ISSN:0144-557X    

DOI:10.1039/AP99330FX001

出版商:RSC    

年代:1993

数据来源: RSC

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111 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 Analytical Division Distinguished Service Award Nominations are invited for the Division’s Distinguished Service Award, the Rules for which are as follows: ... tical Division, which shall recommend to Council of the Division (a) to whom an award should be made, ( b ) the nature of the award, or (c) that no tional recognition as Council of the division shall agree. Nominations for the Award will be invited annually from members of Council of the Division, and may be award should be made. received from any member of the 5. The Award shall be made by the Division. They shall be made in writ- Council of the Analytical Division, ing, with supporting evidence, to the which must approve any alteration of President of the Analytical Division, these Rules. 3. The aim of the Award is to recognize exceptional voluntary service over a period of years to the Analytical Div- ision of The Royal Society of Chemistry (including that to the Society for Analytical Chemistry). ingion house^ London W1V OBN. The Award shall normally be in the Royal Society of Chemistry, Burl- Nominations for the Award should be form of an illuminated address which 4. Nominations shall be considered by sent to the President of the Analytical may be accompanied by such addi- the Honours Committee of the Analy- Division before March 12th, 1993.

ISSN:0144-557X    

DOI:10.1039/AP993300P002

出版商:RSC    

年代:1993

数据来源: RSC

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January 1993 ANPRDI 30(1) 1-64 (1993) Ana I yt ica I Proceedings Proceedings of the Analytical Division of The Royal Society of Chemistry CONTENTS 1 VAM Viewpoint ’The Use of Statistical Methods in Industrial Chemistry’ 2 Reports of Meetings 2 Recent IUPAC Recommendations 3 Analytical Chemistry in the EC ’The Current Status of Analytical Chemistry in Denmark. A Personal View’ by Elo Harald Hansen 10 Analytical Viewpoint ’Overhead Projector Flow Injection Analysis‘ by Kate Grudpan and Thanboosak Thanasarn 19 27 28 31 13 Surface Analysis 13 ’Fourier Transform Infrared Reflection Spectroscopy for Surface Analysis’ by J. Yarwood ’Surface Analysis in an Industrial Environment’ by Robert K. Wild ’Surface Analysis and Adhesive Bonding’ by J.Comyn ‘Surface Enhanced Raman Spectroscopy’ by J. Alan Creighton ‘Surface Reactivity of Anti-wear Additives‘ by D. Landolt, H. J. Mathieu and R. Schumacher 35 Retiring President’s Address ’Bridges in Analytical Chemistry’ by J. D. R. Thomas 35 39 SUMMARIES OF PAPERS 41 44 46 51 39 Short Papers in Pharmaceutical Analysis 39 ’Development and Application of an Assay Procedure for Hyoscine in Urine Using Solid-phase Extraction and High-performance Liquid Chromatography with Electrochemical Detection’ by Peter R. Hurst ’Direct Non-destructive Colour Measurement of Pharmaceuticals’ by Nicola Stock ‘Trace Analysis of Acyclovir Esters‘ by David S. Ashton and Andrew Ray ’Investigation into the Reversed Phase High-performance Liquid Chromatographic Behaviour of Tipredane Metabolites and Related Substances’ by Melvin R.Euerby, Christopher M. Johnson and Steven C. Nichols ’Studies on the Use of Base-deactivated and Polymeric Stationary Phases in Reversed-phase High-performance Liquid Chromatography of Anthracyclines and Their Metabolites’ by Glynis Nicholls, Brian J. Clark and J. E. Brown J 55 Biological Applications of Scanning Tunnelling Microscopy 57 Equipment News 61 SAC Silver Medal (Rules) 62 Conferences and Meetings 62 Courses 63 Analytical Division Diary iii Analytical Division Distinguished Service Award (Rules) I Ill I Typeset and printed by Black Bear Press Limited, Cambridge, England 0144-557XC199311:l-B ... 111 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 ROYAL SOCIETY OF CHEMISTRY Spectrochemical Analysis by Atomic Absorption and Emission By Lauri H.J.Lajunen University of Oulu, Finland This new book describes both the theory of atomic spectroscopy and all the major atomic spectrometric techniques (AAS, Flame- AES, Plasma AES, AFS, and ICP-MS), including basic concepts, instrumentation and applications. Contents : Introduction; Theory of Atomic Spectroscopy; Atomic Absorption Spectrometry; Flame Atomic Emission Spectrometry; Plasma Atomic Emission Spectrometry; Inductively Coupled Plasma Mass Spectrometry; Atomic Fluorescence Spectrometry; Sample Preparation; Advantages and Mutual Comparison of Atomic Spectrometric Methods; Further Reading; Subject Index Spectrochemical Analysis by Atomic Absorption and Emission is very wide in scope and will be extremely useful to both undergraduates and lecturers undertaking modern analytical chemistry courses.It contains many figures and tables which illuminate the text, covers various sample preparation methods and gives suggestions for further reading. xii + 242 pages Price €18.50 Sof tcover ISBN 0 85186 873 8 RSC Members Price €1 3.50 To Order, Please write to the: Royal Society of Chemistry, Turpin Distribution Services Limited, Blackhorse Road, Letchworth, Herts SG6 1 HN, United Kingdom, or telephone (0462) 672555 quoting your credit card details. We can now accept Access/Visa/MasterCard/Eurocard. Turpin Distribution Services Limited is wholly owned by the Royal Society of Chemistry. For information on other books and journals, please write to : Royal Society of Chemistry, Sales and Promotion Department, Thomas Graham House, Science Park, Milton Road, Cambridge CB4 4WF, UK. The Membership Affairs Department at the Cambridge address above. RSC Members should obtain members prices and order from : June 1992 ROYAL SOC,ETY CHEMISTRY / information Services

ISSN:0144-557X    

DOI:10.1039/AP99330BX003

出版商:RSC    

年代:1993

数据来源: RSC

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10 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 Analytical Viewpoint ~~~~~ The following is a member of a continuing series of articles providing either a personal view of part of one discipline in analytical chemistry (its present state, where it may be leading, etc.), or a philosophical look at a topic of relevance t o chemists in general or analytical chemists in particular. These contributions need not have been the subject of papers at Analytical Division Meetings. Persons wishing to provide an article for publication in this series are invited t o contact the editor of Analytical Proceedings, who will be pleased to receive manuscripts or to discuss outline ideas with prospective authors. Overhead Projector Flow Injection Analysis Kate Grudpan Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai, 50002, Thailand Thanboosak Thanasarn Theong Witta yakom School, Chiang Rai, Thailand Flow injection analysis (FIA) is based on injecting a sample into a continuously moving non-segmented carrier stream yielding a change which can be continuously monitored. The observed change is related to the concentration of analyte in the sample. Three processes are involved: sample injection residence time; and controlled dispersion.Measurements are made without equilibrium being reached. This allows a high sampling frequency with economic sample and reagent consumption. Residence time, the period for the sample to travel from the injection point to the detector, can be optimized for any particular system. During this period the sample plug is physically dispersed so that diffusion and possible reaction occur between sample and carrier.Dispersion coefficient, the ratio of concentration of analyte in the injected solution to concentration of analyte at the point of measurement, depends upon flow rate, tube length and sample size. FIA is now an important area of modern analytical chemistry4" and laboratory exercises for students have been de~cribed.~-" Some of these can be easily set up using existing equipment. FIA systems using low-cost instrumentation, which can be adapted for student exercises, have also been devel- oped. 1s-19 The principles of FIA have been demonstrated by means of a capillary tube" or a microconduit slide2* in a 35 mm projector, allowing the shape and size of the sample zone, dispersion processes, stopped-flow FIA and the effects of intermittent pumping to be observed.The operation of a multi-line manifold has been demonstrated by using a microconduit and an overhead projector.21 Microconduit FIA systems are not readily available in developing countries: we have therefore developed a system using low-cost materials to demonstrate the principles and applications of FIA by means of an overhead projector. The system can also be used with a white background instead of a projector for student laboratory exercises. Experimental FIA System The components of the system were placed on a 30 X 30 x 0.2 cm piece of clear acrylic sheet (Fig. 1). A plastic bottle (100 ml) was used as a reservoir for the reagent. Clear polyethylene tubing (1.3 mm i.d., 2.5 mm 0.d.) was used for all reagent lines.An injection port of the Betteridge typeI5 (Fig. 2) was made of acrylic pieces. Injections were made with a plastic syringe (1 ml) without a needle. A transparent plastic tube 1 cm in diameter and 30 cm long (the protecting sleeve for a thermometer) was used as a former, around which 200 cm of the polyethylene tubing were coiled at spaced intervals of 5 mm (approximately 40 turns in 22 cm). Both ends of the tubing were fixed to the former with transparent adhesive tape. The spacing allows clear viewing of the diffusion process and the sample zone can easily be observed when a coloured solution passes through the coil. A mixing chamber (Fig. 3) was assembled from acrylic sheet, microscope slide covers, epoxy glue and opaque tape.A 0.8 cm diameter hole was drilled in the centre of a 3.5 X 3.5 x 0.2 cm sheet. A 0.4 cm wide slot was cut from the hole to one edge of the sheet in order to insert the inlet and outlet tubing and the sheet was then sandwiched with glue between two cover slips. Finally, the mixing chamber was covered with tape cut to expose only the aperture. - T R ~ ~ ~ ~ ~ _ _ _ _ Fig. 1 Arrangement of the FIA system on an acrylic sheet (A) (30 X 30 x 0.2 cm) for placing on an overhead projector or white background: R, reagenucarrier bottle; T, polyethylene tubing (1.3 mm i.d.; 2.5 mm 0.d.); C, former made of clear plastic; I , injection port; M I , mixing coil; MZ, mixing chamber; D, detection point; P, receiverANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 11 Side view Fcm 1.3 cm I I I Fig.2 screws; 4, syringe socket Injection port: 1 , acrylic body; 2, rubber septum; 3 , retaining A chamber of similar design to the one just described, but with a 0.3 cm diameter hole, was used as an observation point for the visual discrimination of coloured products or for timing the period of a colour change. From the detection point, the flowing solution was collected in a 100 ml plastic bottle for possible re-use. In order to start an experiment the tubing at the exit (P in Fig. 1) was connected to a syringe (10 o r 20 ml). The system was filled with the reagent solution by suction via the syringe, and allowed to siphon. The flow rate was regulated by suspending the reagent bottle at various heights.To wash the system, the tubing was removed from the reagent solution (R in Fig. 1) and suction was applied at the exit. The reagent bottle was replaced by a bottle containing water and the system flushed by siphoning until clean. / OT / l _ . Fig. 3 Mixing chamber: A, acrylicsheet (3.5 x 3.5 x 0.2 cm); H, hole (0.8 cm diameter); T, polyethylene tubing for inlet and outlet. fixed to A with epoxy glue; C, cover glass; OT, opaque tape FIA Demonstrations Dispersion and Residence Time A solution of sodium hydroxide (0.02 or 0.1 rnol 1-' in 1% bromothymol blue) is used as the carrier. Injection of a volume (0.15 ml) of 0.6 mol 1- ' hydrochloric acid into the moving blue stream yields a yellow plug which is observed as it moves along the mixing coil. The further the plug moves, the greater its elongation, illustrating the effect of coil length on dispersion.When the plug reaches the mixing chamber the yellow colour disperses further before passing to the detection point. The effect of sample size on dispersion can be demonstrated by injecting a series of volumes (0.07-0.25 ml) of 0.6 rnol I-' hydrochloric acid. The effect of flow rate on dispersion can be investigated by varying the height of the reagent bottle and injecting 0.15 ml of acid. Residence time and its reproducibility can be studied by recording the time from the moment of injection to the appearance of the first colour change at the detection point. Students using wrist watches have recorded residence times of, for example, 18 s with standard deviations of +2 s. Acid-Base Titrations When acid is injected into the carrier, the elapsed time, At, between the colour changes, blue to yellow and back to blue, at lo-' 2 3 4 5 6 lo" 2 3 10-2 2 3 4 5 6 7 8910-l 2 3 Injection volume/ml Fig.4 Elapsed time ( A t ) between colour changes as a function of log(HC1 injection volumes) (curve A) or of log(acid concentration) (curves B, C and D): height of reagent bottle, SO cm; flow rate, 4.8 ml min-I. A, 0.6 mol I-' HCI versus 0.02 mol 1-' NaOH; B, 0.10 ml HCI standard versus 0.02 mol I-' NaOH; C, 0.15 ml HCI standard versus 0.1 mol I-' NaOH; D, 0.15 ml CH,COOH standard versus 0.1 rnol I-' NaOH Acid concentration/mol I-' I I I I I I I I I I I I the detection point corresponds to the peak width at half height in the equation' where V is the volume of the mixing chamber, v the flow rate, C, is the concentration of standard injected, S, the volume injected and CNaOW the concentration of the carrier stream.Plotting At versus log C, should yield in a straight line calibration with a slope of Vlv-lnlO. Results obtained by the authors, displayed in Fig. 4, confirm the logarithmic relationships of Equation 1. At constant flow12 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 Table 1 Determination of acetic acid in vinegar: a comparison of flow injection with conventional titrimetry Elapsed time/s Acetic acid concentration Actual Mean FIA Conventional titrimetry Nominal mol I-' (%w/v) mol I-' (./w/v> (%w/v) Standards - 0.49 mol I-' 15.4,15.5,15.5 15.5 0.58 mol I-' 16.6,16.2.16.6 16.5 0.78 mol I-' 18.4.18.3.18.3 18.3 0.98 rnol I-' 19.5,19.5.19.7 19.6 1.17 mol I-' 21.1,21.0,21.0 21.0 Samples * - 1 17.1,17.3,17.3 17.2 0.655 3.93 0.710 4.26 4 2 19.8,19.7,20.6 20.0 0.930 5.58 0.883 5.28 5 3 19.2,19.3,19.3 19.3 0.990 5.40 0.883 5.00 5 4 19.0,19.1,19.0 19.0 0.880 5.28 0.936 5.62 5 5 16.9,16.3,16.3 16.5 0.550 3.30 0.581 3.48 - * Different commercial vinegars.rate and carrier concentration, plots of t versus log injection volume (A) and t versus log analyte concentration (B) are parallel lines. The slope of the calibration is governed by the injection volume and the carrier concentration (C), and also by the dissociation constant of the acid analyte (D), the sensitivity of response from a weak acid such as acetic being greater than that from a strong acid. From the slope of a calibration, a value for the volume of the mixing chamber can be calculated.From lines A and B, a value of 0.1 ml was obtained, which corresponded closely to the true one. An estimate of detection limit under a particular set of conditions can be made by extrapolating a calibration to At = 0. Conditions for line C for example correspond to a limit of 0.1 rnol 1-' of acid analyte. The calibration line for acetic acid (D) was used to determine the acid content of commercial vinegar (Table 1). FIA for Chloride By using the manifold without the mixing chamber, 0.20 ml of 0.3,0.8 and 1.2 rnol 1-' solutions of chloride (HCI or NaCl) are injected into a carrier stream of a methanolic (10.8%) aqueous solution containing mercury(I1) thiocyanate (0.002 rnol 1 - I ) , iron(II1) nitrate (0.075 rnol I-') and nitric acid (0.075 rnol I-').Visual comparisons of the colour intensities of the red [Fe(SCN)]*' complex are made. With practice, the approximate concentration of an unknown chloride solution can be estimated by matching the intensities. Discussion The effects of tube length, sample size and flow rate on dispersion can be clearly shown using the overhead projector in a lecture room for up to 100 students. The set up can also be used to illustrate the principle of stopped-flow FIA by lowering the carrier reagent bottle to the plane of the overhead projector screen when the colour appears in the mixing chamber. An increase in colour intensity with time is then observed. This set up has been used at Chaing Mai University for training high-school students in a science camp project.Without previous experience, the students obtained results for the determination of acetic acid in vinegar which agree well with those obtained by conventional titrimetry (Table 1). In a lecture period of 1-2 hours the system reported here can provide a simple illustration of the principles of FIA and its application within a budget of less than &lo. Without the projector, students themselves can perform the experiments described within a laboratory session of 2-3 hours. This system has been found to be of value in a developing country. Rather than accepting Nernst's statement, 'Believe me my dear colleague, when I say it is so then it is so',' students of FIA can be convinced by practical demonstrations.We thank Ian McKelvie and Ian Campbell (Monash Univer- sity, Melbourne, Australia), and Colin Taylor (Liverpool John Moores University, UK) for useful discussions. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 References RSii-ka, J . and Hansen, E.. Flow Injection Analysis, Wiley, New York. 2nd edn., 1988. Valcarcel, M.. and Luque de Castro, M. D.. Flow Injection Analysis: Principles and Applications, Ellis Horwood, Chiches- ter, 1987. Stewart, K. K . , Beecher, G. R., and Hare, P. E., Anal. Biochem., 1976, 70, 167. Skoog, D. A., Principles of Instrumental Analysis, Saunders College Publishing, Tokyo, 3rd edn., 1985. Braun, R. D., Introduction to Instrumentational Analysis, McGraw-Hill Book Co., Singapore, 1987, pp. 957-967. Skoog, D. A., West, D.M., and Holler, F. J . , Fundamentals of Analytical Chemistry, Saunders College Publishing, Tokyo, 5th edn., 1988. Hansen, E., and R%ii-ka, J., J . Chem. Educ., 1979, 56, 677. Meyerhoff, M. E., and Kovach. P. M., J. Chem. Educ., 1983, 60, 766. McClintock. S. A., Weber, J . R.. and William, C. P., J . Chem. Educ., 1985. 62. 65. Keller. J . W., Gould, T. F., and Aubert, K. T., J . Chem. Educ., 1986. 63, 553. Rios, A.. Luque de Castro, M. D . , and Valcarcel, M., J. Chem. Educ., 1986, 63. 553. Stults, C. L. M., Wade, A. P.. and Crouch, S. R., J . Chem. Educ., 1988, 65, 645. Stults, C. L. M., Kraus, P. R., Ratanathanawongs, S. K., Patton, C. J . , and Crouch, S. R., J. Chem. Educ., 1989, 66, 1060. RSitka, J., Hansen, E. H., and Ramsing, A. U., Anal. Chim. Acta, 1982, 134, 55. Grudpan, K., in Proceedings of UNESCO South East Asia Regional Workshop on Low Cost Instrumentation, August I I th 1988, Johore Bahru, Malaysia, ed. Mohinder Singh, M., Institute Kimia Malaysia, Kuala Lumpur, pp. 7-15. Grudpan. K.. and Nacapricha, D., Proceedings of the 3rd Asian Chemical Congress: Chemistry International Conference, Bris- bane, Australia, 1989, p. A28. Orprayoon, P., Leelasart, B., and Grudpan, K., Microbial Utilization of Renewable Resources, 1989, vol. 6, pp, 38-43. Grudpan, K., Nacapricha, D., and Wattanakanjana, Y . , Anal. Chim. Acta, 1991, 246. 325. Grudpan. K., and Nacapricha, D.. Anal. Chim. Acta, 1991, 246. 329. Tyson. J., Fresenius Z. Anal. Chem., 1988, 329, 657. McKelvie, I . D., Cardwell, T. J.. and Cattrall, R. W., J . Chem. Educ., 1990, 67, 262.

