were in good agreement with the certified value for NIST Citrus Leaves 133 Biological tissues and botanical samples Ag, Ba, Cd, Cs, Hg, Mo, Pb, Rb, Sb, Sn, Sr — HNO3 PTFE bomb digestion for 40 min ICP-MS Good results were obtained for Ag, Mo, Pb and Rb in NIST Bovine Liver (Sr slightly high), for Rb in NIST Wheat Flour (Mo slightly high) and for Cd and Hg in BCR Pig Kidney 134 Marine biological tissues and botanical samples Al — HNO3 PTFE bomb digestion with seven heating steps (total 56 min), with cooling between each step ETAAS Results were in good agreement with the certified values for Al in NIES Mussel, NIST Citrus Leaves and Oyster Tissue.Spike recoveries of 92–104% were also obtained 135 Analyst, July 1998, Vol. 123 115RTable 2 Microwave digestion procedures for geological samples Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref.Sediment samples Hg Prolabo Microdigest 301 On-line digestion of 0.15% slurries (in 50% HCl), including Br2–BrO32 oxidation of organomercury species On-line analysis by CV-AAS A recovery of 97% was obtained for a methylmercury standard. Good results were obtained for Hg in NRCC PACS-1 Harbour Marine Sediment 22 Sediment samples Hg Prolabo Microdigest 301 Open focused HNO3–H2SO4– H2O2 digestion for 10 min (n = 1) CV-AAS Results were in agreement with the certified value for NRCC PACS-1 Harbour Marine Sediment 22 Soil and sediment samples Hg Prolabo Microdigest 301 On-line digestion of slurries prepared in nitric acid On-line FI–CV-AFS analysis Good results were obtained for State Bureau of Metrology (China) Polluted Farm Soil and Canadian Centre for Mineral and Energy Technology Lake Sediment 24 Soil and sediment samples As, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Zn Prolabo Microdigest A300 and CEM MDS-81D (a) Closed vessel HCl–HNO3–HF digestion.Open focused digestion with: (b) HNO3– H2O2; (c) HCl–HNO3–H2O2 ICP-AES and ICP-MS Good results were obtained for BCR Amended Soil using procedure (b) and for Estuarine Sediment (except high Hg and Pb) using procedures (a) and (c) 25 Soil samples Hg Prolabo Maxidigest MX350 and CEM MDS-81D Open and closed digestion with (a) 1 m HCl, (b) 50% HNO3, (c) HNO3, (d) Aqua regia CV-AAS and ETAAS For the open digestion, results were low for NIST Montana Soil whereas good results were obtained for the closed digestion [procedures (a), (b) and (d)] 136 Sediment samples As Prolabo A300 and CEM MDS-81D Open focused digestion with (a) HCl–HNO3–H2O2 for 35 min (total As), (b) HNO3–HCl for 12 min (As speciation).Closed digestion with: (c) HNO3– HClO4–HF–HCl for 1h (total As) ICP-MS and ICP-AES (for total arsenic) HPLC–ICP-MS (for As speciation) In the lake sediment analysed, the As species DMA, MMA and AsV were stable during the microwave extraction procedure.AsIII was oxidised to AsV, but this was reduced by extraction with orthophosphate or ammonium oxalate. An extraction yield for total As of 100% was obtained for the microwave extraction procedure (calculated as % of the yield obtained by the total As procedures) 137 Sediment samples Hg Prolabo Maxidigest (300 W) On-line digestion of slurries prepared in aqua regia– KMnO4 On-line analysis by FI mercury system Good results were obtained for NIST Buffalo Sediment, although those for NRCC BCSS-1 Marine Sediment were slightly high. Good spike recoveries were also obtained 138 Soil samples Cu, Fe, Zn Prolabo Microdigest 301 Automated DTPA–CaCl2– triethanolamine extraction using a robotic station (5 samples h21) Automated centrifugation and transport to FAAS The extraction efficiency of Zn is comparable to the conventional technique, whereas a greater efficiency resulted for Fe and Cu 139 Sediment samples Hg Prolabo Microdigest A301 Open focused HNO3 digestion for 5 min, followed by 5 min cooling and heating with H2O2 (5 min) FI–ICP-MS Good results were obtained for Hg in NRCC PACS-1 Harbour Marine Sediment, IAEA-356 and BCR S19 sediment samples. Samples from Arcahon Bay were also analysed 140 Dust and air filters Pb CEM SpectroPrep On-line microwave digestion of slurried samples (prepared in 3 m HNO3); 10 min per sample Off-line analysis by ID– ICP-MS Good results for Pb were obtained in NIST SRM 2676d Toxic Metals on Filters 32 Coal samples Ca, Cd, Fe, Mg, Zn CEM MDS-81 On-line stopped-flow digestion of slurries (in Triton X-100 and HNO3) for 5 min per sample Off-line analysis by AAS Incomplete digestion of coal resulted 35 Soil samples As, Se CEM MDS-2000 H2O–HNO3–HCl–HF PTFE bomb digestion for 30 min HG–AAS Good results for MRG-1 silicated rocks (Canada Centre for Mineral and Energy Technology) and spike recoveries of 95% were obtained 38 116R Analyst, July 1998, Vol. 123Table 2 Continued Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref. Geological samples As, Ba, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Tl, V, Zn CEM MDS-81D HNO3 Teflon bomb digestion (18 min) followed by 21 min heating with HF. H2O2 is then added and heating continued in a water-bath (15 min) before adding H3BO3 for a further 5–10 min (n = 12) AAS and ICP-AES Good results were obtained for NIST SRM 2704 Buffalo River Sediment, except for high Sb and Tl, slightly low Co and Cu and slightly high As, Cr and Mn.The method was developed to analyse fertilizers and soil amendments including rock phosphates, liming materials organic material and sandy loam soil 45 Soil samples As, Se CEM MDS-81D PFA bomb digestion for 1–2 h with: (a) HNO3–HCl, (b) H2O2–HCl–H2SO4, (c) H2O2– H2SO4, (d) HNO3–H2SO4 FI–HG–AAS Method (d) gave the best recoveries (validated using NIST San Joaquin Soil). Good results were obtained for As, but Se recoveries were slightly high.Method used to analyse sewage sludge (see Table 1) 51 Sediment samples As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Zn CEM SpectroPrep system On-line digestion of 1% slurries (in 20% HF, 50% HNO3 and 10% HCl) ICP-MS, ICP-AES (for Fe and Al) and ETAAS (for As) Good results were obtained for As, Cd, Co, Cu, Fe, Mn, Ni, Pb and Zn in NRCC BCSS-1 Marine Sediment, however, results for Cr and Al were low 54 Sediment samples Hg CEM MDS-2000 HNO3 PTFE bomb digestion for 70 s CV-AAS Results were in good agreement with the provisional value for NIES No. 2 Pond Sediment 121 Phosphatic fertilisers and animal feedstuffs As, B, Ba, Bi, Cd, Co, Cr, Cu, Hg, Mn, Mo, Ni, Pb, Se, Sb, V, W, Zn CEM MDS-81D PTFE bomb digestion with (a) HNO3, (b) HCl–HNO3– HClO4–HF ICP-MS For NIST Buffalo River Sediment: (a) Results for As, Cd, Co, Cu, Ni and Pb were good, but those for Ba, Cr, Mn, Sb, V, Zn were low and Hg was high; (b) Results for As, Co, Cu and Ni were good; Ba, Hg and V results were better than for (a), but Ni, Zn and Sb were high and Cr low.Results for NBS Florida Phosphate Rock by procedure (a) generally agreed well with the certified and informational values 141 Sediment samples Co, Cu, Mn, Pb, Zn CEM MDS-81D 18 digestion procedures with different combinations of HNO3, H2O2, HF and HCl FAAS and L’vov platform (for Co) PCA and multicriteria decision making methods PROMETHEE and GAIA selected an HCl–HNO3–HCl digestion as the best for NBS Buffalo River Sediment 142 Sediment and rock samples Co, Cr, Cu, Ni, Pb, Zn CEM MDS-81D 14 digestion procedures with different combinations of HF, HNO3, HCl, H2O2 and acetic acid FAAS PCA, SIMCA, PROMOTHEE, GAIA and Fuzzy Clustering chemometric techniques selected an HNO3–HF digestion as the best procedure for NBS Buffalo River Sediment and ‘In House’ secondary rock standard 143 Sediment samples Cd CEM MDS-81D HNO3 Teflon bomb digestion for 80 min (n = 12) ETAAS Good results were obtained for NIST Sediments 1646 and 2704.Results were in good agreement with those obtained from a conventional HF–HClO4 digestion undertaken in platinum crucibles 144 Sediment samples Cr (a) CEM MDS-81, (b) Floyd RMS-150, (c) Milestone MLS 1200 MEGA HCl–HNO3–HF–HClO4 PTFE bomb digestion: (a) heated to 1200 psig, cooled and repeated 32, (b) heated for 20 min at medium pressure, (c) heated for 26.5 min at high pressure. After cooling all samples were evaporated to dryness on a hotplate and solubilised in HNO3 FAAS and ICP-AES Using procedure (b), results were obtained in agreement with the certified value for NIST SRM 2704 Buffalo River Sediment.A mean spike recovery (for all procedures) of 98% was obtained. However, low results were obtained for Cr in NRCC BCSS-1 Marine Sediment following procedures (a), (b) and (c) and also by an open hot-plate method. It was concluded that no acid dissolution procedures are adequate for the determination of Cr in this sample 145 Sediment samples Cr, Cu, Hg, Mn, Ni, Pb, Zn CEM MDS-81D PTFE bomb digestion (n = 12) with (a) aqua regia (80 min), (b) aqua regia–HF (80 min) AAS and CV-AAS (for Hg) For procedure (a), results were generally low for NRCC MESS-1 Estuarine Sediment and PACS-1 Harbour Marine Sediment.Results were slightly improved after digestion by procedure (b), but most results were still not in good agreement with the certified values 146 Analyst, July 1998, Vol. 123 117RTable 2 Continued Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref. Dust, ashes and sediments Al, As, Ba, Be, Ca, Co, Cr, Cu, Fe, Mg, Mn, Ni, Pb, S, Sb, Ti, V, Zn CEM MDS-81D Teflon PFA bomb digestion for 22 min (n = 6) with (a) HNO3–HCl (acid soluble elements), (b) HNO3–HCl–HF with H3BO3 neutralisation (total digestion) ICP-AES (a) Recoveries obtained for different elements in the range: NRCC MESS-1 Estuarine Sediment 25–103%, NRCC PACS-1 Harbour Marine Sediment 38–99%, NIST Coal Fly Ash: 60–103%, ‘in-house’ dust: 23–100%.(b) Good results were obtained for Coal Fly Ash (except low Co) and for MESS-1 (except low Ti). For PACS-1 results were good, except for Al, Fe, Ca, Mg, Ni and S (just outside certified range) 147 Sediment samples As, Hg, Se CEM MDS-81D PFA bomb digestion with (a) H2SO4–HNO3–HCl (As and Se), (b) HNO3 (Hg) FI–AAS (As and Se) and CV-AAS (Hg) Good results were obtained for proposed NIST SRM 1646a Estuarine Sediment and SRM 2704 Buffalo River Sediment.Results were in good agreement with those of a traditional reflux digestion 148 Molybdenite mineral Os CEM MDS-81D HNO3–H2SO4 PTFE bomb digestion for 45 min followed by heating with K2Cr2O7. Os distillation prior to ICP-MS analysis The technique was applied to Re–Os age determination in a natural molybdenite sample. Results were in agreement with those obtained by a U–Pb method for zircon (associated mineral) 149 Marine sediment samples Al, Ca, Cr, Cu, Fe, Mn, Ni, Pb, Zn CEM MDS-2000 and Milestone LAVIS-1000 multiMOIST microwave drying system HNO3 bomb digestion following microwave drying of the sample by the two methods (15 min) ICP-AES and FAAS (for Pb) No significant differences were found between the moisture content of marine sediment dried by traditional oven and vacuum microwave drying techniques.Similar results were obtained for the trace metal and total carbon content of marine sediments dried by the two methods 150 Geological samples Ba, Be, Co, Cr, Cs, Cu, Hf, Mo, Ni, Nb, Pb, Rb, Sb, Sc, Sn, Sr, Ta, Th, Tl, U, W, Zn, Zr and REEs CEM MDS-81D PFA bomb HNO3–HF–HClO4 digestion for 63 min (n = 4), followed by hot-plate evaporation to dryness with HClO4 and dissolution in HNO3 ICP-AES and ICP-MS Generally acceptable results were obtained for Ba, Be, Co, Cs, Cu, Ni, Nb, Pb, Rb, Sb, Sn, Sr, Ta, Th, Tl, U, W, Zn and for most of the REEs in a range of geological CRMs (sediments and rocks).The accuracy of Cr, Hf, Mo, Sc, Zr determinations varied with the sample type, whereas Y recoveries were low in the nine CRMs analysed 151 Geological samples Au, Ir, Pd, Rh, Ru, Pt CEM MDS-81D (a) Low pressure HNO3–HCl– HF–HClO4 digestion. (b) High pressure aqua regia– HF digestion. Residues were fused with Na2CO3–Na2O2– ICP-MS Procedure (a) was employed for the digestion of sulfide-rich samples and procedure (b) for silicate, sulfide and chromitite samples.CRMs were also analysed 152 Airborne particulates on Teflon filters Al, As, Ba, Cd, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Sb, V, Zn CEM MDS-2000 HCl–HNO3–HClO4–HF digestion (32 min) followed by open vessel evaporation of HF (25 min) (n = 12) ICP-MS Generally low results were obtained for NIST Urban Particulate Matter [accredited in part to the low mass of sample used (0.1 mg)]. Higher recoveries were obtained with a conventional pressure bomb digestion although the digestion time was 10 times higher 153, 154 Airborne particulates on glassfibre filters Al, Fe, K, Mg, S, Zn CEM MDS-2000 HNO3–HClO4 PTFE bomb digestion for 9 min followed by cooling, removal of filter residue, heating with HF (9 min) and H3BO3 neutralisation of HF ICP-AES Recoveries of 90–101% were obtained for Al, Fe, K, Mg, S and Zn in NIST Urban Particulate Matter 155 Airborne particulate matter on filters As CEM MDS-2000 HNO3–HClO4 PTFE bomb digestion, followed by cooling, removal of filter and further heating with HClO4–HF (total 18 min).HF removed by evaporation ICP-MS A recovery for As of 107.4% was obtained in NIST (SRM 1648) Urban Particulate Matter 156 118R Analyst, July 1998, Vol. 123Table 2 Continued Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref. Rock samples 59 major and trace elements CEM MDS-2000 HNO3–HF–HClO4 Teflon bomb digestion for 0.5–1 h (n = 9). After cooling samples are transferred to beakers for hotplate evaporation to dryness.HNO3 is then added, and samples taken to dryness again ICP-MS Results were presented for 59 elements in Geological Society of Japan rock reference materials JB-1 Basalt, JB-3 Basalt, JG-1 Granodiorite, JR-2 Rhyolite, JGb-1 Gabbro and JA-2 Andesite 157 Sediment samples Cd, Cr, Co, Cu, Fe, Mn, Ni, Pb, Se, Zn CEM MDS-2000 HF–HCl–HNO3 PTFE bomb digestion for 15 min (n = 12) FAAS and ETAAS Orthogonal array design was applied to the optimisation of digestion parameters.Generally good results were obtained for NBS Buffalo River Sediment, NRCC BCSS-1 Marine Sediment and NIES Pond Sediment 158 Sediment samples Hg CEM MDS-2000 PTFE bomb closed digestion for 30 min (n = 12) with (a) HNO3–H2SO4, (b) HNO3– HClO4 (c) HNO3–HCl, (d) HNO3–HCl–HF FI–CV-AAS Good results were obtained for NIST SRM 1645 River Sediment, NRCC BCSS-1 Marine Sediment, NIES CRM No. 2 Pond Sediment and in a mixture of the latter and NIST SRM 1515 Apple Leaves (chosen to give the sample an organic matter content of 10%).Procedures (c) and (d) were the most effective. Spike recoveries of 94-104% were obtained for sediment and soil samples 159 Soil and sediment samples Cu, Cr, Mn, Pb, Zn CEM MDS-2000 HCl–HNO3–HF PTFE bomb digestion for 30 min (n = 12) FAAS and ETAAS Mixed-level orthogonal array design was applied to the optimisation of digestion parameters. For the optimised procedure, generally good results were obtained for NBS SRM 1645 River Sediment, NIES No. 2 Pond Sediment (except low Cr) and NRCC BCSS-1 Marine Sediment 160 Sediment samples As, Se CEM MDS-2000 Closed digestion for 30 min (n = 12) with (a) HNO3– H2SO4, (b) HNO3–HClO4, (c) HNO3–HCl, (d) HNO3–HCl– HF, (e) HNO3–H2SO4–HClO4 FI–HG–AAS Good results were obtained by all procedures for NIST SRM 1645 River Sediment, however procedures (a), (b) and (e) were not recommended owing to the potential hazardous nature of the digestion environment.No effect to the digestion was observed following the addition of SRM 1515 Apple Leaves (to give an organic content of 10%). Good results were obtained by procedures (c) and (d) for NRCC BCSS-1 and NIES CRM No. 2 Pond Sediment. Spike recoveries of 94–104% were obtained for sediment samples 161 Coral soil samples Si Floyd RMS-150 HNO3–H2O2–HF Teflon bomb digestion for 25 min ICP-AES following addition of H3BO3 and a tertiary amine mixture Acceptable agreement with the results obtained from an LiBO2 fusion procedure was obtained following microwave digestion of five coral soil samples.The average precision of the method was 7% 19 Soil and dust As Floyd RMS-150 HNO3–H2O2 bomb digestion for 32 min (n = 6) ICP-MS Good results were obtained for NIST Urban Particulate Matter and IAEA Soil 7 77 Sediment samples Cd, Cr, Cu, Pb, Ni, Sb, Sn, Th Floyd Samples were digested with HNO3–HF for 52 min, evaporated to dryness on a hotplate and dissolved in HNO3– H2O ID–ICP-MS, ICP-MS and ETAAS Good results were obtained for Cd, Cr, Cu, Pb, Ni, Sb, Sn and Th in a Mississippi River delta sediment sample (NOAA/7) 78 Dust samples Cd, Pb Floyd RMS-150 Teflon bomb digestion for 20 min with (a) HNO3, (b) HCl– HNO3, (c) HNO3–HF DP-ASV and FAAS Digestion efficiencies of 85–95% were obtained (RSD = 10%) for NBS Urban Particulate Matter, BCR City Waste Incineration Ash and River Sediment.No significant differences were found between the results of the three microwave methods and a standard hot-plate digestion method 162 Analyst, July 1998, Vol. 123 119RTable 2 Continued Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref. Geological samples Al, Fe, K, Mg, Na, Si Milestone MLS-1200 PTFE bomb digestion. Coal: HNO3–H2O2–HF–HClO4. Limestone: HCl–HF. Iron Ores: HCl ICP-AES and AAS Results for limestone samples were in good agreement with those obtained by XRF.Fe levels in BCS NIMBA Fe Ore, Fe Ore Sinter and Lincolnshire Fe Ore were within the certified range 72 Sediment samples As, Al, Cd, Cr, Co, Cu, Fe, Mn, Ni, Pb, Sn, Ti, Zn Milestone MLS-1200 MEGA Teflon bomb digestion for 25 min (n = 6) with (a) HNO3–HF– H3PO4–H3BO3, (b) HNO3– HCl–HF–H3BO3, (c) Extraction with HNO3 ICP-AES [for (a) and (b)] and TXRF [for (c)] Results for BCR 320 River Sediment were follows: (a) good for Al, Fe, Mn, low for Cr, Cu and Zn and high for Co; (b) good for Al, Fe, Cu; high for Co and slightly low for Cr, Mn, Zn; (c) good for As, high for Ni, slightly low for Cu, Fe, Pb and Zn and very low for Cr and Sn 163 Sediment samples 50 elements Milestone MLS-1200 HNO3–HF Teflon bomb digestion for 19 min, followed by evaporation to dryness (90 min) and dissolution in HCl ICP-MS and TXRF Good results were obtained for Al, Ca, Fe, K, Mg, Na, Pb, Rb, Sr, Ti and V in NRCC MESS-1 Estuarine Sediment; however, Ba results were high and Zn low 164 Rock and sediment samples Th, U, Y and lanthanides Milestone MLS-1200 MEGA HF–HNO3–HCl Teflon bomb digestion for 16 min, followed by heating with H3BO3 and EDTA for 8 min ICP-MS Results were presented for USGS Andesite (AGV-1), Basalt (BCR-1, BHVO-1), Diabase (W-2, DNC-1), Granite (G-2), Marine Mud (MAG-1), for CCRMP Syenite (SY-2), Gabbro (MRG-1), Lake Sediments (LKSD-1,4), Stream Sediments (STSD-1,4) and for NIM-G Granite, BE-N Basalt, GSD-1,5,6 Stream Sediment and NBS SRM 1645 River Sediment 165 Airborne particulates on PTFE filters Al, As, Cd, Cr, Cu, Fe, K, Mg, Ni, Pb, S, Sb, V, Zn Milestone MLS-1200 HNO3–HClO4–HF PTFE bomb digestion for 8 min FAAS, ETAAS, ICP-AES and ICP-MS Good results were obtained for As, Cd, Cu, Fe, Mg, Ni, S, Sb and Zn in NIST Urban Particulate Matter; however K, Pb and V results were slightly low and Al and Cr very low 166 Coal samples REEs Milestone MLS-1200 PTFE bomb digestion with HNO3–H2O2–HF–HCl HPIC with UV/VIS detection and on-line preconcentration Acceptable agreement was obtained with the published values for NBS 1632a, SARM-18, 19 and 20 coal CRMs (not certified) 167 Baghouse dust Hg Questron Q-Wave 1000 HNO3 Teflon bomb digestion CV-AAS No statistical difference was observed between the results for a traditional water bath method and the microwave digestion method 168 Geological samples Al, Ag, As, Ba, Bi, Cd, Co, Cr, Cu, Fe, Li, Mg, Mn, Mo, Ni, Pb, Sb, Sn, Sr, Th, Ti, Tl, U, V, Zn Questron Q-Wave 1000 HNO3–HF–H2O2 Teflon bomb digestion for 25 min.After cooling, H3BO3 is added and heating continued for a further 15 min (n = 12) ICP-MS Results were presented for NIST SRM 1633b Coal Fly Ash, SRM 1648 Urban Particulate Matter, SRM 1646 Estuarine Sediment, SRM 2704 Buffalo River Sediment, SRM 2709 San Joaquin Soil Baseline Trace Element, SRM 2711 Montana Soil Moderately Elevated Trace Element and SRM 2700 Montana Soil Highly Elevated Trace Element 169 Marine sediments Pb Portland DMR-140 HNO3–HCl PTFE bomb digestion for 10 min ETAAS Results for NRCC PACS-1 Harbour Marine Sediment were good and spike recoveries of 95–99% were obtained.Results were compared with those obtained by a slurry method 170 Coal fly ash As, Se Commercial oven (650W) HCl–HNO3–H2SO4 Parr bomb digestion for 2 3 3 min with interim cooling step (15 min). Sample then heated in a waterbath for 30 min to remove nitrates HG–AAS analysis following pre-reduction with KI and ascorbic acid Results for As and Se in NIST 1633b Coal Fly Ash were in good agreement with the certified values and with the results obtained by an NaOH fusion procedure 171 Sediments, geological samples Hg Domestic oven (800 W) HNO3 PTFE bomb digestion for 3 min at 800 W (n = 5) FANES after reduction with SnCl2 and in situ preconcentration Results for NIST River Sediment were slightly low 85 120R Analyst, July 1998, Vol. 123Table 2 Continued Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref.Sediments samples Cr Domestic oven (650 W) Aqua regia–HF–H2O2 PTFE bomb digestion, followed by 10 min heating with H3BO3 in a water-bath ETAAS (no modifier) Results were in good agreement with the certified value for Cr in BCR River Sediment. Spike recoveries of 101–102% were obtained 95 Sediment and soil samples Ni Domestic oven (1100 W) Aqua regia–HF–H2O2 PTFE bomb digestion for 12 min (4 steps) with cooling between each step.After adding H3BO3 the sample is heated in a boiling water-bath for 10 min ETAAS Good results were obtained in BCR CRM 143R Sewage Sludge Amended Soil, BCR CRM 320 River Sediment, NIES No. 2 Pond Sediment; Canada Centre for Mineral and Energy Technology LKSD 4 Lake Sediment; MAFF North Sea Sandy Sediment COR 5A/91 and Geological Survey of Japan JB 2 Tholeiitic Basalt 97 Sediment samples As Domestic oven (700 W) On-line potassium persulfate– NaOH oxidation following HPLC separation of As species (prior digestion of samples is necessary) On-line analysis by HG–AAS AsV, MMA and DMA species were determined in a sediment extract. No CRMs were analysed 107 Sediment samples Hg Domestic oven (750 W) PTFE bomb HNO3–H2SO4–H2O2 digestion for 4 min following pre-digestion overnight CV-AAS Good results were obtained for Hg in MESS-2 Estuarine Sediment.Spike recoveries of 91–108% were obtained in River Mersey sediment samples 112 Dust wipe and air filters Pb Domestic oven (800 W) Teflon PFA HNO3 digestion for 6 min (n = 12) ETAAS Recoveries of 96–114% were obtained for the NIOSH and ELPAT wipe samples.For air filter samples, spike recoveries of 94–103% were obtained 172 Soil and sediment samples Cd, Cu, Pb — Open HNO3–H2O2–aqua regia– HF digestion following overnight pre-digestion ETAAS Generally low results were obtained for BCR Calcareous Loam Soil, River Sediment and NBS Urban Particulate Matter 128 Silicate rocks Fe oxidation states — HF–H2SO4 PTFE bomb digestion under Ar atmosphere Absorbance at 560 nm of FeIII–Tiron complex followed spectrophotometrically with time FeO and Fe2O3 results were compared with those from the static o-phenanthroline method 173 Soils and clays Al, Ca, Fe, K, Mg, Mn, Na, P, Si, Ti — HF–aqua regia Teflon bomb digestion for 10 min ICP-AES Generally good results were obtained for NIST SRM 1646 Estuarine Sediment, SRM 278 Obsidian and SRM 688 Basalt 174 Analyst, July 1998, Vol. 123 121RTable 3 Microwave digestion procedures for water and waste water samples Matrix Elements Microwave system Digestion method Analysis Technique Comments Ref. Water samples SeIV and SeVI Prolabo Microdigest 301 On-line microwave assisted HCl pre-reduction of SeVI to SeIV On-line analysis by FI–CSV SeVI was calculated as the difference between the SeIV concentration after pre-reduction and the initial SeIV concentration. For a BCR candidate water sample, results were low for SeIV and therefore high for SeVI.The total Se concentration was however in good agreement with the proposed value 10 Water and waste water samples Hg Prolabo Maxidigest On-line digestion of samples prepared in H2SO4–HNO3– KMnO4–K2S2O8 with HCl carrier FI–mercury system Generally good spike recoveries were obtained for inorganic and methylmercury in drinking water and waste water samples 138 Water samples Se Prolabo Microdigest M301 On-line HBr–KBrO3 pre-treatment of SeVI to SeIV (30 samples h21) On-line analysis by HG–AAS Total Se was determined following application of microwave energy to the sample, whereas SeIV was determined in its absence.SeVI was calculated by difference. No CRMs were analysed 175 Water samples Se Prolabo Microdigest M301 On-line HBr–KBrO3 pre-treatment of Se following HPLC separation of species On-line analysis by HG–AAS The determination of selenite, selenate and selenoamino acids (selenocystine, selenomethionine and selenoethionine) in urine was undertaken 176 Water samples Se Prolabo Microdigest 301 On-line HCl (6 m) reduction of SeVI to SeIV On-line analysis by HG–AAS The SeVI concentration was calculated as the difference between the total Se content (microwave on) and the SeIV content (microwave off).Good results were obtained for NIST 1643c Trace Elements in Water 177 Water samples Se Prolabo Microdigest 301 On-line HCl reduction of SeVI to SeIV following HPLC separation of SeVI and SeIV On-line analysis by HG–AFS Results were in good agreement with the total Se content of NIST 1643c Trace Elements in Water 178 Water samples As, Bi, Hg, Pb, Sn Prolabo Maxidigest MX-350 On-line digestion of samples mixed with a suitable oxidising agent (dependent on element) FI–CV-AAS and HG–AAS Good Bi and Hg results were obtained, although problems were experienced for As, Pb and Sn determinations 179, 180 Water samples Hg Prolabo Maxidigest MX-350 On-line KBrO3–KBr digestion (7 samples h21) FI–CV-AAS in amalgamation mode Good recoveries were obtained for the seven Hg species investigated. The results for 22 water samples were in acceptable agreement with those obtained from an external laboratory 181 Water samples Total N and P CEM MDS-81D Teflon bomb potassium persulfate and NaOH digestion for 45 min (n = 12) Colorimetric determination Good recoveries were obtained for all P and N compounds tested except for animoantipyrine (60–73%).Spike recoveries of 98.4–105.9% were obtained in real samples 182 Waste water samples Total P CEM MDS-81D On-line HNO3 digestion with prior addition of pyrophosphatase (25 samples h21) Colorimetric detection of molybdenum blue complex Results were in good agreement with those obtained form a batch ‘block’ digestion (3 h). Complete recoveries of tetrameta-, trimeta-, ortho- and pyrophosphate as orthophosphate were obtained 183 Water samples COD Milestone MLS 1200 On-line K2Cr2O7–H2SO4 oxidation (3 min) FI–spectrophotometric detection Results were in good agreement with those of the standard COD method for a range of water samples and food industry waste 64 Water samples Se Milestone MLS-1200 MEGA On-line KBr–HCl pre-treatment of SeVI, SeIV and Se-Met, following seperation by anionexchange chromatography HG–AAS The seperation of SeIV, SeVI and Se-Met was demonstrated; however, no CRMs were analysed 184 Water samples As Domestic oven (700 W) On-line potassium persulfate– NaOH oxidation, following HPLC separation of As species On-line analysis by HG– AAS AsIII, AsV and arsenobetaine were determined in mineral, sewage and harbour sea water samples although no CRMs were analysed 107 Water, waste water and sewage effluents Total P Domestic oven (700 W) On-line potassium peroxodisulfate digestion FI–colorimetric detection of phosphomolybdenum blue complex Complete digestion of all P compounds tested was achieved, except for condensed phosphates.No significant differences were observed between the results of the on-line and batch methods, although the former had a small positive bias 185 Waste water samples COD Domestic oven (662 W) On-line oxidation of sample, previously mixed with K2Cr2O7 (up to 50 samples h21) On-line FAAS analysis of unoxidised Cr following anion-exchange seperation The COD values obtained for 12 different organic compounds and for urban and industrial waste water samples were not significantly different to those obtained by a closed reflux method 186 122R Analyst, July 1998, Vol. 123Reid et al.187 described a method for the rapid cooling of Teflon pressure vessels using liquid nitrogen. Cooling in the microwave unit itself, although considerably decreasing reaction rates, was useful in some cases to prevent uncontrollable increases in pressure. However, a more effective method involved cooling, subsequent to or between heating cycles.This approach saved considerable time and additionally prevented pressure build-up occurring after the microwave process had ceased, which could otherwise lead to venting of the vessel. Open digestion techniques Open digestion systems operate at atmospheric pressure and so do not suffer from the problems associated with pressure build-up. However, they do require an effective fume removal system. Most open vessel work has been carried out using monomode (focused) microwave systems.10–28,136–140,175–181 Heating is more efficient than with conventional microwave designs (multimode) because the sample is placed within the waveguide, and thus directly within the path of the microwave energy.The potential loss of volatile species is controlled by condensation of vapours in a reflux column situated above the sample flask. The open vessel approach can generally accommodate larger samples (up to 15 g) than the closed technique and allows the delivery of digestion reagents at any stage of the procedure.The latter may be beneficial for the effectiveness of the digestion, and is a distinct advantage over closed methods where the addition of reagents cannot readily be achieved without cooling and opening the vessels. Also, the system can quickly and effectively evaporate to dryness, a particular advantage for the removal of HF during the digestion of geological samples. Another advantage is that the power output of the magnetron can be controlled more readily than for domestic ovens.For example, at 50% power the output of the magnetron is actually reduced to 50% rather than pulsing on and off to produce an overall mean of 50% power (see the previous section). Direct temperature measurements and temperature control to follow a previously defined programme are also possible.188 A potential disadvantage of the open monomode system is that only one sample can be digested at a time, although this can be overcome by use of an autosampler unit with the ability to run up to 16 samples.189 A multicavity monomode system is also available for the digestion of up to four samples for the determination of Kjedahl nitrogen.190 A more recent addition to the commercial market is a two- or six-cavity open microwave digestion unit with the ability to programme the power output/ desired temperature to each sample independently.191 Another point for discussion is that many open digestion procedures, by virtue of operating at atmospheric pressure, must use a high boiling-point acid, such as sulfuric acid, in order to decompose completely organic material in the sample.Many workers overcome the use of highly corrosive sulfuric acid by utilising perchloric acid or hydrogen peroxide instead. A less common approach is the use of conventional microwave ovens87,109,128 for open digestions. Burguera et al.87 demonstrated that the digestion of biological samples could be effectively achieved for 100 samples placed in polyethylene test-tubes (covered with a Teflon sheet) in only 14 min.However, no comment was made as to the lifetime of the oven. Table 4 Key to abbreviations used Analysis Techniques Reference Material Suppliers AFS Atomic fluorescence spectrometry BCR Community Bureau of Reference CIE Capillary ion electrophoresis CCRMP Canadian Certified Reference Materials Project CV-AAS Cold vapour atomic absorption spectrometry CGC Canadian Grain Commission DCP-AES Direct current plasma atomic emission spectrometry ELPAT Environmental Lead Proficiency Analytical Testing Program DP-ASV Differential-pulse anodic stripping voltammetry IAEA International Atomic Energy Agency DPP Differential-pulse polarography ISS/MMM Istituto Superiore di Sanit`a, Rome, Italy ETAAS Electrothermal atomic absorption spectrometry KRISS Korea Research Institute of Standards and Science FAAS Flame atomic absorption spectrometry MAFF Ministry of Agriculture, Fisheries and Food, UK FAES Flame atomic emission spectrometry NBS National Bureau of Standards, USA FANES Furnace atomic non-thermal excitation spectrometry NIES National Institute of Environmental Studies FI Flow injection NIOSH National Institute for Occupational Safety and Health HG–AAS Hydride generation atomic absorption spectrometry NIST National Institute of Standards and Technology, USA HPIC High-performance ion chromatography NRCC National Research Council of Canada HPLC High-performance liquid chromatography NRCCRM National Research Centre for Certified Reference Materials, Beijing IC Ion chromatography USGS United States Geological Survey ICP-AES Inductively coupled plasma atomic emission spectrometry ICP-MS Inductively coupled plasma mass spectrometry Others ID Isotope dilution COD Chemical oxygen demand IR Infrared spectrometry CRM Certified reference material NAA Neutron activation analysis MW Microwave SW-CSV Square-wave cathodic stripping voltammetry REEs Rare earth elements TLC Thin-layer chromatography RM Reference material TXRF Total reflection X-ray fluorescence spectrometry XRF X-ray fluorescence spectrometry Compounds APDC Ammonium pyrrolidin-1-yldithioformate BPTH 1,5-Bis[phenyl-(2-pyridyl)methylene]thiocarbonohydrazide DPTH 1,5-Bis(di-2-pyridylmethylene)thiocarbonohydrazine DTPA Diethylenetriaminepentaacetic acid EDTA Ethylenediaminetetraacetic acid HIPT 2-Hydroxy-4-isopropylcycloheptatrienone IBMK Isobutyl methyl ketone PSAA 2-(5-Bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino) aniline TMAH Tetramethylammonium hydroxide Analyst, July 1998, Vol. 123 123ROn-line digestion techniques There is a growing trend towards the development of on-line microwave digestion and analysis techniques, for both solid22,24,32,35,36,54,83,84,86,88,89,93,94,98,138 and liquid10,21,107,138,1752181,1832186 samples. Such techniques can lead to considerable time savings compared with batch microwave digestions and thus the benefits over conventional techniques are even more impressive.However, for solid samples it is usually necessary to prepare a slurry of the sample before analysis. This is undertaken to ensure effective sample transport into the digestion manifold. Most workers have reported the necessity for further grinding of the sample, sometimes with the addition of surfactants,35,89 in order to produce a stable slurry. Samples can then be digested in either a continuous or stopped flow system for on-line analysis22,24,35,36,86,88,89,93,98,138 or collected for separate treatment. 32,35,54,83,84,94 Hulsman et al.192 investigated the dispersion behaviour of solid particles in flow injection analysis in order to help achieve reproducible pre-treatment procedures. On-line microwave digestion of slurries has been successful for the determination of Al, As, Cd, Cu, Co, Cr, Fe, Hg, Mg, Mn, Ni, Pb and Zn in biological, soil and sediment samples.However, incomplete digestion has been reported for coal samples.35 It has also been noted that for some detection systems, e.g., flame atomic absorption spectrometry (FAAS), that the mass of sample required for trace analysis may be incompatible with the slurry approach.35,36 Other workers have reported blocking of the transfer lines for some samples, therefore necessitating a pre-digestion before on-line treatment. 