ISSN:0144-557X    

DOI:10.1039/AP9933000010

出版商:RSC    

年代:1993

数据来源: RSC

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ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 Surface Analysis 1 13 ATR The following are five of the papers presented at the Industrial Division symposium of the Annual Chemical Congress of the Royal Society of Chemistry, held on April 13-16, 1992, in the University of Manchester Institute of Science and Technology. ~~ Reflection-absorptlon(RAIRS) Emission (EMS) Fourier Transform Infrared Reflection Spectroscopy for Surface Analysis Monolayer Thin films on metals. Electrode surfaces ~ 100A Metal (wire, rough surfaces, etc.,) catalysts, flames J. Yarwood Department of Chemistry, University of Durham Science Laboratories, South Road, Durham DH 1 3LE Introduction Infrared spectroscopy is already extensively used in analysis, both qualitatively (for compound identification)l and quanti- tatively2 (in order to determine concentration).It is less commonly used in ‘surface’ analysis. Indeed it may be thought by some that, because one is dealing with photons of relatively long wavelength (typically 5-25 pm), true ‘surface’ analysis is not possible. It is worth recapping, therefore, on the attributes 1 Substrate-molecule and molecule-molecule interactions (from band multiplicity, bandwidths and frequency shifts) 2 Molecular orientation and packing; degree of ‘chain‘ ordering and ‘tilting’ from linear dichroism measurements 3 Chemical changes (‘finger printing’) e.g., proton transfer, surface degradation, chemical reactions) 4 Electronic changes (conductivity, pyroelectricity, etc.) 5 Molecular arrangement and re-arrangements (crystallinity, fluidity, phase changes, efc.) Major Advantages of FflR ( a ) Non-destructive and ‘universally’ available ( 6 ) ng sensitivity (monolayer and upwards) ( C ) ’in situ’ measurements possible (in devices) (d Relatively cheap (= f20K) Fig. 1 Information available using Fourier transform infrared spectroscopy 1000 8, 500 A Surface ~1 pm XPS SERS 4 Depth profile 4 SIMS Fig.2 Comparison of sampling depths of various ‘surface’ sensitive techniques. ATR (attenuated total reflection); XPS (X-ray photoelec- tron s ectroscopy); SERS (surface-enhanced Raman scattering); SIMS gecondary ion mass spectrometry) of the infrared technique (Fig. 1) in a form which emphasizes that surface sensitivity may be achieved down to S 1 mono- layer (typically 10-20 A of surfactant or polymer or a few tens of nanograms).Surface selectivity is more problematic because, as indicated above, penetration depths are of the order of the wavelength (up to a few micrometres). This is, of course, very high compared with some other techniques (Fig. 2) but infrared reflection techniques do offer opportuni- ties for depth profiling if the sample is suitably prepared and if the correct spectral parameters are measured (see below). Infrared spectra can at present be collected in a wide variety of ways. Fig. 3 gives a summary of the sampling procedures which have been employed. Some of the techniques have been devised specifically to cope with particular sample types [e.g., diffuse reflectance infrared Fourier transform spectroscopy (DRIFT) for powders, photoacoustic spectroscopy (PAS) for ‘black’ materials such as coal, surface electromagnetic wave (SEWS) for organic thin films on metals].This paper deals only with reflection techniques [attenuated total reflectance (ATR) and reflection-absorption infrared spectroscopy (RAIRS)] which may be employed to examine a wide variety of samples but focusing here on the ways in which surface sensitivity and selectivity may be achieved in a (well-founded) analytical laboratory. Fig. 4 compares the conventional transmission experiment with RAIRS and ATR techniques for an organic film with a thickness that may vary from <20 A to several micrometres. The key to obtaining information about molecular orientation and/or ordering lies in the ability to choose a particular electric field polarization (see later). First, the method of obtaining structural and quantitative information is outlined using these techniques and then some examples are presented from our recent work (and that of others) to illustrate the principles involved.paint, mbbers, cloth, etc. Diffuse reflectance G S T i c ( P A S ) I -1 ktm I Carbon, coal, ceramics 1 Surface electromagnetic wave (SEWS) = 1oA Polymer films, organic thin films on metal Fig. 3 FTIR spectroscopy Summary of the most common sampling techniques used in14 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 (a) Transmission or absorption Samples dipoles parallel to substrate surface ( b ) Reflection-absorption (RAIRS) dipoles perDendicular to substrate surface Uses reflection from a metallic surface.Samples EL I s Thin film sample (c) Attenuated total reflection (ATR) interface to increase sensitivity. Samples dipoles both parallel and perpendicular to substrate surtace Uses many reflections at solidiair or solid/liquid Silicon ATR crystal Fig. 4 Three infrared sampling techniques compared Theoretical Background The infrared intensity of the band (corresponding normal mode Qi) which arises from the transition v = k t m depends on the interaction between the incoming photons and the vibrating molecule .3-4 This interaction is computed via the transition moment <rnlp.Elk> where 1.1. is the total dipole moment and E is the external electric field. The transition probability (the probability per unit time for the k c m transition to occur) is given by Pmk 0~ [<mi P.EI k >I2 (1) Expanding the dipole moment in a Taylor series about the equilibrium atom positions for a given normal mode gives 1.1.= PO + (31.1.IaQi) Qz (2) in the harmonic approximation.5 Thus the transition probabil- ity Pmk becomes? ( 3 ) (4) There is a population factor ( N , - N,) involved also but for fundamental transitions (v = 1 t 0) in absorption this is virtually constant. Eqn. (4) indicates that the infrared intensity depends on the square of the cosine of the angle between the transition dipole Mi = ap/aQj and the incident electric field E (see Fig. 5 ) . As Mi directions depend on the t The integral < r n / ~ l k > is zero as Vrm and Yk are orthogonal wave tz (a) RAIRS Sh bstrate t' (b) ATR \ Substrate Fig. 5 Orientation of electric field components Ex, E , and E, relative to the substrate and a typical transition dipole (acl/aQi) of the molecule ( a ) for a RAIRS experiment, ( b ) for an ATR experiment ( a ) Incident beam Reflected beam Thin organic -n b Ere,.film einc 4 45 90 I I 68 Fig. 6 ( a ) Incident and reflected infrared beam polarization com- ponents (p, polarized light is in the plane of incidence). ( 6 ) Corresponding phase shifts as a function of Oinc on a metal surface way in which molecules are oriented (or arranged) at a surface or interface (Fig. 5 ) , and as the Cartesian components of E; El., Ey and EZ can be varied using various optical techniques (including polarization), eqn. (4) forms the basis for surface- functions. sensitive analysis. Clearly, for a given molar absorptivity, theANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 15 observed intensity depends on the size of E* and the value of cos $.Results and Discussion The easiest technique to understand in this context is RAIRS3.6.7 in which infrared radiation is incident at 'grazing' incidence on a metal surface [Fig. 6(a)]. It may be seen [Fig. 6(6)] that s polarized light suffers a 180' phase shift (8) on reflection from a metal surface and so E,(l) and E,(r) cancel at the metal/sample interface. On the other hand E,(l) and E,Cr) differ by -90" at large 8 and so vectorially add together in such a way that maximum field (and maximum intensity) occurs near grazing incidence Oinc = 85-88'. This is the origin of the surface selection rule6-8 which results in the very useful ability to distinguish vibrations which have a transition dipole ( M i ) with a large component perpendicular to the substrate. Vibrations parallel to the substrate have zero intensity.Furthermore, there should be no spectrum for s polarized light (see Fig. 7). Fig. 8 shows that for methylene chains oriented perpendicular to the substrate the va(CH2) and vs(CH2) transition dipoles ( M , ) are parallel to the substrate. Fig. 9 shows16 then that such bonds are extremely weak in RAIRS 0.04 0.03 Q, C m I) v) I) 0.02 a 0.01 0 2918, / mp 285 1 1639 1012 ! 1470 I I 1 I I 3500 3000 2500 2000 1500 1000 Waven u m be rlcm - Fig. 7 Demonstration that the s polarized RAIRS spectrum (a,) has zero intensity. The sample is 10 Langmuir-Blodgett monolayers of a dye JT-11 which has a CZ2 chain attached to an acetylenic head group M - SH 0) t m I) 0.40 0.30 0.20 0.10 0 L 0, a n 1 .oo 0.90 0.80 0.70 0.60 3000 2800 3000 2800 0.20 0.16 0.1 2 0.08 0.04 1700 1500 1300 1.60 1.40 1.20 1 .oo 0.80 0.60 1700 ,1500 1300 Wave n u m be r/cm - 1 Fig.9 Spectra of 21 Langmuir-Blodgett layers of cadmium docosan- oate ( ~ 4 0 0 8, thick) on a silicon ATR crystal; (a) and ( b ) show the spectra in ATR mode. The v,(CH2) and v,(CH2) bands are intense; (c) and (d) show the spectra in RAIRS mode (with an overcoating of metal). Now the v,(CH2) and v,(CHZ) bands are weak, relative to those due to CH3 vibrations. (For details see ref. 16) Substrate Fig. 8 Orientation of the va(CHZ) and v,(CH2) transition dipoles parallel to the substrate for an alkyl chain oriented perpendicular to the substrate.The v,(CH3) and v,(CH3) transition dipoles have components perpendicular to the substrate 1 .o 0 El €0 Fig. 10 Schematic representation of the basic ATR experiment. The evanescent wave penetrates the sample and decays exponentially with distance in the z direction16 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 (compared with ATR, where two components of the field are parallel to the substrate) [Fig. 5(b)]. Such comparisons enable the degree of orientation of molecular monolayers to be assessed semi-quantitatively.6 One disadvantage of RAIRS is that a single reflection leads to relatively poor sensitivity, although monolayer spectra have been reported .9JO The ATR experiment3.12-14 is more complex (Fig. 10) because the incident electric field will have non-zero com- ponents in all three directions (for unpolarized light) [Fig.5(b)] and the use of linear dichroism is more difficult although not impossible.3,10-14 The principal advantages of ATR are (a) sensitivity and ( b ) the ability to carry out depth profiling,l5 (see below). Sensitivity enhancement is achieved by multiple internal reflection (up to 50 reflections are possible).l6 Depth Profiling With ATR Spectroscopy Depth profiling possibilities arise because the evanescent wave falls off exponentially into the rarer medium (usually the sample). Thus the field (E/Eo)2 varies rapidly as a function of distance z away from the substrate surface (Fig. 10). Varia- tions in concentration and crystallinity, for example, might then be detected by a variation of the penetration depth, d,.with E = Eo exp(- zld,) (6) where h is the wavelength of the band of interest, and Eo is the electric field at the surface of the ATR crystal. Clearly d, can be varied by changing A, Oinc or n2 (and therefore nz1). Figs. 11-13 illustrate that this does work. In principle, at least, one can adjust d, so that the evanescent wave samples material near an interface between (say) two polymers. By spectral subtraction of the data at two or more d, makes it is possible for interfacial interactions (or structure) to be examined. Convincing results have yet to be reported. Very recently, however, there has been a report17 of depth profiling using the method originally advocated by Hirschfeld. 18 This involves the recognition that absorbance measurements as a function of €lint may be converted to a concentration profile C(z) via the Laplace transform where R(0) is the reflectivity at incident angle 8 and a(z) is the absorption coefficient as a function of z away from the crystal surface.The sample transmission is Z/Zo = exp(- ad,), where d, is the thickness in transmission which is equivalent to the amount of material probed in the ATR experiment. Fina and Chen17 have shown how eqn. (7) may be solved to yield depth-dependent information about the crystallinity of PET (polyethylene terephthalate) (Fig. 14). Bands attributed to trans-conformers (of the PET chains) decrease in intensity as a function of z. This is because a high level of all-trans chains is found (as expected) for highly crystalline material formed by quenching from the melt at the ATR crystal surface. In a corresponding way, the bands attributed to gauche-conform- ers increase with z reflecting a higher proportion of amor- phous polymer further from the surface.This work promises to provide a basis for important future work in this area. Determination of Adsorption Isotherms Using ATR Spectroscopy For in situ adsorption studies on to an ATR crystal (or a surface layer on an ATR crystal) it is usual to bring the crystal into contact with a solution containing the adsorbate. The resulting spectrum will, of course, reflect both adsorbed and bulk species (after solvent spectrum removal). The procedure for separation of such components in order to measure the surface excess concentrationlg has been described by Sperline and co-workers.20921 The surface excess is defined as Ti = $/o 0 C m e 8 a B I I I I I I 4000 3500 3000 2500 2000 1500 1000 Wavenum bedcm - 1 Fig.11 45". ( b ) Difference spectrum after subtraction of the underlying PMMA spectrum FT infrared spectra of ( a ) a poly(methy1 methacrylate)-poly(viny1 alcohol) polymer laminate on a ZnSe ATR crystal with @inc =ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 17 Fig. 12 Difference spectra of PVA [poly(vinyl alcohol)] at different 'barrier film' thicknesses of PMMA [poly(methyl methacrylate)] in the laminate (€Iinc = 45") showing smaller effective penetration depths into the PVA as the PMMA layer is made thicker; d = A, 0.345; B, 0.396; C, 0.646; D, 1.065; and E, 1.900 pm 0 1 .o N u7 s 0 I I I I I 1 0.4 0.8 1.2 1.6 0.4 0.8 1.2 1.6 Distance z/pm Fig.13 Comparison of (LIE# fields and PVA (1100 cm-l band) infrared intensities as a function of distance away from the ATR crystal surface. (a) A, €Iinc = 39"; B, €Iinc = 45"; and ( b ) A, Oinc = 60" where qi' is the number of moles adsorbed at the interface and <T is the surface area of the interface. In order to measure T$' separately (from the bulk concentration) one needs to integrate the equation for ATR intensity. The absorbance per reflection, AIN of a particular ATR band of interest, is, then where E is the molar absorpthity of the band and 8 the incident angle. [The other parameters are as defined in eqn. (5).] The n g l i 0 0 2.0 4.0 6.0 8.0 10.0 5 0.025 .- + sl s: $ 0.022 0.019 0.016 0.01 3 0.010 0 2.0 4.0 6.0 8.0 10.0 Depth from interface/pm Fig.14 Concentration profiles of trans (T) and gauche (G) conform- ers in a film of quenched PET (or an ATR crystal) as a function of distance from the interface. (a): A, The 1340 cm-1 band (T) and B, the 1505 cm-1 band (T and G). ( b ) : A, The 1370 cm-1 band (G) and B, the 1455 cm-1 band (G). (Reproduced, with permission, from ref. 17) integral has been performed for 'bulk' and surface phase to give AIN = E Cb d, + E (2 d,Id,) (tit) (9)18 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 € 3 I I I I 1 I t 0 0.01 0.02 0.03 0.04 0.05 B u I k co nce nt rat ion/mol d m - 3 Fig. 15 Adsorption isotherm of sorbitan monopalmitate on an Si-SiOH surface from carbon tetrachloride at 298 K 0 II CH2-0-C-C H2 ( CH2 ) 1 jC H3 I HO-CH OH Fig. 16 Sorbitan monopalmitate, SPAN 40 w 4.4 4.6 4.8 5.0 5.5 6.0 6.5 7.0 I I I I I I I I 2200 2000 1800 1600 1400 Wave n u m be rlc m - Fig.17 Detection of diffusion across a polymer-polymer interface: A, the spectrum of an epoxy resin (4 ym); B, the spectrum of an epoxy-urethane laminate; and C, the difference spectrum A - B showing the two bands (marked) due to the isocyanate-urethane product diffusion into the epoxy layer where cb and ci are the bulk and surface concentrations, t is the adsorbed layer thickness and d, is the effective depth into the bulk sample.l3%14 Note that the molar absorptivity is assumed to be unchanged between ‘bulk’ and ‘surface’ species. This is expected to be a good approximation for v(CH2) bands of surfactant chains but it is probably a very poor approximation for ‘head group’ vibrations.Fig. 15 shows the adsorption isotherm of sorbitan monopalmitate (SPAN-40) (Fig. 16) or Si-SiOH from carbon tetrachloride. Note that a calibration of the ATR cell is required to determine N and Oinc accurately and that some ATR cell types22 are unsuitable for such quantitative measurements. Studies of Molecular Diffusion Using ATR Spectroscopy From Fig. 10 it can be seen that diffusion of one material into another across an interface or from the external surface into an organic thin film can be detected by the evanescent field. Fig. 17 shows an example of such detection. In this case a 4 pm polymeric film, a cross-linked epoxy resin, has been ‘over- coated’ onto a ZnSe ATR crystal with a polyurethane containing both isocyanate and amide chemical groupings.It may be immediately observed that bands at 2260 and 1680 cm-1 corresponding to v(N=C=O) and v(NHC=O) , respec- tively, arise (and increase with time) in the subtracted spectrum. As the epoxy film thickness is much higher than 3 x 4 1 3 such bands must arise from diffusion across the polymer/ polymer interface. From this, and the other examples, given in this paper it is clear that there is much scope for development of the infrared surface sensitive techniques into areas impor- tant to, although rarely tackled by, analysts and other industrial scientists. Thus, areas such as depth profiling and surface excess measurements, although known to be feasible for some time, have not been used extensively in our industrial context.It is hoped that this review will have whetted a few appetites. The author acknowledges and thanks the collaborators who performed the innovative spectroscopy described here: M. Pereira, Y. Song, J. Tait, G. Davies and S. Nunn. Thanks are also due to Dr. M. Petty for access to Langmuir-Blodgett dipping facilities and to the SERC, Shell (UK) and Courtaulds Coatings for financial support. 1 2 3 4 5 6 7 .8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 References Bellamy, L. J., Infrared Spectra of Complex Molecules, Chap- man and Hall, London, 1973, vols I and 11. Haaland, D . H . , in Practical Fourier Transform Infrared Spectroscopy, eds. Ferraro, J . R., and Krishnan, K., Academic Press, New York, 1990, ch. 8 (and references cited therein).Debe, M. K., Prog. Surface Sci., 1987, 24, 1. Steele, D . , Theory of Vibrational Spectroscopy, W. B. Saund- ers, London, 1971. Atkins, P. N., Molecular Quantum Mechanics, Oxford Univer- sity Press, Oxford, 1984, p. 50. Golden, W. G. in Fourier Transform Infrared Spectroscopy, eds. Ferraro, J. R., and Basile, L. J., Academic Press, New York, 1985, ch. 8. Hayden, B. E., in Methods of Surface Characterisation, eds. Yates, J. T., and Madey, T. E., Plenum Press, New York, 1987, vol. 1, p. 287. Greenler, R. G . , J. Chem. Phys., 1966, 44, 310. Rabolt, J. R., Jurich, M.. and Swalen J. D . , Appl. Spectrosc. 1985, 39, 281. Chabal, Y. J., Surface Sci. Reprints, 1988, 8, 211. Wendlandt, W. W., and Hecht, H. G., Reflectance Spectro- scopy, Interscience, New York, 1966. Ishida, H . , Rubber Chem. Technol., 1987, 60, 497. Harrick, N. J., Internal Reflection Spectroscopy, Harrick Science Corporation, New York, 2nd edn 1979. Mirabella, F. M., Appl. Spectrosc. Rev., 1985, 21, 45. Urban, M. W., and Koenig, J. L., Vib. Spectra Struct., 1990,18, 127. Davies, G. H., and Yarwood, J., Langmuir, 1989, 5 , 229. Fina, L. J., and Chen, G . , Vib. Spectrosc., 1991. 1, 353. Hirschfeld, T., Appl. Spectrosc., 1977. 31, 289. Hunter, R. J., Foundations of Colloid Science, Oxford Univer- sity Press, Oxford, 1989, vol. 1, ch. 8. Sperline, R . P.. Muralidharan. S . , and Freiser, H., Langmuir, 1987, 3, 198. Sperline, R. P., Muralidharan, S., and Freiser, H . , Appl. Spectrosc., 1986, 40, 1019. Afran, A . , SPECAC Applications Note No. 17.