54 An alternative method to the slurry approach has been reported by Legere and Salin,193 who proposed encasing the sample in a digestible capsule for easy transfer into the digestion tube.Once in place the reagents could be added, the tube sealed and the digestion allowed to continue in a fully automated system. Torres et al.139 developed a microwave-assisted robotic method for the extraction of Cu, Fe and Zn from soil samples. The system was capable of the weighing, extraction, centrifugation and transport of the sample to the flame atomic absorption spectrometer. In contrast to the problems associated with solids, water samples are more compatible with the on-line digestion process.Techniques suitable for the determination of the chemical oxygen demand (COD),64,186 total P,183,185 As,107 Bi179,180 and Hg138,179–181 and the speciation of Se175–178,184 have been developed. These procedures result in considerable time savings on the batch techniques, especially if the system can be combined with an autosampler.Open monomode (focused) microwave digestion systems are particularly suited to on-line applications, having been successfully used by a number of workers for this purpose.21,22,138,175–179,181 Chemometrics Chemometrics and factorial designs78,142,143,159,160 have been used to select the best digestion technique for a particular purpose, i.e., to chose the best combination of reagents, reagent volumes, digestion times and power settings. This is of particular value in a multi-element situation when no single digestion procedure gives good results for all the elements required and a method is needed to obtain the best overall performance. In addition, Feinberg et al.67 related digestion programmes with the nature of the sample matrix using an empirical modelling approach.A preliminary study using Kjedahl nitrogen determinations in food samples to define reference digestion procedures was found to be very effective for precisely defined samples.However, for complex foods the model needed further development. Universal digestion procedures This section discusses the use of microwave digestion systems for the digestion of biological, geological and water samples, in order to identify potential ‘universal’ digestion procedures for a particular matrix or element. Tables 5, 6 and 7 summarise the different reagent combinations that have been used for the determination of different elements in biological, geological and water samples respectively.Biological samples Many papers have been published reporting the use of microwave digestion procedures for biological samples10–135 (Table 1). A wide range of samples have been investigated, the diversity of which is nearly matched by the number of different digestion methods used. A wealth of different combinations of acids and oxidising agents are commonly employed for the determination of different elements in biological samples (Table 5).Few trends seem to exist with good results being obtained for the same element in the same matrix after digestion with a range of different reagents. Conflicting evidence also exists as to the efficacy of the same reagent combination for the digestion of a particular matrix. For the determination of aluminium in biological samples there is disagreement as to whether digestion with HF is necessary. In support is the work of Lajunen and Piispanen,39 who reported low Al recoveries in NIST Citrus Leaves and Pine Needles and IAEA Mixed Diet after a closed nitric–hydrochloric acid digestion.Recoveries were improved by employing nitric and hydrofluoric acid in combination with hydrogen peroxide. Low recoveries were also obtained in BCR Spruce Needles following a nitric acid–hydrogen peroxide digestion25 and in shellfish,83 total diet samples120 and NIST Apple and Peach Leaves80 following closed digestion procedures with just nitric acid.For marine biological tissue (NIST Oyster Tissue), closed digestion procedures with nitric and perchloric acid, and nitric acid and hydrogen peroxide, also resulted in low results for Al.56 However, good results were again achieved by the addition of hydrofluoric acid to the nitric acid and hydrogen peroxide digestion mixture. Evidence also exists, however, to suggest that digestion with HF is unnecessary for some samples. For example, good results have been obtained for Al in NIST Total Diet116 and in NIST Citrus Leaves135 after a simple nitric acid digestion.A combination of nitric acid and hydrogen peroxide was used for the digestion of NIST Wheat Flour, although results for NIST Rice Flour were slightly high.74 Recoveries were not increased, however, by the inclusion of HF in the procedure. We have reported the successful determination of aluminium in tea leaves following an open nitric and perchloric acid digestion, although low recoveries were obtained with nitric acid, alone and in combination with hydrogen peroxide.20 Similar discrepancies exist for the determination of iron.A simple nitric acid digestion was used successfully for the determination of iron in cocoa,33 IAEA Horse Kidney,34 NIST Total Diet,116 NIST Bovine Liver,36,70,119 and NIES Mussel,36 Chlorella,36 Sargasso36 and Pepperbush36 samples. However, Mingorance et al.69 obtained low and imprecise results for botanical samples using a similar method.A slight improvement was obtained with a nitric and hydrofluoric acid digestion, but the results were still outside the certified range. Good results were obtained with nitric acid and hydrogen peroxide for NIST Total Diet and with nitric and perchloric acid for NIES Pepperbush and Mussel samples.69 However, low results were obtained following both procedures for the determination of Fe in BCR Wholemeal Flour. A nitric acid and hydrogen peroxide digestion was also employed by Burguera et al.87 to give good results for NBS Bovine Liver, but for BCR Bovine Muscle the results were slightly high.Using a similar procedure, good 124R Analyst, July 1998, Vol. 123Table 5 Reagents for the microwave digestion of biological samples Samples Reagents used Elements determined Ref. Marine Biological Tissues HNO3 Al, As, Ca, Cd, Co, Cr, Cu, Fe, Hg, Mg, Mn, Ni, Pb, Se, Sr, Zn 23, 27, 29, 52, 58, 59, 60, 70, 74, 83, 84, 92, 100, 102, 114, 117, 135 HNO3 with V2O5 catalyst As 113 HNO3–H2O2 Ag, Al, As, B, Cd, Cr, Cu, Hg, Mg, Mn, Ni, Pb, Se, Sr, Zn 16, 17, 21, 25, 37, 54, 57, 62, 65, 66, 76, 112 HNO3–HCl Ni 81, 82 HNO3–H2SO4 As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Se, Sr, Zn 23 HNO3–HF Ag, Al, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, Sn, Th, Zn 54, 77 HNO3–HF–H2O2–H3BO3 Al 56 HCl–HNO3–H2O2 Cd, Fe, Zn 25 HNO3–HClO4 Co, Cu, Fe, Pb 79 HCl–HNO3–HF Ca, Cu, Fe, Zn 78 HNO3–H2SO4–H2O2 As, Hg, Se 22, 105, 106, 129 HNO3–H2SO4–HNO3–H2O2 Hg 11, 27 HNO3–HClO4–HCl–HF Ca, Cd, Co, Cu, Fe, Mg, Mn, Zn 19, 103, 125 HNO3–H2SO4–H2O2–NH4EDTA Ca, Cd, Cu, Fe, K, Mg, Mn, P, Sr, Zn 19 HNO3–H2O2–HF Si 75 HCl–Br2/BrO32 Hg 22 Aqua regia–HF–H2O2 Ni 97 K2S2O8–NaOH As 21, 107 TMAH Hg, methyl-Hg 28 Methanolic KOH Hg, methyl-Hg 28 Other biological tissues HNO3 As, Ag, Cd, Co, Cu, Fe, Hg, Mg, Mn, Mo, Po, Pb, Rb, Se, V, Zn 27, 34, 35, 36, 70, 119, 124, 134 HNO3–HCl Ni, Pb 81, 88 HNO3–HClO4 Cd, Cu, Pb, Se 79, 118, 122 HNO3– H2O2 B, Bi, Ca, Cd, Co, Cs, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, P, Pb, Rb, Ru, S, Sb, Se, Sn, Sr, Tl, Zn 10, 12, 17, 18, 45, 50, 57, 67, 72, 87, 96, 99, 128 HNO3–HClO4–HCl–HF Ca, Cu, Fe, Mg, Mn, Zn 103 HNO3–H2SO4–H2O2 As 110 HNO3–H2SO4–HNO3–H2O2 Hg 27 HNO3–H2SO4–H2O2–NH4EDTA Ca, Cd, Cu, Fe, K, Mg, Mn, P, Sr, Zn 19 HNO3–HF–H2O2–H3BO3 Al 56 H2SO4–KI Sb 25 Acetic acid SbIII 25 Botanical samples (terrestrial) HNO3 Al, As, B, Ba, Be, Bi, Ca, Cd, Ce, Co, Cr, Cu, Eu, Fe, Hg, K, La, Mg, Mn, Mo, Na, Ni, P, Pb, Po, Rb, S, Se, Sm, Sr, Tb, Te, Th, U, V, Zn 15, 32, 33, 35, 41, 47, 49, 71, 74, 80, 85, 89, 90, 116, 119, 134, 135 HNO3–H2O2 Al, As, B, Ca, Cd, Ce, Cr, Co, Cu, Eu, Fe, K, Mg, Mn, Na, Ni, P, Pb, S, Se, Sm, Tb, 232Th, 238U, Zn 12, 13, 15, 17, 25, 31, 38, 40, 41, 45, 63, 72, 73, 76, 93, 98, 99, 101, 116, 128 HNO3–HCl Ca, Co, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, Zn 30, 39, 81, 82, 88, 116, 127 HNO3–HClO4 Al, Ba, Ca, Cd, Cu, Fe, K, Mg, Mn, P, Pb, S, Zn 20, 53, 79, 109, 121, 122 HNO3–HClO4–HCl–HF Ca, Cd, Cu, Fe, Mg, Mn, Pb, Zn 103, 104, 125 HCl–HNO3–HF Ca, Fe 78 HNO3–HF–H2O2 Al, Fe, Mg, Si 39, 130 HNO3–HF–H2O2–H3BO3 As, Ba, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Tl, V, Zn 46 HNO3–HF–HClO4–HF Cr, Fe 71 HNO3–H2SO4 Al, Ca, Cu, Fe, Mg, Na, Zn 116 H2SO4–H2O2 Kjeldahl N 14 HNO3–HF Ba, Ca, Mg, Mn, Zn 42 HNO3 with V2O5 catalyst As, Cd, Cu, Fe, Pb, Se 91, 115 HNO3–H2O2–HCl P 133 HNO3–HF–H2O2–SiO2 Al, B, Ba, Ca, Cr, Cu, Fe, K, Mg, Mn, Na, P, S, Sr, Zn 55 Aqua regia–HF–H2O2 Ni 97 Aqua regia–H2O2 Cr 95 NaOH–H2O2 Br2 111 Botanical samples (marine) HNO3 Cd, Fe, Mg 36 HNO3–H2O2 Co, Cr 101 HNO3–HClO4–HCl–HF Cd, Cu, Pb 103, 125 Analyst, July 1998, Vol. 123 125Rresults have been obtained for BCR Pig Kidney,128 Bovine Muscle128 and Liver18,50 and NBS Citrus Leaves,128 Pine Needles128 and Wheat Flour.128 However, low iron recoveries have been reported using the same reagent combination for NBS Orchard Leaves, Spinach Leaves and Bovine Liver,72 BCR Rye Grass,40 IAEA Hay40 and for fat-rich foods.13 For the last samples, recoveries were improved by use of a closed nitric–sulfuric–nitric acid digestion.Sulfuric acid has also been used by Krushevska et al.19 in combination with nitric acid, hydrogen peroxide and NH4EDTA to give good results for NIES Mussel Tissue, IAEA Horse Kidney and NIST Bovine Liver and Oyster Tissue. For NIST Citrus Leaves the use of nitric and hydrochloric acid gave low recoveries for Fe, but good results were obtained for a variety of other metals.30 A number of workers, however, have reported that to obtain complete iron recoveries, digestion with HF was necessary.For example, for the digestion of NIST Citrus Leaves, Lajunen and Piispanen39 found hydrogen peroxide, nitric and hydrofluoric acid to be more effective than a simple nitric and hydrochloric acid digestion. Park and Suh71 reported that a nitric acid digestion was unsuitable for the determination of Fe and Cr in rice flour samples, but in combination with hydrofluoric and perchloric acids complete recoveries were obtained.A nitric, perchloric, hydrochloric and hydrofluoric acid digestion was successfully developed by Kojima et al.103 for the determination of iron in NIST Bovine Liver and NIES Pepperbush and Mussel samples, although for NIES Tea Leaves the results were slightly high. Sun et al.55 obtained complete recoveries for iron in NIST Pine Needles and Apple, Tomato and Peach Leaves following digestion with nitric acid, hydrofluoric acid and hydrogen peroxide.Mohd et al.78 used a chemometrics technique to select the best reagent combination for the digestion of NRCC TORT-1 and NIST Pine Needles. The chosen procedure involved digestion with hydrochloric, nitric and hydrofluoric acid in which the hydrochloric and nitric acid were present in equal proportions rather than as aqua regia. A number of different techniques for the determination of selenium have also been suggested. Banuelos and Akohoue31 investigated a number of different reagent combinations with and without a pre-digestion stage.Using a simple nitric acid digestion, a selenium recovery of only 23% was obtained for NIST Wheat Flour. Recoveries were improved to 80% after a nitric acid and hydrogen peroxide digestion with a 4 h predigestion step (57% without pre-digestion). However, further heating or the addition of hydrochloric acid did not increase the recoveries. In another publication,70 the determination of selenium in NIST Bovine Liver was successfully achieved by digestion with nitric acid, but results for IAEA Fish Flesh were low.Selenium determinations have also been successfully carried out using open nitric acid and hydrogen peroxide digestion procedures for BCR Lyophilised Pig Kidney,10 cereal reference materials15 and NIST Bovine Liver12 and Mixed Diet12 samples. However, results for NIST Total Diet12 were slightly low. Good results have also been obtained in closed digestions using nitric acid and hydrogen peroxide for BCR Maize Leaves38 and lyophilised fish samples.37 An alternative technique, for the digestion of NIST Bovine Liver, was offered by Prasad et al.118 This involved a closed nitric acid digestion, followed by evaporation to dryness with perchloric acid to remove organics and analysis by square-wave cathodic stripping voltammetry.However, for analysis by hydride generation –atomic absorption spectrometry (HG–AAS) a similar procedure was found to be ineffective.This was also the case when phosphoric acid or potassium persulfate was added.106 Good results were obtained, however, for NIES Mussel Tissue by using nitric and sulfuric acid with hydrogen peroxide;105,106 for NRCC DORM-2 and DOLT-2 after digestion with nitric and hydrofluoric acid66 and by a semi-on-line nitric acid digestion method for NIST Oyster Tissue84. For arsenic, good results have been obtained using a nitric acid digestion for BCR Cod Muscle,11 NIST Oyster Tissue,117 NIST Orchard Leaves,49 NIST Bovine Liver70 IAEA Fish Flesh70 and NRCC DORM-1,52 although for NRCC TORT-1 results were slightly high.52 Yusof et al.60 and Liu et al.23 obtained good results for TORT-1 using a similar digestion procedure.A nitric acid digestion was also carried out by Navarro and co-workers,113,115 in combination with a catalyst of V2O5, to obtain good results for BCR Mussel Tissue and NBS Citrus Leaves.A nitric acid and hydrogen peroxide digestion was successfully employed for the digestion of NIST Oyster Tissue and Orchard Leaves,76 for NRCC TORT-1 and DORM- 1,21 for BCR Maize Leaves38 and for BCR Spruce Needles, White Clover, Cod Muscle and Plankton samples.25 However, El Moll et al.129 required the use of a more vigorous multi-step procedure with nitric and sulfuric acid and hydrogen peroxide for the open vessel digestion of a range of fish samples. Krushevska et al.19 also employed a mixture of nitric acid, sulfuric acid and hydrogen peroxide, but in combination with NH4EDTA, for a range of biological samples.Schramel and Hasse79 reported that a nitric acid procedure was successful for the digestion of NIST Orchard Leaves, although for the determination of arsenic in fish by HG–AAS a nitric, perchloric and sulfuric acid digestion was necessary, presumably to break down organoarsenic compounds in the sample. In the work of Edwards et al.66 the very low recoveries obtained for As in marine biological samples, following digestion with nitric acid and hydrogen peroxide and HG–AAS analysis, was attributed to the incomplete breakdown of organoarsenicals.For analysis by HG–AAS, Mayer et al.110 found a nitric acid, sulfuric acid and hydrogen peroxide mixture to be successful for the determination of As in NIST Bovine Liver. McLaren et al.77 developed a nitric and hydrofluoric acid digestion followed by hot-plate evaporation to dryness and dissolution of the sample in nitric acid for the determination of a range of elements, including arsenic, in NRCC DORM-2 and DOLT-2.For the determination of As and Se sewage sludge51 by HG–AAS, digestion with nitric and sulfuric acid has been found to be the most effective procedure. An alternative approach for the breakdown of organoarsenic compounds is to undertake an on-line potassium persulfate–sodium hydroxide digestion.21,107 Speciation of arsenic may then be achieved by the coupling of an HPLC column to the system.Using the former system we have also Table 5 Continued Sewage sludge samples HNO3 Cd, Cr, Cu, Hg, Ni, Pb, Zn 24, 32, 44, 86 HCl–HNO3 Ag, Al, Ba, Be, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, Pb, Sn, Ti, V, Zn 44, 123 HNO3–H2O2 Cu, Mn, Pb 93, 98 HNO3–H2SO4 As, Se 51 Aqua regia–H2O2 Cr 95 Aqua regia–HF–H2O2 Ni 97 Aqua regia–HF–H2O2–H3BO3 Cd 96 K2Cr2O7–H2SO4 COD 64 126R Analyst, July 1998, Vol. 123shown that the determination of total arsenic was possible by virtue of an l-cysteine pre-reduction step.21 The more readily released elements from biological matrices such as Cu and Zn have been determined after digestion with a vast number of different reagents ranging from nitric acid alone19,23,29,33–36,40,41,49,60,70,91,98,119,126 and in combination with hydrogen peroxide13,15,16,18,25,41,50,72,87,93,98,101,116 to combinations of nitric, perchloric, hydrochloric and hydrofluoric acids.103,122 Methods for the determination of mercury are, however, slightly in agreement, with many workers employing a closed nitric acid digestion procedure.Good results have been obtained Table 6 Reagents for the microwave digestion of geological samples Samples Reagents used Elements determined Ref. Sediments HNO3 Al, As, Ca, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Zn 24, 85, 121, 141, 144, 148, 150, 162 HNO3–H2O2 Cd, Hg 140 HCl–HNO3 As, Cd, Hg, Pb, Se 161, 162, 170 HNO3–HF Al, Ca, Co, Cr, Cu, Fe, K, Mg, Na, Ni, Pb, Rb, Sr, Ti, V, Zn 143, 162, 164 HCl–HNO3–H2O2 As, Cd, Cr, Cu, Ni, Zn 25, 137 H2SO4–HNO3–HCl As, Se 148 HNO3–H2SO4–H2O2 Hg 22, 112 HNO3–HClO4-HF As, Ba, Co, Cr, Cu, Ni, Nb, Pb, Rb, Sb, Sn, Sr, Ta, Th, Tl, W, Zn, REEs 141, 145 HNO3–HCl–HF Al, As, Ca, Cd, Co, Cr, Cu, Fe, Hg, Mg, Mn, Mo, Ni, Pb, S, Se, Th, Ti, U, V, Y, Zn, lanthanides 25, 54, 142, 147, 158, 159, 160, 161, 163, 165 HNO3–HF–HNO3 Cd, Cr, Cu, Ni, Pb, Sb, Sn, Th 78 HNO3–HClO4–HF–HCl As 137 HNO3–HF–H2O2–H3BO3 Ag, Al, As, Ba, Bi, Cd, Co, Cr, Cu, Fe, Hg, Li, Mg, Mn, Mo, Ni, Pb, Sb, Sn, Sr, Th, Ti, Tl, U, V, Zn 45, 169 HF–aqua regia Al, Ca, Fe, K, Mg, Mn, Na, P, Si, Ti 174 Aqua regia–KMnO4 Hg 138 Aqua regia–HF–H2O2 Cr, Ni 95, 97 HCl–Br2/BrO32 Hg 22 Rocks/minerals HNO3 Co, Cr, Cu, Mn, Mo, Ni, V 141 HCl Fe 72 HNO3–HF Co, Cr, Cu, Ni, Pb, Zn 143 HCl–HF Al, K, Mg, Si 72 HNO3–HClO4–HF Al, Ba, Be, Bi, Cd, Ce, Co, Cr, Cs, Cu, Dy, Er, Eu, Fe, Ga, Gd, Ge, K, Hf, Ho, In, La, Li, Lu, Mg, Mn, Mo, Na, Nb, Nd, Ni, Nb, Pr, Pb, Rb, Sb, Sc, Sm, Sn, Sr, Ta, Tb, Th, Ti, Tl, Tm, U, V, W, Y, Yb, Zn, Zr, REEs 151, 157 HF–H2SO4 Fe 173 HNO3–H2SO4–K2Cr2O7 Os 149 HF–HNO3–HCl As, Se, Th, U, Y, lanthanides 38, 165 HNO3–HCl–HF–HClO4 Au, Ir, Pb, Pt, Rh, Ru 152 Aqua regia–HF–H2O2 Ni 97 HF–aqua regia Al, Ca, Fe, K, Mg, Mn, Na, P, Si, Ti 174 Soil HNO3 Hg 24, 136 HNO3–H2O2 As, Cd, Cu, Ni, Pb, Zn 25,77 HNO3–H2SO4 As 51 HCl Hg 136 HNO3–H2O2–HF Si 19 HNO3–HF–H2O2–H3BO3 Al, Ag, As, Ba, Bi, Cd, Co, Cr, Cu, Fe, Li, Mg, Mn, Mo, Ni, Pb, Sb, Sn, Sr, Th, Ti, Tl, U, V, Zn 169 Aqua regia Hg 136 Aqua regia–HF–H2O2 Ni 97 Dust, ashes and airborne particulate matter HNO3 Cd, Hg, Pb 32, 162, 168, 172 HNO3–H2O2 As 77 HNO3–H2SO4 As, Se 171 HCl–HNO3 Cd, Pb 162 HNO3–HF Cd, Pb 162 HNO3–HCl–HF Al, Ba, Ca, Cr, Cu, Fe, Mg, Mg, Ni, Ti, V, Zn 147 HNO3–HClO4–HF Al, As, Ba, Cd, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, S, Sb, V, Zn 153, 154, 155, 156, 166 HNO3–HF–H2O2–H3BO3 Al, Ag, As, Ba, Bi, Cd, Co, Cr, Cu, Fe, Li, Mg, Mn, Mo, Ni, Pb, Sb, Sn, Sr, Th, Ti, Tl, U, V, Zn 169 Coal HNO3–H2O2–HF–HCl REEs Analyst, July 1998, Vol. 123 127Rfor BCR Pig Kidney,27,134 Mussel Tissue114 and Cod Muscle92 NIST Citrus Leaves,85 Pine Needles85 and Albacore Tuna,59 IAEA Fish Tissue27 and NRCC TORT-162 using this procedure. However, a number of workers have reported the use of strong oxidising reagents such as sulfuric acid and hydrogen peroxide for the determination of mercury using open22,27 and closed microwave digestion systems.62,66,111 An on-line system developed in our own studies for the determination of Hg in biological and sediment samples has been reported.22 The system was suitable for the analysis of samples containing organomercury compounds by the utilisation of a bromide– bromate oxidation reaction.For the speciation of mercury, Tseng et al.28 have developed an open focused microwave assisted extraction procedure for analysis by HG–CT–GC– ETAAS.Geological samples Less work has been carried out for the digestion of geological samples than for biological samples, although a wide range of matrices have been digested by a number of different digestion procedures (see Tables 2 and 6). Included are sediment, soil, rock, coal, ash and dust samples. For the determination of some elements simple nitric or hydrochloric acid digestions will suffice for some samples. For example, good Fe recoveries in Fe ore samples were obtained using a simple hydrochloric acid digestion, but for limestone samples the additional use of hydrofluoric acid was required.72 Lead in dust wipe and air filters172 has been determined after a nitric acid digestion. Mercury was also determined using a similar procedure in NIES Pond Sediment,121 baghouse dust168 and NIST SRM Estuarine Sediment and Buffalo River Sediment. 148 However, other workers reported high results for NIST Buffalo Sediment141 and slightly low results for NIST River Sediment.85 Following both an open and closed nitric acid digestion procedure, results were also low for mercury in NIST Montana Soil.136 However good results were obtained for Hg in NRCC PACS-1, IAEA-356 and BCR S19 sediment samples following open digestion with nitric acid.140 Morales-Rubio et al.24 have found the on-line digestion of slurries prepared in nitric acid to be successful for the determination of Hg in soil and sediment samples.An alternative system for the determination of mercury has been proposed by Hanna and McIntosh.138 Sediment slurries, prepared in aqua regia and potassium permanganate, were digested on-line for analysis in a flow injection mercury system.In addition, an on-line system employing a bromide–bromate oxidation reaction has been developed in our studies for the determination of mercury in sediment samples.22 Feng and Barratt162 observed no significant differences between the results obtained after digestion with nitric acid, with hydrochloric and nitric acid or with nitric and hydrofluoric acid for the determination of Cd and Pb in BCR City Waste Incineration Ash, BCR River Sediment and in NBS Urban Particulate Matter.However, using a nitric acid and hydrogen peroxide digestion, low Cu and Pb (but good Cd) results were obtained in the last two samples and in BCR Calcareous Loam Soil by Chakraborti et al.128 The results were improved by employing an extra heating step with aqua regia and HF.Good results for cadmium in sediment samples were obtained following digestion with just nitric acid.144 A nitric acid digestion was also employed by Averitt and Wallace141 to obtain good results for As, Cd, Co, Cu, Ni and Pb in NIST Buffalo River Sediment, although the Ba, Cr, Mn, Sb, V and Zn results were low and Hg results were high. Barium, Hg and V results were improved using a nitric, perchloric, hydrofluoric acid digestion procedure, but Cr was still low and Zn and Sb high.Marr et al.146 reported a benefit from the addition of HF to an aqua regia digestion for the analysis of the sediments NRCC MESS-1 and PACS-1, but again the results for Cr and Mn were low. In environmental analysis, it is often useful to acquire information on the bioavailable rather than the total elements present. This can often be achieved by using a mild leaching procedure. Paudyn and Smith147 carried out such a procedure for NRCC MESS-1 and PACS-1 and NIST Coal Fly Ash.This work also demonstrated how the digestion conditions required for the total release of different elements varies from sample to sample. For example, complete recoveries of Mn were obtained for NIST Fly Ash, compared with only 63% for MESS-1 sediment. Total recoveries were also obtained for Cu and Zn in all samples whereas for other elements recoveries were much lower, e.g., 35–60% for chromium, thus warranting a more vigorous digestion procedure.Low Cr and Al values were obtained for NRCC BCSS-1 sediment following an on-line hydrofluoric, nitric and hydrochloric acid digestion, although recoveries for As, Cd, Co, Cu, Fe, Mn, Ni, Pb and Zn were good. Using a similar combination of reagents, good results were obtained for Cr in NRCC BCSS- 1, although the results for NIES No. 2 Pond Sediment were low. Following disappointing results using a number of medium- and high-pressure digestion procedures for Cr in NRCC BCSS-1, Liu et al.145 concluded that no acid dissolution procedures were adequate for this sample.Low Al and Cr results have also been reported for NIST Urban Particulate Matter after digestion with nitric, perchloric and hydrofluoric acids, however results for As, Cd, Cu, Fe, Mg, Ni, S, Sb and Zn were good.166 Complete recoveries of Cr have been obtained for NIST Fly Ash, NRCC MESS-1 and PACS-1 following a nitric, hydrochloric and hydrofluoric acid digestion.147 Good recoveries were also obtained for Cr in Mississippi River delta sediment following a nitric and hydrofluoric acid digestion77 and by an aqua regia, hydrofluoric acid and hydrogen peroxide digestion for BCR River Sediment.95.However, good Cr recoveries were obtained without the use of HF following an open hydrochloric, nitric acid and hydrogen peroxide digestion (and in a closed hydrochloric, nitric and hydrofluoric acid digestion) in BCR Estuarine Sediment.132 Good As, Cd, Cu, Ni and Zn recoveries were also obtained, although Hg and Pb levels were high.Totland et al.151,152 also employed HF in combination with nitric and perchloric acid for the successful digestion of nine rock and sediment samples. For the determination of As and Se in soil51 and in coal fly ash171 by HG–AAS, digestion with nitric and sulfuric acids has been found to be the most effective procedure. Lasztity et al.76 employed a nitric acid and hydrogen peroxide digestion for the determination of As in NIST Urban Particulate Matter and IAEA Soil 7, whereas a nitric, hydrochloric and hydrofluoric acid digestion was successfully employed by Jimenez de Blas et al.38 for the determination of As in soil samples.The determination of As in airborne particulate matter has been Table 7 Reagents for the microwave digestion of water and waste water samples Reagents used Elements determined Ref. K2S2O8 Total P 185 K2S2O8–NaOH As, total P and N 107, 182 H2SO4–HNO3–KMnO4–K2SO8 Hg 138 HNO3 and pyrophosphatase Total P 183 KBrO3–KBr Bi, Hg 179, 180, 181 KBrO3–HBr Se 175, 176 HCl Se 10, 177, 178 K2Cr2O7–H2SO4 COD 64, 186 KBr–HCl Se 184 128R Analyst, July 1998, Vol. 123undertaken by digestion with nitric, hydrofluoric and perchloric acids156. For the determination of rare earth elements in coal, Watkins et al.167 employed a closed nitric acid, hydrogen peroxide, hydrofluoric and hydrochloric acid digestion. Sen Gupta and Bertrand165 developed a hydrofluoric, nitric and hydrochloric acid digestion for the determination of Th, U, Y and the lanthanides in a large number of sediment and rock samples.The use of a chemometrics technique for the selection of the best technique for the determination of Co, Cu, Mn, Pb and Zn in NBS Buffalo River Sediment was reported by Kokot et al.142 A digestion procedure with hydrofluoric, nitric and hydrochloric digestion was selected, whereas for Co, Cr, Cu, Ni, Pb and Zn a nitric and hydrofluoric acid digestion was found to be most effective.143.A hydrofluoric, hydrochloric and nitric acid digestion procedure was selected by an orthogonal array design for the digestion of sediment samples.158,160 Water Samples Relatively few publications have reported the application of microwave digestion to the determination of elements in water samples (see Table 3).10,107,138,175–185 Benson et al.185 successfully employed an on-line potassium persulfate digestion for the determination of total phosphorus, although incomplete digestion of condensed phosphates was observed. A similar digestion procedure, but in batch mode, was applied for the determination of total phosphorus and nitrogen by Johnes and Heathwaite.182 Complete recoveries for phosphorus were obtained but for nitrogen the breakdown of aminoantipyrine was incomplete.The determination of phosphorus was also carried out using a nitric acid digestion with prior addition of pyrophosphate to give complete recoveries of tetrameta-, trimeta-, ortho- and pyrophosphate.183 Arsenic speciation has been achieved by online HPLC separation followed by a potassium persulfate and sodium hydroxide microwave digestion and analysis by HG– AAS.107 Pitts et al.177 developed an on-line microwave reduction system for the conversion of SeVI to SeIV prior to analysis by HG–AFS.In a subsequent study by the same authors, the system was used for the speciation of SeVI and SeIV following separation by HPLC.178 A similar system for the prereduction of SeVI to SeIV, but for analysis by FI–CSV, was reported by Bryce et al.10 The determination of Se has also been addressed using on-line HBr–KBrO3 pre-reduction methods, developed by Gonzalez LaFuente et al.175 and coupled with HPLC separation by Marchante-Gayon et al.176 Ellend et al.184 have also developed an on-line separation and pre-treatment method for the speciation of Se.For the determination of mercury by CV-AAS, digestion with KBrO3–KBr is commonly used.Welz et al.181 developed an on-line system for Hg and Bi, although problems were encountered in the determination of As, Pb and Sn. As described previously, a system for the on-line determination of Hg in sediments, water and waste water samples was proposed by Hanna and McIntosh.138 For COD determinations, Balconi et al.64 and Cuesta et al.186 developed on-line methods employing a K2Cr2O7–H2SO4 digestion.Conclusions Biological samples consist of a complex mixture of carbohydrates, proteins and lipids and so are not completely soluble in water or organic solvents. Before analysis it is therefore necessary to decompose the organic matter and release the metals from the sample matrix. The majority of the digestion procedures used to date involve the initial use of strong oxidising agents such as nitric acid to decompose the organic matrix of the sample. Many elements are then liberated as soluble nitrate salts.Other acids can be employed to break down the sample further, according to the elements that need to be determined and the analysis technique chosen. For example, hydrochloric acid is a good solvent for many metal oxides, for metals that are oxidised more easily than hydrogen and for some organometallic compounds. The use of hydrofluoric acid is necessary for the determination of a number of elements which are associated with siliceous minerals.For the determination of iron and aluminium in biological samples, a wide variety of reagents have been successfully employed. For the former, digestions with nitric acid alone and in combination with hydrogen peroxide are very common and generally effective for the digestion of biological samples. However, the requirement for HF during the digestion of some botanical samples has been reported. For the determination of aluminium many workers have reported that the low results generated from digestion with just nitric acid or nitric acid and hydrogen peroxide can be improved by the addition of HF.However other workers have found this step unnecessary. Therefore, no steadfast rules regarding the need for HF during the determination of Al and Fe can be made, since it depends on the exact sample type and the amount of siliceous material present. The determination of Al is also hindered by high background levels, which can be prohibitive for trace analysis.Hence measures to control background contamination, in addition to the preparation of sample blanks, should be routinely adopted to minimise this problem. For the determination of arsenic, digestion with nitric acid may be used successfully in many cases, although for the analysis of fish samples by HG–AAS more vigorous conditions are usually required. Sulfuric acid is often employed to break down organoarsenic compounds which are not reduced and thus not hydride forming upon reaction with sodium borohydride.Similar findings have also been observed for the determination of Hg and Se. For the latter, nitric acid and hydrogen peroxide digestions have been used successfully to replace the conventional nitric and perchloric acid and sulfuric acid procedures. However, when analysis by hydride generation is desired, sulfuric acid is still required to break down the more resistant organoselenium compounds. For the less strongly bound elements in biological materials, such as Cu and Zn, the digestion procedure is less critical with a wide range of different reagent combinations giving good results.The wide range of sample compositions represented by geological materials preclude the use of any single digestion procedure. For example, sediment samples consist of a combination of different materials, e.g., clay, organic material, siliceous and other minerals, and are therefore one of the most difficult sample matrices to digest.Hence in many cases, to attain complete digestion the use of HF is necessary to decompose resistant minerals, in addition to strong oxidising reagents such as nitric acid and sometimes perchloric acid to break down organic matter. In the literature there is evidence to suggest that a number of elements such as Cd, Cu, Hg, Pb and Zn can be easily released after digestion with just nitric or hydrochloric acid. This is because in many cases they are sorbed on clay minerals or are in other readily decomposed phases, rather than within the resistant framework-lattice silicates.However, other elements are more strongly bound, either as part of resistant minerals or associated with other minerals, and so in such cases the use of HF may be required. For example, some Cr bearing minerals, notably chromite, are very difficult to decompose even with the use of HF–HClO4 under pressure, although complete Cr recoveries have been reported without the use of HF.The need for HF is therefore very much dependent on the nature of the minerals present in the samples. Because real samples will vary in composition from that of the certified reference materials used for validation of a procedure, it would seem prudent to suggest that for the determination of all but the most weakly bound elements in sediments, digestion with HF is recommended. Analyst, July 1998, Vol. 123 129RThe digestion reagents required for the efficient digestion of a particular sample type are very much dependent on the exact sample matrix and the elements to be determined.Excluding water samples there is generally little agreement in the literature, with often conflicting evidence as to which reagent combinations are most effective for the same matrix. This may reflect the fact that the digestion is influenced by factors other than just the choice of reagents, e.g., the relative proportions of each reagent, heating times and the pressure and temperature reached during the procedure.In many cases good results for the same matrix have been reported by a number of different methods. In addition, the literature suggests that no standard digestion procedures can be employed for the determination of a specified element in all samples of the same type, e.g., Fe in all biological samples, or for the determination of all the elements in a particular sample, e.g., all the elements in mussel tissue.Therefore, it may not be justified to extrapolate a technique designed for the determination of just a few elements to a multi-element determination. The choice of sample preparation method may, however, be influenced by a number of practical considerations in addition to the type of sample and elements to be determined. These may include the number of samples to be analysed, method of analysis, safety aspects, capital and operating costs of equipment, operator skill and the degree of accuracy and precision required.The method of final analysis is an important factor, influencing the extent of the digestion required, e.g., for electroanalytical techniques complete breakdown of the organic components is necessary, whereas ICP-AES can tolerate dissolved solid contents of up to 1–2%. Also, the addition of certain reagents during the reaction can be considered. For example, the addition of boric acid for HF neutralisation may cause the final solution to possess a high solids content, which can give problems in sample introduction systems in addition to increasing the background signal and thus degrading sensitivity.ICP-MS suffers from a number of interferences, particularly polyatomic ion interferences. Hence the presence of a number of acids, including hydrochloric and sulfuric acid, in the final solution is not recommended for the determination of some elements. Therefore, adapting a digestion method for analysis by a different technique to that originally intended may not prove successful.When considering the speed of a particular procedure, it is not just the time for the actual digestion that should be considered. Other factors should also be taken into account, e.g., sample preparation before analysis, including grinding and slurry formation; pre-digestion and cooling times, including those necessary between reagent additions/heating cycles; and the washing of digestion vessels. These factors are often overlooked.A standard method of validating a procedure is to use a suitable certified reference material. However, as discussed previously, there seems to be a lack of consistency in the ‘grading’ of these results. Often results are classed as ‘good’ even though they do not lie within the uncertainty limits of the certified values. As mentioned above, it is not just digestion procedures which lend themselves to automation, but also the choice of digestion method. Chemometrics and factorial designs have been used effectively to help choose the best digestion procedure for a particular purpose.Models have also been developed to predict digestion methods depending on the composition of the sample of interest. This undoubtedly is a useful step forward, especially to predict digestion conditions for new samples. For batch digestions, many closed digestion procedures are developed in terms of heating at a particular power setting for a certain period of time, usually optimised to maximise energy input without causing venting of the vessels.These procedures are therefore operational, i.e., specific to the particular microwave system and bomb design used. Adaptation for use in a different laboratory may not be straightforward unless the same equipment is used, as re-optimisation of the original power settings and heating times may be necessary. The optimum power and time settings may also vary considerably according to the exact nature of the sample owing to the amount of organic matter present, which influences the amount of gaseous products evolved during the reaction.It has been shown that direct temperature and pressure measurements during the course of the digestion are possible. Such measurements can then be fed to a computer controlling the magnetron to achieve a pre-set temperature or pressure programme. This technology offers the potential to produce far more reproducible and controllable procedures, reducing the possibility of venting of digestion vessels.In closed systems it also enables the system to be operated to its full digestion potential, i.e., at its maximum pressure level without venting, regardless of the exact level of organic matter. Following this approach, digestion procedures can be transferred far more easily between similar samples and between different workers, and could potentially lead to the establishment of standard digestion procedures, in addition to improving the overall safety of this technique.There has been a growing trend in recent years towards the development of fully automated on-line microwave digestion and analysis techniques. This area is well suited to water and waste water samples, of which a large number are routinely analysed in many laboratories. Open monomode (focused) systems have been found to be particularly useful for on-line applications. Further developments are likely through both the adaptation of standard batch digestion methods to on-line applications and in the development of new chemistries suited to the on-line approach.The digestion of solids is complicated by the method of introducing the sample into the system. Introduction in the form of a slurry is one approach; however, the determination of low levels of analyte may be problematic owing to limitations in the maximum stable slurry concentration. However, despite the initial problems encountered with on-line microwave digestion systems for solid samples, good results have been obtained for a number of biological and geological materials.This approach to sample digestion would seem to offer much potential for further development and could result in dramatic time savings over batch microwave and conventional digestion techniques. 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A., and Preston, G., Analyst, 1993, 118, 245. 184 Ellend, N., Rohrer, C., Grasserbauer, M., and Broekaert, J. A. C., Fresenius’ J. Anal. Chem., 1996, 356, 99. 185 Benson, R. L., Mckelvie, I. D., Hart, B. T., and Hamilton, I. C., Anal. Chim. Acta, 1994, 291, 233. 186 Cuesta, A., Todoli, J. L., and Canals, A., Spectrochim. Acta Part B, 1996, 51, 1791. 187 Reid, H. J., Greenfield, S., and Edmonds, T. E., Analyst, 1993, 118, 443. 188 Prolabo Synthewave Product Literature, Prolabo, Paris, 1996. 189 Prolabo A301 Product Literature, Prolabo, Paris, 1993. 190 Prolabo Maxidigest Product Literature, Prolabo, Paris, 1995. 191 CEM Star System Product Literature, CEM, Matthews, NC, 1996. 192 Hulsman, M., Bos, M., and van der Linden, W. E., Anal. Chim. Acta, 1997, 346, 351. 193 Legere, G., and Salin, E. D., Appl. Spectrosc., 1995, 49, 14A. Paper 8/00776D Received January 28, 1998 Accepted April 7, 1998 Analyst, July 1998, Vol. 123 133R