ISSN:0144-557X    

DOI:10.1039/AP9933000013

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 19 Surface Analysis in an industrial Environment Robert K. Wild Nuclear Electric plc, Berkeley Technology Centre, Berkeley, Gloucestershire GL 13 9PB Introduction Industry uses surface analytical techniques to ensure quality, to improve productivity, to solve problems and to increase safety. A high-pressure steam pipe may have failed causing considerable damage, possible risk to staff and perhaps the public, and loss of production. Surface analytical techniques can be used to aid the investigation into the cause of the failure and could subsequently be used to monitor the quality of components to be used in future to prevent the recurrence of such a failure. Quality control is particularly important in the semiconductor industry where the surface condition of the component is of vital importance to the performance of the resulting electronic device.There are many surface analytical techniques available at present and decisions must be made as to which is the most suitable for the particular application. This is more important in surface studies because these techniques tend to be expensive and very few industrial companies are large enough to run a department which operates all the possible techniques. Increasingly, use is being made of the facilities and expertise that exist in many universities and much of the specialized analytical work is being undertaken under contract. As a result the relationship between industry and the universities has become much closer in recent years. This paper attempts to identify the different surface techniques, illustrate their strong and weak points and give some examples of where such techniques can be useful. For a complete description of the techniques available the reader is referred to Rivikre.1 Surface Analytical Equipment When deciding which technique is the most suitable to solve a particular problem or which piece of equipment should be purchased by a laboratory, careful consideration needs to be given to the requirements of the job and these then need to be matched to the techniques and equipment.If it is important to be able to obtain a good image of the surface and the analytical information is required from small areas then good spatial resolution is essential; if high sensitivity to certain elements is required or the chemical state of the surface must be determined then a different technique may be necessary.Some of the major techniques available in commercial form are listed in Table 1. In this table the type of probe, the species detected and the process involved in the technique are given. In any investigation it is important to cause as little damage, to the surface that is being probed, as possible. For this reason the least damaging technique should be considered first. In general, photons cause less damage than electrons which are, in turn, less damaging than ions or atoms. The energy and intensity of the beam must also be considered, an intense photon beam such as encountered in some lasers can be far more damaging than a low intensity ion beam, while a photon with a wavelength of a few hundred nanometres would cause less surface perturbation than an X-ray photon with a wavelength of a fraction of a nanometre.Infrared spectro- scopy (IRS), laser Raman spectroscopy (LRS) and X-ray photoelectron spectroscopy (XPS) all probe with photons and can identify elements and give some chemical state informa- tion. They do, however, have the drawback that the spatial resolution is limited to approximately 1 pm for the IRS and LRS techniques and to approximately 10 pm for XPS. There is also the difficulty of ensuring that the surface is being analysed. Both visible and infrared radiation penetrate several hundred nanometres into most solids and even when using reflection infrared, sampling is from a relatively large volume.If spatial resolution is an important criterion then electron or ion probes must be employed. Both Auger electron spectro- scopy (AES) and secondary ion mass spectrometry (SIMS) can give spatial resolutions of a few tens of nanometres while at the same time giving good element detection with some Table 1 Surface techniques available to industry Technique Infrared Raman (laser) X-ray photoelectron spectroscopy Low energy electron diffraction (LEED) Auger electron spectroscopy (AES) Secondary ion mass spectrometry (SIMS) Sputtered neutral mass spectrometry (SNMS) Scanning tunnelling microscopy (XPS) (STM) Probe Photon Photon Photon Electron Electron Ion Ion High field Detected Photon Photon Electron Electron Electron Ion Atom Electron current Process Bond vibrations Electronic excitation Ionization Elastic scattering Auger Sputtering Sputtering Electron tunnelling Table 2 Relative merits of surface analytical techniques Element Technique detected Sensitivity Quantitative Infrared Raman (Laser) XPS LEED AES SIMS SNMS STM Most Most All None All >He All All Some Few % Few YO 1 Yo N/A 1 Yo PP" NIA PPb Semi Semi Yes NIA Yes No Yes NIA Spatial resolution ym ym 10 ym <1 ym 20 nm 50 nm 50 nm 0.1 nm Chemical state Yes Yes Yes No Some Yes Yes Some Structural information No No No Yes No No No Yes NIA = Not applicable.20 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 Fig.1 Failure of a weld in a power station steam chest chemical state information. The relatively new technique of scanning tunnelling microscopy (STM) and the atomic force microscope (AFM) are, in principle, non-damaging and can give better resolution than any of the other techniques.In practice, considerable expertise is required to obtain the best from these instruments in a non-destructive manner. The STM can also give valuable structural information which together with low energy electron diffraction (LEED) can be useful in determining defect concentrations and surface damage. Finally, if sensitivity is important then the ion bombardment techniques should be employed. Most surface analytical techniques have a detection limit in the region of 0.1 at.-% but SIMS and sputtered neutral mass spectrometry (SNMS) can achieve ppm and in some cases ppb detection levels, often with high spatial resolution.The relative merits of the various techniques are listed in Table 2. Here the elements detected, the sensitivity, quantita- tive analysis, spatial resolution, chemical state and structural information that can be obtained are included. Applications Industry has a wide range of needs for surface analytical techniques. These range from control and development of semiconductors to mechanical properties of superalloys exposed at high temperature to large doses of radiation, from corrosion in both gaseous and aqueous environments to the performance of new materials such as carbon-fibre composites and metal-matrix composites and from catalysis in the chemical industry to adhesion, or the lack of it, between materials. Attempts will be made to give some examples of the use of surface techniques in a few of these industrial applications.The examples will come mainly from the nuclear or power generating industries but the principles apply to a much wider range of industries. North HP steam chest Fig. 2 Schematic diagram showing location of a steam chest failureANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 21 7 , I 1 t Crack tip Fig. 3 SAM Fracture surface of the crack from the steam chest opened in Mechanical Properties The mechanical properties of steels are frequently determined by the segregation of trace elements to grain boundaries together with the build-up of particles, typically carbides, at these boundaries. An example where a steel component has failed in a catastrophic manner is shown in Fig. 1. This is a steam chest in a conventional power station and is a relatively large component used to transport high-pressure steam between the boiler and the turbines and is shown schematic- ally in Fig.2. It is constructed from a ferritic steel and has failed in the vicinity of a weld. Here the history of the failure can be determined from crack dating but to find the cause of the failure we must look to the state of the bulk metal. Ferritic steel can be induced to fracture in an intergranular manner at liquid nitrogen temperatures. It is, therefore, a relatively straightforward matter to produce a fracture surface for analysis in a scanning Auger microscopy (SAM) or SIMS instrument. The specimen has to be cooled to close to 77 K and a sharp impact applied to the specimen. A typical fracture surface is shown in Fig.3. This was obtained by cutting a specimen in such a way that the pre-existing crack could be used as a notch to initiate the fracture path. The grains, ahead of the crack tip, are clearly visible as smooth surfaces with sharp boundaries between adjacent grains and the tip of the crack can also be seen running across the image. This problem requires a technique with good spatial resolution to identify 6 5 - w X Z F m F L3 c w 4 1 I x 3 2 1 n 100 200 300 400 500 600 700 800 900 1 ~ 0 0 Kinetic energyIeV Fig. 4 Auger spectrum recorded from the fracture surface in Fig. 3 Fig. 5 Example of intergranular fracture in alloy PE16 elements on the grain boundary. Auger spectroscopy has been used but SIMS could have been used if very high sensitivity were required.An Auger spectrum recorded from a grain is shown in Fig. 4. In this instance two embrittling elements, tin and copper, are observed to be present on the grain surface and these were undoubtedly, at least in part, responsible for the failure of this component. Phosphorus is a much more common segregant that causes embrittlement in ferritic steels and has resulted in the cracking of countless bolts in industrial plants. Consideration is being given to the use of this technique to determine the condition of components so that those that are susceptible to failure could be replaced and research is underway to predict more accurately the failure of steels from a knowledge of phosphorus content and grain size. The mechanical properties of nickel-based alloys are also determined by grain boundary segregants but these alloys are ductile at liquid nitrogen temperatures and it is necessary to hydrogen charge the specimen and then fracture by using a slow tensile strain.A nickel-based alloy PE16 (44 + 34 + 16% m/m, Ni + Fe + Cr, respectively) is used to support the heavy fuel assembly in our advanced gas cooled reactors (AGR) and it is important that the integrity of this rod is guaranteed throughout its life. The alloy becomes radioactive during its life yet must, of course, be examined. We have, therefore, developed a tensile fracture stage that is capable of fracturing small active specimens in the form of matchsticks less than 8 X 1 X 1 mm to reduce the operator dose.* The fracture surface of one of these alloys is reproduced in Fig.5 and looks similar to the ferritic impact surface. The Auger spectra from a grain boundary in a specimen in the ‘as manufactured’ condition is compared with the spectrum from a specimen following22 r: 0 .- .I.- F al 2 25 0 0 4- ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 - 7 (a) 6 - I I tNi I - Fe Fe 7- Ni I A 0 100 200 300 400 500 600 700 800 900 1000 - Z - 7 ] ( b ) 6 t "4 0 100 200 300 400 500 600 700 800 900 1000 Kinetic energylev Fig. 6 Auger s ectra from grain boundary of ( a ) as manufactured alloy PE16 and 8) irradiated alloy PE16 irradiation (Fig. 6). It is clear that there has been an enrichment of nickel and a depletion of chromium and iron together with segregation of phosphorus and silicon. The nickel enrichment is considered to be the result of the diffusion rates of the major alloying species.Irradiation produces a large number of point defects (vacancies and interstitials). These move through the lattice in a random manner but on encountering a sink such as a grain boundary will be annihilated. This produces a net flux of point defects towards the grain boundaries. A flow of vacancies towards grain boundaries implies a flow of atoms in the opposite direction; however, the rate of flow will depend on the jump frequency of the particular atom.3 It has been determined that the jump frequency, J , of chromium > J(iron) > J(nickel);4 hence, as chromium and iron flow away from the boundary at a greater rate than nickel the boundaries become enriched in nickel by default.The effect can be reversed by annealing the alloy at 600 "C for 1 h. Fig. 7(a) and ( b ) show the levels of grain boundary components for the major and trace elements, respectively, in the 'as received', irradiated and annealed conditions. This indicates that the annealing returns the alloy at least partially to its original condition. Corrosion In the nuclear industry the 20% Cr-25% Ni/Nb stabilized stainless steel is used to clad the uranium dioxide fuel and must operate at temperatures of up to 1000 K in a C02-1% CO gas environment at a pressure of 40 atm (4.05 MPa). A potential problem was envisaged with this alloy. During thermal cycling it was thought that the oxide which formed on this alloy might J. o t 1 1 I I Arch Matrix Arch G.B. lrrad Anneal Treatment 4 3 s 1 C 0 .