ISSN:0003-2654    

DOI:10.1039/a800776d

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Optimization of confocal epifluorescence microscopy for microchip-based miniaturized total analysis systems Gregor Ocvirk, Thompson Tang and D. Jed Harrison* University of Alberta, Department of Chemistry, Edmonton, Alberta, Canada A confocal epifluorescence detection scheme, optimized to give sub-picomolar detection within planar glass substrates etched to a 30 mm depth, is described. A 340, 0.6 numerical aperture (N.A.) lens with a 3.7 mm working distance was used to create a focused laser spot about 12 mm in diameter, by under-filling the lens aperture to give an effective, measured N.A.of 0.22 for the laser beam. The sectioning power (optical axis field of view) of various pinholes and the corresponding detector probe volumes (overlap of the excitation and observation volumes) were: (pinhole diameter, sectioning power, probe volume): 100 mm, 18 mm, 0.1 pl; 200 mm, 20 mm, 0.4 pl; 400 mm, 26 mm, 1.7 pl; and 600 mm, 36 mm, 2.4 pl. A log–log plot of fluorescence intensity versus fluorescein concentration, measured in continuous-flow mode using the optimum 400 mm pinhole, showed a correlation coefficient of 0.996 and a slope of 0.85.In this mode, 300 fm fluorescein gave a signal of 34.6 ± 8.1 mV over background with an S/N of 6.1, representing the lowest measured fluorescein dye concentration reported on-chip. Capillary zone electrophoresis of 1 pm fluorescein resulted in a mean S/N of 5.8. The injection plug, estimated to be about 5470 molecules, corresponds to 570 detected molecules on average.The design and use of quick-fit, flangeless fittings for interfacing tubing, fused-silica capillaries or pressurized systems to microfluidic channels etched in planar glass chips is briefly presented. Keywords: Microchip; confocal epifluorescence detection; capillary electrophoresis; microfluidics; fluidic interface Performing chemical analysis on microchips utilizing electrokinetic pumping for fluid control has been shown to offer many advantages.These devices have been combined with capillary electrophoresis (CE) and applied to a variety of biochemical analyses such as immunoassay1–5 and DNA sequencing.6,7 However, highly sensitive detection methods such as laserinduced fluorescence8 (LIF) have to be employed in order to detect the inherently minute amounts of analyte. Several groups have performed on-channel LIF detection by launching a laser beam at the separation channel and collecting fluorescence with microscopes mounted perpendicular to the chip.9–12 Typically, a microscope objective with moderate numerical aperture (N.A.) has been used, so as to accommodate both the lens and the beam near the chip.In order to reduce the field of view, a relatively large circular aperture is mounted in the image plane. This approach suffers from fairly high background levels resulting from reflections and scattered light from the cover glass and channel walls, although detection in the 10 pm range is possible.Our goal here was to extend on-chip detection to lower concentration detection limits, which for clinical analyses is often more relevant than the mass detection limit. One of several approaches towards high sensitivity employs hydrodynamic focusing of the eluent within sheath flow cuvettes for post-column detection.13–15 Adapting this method to microchips will require new microchip device designs. Another approach to enhance signal-to-background and signalto- noise ratios (S/N) relies on background rejection by confocal epifluorescence microscopy.High N.A. microscope objectives allow for small detection volumes and high collection efficiencies, while a smaller pinhole at the image plane allows vertical discrimination against reflections and scattered light from capillary walls. This technique has the advantage that it can be used with present chip designs. Mathies and coworkers6,16 introduced the use of confocal fluorescence scanners to detection in gels, on capillary arrays and subsequently on microfabricated capillary array electrophoresis chips.They demonstrated the high sensitivity of this approach by detecting a 5 3 10212 m fluorescein derivative in microtiter plates.17 Several workers have also used epifluorescence detection schemes for on-column detection in fused-silica capillaries, showing detection limits in the 1–10 pm range.18,19 Single molecule detection has been shown for dyes in the 10 pm range or higher in the fused-silica capillary format,20–22 and more recently was demonstrated on planar devices.23 However, the probe volume for single molecule detection has typically been decreased to the femtoliter range in order to reduce the magnitude of the background,24 resulting in 10–100 pm concentration detection limits.Concentration detection limits can be improved by detecting a larger number of injected molecules through an increase in the probe volume.The detected spot size-to-channel width ratio should be maximized, while fluorescence generated over the total depth of the separation channel should be collected. Hence, the excitation laser spot size at the focal plane should be large for a given channel width. This can be achieved by reducing the excitation aperture, i.e., under-filling of the back aperture of the microscope objective, while fluorescence is still collected with the full N.A.of the lens.25 In order to evaluate this approach in our chip, the effect of essential parameters such as pinhole size, channel depth, excitation intensity and laser spot size was measured. The aim of this work was to provide the concentration detection sensitivity that is necessary for a variety of biological assays on-chip. Experimental Materials and reagents A 1 mm stock solution of fluorescein disodium salt (Sigma, St. Louis, MO, USA) was prepared in a 1.9 mm borate (J.T. Baker, Phillipsburg, NJ, USA)—6.3 mm Tris [tris(hydroxymethyl)methylamine, Sigma] buffer of pH 9. All buffers were prepared in doubly distilled, de-ionized water. Further solutions were prepared by serial dilution of the 1 mm fluorescein stock solution with the same running buffer. Sets of 100 ml calibrated flasks were used for all dilutions. All glass flasks and vials were cleaned with H2SO4–H2O2 (3 + 1), followed by thorough rinsing with doubly distilled, de-ionized water.Using this cleaning method, reproducible fluorescence signals were obtained from replicate solutions prepared in different sets of flasks. Solutions were filtered [Millipore (Bedford, MA, USA) 0.22 mm filters] before introduction into the chip. Analyst, July 1998, Vol. 123 (1429–1434) 1429Devices Devices were fabricated in 3 3 3 in 540 mm thick cover slip glass (Corning 0211, Corning Glass, Parkride, IL, USA) by the Alberta Microelectronic Centre (Edmonton, Canada).A previously employed manifold of injection and separation channels5 was etched in a bottom plate. Access holes, 1.9 mm in diameter, were drilled mechanically into the top plates; these were then thermally bonded to the etched plates for 6 h at 595 °C.5,9,12 Channels were 30 mm deep, 102 mm wide at the top and 50 mm wide at the bottom. The separation channel was 80.2 mm long, and the injector-to-detector distance used was 45 mm. The injector design used the double-T format to define geometrically the sample plug volume.3,5,10 Instrumentation The high-voltage control system for the chips has been described previously.9 High-voltage (HV) supplies used in the system were from Gamma High Voltage Research (RMC15/ 0.12P, Ormond Beach, FL, USA). The diagram in Fig. 1 illustrates the optical set-up. In order to mount the device rigidly, the CE chip was clamped in a 1/2 in. thick Plexiglas holder, allowing for connection of flangeless fittings (P202&P200, Upchurch Scientific, Oak Harbor, WA, USA) to the chip as illustrated in Fig. 2(a). This provides a reservoir of up to 180 ml volume. The chip connector was then placed on a Plexiglas block that was mounted parallel to the optical table, providing simple connection of the electrodes to HV power supplies. Three translation stages (Newport 423, Irvine, CA, USA) were used for xyz translation of the device, employing microactuators (Newport AJS-1) for horizontal (i.e., xy, orthogonal to the optical axis) and a differential micrometer (Newport DM13) for vertical (i.e., z, along the optical axis) translation.Excitation light from an air-cooled argon ion laser (488 nm, Uniphase 2014, San Jose, CA, USA) was passed through a 485 nm bandpass filter (485DF22, Omega, Battleboro, VT, USA), reflected by a dichroic mirror (505DRLP02, Omega) and focused onto a glass device to a Å 12 mm spot. A 0.6 N.A., 340 long working distance, infinite conjugate, microscope objective (Planachromat LDN 1.2-A, Carl Zeiss Jena, Jena, Germany) with a cover slip correction collar (0.5–1.6 mm) was used.The fluorescence emission was collected by the same objective, passed through the dichroic mirror and focused with a tube lens (achromat, Newport PAC064, f = 200 mm) onto a pinhole located at the focal point. Pinholes of 100–600 mm aperture (Melles Griot, Irvine, CA, USA) were aligned with an xyz translation stage (Newport LP-1-XYZ). A photomultiplier tube (PMT, Hamamatsu R1477, Tokyo, Japan; bias: 800–1000 V). was mounted on top of the microscope tube with a 514.5 nm bandpass filter (9 nm bandpass, Melles Griot) and a 488 nm rejection bandpass filter (Beckman Instruments, Fullerton, CA, USA) used for spectral filtering.The analog signal was filtered with a Butterworth 15 Hz cut-off filter (Krohn-Hite 3442), giving a 150 ms rise time. Electropherograms were recorded on a PowerMac 7100/66 with an NB-MIO16 A/D board and a program written in Labview (National Instruments, Austin, TX, USA) at a sampling rate of 50 Hz.All data were smoothed using a seven-point box smooth algorithm, included in Igor Pro (Wavemetrics, Lake Oswego, OR, USA). Procedure In order to align the detection set-up, the microscope objective was first removed and a mirror was placed on the chip. The reflected beam was superimposed on the incoming beam by adjustment of the beam steering mirrors. The microscope objective was then mounted, the beam was focused on the mirror by vertically translating the chip holder, and a pinhole was aligned. Subsequently, the mirror was removed, the pinhole was replaced by a 310 Huygens eyepiece (Melles Griot) and the dry separation channel was brought into focus.A 1 nm fluorescein solution was then flushed through the channel by application of a vacuum at the buffer waste port, the eyepiece was replaced with the filter and PMT housing, and the position of the pinhole was re-optimized. The corrected fluorescence signal was obtained by correcting for background, i.e., subtraction of the blank signal.The blank signal was measured by flushing the dilution buffer through the separation channel at the same flow velocity as the sample. The maximum background-corrected fluorescence signal at a given fluorescein concentration was found by monitoring the response for both fluorescein and buffer solutions while vertically translating the chip in 1 mm steps. For continuous-flow experiments, the flow velocity in the separation channel was determined to be 3.5 mm s21, by measuring the time required for the dye front to travel from the injector to the detector.For calibration graphs, sample and buffer solutions were alternated, in the order of increasing Fig. 1 Confocal epifluorescence apparatus for ultrasensitive detection on a CE chip. The chip is mounted on xyz stages for translation in horizontal (xy) and vertical (z) directions.The vertical direction is the optical axis. Dimensions between optical elements are given. Fig. 2 Chip connectors: (a) Quick-connect coupling with inserted electrode allowing for connection of tubing or capillary between chip and vacuum or pump. In this study volumes of 100 ml or less were directly inserted into the fitting; (b) Commonly used pipette tips inserted into drilled access holes. 1430 Analyst, July 1998, Vol. 123fluorescein concentration, to ensure that the background levelcorrection was accurate and to validate the sample signals.Solutions were introduced by rinsing the reservoirs three times,and flushing the channels under vacuum for several minutes.Data were obtained for 25 s, and averaged to give the meansignal, background and corrected signal.The noise was taken asthe standard deviation (s) estimated from the overall variance ofthe corrected fluorescence signal, obtained by adding thevariances of the sample and the buffer background signals.Capillary zone electrophoresis was performed with 10 sdouble-T style injections at 2 kV (as discussed previously3,5,10),followed by separation at 7.5 kV (93.5 V mm21).The injectorto-detector distance, did, was 4.5 cm. The S/N for eachelectropherogram was calculated by dividing the average peakheight by the s value in the background, determined from theportion of the electropherogram before the peak. The sevenpointsmooth did not broaden or alter the shape of the peaks,which are defined by about 30 points.Results and discussionFluidic interfaceContacts to the introduction channels on the chip have mostoften been made with pipette tips glued in place, as depicted inFig. 2(b).5,9¡V12,26¡V28 In order to overcome the limitations of thisinitial approach, a chip connector consisting of quick-connectfittings and a Plexiglas chip holder was developed as shown inFig. 2(a). Buffers and samples were injected into the throughhole of the fitting, and Pt electrodes were inserted into thefittings to apply high voltages.Ferrules provide a seal to the topof the chip. These fittings allow for tubing connections tonegative or positive pressure sources (vacuum or pumps). Theglass-to-ferrule seal withstood working pressures of up to atleast 20 atm when coupled to an HPLC pump with PEEK tubing(od = 1.6 mm, id = 0.8 mm) for rinsing purposes. Fused-silicacapillaries can be connected to the chip using flangeless fittingsand capillary sleeves, to allow for electrokinetic transfer fromoff-chip and for 1¡V2 ml dead volume inter-chip connections.Optical characteristicsThe choice of microscope objective is of crucial importance, ashas been shown by Hernandez et al..29 The thickness of the twoglass plates of the devices limits the minimum workingdistance, and hence the N.A.of the lens. Given that the glassoften used5,9,12,27 is as thick as 2 mm, an objective with aworking distance of up to 3.7 mm and an N.A.of 0.6 waschosen. While the collection efficiency scales with N.A.2, thespot size is inversely proportional to the N.A., which reduces theexcitation volume. However, by under-filling the back apertureof the microscope objective with the laser beam the numericalaperture used for excitation can be made smaller than thenominal N.A., giving a larger laser probe volume. In order tomeasure the actual numerical aperture of excitation, a mirrorwas scanned vertically through focus and the laser spot size wasdetermined at various out-of-focus planes.From a plot of laserspot size versus vertical displacement, Dz, the angular semiaperture,a, was obtained. The sine of a (nair = 1) gives an N.A.of 0.22 for the excitation beam. Assuming a Gaussian beam, thelaser beam waist (1/e2) can be calculated from:w z w zzw z2 2220 10( ) ( )( )= = += ÆÙ ¢X£»£»lp (1)where z is the distance from the focal plane, w the beam radiusand l the wavelength. For 488 nm excitation light and a focallength of 4.9 mm for the objective, the calculated 1/e2 waist is9.1 mm in diameter at the focal plane.This value can becompared with an observed spot diameter of 12 mm seen inFig. 3.The strength of the optical sectioning is the rate at which thefluorescence intensity decreases with vertical distance betweenobjective and object, and is known as the axial response of aconfocal microscope. Analogous to Wilson,30 we define thevertical displacement given by the full-width-at-half maximum(FWHM) of the axial response as a measure of a microscope¡¦ssectioning power. To evaluate our design, a CE chip wasscanned vertically through focus in steps of 1 mm.Solutions of1 nm fluorescein or buffer were alternately flushed through the30 mm deep flow channel in the chip using a vacuum. In Fig. 4,the corrected fluorescence signals were ratioed to the estimatednoise, as described under Experimental, then plotted versus thevertical displacement, Dz.The 400 mm pinhole was clearly theoptimum choice. A plot of corrected fluorescence signals versusDz was similar, except that the signals increased monotonicallywith pinhole size and the curves showed less scatter. Theconfocal sectioning power and its uncertainty were estimatedgraphically from the FWHM displacements, using the plots ofcorrected fluorescence versus Dz. For the 100, 200, 400 and 600mm sized pinholes, the FWHM displacements were 18 ¡Ó 0.7, 20¡Ó 0.7, 26 ¡Ó 0.7 and 36 ¡Ó 0.7 mm, respectively.Estimates fromWilson¡¦s model31 for a lem/lexratio of 1 (8, 14, 28 and 41 mmFWHM displacements for 100, 200, 400 and 600 mm pinholes,respectively, at 340 magnification, 0.6 N.A.) compare wellFig. 3 On-chip laser-induced epifluorescence detection illustrating laserbeam spot size, showing digitized video image of 1 mm fluorescein solutionflowing through a 30 mm deep and 102 mm wide separation channel.Fig. 4 Signal-to-noise ratio (signal corrected for background fluorescence,S 2 B/N) versus vertical displacement of chip (Dz) for variouspinhole diameters.A 1 nm fluorescein solution in pH 9 buffer wascontinuously flushed through the separation channel by vacuum (flowvelocity:3.5 mm s21); excitation: 488 nm, 3.75 mW, PMT voltage: 850 V.Analyst, July 1998, Vol. 123 1431with the measured values for the two larger pinholes. The 100 and 200 mm pinholes resulted in less axial resolution than predicted theoretically. As all of the pinholes tested were outside the range of calculations presented by Wilson, a linear extrapolation of his data from the largest pinholes he analyzed was used, which may account for some of the discrepancy. In order to determine the number of detected fluorescein molecules over a given acquisition time, the probe volume has to be known.The probe volume is given by the overlap of the detection volume and the excitation volume. From eqn. (1), the 1/e2 Gaussian beam radius at the focal plane is 9.1 mm and at the bottom of the channel is 9.2 mm in diameter.While the actual beam size may be slightly larger, given the measured laser spot size of 12 mm, the calculation shows that the excitation volume can be approximated as a cylinder, with a 30 mm height resulting from the channel depth. Using the 1/e2 radius gives an excitation volume of 1.9 pl. The detection volume can be estimated from the pinhole diameter, the magnification and the known sectioning strength.Pinholes of 100, 200, 400 and 600 mm were used with a 340 objective, corresponding to observation spot diameters of 2.5, 5, 10 and 15 mm. For the 400 mm pinhole, the measured axial sectioning was 26 ± 0.7 mm. Approximating the shape of the detection zone as a cylinder gives a detection volume of Å 2.0 pl for this pinhole. These two volumes lead to a probe cylinder for the 400 mm pinhole that is 9.1 mm in diameter and 26 mm in height, resulting in an approximate volume of 1.7 pl and a cross-sectional area for the probe of 237 mm2.The probe volumes for the 100, 200 and 600 mm pinholes are estimated to correspond to 0.1, 0.4 and 2.4 pl. The 30 mm deep channel was 102 mm at the top and 50 mm wide at the bottom, giving a cross-sectional area of 2280 mm2. Hence, the probing efficiency is 10.4% with a 400 mm pinhole and 0.22 N.A. for the excitation beam. Fig. 4 shows that an increase in pinhole diameter from 100 to 400 mm results in an improvement in S/N by a factor of 10.This improvement is consistent with the increased probe volume discussed above, although the fact that it is smaller than the 17-fold volume increase may indicate that the 400 mm pinhole does not completely discriminate the background arising from the channel walls. For the 600 mm pinhole, the depth of field exceeds the channel depth and the observed spot size is larger than the excitation diameter, so that significant background from the channel walls will be measured, causing a decreased S/ N, consistent with the data in Fig. 4. Hence 400 mm diameter pinholes were used for detection in 30 mm deep channels. Confocal epifluorescence detection schemes for use with channel arrays on microdevices have been demonstrated.6 A major practical concern is the positioning tolerance required when scanning the chip horizontally: displacement of the focal point from the channel center can result in collection of scattered light from the channel walls and increased background and noise levels.Since the 30 mm deep channels are 50 mm wide at the bottom, a horizontal displacement of more than 25 mm from the channel center will result in decreased S/N. Experimentally, vertical displacement of the chip by 5 mm results in a decrease of Å 18% in S/N for a 400 mm pinhole size with a 30 mm channel depth. It should be noted that the decreased sensitivity of the 600 mm pinhole to Dz translation (i.e., focusing) might be convenient in many applications.Detection performance Mathies et al.32 emphasized the importance of illumination time and incident laser power for high sensitivity fluorescence detection. Owing to ground state depletion and photobleaching, the S/N will decrease when exceeding the optimum incident laser power for a given flow rate. In the present work, measurements of the optimum power were made with both pressure driven (3.5 mm s21) and electrokinetically driven flow (3.75 mm s21 at 7.5 kV).In electrokinetic flow the flow rate of fluorescein is the vector sum of the electrophoretic and electroosmotic flow rates. The overall linear flow rates of fluorescein were matched in this work, rather than the linear solvent flow rate, in order to ensure equivalent fluorescein flux for pressure and electrokinetically pumped studies. The fluorescein velocity used was the highest attainable within these devices, as arcing became a problem above 7.5–8 kV. In our case the optimum laser output power for linear flow velocities of 3.5–3.75 mm s21 was measured to be 4.5 mW, with a range of ±1 mW because the maximum was very broad.This results in Å 2.2 ± 0.5 3 104 W cm22 at the 1/e2 diameter at the focal plane. The fluorescence signal was studied as a function of electro-osmotically driven flow rate at 4.5 mW, and found to vary by no more than 10% between 1 and 3.75 mm s21. Consequently, excluding dispersion effects, the data obtained with pressure driven flow can be readily compared with those for electro-osmotically driven flow.The observed background depended on both the buffer and the buffer concentration used. Photobleaching of fluorescent contaminants in the buffer was evaluated by measuring the background intensity with the flow on and then off. Stopping the electro-osmotic or vacuum driven flow typically reduced the background by 4% or less. Decreasing the concentration of the Tris–borate buffer by a factor of 4 decreased the background by an average of 2%.Owing to adsorption of fluorescein on channel walls, background levels tended to increase after frequent use. However, rinsing with 0.1 m NaOH for 1 h, then running buffer solution, was successful in reducing the background signal to the initial levels in most cases. In order to determine the instrument response to concentration, various fluorescein solutions were continuously flushed through the separation channel under vacuum.The average blank signal from the buffer for a typical data set was 369.8 ± 5.7 mV, using a laser power of 3.75 mW, 850 V PMT bias, a 25 s collection time and a seven point box smoothing. Fig. 5 shows fluorescence signals for buffer, 300 fm and 10 pm fluorescein solutions; 300 fm fluorescein gave a signal 34.6 ± 8.1 mV over background, corresponding to a S/N of 6.1 for this data set. At the linear fluorescein migration velocity of 3.5 mm s21 and hence a volumetric flow rate of 8 nl s21, 22 molecules were detected on average over the detector response time of 150 ms, given a probing efficiency of 10.4%.These data correspond to detection of 3750 molecules over 25 s, or 225 molecules over 1.5 s, a typical peak width for the electropherograms discussed below. This is, to the best of our knowledge, the lowest detectable fluorescein concentration ever reported on-chip. A log–log plot of background-corrected fluorescence signals of 300 fm–3 nm fluorescein is shown in Fig. 6 for data obtained with continuous vacuum driven flow. The signal for 300 fm fluorescein remains well above the signal detection limit illustrated in Fig. 6. A weighted linear fit of all data points gives a slope of 0.85 and a regression coefficient of 0.996 for replicate Fig. 5 Signal levels for vacuum driven continuous flow of pH 9 buffer, 300 fm and 10 pm fluorescein solutions in the same running buffer. Flow velocity: 3.5 mm s21; pinhole: 400 mm; excitation: 488 nm, 3.75 mW, PMT voltage: 850 V. 1432 Analyst, July 1998, Vol. 123calibration graphs. The weighting factor wi for each datapoint was given by the reciprocal of the variance. Deviations from unity slope have been reported for fluorescence detection at low concentrations by Hoon Hahn et al.,14 Mathies et al.,17 and Ingle and Wilson.33 The effects were ascribed to adsorption on glass, either in the detection system or the preparation flasks, to impurities in solution, or to statistical fluctuations in the numbers of molecules at the very lowest concentrations.On-chip detection of samples under continuous-flow conditions is a realistic application of microchip reactor devices, as was recently illustrated by Hadd et al.,34, who performed enzyme assays on-chip by diluting and mixing reagents in nanoliter volumes with subsequent detection of the enzymatic products in continuous-flow mode. However, CE-based separations are also an important application area for the detector. By performing studies of both methods at similar flow rates (3.5–3.75 mm s21), under conditions that are nearly flow rate independent, the results of both types of experiment are readily comparable after accounting for dispersion.12 Because of dispersion of the injected sample plugs the peak sample concentration will be decreased 3-fold or more by the time it reaches the detector,12 making the demands on a detector more stringent in CE than in continuous-flow measurements.Several electrokinetic injections and capillary zone electrophoretic separations of 1 pm fluorescein solutions were performed utilizing a previously described double-T injection system,5 as illustrated in Fig. 7. S/N values of 6.5 ± 0.2, 5.3 ± 0.2, 5.6 ± 0.2 and 5.7 ± 0.2 (±1s of each electropherogram, mean 5.8) were obtained. Subsequent injections of buffer solution alone did not show peaks above the noise level, excluding the possibility of false-positives due to fluorescent impurities or dust scattering.The same running buffer solution was used as had been used to dilute the fluorescein stock solution, in order to avoid any sample stacking effects. No peaks above background were obtained after double-T injection of 300 fm fluorescein into uncoated channels. We attribute this decreased detection limit relative to the continuous-flow study to peak dispersion during the separation.12 In order to determine the mass detected, the injection volume and the probe volume have to be known.In principle, the injection volume is confined by the geometry of the device layout. The double-T injector is 202 mm in length and has a cross-sectional area of 2280 mm2, resulting in a 461 pl volume. In typical applications of the double-T at higher concentrations, we have found that leakage effects can, on occasion, significantly increase the length of the plug formed.27,28 Since it is not possible to monitor visually the plug shape at these low concentration levels, an upper limit estimate of the injection volume Vinj was made from the band broadening of the peaks.Assuming no non-ideal effects, the band broadening contributed by the injector Vinj can be calculated from: V A inj inj = 12s (2) where sinj is the injector standard deviation and A the crosssectional area of the channel. The value of s2 inj can be estimated using a procedure discussed previously.27 The important parameters required are the detector size of 9.1 mm, the average migration time of 10.1 s, and a diffusion coefficient27 of 3.3 3 1026 cm2 s21.Longitudinal diffusion contributes a variance of 6.68 3 1025 cm2, so that s2 inj is calculated to be 1.32 ± 0.2 3 1022 cm2 and so Vinj is 9.1 nl. The maximum volume of the injected plug in this study has an upper limit about 20 times larger than a geometrically defined plug. The corresponding upper limit on the number of injected molecules is 5470, corresponding to Å 570 detected molecules.This study demonstrates the excellent concentration detection limits obtainable by confocal epifluorescence detection for microchip-based analysis systems. Concentration detection limits in the 0.3–1 pm range will allow the advantages of biological assays on-chip, such as high speed, low reagent consumption and high throughput, to be fully exploited for trace biochemical analytes. The measured sectioning powers for the pinholes indicate that for larger apertures a linear extrapolation of Wilson’s model, along with the use of eqn.(1), is predictive for the optimum pinhole size. The probing efficiency of 10.4% obtained here for 30 mm deep channels with a 400 mm pinhole could be improved if the channel width could be reduced. However, this is not feasible because the etching depth determines the width of chemically etched channels in glass, since the etching process is isotropic.The measured displace- Fig. 6 Log–log plot of fluorescence intensity, S, from continuous flow, corrected for background fluorescence, B, as a function of fluorescein concentration. Solutions were hydrodynamically flushed at 3.5 mm s21. Pinhole: 400 mm, excitation: 488 nm, 3.75 mW, PMT: 850 V. Error bars are ±1 s, calculated as indicated under Experimental, and are smaller than the points where not shown. A line 3s above the background signal (estimated from the s value for 300 fm) is shown to illustrate the signal detection limit floor.Fig. 7 Four separate electrophoregrams of 1 pm fluorescein solutions: Injection time: 10 s at 2 kV injection voltage, separation field strength: 93.5 V mm21, injector–detector distance: 45 mm, pinhole: 400 mm; laser power: 3.75 mW, PMT voltage: 1000 V. Analyst, July 1998, Vol. 123 1433ment curves allow an estimate of the vertical (optical axis) positioning tolerance of the microscope as less than 5 mm, meaning that, for physically scanning a multichannel device, the distance between channels must be minimized to alleviate positioning tolerance demands.The finger-tight, quick-fit fluid interface used here proved to be highly convenient, reducing many problems associated with the world-to-chip interface, and should also be useful in applying chips to biochemical analyses. We thank the Natural Sciences and Engineering Research Council of Canada for support, P. Andersson, E. Arriaga and M.D. Morris for helpful discussions, and the Alberta Microelectronic Centre for device fabrication. References 1 Harrison, D. J., Fluri, K., Chiem, N., Tang, T., and Fan, Z., Digest of Technical Papers, Transducers 95, The 8th International Conference on Solid-State Sensors and Actuators—Eurosensors IX, June 25–29, 1995, Stockholm, Sweden, Royal Swedish Academy of Engineering Sciences, Stockholm, 1995, pp. 752–755. 2 Harrison, D. J., and Chiem, N., Technical Digest, Solid State Sensor Actuator Workshop, Hilton Head Island, SC, June 3–6, 1996, Transducers Research Foundation, Cleveland Heights, OH, 1996, pp. 5–8. 3 Koutny, L. B., Schmalzing, D., Taylor, T. A., and Fuchs, M., Anal. Chem., 1996, 68, 18. 4 Vonheeren, F., Verpoorte, E., Manz, A., and Thormann, W., Anal. Chem., 1996, 68, 2044. 5 Chiem, N., and Harrison, D. J., Anal. Chem., 1997, 69, 373. 6 Woolley, A. T., and Mathies, R. A., Anal. Chem., 1995, 67, 3676. 7 Jacobson, S. C., and Ramsey, J.M., Anal. Chem.,1996, 68, 720. 8 Cheng, Y. F., and Dovichi, N. J., Science, 1988, 242, 562. 9 Seiler, K., Harrison, D. J., and Manz, A., Anal. Chem., 1993, 65, 1481. 10 Effenhauser, C. S., Manz, A., and Widmer, H. M., Anal. Chem., 1993, 65, 2637. 11 Jacobson, S. C., Hergenroeder, R., Moore, A. W., and Ramsey, J. M., Anal. Chem., 1994, 66, 4127. 12 Liang, Z. H., Chiem, N., Ocvirk, G., Tang, T., Fluri, K., and Harrison, D. J., Anal. Chem., 1996, 68, 1040. 13 Nguyen, D. C., Keller, R. A., Jett, J. H., and Martin, J. C., Anal. Chem., 1987, 59, 2158. 14 Hoon Hahn, J., Soper, S. A., Nutter, H. L., Martin, J. C., Jett, J. H., and Keller, R. A., Appl. Spectrosc., 1991, 45, 743. 15 Chen, D. Y., Adelhelm, K., Cheng, X. L., and Dovichi, N. J., Analyst, 1994, 119, 349. 16 Huang, X. C., Quesada, M .A., and Mathies, R. A., Anal. Chem., 1992, 64, 2149. 17 Mathies, R. A., Scherer, J. R., and Quesada, M .A., Rev. Sci. Instrum., 1994, 65, 807. 18 Hernandez, L., Escalona, J., Joshi, N., and Guzman, N., J. Chromatogr., 1991, 559, 183. 19 Beale, S. C., and Sudmeier, S. J., Anal. Chem., 1995, 67, 3367. 20 Nie, S., Chiu, D. T., and Zare, R. N., Science, 1994, 266, 1018 21 Haab, B. B., and Mathies, R. A., Anal. Chem., 1995, 67, 3253. 22 Castro, A., and Shera, E. B., Appl. Opt., 1995, 34, 3218. 23 Effenhauser, C. S., Bruin, G. J. M., Paulus, A., and Ehrat, M., Anal. Chem., 1997, 69, 3451. 24 Barnes, M. D., Whitten, W. B., and Ramsey, J. M., Anal. Chem., 1995, 67, 418A. 25 Brakenhoff, G. J., Visscher, K., and van der Voort, H. T. M., in Handbook of Biological Confocal Microscopy, ed. Pawley, J. B., Plenum Press, New York, 1990, pp. 87–91. 26 Harrison, D. J., Manz, A., Fan, Z. H., Luedi, H., and Widmer, H. M., Anal. Chem., 1992, 64, 1926. 27 Fan, Z., and Harrison, D. J., Anal. Chem., 1994, 66, 177. 28 Seiler, K., Fan, Z., Fluri, K., and Harrison, D. J., Anal. Chem., 1994, 66, 3485. 29 Hernandez, L., Marquina, R., Escalona, J., and Guzman, N. A., J. Chromatogr., 1990, 502, 247. 30 Wilson, T, in Handbook of Biological Confocal Microscopy, ed. Pawley, J. B., Plenum Press, New York, 1990, pp. 113–126. 31 Wilson, T., J. Microscopy, 1989, 154, 143. 32 Mathies, R. A., Peck, K., and Stryer, L., Anal. Chem., 1990, 62, 1786. 33 Ingle, J. D., and Wilson, R. L., Anal. Chem., 1976, 48, 1641. 34 Hadd, A. G., Raymond, D. E., Halliwell, J. W., Jacobson, S., and Ramsey, J. M., Anal. Chem, 1997, 69, 3407. Paper 8/00153G Received January 5, 1998 Accepted March 30, 1998 1434 Analyst, July 1998, Vol. 123