- c c F 2 al Z ' 0 0 -1 I I I I Anneal Arch Matrix Arch G.B.lrrad Treatment Fig. 7 Grain boundary composition of alloy PE16 as a function of various treatments. (a) For bulk elements: A, nickel; B, iron; and C, chromium. ( b ) For trace elements: A, silicon, B, phosphorus; and C , molybdenum spa11 off and be transported around the coolant circuit. The spalled oxide would be radioactive and could lead to high levels of radiation at points around the coolant circuit. It is, therefore, important that the mechanisms of corrosion and spallation are understood and that the composition of the oxide is determined. Several techniques can be utilized to determine the oxide composition and to determine the mode of spallation. The XPS, AES, SIMS, SNMS, Raman and laser Raman tech- niques can be used to characterize the outer oxide surface.Laser Raman spectroscopy has been applied to the outer atom layers of the oxide that forms on this alloy. Fig. 8 shows a series of Raman spectra obtained from oxide films formed on 20% Cr-25% Ni/Nb stabilized stainless steel following 24 h oxidation in C02-4% CO-300 ppm v/v CH4-300 ppm v/v H20-400 ppm v/v H2 at temperatures between 600 and 900 "C.s At the lower temperature only the spinel oxide forms but as the temperature increases the rhombohedra1 Cr203 phase begins to form. To characterize the oxide the composition must be obtained as a function of depth. This can be carried out by depth profiling through the oxide using ion bombardment while using a surface-sensitive technique to monitor the composi- tion.As spatial resolution was not important and the chemical state of the oxide was required XPS was used in combination with argon ion bombardment. The results of this are shown in Fig. 9 and indicate that the oxide is duplex in nature with an outer layer approximately 0.4 pm thick consisting of a chromium manganese layer on top of a chromium-rich layer also 0.4 pm thick. As a large area has been depth profiled, X-ray diffraction (XRD) could be used to obtain structural information concerning the oxide layers. X-ray diffractionANALYTICAL PROCEEDINGS. JANUARY 1993, VOL 30 23 traces taken before depth profiling and after 0.4 pm has been removed are shown in Fig. 10. The spectrum recorded before etching showed both spinel and rhombohedral oxide peaks but the diffraction pattern recorded after profiling to a depth of II I /I Fe304/spinel Spine' (broad feature) c 4- - 1000 900 800 700 600 500 400 300 Raman shiftkm-' Fig.8 Raman spectra obtained from oxide film formed on 20% Cr-25% Ni/Nb stabilized stainless steel for 24 h in C 0 2 at various temperatures Oxygen Ox id e/oxid e Oxide/metal Oxide'gas interface - interface i n te rface , . . . ...- 0 0.2 0.4 0.6 0.8 1 .o 1.4 Depth profiled/pm Fig. 9 Cr-25% Ni/Nb stabilized stainless steel XPS depth profile through the oxide formed on a 20% 0.4 pm showed only rhombohedral peaks, indicating that the outer layer was the spinel oxide. The bulk of the oxide could therefore, be characterized as an outer chromium-manganese spinel superimposed on a rhombohedral chromium oxide.If one is attempting to determine very low levels of certain trace elements then SIMS should be used. A low level of yttrium was added to an experimental alloy to determine if it could usefully be used to reduce the rate of corrosion and SIMS was then used to determine the position of the yttrium in the oxide. bSpinel+Cr203e- 20-25/Nb Steel 106 105 v) 104 103 F I v) c 3 c1 .- v) S g 102 - . - 0 10 20 Sputtering time/min 30 Fig. 11 20% Cr-25%0 Ni/Nb stabilized stainless steel SIMS depth profile through oxide formed on yttrium doped Stage / I l l 1. Vapour deposit nickel foil at -300 "C in glow cut -,-A discharge Stage 3. Cut along end of nickel dated foil stresses between oxide and nickel cause nickel Cut - to peel away exposing metal/oxide Nickel ' Oxide Foil interface Stage 2.Cut along edges of nickel plated foil Fig. 12 Sputter ion placing method to reveal the rnetal/oxide interface 65 60 55 50 45 40 35 30 25 2H Fig. 10 XRD Datterns recorded from the surface of the oxide in Fig. 8:Ta) before de'pth profiling and ( b ) after profiling through 0.4 pm. 8, austenite: R, rhombohedral phase (= Fe203 in Crz03): S, manganese spinel (MnFe, Cr2-, 0,: 0 d x d 2) Cr-25% Ni/Nb stabilized stainless steel Fig. 13 ( a ) Metal and ( b ) oxide sides of the interface on 20%24 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 Fig. 14 Auger element maps from the metal side of the metalloxide interface: ( a ) SEI; ( b ) Fe; ( c ) Cr; and (d) Si An example of a SIMS depth profile through an oxide on this steel that had been implanted with yttrium is reproduced in Fig.11.6 The level of yttrium can be seen to vary by more than four orders of magnitude across the oxide layer. An attempt was made to simulate spalling of the oxide layer using a technique of sputter ion coating (Fig. 12).7 Nickel was plated onto the outer oxide surface in the presence of an argon ion plasma discharge at a temperature of 500 K. The plasma discharge cleans the outer surface and aids the bonding of the nickel to the oxide while the elevated temperature causes strains to be set up on cooling to room temperature between the layer and the oxide which in turn causes the oxide to spa11 away from the substrate alloy. Scanning Auger microscopy was used to analyse the two sides of the metal/oxide interface. Secondary electron images of the interface are shown in Fig.13.8 The oxide appears to have pulled away from the metal pulling out plugs of oxide from the positions of the grain boundaries. The spatial resolution of SAM is of the order of 100 nm and is therefore more than adequate to study boundaries of 10 pm in size. Spectra can be recorded from various points on the grain boundary surface but it is qualitatively more instructive to produce element maps across the surface. Typical examples are shown in Fig. 14 for the metal side of the interface for chromium, silicon and iron. It can be seen that chromium and silicon concentrate at the grain boundaries on the metal side. Similar studies on the oxide side of the interface indicate that chromium and silicon concen- trate at the grain boundaries but that silicon alone is present at the grain centre.Depth profiling through this silicon layer at the centre of the grain shows that the silicon layer is only 20 nm thick. Laser Raman spectroscopy can be used to study the change in stresses at points across the oxide and this may be important in determining the adherence of the oxide during thermal cycling. Stress in solids affects the molecular vibra- tions, generally moving them to higher frequency and thus producing a Raman shift to higher frequencies. This change in Raman frequency with stress has been used to determine the stress across a Cr203 oxide formed on stainless steel.9 Fig. 15 shows the appearance of the corrosion scale with low ridges formed over the grain boundaries of the substrate metal.The stress-induced shift in the Raman band of Cr203 is plotted against position in Fig. 15 and reveals significant stress relief at the grain boundaries. 560 ""I 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Dista nce/ym Fig. 15 laser Raman Determination of stress in oxide using frequency variation in Combining these results allows the oxide to be charac- terized and the mode of spallation to be determined. Indeed the oxide spalls by fracturing on the metal side of the silicon oxide layer at the grain centres but it fractures through the oxide at the grain boundaries. This has the effect of leaving a plug of silicon-chromium oxide in the grain boundary whichANALYTICAL PROCEEDINGS, JANUARY 1993. VOL 30 25 . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . ' oxide Outer) Spinel Carbon rich layer Silicon rich layer ri::} Rhombohedral Fig. 16 Schematic diagram of oxide on 20y0 Cr-25y0 Ni/Nb stabilized stainless steel Fig. 17 Low energy diffraction pattern from silicon(ll1) surface reduces cation diffusion along the boundary and hence reduces the subsequent rate of oxidation. This is summarized in Fig. 16 where the oxide composition is shown in schematic form and the spallation path indicated. Semiconductors Semiconductor production and development have become fundamental to the running of the world as we now know it. Almost all electronic equipment contains many of these components. The level of reliability must be high and to achieve this, manufacture must be carried out in conditions of maximum cleanliness to prevent surface contamination.Sur- face analytical techniques are used extensively to monitor cleanliness, to determine dopant concentrations and to aid in developing new products. The quality of the silicon can be determined in a number of ways, the bulk structure can be determined using conventional XRD techniques but the structure of the surface requires surface diffraction and here LEED can identify the structure. In one example the LEED pattern from an Si(ll1) surface is reproduced in Fig. 17. This diffraction pattern might imply that the surface structure was perfect as it appears to give only reflections in the positions expected from a good silicon crystal. However, if the same surface is examined using STM it becomes clear that the surface is far from perfect and in fact contains many defects (Fig.18). 10 Analysis of the material can be carried out using a technique with spatial resolution better than 1 um as this will allow the various pathways in the device to be resolved. This makes XPS a borderline technique although some good work has been carried out using the imaging ESCASCOPE, but AES and SIMS are most frequently used. The former is generally preferred if accurate quantitative determinations are required but SIMS must be used to detect low levels of impurities and dopants. Frequently, several layers of material are deposited onto a silicon substrate and these may be only a few tens of nanometres thick and it is important to be able to measure the thickness of the layers following deposition.Depth profiling techniques must be used, either in conjunction with AES or SIMS. Fig. 19 shows a depth profile through an Fig. 18 Scanning tunnelling microscope image of silicon( 11 1) surface AlGaAs multilayer quantum well structure. It contains 30 repeating layers of GaAs substrate each 14 nm thick with 6 nm thick layers of A1,Gal-,As between them all, on top of a 400 nm thick layer of GaAs on 9 layers of 4 nm GaAs, between 4 nm thick A1,.Gal-,.As, on GaAs substrate. This demonstrates how many very thin layers can be successfully resolved. Many semiconductor devices are encapsulated in plastics which presents the analyst with particular problems, particularly if he/she is attempting to identify a cause of failure. In these cases the non-destructive technique known as infrared micro- scopy (IRM) can be utilized.This technique can be used to identify failures in encapsulated devices." The trick is to mount the complete device and then polish back mechanically to the silicon substrate (Fig. 20). As silicon is transparent to infrared radiation and IRM has a spatial resolution of 1 pm the surface of the device may be investigated through the silicon substrate. In particular, corrosion at pin locations can be detected and the effect of operating the device can be monitored using this technique. In the example shown here a 14 pin CMOS chip is studied. Fig. 21 shows this component26 0 50 100 150 200 250 300 350 400 450 500 Sputtering time/mi n Fig. 19 Depth profile through an MBE grown AlGaAs multilayer quantum well structure Fig.20 operating surface Polishing a CMOS device to allow infrared studies of the ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 (4 after being polished to the back side of the silicon substrate and viewed by illuminating with infrared radiation. By connecting the device and applying a voltage to the approp- riate pin the device could be triggered to operate. By raising the voltage further it was possible to increase the dissipation in the polysilicon input resistor until the associated thermal emission was clearly identifiable as the light coloured region in Fig. 21(b). Continued application of such power, for even short periods of time, would be likely to result in permanent damage to the device. In this study, fusion from the input resistor occurred after approximately 1 s as can be seen in Fig.21(c). Clearly this method of viewing an interface from below has considerable potential and may be applicable to IR spectroscopy. Conclusions Surface analytical techniques are being used increasingly by industry to solve problems and maintain quality. This has become possible as a result of the basic work undertaken at universities and other major research establishments to understand the techniques fully and as a result of manufactur- ers producing reliable, easy to use equipment that can give results for a large number of specimens quickly. Some techniques are used less than they might be by industry because they have not yet reached this level of sophistication. Laser Raman instruments have only recently been developed to the stage where they are sufficiently robust to be used by industry while STM is a technique with potential that is still to be realized. These and some other techniques will remain in universities for some years yet, but will be utilized by industry Fig. 21 Infrared microscopy of a 14 pin CMOS chip: ( a ) after polishing: ( b ) during operation: and (c) after operation to a level which caused failure on a contractural basis thus maintaining the important link between the two areas. References 1 Riviere. J. C.. Surface Analytical Techniques, Clarendon Press, Oxford, 1990.ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 27 2 3 Nettleship, D. J.. and Wild, R. K., Surface Interface Anal., 1990, 16. 552. Perks, J. M., Marwick, A. D., and English, C. A., Proc. Radiation Induced Sensitisation of Stainless Steels, Berkeley Nuclear Laboratories, Berkeley, 1986, pp. 86-98. Marwick, A. D., Piller, R. C., and Horton, M. E., AERE Report 10895, Atomic Energy Research Establishment, Har- well, 1983. Bennett, M. J., Proceedings of the Conference on UK Corro- sion, 1984, p. 43. 4 5 6 Bennet, M. J., Bishop, H. E . , Chalker, P. R., and Tuson, A. T., United Kingdom Atomic Energy Authority Report A E R E R 12540, Atomic Energy Research Establishment, Harwell, 1987. 7 Coad, J . P., and Wild, R. K., Appl. Surface Sci., 1983,14,321. 8 Wild, R. K., Spectrochim. Actu, Part B , 1985, 40, 827. 9 Gardiner, D., and Bowden, M., Microsc. Anal., 1990, 20, 27. 10 Tear, S. P., Microsc. Anal., 1990, 19, 7 . 11 Ford, D. J., Microsc. Anal., 1990, 17. 39.