ISSN:0003-2654    

DOI:10.1039/a800153g

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Piezoelectric mechanical pump with nanoliter per minute pulse-free flow delivery for pressure pumping in micro-channels Satyajit Kar, Scott McWhorter, Sean M. Ford and Steven A. Soper* Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803-1804, USA. E-mail: steve.soper@chemgate.chem.lsu.edu A novel computer-controlled mechanical syringe pump is described which uses a piezoelectric actuator and a pivoted lever for amplification of the linear displacement of the piezo-actuator to deliver solvents free from pump pulsations at volumetric flow rates approaching 1 nl min21 even at high loading levels (high output pressures).The flow patterns can be programmed by controlling the voltage waveform to the piezo-actuator to produce a linear displacement of 72 mm. By using the pivoted lever, a ninefold amplification of the piezo-expansion was achieved producing a total linear displacement of 648 mm. When a gas-tight glass syringe of 1.0 mm diameter was interfaced to the piezo-pump, the total volume delivered in a single pump stroke was 511 nl.Whereas the pumping profile was governed by the expansion behavior of the piezoelectric actuator, the flow rate was also slightly affected by the loading pressure on the pump as well. The piezo-pump was found to deliver adequately a stable flow of solutions with loading pressures as high as 3.79 3 105 Pa (actual loading pressure at the piezo-actuator = 3.41 3 106 Pa). Monitoring the flow stability using fluorescence indicated that the volume flow was fairly noise free at pumping rates from 4 to 150 nl min21.Below a volume flow rate of 4 nl min21, the pump exhibited extensive noise characteristics due to the step resolution of the DAC driving the piezo-actuator. A diffuser–nozzle system was fabricated which allowed automatic refilling of the syringe pump and was micromachined into Plexiglas (PMMA) using X-ray lithography. The diffuser–nozzle system contained channels that were 50 mm in depth and tapered from 300 to 30 mm.The diffuser–nozzle system was interfaced to the syringe pump by connecting conventional capillary tubes to the PMMA-based diffuser–nozzle, the piezo-pump and the chemical analysis system. Keywords: Piezoelectric actuator; syringe micropump; diffuser/nozzle; micromachining There is currently a pressing need for mechanical pumping devices that can deliver small volumes of materials (liquids or gases) at controlled rates with volumetric delivery rates in the low to sub nl min21 range. Not only is it necessary to possess the ability to pump at low volumetric flow rates, but also the pump should be able to operate effectively when the pressure drop is large.For example, in the biomedical area, small and compact pumps can be used for administering drugs for the treatment of such diseases as diabetes. In analytical chemistry, pumps which can deliver low volumetric flow rates can be used for microliquid chromatography1 and micro-dialysis.2 Recently, several research groups have focused on the development of miniaturized chemical analysis systems that can perform several different analytical techniques, such as separation via electrophoresis or chromatography, sample preparation and, finally, sample detection.3–18 These devices consist of a series of channels with dimensions in the range 20–40 mm lithographically fabricated in glass or plastic materials.While most of the micro-fluidics in these devices have been accomplished via electropumping produced by electroosmotic flows generated in these micro-devices when fabricated in glass substrates, mechanical pumps will need to be developed which can be used in devices where the magnitude of the electroosmotic flow is insufficient to allow facile pumping. For example, when predominantly organic run buffers are used in the micro-electrophoretic format, the magnitude of the electroosmotic flow may be insufficient to allow facile electropumping. 19 In addition, we have developed miniaturized chemical analysis systems in Plexiglas [PMMA, poly(methyl methacrylate)] using X-ray lithography and determined that the small electroosmotic flow generated in these devices will require an external pumping source in some cases to move fluids at reasonable linear velocities.20 Since these miniaturized chemical analysis systems possess ultra-small channels, micro-pumps must be developed which can deliver fluids at very low volumetric flow rates to obtain reasonable linear velocities.In Fig. 1 is shown the calculated volumetric flow rate versus linear velocity through circular tubes with various diameters (10, 20 and 50 mm id). As can be seen, to obtain a linear velocity of 0.01 cm s21 in a 10 mm id channel, a volumetric flow rate of 450 pl min21 is required. Fig. 1 Volume flow rate (solid lines, closed symbols) and pressure drop (dashed lines, open symbols) as a function of linear flow velocity.The volume flow rate and pressure drop were calculated assuming that pumping was occurring through a circular capillary tube of id 10 mm (circles), 20 mm (triangles) and 50 mm (squares) with a length of 10 cm. The solution viscosity was assumed to be 1 cP. Analyst, July 1998, Vol. 123 (1435–1441) 1435Another concern associated with pumping liquids through narrow bore channels is the pressure drop created by the small diameter and, as such, the micro-pump should be able to drive solutions adequately through these tubes when operated with high load pressures.In Fig. 1 is also shown the pressure drop across a micro-tube generated under different linear flow velocities. For example, a micro-tube possessing 10 mm id with a total length of only 5 cm and pumping water (viscosity = 1 cP) at a linear velocity of 0.01 cm s21 results in a pressure drop of approximately 450 Pa. Therefore, one can see that microfluidic pumping in small id channels requires a mechanical pump that can operate reasonably well at high load pressures and also demonstrate stable flows at low volumetric rates.Micro-mechanical pumps can also find applications in systems where it becomes necessary to transfer small volumes (picoliter to nanoliter) of material into micro-reactors for sample preparation utilized in miniaturized chemical analysis systems. An example is a genetic analysis system that we are currently constructing which involves the restriction digestion of DNA samples in an enzyme micro-reactor possessing a total volume of approximately 20–30 nl and transferring this minute sample to an electrophoresis separation channel for fractionation.Since the handling of nanoliter volumes of samples can be problematic, the enzyme reactor is incorporated on-line with the electrophoresis separation device. Prior to separation, intact DNA molecules are subjected to enzyme catalyzed fragmentation.Based on our experience, the residence time of the DNA sample in the micro-reactor required to achieve exhaustive digestion ranges from 5 to 10 min. Therefore, with a microreactor possessing a volume of 30 nl, a volumetric flow rate of 3–6 nl min21 is required to obtain the required residence time. Several groups have constructed micro-pumps which are based upon a reciprocating or peristaltic mode of operation and have used piezo-actuators to produce the pumping action.21–25 In most cases, these pumps use a bimorph which bends when a voltage is applied to the piezo-actuator.By inserting valves into the device, the pump can operate continuously with the volumetric pumping rate determined by the frequency of the driving voltage to the pump chamber. Unfortunately, many of these can operate only at low loading pressures and pump at volume flow rates in the ml min21 range. Also, since the pumping action is based on a reciprocating process, pump pulsations may be evident which could present problems when used in particular applications, e.g., in micro-chromatographic systems where the detector is sensitive to such noise sources.In this paper, we describe the construction and operational characteristics of a computer-controlled mechanical pump constructed from a piezoelectric actuator (PA), pivot lever and micro-syringe (see Fig. 2). By applying a voltage to the PA, the crystal can be expanded pushing the plunger of the microsyringe, causing fluid displacement.The expansion of the PA is amplified by means of the pivoted lever, which provides a higher volume displacement per pump stroke. The volumetric flow rate is controlled by specifying the slope on the driving voltage ramp to the PA. Owing to the precise positioning capabilities of the PA, low nl min21 volumetric flow rates can be generated with this device, making it appropriate for fluid pumping in miniaturized chemical analysis systems.In addition, we discuss the micro-fabrication of a diffuser–nozzle system which would allow automatic refilling of the syringe pump without requiring disconnection of the pump from the supply source and the chemical analysis system to which it is interfaced.26 Recently, several groups have shown that the diffuser–nozzle geometry is a viable choice for valveless operation in micro-mechanical-based pumps using reciprocating type action.27–30 The diffuser–nozzle system was micromachined in PMMA using X-ray lithography.The PMMAbased diffuser–nozzle was interfaced to the syringe pump using small diameter fused silica capillary tubes of 50 mm od and 20 mm id. Experimental Construction of PA pump In Fig. 2 is shown a schematic diagram of the micro-pump. All components were mounted on a Plexiglas stage (14.0 3 8.0 3 1.3 cm). The expansion of the PA (Model P-178.50, from Polytec PI, Costa Mesa, CA, USA) was controlled by a computer (Gateway 2000, P5-120) equipped with a 12-bit analog output voltage (0 to ±10 V, dc) from a multi-function I/O board (Model AT-MIO-16XE-50, National Instruments, Austin, TX, USA).The maximum linear displacement for this PA was stated to be 80 mm by the manufacturer when a voltage of 21000 V (negative polarity) was applied from a high voltage power source (Model K10N, from Emco High Voltage, Sutter Creek, CA, USA) which was driven by the output of the DAC. With 4096 (12-bit) step resolution over 10 V (2.44 mV per step), the minimum linear displacement of the PA was 19.5 nm per step (assuming a total linear expansion of 80 mm).The piezoexpansion was amplified using an in-house constructed lever (5.8 3 1.5 3 0.64 cm) and a pivot. The lever sits horizontally on a 0.8 mm thick 3 2.5 mm id 3 12.0 mm od copper disk that surrounds the pivot and can move without touching any part of the Plexiglas stage. The PA was placed in a rectangular slot (8.1 31.8 30.4 cm) made on the Plexiglas stage such that the center of the front face of the PA head was butted against the tip of one arm (A-1, see Fig. 2) of the lever. As shown in Fig. 2, the distance between A-1 and the center of the pivot was 5.0 mm, whereas the distance between the center of the pivot and the second arm (A-2) of the lever was 50.0 mm. A spring (S-1) maintained contact between A-1 and the PA head. A microsyringe (gas-tight, Model RN80230, from Hamilton, Reno, NV, USA) made of glass was situated horizontally on the Plexiglas stage and mounted (at a right-angle with respect to the long axis of the lever) on a drilled slot in this stage.Another spring (S-2) Fig. 2 Schematic representation of the piezo-driven micro-syringe pump. 1436 Analyst, July 1998, Vol. 123Pump Nozzle Nozzle Device Sample Device Sample Diffuser Pump Pump supply Pump chamber Pump delivery d 2 q (A) (B) maintained contact between A-2 of the lever and the syringe plunger.The syringe was connected directly to a fused silica capillary (Polymicro Technologies, Phoenix, AZ, USA) with 2.0 cm 3 0.010 in id poly(vinyl chloride) (PVC) tubing (Elkay Products, Shrewsbury, MA, USA). Measurement of absolute expansion of PA In order to measure the absolute expansion of the PA and subsequent amplification by the lever, one end of a short piece of fused silica capillary (20 cm 3 150 mm od) was attached to the PA head and A-2. The respective expansions were determined by focusing the other end of the capillary under a stereomicroscope (1003 magnification, Optiphot-2 microscope, Nikon, Tokyo, Japan) equipped with a CCD video camera and monitored by measuring the movement of this capillary on a calibrated TV screen.The small size of the capillary (low mass) minimized the loading back-pressure on the PA. Measurement of absolute flow rates To measure the absolute linear fluid velocity under pump operation (applied voltage), the micro-syringe was filled with a suspension of 0.0005% uniform latex micro-beads (0.203 mm polystyrene micro-spheres, Duke Scientific, Palo Alto, CA, USA) in water.Fused silica capillaries of various lengths were connected to the syringe and movement of the latex beads through a small segment of the capillary window was monitored under the microscope. A window for optical viewing was made by removing about a 1.0 cm portion of the polyimide coating situated about 20 cm from the capillary inlet. The entire capillary was filled with the bead solution manually from the syringe. Care was taken to avoid hydrostatic flows generated by a height difference between the two ends of the capillary causing siphoning action.A timer was used to record the time necessary for the beads to travel through the known viewing segment of the capillary when various voltage ramps were applied to the PA. Since beads were chosen randomly throughout the internal diameter of the capillary tube, the stated flow velocities represent the average linear flow rate of the parabolic flow profile produced by this pressure driven system.Laser-induced fluorescence determination of flow stability The flow stability was determined using laser-induced fluorescence (LIF) signals by flowing a solution of 50 nm fluorophoric dye (IR-144, from Kodak Chemicals, Rochester, NY, USA). Owing to the photobleaching experienced by a fluorogenic dye when flowing into a focused laser beam with high irradiance, the fluorescence signal becomes a sensitive probe of flow rate.31,32 LIF was performed using a 750 nm diode laser (GaAlAs) as the excitation source.This beam was focused on to a 75 cm long multimode glass fiber [50 mm core, 125 mm cladding, 0.30 numerical aperture (NA), from 3M (St. Paul, MN, USA)] with a 203 microscope objective. The distal end of the fiber was situated in close proximity to the observation window of a 10 cm (100 mm id 3 365 mm od) fused silica capillary which was affixed to a laboratory made Plexiglas capillary holder.The fluorescence was collected at right-angles (with respect to the laser light) with a 403 (0.85 NA) microscope objective and imaged on to a slit serving as a spatial filter to reduce the amount of scattered photons generated at the air–glass and glass–liquid interfaces of the capillary from reaching the photon transducer. The fluorescence was further isolated from the scattering photons by a 780 nm (±10 nm) bandpass filter and a 780 nm long pass filter and finally focused on to the photoactive area of the detector (single photon avalanche detector, EG&G Optoelectronics, Vaudrieulle, Canada) with another microscope objective (203).The fused silica capillary was filled with a 50 nm solution of IR-144 fluorescent dye solution (Kodak Chemicals) and connected to the pump. Construction of diffuser–nozzle system The principle operational modes of the diffuser–nozzle system is depicted in Fig. 3(A). There are basically two modes of operation, pump supply and pump delivery. During pump supply, the plunger of the syringe pump is retracted, causing sample to fill the pump chamber. During the pump delivery mode, the sample in the pump chamber is delivered to the chemical analysis device. The proper direction of fluid flow during the appropriate pump cycle is determined by two elements, a diffuser and nozzle, which have different pressure drops across them causing maximum flow from one port (diffuser) and minimizing the flow in the other (nozzle).For optimum performance, the pressure drop across the diffuser should be much less than that for the nozzle so that the majority of the solution movement occurs preferentially through the diffuser. The pressure drop across the diffuser (DPd) and nozzle (DPn) can be calculated from26 DPd d d = rn x 2 2 (1) DPn n n = rn x 2 2 (2) Fig. 3 (A) Operational modes of diffuser–nozzle system.The arrows represent the direction of fluid flow and the magnitude of fluid flow through the device during operation. (B) Scanning electron micrographs of the diffuser–nozzle system micromachined into PMMA using X-ray lithography. The channels were machined to a depth of 50 mm. Analyst, July 1998, Vol. 123 1437where r is the density of the solution, n is the linear flow velocity through the narrowest part of the diffuser or nozzle element and x is the pressure loss coefficient.To achieve efficient filling of the pump chamber xd/xn > 1, which can be accomplished by minimizing the pressure loss coefficient associated with the diffuser.33 The pressure loss coefficients depend on a number of design parameters associated with the diffuser and the general topology of the diffuser (flat-walled versus conical). For a flat-walled diffuser, the minimum pressure loss occurs in a flow regime classified as transitory stall, where flow separation occurs at the wall of the diffuser. The physical dimensions of the diffuser which must be considered in order to operate in this regime are 2q and the ratio, L/d, where these dimensions are defined in Fig. 3(A). In the present case, L/d = 41 and 2q = 8°, which put the diffuser in the transitory stall regime. The required channels were machined into the PMMA substrate using X-ray lithography following previously described procedures.20,34–36 Briefly, an optical mask containing the required device topography was situated on a 5 cm square piece of PMMA which was coated with a 5 nm layer of Au–Cr, which served as a plating base, and a positive resist.After UV exposure, the resist was developed and a thick overlayer of Au (3 mm) was applied electrolytically to the developed areas to serve as the X-ray absorber during exposure. After the required Au layer was applied to the device, the remaining resist was removed and the device was placed in the X-ray beam and exposed to soft X-rays at our Center for Advanced Microstructure and Device facility.After exposure, the PMMA substrate was developed to remove exposed PMMA and then, after thorough cleaning, another piece of PMMA was thermally bonded to the diffuser–nozzle device. In Fig. 3(B) is shown a scanning electron micrograph of the micromachined PMMA. The depth of the channels was found to be 50 mm and the narrowest portion of the diffuser–nozzle was 30 mm with the widest part being 300 mm.Owing to the ability to machine in PMMA using X-rays with high aspect ratios, the channel topography in the diffuser–nozzle was considered to be flatwalled and not conical. After sealing the PMMA top sheet to the diffuser–nozzle, small diameter capillary tubes were inserted into the three ports of the diffuser–nozzle, one interfaced to the syringe pump, the second to the sample supply and the third port to the chemical analysis system. The capillary tubes were of 20 mm id and 40 mm od.These capillary tubes were inserted into the narrow PMMA channels by placing the tube on an XYZ micropositioner and then carefully inserting the capillary tube into the PMMA channel using an optical microscope for visual inspection. Once the capillary tube had been properly inserted into the device, it was sealed to it using epoxy. Results and discussion Piezo-pump characteristics The PA used in the fabrication of the pump was made from a thin-layered ceramic stack which allowed greater expansion. The translator expands between the casing and the magnetic top piece (PA head). This particular piezo-stack is expected to produce a linear expansion of 80 mm and operate with a maximum pushing force of 2000 N (pressure = 6.37 3 106 Pa for head area of 3.14 cm2).Our microscopic observations revealed that the maximum expansion of the PA was 72 mm at an applied voltage of 21000 V when approximately 68.9 Pa (0.01 psi) back-pressure was loaded on to the syringe.This amounts to a 10% loss in linear displacement when little or no load was applied against the PA. The main purpose of using the pivoted lever was to amplify the PA expansion in order to permit higher volume displacements per pump stroke. The output displacement of the single-lever expansion unit was determined to be 648 mm, which resulted in an expansion gain of 9.0. Expansion and amplification of PA movement during voltage ramp The absolute expansion of the piezoelectric actuator and its amplification by the pivoted lever (with no load) were monitored as a function of a linear voltage ramp.A plot of the observed PA and PA–lever linear displacement with respect to the progression of the voltage ramp is shown in Fig. 4. In both the amplified and non-amplified linear displacements, no expansion was observed until the applied voltage reached about 2100 V. As can be seen from this plot, the displacement of the PA was fairly linear with applied voltage except at low applied voltages (2100 to 2300 V) and then at the high end of the voltage ramp also (2800 to 21000 V).This type of expansion profile can be expected owing to hysteresis effects from this type of piezoelectric translator, particularly when operated without a position sensor. To obtain a linear expansion of the PA, a non-linear voltage ramp can be used to compensate for these effects. It can also be observed from Fig. 4 that the amplified displacement by the lever tracked the displacement of the PA without amplification fairly well with non-linearities at the high and low ends of the applied voltage ramp. Assuming a ninefold amplification factor by the lever, it can be seen from this plot that the amplification is less than ninefold below an applied voltage of 2200 V and then exceeds ninefold above an applied voltage of 2600 V. Pump flow profile during voltage ramp The volumetric flow rate (nl min21) is basically a function of two parameters: the slope of the applied voltage ramp (V s21) and the diameter of the micro-syringe.Since we used a linear voltage ramp, the pivoted lever amplified expansion was expected to be dependent on the absolute expansion behavior of the PA and the load pressure also. The flow rate of latex beads was followed in a 2.56 mm long window of a 30 cm 3 50 mm id fused silica capillary (the viewing window was in the middle of the capillary).Based on our microscopic studies, no pumping action was seen until the voltage reached approximately 2100 V with a load pressure (DP) of 0.22 psi (1.5 3 103 Pa), which was calculated using the expression Fig. 4 Linear displacement of piezo-head, non-amplified (squares) and amplified (circles), versus applied voltage. The linear displacement was determined by placing a short piece of capillary tube against the PA head and then monitoring the movement of the capillary tube under a stereomicroscope with a video screen that was calibrated. 1438 Analyst, July 1998, Vol. 123DP LQ R = 8 4 h p (3) where L is the length of the capillary tube (m), Q is the volume flow rate (m3 s21), h is the solution viscosity (Pa s) and R is the radius of the capillary (m). In this case, the solution viscosity was assumed to be near that of pure water (1 cP or 0.001 Pa s). It should also be noted that owing to the amplification by the pivoted lever, this load pressure is back-amplified to the PA so that the actual load pressure at the PA head is 1.98 psi (1.35 3 104 Pa). In addition, this load pressure was calculated at the highest volume flow rate investigated (47.8 nl min21) and is expected to decrease at the lower volume flow rates.The observed flow patterns at three ramp speeds are shown in Fig. 5. Qualitatively, we did not notice any pulsing movement of the microbeads resulting from pump steps in the ramp-speed range 0.25–4.4 V s21 in this 50 mm id capillary.However, in these experiments we did observe the beads close to the wall moving more slowly than those in the center of the capillary owing to the parabolic nature of the laminar flow. As is apparent from Fig. 5, the volume flow rate requires a fixed time period (applied voltage) to reach a constant value with the duration dependent upon the ramp rate (i.e. load pressure). For example, at a ramp rate of 1.96 V s21, the volume flow rate does not become constant until an applied voltage of about 2400 V is reached, whereas for a voltage ramp of 0.49 V s21, the volume flow rate reaches a constant value at an applied voltage of approximately 2150 V.As shown in the inset in Fig. 5, the volume flow rate was linear (r = 0.9998) with the applied linear voltage ramp over the range investigated (0.49–1.96 V s21). Effect of applied back-pressure The dependence of liquid flow rate on applied back-pressure at different voltage ramps was determined from microscopic experiments.A 2.0 m 3 20 mm id fused silica capillary was connected to the pump and filled with a latex bead solution. The liquid was pumped through the capillary at different flow rates which were controlled by varying the slope of the applied voltage ramp to the PA. This operation was repeated after reducing the length of the capillary in 0.3 m increments. The observed flow rates increased linearly with increase in the slope of the voltage ramps for this series of capillaries, which is illustrated in Fig. 6(A). If the load pressure did not exert a perturbation on the volume flow rate, then these lines should exhibit similar slopes, independent of tube length. However, as can be seen from this graph, deviations are observed at high pumping rates (large pressure drops) for different lengths of capillaries, indicating that the volume flow rate will show some dependence on load pressure. The effect of pressure drop across the capillary on the observed flow rate is presented in Fig. 6(B), in which the pressure was calculated from eqn. (3). As can be seen, the volumetric flow rate decreased with increasing pressure drop. Given a fixed diameter and length of capillary, the magnitude of this effect (slope of volume flow rate versus load pressure) increases with increasing pumping rate since the load pressure also depends on the volume flow rate. These effects can easily be rationalized based on the fact that the PA can be considered to act as an elastic body with a given stiffness and a changing load during the expansion.The load pressure changes during the expansion since the flow velocity increases during each voltage step, which results in reductions of expansion under load conditions (DL). This loss can be determined from the expression Fig. 5 Volume flow rate versus applied voltage to the amplified PA head. The linear velocity was determined by observing the movement of the micro-beads in water moving through a 30 cm 3 50 mm id capillary tube using a stereomicroscope.The volume flow rate was calculated by multiplying the observed linear velocity by the cross-sectional area of the capillary tube. Fig. 6 (A) Volume flow rate as a function of ramp speed (V s21) for 20 mm id tubes of various lengths. The lengths of the capillary tubes were changed in 0.3 m increments so as to alter the load pressure on the PA head. (B) Volume flow rate versus load pressure at five different ramp speeds (V s21).If the volume flow rate was not dependent on load pressure, then the slopes of these plots should be zero. The load pressure shown here does not account for the amplification factor associated with the pivoted lever. Therefore, the actual load pressure at the PA head should be multiplied by nine. The volume flow rate was calculated using the same procedure as described in Fig. 4. Analyst, July 1998, Vol. 123 1439DL L c c c = + 0 T T S (4) where L0 is the linear displacement under no load conditions (72 mm), cT (N mm21) is the stiffness of the PA (45 N mm21) and cs (N mm21) is the stiffness or spring constant of the load.For a capillary of 2.0 m 3 20 mm id and a pump volume flow rate of about 47 nl min21, the pressure drop at the PA head is 513 psi (57 psi 3 9, where 9 is the amplification factor of the pivoted lever) or 3.54 3 106 Pa. This results in a value of cs = 15.4 N mm21. Under these load conditions, the actual linear expansion is 54 mm or 13.6 nm per step (12-bit DAC) compared with 17.6 nm per step under no-load conditions.At a driving voltage ramp of 2.44 V s21 the volume flow rate with this load was calculated to be 55.5 nl min21, which compared favorably with the observed 47.8 nl min21 measured under these load conditions [see Fig. 6(B)]. Under no-load operation, the volume flow rate expected would have been 74.6 nl min21. Flow stability In order to examine the stability of the pump when operated at low volume flow rates, a dye solution was pumped through a 100 mm id capillary tube and the fluorescence was monitored during the driving voltage ramp at three different ramp rates.When the dye molecules travel through an intense Gaussian laser beam, the integrated fluorescence intensity depends on the photoalteration parameter (F), which was determined using the expression31 F = PFds/(p1/2wn) (5) where P is the average laser irradiance (photons s21), Fd is the dye photodestruction quantum efficiency, defined as the probability that a molecule photodegrades once in the excited state, s is the absorption cross-section (cm2), w is the 1/e2 laser beam radius (cm) and n is the linear flow velocity of the fluorescent dye molecule (cm s21).As can be seen from this expression, F depends inversely on the linear flow velocity and hence will affect the integrated fluorescence intensity. Therefore, monitoring the fluorescence intensity will be a sensitive indicator of flow fluctuations produced by the pump.However, it should be pointed out that only under the conditions of 0.1 @ F@100 does the integrated fluorescence depend directly on the photoalteration parameter. When F < 0.1, no dye bleaches during its travel through the beam, and when F > 100, all dye molecules are immediately bleached upon entering the sampling volume. In the present case for the dye used in these experiments (IR-144), P = 1.88 3 1016 photons s21 (5 mW at 750 nm), s = 2.4 3 10216 cm2, w = 25 3 1024 cm, Fd = 9 3 1027 and variation of n from 1.9 3 1025 to 8.2 3 1024 cm s21 resulted in a photoalteration parameter which ranged from 47 to 1.1.The fluorescence intensity as a function of three different volumetric flow rates is displayed in Fig. 7. As can be seen, the average intensity is a function of the flow rate, indicating that the photoalteration parameter is within the range where the fluorescence intensity does depend upon the linear flow velocity.Careful inspection of the data when the pump has reached a level where the average fluorescence intensity is constant demonstrates the lack of large fluctuations in the intensity which could arise from pulsations in the pumping action. However, there is some noise superimposed on these traces, most of which arises from Poisson noise (shot noise) in the counting experiment. This is particularly evident at the very low pumping rate (9.18 nl min21) where the average fluorescence intensity is low and the degree of photobleaching is high (F = 47).Conclusions We have fabricated a micro-syringe pump which consisted of a piezo-pusher and pivoted lever for amplifying the displacement of the PA. This pump can deliver volume flow rates in the low nl min21 range, even under the conditions of high load pressures where the peristaltic pumps may display difficulties. Since the pump will operate under high loads, it will be an important device for micro-fluidic applications where solutions must be pumped through narrow bore channels.In addition, the low volume flow rates that are achievable will allow manipulation of fluids in narrow channels which require long residence times, such as in micro-chromatographic techniques or micro-based flow injection analysis. Another advantage associated with the present device is that pump-dependent noise resulting from pulsations is absent.However, a difficulty associated with this pump in its present format is the limited volume it can deliver per pump stroke. At a pump volume of 560 nl and a volume flow rate of 9.2 nl min21, it can effectively operate for 60 min before requiring to be refilled, but at a flow rate of 368 nl min21 the pump can operate for only 1.5 min. Another potential problem is the ruggedness of the device, owing to the need for the sophisticated pivoted lever system.An alternative format for amplifying the linear displacement of the PA could reduce this difficulty, e.g., the implementation of a pulley system. In order to refill the pump automatically without requiring disconnection of the pump from the chemical analysis system, a diffuser– nozzle device was micromachined into PMMA to create a low volume pump chamber and channel network to allow ease of use. The micromachined diffuser–nozzle contains no moving parts and can allow directing flows in hydrodynamically driven systems.However, it should be pointed out that during operation of the diffuser–nozzle for pump refilling, discontinuities in the flow do result. The authors thank the Whitaker Foundation for financial support of this reseach. They also like to thank Professor Robin McCarley for helpful discussions during the course of this work. References 1 Rothman, L. D., Anal. Chem., 1996, 68, 587R. 2 Lunte, C. E., Scott, D. O., and Kessinger, P.T., Anal. Chem., 1991, 63, 773A. 3 Harrison, D. J., Fluri, K., Seiler, K., Fan, Z., Effenhauser, C. S., and Manz, A., Science, 1993, 261, 895. 4 Effenhauser, C. S., Manz, A., and Widmer, H. M., Anal. Chem., 1993, 65, 2637. 5 Harrison, D. J., Glavina, P. G., and Manz, A., Sens. Actuators B, 1993, 10, 107. Fig. 7 Pump stability at three different volume flow rates. The flow stability was determined by monitoring the fluorescence produced by the dye IR-144. The fluorescence was excited with 5 mW of laser power at 750 nm.The fluorescent dye was dissolved in methanol at a concentration of 50 nm 1440 Analyst, July 1998, Vol. 1236 Fan, Z. H., and Harrison, D. J., Anal. Chem., 1994, 66, 177. 7 Jacobson, S. C., Hergenroder, R., Koutny, L. B., Warmack, R. J., and Ramsey, J. M., Anal. Chem., 1994, 66, 1107. 8 Jacobson, S. C., Hergenroder, R., Koutny, L. B., and Ramsey, J. M., Anal. Chem., 1994, 66, 1114. 9 Effenhouser, C. S., Paulus, A., Manz, A., and Widmer, H.M., Anal. Chem., 1994, 66, 2949. 10 Jacobson, S. C., Koutny, L. B., Hergenroder, R., Moore, A. W., and Ramsey, J. M., Anal. Chem., 1994, 66, 3472. 11 Seiler, K., Fan, Z. H., Fluri, K., and Harrison, D. J., Anal. Chem., 1994, 66, 3485. 12 Woolley, A. T., and Mathies, R. A., Proc. Natl. Acad. Sci. USA, 1994, 91, 11 348. 13 Jacobson, S. C., Moore, A. W., and Ramsey, J. M., Anal. Chem., 1995, 67, 2059. 14 Effenhauser, C. S., Manz, A., and Widmer, H. M., Anal. Chem., 1995, 67, 2284. 15 Woolley, A. T., and Mathies, R. A., Anal. Chem., 1995, 67, 3676. 16 Raymond, D. E., Manz, A., and Widmer, H. M., Anal. Chem., 1996, 68, 2515. 17 Jacobson, S. C., and Ramsey, J. M., Anal. Chem., 1996, 68, 720. 18 Fluri, K., Fitzpatrick, G., Chiem, N., and Harrison, D. J., Anal. Chem., 1996, 68, 4285. 19 Schwer, C., and Kenndler, M. G., Anal. Chem., 1991, 63, 1801. 20 Ford, S. M., Kar, B., McWhorter, S., Davies, J., Soper, S. A., Klopf, M., Calderon, G., and Saile, V., J. Microcol. Sep., in the press. 21 van Lintel, H. T. G., van de Pol, F. C. M., and Bouwstra, S., Sens. Actuators, 1988, 15, 153. 22 van de Pol, F. C. M., Wonnink, D. G. J., Elwenspoek, M., and Fluitman, J.H. J., Sens. Actuators, 1989, 17, 139. 23 Smits, J. G., Sens. Actuators, 1990, 21, 203. 24 Schomburg, W. K., Fahrenberg, J., Maas, D., and Rapp, R., J. Micromech. Microeng., 1993, 3, 216. 25 Korenaga, T., Zhou, X., Moriwake, T., Muraki, H., Naito, T., and Sanuk, S., Anal. Chem., 1994, 66, 73. 26 Stemme, E., and Stemme, G., Sens. Actuators A, 1993, 39, 159. 27 Olsson, A., Enoksson, P., Stemme, G., and Stemme, E., J. Micromech. Microeng., 1995, 6, 87. 28 Heschel, M., Mullenborn, M., and Bouwstra, S., J. Microelectromech. Syst., 1997, 6, 41. 29 Olsson, A., Enoksson, P., Stemme, G., and Stemme, E., J. Microelectromech. Syst., 1997, 6, 161. 30 Olsson, A., Larsson, O., Holm, J., Lundbladh, L., Ohman, O., and Stemme, G., Sens. Actuators A, 1998, 64, 63. 31 Mathies, R., Oseroff, A. R., and Stryer, L., Proc. Natl. Acad. Sci. USA, 1976, 73, 1. 32 Soper, S. A., Nutter, H. L., Keller, R. A., Davis, L. M., and Shera, E. B., Photochem. Photobiol., 1993, 57, 972. 33 White, F. M., Fluid Mechanics, McGraw-Hill, New York, 1986, pp. 348–352. 34 Vladimirsky, Y., Vladimirsky, O., Saile, V., Morris, K. J., and Klopf, J. M., Proc. SPIE, 1995, 2621, 399. 35 Vladimirsky, Y., Vladimirsky, O., Saile, V., Morris, K. J., and Klopf, J. M., Proc. SPIE, 1995, 2437, 391. 36 Ford, S. M., Davies, J., Kar, B., Owens, C. V., Klopf, M., Calderon, G., Saile, V., and Soper, S. A., Anal. Chem., submitted for publication. Paper 8/00052B Received January 2, 1998 Accepted March 17, 1998 Analyst, July 1998, Vol. 123 1441

ISSN:0003-2654    

DOI:10.1039/a800052b

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Holographic refractive index detector for application in microchip-based separation systems Norbert Burggraf‡a,b, Beat Krattiger†a, Andrew J. de Melloc, Nico F. de Rooijb and Andreas Manz*a,c a Ciba-Geigy, Corporate Analytical Research, CH-4002 Basle, Switzerland b Institute of Microtechnology, University of Neuch�atel, Rue A. L. Breguet 2, CH-2000, Neuch�atel, Switzerland c Zeneca/SmithKline Beecham Centre for Analytical Sciences, Department of Chemistry, Imperial College of Science, Technology and Medicine, Exhibition Road, South Kensington, London, UK SW7 2AY A novel detection scheme for capillary electrophoresis on planar glass microchips is presented.The application of a holographic-based refractive index detector to the electrophoretic separation of carbohydrates is described. The microchip device consists of a cyclic (square) separation channel having a circumference of 80 mm, a width of 40 mm and a depth of 10 mm. The volume of the injection scheme is approximately 16 pl.Separation and refractive index detection of a mixture of sucrose, N-acetylglucosamine and raffinose, each at a concentration of 33 mm, was achieved within 17 s of injection. Preliminary results demonstrate the feasibility of using hologram-based refractive index detectors in microchip separation systems. Although the initial detection limits are poor in comparison with alternative techniques, the potential of a universal detector of this kind is clear.Keywords: Holographic refractive index detector; microchips; capillary electrophoresis; carbohydrates Miniaturization of conventional laboratory instrumentation has been the focus of much attention during the last decade. The advantages of ‘downsizing’ analytical processes lie in improved efficiency with respect to sample size, response time, cost, throughput and automation. In particular, miniaturization of liquid phase separation methods has proved highly successful in the manipulation and analysis of small amounts of samples.This new generation of analytical instrumentation has been termed miniaturized total analysis systems (m-TAS).1,2 A m- TAS aims to combine all steps of a complete, chemical analysis (sample handling, chemical reactions, sample separation, detection and product isolation) on a single, integrated device. Normally, such devices are fabricated on glass or silicon substrates using standard micromachining methods (photolithography, etching, thin-film deposition and bonding).The result is a planar chip containing an enclosed channel manifold through which the sample can be maneuvered. In addition, more complex components such as heaters, electrodes and mixers can be fabricated within the channel network. A diversity of chemical separation techniques have been successfully integrated into the concept of a m-TAS. These include capillary electrophoresis (CE),3–6 free-flow electrophoresis, 7,8 open-channel electrochromatography,9,10 openchannel liquid chromatography,11–13 gas chromatography,14 micellar electrokinetic capillary chromatography15,16 and synchronized cyclic capillary electrophoresis (SCCE).17–19 The adaptation of conventional detection protocols to measurement in small volumes has closely accompanied the development of m-TAS.Indeed, it has long been realized that size limits for m-TAS are primarily set by the system detector. For example, when performing CE on a typical microfabricated device, injection volumes typically range between 10214 and 10210 dm3.At a diagnostically relevant target concentration of 1 nm, this results in between 10 and 104 detectable molecules. Consequently, it is clear that high-sensitivity detection is essential when performing micro-separations. Small volume detection in analytical technology has generally been based around optical measurements (either absorption or fluorescence). Unfortunately, small volume absorption measurements are compromised owing to the difficulty in probing small volume cells whilst maintaining a sufficiently long pathlength.20,21 With microfabricated devices this problem is exacerbated (owing to reduced channel dimensions) and, to date, fluorescence methods have proved superior.Detection limits for fluorescence based measurements are extremely low.22 As few as 105 molecules are generally detectable in most laboratories utilizing laser-induced fluorescence (LIF).Furthermore, recent developments in ultra-high sensitivity fluorescence detection have allowed single molecule detection to be performed ‘on-chip’.23 Although fluorescence techniques are inherently sensitive, they are costly and not applicable for all molecular systems (i.e., not all species that absorb radiation fluoresce). Other approaches to detection have included electrochemiluminescence, 24 electrochemical25 and thermal conductivity methods.14 For example, Arora et al.24 recently demonstrated the detection of only 3 3 104 tris(2,2A-bipyridyl)ruthenium(ii) molecules in a 100 nl probe volume ‘on-chip’.In addition, elegant experiments have demonstrated the feasibility of coupling electrophoresis microchips with the technique of electrospray ionization mass spectrometry (ESI-MS). Xue et al.26 demonstrated high sensitivity (low nanomolar) microchip ESI-MS in the analysis of proteins whilst Ramsey and Ramsey27 reported a method of generating electrospray from solutions emerging from microchannels etched on planar glass substrates.Both approaches extend the applicability of m-TAS to molecules that are non-fluorescent, and lead to the possibility of high-throughput MS analysis in screening and diagnostic applications. The need for alternative detection protocols that are both sensitive and universal is apparent. Refractive index (RI) detection has been successfully applied to many analytical techniques (including HPLC28 and CE29,30).RI based detectors have important characteristics which make them attractive alternatives to fluorescence and absorption methods. First, RI detection is a relatively simple technique useful in the micro- to millimolar range. It can be used with a wide variety of buffer systems, is universal in nature and is concentration sensitive † Present address: Storz Endoskop GmbH, Schneckenackerstrasse 1, CH-8200 Schaffhausen, Switzerland. ‡ Present address: EG&G IC Sensors, 1701 McCarthy Blvd., Milpitas, CA 95035, USA.Analyst, July 1998, Vol. 123 (1443–1447) 1443(not mass sensitive). In addition, RI detectors have been shown to be successful in performing sensitive absorption detection in narrow bore capillaries (< 50 mm id) using thermo-optical methods.29,31 The major challenge when applying RI detection to CE is controlling Joule heating. The variation of RI with temperature (dn/ dT) for most solvent systems is of the order of 1024 RI units (RIU) K21 at room temperature.Since most applications require a sensitivity below Dn = 1026 RIU, thermal stability becomes the dominant factor in lowering detection limits. This limitation is partly offset when moving to a microfabricated format. m-TAS for CE are conventionally fabricated by etching channels on a planar glass substrate. Channel dimensions are typically 10–20 mm deep, 20–50 mm wide and 10–100 mm long. These dimensions are significantly smaller than those of typical capillaries used in CE.During electrophoresis, solution resistance to current transport causes Joule heating. This can be considerable under normal conditions. Simple theory defines the heat generated per unit length (Q) in rectangular channels as Q U h a L c = 2 2 2 l (1) where U (V) is the applied potential, h (m) is the channel height, a is the aspect ratio, l (m2 mol21 W21) is the (pH-dependent) molar conductivity of the buffer and c (mol dm23) its concentration. Eqn.(1) clearly demonstrates that a reduction in the cross-sectional area (and channel length) will minimize heat generation and thus temperature variations within the channel. This in turn will reduce variations in n and improve detector sensitivity. A variety of RI detectors have been used in separation based analyses. Early attempts utilized interference patterns arisfrom side-illuminated capillaries. Optical interfaces within the capillary system separate the incident beam into three components (reflected, refracted and transmitted parts) which interfere in the far field and yield an interference pattern characteristic of sample in the detector volume.This method has been successfully applied to HPLC, CE and flow-injection analysis.29 The ‘off-axis’ method for CE was also demonstrated by Bruno et al.30 in the analysis of underivatized sugars in a 50 mm id capillary. They identified Joule heating as the major source of experimental noise.A more recent study by the same group used a holographic optical element to generate the interference pattern (rather than the capillary).32 In this case the sampling arm of the interferometer traverses the capillary at its centre, where the optical path is longer and diffraction effects are smaller. This approach allowed the use of capillaries as narrow as 5 mm id. This paper extends the application of holographic optical elements (HOEs) and reports the first demonstration of a holographic RI detector applied to a microchip-based separation system.Preliminary results established the feasibility of RI detection for CE separations on planar glass microchips. Principle of refractive index detection The principle of hologram-based RI measurement is shown in Fig. 1. A collimated, coherent beam of radiation is partially deflected by an HOE placed at a given angle (approximately 30°) with respect to the illumination beam.The HOE acts as a set of two nearly superimposed focusing lenses, laterally displaced by a distance of 14 mm. It acts to divide the incident beam into two coherent beams, and focus them on to the plane of the channel within the planar, glass substrate. One beam (probe beam) is directed through the electrophoresis channel and the second (reference beam) acts as a control and passes through the glass substrate only. As indicated, both beams subsequently diverge in the far field and interfere.This results in the generation of an interference pattern consisting of equally spaced fringes, which are detected on a photodiode array (PDA). Interference occurs since both beams originate from the same source. A change in RI within the channel (e.g., when an analyte band passes through the detector volume) induces a phase change in the probing beam, resulting in a lateral shift of the fringe pattern. A detailed description of theory is given elsewhere.32 Experimental CE microchip SCCE glass structures were used in these studies to perform CE owing to their availability.19 All devices were fabricated under contract by Baumer IMT (Greifensee, Switzerland) using proprietary processes.Structures used in this work were made from soda lime glass (4012 SLW 5, Hoya, Tokyo, Japan). Channels were formed in the substrate material by etching with glycerol–HF solution. All channels were 10 mm deep and 40 mm wide. The isotropic etching procedure resulted in a rounded channel profile, with a channel bed width of 20 mm.A cover plate was thermally bonded to the substrate by heating the assembly at 500 °C for 3 h, followed by 580 °C for 3 h. The complete device was then allowed to cool for at least 12 h. Ten holes drilled in the top plate allowed access to the fluidic network. The chip layout used is illustrated in Fig. 2. The volume of the injection scheme was approximately 12 pl. As noted previously, the construct was only used to perform CE and not SCCE.Consequently, RI detection was performed in the locality of the asterisk. This corresponds to a separation length of approximately 20 mm or one quarter of the loop. The lengths of the inlet and injection channels were 10 and 12 mm, respectively. Fig. 1 Principle of holographic RI detection: the HOE generates both reference and probe beams. Beams ‘fan out’ and interfere in the far field producing an evenly spaced fringe pattern. A change in the refractive index within the channel results in a shift of the fringe pattern. 1444 Analyst, July 1998, Vol. 123Optical system The optical set-up for RI detection is shown in Fig. 3. Briefly, a diode laser module (LDM 145; Imatronic, Batavia, IL, USA) operating at 670 nm and 3 mW was used as the light source for all experiments. A lens was mounted at the front of the laser diode to collimate the laser output. A precision current source was used to drive the laser diode.The HOE was glued at an angle of 30° on a machined plastic pipe and connected to the laser diode. This acts to shield the laser output and to avoid Schlieren effects. All HOEs were manufactured according to standard photolithographic procedures, and had detection efficiencies ranging from 16 to 23% batch to batch. The laser diode was maintained at a constant temperature to ensure a stable laser output. This was achieved using a Peltier element (Melcor, Trenton, NJ, USA) and temperature controller (LDT 5901B; ILX Lightwave).The laser diode and HOE were mounted on a motor controlled x–y translation stage (Spindler Hoyer, G�ottingen, Germany). This allows for facile alignment of the probe and reference beams. A cylindrical lens (Newport, Fountain Valley, CA, USA) was used to compress the interference pattern along one direction. The fringes were equally spaced and thus easily monitored by a PDA (KOM 2045; Siemens, F�urth, Germany) mounted on an x–y translation stage (Microbench; Spindler Hoyer).The PDA consisted of eight single diodes (four pairs) and was wired in parallel to combine the signals of the four fringes. With this setup, the lateral shift of the fringe pattern caused by sample passing though the detection volume was converted into a voltage. An Apple Quadra computer was used to perform data acquisition and analysis. Capillary electrophoresis Four (where necessary) high voltage power supplies (HCN 12500; FUG Elektronik, Rosenheim, Germany) were used to provide the high potentials for electrophoresis. They were connected to external electrolyte reservoirs by Pt electrodes. Current was monitored by an auto-ranging picoammeter (Model 485; Keithley, Cleveland, OH, USA) between a solvent reservoir and ground.All buffer reservoirs were connected to a high voltage power supply. Both the sample and waste reservoirs (of the injection channel) can be connected to high voltage power supplies in addition to ground.High voltage relays (Magnecraft, Chicago, IL, USA) were placed between the power supplies and electrodes. All relays were controlled by TTL signals generated by an Apple Quadra computer with a DAC (NB MIO 16 XH 18; National Instruments, Austin, TX, USA). To obtain an evenly charged channel surface, the complete fluidic network was washed with 0.1 m NaOH prior to use. For separation, channels were pressure filled with buffer using nitrogen at 0.5 MPa.Injection was effected by electroosmotically filling the intersection zone of the square channel and the injection channel (expanded portion of Fig. 2). This was achieved by applying a voltage of 1000 V between the ‘sample’ and ‘waste’ reservoirs for 15 s. Subsequently, an applied voltage of 5 or 2.5 kV between reservoirs 9 and 5 allowed electrophoretic movement of sample along the separation channel. Reagents Two buffer systems were used: a pH 9.0 buffer of 20 mm boric acid and 100 mm tris(hydroxymethyl)aminomethane (AnalaR grade) and a pH 9.0 buffer of 100 mm B(OH)3 and 0.1 m NaOH.Sucrose, N-acetylglucosamine and raffinose (Fluka, Buchs, Switzerland) were used without further purification. Results The feasibility of holographic RI detection for microchip based separation systems was established in two ways, as follows. Injection and detection of a single component Using the protocol outlined previously, a sample of 30 mm sucrose solution was injected on to the SCCE chip.Subsequent movement around the square channel was driven by an applied potential between reservoirs 9 and 5. The RI detector was positioned 20 mm along the separation channel (marked by an asterisk in Fig. 2). Fig. 4(a) shows the RI detector response as a function of time after injection for an electric field strength of 500 V1. Although the S/N is poor, a distinct voltage peak at 6.68 s is observed. Fig. 4(b) shows a similar injection, but at an applied field strength of 1000 V cm21.In this case the detection of the sucrose sample plug is observed after 3.54 s. Comparison of the figures demonstrates the fidelity of electroosmotic flow, since the detection time should be inversely proportional to the applied voltage. Repetitive injection of four samples at each electric field strength (500 and 1000 V cm21) results in measurable values Fig. 2 Layout of cyclic CE chip. The square separation channel has a circumference of 80 mm, a width of 40/20 mm at the top/bottom and a depth of 10 mm.The volume of the injection scheme is approximately 16 pl. Fig. 3 Schematic diagram of the experimental set-up showing the glass chip, reservoirs, electrodes, diode laser, HOE and diode array (not drawn to scale). Analyst, July 1998, Vol. 123 1445describing the reproducibility of the geometrically defined injection process. These values are given in Table 1. It can be seen that the reproducibility of the detection time is excellent and that the number of theoretical plates generated is satisfactory.Separation of a multi-component mixture Fig. 5 shows the separation of a multicomponent mixture using an applied field strength of 500 V cm21 and a separation length of 20 mm. The sample consists of a mixture of sucrose, Nacetylglucosamine and raffinose, each at a concentration of 33 mm and a pH of 9.0. All components are detected within 17 s of injection (sucrose, 13.3 s; N-acetylglucosamine, 14.8 s; raffinose, 16.3 s).Although the S/N is still unimpressive, all three components have been discriminated on the basis of their electrophoretic mobilities. Discussion These preliminary results demonstrate the feasibility of using hologram-based RI detectors in microchip separation systems. It is clear that detection limits in the current system are barely sufficient for routine analysis (10 mm carbohydrate). However, it should be noted that this is a prototype system.In addition, injection of 20–30 pl of sample corresponds to the detection of at most 600–900 fmol of carbohydrate. Since RI detection is a concentration-sensitive protocol, the HOE RI detector should prove far more sensitive when applied to small volumes (picoliter and sub-picoliter) with time constants less than 10 ms. Current S/N levels will be improved through the use of electronic filtering and possibly temperature pulsing methods. It should also be noted that thermal lensing techniques have generated detection limits of single molecules in femtoliter volumes.33 Assuming that the RI detector is ideally concentration sensitive, significantly improved detection limits should be attainable (through better chip design). The hologram-based RI detection system presented in this paper is a most cost effective detection protocol.The complete detector is valued at a few hundred dollars, which compares well with current LIF and confocal detection schemes. More importantly, it should be possible to integrate the HOE on to the microchip. This would aid miniaturization and obviate the need for complex alignment of the detector and separation channel.In this way, multiple detectors could be integrated (e.g., absorption, fluorescence and RI detection) to provide a truly universal detection unit. The studies presented here are intended to be the first step towards a universal, small volume detector for chip-based separation systems.To that end, RI detection has been proved to be a feasible candidate for that eventual application. We thank Alfredo Bruno, Sabeth Verpoorte (Novartis, Switzerland) and the group of Rene Dandliker (IMT Neuch�atel, Switzerland) for supplying us with the HOE for the experiments and the late H. Michael Widmer for long term support of research activities. References 1 Manz, A., and Widmer, H. M., Sens. Actuators, B, 1990, 1, 244. 2 Manz, A., and Becker, H., Microsystem Technology in Chemistry and Life Sciences, Springer, Berlin, 1998. 3 Manz, A., Harrison, J. D., Verpoorte, E., Fettinger, J. C., Paulus, A., Luedi, H., and Widmer, H. M., J. Chromatogra., 1992, 593, 253. Fig. 4 Injection of a sample of sucrose (30 mm) in 20 mm boric acid–100 mm Tris buffer (pH 9.0). RI detection with two different electric field strengths in the separation channel. Injection was performed using 1000 V for 15 s: (a) electric field strength, 500 V cm21, L = 20 mm; (b) electric field strength, 1000 V cm21, L = 20 mm.Table 1 Reproducibility of the geometrically defined injection and separation process. No. of Applied electric theoretical field/V cm21 s/s tmax/s plates 1000 0.02 ± 0.002 3.54 ± 0.01 31 300 ± 6 300 500 0.05 ± 0.001 6.68 ± 0.01 17 800 ± 700 Fig. 5 Electrophoretic separation of a mixture of three sugars (sucrose, Nacetylglucosamine and raffinose) at a concentration of 33 mm each. Electric field strength, 500 V m21; L = 20 mm. 1446 Analyst, July 1998, Vol. 1234 Jacobson, S. C., Hergenr�oder, R., Koutny, L. B., and Ramsey, J. M., Anal. Chem., 1994, 66, 1114. 5 Woolley, A. T., and Mathies, R. A., Proc. Natl. Acad. Sci. USA, 1994, 91, 11 348. 6 Harrison, J. D., Manz, A., Fan, Z., Luedi, H., and Widmer, H. M., Anal. Chem., 1992, 64, 1926. 7 Raymond, D. E., Manz, A., and Widmer, H. M., Anal. Chem., 1994, 66, 2858. 8 Raymond, D. E., Manz, A., and Widmer, H. M., Anal. Chem., 1996, 68, 2515. 9 Jacobson, S.C., Hergenr�oder, R., Koutny, L. B., and Ramsey, J. M., Anal. Chem., 1994, 66, 2369. 10 He, B., and Regnier, F., J. Pharm. Biomed. Anal., in the press. 11 Manz, A., Miyahara, Y., Miura, J., Watanabe, Y., Miyagi, H., and Sato, K., Sens. Actuators, B, 1990, 1, 249. 12 Ocvirk, G., Verpoorte, E., Manz, A., Grasserbauer, M., and Widmer, H. M., Anal. Methods Instrum., 1995, 2, 74. 13 Cowen, S., and Craston, D. H., Anal. Methods Instrum., 1996, Special Issue m-TAS ’96, 196. 14 Terry, S. C., Jerman, J. H., and Angell, J. B., IEEE Trans. Electron Devices, 1979, 26, 1880. 15 von Heeren, F., Verpoorte, E., Manz. A., and Thormann, W., Anal. Chem., 1996, 68, 2044. 16 Moore, A. W., Jacobson, S. C., and Ramsey, J. M., Anal. Chem., 1995, 67, 4184. 17 Burggraf, N., Manz, A., Effenhauser, C. S., Verpoorte, E., de Rooij, N. J., and Widmer, H. M., J. High Resol. Chromatogr., 1993, 16, 594. 18 Burggraf, N., Manz, A., Verpoorte, E., Effenhauser, C. S., and Widmer, H. M., Sens. Actuators, B, 1994, 20, 103. 19 von Heeren, F., Verpoorte, E., Manz, A., and Thormann, W., J. Microcol. Sep., 1996, 8, 373. 20 Verpoorte, E., Manz, A., L�udi, H., Bruno, A. E., Maystre, F., Krattiger, B., Widmer, H. M., van der Schoot, B. H., and de Rooij, N. F., Sens. Actuators, B, 1992, 6, 66. 21 Liang, Z. H., Chiem, N., Ocvirk, G., Tang, T., Fluri, K., and Harrison, D. J., Anal. Chem., 1996, 68, 1040. 22 Barnes, M. D., Whitten, W. B., and Ramsey, J. M., Anal. Chem., 1995, 67, 418A. 23 Fister, J. C., Jacobson, S. J., Davis, L. M., and Ramsey, J. M., Anal. Chem., 1998, 70, 431. 24 Arora, A., de Mello, A. J., and Manz, A., Anal. Comm., 1997, 34, 393. 25 Woolley, A.T., Lao, K., Glazer, A. N., and Mathies, R. A., Anal. Chem., 1998, 70, 684. 26 Xue, Q., Foret, F., Dunayevskiy, Y. M., Zavracky, P. M., McGruer, N. E., and Karger, B. L., Anal. Chem., 1997, 69, 426. 27 Ramsey, R. S., and Ramsey, J. M., Anal. Chem., 1997, 69, 1174. 28 Bornhop, D. J., and Dovichi, N. J., Anal. Chem., 1986, 58, 504. 29 Bornhop, D. J., and Dovichi, N. J., Anal. Chem., 1987, 59, 1632. 30 Bruno, A. E., Krattiger, B., Maystre, F., and Widmer, H. M., Anal. Chem., 1991, 63, 2689. 31 Bruno, A. E., Paulus, A., and Bornhop, D. J., Appl. Spectrosc., 1991, 45, 462. 32 Krattiger, B., Bruin, G. J. M., and Bruno, A. E., Anal. Chem., 1994, 66, 1. 33 Takehiko, K., paper presented at the Eleventh International Symposium on High Performance Capillary Electrophoresis and Related Microscale Techniques, 1998. Paper 8/01478G Received February 20, 1998 Accepted June 2, 1998 Analyst, July 1998, Vol.