ISSN:0144-557X    

DOI:10.1039/AP9933000019

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 27 Surface Analysis and Adhesive Bonding J. Comyn Centre for Surface and Materials Analysis, Armstrong House, Oxford Road, Manchester M 7 7ED For work on adhesive bonding there are four experimental techniques that are used on a routine basis and which provide much useful information. The purpose of this paper is to review briefly these techniques. There are other techniques that are not yet routine, but which have been applied to adhesive bonding; they include inelastic electron tunnelling spectroscopy (IETS)1-3 and surface enhanced Raman spectro- scopy (SERS) .4 Contact Angle Measurement A useful and simple way of assessing solid surfaces is to measure the contact angle made by small liquid drops. At the centre of the method is an equation due to Fowkess which is Here 0 is the contact angle and yL is the liquid surface tension.The dispersive and polar components of the surface energy of the liquid are yLd and yLP, and ysd and ysP are those of the solid surface. If contact angles are measured for a number of liquids and yL (1 + c0s6)/2(y~d)~/2 is plotted against ( ~ ~ P / y ~ d ) l / ~ , the graph should be linear with intercept (ysd)l/2 and slope (ysP)l/*. A list of available test liquids, basically arranged in decreasing order of the x-parameter of the plot, is shown in Table 1. For a liquid the surface tension and surface free energy are numerically the same, but have units of mN m-1 and mJ m-2, respectively. This information is of value in the selection of adhesives in that the optimum condition is to match the dispersive and polar components of the adhesive and substrate.Surfaces that are difficult to bond such as the polyalkenes have low surface energies which are almost entirely dispersive in character. Surface treatments which render such materials bondable cause increases in the polar component of the surface energy. The thermodynamic work of adhesion6 can be calculated from the polar and dispersive components of the two phases. More important, however, is that the thermodynamic work of adhesion in the presence of water can also be calculated; this will indicate whether a bond is stable or not in wet conditions. Water is a polar liquid which has a tendency to displace adhesives and paints from metal surfaces. Water displacement is not generally a problem for paints or adhesives on plastics because the surfaces are much less polar than metals.However, if one surface has polar groups such as carboxylates, then in the presence of water there may be a significant reduction in the work of adhesion and hence in the stability of the bond. Surface Infrared Spectroscopy In multiple internal reflectance (MIR) the sample is placed in contact with a prism of either germanium or thallium bromide iodide, which can be placed in either a dispersive or Fourier transform spectrometer. The spectra obtained closely resemble conventional transmission infrared (IR) spectra. The sampling depth is about the wavelength of the radiation used. As this is many thousands of molecular layers, it is thus much less surface sensitive than X-ray photoelectron spectro- scopy and static secondary ion mass spectrometry (SSIMS).It is important that the sample makes good optical contact with the prism, and for this reason it is easier to work with rubbery polymers. Multiple internal reflectance has been applied to the oxidative surface treatment of polyalkenes, where it shows the formation of carbonyl groups. However, because of the relatively large sampling depth, the samples were probably treated to a greater depth than is actually required for the purposes of adhesive bonding or printing. Diffuse reflectance in Fourier transform (DRIFT) is parti- cularly useful in that powdered samples can be examined without further preparation; the method has been used extensively by Koenig to study silane coupling agents on powdered silica.With reflection-absorption infrared spec- trometry (RAIR) a beam that is polarized parallel to the plane of incidence is reflected from a polished metal mirror at a high angle of incidence (about SOo). It has been employed by Boerio in studying silane coupling agents on a number of metals. The application of these two techniques to silane coupling agents has been reviewed by the author? Great advantages are conferred on the Fourier transform infrared (FTIR) spectrometer by the incorporation of a computer. These include multiple scanning to improve signal- to-noise ratio, and the ability to produce difference spectra. The latter has been used by Naviroj et a1.8 to study the adsorption of 3-aminopropyldimethylethoxysilane on titan- ium and aluminium oxides.The difference spectra (metal oxide treated with silane-metal oxide) revealed weak bands at 950 and 963 cm-1, which are respectively assigned to Si-0-Ti and Si-0-A1 groups. X-ray Photoelectron Spectroscopy (XPS) In this technique a monochromatic beam of soft X-rays from an A1 or Mg target is directed at a surface in an ultra-high vacuum chamber. Electrons are ejected from the core levels of atoms in the surface, and their binding energies which are characteristic, are determined. An XPS spectrum can have a number of peaks arising from electrons emitted from different core levels, and when the relative atomic sensitivities are taken into account, this can give an atomic analysis of the surface. Hydrogen is not detected by XPS because it has no core electrons.Although the X-rays penetrate deep into the sample, only electrons from near the surface are able to escape and be detected; the sampling depth is about 5-10 nm. This technique was used by Brewis and Briggs9 to demon- strate which chemical groups are introduced on to polyalkene28 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 Table 1 Liquids for contact angle measurements Liquid Water Glycerol Ethane-1,2-diol Formamide Ethanol Dimethyl sulfoxide 2-Ethoxyethanol Dimethylformamide Dime thylsiloxanes Methylene diiodide Tricresyl phosphate Trichlorobiphen yl Pyridine Hexadecane 1-Bromonaphthalene yLd/mJ m-2 21.8 f 0.7 37.0 k 4 29.3 39.5 _+ 7 17.0 34.86 23.6 32.42 16.9 k 0.5 48.5 k 9 50.76 39.2 f 4 44 k 6 37.16 27.6 47 _+ 7 yLP/mJ m- 51.0 26.4 19.0 18.7 5.4 8.68 5.0 4.88 2.1 2.3 1.7 1.3 0.84 -2.4 -2 IL/mJm-2 72.8 63.4 48.3 58.2 22.4 43.54 28.6 37.30 19.0 50.8 50.76 40.9 45.3 38.00 27.6 44.6 (YL~/Y")"' 1.529 2 0.035 0.845 -t 0.11 0.805 f 0.14 0.688 2 0.19 0.563 0.499 0.460 0.388 0.352 0.218 k 0.446 0.0 0.208 0.172 0.150 0.0 0.2261 _+ 0.14 surfaces by flame and corona treatment.It is particularly useful at detecting contamination involving a heteroatom, such as might be the case with silicone and fluorocarbon mould release agents. Static Secondary Ion Mass Spectrometry This technique places the sample in an ultra-high vacuum chamber and bombards it with ions ( A r f , Xe+ or G a t s 4 keV). The surface then emits secondary ions which are analysed in a mass spectrometer, and in separate experiments both the positive and negative ion spectra can be obtained.In static, as opposed to dynamic, SIMS the primary ion current is so low (=1 nA cm-2) that the surface is not significantly ablated. Data are obtained from the top few molecular layers. A major problem with insulators is the build up of surface charge. This can be avoided by flooding the surface with low energy ( ~ 7 0 0 eV) electrons. The SSIMS spectra are complicated and it is difficult to assign all the mass peaks. However, there have been a number of instances where it has been very successful in identifying surface contamination. One is the detection of ethylene bis(stearamide) on the surface of a polyurethane,lO from the presence of peaks at 282 and 310 u, which are due to the following cleavage reaction: C 17H35-CO-NH-CH2-CH2-NH-CO-C 17H35 + C17H35-CO-NH+ + C17H35-CO-NH-CH2-CH2f 282 u 310 u Silicone contamination is a common problem in adhesive bonding, and polysiloxane contaminants can be readily identified by their fragmentation patterns.1 2 3 4 5 6 7 8 9 10 References Comyn, J., Oxley, D. P., Pritchard, R. G., Werrett, C. R., and Kinloch, A. J., Int. J. Adhes. Adhes., 1989 9, 201. Comyn, J . , Horley, C. C., Pritchard, R. G., and Mallik, R. R., Adhesion, 1987, 11, 38. Comyn, J., Horley, C. C., Oxley, D. P., Pritchard, R. G., Werrett, C. R., and Tegg, J. L., Br. Polym. J . , 1983, 15, 50. Creighton, J. A., in Advances in Spectroscopy, eds. Clark, R. J. H., and Hester, R. E., Wiley. Chichester, 1988, vol. 16, Fowkes, F. M., Ind. Eng. Chem., 1964, 56,40.Comyn, J., in Durability of Structural Adhesives, ed. Kinloch, A. J., Applied Science Publishers, Barking, 1983, ch. 3, pp. Comyn, J.. in Structural Adhesives: Developments in Resins and Primers, ed. Kinloch, A. J., Elsevier, Barking, 1986, ch. 8, pp. 269-3 12. Naviroj, S . , Koenig, J. L., and Ishida, H.. J. Adhesion, 1985,18, 93. Brewis, D. M., and Briggs, D., Polymer, 1981, 22, 7. Briggs, D., Surface Interface Anal., 1986, 9, 391. ch. 2, pp. 37-89. 85-131. Surface Enhanced Raman Spectroscopy J. Alan Creighton Chemical Laboratory, University of Kent, Canterbury CT2 7NH Surface-enhanced Raman spectroscopy (SERS) is becoming recognized as a powerful method for obtaining detailed information on the first monolayer of molecules at certain surfaces. It combines the high structural information content of vibrational spectroscopy with the very high sensitivity and surface specificity which come from the special enhancement mechanism of SERS.Moreover, it is a non-UHV (ultra-high voltage) technique. It is thus very well suited to in situ analyses of surfaces in a wide range of ambient media, and is beginning to find use in fields as diverse as heterogeneous catalysis, electrochemistry and corrosion, adhesion, surfactants, or the interaction of bio-molecules with metal surfaces. Unfortu- nately, it has the limitation that it is only applicable to certain surfaces, namely those of free-electron metals, particularly copper, silver and gold, although some progress has recently been made towards partially overcoming this limitation, as is described below.There have been several reviews describing applications of SERS,1-3 and a number of recent reviews give detailed accounts of its mechanism.4 A more general account of SERS is included among the papers presented at the previous SATA symposium.-5 In its experimental set-up SERS does not differ greatly from normal Raman spectroscopy. The surface is irradiated with monochromatic light from a laser and the scattered light is collected and analysed. The inelastically scattered component in this scattered light gives the vibra- tional spectrum of the molecules adsorbed at the surface. For most surfaces the intensity of Raman bands from the surface monolayer is very low, and indeed, is normally undetectable. If the surface is a suitably roughened or finely divided surfaceANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 29 of copper, silver or gold, however, the SERS effect then amplifies the first monolayer Raman scattering by a factor of up to 105, giving easily detectable Raman signals specifically from the first monolayer.The objective of this paper is to highlight some recent applications of SERS which relate to problems of applied interest. Before doing this, however, it is helpful to examine, briefly, the mechanism of the enhancement in order to understand some of the special features of SERS, such as its surface specificity and its restriction only to certain surfaces. There appear to be two enhancement effects, each of which contributes 2-3 orders of magnitude to the over-all Raman intensity enhancement. One of these, often called the electromagnetic SERS effect, depends on the response of the conduction electrons of the metal surface to the electromag- netic field of the incident and scattered light.It is necessary for SERS that the metal surface is very finely divided, and the most widely used surfaces have thus been metal electrodes that have been roughened by electrochemical oxidation- reduction cycling, evaporated metal films deposited at very low temperatures in high vacuum, or colloidal metal disper- sions. The essential feature of this fine state of division is that the conduction electrons of the metal then have localized ‘plasma’ oscillations at optical frequencies, and these oscilla- tions radiate a secondary electromagnetic field close to the surface which augments the incident and scattered electro- magnetic fields.In the electromagnetic SERS effect this augmenting of the surface electromagnetic field can become fairly large because the frequency of the incident light is chosen so that there is a resonant excitation of the plasma oscillations at the surface. Because the large surface electro- magnetic fields depend on this resonant excitation it follows that there is a laser frequency- and surface morphology- dependence in the magnitude of the electromagnetic SERS effect, and that only metals which give undamped plasma resonances in the visible or near-infrared range, namely the essentially free-electron metals, can show the effect. The other enhancement mechanism, often referred to as the chemical SERS effect, arises if the incident radiation is in resonance with a charge transfer transition between the surface and the adsorbed molecules.The effect of such a resonance is to enhance the polarizability of the first layer of molecules at the surface at that frequency, and thus to give rise to enhanced light scattering by the well established resonance Raman mechanism. It is this ‘chemical’ effect, rather than the electromagnetic contribution, which gives the SERS effect its high degree of specificity to the first monolayer. The requirement that there is a surface-adsorbate charge transfer transition in resonance with the incident frequency restricts the adsorbates to some extent, and also the surface sites, which exhibit the chemical SERS effect at a given excitation wavelength, although this restriction is not as limiting as might at first be expected (presumably because of the breadth of the metal bands which are the donor or acceptor levels in such surface-adsorbate charge transfer transitions).Thus it is found that molecules such as alkanes which do not have suitable orbitals to participate in surface-adsorbate charge transfer transitions give very much weaker SERS spectra than alkenes or aromatics with filled low-energy n and empty n* orbitals. It has also become increasingly apparent that there may be sites of widely differing SERS activity on a given surface, and there is currently considerably interest in elucidating the nature of these SERS-active sites (see below).4 A straightforward application of SERS which demonstrates its unique capability for in situ analysis is in competitive adsorption studies at surfaces under normal ambient condi- tions.An example of such a study is a recent measurement by Musiani et a1.6 of the competitive adsorption of corrosion inhibitors at copper surfaces under aqueous conditions. An interesting feature of this work is that it was carried out using a Fourier transform (FT)-Raman spectrometer with near- infrared excitation at a wavelength of 1.064 ym. Most SERS measurements to date have been made with visible-range excitation. It has been known for some time, however, that with copper and gold surfaces it is necessary to use excitation wavelengths no shorter than about 600 nm, and that with these metals the SERS intensity continues to increase into the far red.Recent measurements by Chase and Parkinson7 have shown that the maximum SERS enhancement for copper and gold surfaces, at least for the electrochemically roughened surfaces which they investigated, is obtained at excitation wavelengths even longer than 1.064 pm in the near-infrared, and the excellent quality of SERS spectra which may be obtained for copper surfaces with an FT-Raman spectrometer and 1.064 pm excitation is illustrated by recent work by Sockalingum et al.8 The high degree of surface specificity of SERS has recently been exploited to provide a new method for measuring thin-film diffusion. The thin film is deposited on an SERS- active silver or gold surface and the film is exposed to the diffusate, the spectrum for which appears when the diffusate reaches the SERS-active surface.This method has been applied by Hong et al.9 to the measurement of the self- diffusion of polystyrene at 170°C (the diffusion of per- deuteriopolystyrene through polystyrene), and by Blue et al.,10 who developed a more complete analysis of the experimental data to obtain accurate values for the diffusion coefficient of ethylene through pyrazine over a range of very low temperatures. An earlier use of the surface specificity of SERS to give an analytical measurement relating only to the metal interfacial side of a surface film is that of Hutchinson et al.,” who compared the extent of an electrochemical reaction (the interconversion of thionine and leucothionine) at the metal-film interface measured by SERS with the extent of the reaction in the film as a whole, measured by coulometry.From the hysteresis between the two measure- ments as the electrode potential was cycled, it was deduced that the electrochemical oxidation or reduction began at the solution side rather than at the metal side of the film, and is thus essentially a solution- rather than a metal-interfacial phenomenon. An application of SERS to perhaps a more conventional thin film analytical problem is that of Knight et al. ,12 in which diamond films deposited on silicon by cold vapour deposition (CVD) were characterized by Raman spectroscopy. For very thin films the detection of the characteristic Raman band of diamond was greatly improved by sputtering on to the films a silver overlayer of thickness about 5 nm.This is one of several papers which illustrates a recent important development in SERS, namely the use of the enhanced electromagnetic fields which extend out by 10 A or more from an SERS-active silver or gold surface by virtue of the electromagnetic SERS effect, to obtain enhanced Raman scattering from the adjacent non-SERS-active medium. This method of depositing a particulate silver overlayer has been used, for example, by Alsmeyer and McCreeryl3 to sharpen up the sampling depth in the Raman measurement of the surface defect density induced in graphite electrodes by laser irradiation or electro- chemical anodization. The electron transfer rate between these electrodes and solution species is found to correlate with this surface defect density, but only the first few atomic layers control the electrochemical behaviour.This sharpening of the sampling depth comes about because of the short range of the enhanced electromagnetic fields induced by the silver over- layer, and the correlation with the graphite electrode electron transfer rate was thus better in the presence of the silver overlayer (sampling depth about 20 A) than with the normal Raman sampling depth for graphite of about 135 A. There have also been SERS studies of indium phosphide14 and tin oxide15 surfaces where the SERS is similarly induced by the deposition of a discontinuous thin silver overlayer. An important group of such studies is where the non-SERS-30 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 active surface is that of another metal.For these surfaces the use of a roughened silver or gold substrate, with the non-SERS-active metal deposited as a thin continuous film, has been found to be the most effective sample arrangement. Several studies of this type have been directed to iron films in an aqueous electrolyte.16J7 In one such study by Mengoli et al. , I 6 approximately 30 atomic layers of iron were deposited electrochemically on to a pre-roughened silver electrode. On exposure to the adsorbate pyridine, this electrode showed an SERS spectrum attributed to pyridine adsorbed on iron, which was clearly different to that of pyridine adsorbed on the underlying silver, and there was also a band attributed to chloride ions adsorbed on iron. Another group of recent papers concerns SERS studies by Weaver et al.18-20 of various adsorbates and electrochemical reactions at the surface of thin continuous films of platinum-group metals deposited on roughened gold substrates. An example is an investigation by SERS of the electrochemical behaviour of CO on platinum. 18 Approximately two atomic layers of platinum were deposited electrochemically on the gold substrate, and the completeness of the film was checked by cyclic voltammetry, which showed predominantly oxidation or reduction waves at potentials corresponding to the formation or removal of platinum oxide. On exposure of the electrode to CO, the SERS spectrum showed well-resolved bands due to CO adsorbed at sites both on platinum and on gold, and the loss of these bands due to electrochemical oxidation at positive potentials was also observed.A parallel study of CO adsorption on a palladium thin-film electrode gave similar results, but showed the greater tendency for adsorption of CO on palladium which has long been known from infrared studies on palladium in UHV. A very interesting feature of this work is the observation that the stretching frequency of CO adsorbed on the platinum electrode is about 10 cm-1 lower in the SERS spectrum than in the surface infrared spectrum, and the potential at which the SERS band disappears due to electro-oxidation of adsorbed CO is about 0.3 V more positive than the potential at which the surface infrared band disappears or at which electro- oxidation is observed by cyclic voltammetry. This suggests that SERS is detecting a sub-set of the totality of adsorbed CO molecules and that these SERS-active sites are particularly tightly binding (and therefore particularly reactive).There are other pieces of evidence which lead to similar conclusions. Among these are some recent results of Gu et a1.21.22 relating to the epoxidation of ethylene in which data from SERS and electron energy loss spectroscopy (EELS) are compared. In one of these studies21 the decomposition of ethylene oxide at a silver surface at 160 K was observed to give rise to a strong SERS band of CO, but in the EELS spectrum no CO was detected at this temperature. In the other study22 a silver surface was exposed to 1,2-dichloroethane at 55 K. From the SERS spectrum it appeared that the adsorbed dichloroethane was completely converted into ethylene at this temperature, whereas the EELS spectrum of the same surface showed only unchanged dichloroethane.This seems clear evidence that the SERS-active sites are particularly reactive, and it was sugges- ted that they may indeed be the sites on the silver surface which are catalytically the most effective. Other evidence relating to the existence of particular SERS-active sites has been reviewed by Otto$ and includes the quenching of SERS from silver surfaces at low temperatures by small amounts of adsorbed oxygen, which adsorbs only at high index surface sites and which thus further indicates that high index sites are the sites of the SERS activity. The fact that there is now strong evidence that SERS gives information that relates only to minority sites and is not representative of a surface as a whole is an intriguing result.There are already several other analytical techniques which fulfil the requirement of giving information which is represen- tative of surfaces as a whole, and it could turn out to be an advantage that SERS is able to probe only certain of the surface sites.4 It will be necessary to identify more clearly the nature of these sites, but the possibility that SERS is not only highly surface specific, but also on some catalytically active surfaces may be specific to the actual catalytic sites them- selves,22 is an exciting idea. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 References Chang, R. K., and Laube. B. L., CRC Crit. Rev. Solid State Muter.Sci., 1984, 12, 1. Pockrand, I.. Springer Tracts in Modern Physics, 1984, 104, 1. Cotton, T. M., Kim, J.-H., and Chumanov, G. D., J . Raman Spectrosc., 1991, 22, 729. Otto, A., Mrozek, I., Grabhorn, H., and Akemann, W., J. Phys.: Condens. Matter, 1992, 4, 1143, and references cited therein. Creighton, J. A , , in Surface Analysis Techniques and Applica- tions, eds. Randell, D. R., and Neagle, W., Royal Society of Chemistry, Cambridge, 1990, p. 13. Musiani, M. M., Mengoli, G . , Fleischmann, M., and Lowry, R. B., J . Electroanal. Chem., 1987, 217, 187. Chase, B., and Parkinson, B., J. Phys. Chem., 1991, 95,7810. Sockalingum, D., Fleischmann, M., and Musiani, M. M., Spectrochim. Acta, Part A , 1991, 47, 1475. Hong. P. P., Boerio, F. J., Clarson. S . J., and Smith, S .D., Macromolecules, 1991, 24, 4770. Blue, D., Helwig, K., and Moskovits, M., J . Phys. Chem., 1989, 93, 8080. Hutchinson, K., Hester, R . E., Albery, W. J., and Hillman, A. R., J . Chem. SOC., Faraday Trans. I , 1984, 80, 2053. Knight, D. S . . Weimer. R., Pilione L., and White, W. B., Appl. Phys. Lett.. 1990, 65, 1320. Alsmeyer, Y. W., and McCreery, R. L., Langmuir, 1991, 7, 2370. Feng, Q., and Cotton, T. M., J . Phys. Chem., 1986, 90, 983. Kaul, B. B.. Holt, R. E., Schlegel, V. L., and Cotton, T. M., Anal. Chem., 1988, 60, 1580. Mengoli, G., Musiani, M. M., Fleischmann, M., Mao, B., and Tian, Z. Q., Electrochim. Acta, 1987, 32, 1239. Uehara. J., Nishihara, H., and Aramaki, K., J . Electrochem. SOC.. 1990, 137, 2677. Leung, L.-W. H., and Weaver, M. J ., J . Am. Chem. Soc., 1987, 109. 5113. Wilke, T., Gao, X., Takoudis, C. G., and Weaver, M. J . , Langmuir, 1991, 7, 714. Feilchenfeld, H.. and Weaver, M. J . , J . Phys. Chem., 1991,95, 7771. Gu, X. J.. Akers, K. L., and Moskovits, M., J . Phys. Chem., 1992, 96, 383. Gu, X. J., Akers, K. L., and Moskovits, M., J . Phys. Chem., 1991, 95, 3696.ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 31 Surface Reactivity of Anti-wear Additives D. Landolt and H. J. Mathieu Labomtoire de Metallurgie Chimique/Departement des Materiaux, Ecole Polytechnique Federale de Lausanne (EPFL), MX-C Ecublens, 1015 Lausanne, Switzerland R. Schumacher Ciba-Geig y Research Laboratories, Marly, Switzerland Introduction Three lubrication regimes can be distinguished in lubricated contacts. 1 In the hydrodynamic lubrication regime, the sliding surfaces forming the contact are separated by a liquid lubricant film, leading to low friction and wear.In the mixed lubrication regime, the surface asperities touch intermittently. Elastic and plastic deformation of the asperities and the formation of microscopic junctions lead to increased friction and wear. In the boundary lubrication regime the surfaces are in direct contact, leading usually to seizure and failure of the contact. Anti-weadextreme pressure (AW/EP) additives improve friction and wear in the mixed regime, mostly, by chemical reaction with the rubbing surfaces. For the formulation of improved lubricants it is important to understand the reaction mechanisms of AW/EP additives. For this the chemical composition of surface layers formed under different conditions must be characterized and correlated with observed friction and wear behaviour.The application of surface analysis methods such as Auger electron spectroscopy (AES), photoelectron spectroscopy (XPS) and secondary ion mass spectrometry (SIMS) to tribological problems is des- cribed in two reviews.2.3 These methods are particularly well suited for studying the reactions of AW/EP additives with rubbing metal surfaces.4 The purpose of the present paper is to present experimental results obtained in these laboratories with model additives on steel surfaces under sliding wear conditions and to propose a mechanistic interpretation of the observations. The data illustrate the usefulness and limits of surface analytical methods in the area of tribology and lubrication.Experimental The most commonly used AW/EP additives contain phospho- rus and sulfur, zinc dialkyldithiophosphate (ZDTP) being a well known example. In our laboratory, zinc-free phosphorus esters based on the molecular structure shown below were used as model compounds for the systematic investigation of the relation between the molecular structure of the additive and its effect on friction and wear.5 The groups X , Y , R1 and R2 were varied systematically. The solvent was a sulfur-free synthetic hydrocarbon. Prl x = s , o C H 2-CO-N-( C3 H7 12 Y = S , O Wear tests were carried out with a ball-on-plate test device (SRV) shown in Fig. 1. This device, described in more detail in ref. 5 , allows one to perform tests with small amounts of lubricant (typically two drops) and the sample size is well adapted for surface analytical investigations.Furthermore, the mechanical quantities, normal force and tangential force, which determine the friction coefficient, can be measured easily. The apparatus allowed the variation of oil temperature in a controlled manner. Thus the friction coefficient and the extent of wear could be determined as a function of temperature for different load conditions. The latter was calculated from the wear scar sectional area, measured by a Talysurf instrument. Typical experimental conditions used are indicated in Fig. 1. In order to obtain reproducible results a running-in period of 10 min under standard load (100 N) was used in all wear tests before starting an experiment under any particular conditions.After the running-in period the friction coefficient varied relatively little with time. Normally the value measured after 2 h was taken. After a wear test the samples were carefully cleaned in an ultrasonic bath containing a 1 + 1 mixture of chemically pure acetone and white spirit. They were then stored over silica gel under argon gas before being introduced into the ultra-high voltage (UHV) system for surface analysis. The described cleaning procedure removed all soluble organic matter, such as residual oil films from the surface, leaving only adherent insoluble matter. Auger depth profiling was carried out in the PHI 550/590 AEYXPS apparatus equipped with a cylindrical mirror analyser and two differentially pumped ion guns.The electron beam of approx 1 pm diameter (2 keV, 0.5 WA) was scanned typically over 200 x 200 vm, the ion beam (2 keV) over 2 x 2 mm. After an initial survey, a depth profile was measured. The sputtered depth was approximately 20 nm. To estimate the concentrations, elemental sensitivity factors were employed throughout. The concentration values at a depth of 1.8 and 4.3 nm were computed (in some cases also at 17 nm). These values were found to be more representative of the reaction layer composition than the surface concentration values measured without sputtering which are strongly af- fected by adsorbed contaminants. The reproducibility of the AES concentration values so determined was good.4 FN 0.5 mm - H 1.2 mm Fig. 1 Schematic diagram of the ball-on-plate wear test apparatus.Operating conditions: load, 100 or 200 N; frequency; 50 Hz; amplitude, 0.5 mm; temperature, 40-120°C. Plate and Ball: Steel 100 Cr 6 (1% C, 1.5% Cr) HRC = 6232 A ANALYTICAL PROCEEDINGS, JANUARY 1993. VOL 30 I I3 1 0.14 4- S a, 0 .