ISSN:0003-2654    

DOI:10.1039/a801478g

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Capillary electrophoresis–time-of-flight mass spectrometry of drugs of abuse Iulia M. Lazara, Gary Naisbittb and Milton L. Lee*a a Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602-5700, USA b Utah State Crime Laboratory, Salt Lake City, UT 84114-8285, USA The use of capillary electrophoresis (CE) for the determination of drugs of abuse was explored. A commercial CE system was interfaced with a laboratory-built time-of-flight mass spectrometer (TOFMS) which was equipped with a high-speed data acquisition system to provide accurate monitoring of efficient separations.Ionization of the CE eluent was achieved with an electrospray ionization source. Standard mixtures and seized samples were analyzed either by direct infusion of the analyte solutions or after separation by CE. Detection at the low femtomole level was obtained using CE–TOFMS. Keywords: Capillary electrophoresis; mass spectrometry; time-of-flight; drugs of abuse Capillary electrophoresis (CE), which was developed in the 1980s and rapidly expanded in the 1990s, has become a useful, complementary separation technique.1,2 The fast development of CE is due to its unique characteristics, such as broad applicability (ionic/non-ionic, and low/high molecular mass compounds), high separation efficiency in a short analysis time, very good mass limits of detection and relatively low cost and simplicity. Rapid progress has also been made in interfacing CE with mass spectrometry (MS).3 There are several ionization sources in use for on-line CE–MS, such as electrospray (ESI), inductively coupled plasma (ICP) and continuous flow fast atom bombardment (CF–FAB).However, it is the development of the ESI technique4–6 which greatly facilitated CE–MS interfacing. ESI, much like CE, is applicable to both small and large molecules and ionic or ionizable compounds. CE–ICP is used in trace metal analysis, while CF–FAB is less sensitive than ESI and has less applicability to high molecular mass compounds.A limitation of the most common mass spectrometers used as CE detectors (quadrupole, ion trap and Fourier transform ion cyclotron resonance) is the relative long time (0.1–3 s) that they need to produce a full mass spectrum, which is a direct result of the requirements of scanning or ion trapping. Time-of-flight (TOF) MS is able to produce a full mass spectrum in 50–200 ms depending on the pre-set pulsing rate.Along with speed, high ion transmission efficiency (about 10%) and high duty cycle ( > 50% for the off-axis configuration) result in increased sensitivity.7,8 For fast and high-speed separations,9–11 interfacing with MS detection will require the high speed attributes of TOFMS. Illegal drugs normally encountered at the local or state levels are amphetamine, methamphetamine, marijuana, cocaine and heroin. Less frequent, but not uncommon, are the hallucinogens, psilocybin from mushrooms, mescaline from cactus and lysergic acid diethylamide (LSD).A wide variety of designer and commercial preparations are also encountered. Although drugs are typically found in all developed societies, their distribution and form vary from region to region. For example, in the southwest USA, methamphetamine and cocaine are the most commonly abused drugs, whereas in the eastern states, cocaine and heroin are far more common than methamphetamine. Heroin distributed in the East is usually a white powder thought to come from the Middle East, but in the western USA ‘black tar heroin’ from Mexico predominates.Drugs of abuse are typically analyzed using gas chromatography (GC) or high-performance liquid chromatography (HPLC).12–18 Certain compounds are thermally labile (e.g., LSD, anabolic steroids and psilocybin), require derivatization (e.g., morphine, adulterants and diluents such as sugars) or display poor chromatographic performance (e.g., amphetamine and methamphetamine in their salt forms), and are not amenable to GC analysis.19 HPLC has benefits of rugged instrumentation and methodology and good separation selectivity and it is applicable to a broad range of compounds; however, the separation times are often too long and efficiencies are much lower than in CE.Since the analytical methods used in forensic analysis must rely on widely accepted analytical techniques, and must be confirmed by at least two methods based on different physico-chemical principles with similar sensitivities, 20 CE can be very suitable for cross-validation of GC or HPLC.In the past few years, CE and especially micellar electrokinetic chromatography (MEKC) have gained interest for the analysis of illicit drugs. Numerous papers have described methodologies that exemplify increased resolving power and shorter analysis times compared with conventional methods. 19–32 Interfacing with MS offers further specificity and sensitivity.CE–MS analysis of illicit drugs has the potential to become an indispensable tool for the forensic scientist.33 We have recently developed an electrospray time-of-flight mass spectrometer (ESI-TOFMS) for use as a detector for fast and efficient liquid phase separations. The main features of this instrument are speed and sensitivity. Low attomole detection limits have been achieved with continuous infusion experiments, and the acquisition rate can be as high as 10 000 spectra per second.34 This paper describes the analysis of various mixtures of illicit drugs using CE–TOFMS.Seized drug samples were analyzed either by continuous infusion or CE–TOFMS. Using CE– TOFMS, low femtomole detection was achieved, even in the presence of non-volatile buffers. Experimental Reagents Standard mixtures were prepared using HPLC grade solvents. High purity methanol and water were purchased from Mallinckrodt (Chesterfield, MO, USA).Anhydrous (99.5%) citric acid glacial acetic acid, and ammonia solution (28–30% ammonia) were obtained from EM Science (Gibbstown, NJ, USA). Tetracaine hydrochloride, procaine hydrochloride, methamphetamine hydrochloride and cocaine hydrochloride were purchased from Sigma (St. Louis, MO, USA) and amphetamine hydrochloride from Alltech (Deerfield, IL, USA). We were not able to obtain a heroin standard; however, a seized heroin Analyst, July 1998, Vol. 123 (1449–1454) 1449sample contained enough material (about 30% m/m) to allow positive identification.Instrumentation Capillary electrophoresis A Crystal CE 300 system (ATI, Madison, WI, USA) and an Applied Biosystems (Foster City, CA, USA) Model 785A UV absorption detector were used. Uncoated fused silica (50 mm id and 190 mm od) was used to perform the separations (Polymicro Technologies, Phoenix, AZ, USA). Prior to analysis, the capillaries were conditioned by rinsing for 10–15 min with sodium hydroxide solution (1 m), followed by HPLC grade water (10–15 min).Between analyses the capillaries were rinsed with running buffer for 5 min. The CE analysis of seized samples necessitated additional rinsing with sodium hydroxide solution (0.1 m) between runs in order to prevent the deterioration of the fused silica inside wall. The basic solution treatment was followed by water and buffer rinses. Samples were prepared by dissolving the appropriate amount of compound in CH3OH or CH3OH–CH3COOH (100 + 0.1 v/v).Seized sample solutions (0.04 mg ml21) were filtered through Acrodisc PF 0.2 mm filters (Gelman Sciences, Ann Arbor, MI, USA). Time-of-flight mass spectrometry An in-house built ESI-TOFMS with orthogonal extraction was used as a detector. Using this system, the analytes from CE are brought from the liquid phase into the gas phase with the aid of an ESI source. A countercurrent heated curtain gas, fed between the sampling nozzle and a focusing lens (interface plate) placed in front of the ESI needle, is used to dry the electrosprayed droplets.Sampling of the ions from the source is accomplished using a nozzle–skimmer arrangement. The ion beam is preserved and focused by two sets of radiofrequency (rf)-only quadrupole ion optics before the pulsing region. The mass spectrometer was operated in a linear mode (non-reflecting) with detection occurring at the end of the flight tube. Both electrospray configurations, microspray and liquid sheath, were utilized.Microspray tips were prepared by pulling 50 mm id 3 190 mm od fused silica capillaries to 10–15 mm id 3 40–60 mm od, which promoted the onset of electrospray at relatively low voltages (1600–1800 V). The ESI voltage was applied through a metal union placed 1–1.5 cm away from the microspray tip. For CE separations, the liquid sheath ESI source was used. The liquid sheath contained CH3OH–H2O– CH3COOH (80 + 20 + 0.1 v/v) supplied through a stainless steel needle (gauge 26), which had an id which closely matched the od of the fused silica capillary.Detailed descriptions of the TOFMS and the ESI source were given in previous papers. 34,35 Continuous infusion of analytes was performed using a Harvard (South Natick, MA, USA) Model 22 syringe pump. The analyte solutions, the CE buffer and the sheath liquid were de-gassed by sonication for 5 min. Results and discussion Most drugs of abuse are relatively small molecules which contain basic amino functional groups that can be easily protonated in acidic aqueous solutions.If there is sufficient difference between their electrophoretic mobilities, efficient and rapid CE separations can easily be achieved. Several publications have reported the CE separation of similar drug mixtures using various buffer systems: phosphate or citrate at low pH (2.35), or borate, borate–sodium dodecyl sulfate (SDS), phosphate–borate–SDS and cyclodextrin–SDS (the last three used for MEKC) at high pH (8–9).The composition of the CE eluent can significantly alter the ionization efficiency of the ESI source. The coaxial liquid sheath flow, supplied at the CE capillary terminus, which has the combined purpose of completing the CE circuit and ensuring the ESI voltage, dilutes the CE eluent and allows it to be electrosprayed. It is especially difficult to deal with MEKC buffers in which the high concentration surfactant can result in complete analyte signal suppression. In CE, even though at low concentration, volatile buffer systems such as ammonium acetate or formate (for acidic pH conditions) are recommended; non-volatile buffer systems used at low concentrations can be electrosprayed if needed at the expense of some loss of ionization efficiency.We performed the separation of a mixture of amphetamine (A), methamphetamine (MA), cocaine (C) and heroin (H) in 25 mm citrate buffer. The pH of the citric acid solution was adjusted to 3 with ammonia solution.The eluent flow rate was approximately 150 nl min21 in the presence of a 10 mbar pressure applied to the CE inlet vial. For the 88 cm separation capillary used, this inlet pressure usually decreased the analysis time by 10–13%. The CE–UV and CE–TOF separations of this mixture are shown in Fig. 1. UV detection was applied at 210 nm, 70 cm from the inlet end. TOF detection occurred at the end of the column (90 cm from the inlet), which explains the greater migration time for the CE–TOF separation.The relative peak intensities in the two electropherograms differ for two main reasons. One is the different responses of the two detectors for the analytes. The second is that amphetamine and methamphetamine break down in the ion source as a result of the nozzle voltage setting, and the nozzle voltage had to be maintained at a lower value (lower acceleration voltage between the nozzle and skimmer), which did not produce the maximum signal intensity for all of the compounds. For later eluting components, the nozzle voltage was changed to the optimum value.These changes in nozzle voltage had to be performed with great care in order to not disturb the stability of the electrospray. The liquid sheath was used at a flow rate of 1 ml min21. With careful adjustment of the positions of the CE capillary inside the ESI needle and the ESI needle in the ion source, a stable spray was produced even in the presence of citrate buffer.The ion source was maintained at 90 °C and the drying gas was set at a flow rate of 1200 ml min21. The CE separation efficiency was not affected by the ESI source, but was preserved at its original value of between 200 000–300 000 plates. The inconvenience of using the citrate buffer was that it produced a noisy background. Fig. 1 CE–UV and CE-TOFMS electropherograms of drugs of abuse. Conditions: 85 cm 350 mm id uncoated fused silica capillary, 25 mm citrate buffer (pH 3), 30 mbar 3 0.2 min injection ( ~ 650–810 fmol).A, CE–UV: 27 kV, 10 mbar, 8.9 mA, 210 nm at 70 cm; B, CE-TOFMS: 30 kV, 10 mbar, 7.8 mA, 1 ml min21 CH3OH–H2O–CH3COOH (80 + 20 + 0.1 v/v) liquid sheath; ESI (3000 V), 90 °C; MS data acquisition at 5000 Hz, 1000 spectra averaged, 5 data points s21. Peak identifications: 1, amphetamine; 2, methamphetamine; 3, cocaine; 4, impurity; 5, heroin. 1450 Analyst, July 1998, Vol. 123A series of strong citric acid clusters (an intense ammonium citrate ion followed by its citric acid clusters) were present at m/z < 1200 (Fig. 2). A background spectrum showing the ions present during the elution of amphetamine and methamphetamine [Fig. 2(A)] was acquired at a nozzle–skimmer voltage difference of 22 V, whereas a different background spectrum [Fig. 2(B)], which was acquired at a nozzle–skimmer voltage difference of 52 V, was obtained during the elution of the later components.The flight time region in the spectrum between 16 and 24 ms became crowded with solvent–buffer cluster ions at the higher voltage; however, these ions did not overlap with the higher m/z ions. At greater liquid sheath flow rates (2–3 ml min21), the intensities of the citrate clusters decreased significantly, but the analyte signals also diminished. Owing to the negative effects of the citrate buffer on ionization efficiency, a larger sample (650–810 fmol) was initially injected into the CE column.A similar mixture containing two additional compounds, procaine (P) and tetracaine (T), was analyzed using CE–TOFMS at two injection levels, one at 610–850 fmol [Fig. 3(A)] and the other much lower (110–135 fmol) [Fig. 3(B)]. Intense peaks were still observable. It was estimated from observing the background noise and signal intensities for the 110–135 fmol injection that detection limits of approximately 30–50 fmol in the presence of citrate buffer would be obtained (S/N = 3).All injections were performed by the hydrodynamic technique by applying the necessary pressure to the inlet vial. The TOF mass spectra for all of these analytes displayed the protonated molecular ion along with the background citrate clusters (Fig. 4). Attomole, or more commonly, low femtomole (i.e., < 10 fmol) detection limits are about the best that have been reported for CE–MS; however, most of these reported separations were performed with volatile buffer systems that permit the use of a microspray source, which leads to increased ionization efficiency.In the presence of a liquid sheath, depending on the operating conditions, the signal may decrease 10–100-fold. CE displays excellent mass detection limits; however, its poor concentration detection limits are a result of the very small injection volume required when using small CE columns (i.e., < 10 nl for 50 mm id capillaries). With proper preconcentration techniques, such as membrane preconcentration, transient isotachophoresis (ITP), stacking or field amplified injection, concentration detection limits can be improved 100–1000-fold.Selected-ion traces for CE–TOFMS analysis of drugs of abuse were obtained by integrating ion intensities within narrow m/z ranges that corresponded to the protonated molecular ions of the analytes (i.e., m/z AH+ 136, MAH+ 150, PH+ 237, TH+ 265, CH+ 304, HH+ 370). Spectra were recorded at the rate of 5 Hz and data collection usually started approximately 30 s prior to the elution of analytes.This procedure allowed for a significant reduction in the amount of stored data and in the time required for data work-up. It is very important that data be acquired at a high enough speed to preserve the separation efficiency. A minimum of 10–15 data points should be acquired across the peak to allow precise quantification. Since each data point corresponds to a full mass spectrum, it is this instant where the ability of the mass spectrometer to produce the spectrum in a short enough time is seriously challenged.Seized samples could easily be analyzed by continuous infusion using the microelectrospray, since the main objective in such analyses is to identify the drug present in the confiscated material. The analysis time depended only on the time needed to dissolve and filter the sample, and it was very fast, taking about 5 min. Fig. 5 shows TOF mass spectra for a standard cocaine sample and for three seized samples: cocaine, heroin and a mixture of amphetamine and methamphetamine.The spectra of the seized samples displayed mainly the protonated molecular ion of the drug. Weak signals corresponding to polymeric contaminants were also present in each of the spectra. Other components, eventually present, did not give a measurable signal in the positive ion electrospray mode. The seized sample containing a mixture of amphetamine and methamphetamine was also analyzed using CE–TOFMS (Fig. 6) under conditions identical with those used for the previously described separations.The total analysis time, including sample preparation, was 15 min. The configuration of the commercial CE system did not permit the use of short capillaries, and a minimum length of 85 cm was necessary to achieve the interfacing through the liquid Fig. 2 TOFMS background spectra at two nozzle–skimmer voltage differences, DV = (A) 22 and (B) 52 V.Conditions as in Fig. 1. Asterisks indicate citrate clusters. Fig. 3 CE–TOFMS electropherograms of drugs of abuse. Conditions: 85 cm 3 50 mm id uncoated fused silica capillary, 25 mm citrate buffer (pH 3), 30 kV, 10 mbar, 7.8 mA; 1 ml min21 CH3OH–H2O–CH3COOH (80 + 20 + 0.1 v/v) liquid sheath; ESI (3000 V), 90 °C; MS data acquisition at 5000 Hz, 1000 spectra averaged, 5 data points s21. A, 30 mbar 3 0.2 min injection ( ~ 650–810 fmol); B, 10 mbar 3 0.1 min injection ( ~ 110–135 fmol).Peak identifications: 1, amphetamine; 2, methamphetamine; 3, procaine; 4, tetracaine; 5, cocaine; 6, impurity; 7, heroin. Analyst, July 1998, Vol. 123 1451sheath source. This resulted in relatively long migration times. Unfortunately, much shorter capillaries would be necessary to conduct separations in less than about 2 min. For small molecules, ESI produces primarily the protonated molecular ion (in the positive ion mode), and the resultant ESI mass spectra are very simple and easy to interpret.The identification of the main components of an unknown sample can be achieved simply by direct infusion without prior separation of the individual components. On the other hand, for the investigation of complex mixtures, CE is an ideal technique to use prior to MS, since efficient separations can be achieved in a short time without extensive efforts at optimization. The US Environmental Protection Agency (EPA), through its Office of Research and Development (ORD), funded this research under an assistance agreement (Agreement No.CR Fig. 4 TOF mass spectra of drugs of abuse (standard compounds) obtained from a CE run. Conditions as in Fig. 3. The labeled ions are the protonated molecular ions (MH+) of the specific drug. 1452 Analyst, July 1998, Vol. 123824316-01-0) to Brigham Young University. This paper has been subjected to the EPA’s peer review and has been approved as an EPA publication. Mention of trade names or commercial products does not constitute endorsement or recommendation by EPA for use.References 1 Jorgenson, J. W., and Lukacs, K. D., Anal. Chem., 1981, 53, 1298. 2 Jorgenson, J. W., and Lukacs, K. D., Science, 1983, 222, 266. 3 Cai, J., and Henion, J., J. Chromatogr. A, 1995, 703, 667. 4 Yamashita, M., and Fenn, J. B., J. Phys. Chem., 1984, 88, 4451. 5 Yamashita, M., and Fenn, J. B., J. Phys. Chem., 1984, 88, 4671. 6 Whitehouse, C.M., Dreyer, R. N., Yamashita, M., and Fenn, J. B., Anal. Chem., 1985, 57, 675. 7 Price, D., and Milnes, G. J., Int. J. Mass Spectrom. Ion Processes, 1990, 99, 1 8 Sin, C. H., Lee, E. D., and Lee, M. L., Anal. Chem., 1991, 63, 2897. 9 Monnig, C.A., and Jorgenson, J. W., Anal. Chem., 1991, 63, 802. 10 Cai, J., and El Rassi, Z., J. Liq. Chromatogr., 1992, 15, 1193. 11 Kleibohmer, W., Cammann, K., Robert, J., and Mussenbrock, E., J. Chromatogr., 1993, 638, 349. 12 Lurie, I.S., Sperling, A. R., and Meyers, R. P., J. Forensic Sci., 1994, 39, 74. 13 Lurie, I. S., and Carr, S. M., J. Liq. Chromatogr., 1986, 9, 2485. 14 Lurie, I. S., Moore, J. M., Cooper, D. A., and Kram, T. C., J. Chromatogr., 1987, 405, 273. 15 Clauwaert, K. M., Van Bocxlaer, J. F., Lambert, W. E., and De Leenheer, A. P., Anal. Chem., 1996, 68, 3021. 16 Makino, Y., Ohta, S., and Hirobe, M., Forensic Sci. Int., 1996, 78, 65. 17 Virag, L., Mets, B., and Jamdar, S., J. Chromatogr. B, 1996, 681, 263. 18 Nishikawa, M., Nakajima, K., Tatsuno, M., Kasuya, F., Igarashi, K., Fukui, M., and Tsuchihashi, H., Forensic. Sci. Int., 1994, 66, 149. 19 Lurie, I. S., J. Chromatogr. A, 1997, 780, 265. 20 Tagliaro, F., and Smith, F. P., Trends Anal. Chem., 1996, 15, 513. 21 Salomon, K., Burgi, D. S., Helmer, J. C., J. Chromatogr., 1991, 549, 375. 22 Wainright, A., J. Microcol. Sep., 1990, 2, 166. 23 Weinberger, R., Lurie, I. S., Anal. Chem., 1991, 63, 823. 24 Trenerry, V. C., Wells, R.J., and Robertson, J., J. Chromatogr. Sci., 1994, 32, 1. 25 Trenerry, V. C., Robertson, J., and Wells, R. J., Electrophoresis, 1984, 15, 103. 26 Lurie, I. S., and Wan, T. S. M., J. Chromatogr., 1993, 612, 172. 27 Altria, K. D., J. Chromatogr., 1993, 646, 245. 28 Páez, X., Rada, P., Tucci, S., Rodríguez, N., and Hernández, L., J. Chromatogr. A, 1996, 735, 263. 29 Naylor, S., Benson, L. M., and Tomlinson, A. J., J. Chromatogr. A, 1996, 735, 415. 30 Tagliaro, F., Turrina, S., and Smith, F.P., Forensic Sci. Int., 1996, 77, 211. Fig. 5 TOF mass spectra of drugs of abuse (seized samples) from direct infusion. Conditions: continuous infusion (0.3 ml min21); ESI (1850 V), 80 °C; MS data acquisition at 5000 Hz, 5,000 spectra averaged, 1 spectrum s21. A, Cocaine standard [22 mm in CH3OH–CH3COOH (100 + 0.1 v/v)]; B, seized cocaine (0.04 mg ml21 in CH3OH); C, seized heroin (conditions as in B); D, seized amphetamine and methamphetamine (conditions as in B). The labeled ions are protonated molecular ions (MH+). The flight time range corresponds to 0–1545 m/z. Fig. 6 CE–TOFMS electropherogram of a seized sample containing amphetamine and methamphetamine. Conditions: 85 cm 3 50 mm id uncoated fused silica capillary, 25 mm citrate buffer (pH 3), 30 kV, 10 mbar, 7.8 mA, 30 mbar 3 0.2 min injection (0.04 mg ml21 in CH3OH); 1 ml min21 CH3OH–H2O–CH3COOH (80 + 20 + 0.1 v/v) liquid sheath; ESI (3000 V), 90 °C; MS data acquisition at 5000 Hz, 1,000 spectra averaged, 5 data points s21. Peak identifications: 1, amphetamine; 2, methamphetamine. Analyst, July 1998, Vol. 123 145331 Tagliaro, F., Smith, F. P., Turrina, S., Equisetto, V., and Marigo, M., J. Chromatogr. A, 1996, 735, 227. 32 Lurie, I. S., Am. Lab., 1996, January, 26. 33 Curcuruto, O., Zaramella, A., Hamdan, M., Turrina, S., Tagliaro, F., Rapid Commun. Mass Spectrom., 1995, 9, 1487. 34 Lazar, I. M., Lee, E. D., Rockwood, A. L., Fabbi, J. C., Lee, H. G., and Lee, M. L., Anal. Chem., 1997, 69, 3205. 35 Lazar, I. M., Lee, E. D., Rockwood, A. L., and Lee, M. L., J. Chromatogr. A, 1997, 791, 269. Paper 7/08903A Received December 9, 1997 Accepted Febraury 9, 1989 1454 Analyst, July 1998, Vol. 123

ISSN:0003-2654    

DOI:10.1039/a708903a

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Use of affinity capillary electrophoresis for the study of protein and drug interactions Jinping Liu*a, Sadia Abidb, Mark E. Haila Mike S. Leea, Jon Hangelandc and Nada Zeind a Analytical Research and Development, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA b Analytical Research and Development, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT 06492, USA c Discovery Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA d Oncology Department, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA Protein–drug interactions were studied using affinity capillary electrophoresis (ACE).The initial study was performed using a model system, fibronectin–heparin interaction. Two distinct binding constants, 21 and 641 nm, were derived from the Scatchard plots. The results are consistent with reported data obtained using other analytical techniques.The ACE binding assay was applied for studying molecular interactions between kedarcidin chromophore and apoprotein. Conditions with an organic solvent as buffer component were examined to establish a suitable binding assay. It appears that the electrophoretic behavior of the protein shows little distortion in the presence of either dimethyl sulfoxide (up to 10%) or acetonitrile (ACN) (up to 30%). The binding assay was initially conducted in aqueous buffer phase. The saturation concentration of chomophore was found to be around 15 mm.A linear Scatchard plot was derived from binding data with a correlation coefficient of 0.94. The binding constant was determined as Kd = 5.6 mm. The effects of organic solvent content ranging from 0 to 30% ACN on the constant were examined. The binding constants were determined as Kd = 11, 12.5 and 16.2 mm for 5, 10 and 30% ACN, respectively. It appeared that the binding affinity between kedarcidin chromophore and apoprotein is reduced as the organic solvent content in the aqueous phase is increased.Keywords: Affinity capillary electrophoresis; protein–drug interactions; kedarcidin; apoprotein The interaction of proteins with specific ligands such as drugs and toxins is among the important aspects of biological studies in drug discovery and pharmaceutical development processes. The characterization of the binding phenomenon and the determination of binding parameters such as dissociation constant and stoichiometry are essential for the evalution of bioaffinity and the understanding of receptor–ligand interactions.Traditional biological binding assays generally require the measruement of bound or free species in an equilibrium system, or radioactively labeled ligands and relatively large amounts of sample, which is not always possible owing to insufficient labeling and sensitivity, NMR, the transition temperature in differential scanning calorimetry and chromatographic techniques, have been utilized to study molecular interactions. The recent advances in capillary electrophoresis (CE) have generated tremendous interest in the area of biological research.As an alternative approach in the study of molecular interactions, affinity capillary electrophoresis (ACE) has been introduced for the analysis of receptor–ligand interactions and the determination of binding constants.1–12 Although several CE-based approaches have been presented for binding studies such as the quantification of bound and free ligands after reaching an equilibrium state,4,6 CE–fronal analysis for drug– protein interactions10 and other similar approaches,5 with the most recent studies dealing with receptor–ligand interactions such as protein–drug ligand, protein–sugar, DNA–peptide, peptide–drug and antibody–antigen, were accomplished using an ACE approach.1–3 The ACE assay as an alternative screening technique for identifying a tight-binding ligand for a receptor in a small peptide library has also been described.3 Most recently, an ACE-based binding assay has been combined with mass spectrometry (MS) for screening combinatorial libraries for drug discovery research.12 The basic principle involves the measurement of an altered electrophoretic mobility (or migration shift) of the complexed species as compared with the free ligand.This novel affinity binding assay also inherits various advantages of CE such as high resolving power, high speed, sensitive detection and ease of automation.In addition, it requires no radiolabeling and allows the simultaneous analysis of each individual component within a sample mixture. Scatchard analysis generally can be performed by measuring the migration shift resulting from the change in charge status before and after the formation of the complex to derive the binding constant and other related interaction parameters. Our recent efforts in studying biomolecular interactions, especially dealing with protein–drug binding, led us to utilize the ACE binding assay for the characterization of binding profiles in our drug discovery programs.One of the interesting studies involves the interaction between a chromoprotein, kedarcidin, and its chromophore. Among the enediyne class of antitumor agents, kedarcidin has been identified as a highly potent and very active chromoprotein antitumor antibiotic in both murine tumor models and in human tumor xenografts.13 Much of the discovery and development efforts have been conducted in recent years.The structural characterization and elucidation of the molecule and related compounds and their biological relevance have been extensively studied.14–17 The chromophore is chemically labile when free in solution. The antitumor activity of the chromoprotein is due primarily to the chromophore. The apoprotein’s function is to stabilize the chromophore and appear to direct the delivery of the chromophore to the DNA of intact cells.Detailed studies on the molecular interaction between chromophore and apoprotein would provide useful information in understanding the mechanism of action. In this work, we established a CE-based method for the separation of the apoprotein and other minor species and examined the feasibility of using ACE for studying the interaction between kedarcidin chromophore and apoprotein under various conditions, including organic solvents as media additives in the background electrolyte.We demonstrate the Analyst, July 1998, Vol. 123 (1455–1459) 1455utility of a rapid binding assay using the highly sensitive ACE approach for the determiantion of thermodynamic association constants without radiolabeling. Experimental Apparatus All ACE experiments were carried out with a Beckman (Fullerton, CA, USA) P/ACE 5010 CE equipped with either a UV or a diode-array detector. For fibronectin–heparin interaction experiments, a polymer-coated fused-silica capillary obtained from Beckman was used (360 mm od, 50 mm id, total length 27 cm).For other experiments, a sulfonic acid-coated fused-silica capillary (360 mm od, 75 mm id) with a total length of 34–56.7 cm (27–50 cm effective separation length) obtained from Scientific Resources (Eatontown, NJ, USA) were used for the separation. Pressure injection was used for sample introduction with a duration of 5–10 s.A high voltage of 29 kV set at reverse polarity was applied for electrophoretic runs. The protein was monitored at a wavelength of either 280 or 214 nm. Data were collected and analyzed with System Gold software. Materials and methods The protein sample fibronectin was purchased from Boehringer Mannheim (Indianapolis, IN, USA) and a low moelcular mass heparin (Mr 6000) was obtained from Sigma (St. Louis, MO, USA). The kedarcidin apoprotein and chromophore sample were obtained in-house.The ACE binding assays were performed based on previously described methods.1–3,7,8 Generally, an electrophoretic buffer was selected for a specific binding system to establish the separation of the protein from other sample species. A ligand molecule was subsequently added to the running buffer at various concentrations. Upon introduction of the protein sample, affinity interaction between the protein and ligand may take place during electrophoresis. In these studies, a buffer consisting of 20 mm Tricine (pH 8.0) was used for fibronectin– heparin binding.For kedarcidin chromophore and apoprotein interaction, the running buffer consisted of citrate–MES (pH 6.0) with appropriate concentrations of organic solvents, either dimethyl sulfoxide (DMSO) or acetonitrile (ACN). The concentration of the original kedarcidin apoprotein sample was 0.85–1.0 mg ml21 in 50 mm TRIS–HCl buffer (pH 7.5). The kedarcidin chromophore solution was diluted to 2.91 mm.Affinity buffers were freshly prepared by adding the appropriate concentration of kedarcidin chromophore, which was stored in a refrigerator prior to injection to minimize room temperature exposure. An aliquot of the protein sample was mixed with a negatively charged small molecule (salicylic acid) as internal reference for injection. Affinity interactions between protein and ligand molecules were examined by measuring the migration time of the internal reference and protein as a function of ligand concentrations.The analysis was duplicated and the average was taken for further calculation. Several methods have been proposed for the determination of binding constants.1–6 to simplify the process, Scatchard analysis was performed based on a slightly modified dimensionless number, the shifting coefficient R [R = (M0 2 M)/M0],18 where M0 is the mobility of free protein and M is the observed protein mobility during interaction. In a CE format, the determination of mobility can be converted to the measurement of migration time.A plot of R/[ligand concentration] versus R should yield a straight line with a slope of 21/Kd for the determination of the binding constant. Results and discussion To validate the ACE-based binding assay for protein interaction with drug molecules, we initially evaluated a model system, protein–glycosaminoglycan (GAG) binding, studied with affinity gel retardation electrophoresis,18 a technique similar to the ACE approach. The initial protein selected was fibronectin, a glycoprotein of two similar polypeptide chains connected by two disulfide bridges.It has been reported that fibronectin binds heparin and heparin sulfate with high affinity.18 Analysis of the fibronectin–heparin interaction using ACE is shown in Fig. 1. The interaction was reported to be related to the structural features of GAG such as size, charge density and disaccharide unit. Therefore, a low molecular mass heparin (Mr 6000) was chosen in this case.Instead of labeling heparin as described in the literature, the molecule was mixed with the background electrolyte at various concentrations. The addition of heparin to the buffer has virtually no effect on the UV background owing to the lack of a chromophore in the molecule. The protein peak was monitored at a wavelength of 280 nm and the migration shift was determined as a function of heparin concentration in the range 0.05–100 mm.The binding profile clearly indicated that the increased concentration of heparin resulted in a significant change in the protein mobility, similar to the retardation in gel electrophoresis, implying a high affinity interaction between heparin and fibronectin. Graphical analysis of the profile is shown in Fig. 2, resulting in two distinct linear plots. This phenomenon suggests that more than one binding site is associated with the interaction. Based on a similar method in affinity gel retardation studies, two binding constants were derived from the Scatchard plots as 21 and 641 nm.The results obtained from the ACE approach are consistent with the reported data obtained by the conventional affinity gel electrophoretic technique.18 It has been reported that kedarcidin is composed of a single polypeptide chain consisting of 114 amino acid residues and a cytotoxic, highly labile, non-protein chromophore.14 The noncovalently bound apoprotein and chromophore form a 1 : 1 complex and are separable from each other.The molecular interaction between the apoprotein and the chomophore appears to be crucial for maintaining the antitumor activity, which is due primarily to the chromophore, whereas the apoprotein is believed to play a role in the stabilization and transport of the chromophore. The proposed structure of kedarcidin chromo- Fig. 1 Binding profiles of fibronectin–heparin interaction using ACE. Column, Beckman coated fused-silica capillary (360 mm od 3 50 mm id), 27 cm in length (20.3 cm affective separation length); buffer, 20 mm Tricine (pH 8.0); pressure injection, 20 s duration; detection wavelength, 280 nm. 1456 Analyst, July 1998, Vol. 123O H3CO OCH3 OH NH O H3C CH3 O N Cl O O O O CH3 N(CH3)2 OH O O HO HO H3C H3C O Chromophore ASAAVSVSPA TGLADGATVT VSASGFATST SATALQCAIL ADGRGACNVA EFHDFSLSGG EGTTSVVVRR SFTGYVMPDG PEVGAVDCDT APGGCEIVVG GNTEEYENAA ISFE 114 Apoprotein phore and the primary sequence structure of apoprotein are shown in Fig. 3. The apoprotein is water soluble but the chromophore is only solvent extractable and has a very limited solubility in the aqueous phase. The common aqueous buffer used in most CE applciations appears to be problematic for further ACE studies dealing with the chromophore and apoprotein interaction. The initial focus of these studies was therefore to establish appropriate conditions suitable for the apoprotein separation and the subsequent determination of the binding constant, especially in the presence of organic solvents.With the use of a solfonic acid-coated capillary column and a buffer consisting of citrate–MES (pH 6.0), a major protein peak and two minor peaks were observed at a wavelength of 280 nm. This appears to be consistent with bands observed by SDS-PAGE. A characteristic UV spectrum was acquired for the major peak with the use of a diode-array detector. Since UV spectroscopy is generally considered not to be an ideal method for protein characterization, the apoprotein was further confirmed using electrospray ionization (ESI) MS.The CE separation and a deconvoluted (zero charge state) mass spectrum are shown in Fig. 4. The molecular mass determined (11 181) is consistent with the calculation from the sequence structure. It has been reported that the hydrophobic chromophore dissolves only in DMSO-containing buffers, so a 10% DMSO buffer solution was initially used in these studies.It was observed that the background noise increases upon addition of 5 mm of kedarcidin chromophore to the running buffer. As the concentration of chromophore reached 50 mm, the electropherogram became unacceptable for further binding studies owing to the appearance of multiple spike peaks. This phenomenon is most likely due to light scattering effects caused by chromophore particles that are perhaps not entirely dissolved in the buffer solution.Increasing the DMSO concentration in the buffer seems to reduce the spiking but causes distortion of the protein peaks, perhaps as a result of the conformational change caused by the strong organic solvent. An alternative approach is to use other moderate organic solvents. In these studies, ACN at concentrations ranging from 5 to 50% in buffer was evaluated. The electropherograms appear to be acceptable with ACN concentrations < 35%. Although the poor solubility of the kedarcidin chromophore in the aqueous phase appears to be the key factor that prevents the use of other conventional binding assays, this problem may not be that critical if a CE-based assay is utilized.The unique capability of handling small sample volumes and the high sensitivity detection offered by CE may compromise some of the experimental difficulties in other traditional methods. Based on the established experimental conditions with or without ACN as organic additive, binding interaction between kedarcidin chromophore and apoprotein was conducted.The initial ACE binding assay was performed using an aqueous buffer without an organic solvent. A representative binding profile showing the migration shift of the protein peak as a function of ligand concentration is displayed in Fig. 5. A negatively charged small molecule, salicylic acid (SA), was used as an internal reference for the measurement of relative mobility of the protein.A series of buffers containing kedarcidin chromophore at concentrations ranging from 0 to 20 mm were used for the ACE binding experiments. As the chromophore concentration in buffer solution increases, the protein peak gradually shifts away from the reference peak, indicating that a binding interaction between the chromophore and apoprotein takes place. As displayed in Fig. 5, the internal reference has a migration time that is independent of the concentration of the chromophore. Under the above ACE conditions with a buffer of pH 6.0, Fig. 2 Scatchard plot of bibronectin–heparin interaction derived from binding profile data using ACE. R is defined in the Experimental section. Fig. 3 Proposed structure of kedarcidin chromophore and primary structure of apoprotein. Fig. 4 Separation of kedarcidin apoprotein (KA) sample using CE and the deconvoluted (zero charge state) ESI mss spectrum of KA obtained via LC– ESI–MS (inset). Column, zero EOF sufonic acid-coated fused-silica capillary (360 mm od 3 75 mm id), 56.7 cm in length (50 cm effective separation length); buffer, citrate–MES (pH 6.); pressure injection, 10 s duration; applied voltage, 29 kV with reverse polarity; detection wavelength, 280 nm.Mass spectra measurement of kedarcidin was performed by LC–MS using the negative ion mode. A 50 3 0.5 mm id PLRP-s 4000 A polymeric column was used at a flow rate of 20 ml min21 with a gradient from 20 to 95% B in 20 min (A = H2O–ACN–aq.NH3 (98 + 2 + 0.1), B = H2O–ACN–aq. NH3 (10 + 90 + 0.1). Analyst, July 1998, Vol. 123 1457the ligand that most likely is positively charged has a tendency to migrate towards the cathode inlet which is opposite the detector (anode outlet). When the ligand binds to the protein, the complex has a reduced electrophoretic mobility and hence a slower migration time than the protein alone. The change in protein peak shape and the slight peak split indicate that a complex binding process occurs.This phenomenon is generally related to the affinity interaction between the two moelcules as a result of the complex formation and the on and off binding kinetic process. Attempts to increase the chromophore concentration in a non-organic buffer solution further appear to be problematic owing to its limited solubility in the aqueous phase. The correlation between relative mobility of the protein and chromophore concentration indicates that the saturation is reached at a concentration of around 15 mm.A Scatchard plot was derived from the binding data as shown in Fig. 6. The binding constant was calcualted to be Kd = 5.6 mm with a correlation coefficient of 0.94. This indicates that tight binding between kedarcidin chromophore and apoprotein is expected in the aqueous buffer phase. Since most biological studies related to the complexation of the chromophore and apoprotein were conducted under conditions of using a certain concentration level of organic solvent owing to concerns about the limited solubility of the kedarcidin chromophore in the aqueous phase,16,17 further investigations of the effects of organic solvent content on binding constants were performed.This approach will possibly provide further information for the understanding of the affinity interaction and the mechanism of action. ACN was selected as the additive in the binding buffer for these studies. A separation profile similar to that in aqueous buffer conditions was obtained with 5% ACN in the binding buffer.It was noted that the workable chromophore concentration increased to more than 30 mm as compared with previous studies without organic solvent. Further increases in chromophore concentration appear to cause baseline noise problems owing to its limited solubility. A correlation plot of relative mobility of the protein versus chromophore concentration was obtained and a saturation point seems to be reached at around 30 mm.The Scatchard plot was derived from the binding data as shown in Fig. 7(A). The binding constant was calculated to be Kd = 11 mm with a correlation coefficient of 0.90. Similar experiments were also conducted with further increased organic content. With 10% ACN in the binding buffer, the correlation between the relative mobility of the protein and chromophore concentration indicated a slightly increased chromophore concentration range of up to 40 mm.This is simply due to the increased solubility of chromophore in the aqueous buffer solution. A linear Scatchard plot was derived from the binding data as shown in Fig. 7(B). The binding constant was calculated to be Kd = 12.5 mm with a correlation coefficient of 0.86. The ACE binding assay could also be accomplished as the organic solvent content increased to 30% ACN in the buffer. There appears to be little indication of peak distortion related to protein conformational change at this level of organic solvent concentration.The workable chromophore concentration range has been extended to around 90 mm. From the linear Scatchard Fig. 5 ACE profiles for the interaction of kedarcidin chromophore and apoprotein in aqueous buffer phase without organic additive. IR, internal reference; KA, kedarcidin apoprotein. Concentration range of kedarcidin chromophore in the buffer, 0–20 mm; column, zero EOF sulfonic acidcoated fused-silica capillary (360 mm od 3 75 mm id), 34 cm in length (27.3 cm effective separation length); buffer, citrate–MES (pH 6.0); pressure injection, 2 s duration; applied votlage, 29 kV with reverse polarity detection wavelength, 280 nm.Fig. 6 Scatchard plot of kedarcidin chromophore and apoprotein binding obtained using ACE. Data derived from binding profiles under conditions of aqueous buffer without organic additive. R is defined in the Experimental section. Fig. 7 Scatchard plots of kedarcidin chromophore and apoprotein binding obtained using ACE under conditions with various concentrations of ACN additive (R is defined in the Experimental section).Capillary column, zero EOF sulfonic acid-coated fused-silica capillary (360 mm od 3 75 mm id), 46.9 cm in length (40.2 cm effective separation length); buffer, citrate– MES–5% ACN (pH 6.0); pressure injection, 5 s duration; applied voltage, 30 kV with reverse polarity; detection wavelength, 280 nm. A, 5% ACN, concentration range of kedarcidin chromophore in the buffer 0–32 mm; B, 10% ACN, chromophore concentration range 0–40 mm; and C, 30% ACN, chromophore concentration range 0–90 mm. 1458 Analyst, July 1998, Vol. 123plot shown in Fig. 7(C) (correlation coefficient 0.981) the binding constant was determined to be Kd = 16.2 mm. The effects of ACN concentration ranging from 0 to 30% on the binding constant were investigated and the results are summarized in Table 1. It appears that the dissociation constant (Kd) increases, or conversely there is a decrease in binding affinity between the chromophore and apoprotein, as the organic solvent content in the buffer is increased.The higher Kd values observed in the presence of organic solvent are perhaps related to several factors. Any protein conformational change could affect the specific binding pocket where the chromophore is known to have strong interactions. The addition of an organic solvent will also possibly reduce the hydrogen bond interactions between the protein and the chromophore.We generally observed a lower association constant (higher Kd) in this binding system involving the use of organic solvent, which appears to be consistent with other biological studies. The binding affinity between the chromophore and apoprotein seems not to be compromised by the increased solubility of the chromophore. The addition of an organic solvent may actually cause protein conformational changes or partial denaturation, thus reduced binding.A higher percentage of organic content ( > 35%) even resulted in precipitation of buffer salts. Although the binding assay under such unconventional conditions can be developed, concerns for protein denaturation and other experimental difficulties also arise. These studies may provide a rapid approach for the determination of binding constants under normal and extreme binding conditions and provide some analytical insight into understanding the mechanism of action in the complexation process between an apoprotein and its natural ligand.References 1 Chu, Y.-H., Avila, L. Z., Biebuyck, H. A., and Whitesides, G. M., J. Med. Chem., 1992, 35, 2915. 2 Chu, Y.-H., Avila, L. Z., Biebuyck, H. A., and Whitesides, G. M., J. Org. Chem., 1993, 58, 648. 3 Avila, L. Z., Chu, Y.-H., Blossey, E. C., and Whitesides, G. M., J. Med. Chem., 1993, 36, 126. 4 Heegaard, N. H. H., and Robey, F. A., Anal. Chem., 1992, 64, 2479. 5 Kraak, J. C., Busch, S., and Poppe, H., J. Chromatogr., 1992, 608, 257. 6 Heegaard, N. H. H., and Robey, F. A., J. Liq. Chromatogr., 1993, 16, 1923. 7 Liu, J., Vol, K. J., Lee, M. S., Kerns, E. H., and Rosenberg, I. E., J. Chromatogr., 1994, 680, 395. 8 Liu, J., Volk, K. J., Lee, M. S., Pucci, M. J., and Handwerger, S., Anal. Chem., 1994, 66, 2412. 9 Mammen, M., Gomez, F. A., and Whitesides, G. M., Anal. Chem., 1995, 67, 3526. 10 Ohara, T., Shibukawa, A., and Nakagawa, T., Anal. Chem., 1995, 67, 3520. 11 Xian, J., Harrington, M. G., and Davidson, E. H., Proc. Natl. Acad. Sci. USA, 1996, 93, 86. 12 Chu, Y.-H., Dunayevskiy, Y. M., Kirby, D. P., Vouros, P., and Karger, B. L., J. Am. Chem. Soc., 1996, 118, 7827. 13 Lee, J. E., Schroeder, D. R., Hofstead, S. J., Golik, J., Colson, K. L., Huang, S., Klohr, S. E., Doyle, T. W., and Matson, J. A., J. Am. Chem. Soc., 1992, 114, 7946. 14 Lee, J. E., Schroeder, D. R., Langley, D. R., Colson, K. L., Huang, S., Klohr, S. E., Lee, M. S., Golik, J., Hofstead, S. J., Doyle, T. W., and Matson, J. A., J. Am. Chem. Soc., 1993, 115, 8432. 15 Lee, M. S., Klohr, S. E., Kerns, E. H., Volk, K. J., Lee, J. E., Schroeder, D. R., and Rosenberg, I. E., Biol. Mass Spectrom., 1996, 31, 1253. 16 Zein, N., Colson, K. L., Leet, J. E., Schroeder, D. R., Solomon, W., Doyle, T. W., and Casazza, A. M., Proc. Natl. Acad. Sci. USA, 1993, 90, 2822. 17 Zein, N., Casazza, A. M., Doyle, T. W., Leet, J. E., Schroeder, D. R., Solomon, W., and Nadler, S. G., Proc. Natl. Acad. Sci. USA, 1993, 90, 8009. 18 Lee, M. K., and Lander, A. D., Proc. Natl. Acad. Sci. USA, 1991, 88, 2768. Paper 8/00285A Received January 5, 1998 Accepted April 4, 1998 Table 1 Effects of ACN concentration on binding constants for the interaction between kedarcidin chromophore and apoprotein Concentration of ACN (%) Dissociation constant, Kd/mm 0 5.6 5 11.0 10 12.5 30 16.2 Analyst, July 1998, Vol. 123 1459