- E 0.12 s C .o 0.10 + .- L L L 0.08 S 0 i C D d, t 40 80 120 40 80 120 40 80 120 40 80 120 40 80 120 TemperaturePC Fig. 2 the reaction layer at a depth of 4.3 nm, measured by AES after completion of the respective wear tests Friction coefficient as a function of temperature in the presence of additives A-D. Also indicated is the phosphorus concentration in Auger electron spectroscopy yields atomic concentration, but gives no information on the chemical state of the species present.For this reason, a few complementary experiments were made by XPS, using a Mg Ka source. In addition, SIMS measurements were performed, using a combined AESlSIMS instrument (PHI 54YATOMICA 3000) equipped with a quadrupole mass analyser. This equipment could be used in the static or the dynamic mode.6 Friction and Wear Behaviour of Model Additives The effectiveness of model additives was compared by performing friction and wear experiments at different temper- atures. Increasing temperature decreases the viscosity of the solvent and thus leads to more severe wear conditions. The friction coefficient was plotted as a function of temperature. From this the critical temperature was determined at which lubrication breakdown occurred and the friction coefficient increased markedly.The transition from low to high friction was found to depend on the chemical nature of the additive present. Fig. 2 shows experimentally measured friction coefficients in the presence of different iso-geometrical phosphorus ester additives, present at a concentration of 2%. Below the transition temperature the presence of the additives leads to a lower value of the friction coefficient compared to the pure solvent (PAO). The best additives are those exhibiting the highest transition temperature. The wear rate as a function of temperature (not shown in Fig. 2) exhibits similar behaviour to the friction coefficient. The data clearly demon- strate the effectiveness of AW/EP additives in reducing friction and wear under the present experimental conditions.The worn surfaces were analysed by AES depth profiling. Fig. 2 shows the measured phosphorus concentration at a depth of 1.8 nm. The data correlate well with the friction coefficient. Under conditions of low friction, phosphorus is present at the surface, but at the transition temperature the phosphorus concentration decreases sharply. On the other hand, no such correlation was observed for sulfur. Phosphorus rather than sulfur, is therefore, the active component of the AW/EP additives tested. Chemical Nature of Reaction Layers Information on the chemical nature of the reaction layers was obtained from three independent sources, the oxygen : phos- phorus ratio measured by AES, and XPS binding energy of phosphorus and SIMS fingerprint sprectra. The atomic ratios 0 : P in the reaction layer formed under wear test conditions were found to differ from those observed for steel samples having been simply immersed in the additive containing solvent (Fig.3). The data shown were measured at a depth of 1.8 nm after immersion at 25 “C for 2 min and after a wear test at 200 N and 40°C, respectively. The surface layers formed during the wear test obviously contain much more phosphorus and sulfur than those formed during immersion. Also they are much thicker, an observation not shown in Fig. 3. The atomic ratio P : 0 in the reaction layers formed during wear testing in the low friction regime was approximately 4 : 1. This indicates that the phosphorus in the reaction layer is present mainly as phosphate. The measured XPS binding energy of phosphorus was found to be in agreement with this hypothesis.6 Additional information on the chemical state of the reaction layers can be obtained by SIMS.Negative SIMS spectra from surfaces of iron immersed in phosphoric acid (formation of iron phosphate) and from reaction layers resulting from wear testing were compared and found to contain similar phospho- rus containing fragments (Fig. 4). The data provide further support that the reaction layers formed under friction condi- tions contain iron phosphate. However, a detailed interpreta- tion of the chemical information contained in SIMS spectra was not possible, because too many factors may influence the production of fragments during the analysis.6 100 - s 80 I Er 60 4- m C 0 .- +- 40 4- C a, C s 20 0 Immersion Wear Fig.3 Elemental composition of the reaction layer at 1.8 nm derived from AES measurements performed on samples having been sub- jected to a wear test in the presence of additives A, B and C (see Fig. 2 for formulae) o r simply immersed in the base oil in the presence of 2% of these additivesANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 33 Synergistic Effects of Antioxidants Commercial lubricants contain, in addition to the base oil and AW/EP additives, several other compounds, such as viscosity improvers, emulsifiers, detergents and antioxidants. Some of these components might possibly affect the effectiveness of AW/EP additives. Of particular interest in this respect are antioxidants, because of their chemical reactivity. The effect of selected antioxidants was investigated with additive E the structure of which is indicated in Fig.2. The transition from low to high friction and wear occurred at approximately 120 "C in the presence of this additive. At the transition temperature, the friction coefficient and the wear scar area increase and the phosphorus content of the reaction layer decreases. At the same time the oxygen content of the surface layers increases sharply, indicating that severe surface oxidation takes place.7 The presence of an antioxidant shifts the transition tempera- ture characteristic for this additive to a higher value, as shown by the data in Fig. 5 . The antioxidant in this case was a blend of a sulfur containing sterically hindered phenol and an aromatic amine added at a concentration of 0.75%.The additive concentra- 104 PO2 103 c 102 I v) v) 4- 103 102 0 50 m/z 100 Fig. 4 Negative SIMS spectra of a steel surface having been (a) immersed in phosphoric acid or ( b ) having been subjected to a wear test in a synthetic hydrocarbon containing 2% of additive A 100 I I J E *c)/ 0 B 74 40 50 60 70 80 90 100 110 120 130 140 150 Tern peratu re/"C Fig. 5 Wear scar area as a function of temperature measured in an additive containing synthetic hydrocarbon solvent in A, the presence (1.94% additive E +0.75% A03) and B, the absence (1.94% additive E) of an antioxidant 60 50 N E 40 1 2 3 Antioxidant Fig. 6 Influence of additive E and antioxidants A01, A 0 2 and A 0 3 on the measured wear scar area formed during a wear test at 120°C tion was 2%.The data given in Fig. 5 suggest that the antioxidant improves the efficiency of the additive by inhibit- ing surface oxidation. To verify this hypothesis the wear behaviour at 120 "C (just above the transition from low to high wear in the presence of additive E) was investigated for four different lubricant formulations involving a common base oil. Formulation (a) corresponded to the pure base oil, formula- tion ( b ) to the base oil + additive E , formulation ( c ) to the base oil + antioxidant and formulation (d) to the base oil + additive E + antioxidant. Three different antioxidants were used, a sulfur containing phenol (AOl), an aromatic amine (A02) and a mixture of both (A03). The antioxidant concentration was 0.75% in all cases, while the additive concentration was 1.92%. Results of the different experi- ments are summarized in Fig.6. They show that under the experimental conditions chosen (just above the transition temperature) the base oil with and without additive leads to a similar wear rate. Adding only antioxidant to the base oil does not improve the wear behaviour and in one case even leads to significant deterioration. On the other hand, for all three antioxidants tested, a mixture containing both additive and antioxidant gives a much lower wear rate. The results demonstrate the existence of a synergistic effect between the AW/EP additive and the antioxidants. Reaction Mechanism The most widely accepted wear mechanisms are: adhesive wear, abrasive wear, fatigue (delamination) wear and oxida- tion wear.8 Adhesive wear involves the formation and destruction of welded junctions at asperities touching each other.Material removal is due to tearing off of metal from these junctions. By forming a reaction layer at the rubbing surfaces AW/EP additives prevent welding at the metal-metal contacts. The present data and others are in agreement with such a hypothesis. Abrasive wear involves removal of material by ploughing, which results in plastic deformation and fracture. The wear particles thus formed (oxides, metal) act as abrasives. An influence of the additives on this mechanism has not been demonstrated to date. A possible action on the size and mechanical properties of the wear particles formed cannot be excluded a priori, however. Delamination wear, important in brittle materials, involves formation of fatigue cracks below the surface and periodic rupture.It is unlikely that the additives influence this mechanism in a significant way. Oxidation wear involves periodic formation and removal of34 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 thin oxide layers.3 The AW/EP additive can reduce this type of wear by reacting with the surface in competition with oxygen. The synergistic effect of antioxidants is in favour of such a hypothesis. From the chemical surface and other data presented, three steps can be distinguished in the reaction mechanism of an AW/EP additive with steel. Firstly, the additive molecule dissolved in the oil must be able to adsorb at the metal surface. The molecular structure of the additive, notably its steric effects, may play an important role at this stage.The molecular structure also determines the solubility of the additive in the base oil and its intrinsic thermal stability. These factors are of importance because according to the adsorption isotherm, the surface coverage depends on the bulk concen- tration of the additive. Insufficient thermal stability leads to a decrease in the bulk concentration with time. In a second step, the adsorbed additive molecule is torn apart, by the prevailing shear forces, and highly reactive molecular fragments are formed. This process, called tribo- fragmentation, depends on the prevailing mechanical parameters and the temperature. The occurrence of tribo- fragmentation is supported by the observation that at equal temperature, immersion experiments produce less phos- phorus at the surface than wear experiments.9 In a third step, the molecular fragments react chemically with the metal surface, the end product of the reaction sequence being essentially iron phosphate.In the mixed lubrication regime, under relatively mild conditions, these act as a solid lubricant and prevent adhesive wear. Under more severe conditions they reduce surface oxidation by reacting with surface sites in competition with dissolved oxygen, thus reducing oxidative wear. The thickness of the phosphate reaction layer probably depends on the test conditions. Under the present experimen- tal conditions is was found to be of the order of several tens of nanometres in the low wear regime. Above the transition temperature the phosphorus reaction layer no longer forms, and the reaction between the metal and oxygen becomes dominant. Several factors, not yet well understood, could contribute to the observed change in mechanism at the critical temperature. On one hand, the adsorption equilibrium of the additive is less favourable at higher temperature, decreasing the amount of adsorbed additive and hence the rate of tribo-fragmentation. Another possibility is that the activation energy of the reaction of oxygen with the metal is higher than that of the reaction of additive fragments. In conclusion, the present data show that surface analytical techniques, such as AES, XPS and SIMS, used in combination with well designed wear expqriments, can yield interesting information on surface reactions of additives under wear test conditions, information not accessible by other methods. Many aspects of the reaction mechanism of AW/EP additives need further study. Surface analysis methods should prove very useful for further progress in this field. References Czichos, H., Tribology, Elsevier, Amsterdam, 1978. Buckley, D. H., Surface Effects in Adhesion, Wear and Lubrication, Elsevier, Amsterdam, 1981. Quinn, T. F. J . , Physical Analysis for Tribology, Cambridge University Press, Cambridge, UK, 1991. Mathieu, H. J . , Landolt, D., and Schumacher, R., Wear, 1981, 66, 87. Schumacher, R., Landolt, D., Mathieu, H. J . , and Zinke, H., ASLE Trans., 1983, 26, 94. Mathieu, H. J., Schumacher, R., and Landolt, D.. Wear, 1989, 132, 99. Schumacher, R., Zinke, H., Landolt, D., and Mathieu, H. J., Wear, 1991, 146, 25. Rabinowicz, E., Friction and Wear of Materials, Wiley, New York, 1965. Schumacher, R., Zinke, H., Landolt, D., and Mathieu, H. J . , 5th International Kolloquium Additive fur Schmierstoffe und Arbeitsfliissigkeiten, Technische Akademie, Esslingen, 1986, V O ~ . I, p. 3.9.1-19.