ISSN:0003-2654    

DOI:10.1039/a800285a

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Quantitative assay for epinephrine in dental anesthetic solutions by capillary electrophoresis Philip Britz-Mckibbina, Andrea R. Kranacka, Alison Paprica†b and David D. Y. Chen*a a Department of Chemistry, University of British Columbia, Vancouver, BC, V6T 1Z1, Canada. E-mail: chen@chem.ubc.ca b Astra Pharma Inc., 1004 Middlegate Road, Mississauga, Ontario, L4Y 1Z1, Canada A simple and robust method for the separation and quantification of epinephrine in dental anesthetic solutions was developed.The method allows the direct injection of high salt solutions without sample pre-treatment. Large sample plugs (5.7% of the total capillary length) are used for epinephrine determination by selective analyte focusing in capillary electrophoresis. The concentration detection limit for epinephrine is about 5.0 3 1027 M (90 ng ml21) with a commercial UV detector. The separation protocol was validated in terms of its precision, linearity, accuracy and specificity.Keywords: Capillary electrophoresis; epinephrine; dental anesthetic solutions; analyte focusing Local anesthetic solutions have recently gained widespread acceptance among emergency healthcare personnel.1,2 The ease of use, minimal toxicity and reduced costs are commonly cited advantages of local anesthetic solutions over alternative reagents. Epinephrine, a natural catecholamine, is often a minor component in commonly administered local anesthetic solutions. It primarily acts as a vasoconstrictor to confine the anesthetic to a limited area.Air oxidation is a major problem for epinephrine in pharmaceutical products.3 The addition of antioxidants, such as sodium metabisulfite, to anesthetic formulations serves to minimize epinephrine oxidation. However, regular testing of commercially available anesthetic solutions is essential to ensure the presence of the minimum required epinephrine concentration stated on the label. Previous techniques for the characterization and quantification of epinephrine in anesthetic solutions have relied primarily upon cation exchange4 and reversed phase HPLC methods.3,5 There are only a few reported capillary electrophoretic (CE) methodologies for the determination of epinephrine in formulated products.Peterson and Trowbridge6 developed a quantitative CE assay for the determination of the enantiomeric ratio of epinephrine in a pharmaceutical formulation. However, to our knowledge there are no CE methods for the determination of epinephrine in anesthetic solutions.CE is well suited for such analyses because of its small sample volume requirements, low overall costs and minimal environmental impact. The purpose of this investigation was to develop a quantitative assay for epinephrine by direct injection of dental anesthetic solutions without sample pre-treatment. The method was validated by determining the accuracy, precision, linearity, specificity and ruggedness of the assay.Selective focusing of epinephrine allows for superior concentration detection limits with a commercial UV detector. Experimental Buffer preparation and chemicals The aqueous background electrolyte used for CE separation consisted of 160 mm borate (Sigma Chemical, St. Louis, MO, USA) and 1 mm ethylenediaminetetraacetic acid (EDTA) (BDH Chemicals, Toronto, Ontario, Canada). The pH of the background electrolyte was adjusted to 10.1 by using 0.1 m NaOH (BDH Chemicals).Standard epinephrine [epinephrine (2)-hydrogentartrate, Sigma Chemical) solutions were made by appropriate dilution of a stock standard solution of 50 mg ml21 epinephrine free base. The standard solutions also contained 1 mm EDTA, 3.0 mm sodium metabisulfite (BDH Chemicals) and 155 mm NaCl (BDH Chemicals). Topical dental anesthetic solutions, accuracy and recovery solutions and adenochrome were provided by Astra Pharma (Missisauga, Ontario, Canada). Dental anesthetic solutions consisted of a mixture of lidocaine (local anesthetic: 5–20 mg ml21) and various additives and impurities: sodium metabisulfite, sodium chloride, methylparaben, 2,6-xylidine, o-toluidine and hydroxybenzoic acid.It should be noted that the concentration of epinephrine in dental anesthetic solutions (5–10 mg ml21) is much lower than that of the other components (1–40 mg ml21). Capillary electrophoresis system Separations were performed on a P/ACE 5500 automated CE system (Beckman Instruments, Mississauga, Ontario, Canada).Fused silica capillaries (Polymicro Technologies, Phoenix, AZ, USA) of id 75 mm, od 375 mm and lengths 57 cm were used. New capillaries were first rinsed with 1.0 m NaOH for 5 min under high pressure (20 psi), followed by rinsing with the separation electrolyte for 10 min. The capillary was then left to equilibrate overnight in the separation electrolyte prior to use. Each separation was preceded by a 1.5 min high pressure rinse with 0.1 m NaOH, followed by a 4 min rinse with the separation electrolyte.The samples were introduced using a 25 s low pressure injection (0.5 psi) and the separation was carried out for 15 min at 15 kV and 25 °C. Absorbance was monitored at 280 nm and data were collected and processed using Beckman P/ACE station version 1.0 software. Results and discussion Optimization of separation Borate was selected as the background electrolyte because it selectively complexes with analytes having vicinal dihydroxyl groups.Epinephrine, a catecholamine, should be the only analyte in the anesthetic solution capable of complexing with borate. The separation of epinephrine from the neutral solutes was achieved using 160 mm borate at pH 10.1. It was observed that the addition of 1 mm EDTA to the run buffer resulted in better reproducibility and sharper peaks in epinephrine determination. Fig. 1 depicts two electropherograms obtained from commercially available multi-use and single-use (xylocaine) dental anesthetic solutions.The anesthetic solutions were directly injected into the capillary with no sample pre-treatment. Epinephrine is separated from the excess of neutral components (lidocaine, methylparaben, etc.) which elute with the electroosmotic flow. Excellent concentration sensitivity for epinephrine was observed owing to the large sample volumes injected. † Present address: Genpharm Inc., 85 Advance Road, Etobicoke, ON, M8Z 2S9, Canada.Analyst, July 1998, Vol. 123 (1461–1463) 1461As depicted in Fig. 1, a very high column efficiency (N Å 180 000) was obtained even though a relatively large sample plug (5.7% of the total capillary length or 150 nl) was used. Normally the separation in CE is severely compromised when the plug length is greater than 1% of the capillary (typically less than 10 nl) because of band broadening.7 The sample matrix plays a vital role in CE separation performance.The anesthetic solutions were observed to be relatively acidic with a pH ranging from 3 to 3.5. A possible explanation of epinephrine focusing in this system is the development of a dynamic pH gradient that occurs between the injected acidic sample zone and background basic buffer zone. Epinephrine is a zwitterion that possesses opposing mobilities under acidic (positively charged) and basic (negatively charged) conditions. Hence epinephrine would migrate rapidly to the front edge of the sample plug with a positive electrophoretic mobility until it came into contact with the background electrolyte where it would migrate with a negative electrophoretic mobility. The epinephrine is focused because of the difference in its migration behavior in the low pH sample solution and the high pH background electrolyte.The hydroxide ions in the background electrolyte migrate through the sample plug and raise the pH of the plug, and the focused epinephrine then migrates through the rest of the capillary with a negative electrophoretic mobility.This focusing method does not need ampholytes, and should be generally applicable to all zwiterionic analytes such as amino acids and peptides, as long as the pH difference between the sample solution and the background electrolyte is adjusted according to the isoelectric point of the analyte of interest. Aebersold and Morrison8 utilized a step pH gradient in the focusing of peptides by altering the pH of the sample solution relative to the background buffer.Epinephrine focusing in this case arises naturally owing to the acidic conditions of the anesthetic formulation and the basic background electrolyte. An important aspect of this preconcentration technique is that focusing can still occur with the presence of a high concentration of salt (155 mm NaCl). Most other sample stacking techniques require a plug of low conductivity solution.9213 Oncolumn preconcentration techniques using transient isotachophoresis14216 and sample stacking with acetonitrile–salt mixtures17,18 also permit the use of larger injection plugs to enhance the concentration sensitivity in CE in the presence of high salt in the sample.Method validation Appropriate method validation information concerning the use of a new analytical technique for the analysis of pharmaceuticals is required by regulatory authorities. The precision of the method was evaluated in terms of repeatability (same day) and reproducibility (inter-day and inter-analyst).Six replicate injections of three standard epinephrine solutions of 2, 10 and 32 mg ml21 were used for the repeatability study. The repeatability for epinephrine standards, based on average peak areas, gave an RSD of 2.3%. Six replicate injections of four different anesthetic solutions made on two different days by one analyst (inter-day) and by two analysts (inter-analyst), a total of 48 runs each, were used for the reproducibility study.Both inter-day and inter-analyst reproducibility studies gave an average RSD of 2.0%. The linearity of the detector response versus concentration was tested by constructing a calibration curve from three sets of standard solutions of epinephrine at seven different concentrations, ranging from 2.00 to 32.0 mg ml21. Linear regression of the calibration curve gave a linear equation, with a correlation coefficient (r2) of 0.9994.The limit of detection (LOD, S/N = 3) for epinephrine determination by this CE method was 90 ng ml21 (or 5 3 1027 m). The LOD was estimated based on Knoll’s method.19 The accuracy and recovery of the method were determined by analyzing four different anesthetic solutions fortified with epinephrine at 40, 100 and 160% of the label claim. The average recovery was 95.3% with a r2 0.9987. Fig. 1 Two electropherograms showing the separation of epinephrine (peak 1) from excess solute components by directly injecting (A) multi-use and (B) single-use (xylocaine) dental anesthetic solutions that contain 5 and 10 mg ml21 of epinephrine, respectively.Fig. 2 Comparison of the electropherograms of adenochrome (A) and adenochrome spiked with epinephrine (B) that highlights the specificity of the separation. Peak 1 is epinephrine. 1462 Analyst, July 1998, Vol. 123The method specificity was determined from two lines of evidence: the spiking of pure components in the anesthetic solutions and by comparing the electropherograms of epinephrine and adenochrome (oxidized epinephrine products) to ensure no interference in epinephrine quantification.The spiking of various components in the anesthetic solutions confirmed the separation of epinephrine from the matrix. In addition, the use of a diode array detector confirmed that the absorbance spectrum of the peak in the electropherogram matched that of pure epinephrine. As shown in Fig. 2, the analysis of adenochrome demonstrated that the oxidized products of epinephrine did not interfere with epinephrine quantification.Adenochrome consisted of a mixture of highly charged solutes which migrated after epinephrine. Table 1 summarizes the validation protocol for epinephrine using the CE method. Conclusion A robust method for epinephrine quantification in dental anesthetic solutions by CE was developed. Direct injection of anesthetic solutions into the capillary eliminated the need for tedious sample pre-treatment.Selective epinephrine focusing is believed to arise naturally owing to the development of a dynamic pH gradient between the sample and the background electrolyte. The limit of detection for epinephrine is 5 31027 m when using long injection plugs with a commercial UV absorbance detector. The CE assay for epinephrine was supported by method validation which demonstrated excellent precision, accuracy, linearity and specificity.This work was supported by the Natural Sciences and Engineering Research Council of Canada and Astra Pharma (Canada), Beckman Instruments (Canada) kindly loaned the P/ACE 5500 system. References 1 Larson, T. A., Uden, D. L., and Schilling, C. G., Am. J. Health-Syst. Pharm., 1996, 53, 659. 2 Ernst, A. A., Marvez, E., Nick, T. G., Chin, E., Wood, E., and Gonzaba, W. T., Pediatrics, 1995, 95, 255. 3 Wilson, T. D., and Forde, D. M., Am. J. Hosp. Pharm., 1990, 47, 2504. 4 Fu, C., and Sibley, M.J., J. Pharm. Sci., 1979, 66, 425. 5 Wilson, T. D., J. Chromatogr. , 1990, 498, 402. 6 Peterson, T. E., and Trowbridge, D., J. Chromatogr., 1992, 603, 298. 7 Terabe S., and Otsuka K., T. A., Anal. Chem., 1989, 61, 251. 8 Aebersold, R., and Morrison, H. D., J. Chromatogr., 1990, 516, 79. 9 Quirino, J. P., and Terabe, S., Anal. Chem., 1998, 70, 149. 10 Chien, R., and Burgi, D. S., J. Chromatogr. A, 1991, 559, 141. 11 Burgi, D. S., and Chien, R. L., Anal. Biochem., 1992, 202, 306. 12 Farry, L., Oxspring, D. A., Smyth, W. F., and Marchant, R., Anal. Chim. Acta, 1997, 349, 221. 13 Geldart, S., and Brown, P. R., Am. Lab., 1997, 29(24), 48. 14 Krivankova, L., Vrana, A., Gebauer, P., and Bocek, P., J. Chromatogr. A, 1997, 772, 283. 15 Foret, F., Szoko, E., and Karger, B. L., J. Chromatogr., 1992, 608, 3. 16 Gebauer, P., Thormann, W., and Bocek, P., J. Chromatogr., 1992, 608, 47. 17 Shihabi, Z. K., J. Capillary Electrophoresis, 1995, 6, 267. 18 Shihabi, Z. K., J. Chromatogr. A, 1996, 744, 231. 19 Knoll, J. E., J. Chromatogr. Sci., 1985, 23, 422. Paper 8/00772A Received January 28, 1998 Accepted February 19, 1998 Table 1 Validation protocol for epinephrine determination using CE Parameter Result Repeatability RSD = 2.3% Reproducibility— Inter-day RSD = 1.9% Inter-analyst RSD = 2.0% Linearity* y = 4412x 2 382 (r2 = 0.9994) LOD (S/N = 3) 90 ng ml21 Average recovery 95.3% Accuracy correlation plot† y = 1.017x + 0.2766 (r2 = 0.9987) * y = Peak area; x = epinephrine concentration (mg ml21). † y = Theoretical concentration (mg ml21); x = measured concentration (mg ml21) Analyst, July 1998, Vol. 123 1463

ISSN:0003-2654    

DOI:10.1039/a800772a

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Ion analysis by capillary zone electrophoresis with indirect injection: applications in the nuclear power industry Randall E. Lewisa, Eric S. Ahujab and Joe P. Foleyc a PECO Nuclear, Limerick Generating Station, Sanatoga, PA 19464, USA b American Cyanamid Company, Analytical, Physical & Biochemical Research, P.O. Box 400, Princeton, NJ 08543-0400, USA c Villanova University, Department of Chemistry, Villanova, PA 19085, USA Capillary zone electrophoresis (CZE) was used for the determination of trace level inorganic ions and its applicability in the power industry investigated.Detection of Cl2, SO4 22 and NO32 was accomplished by using a background electrolyte consisting of 7.0 mM CrO4 22, 0.5 mM TTAB and 1.0 mM NaHCO3. To enhance electrokinetic injection, transient isotachophoretic conditions were employed by the addition of 100 µl of 70 mM sodium octanesulfonate to 100 ml of sample. The addition of a 20 µg l21 aliquot of tungstate was used as an internal standard to limit possible electrokinetic injection biases.In addition, 150 µm extended path length capillaries were investigated for improving detection limits. Detection limits obtained via CZE were comparable to those given by ion chromatography (IC) with the use of the extended path capillaries. CZE results obtained were 0.38, 0.54 and 0.75 µg l21 for Cl2, SO4 22 and NO32, respectively, while IC results were 0.10, 0.30 and 0.25 µg l21. The advantages of using CZE as opposed to IC for the analysis of inorganic anions in power plant reactor water are discussed.Keywords: Ion detection; capillary zone electrophoresis; reactor cooling water; boiler feedwater; ion chromatography Ion analysis by capillary electrophoresis was first developed in 1967 by S. Hjerten for the separation of bismuth and copper cations.1 Tsuda and co-workers separated copper and iron cations in 1983 and in 1987 Zare reported the separation of potassium, sodium, lithium and selected alkylamines.2,3 Anion analysis via capillary electrophoresis was first reported in 1979 by Mikkers et al.4 Capillary zone electrophoresis (CZE) has been used to separate inorganic ions, hydrophobic and hydrophilic organic molecules and large macromolecules, such as proteins, peptides, carbohydrates and catecholamines.5 CZE has grown over the last few years into a standard separation method preferred in many applications over conventional liquid chromatographic techniques because of its speed, high efficiency, low operational costs and flexibility. Reported separation efficiencies have been in excess of 500 000 theoretical plates, far beyond the 10 000–20 000 typically reported for liquid chromatography. The increased efficiency has been attributed to the elimination of chromatographic peak spreading due to diffusional contributions.6 Unfortunately, a simultaneous analysis of cations and anions, by either ion chromatography (IC) or CZE, is not feasible in low-level analysis because of the loss of efficiency, and thus resolution.7–9 A detection barrier complicates the analysis of inorganic anions.Owing to the non-absorptive nature of inorganic anions, indirect UV detection is often employed.10 In the analysis of highly mobile anions, such as chloride, sulfate and nitrate, a chromate containing background electrolyte (BGE) has been optimized.11–14 A direct, conductimetric detection scheme, as employed in IC, is often unsuitable without suppression because of the high background conductance of the running buffer.Current research initiatives include the application of suppressed conductivity techniques and direct conductivity measurements based upon low ionic strength running buffers.15 Anion analyses are customarily performed with the assistance of an electroosmotic flow (EOF) modifier, such as tetradecyltrimethylammonium bromide (TTAB).16,17 The addition of an EOF modifier produces a change in the normal direction of the EOF, cathode to anode, thus increasing the speed of the anion separation.Cation analyses are performed under normal anode to cathode EOF, often with complexing agents and BGEs such as citrate, a-hydroxyisobutyric acid (HIBA) and imidazole.5 In boiling water reactors (BWR), the coolant is ultra-pure water. Conductivity is frequently below 0.090 µS cm21. Reactor water chemistry is in a constant state of dynamic equilibrium, dependent on various system inputs and operating conditions.To mitigate detrimental effects, such as intergranular stress corrosion and cracking (IGSCC) and fuel cladding failures due to crud-induced localized corrosion (CILC), contaminants such as Cl2 and SO4 22 must be maintained at a minimum.18,19 Pressurized water reactors (PWR) rely upon a borated coolant in the primary or nuclear side and aminated water for corrosion inhibition in the secondary or turbine side. Amines, such as ethanolamine, 5-aminopentanol, aid in oxygen scavenging and passivation of metal surfaces in the condensate–feedwater system, inhibiting general and localized corrosion of ferrous materials and reducing pitting susceptibility of alloys.20 Controlling the ingress of undesired components is a costly and time consuming endeavor within the power industry.In both BWRs and PWRs, some common analytes are Cl2, SO4 22, NO32, Na+ and various organic acids. These analytes are monitored regularly via ion chromatography and have become indicators of overall plant performance. In this paper we report CZE analytical methodology that uses sample electrostacking procedures in combination with the use of extended path length capillaries for trace analysis of common anions found in nuclear power plant reactor water.The CZE analytical methodology employed was compared with existing IC methodology for the analysis of trace anions in nuclear power plant reactor water.Evaluations of each technique were made with respect to detection limits, operational costs, sample throughput and overall analytical performance. Experimental Apparatus Analyses were performed with a 3-D capillary electrophoresis system (Hewlett-Packard, Wilmington, DE, USA) utilizing a photodiode array detector (DAD) at 375 nm, bandwidth 40 nm. Analyst, July 1998, Vol. 123 (1465–1469) 1465Two polyimide-coated fused silica capillary columns (350 µm) obtained from Hewlett-Packard were employed: (i), an 80.5 cm 3 75 µm capillary equipped with a 150 µm ‘extended’ path length in the detector region; and (ii), a 50 µm ‘normal’ capillary at 64.5 cm 3 50 µm.The lengths from the inlet to the detector were 72.0 and 56.0 cm, respectively. Sample injection was performed electrokinetically at an applied voltage of 25 kV for 45 s. BGE replenishment was performed after each injection to minimize migration variance. Data acquisition and analysis was performed with H-P 3DCE ChemStation software.The detector time constant was set at 0.1 s and data acquisition rate was 10 points s21. The IC instrument used in this study was a Dionex 2020I (Dionex Corporation, Sunnyvale, CA, USA) and a CDM-1 conductivity detector with A-SRS auto-suppression. The separation chemistry employed an AG12 for pre-concentration and AS12 for separation with 2.5 mM Na2CO3–0.5 mM NaHCO3 as the eluent. The sample loading volume was 30 ml, loading being accomplished by a constant load volume apparatus.Shown in Fig. 1, the constant volume apparatus enabled consistent volumes to be preconcentrated onto the guard column (Dionex AG12). Data acquisition and analysis were performed with Dionex AI-450 V3.30 software. The data acquisition rate was 5 points s21. Materials The chromate electrolyte system was prepared from 100 mM sodium chromate tetrahydrate (Mallinckrodt, Paris, KY, USA: analytical grade) and 0.0056 mM sulfuric acid (J.T.Baker, Phillipsburg, NJ, USA; Ultrex grade). The electrooosmotic flow modifier was 20 mM concentrated tetradecyltrimethylammonium bromide (TTAB) obtained from Waters (Waters Corp., Milford, MA, USA). Final working electrolyte concentration employed was 7.0 mM CrO4 22, 0.5 mM TTAB and 1.0 mM NaHCO3 (J.T. Baker), pH 9.1. Sodium octanesulfonate (NaOS) from Fluka (Fluka Chemika-BioChemika, Buchs, Switzerland) was employed as the terminating electrolyte by the addition of 100 µl of 70 mM NaOS to 100 ml of sample.The stock eluent for IC was prepared by dissolving 26.5 g of anhydrous Na2CO3 (J.T. Baker) and 4.15 g of anhydrous NaHCO3 (J.T. Baker) in 1 l of 18 MW water for final concentrations of 250 mM and 50 mM Na2CO3–NaHCO3. This stock eluent solution was further diluted 1:100 for an eluent strength of 2.5 mM Na2CO3 and 0.5 mM NaHCO3. Standards and simulated test samples were prepared from 1000 mg l21 standards obtained from J.T. Baker diluted in 18 MW water.Tungstate was added as an internal standard at 20 µg l21. Standards and samples simulating the secondary PWR system were prepared as the BWR standards and samples with the addition of the specified corrosion inhibitor, such as ethanolamine, at a standard concentration of 10 mg l21. Amines evaluated were ethanolamine, morpholine, 5-aminopentanol and methoxypropylamine. Methods Rinsing with 1 M sodium hydroxide (NaOH) for 20 min followed by subsequent rinses with 0.1 M NaOH and demineralized water for 20 min each activated the capillary.The final rinse was performed with the operating buffer prior to use. The capillary was purged for 2 min and inlet/outlet vials replenished after each run with the BGE. Sample vials were prepared by soaking in demineralized water for a minimum of 24 h. The vials were subsequently rinsed three times each with demineralized water and sample to reduce possible contamination. Internal standardization using tungstate (WO4 22) at 20 µg l21 was employed to decrease possible bias (see Fig. 4) due to electrokinetic injection.21,22 Analyses with recoveries of 90–110% WO4 22 were considered valid, with no associated injection bias. Calibration curves were created by plotting the normalized peak area versus concentration (Fig. 2). A linear regression was performed to determine slope and intercept that would allow for the quantitation of unknowns. Correlation coefficients (r2) were calculated and used to evaluate linearity. Detection limits were calculated by determining 3s of 30 or more data points of the 10 µg l21 standards.IC calibrations were performed with 10, 20 and 40 µg l21 (IC 2) and 2 and 10 µg l21 (IC 3 and 4) standards. Detection limits were calculated based upon 3s of 30 or more data points of 2 µg l21 standards. Theory In CZE, analyte separation occurs based upon the differing electroosmotic mobilities (µep) of the ions in the BGE when an electric field is applied through a capillary.Electroosmotic (µeo) flow reversal is essential in the analysis of anionic species, as the µep of the analyte may exceed the µeo under normal CZE Fig. 1 Constant volume apparatus. The constant volume apparatus enabled consistent volumes to be preconcentrated onto the guard column (Dionex AG-12). When sample loading begins, the effluent stream from the guard column is diverted into an acrylic sample tube. Attached to the sample tube is a capacitive proximity switch and once the sample pump has loaded the sample to the level prescribed by the proximity switch, a signal is sent to the data system, via the advanced computer interface, shutting off the sample pump and beginning sample analysis.After the analysis has been completed, the data system signals the drain valve to open and re-set the system. The sample volume can be increased or decreased according to sample type by moving the position of the proximity switch.For trace analysis, e.g., reactor water, the load volume was approximately 30 ml. This provides reproducible sample loading, which decreases variance and, consequently, decreases the IC LODs. Fig. 2 Calibration curve, extended path detector. Calibration curve prepared using standards of 10, 20 and 40 µg l21. Correlation coefficients for Cl2 and SO4 22 were better than 0.995, denoting linearity for analytes. NO32 shows deviation from linearity and possible contamination based upon intercept value. 1466 Analyst, July 1998, Vol. 123conditions and thus the analyte will not reach the detector. Additionally, increased efficiencies are obtained when the analytes of interest migrate in the same direction as the EOF.15,16 The chromate BGE is used because of similar electrophoretic mobility to the three anionic contaminants studied, Cl2, SO4 22 and NO32, broad UV absorption characteristics and high molar absorptivity. As the mobility differences of the co-ion and analyte increase, peak shape will become less symmetrical.23 As the analyte mobility decreases, peaks tail; as mobility increases, analyte peaks will begin to front.Therefore, chromate satisfies the requirements established by the first and second coion conditions.24,25 Electrokinetic injection with an isotachophoretic terminating analyte is employed as a pre-concentration step in trace analysis.9 The addition of NaOS fulfills the requirement for a terminating electrolyte by allowing the analytes of interest to ‘stack’ in low ionic strength samples by permitting a sufficient electric charge for ionic transfer to occur from the bulk sample solution into the capillary.22,25 Also, the addition of WO4 22 as an internal standard eliminates the reporting of inaccurate data due to electrokinetic injection bias.In an attempt to compare the performances of the different capillaries, a number of parameters are evaluated, which include apparent mobility (µapp), resolution (Rs), theoretical plates (N), capacity (kA) and tailing factors (USP t).Apparent mobility is calculated according to eqn. (1), where Ld = length to the detector, L = total capillary length, tm = migration time and V = applied voltage. mapp d m = ¥ ¥ L L t V (1) USP tailing factors [eqn. (2)] are calculated to further compare the performance of the 150 and 50 µm path width capillaries in BWR and PWR matrices: USP = 2 0.05 w t W t ¥ (2) where tw = distance between peak front and tr at 5% peak height; units are the same as used for W0.05 (width at 5% peak height).Because of the velocity dependence of CZE peaks, the normalized peak areas are used for quantitation.5 A A t norm m = (3) Results and discussion Data analysis According to Beer’s Law, A = ebc, sensitivity and detection ranges can be improved by increasing the detector path length or molar absorptivity. Chromate has a high molar absorptivity at the wavelengths employed, therefore further increases in sensitivity can be achieved by expanding the detector path length.A factor limiting the amount by which the detector can be enlarged is the increase in current and subsequent increase in Joule heating. By increasing the path length only in the detector region, no increase in current is experienced and the flow velocity decreases due to decreasing field strength. This decrease in velocity in essence stacks the analyte zones, thereby increasing the signal to noise ratio and sensitivity.Based upon detection limit calculations in the BWR matrix, a significant improvement in detection limits is realized with Cl2 and SO4 22 by increasing the path length (Table 1). Migration times within the BWR simulated matrix were extremely stable for the ionic strengths employed in both the ‘normal’ and ‘extended’ path capillaries. In both the 150 and 50 µm path capillaries, the % RSD of the migration times for all analytes was less than 0.20%.Specifically, Cl2 was 0.10 and 0.15%, SO4 22 was 0.11 and 0.15% and NO32 was 0.12 and 0.16%. The migration times for analytes in the ETA and 5-AP matrices increased significantly, but were stable with tm % RSDs less than 1.5. The increase in migration time can be attributed to an increase in viscosity or a change in the capillary zeta potential. Unfortunately, migration times within the morpholine and methoxypropylamine matrices were extremely unstable, hence the data was not useful.Detection limits in the BWR matrix were higher than IC detection limits in both capillaries (Table 1). Cl2 limits of 0.38 and 0.58 µg l21 were achieved in CZE, while IC was as low as 0.1 µg l21. Similar detection limits were obtained for both SO4 22 and NO32. Clearly, the use of the extended path length capillary yields detection limits that are lower than those obtainable with the normal capillary (50 µm), as shown in Table 1. The detection limit results shown using the normal capillary are comparable to results previously reported in which similar experimental conditions were used.11 Compared with the BWR matrices, there was a significant decrease in signal to noise ratios in the aminated matrices, drastically reducing the sensitivity (Table 1).Apparent mobilities (µapp) were constant in the BWR matrix (Table 2), regardless of the capillary employed, as predicted by theory [see eqn. (1)]. A shift of greater than 25% was encountered in the varying matrices.Apparent mobilities for the BWR matrix were 0.989 3 1024 to 1.06 3 1024 cm2 V21 s21 and 7.43 3 1025 to 7.94 3 1024 cm2 V21 s21 for the amine matrices. This drastic shift in µapp for the amine matrices indicates either an interaction with the capillary wall or viscosity effects.26 Table 1 Comparison of IC–CE detection limits—BWR and PWR matrices* Method Cl2/µg l21 SO4 22/µg l21 NO32/µg l21 IC BWR matrix— (System 2) 1.26 1.47 1.96 (System 3) 0.10 0.30 0.25 (System 4) 0.14 0.46 0.23 CZE BWR matrix— 50 µm ‘normal’ 0.58 0.82 0.81 150 µm ‘extended path’ 0.38 0.54 0.75 PWR matrices— 150 µm ‘extended path’ 5-Aminopentanol 1.20 1.09 0.97 Ethanolamine 2.00 2.60 4.89 * Ion chromatographs were calibrated at 10, 20 and 40 µg l21 (system 2) and 2 and 10 µg l21 (systems 3 and 4).Capillary zone electrophoresis was calibrated at 10, 20 and 40 µg l21 (system 2). Detection limits were calculated by 3 times the sample standard deviation of thirty 10 µg l21 standards.Table 2 Apparent mobilities (cm2 V21 s21)* Column/matrix Cl2 SO4 22 NO32 50 µm/water 1.07 3 1024 1.04 3 1024 1.01 3 1024 150 µm/water 1.06 3 1024 1.02 3 1024 9.89 3 1025 150 µm/5-AP 7.94 3 1025 7.57 3 1025 7.43 3 1025 150 µm/ETA 7.92 3 1025 7.56 3 1025 7.43 3 1025 * The apparent mobilities in both BWR pure water and PWR amine matrices are shown to demonstrate effects of amines on analysis. Sample pre-treatment of aminated sample matrices is required to remove or lessen effects. Analyst, July 1998, Vol. 123 1467Table 3 illustrates the high separation efficiency values that are attained in CZE. In all the BWR analyses, efficiency exceeded 177 000 theoretical plates for 150 µm capillaries and 270 000 for 50 µm capillaries. Baseline resolution (Rs > 1.5) was obtained in all test matrices. USP tailing factors were less than 2 in all analyses (see Figs. 3 and 4), with NO32 exhibiting the poorest performance, due primarily to its high diffusion coefficient.Multiple BWR samples were analysed under the same conditions as established for calibration and detection limit determination. As shown in Table 4, good correlation exists for analyses performed via IC and CZE. A notable exception is illustrated in the NO32 data. Generally, CZE NO32 data is low in comparison with values obtained via IC. This can be attributed to the contamination of NO32 found in the calibration curves. The NO32 contamination has been traced to organic contaminants in the source water, with subsequent UV oxidation releasing the NO32 ion.Unfortunately, since the detection limits are greater in CZE, samples requiring lower detection limits, such as reactor water, must be analysed via IC at this time. Cost to benefit analysis At the forefront of cost to benefit calculations is analysis time. Current IC analyses require a minimum of 15 min for laboratory and up to 90 min for in-line systems. The analysis via CZE, as illustrated by Figs. 3 and 4, can be completed within 5–7 min. A resultant increase laboratory sample throughput by a minimum factor of three can be realized over IC analyses. Material costs are significantly decreased in CZE compared with IC. Based upon the life expectancy of columns, typical costs per year are $10–200 for CZE and $2100 for IC, assuming two concentrator, guard and separator IC column replacements. Also, self-regenerating suppressors are not required with CZE, reducing costs yet again.Assuming a flow rate of 1.3 ml min21, an IC in continual operation will require 680 l of eluent per year, compared with approximately 52 l of BGE for CZE. Mobile phase costs are reduced substantially, hence radioactive waste generation and reprocessing costs decrease. Two major concerns within the nuclear power industry are personnel exposure and radioactive waste generation. ‘As low as reasonably allowable (ALARA)’ is an important fundamental principle that can be achieved with the diminished sample size of CZE.Also, capillary columns have a significantly longer life expectancy, minimizing waste generation, i.e., columns and suppressors. Finally, CZE systems can be transformed from anion to cation to organic analysis with ease, increasing the flexibility of the laboratory. Inasmuch as an IC is a dedicated system, only one type of analysis may be performed per system without expensive and time-consuming modifications.Table 3 Comparison of chromatographic figures of merit for normal and extended path capillaries* USP tailing Rs N a 50 mm ‘normal’— Cl2 1.10 330 571 SO4 22 1.40 4.58 413 857 1.04 NO32 1.73 3.21 272 829 1.03 150 mm ‘extended path’— Cl2 1.06 317 139 SO4 22 1.35 4.46 371 434 1.04 NO32 1.66 2.58 177 725 1.02 5-AP (150 mm) ‘extended path’— Cl2 0.94 202 795 SO4 22 1.16 5.60 402 621 1.05 NO32 1.18 1.85 199 269 1.02 ETA (150 mm) ‘extended path’— Cl2 1.13 264 746 SO4 22 1.19 5.29 320 191 1.05 NO32 1.40 1.90 210 673 1.02 * Tailing factors, resolution, efficiency and selectivity calculated using 10 µg l21 samples.Fig. 3 Representative electropherograms, water matrix. Top: normal capillary; bottom: extended path capillary displaying analyte peaks 1, chloride, 2, sulfate, 3, nitrate, 4, tungstate and 5, carbonate. Standard concentration was 10 µg l21 in pure water matrix. Fig. 4 Tungstate internal standard electropherogram. Calibration standards overlay with internal tungstate in BWR matrix: 10, 20 and 40 µg l21 for 1, Cl2, 2, SO4 22, 3, NO32 and 4, 20 µg l21 WO42.Table 4 IC–CE data (µg l21) for various sampling points* Sample IC CE IC–CE EDST-A†— Cl2 1.30 1.10 1.18 SO4 22 6.10 5.80 1.05 NO32 8.20 7.50 1.08 CST‡— Cl2 0.50 < 0.40 — SO4 22 5.90 6.10 0.97 NO32 7.40 6.70 1.10 U1RWCU§— Cl2 < 0.10 < 0.40 — SO4 22 0.98 1.05 0.93 NO32 4.30 3.20 1.34 * Comparison of results obtained from capillary zone electrophoresis (CZE), ‘extended’ path capillary (150 µm) and ion chromatograph (IC).† EDST-A Equipment drain sample tank A (aqueous sample). ‡ CST Condensate storage tank. § U1RWCU Unit 1 reactor water cleanup demineralizer effluent (reactor water sample point). 1468 Analyst, July 1998, Vol. 123Conclusion Capillary electrophoresis for the determination of inorganic ions requires extreme care in standard and sample preparation due to diminished injection volumes. Increased separation efficiency and improved selectivity allow for rapid analysis of complex samples compared with liquid chromatographic techniques. Increases in sensitivity and detection ranges, which approach limits of IC, can be realized using ‘extended’ path length capillary columns.Based upon cost to benefit calculations, ion analysis via capillary electrophoresis provides a low cost alternative to ion chromatography in most analyses performed within the nuclear power industry. Depending upon required detection limits, up to 90% of routinely performed analyses may be performed with current capillary electrophoresis systems.Since the detection limits of externally calibrated indirect CZE are factors of three to five greater than IC, increases in detection limits may be achieved through methods of standard additions for quantitation and peak area correction by internal standardization. References 1 Hjerten, S., Chromatogr. Rev., 1967, 9, 122. 2 Tsuda, T., Nomura, K., and Nakagawa, G., J.Chromatogr., 1983, 264, 385. 3 Huang, X., Pang, T. K. J., Gordon, M. J., and Zare, R. N., Anal. Chem., 1987, 59, 2747. 4 Mikkers, F. E. P., Everaerts, F. M., and Verheggen, T. P. M., J. Chromatogr., 1979, 169, 18. 5 Weinberger, R., Practical Capillary Electrophoresis, Academic Press, Boston, MA, USA, 1993, ch. 2, 3. 6 Giddings, J. C., Sep. Sci., 1969, 4, 181. 7 Hayakawa, K., and Miyazaki, M., LC-GC, 1988, 6, 508. 8 Tarter, J. G., J. Chromatagr. Sci., 1989, 27, 462. 9 Saari-Nordhaus, R., Nair, L., and Anderson, J. M., J. Chromatogr., 1992, 602, 127. 10 Jones, W. R., Electrophoretic Capillary Ion Analysis, ed. Landers, J. P., CRC Press, Boca Raton, FL, 1994. 11 Jandik, P., and Jones, W. R., J Chromatogr., 1991, 546, 431. 12 Romano, J. P., Jandik, P., Jones, W. R., and Jackson, P. E., J. Chromatogr., 1991, 546, 411. 13 Romano, J. P., and Krol, J., J. Chromatogr., 1993, 640, 403. 14 Krol, J., Trace (PPB) Anions and Cations Analysis in High Purity DI Water and Process Water Used in the Power Generation Industry Using Capillary Ion Analysis, International Ion Chromatography Symposium, Sept., 1993. 15 Huang, X., Luckey, J. A., Gordon, M. J., and Zare, R. N., Anal. Chem., 1989, 61, 766. 16 Jones, W. R., and Jandik, P., Am. Lab., 1990, 22, 51. 17 Jones, W. R., and Jandik, P., J. Chromatogr., 1991, 546, 445. 18 Guidelines for Chemistry at Nuclear Power Stations, INPO 88-021, Rev. 01, 1991. 19 Intergranular Corrosion of Stainless Steel, Electric Power Research Institute ER-7247, vols. 1, 2, 1991. 20 PWR Secondary Water Chemistry Guidelines, Electric Power Research Institute NP-5056-SR, Rev. 1, 1987. 21 Rose, D. J., and Jorgenson, J. W., Anal. Chem., 1989, 480, 95. 22 Huang, X., Gordon, M. J., and Zare, R. N., Anal. Chem., 1988, 60, 375. 23 Whetham, W. C. D., Phil. Tans., 1893, A184, 337. 24 Yeung, E. S., Acc. Chem. Res., 1989, 22, 125. 25 Rose, D. J., and Jorgenson, J. W., Anal. Chem., 1989, 60, 642. 26 Cohen, N., and Grushka, E., J. Chromatogr. A., 1994, 678, 176. Paper 8/00705E Received January 24, 1998 Accepted June 7, 1998 Analyst, July 1998, Vol. 123 1469

ISSN:0003-2654    

DOI:10.1039/a800705e

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Capillary electrophoresis with linear polymers containing hydrophobic groups for the separation of small molecules Hirokazu Sawada and Kiyokatsu Jinno* School of Materials Science, Toyohashi University of Technology, Toyohashi 441-8580, Japan The capillary electrophoretic separation of small charged molecules using linear (non-cross-linked) polymers containing hydrophobic groups was investigated. First, various compositions of linear copolymers, consisting of the water-soluble monomers, acrylamide and N-isopropylacrylamide (IPAAm), were prepared in a running buffer solution, and the effects of alkyl group content in the copolymer chain on the separation of small molecules were investigated.In order to increase the hydrophobic selectivity, linear copolymers containing more hydrophobic groups (tert-butyl or n-octadecyl) were prepared in methanol as the next step, and were applied to capillary electrophoretic separations after purification. The migration behavior of small molecules in solutions of the copolymers containing hydrophobic groups was different from the separation in a free solution or in polyacrylamide solution.Separations with linear polymers containing hydrophobic groups can be achieved on the basis of the differences in the electrophoretic mobilities and the hydrophobicities of the solutes. Keywords: Capillary electrophoresis; linear acrylamide–N-alkylacrylamide copolymer Capillary electrophoresis (CE) is a rapidly growing, powerful separation method that is being used for the separation of various biological compounds and pharmaceuticals.In capillary zone electrophoresis (CZE), the separation of ionic analytes is based on the differences in their electrophoretic migration velocities under an applied electric field. Although CZE offers a high separation efficiency for such ionic species, mixtures of charged analytes having similar electrophoretic mobilities or of neutral compounds are difficult to separate. Many attempts have been made to alter the separation selectivity and to enhance the resolution in CZE.For instance, in micellar electrokinetic chromatography (MEKC), the separation is based on differential partitioning of solutes between the aqueous phase and micellar phase and on differences in the electrophoretic mobilities of the solutes. Recently, various linear polymers have been applied to the CE separation of small molecules to improve the resolution.1–6 The separations of small molecules in the presence of a linear polymer exhibited an enhancement of the resolution compared with the normal CE separation in a free solution without additives and the linear polymers acted as an interactive additive (or a pseudo-stationary phase).In capillary gel electrophoresis (CGE), which can separate large biomolecules such as DNA fragments according to their molecular size, recent studies have focused on the use of a linear (or non-crosslinked) polyacrylamide solution as a sieving matrix instead of cross-linked polyacrylamide gel-filled capillary.7–15 The use of a linear polyacrylamide solution is free from some of the practical problems associated with cross-linked gel-filled capillaries, including poor reproducibility of the column preparation and limited lifetime of the capillary due to the bubble formation. Moreover, linear polyacrylamide solutions can be refilled after each analysis, if the solution has low viscosity.This is important with regard to reproducibility, reliability of analysis and the cost of capillary columns. We have previously described16 the separations of small molecules such as barbiturates and dansylated (Dns)-amino acids in linear polyacrylamide (PAAm) and acrylamide (AAm)–N-isopropylacrylamide (IPAAm) copolymer solutions at ambient and at elevated temperatures, because it is known that linear polymers or cross-linked gels containing IPAAm have thermosensitive (or thermoresponsive, thermoreversible) properties.The polymers exhibit strong hydrophobic properties above their phase transition temperature. In addition to obvious changes in the migration behavior at elevated temperature, slight differences between PAAm and AAm–IPAAm copolymer- filled columns with respect the migration behavior of barbiturates were observed even at ambient temperature. In this study, in order to introduce hydrophobic selectivity in CZE separations, we prepared linear acrylamide–N-alkylacrylamide copolymers as interactive materials and applied them to the separation of small, charged molecules.To investigate the effects of alkyl group content of the copolymer on the migration behavior, we first prepared columns filled with various compositions of AAm–IPAAm copolymer [designated poly- (AAm-co-IPAAm)] solution and PAAm solution. To incorporate more hydrophobic groups in the polymer network, we prepared linear acrylamide copolymers containing N-tertbutylacrylamide (TBAAm) or N-n-octadecylacrylamide (ODAAm) designated poly(AAm-co-TBAAm) and poly(AAmco- ODAAm)] and investigated the effects of the alkyl groups on the separation of small molecules.The advantages of using nonionic linear polymers are that they do not contribute to the conductivity of the running buffer and they can prevent electrostatic interactions between the charged solutes and the linear polymers. Experimental Apparatus All experiments were performed using a laboratory-made system, consisting of a Model HCZE-30PN0.25 high-voltage power supply (Matsusada Precision Devices, Kusatsu, Japan), a Model CE-970 UV detector (Jasco, Tokyo, Japan), buffer reservoirs and a fused-silica capillary of 0.075 mm id and 0.363 mm od (Supelco, Bellefonte, PA, USA).The UV detector was used at 254 nm. Part of the polyimide coating of the capillary was removed to create the UV detection window. Borwin chromatography software (Jasco) was employed for data acquisition and processing. Sample injection was performed by electromigration from the cathodic end of the capillary tube.Samples were dissolved in the buffer at concentration of 0.05–0.2 mg ml21. No column temperature control was available. Molecular mass measurements of prepared polymers were performed using a gel permeation chromatographic (GPC) system equipped with two TSK-GEL a-M columns (300 mm 3 7.8 mm id) (Tosoh, Tokyo, Japan) connected in series and with Analyst, July 1998, Vol. 123 (1471–1476) 1471a refractive index detector (Tosoh).The mobile phase used in GPC was TRIS-boric acid buffer (pH 8.2). A series of pulluran standards (Tosoh) were employed. as molecular mass standards. Chemicals AAm, ammonium peroxodisulfate (APS), 2,2A-azobisisobutyronitrile (AIBN), N,N,NA,NA-tetramethylethylenediamine (TEMED), boric acid, methanol and tris(hydroxymethyl)aminomethane (TRIS) were obtained from Kishida Chemical (Osaka, Japan), TBAAm, IPAAm and 3-methacryloxypropyltrimethoxysilane were obtained from Tokyo Kasei (Tokyo, Japan).ODAAm from Polysciences (Warrington, PA, USA). AIBN was recrystallized from methanol prior to use and IPAAm was purified with a hexane–toluene mixture prior to use. Dns-amino acids were obtained from Sigma (St. Louis, MO, USA). Distilled, de-ionized water, purified with a Milli-Q water system (Millipore, Bedford, MA, USA), was used for the preparation of running buffers and sample solutions.The running buffers prepared were passed through a hydrophilic filter unit of 0.45 mm pore size (DISMIC-13HP, Tokyo Roshi, Japan) prior to use. Preparation of linear polymer filled-columns All capillaries were first treated with 3-methacryloxypropyltrimethoxysilane and then coated with 4% m/v linear PAAm as described by Hjertén.17 Electroosmotic flow (EOF) was efficiently suppressed by this coating procedure. Preparation of linear polymer containing isopropyl groups Because AAm and IPAAm are water-soluble monomers, copolymers of AAm and IPAAm could be directly prepared in the running buffer using APS and TEMED as the initiator and the catalyst in the same manner as in the preparation of linear polyacrylamides, which are used as a sieving medium in the size separation of large biomolecules.First, a monomer solution containing AAm, IPAAm and TEMED was purged with nitrogen for 15 min, then APS was added.The solution was then filled into the coated capillaries, the capillaries were kept overnight and both ends of the capillaries were dipped into the same polymerization solution. Before use, the capillaries filled with the polymer solution were conditioned by pre-electrophoresis for 2 h to remove any charged catalyst and unreacted monomer. The polymer solutions were characterized by GPC and reversed-phase liquid chromatography (RP-LC) as described by Carrilho et al.15 Preparation of linear polymer containing tert-butyl or octadecyl groups In order to incorporate more hydrophobic groups in a polymer network, we prepared linear polymers containing AAm and TBAAm (or ODAAm).Because TBAAm and ODAAm are water-insoluble monomers, the copolymerization with AAm was conducted in methanol as the polymerization solvent using AIBN as the initiator. A typical copolymerization process for the copolymer was as follows. TBAAm (1.25 g) (or ODAAm), AAm (0.77 g) and methanol (20 ml) were placed in to a flask, then the monomer solution was purged with nitrogen for 15 min.After purging, the solution was heated to 60 °C and AIBN (0.04 g) was added. The reaction was allowed to proceed for 3 h, after which the mixture was cooled to stop the reaction. The crude polymer solution was slowly added to 800 ml of acetone while stirring to precipitate the copolymer. The copolymer was further washed with acetone and finally dried under vacuum for 10 h.The products were characterized by 1H NMR spectroscopy and GPC. For comparison, copolymers consisting of AAm and IPAAm were also prepared by the same method. Before use of the copolymers in CE experiments, each copolymer was dissolved in running buffer and kept for overnight to ensure the dissolution of the copolymer. Results and discussion Separation of small molecules using linear polymers containing isopropyl groups Dns-amino acids were chosen as representatives of small molecules in this study.Fig. 1 shows a CZE separation of Dnsa- aminobutyric acid (a-Aba) and -threonine (Thr) in TRISboric acid buffer (pH 8.2) using a PAAm-coated column. These solutes were negatively charged under these conditions, and therefore migrated towards the anode. They were slightly separated according to the difference in their electrophoretic mobilities. To improve the resolution, these solutes were separated in a 4% m/v solution of poly(AA-co-IPAAm) with 85 mol-% IPAAm [Fig. 2(b)]. For comparison, the same experiment was performed in 4% m/v PAAm solution [Fig. 2(a)]. Compared with the separation in the free solution, no obvious changes in the migration behavior were observed in the PAAm solution. This result indicates that the polymer network of PAAm makes little contribution to the separation of these solutes. On the other hand, these solutes were co-eluted in the 4% m/v solution of poly(AAm-co-IPAAm) with 85 mol% IPAAm (Fig. 2b).Moreover, the separation was improved, and the elution order changed in a 10% (m/v) solution of poly(AAm-co-IPAAm) with 85 mol-% IPAAm [Fig. 2(c)]. These results suggested that the copolymers containing isopropyl groups contributed to the separation of the small molecules. The more hydrophobic solute, Dns-a-Aba, was eluted later than Dns-Thr. Ong et al.18 investigated the MEKC separation of 15 Dns-amino acids, and reported that there was a good correlation between the elution order and the hydrophobicity (log P) of Dns-amino acids and that the separation was based primarily on the differences in the hydrophobicities of the Dns-amino acids.The migration order obtained from Fig. 2(c) was consistent with the results obtained by MEKC18 and RP-LC.19,20 Table 1 shows the effect of IPAAm content on the migration of these two Dns-amino acids at a constant total monomer concentration of 10% m/v. In Table 1, the ratios of the migration time of the solutes represent the selectivities.The selectivity Fig. 1 Separation of Dns-a-Aba and -Thr in free solution using a polyacrylamide-coated column. Conditions: capillary column, 400 3 0.075 mm id (250 mm effective length); buffer, 100 mm TRIS–150 mm boric acid (pH 8.2); field strength, 200 V cm21; injection, electromigration at 5 kV for 2 s at the cathode side; UV detection, 254 nm; temperature, ambient. Peak identification: 1, Dns-a-Aba; and 2, Dns-Thr. 1472 Analyst, July 1998, Vol. 123changed with increasing IPAAm content in the copolymers, the migration order being reversed at 40 and 85 mol-% IPAAm content compared with the migration order in PAAm solution. Table 2 also shows a comparison of the migration of Dns-a- Aba and -Thr in solutions of PAAm and poly(AAm-co- IPAAm). Although the selectivity did not exhibit substantial changes in a series of PAAm solutions of different concentrations, the migration time of Dns-a-Aba in poly(AAm-co- IPAAm) increased with increasing total monomer concentration (i.e., increasing isopropyl group content), relative to the migration of Dns-Thr.From these results, it appears that the separation using a solution of linear polymers containing isopropyl groups is based on differences in the hydrophobicities of the solutes in addition to differences in their electrophoretic mobilities. Fig. 3 shows the separations of three isomeric Dns-leucines (Dns-Leu, -Ile, and -Nle) in 4% m/v solutions of poly(AAm-co- IPAAm) with various IPAAm contents.The migration behavior changed with increasing IPAAm content of the poly(AAm-co- IPAAm). Dns-Leu and -Nle, which could not be separated in PAAm solution, could be slightly separated in poly(AAm-co- IPAAm) with 60 mol-% IPAAm. Table 3 shows the results of the characterization of the linear polymer used in Fig. 3. The linear polymers were characterized in terms of molecular mass and monomer conversion as described by Carrilho et al.15 For poly(AAm-co-IPAAm) with 85 mol% IPAAm, the molecular mass could not be determined because the copolymer was adsorbed on the GPC column.The monomer conversion was determined by measuring the residual AAm and IPAAm in each polymer solution and was found to be > 96% for all polymer solutions. The high conversion efficiency, as shown in Table 3, is important for the use of linear polymer solution-filled columns without further purification. Separation of small molecules using linear polymers containing tert-butyl or octadecyl groups Conducting polymerization in running buffer is very easy and the prepared polymer solution-filled columns can be used for CE experiments without further purification because of the high conversion efficiency of the monomers.However, the applicable monomers are limited to water-soluble compounds, such as IPAAm, unless a solubilizing agent is used. Moreover, as shown in Fig. 3, the separations of the three isomeric Dns-leucines were inadequate in each poly(AAm-co-IPAAm) solution because of its weak hydrophobic selectivity.Therefore, in order to incorporate more hydrophobic groups and to increase the hydrophobic selectivity, we prepared linear copolymers containing N-tert-butylacrylamide (TBAAm) or N-octadecylacrylamide (ODAAm) (and also IPAAm for comparison) using methanol as the polymerization solvent. The compositions and the molecular masses of the copolymers prepared in this study are given in Table 4.The molecular mass was not determined for poly(AAm-co-TBAAm) and poly(AAm-co-ODAAm) because of the possibility of adsorption on the GPC column. For poly(AAm-co-ODAAm), the composition could not be determined since the amount of ODAAm was very small and below the detection limit of the instrument. It is well known that the entanglement of linear polymers takes place above the entanglement threshold, c*. Therefore, it is important to know the c* values for the prepared acrylamide copolymers. According to Heller,21 the entanglement threshold is defined by c* Å 3 M/4 p NA Rg 3 (1) Fig. 2 Separation of Dns-a-Aba and -Thr in solutions of (a) 4% m/v PAAm, (b) 4% m/v poly(AAm-co-IPAAm) with 85 mol-% IPAAm and (c) 10% m/v poly(AAm-co-IPAAm) with 85 mol-% IPAAm. Conditions and peak identification as in Fig. 1. Table 1 Effect of IPAAm content in poly(AAm-co-IPAAm) on the migration of Dns-a-Aba and -Thr. Total monomer concentration, 10% m/v.Experimental conditions as in Fig. 2 IPAAm content (mol-%) Compound Parameter 0 20 40 85 Dns-a-Aba Migration time/min 22.51 21.73 22.81 24.24 Dns-Thr Migration time/min 22.75 21.73 22.60 23.48 Selectivity (tThr/ta-Aba) 1.011 1.000 0.991 0.969 Table 2 Comparison of the migration of Dns-a-Aba and -Thr in solutions of PAAm and poly(AAm-co-IPAAm) with 85 mol-% IPAAm. Experimental conditions as in Fig. 2 Total monomer concentration (%) Polymer Compound Parameter 4 6 8 10 PAAM Dns-a-Aba Migration time/min 15.41 16.98 18.37 22.51 Dns-Thr Migration time/min 15.64 17.20 18.59 22.75 Selectivity (tThr/ta-Aba) 1.015 1.013 1.012 1.011 Poly(AAm-co-IPAAm) with 85 mol-% IPAAm Dns-a-Aba Migration time/min 15.58 17.94 20.29 24.24 Dns-Thr Migration time/min 15.58 17.67 19.80 23.48 Selectivity (tThr/ta-Aba) 1.000 0.985 0.976 0.969 Analyst, July 1998, Vol. 123 1473where Rg, NA and M are the radius of gyration, Avogadro’s number and the molecular mass of the polymer, respectively.The entanglement threshold is the concentration at which the polymer chains touch each other.21 The radius of gyration can be obtained by measuring the intrinsic viscosity (h): [h] Å 2.5 Rg 3 NA / Mw (2) The intrinsic viscosity can be obtained by measuring the viscosity of the polymer solution at various concentrations (Fig. 4). In this study, we roughly estimated the c* values of each copolymer by following equation, which was obtained by combining eqns. (1) and (2).c* Å 0.6 [h]21 (3) The calculated intrinsic viscosity, [h], and entanglement threshold, c*, for each copolymer solution are summarized in Table 4. In each solution, it was estimated that entanglement occurred above approximately 2% m/v. The separations of the three Dns-leucines with the acrylamide –N-alkylacrylamide copolymers are shown in Figs. 5–7. These polymer solutions could be refilled after each run up to at least 8% m/v because of the low viscosity of the polymer solutions.As expected from Fig. 3, the three Dns-leucines were poorly resolved in 4% m/v poly(AAm-co-IPAAm) solution owing to the weak hydrophobic nature of the copolymer, and Dns-Nle and -Ile were co-eluted in 8% m/v poly(AAm-co- IPAAm) solution (Fig. 5). On the other hand, the three Dnsleucines were completely resolved in 6% and 8% m/v poly(AAm-co-TBAAm) solution (Fig. 6), in spite of a smaller alkyl group content than the poly(AAm-co-IPAAm). In a MEKC study using the same running buffer (containing 40 mm SDS),22 the elution order was Dns-Ile, -Leu, and -Nle.This result reflects the difference and the order of their hydrophobicity. In 8% m/v poly(AAm-co-TBAAm) solution, Dns- Nle, probably most hydrophobic solute of the three, was eluted later than others. This result suggests that the copolymers containing tert-butyl groups have stronger hydrophobic selectivity than those containing isopropyl groups (Fig. 5). Fig. 3 Separation of three isomeric Dns-leucines in solutions of poly(AAm-co-IPAAm) with (a) no IPAAm, (b) 60 mol-% IPAAm and (c) 85 mol-% IPAAm.Conditions: capillary column, 650 3 0.075 mm id (500 mm effective length); buffer, 100 mm TRIS- 150 mm boric acid (pH 8.2); field strength, 300 V cm21; injection, electromigration at 8 kV for 2 s at the cathode side; UV detection, 254 nm; temperature, ambient. Peak identification: 1, Dns-Leu; 2, Dns-Nle; and 3, Dns-Ile. Table 3 Characteristics of poly(AAm-co-IPAAm) solutions prepared in running buffer using APS as the radical initiator Monomer conversion (%)† Polymer Apparent M* AAm IPAAm PAAm 4.0 3 105 96.7 — Poly(AAm-co-IPAAm) with: 40 mol-% IPAAm 6.1 3 105 98.9 98.6 60 mol-% IPAAm 5.2 3 105 99.5 98.7 85 mol-% IPAAm — 99.5 99.1 * The molecular mass was determined by GPC using a series of pulluran solutions as the molecular mass standard. † The monomer conversion was determined the residual AAm and IPAAm in each polymer solution by RP-LC.Table 4 Characteristics of acrylamide–N-alkylacrylamide copolymers prepared in methanol using AIBN as the radical initiator Polymer Apparent M N-AlkylAAm content (mol-%) Intrinsic viscosity/dl g21 Entaglement threshold (%) Poly(AAm-co-IPAAm) 7.56 3 104 68.2 0.54 1.12 Poly(AAm-co-TBAAm) — 39.9 0.38 1.58 Poly(AAm-co-ODAAm) — 0.26* 0.36 1.64 * Initial monomer content Fig. 4 Viscosity measurements of acrylamide–N-alkylacrylamide copolymers prepared in methanol using AIBN as the radical initiator as a function of their polymer concentrations.The measurements were performed using a modified Ubbelohde-type viscometer at 25 °C. Each copolymer was dissolved in TRIS-boric acid buffer (pH 8.2). 1474 Analyst, July 1998, Vol. 123The migration behavior further changed in poly(AAm-co- ODAAm) solutions in spite of the incorporation of only a small alkyl group content in its polymer chain ( < 1%). As shown in Fig. 7(b) and 7(c), the solutes were resolved only at 2% or 4% m/v, and Dns-Nle was eluted later than the others.With 8% m/v poly(AAm-co-ODAAm) solution, the migration time of Dns-Nle was prolonged to about 50 min. McCormick et al.23 described the interchain hydrophobic association of copolymers of acrylamide and N-alkylacrylamide with 8–12 carbons in aqueous solution. Therefore, it appears that the copolymers containing octadecyl groups form a strong hydrophobic core by the strong interchain associations and that the solutes strongly interacted with the core.The strong interchain association is also apparent from large increase of the viscosity of poly(AAmco- ODAAm) as shown in Fig. 4.23 Although poly(AAm-co- TBAAm) and poly(AAm-co-IPAAm) did not exhibit substantial increases in viscosity compared with poly(AAm-co-ODAAm), these copolymers might also be associated under the present conditions because they contain a number of hydrophobic groups and touch each other. The results obtained from Figs. 6 and 7 indicated different separation selectivities from MEKC22 and RP-LC separations. 20 This would be caused by the difference in the original electrophoretic mobilities of Dns-leucines, as shown in Figs. 6(a) and (b). Therefore, the separation with acrylamide–Nalkylacrylamide copolymers would be achieved by a combination of the differences in electrophoretic mobilities and hydrophobicities of the solutes. Additionally, at a 1% m/v polymer concentration, a different migration behavior was observed with poly(AAm-co-ODAAm) solution compared with those with poly(AAm-co-IPAAm) and poly(AAm-co-TBAAm) solution.This result could be attributed to the intrachain association of poly(AAm-co-ODAAm) chains provided that the copolymer chains are isolated from each other around that concentration. Conclusion The separations of small, charged molecules using solutions of linear polymers containing hydrophobic groups were investigated and we demonstrated the usefulness of acrylamide–Nalkylacrylamide copolymer-filled columns for small molecule separations.The small molecules, which could not be resolved in free solution or in PAAm solution, were resolved in acrylamide–N-alkylacrylamide copolymer solutions according Fig. 5 Separation of three Dns-leucines in (a) 1, (b) 4 and (c) 8% m/v poly(AAm-co-IPAAm) solution. Conditions and peak identification as in Fig. 3. Fig. 6 Separation of three Dns-leucines in (a) 1, (b) 4 and (c) 8% m/v poly(AAm-co-TBAAm) solution.Conditions and peak identification as in Fig. 3. Fig. 7 Separation of three Dns-leucines in (a) 1, (b) 2 and (c) 4% m/v poly(AAm-co-ODAAm) solution. Conditions and peak identification as in Fig. 3. Analyst, July 1998, Vol. 123 1475to the differences in their hydrophobicities. Therefore, the separations were accomplished by a combination of electrophoretic and hydrophobic selectivities. The degree of hydrophobic selectivity can be manipulated by varying the Nalkylacrylamide content in the copolymer and the length of the alkyl substituent of N-alkylacrylamide.The copolymers investigated here have applicability as interactive materials in small molecule separations. The authors thank Hisato Yasui and Dr. Seigoh Kawaguchi, Laboratory for Polymer Science, Toyohashi University of Technology, for polymer characterization and helpful discussions during the course of this research. References 1 Hjertén, S., Valtcheva, L., Elenbring, K., and Eaker, D., J.Liq. Chromatogr., 1989, 12, 2471. 2 Terabe, S., and Isemura, T., Anal. Chem., 1990, 62, 652. 3 Cruzado, I. D., and Vigh, G., J. Chromatogr., 1992, 608, 421. 4 Ingelse, B. A., Everaerts, F. M., Desiderio, C., and Fanali, S., J. Chromatogr., 1995, 709, 89. 5 Esaka, Y., Yamaguchi, Y., Kano, K., Goto, M., Haraguchi, H., and Takahashi, J., Anal. Chem., 1994, 66, 2441. 6 Soini, H., Riekkola, M.-L., and Novotony, M. V., J. Chromatogr. A, 1994, 680, 623. 7 Heiger, D. N., Cohen, A. S., and Karger, B. L., J. Chromatogr., 1990, 516, 33. 8 Widhalm, A., Schwer, C., Blaas, D., and Kenndler, E., J. Chromatogr., 1991, 549, 446. 9 Ruiz-Martinez, M. C., Berka, J., Belenkii, A., Foret, F., Miller, A. W., and Karger, B. L., Anal. Chem., 1993, 65, 2851. 10 Chiari, M., Nesi, M., and Righetti, P. G., J. Chromatogr. A, 1993, 652, 31. 11 Best, N., Arriaga, E., Chen, D. Y., and Dovichi, N. J., Anal. Chem., 1994, 66, 4063. 12 Manabe, T., Chen, N., Terabe, S., Yohda, M., and Endo, I., Anal. Chem., 1994, 66, 4243. 13 Zhang, J. Z., Fang, Y., Hou, J. Y., Ren, H. J., Jiang, R., Roos, P., and Dovichi, N. J., Anal. Chem., 1995, 67, 4589. 14 Gelfi, C., Orsi, A., Leoncini, F., and Righetti, P. G., J. Chromatogr. A, 1995, 689, 97. 15 Carrilho, E., Ruiz-Martinez, M. C., Berka, J., Smirnov, I., Goetzinger, W., Miller, A. W., Brady, D., and Karger, B. L., Anal. Chem., 1996, 68, 3305. 16 Sawada, H., and Jinno, K., Electrophoresis, 1997, 18, 2030. 17 Hjertén, S., J. Chromatogr., 1985, 347, 191. 18 Ong, C. P., Ng, C. L., Lee, H. K., and Li, S. F. Y., J. Chromatogr., 1991, 559, 537. 19 Wilinson, J. M., CRC Handbook of HPLC for the Separation of Amino Acids, Peptides and Proteins, CRC Press, Boca Raton, FL, 1985, vol. 1, pp. 339–350. 20 Takeuchi, T. and Miwa, T., Chromatography, 1995, 16, 184. 21 Heller, C., J. Chromatogr. A, 1995, 698, 19. 22 Sawada, H., and Jinno, K., unpublished work. 23 McCormick, C. L., Nonaka, T., and Johnson, C. B., Polymer, 1988, 29, 731. Paper 8/00027A Received January 2, 1998 Accepted February 23, 1998 1476 Analyst, July 1998, Vol. 123

ISSN:0003-2654    

DOI:10.1039/a800027a

出版商:RSC    

年代:1998

数据来源: RSC