ISSN:0144-557X    

DOI:10.1039/AP9933000027

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 35 Reti ri ng President’s Address Bridges in Analytical Chemistry J. D. R. Thomas School of Chemistry and Applied Chemistry, University of Wales, PO Box 912, Cardiff CFI 3TB Fifty years ago, come next year, I crossed a bridge in the upper Towy Valley in Wales and travelled along the road beyond the Black Mountain for my further education and life’s work. On looking back from the Mountain, the Carmarthen Vans of the legendary Lady of the Lake and home of the Physicians of Myddfai (who used papaverine-based herbs) are seen on the right, and on the left is the dramatically placed Carreg Cennen Castle. In between, lies rich and scenic agricultural land, which at that time was unpeppered by the new pesticides of the kind discussed in our symposium today (March 27th, 1992) on The Analysis of Fungicides, Herbicides and Insecticides.The Black Mountain itself is one of limestone, silica, peat beds and even an outcrop of coal. From time to time, the silica is quarried for industrial use, and until recently the limestone provided the lime from up to 35 kilns for whitewashing farm buildings and land-liming. The peat, used for conditioning the soil of many a garden, provided me with a topic for analytical chemical study which was later to become the foundation for an academic-industrial bridge. This study linked solvent extrac- tion and ion-exchange chromatographic separation with ultra- violet spectroscopy for monitoring and infrared spectroscopy (Fig. 1) for characterization. The journey fulfilled my ambition to take up chemistry as a career.This ambition was stimulated by my liking for chemistry, and was given direction by an encyclopaedia entry for ‘Chemistry’ which gave The Institute of Chemistry of Great Britain and Ireland as the official organization of the profession (there was little in the way of careers advice in those days). The Institute described the ways forward to its new Student Member. The intervening span from then to the present, the suspension bridge of my life, stretches from the days of classical analytical chemistry to those of the present labour-saving computer-based and data-handling instrumentation with Fig. 1 fractions following solvent extraction and ion-exchange separation Plotting an infrared spectrum in 1962 to characterize peat sophisticated sensors and robotics.Also, by now analytical chemistry often goes froml the laboratory to the bedside, into industrial plant, to the roadside and into rivers and streams. The bridging of analyticial chemistry of the past to that of the present and on to the future will vary in perspective between viewers, but the chasms crossed are essentially the same. To assure the future, the present needs to be used for attracting interest in the young. Analytical chemistry is a superb vehicle for such stimulation and for the teaching of chemistry. It is the central theme used by the Analytical Division in supporting Schools Analysts competitions, and in association with the Education Department of the Society, with sponsorship from the Analytical Chemistry Trust, in producing the brochure ‘Analytical Chemistry’ and the video ‘Chemical Detectives’, each aimed at senior pupils in schools.At present, ‘Anna Lytic’, brainchild of the Division’s Education and Training Committee, again in association with the Society’s Education Department and with sponsorship from the Analytical Chemistry Trust, is being moulded to encourage a younger age group to take interest in chemistry through analytical chemistry. Although the immediate aim is to stimulate interest in chemistry, the ultimate objective is to develop the profession of analytical chemistry, which invar- iably depends on sound training in general chemistry. The transition from school to further education is a significant step; the bridging of this is made no easier by successive government upheavals.On crossing this bridge there are signposts pointing in many directions. For analytical chemistry, there are the three main routes of industry, public service and education. Each of these has bridges, and other bridges interconnect the main trails. Within each trail there may be research orientation, quality control and quality assurance in various guises, diagnostic work, curriculum and other developments, technique specialisms, etc. Rather than philosophize any further, let me now go on and relate to those bridges that have proved prominent in regard to my own life in and for analytical chemistry. These relate to the curriculum, academic-industrial links and international relations. Bridges in the Curriculum Analytical chemistry, by being essentially an experimental study of the composition of solids, liquids and gases, is a keystone for bridging the component parts of chemistry, no matter how they are classified. The teaching of chemistry at tertiary level in the first half of this century embraced practical laboratory instruction geared mainly to classical qualitative and quantitative analysis of inorganic materials, and to synthesis and qualitative analysis of organic compounds.The focus on practical physical chemistry was relatively small. Of course, main courses in chemistry were usually reinforced by subsidiary courses in physics and/or mathematics. Against this background my own view of chemistry could be summarized as one of ‘making’ and analysis, coupled with36 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 characterization through chemical and physical properties.Working in the plastics industry, and job prospects in other industries, reinforced this view, as indeed did working in the food industy and in a public analyst’s laboratory. Within my undergraduate education there were, more by accident than design, the fundamentals and foundation of much that is now to be seen in modern analytical chemistry. Thus, in addition to a great deal on classical reaction analytical chemistry, there was the basis of understanding of spectro- scopic analysis through quantum theory and wave mechanics and its application through the Lambert-Beer relationship. The principles of electrochemical analysis were evident in the Nernstian view of the theory of the electrode process.Certain gems, like the urease-catalysed hydrolysis of urea, were to become the basis of new electrochemical sensors. Polaro- graphy was well set in our curriculum in the 1940s. However, chromatography, which along with spectroscopy is a mainstay of today’s laboratory instrumental analysis, was yet to cross the divide from biochemistry into chemistry curricula. Inciden- tally, The Analyst, in the RSC Sesquicentenary Celebration Issue in December 1991, is the only primary journal to have paid tribute to the pioneers of modern chromatography. The 1940s saw the foundation of a great surge in analytical chemistry. This was reflected in the growth of membership of The Society of Public Analysts and Other Analytical Chemists from fewer than 1000 50 years ago to about 1600 by 40 years ago. These turned out to be years poised for changes in the curriculum.Microchemical techniques revolutionized quali- tative analysis, and their introduction into the curriculum greatly reduced the timc spent on such analysis (and on organic syntheses), but the time freed was rarely used to promote the newer and instrumental methods of analysis. However, the scene was set for new bridge building, an ever present need in analytical chemistry because of progress, new developments and concerns. Regarding concerns, J. R. Nicholls, who was President of The Society of Public Analysts and Other Analytical Chemists at the time of my admission as a Member in 1952, in his Presidential Address talked of ‘Public Health Hazards and the Analytical Chemist’.He referred to hazards arising from air, water, food, clothing, cosmetics and household materials. It was a far-reaching address, and after considering the hazards he went on to classify them as being due to lack of personal hygiene, contamination and ‘chemicals’ in the widest sense. On chemicals, he made the point that ‘Whatever control measures are envisaged, it seems generally agreed that provision must be made for periodical review.’ Analytical chemistry has to keep pace. Industry and the public service gave me salutary experience on the role of analytical chemistry at work and in daily life (Fig. 2). Up to then, that is, 40 years ago, there was by today’s standards, apart from analytical balances, and perhaps an odd refractometer, polarimeter and simple photometer like the ‘Spekker’, relatively little in the way of instrumentation in chemical laboratories that was not electrochemical.Even the electrochemical equipment tended to be simple units for potcntiometry, conductimetry and electrolysis. The sophistica- tions of pH meters and polarographs were the prerogative of the privileged few. Advances in electronic technology, and micro-electronic solid-state circuitry, had yet to take effect. The local authority controlled colleges of technology (or technical colleges as they were earlier known), through their involvement in National and Higher National Certificates in Chemistry and associated links with industry, came to be equipped in the early 1950s with a range of physicochemical instrumentation.As a procurer of such instruments at the Cardiff College of Technology and Commerce I introduced instrumental analysis alongside microanalytical approaches to undergraduates of chemistry and pharmacy. This was in addition to fulfilling the needs of day-release Higher National Certificate and other students. It was also possible to innovate with undergraduate student projects, such as by the construc- tion of a gas chromatograph , separations by electrophoresis and X-ray characterizations. Although those were days of relative rigidity in curricula, governed by entrenched views in universities, and by ex- aminers of external University of London degrees and of examinations for The Royal Institute of Chemistry, it was still possible to elucidate the more theoretical concepts effectively by bridging with pertinent analytical chemistry. Such bridging became easier as the years wore on.The experience proved invaluable for designing dedicated courses in analytical chemistry as new qualifications emerged, that is, courses like the Diploma in Technology and Endorsements to Higher National Certificates. The latter was soon to lead on to Licentiateship of the Royal Institute of Chemistry. Since then there have been many changes, such as the Diplomas in Technology becoming degrees of the new technological universities and of the CNAA, and further changes are afoot with the recent passing of the new Higher and Further Education Bill. Among all this, it was my privilege in 1958 to set up the first ever Higher National Certificate (HNC) Endorsement Course in Analytical Chemistry in the United Kingdom at the South East Essex Technical College, and later to have analytical chemistry incorporated in its own right into undergraduate curricula.To me, as a member of the Society that in 1954 became the Society for Analytical Chemistry, much of the inspiration and indeed tacit endorsement of my actions came from the papers, published in The Analyst of November and December 1952, presented at the First International Congress on Analytical Chemistry held under the Patronage of IUPAC at Oxford in September 1952. The presentations were in eight sections devoted, respectively, to microchemical methods, biological methods, electrical methods, optical methods, radiochemical methods, organic complexes, presentation of data, and adsorp- tion and partition methods.These papers succinctly bridged the theoretical principles of chemistry and science in general with the analytical approaches of such diverse methods as solvent extraction, square wave polarography, equivalence point determination (Gran’s plots), chelating ligands, infrared spectroscopy, statistics, gas-liquid chromatography, and ion exchange. The IUPAC Congress was one that blazed the trail for Analytical Chemistry, and laid hopes. Sir Robert Robinson, President of the Congress, is recorded to have ‘deplored the fact that the University (Oxford) had not yet got a Chair of Analytical Chemistry. At least four Chairs of Chemistry were needed: Physical , Organic, Inorganic and Analytical’. We are still waiting! Fig.2 At work (right) in an industrial control laboratory in 1950. when modern physicochemical instrumentation was yet to be in common uscANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 37 In the interim there seems to have been a lag in the developing perception of analytical chemistry. At the Con- gress, Norman Sheppard gave account of advances in the design of infrared spectrometers for incorporation in instru- ments designed for analytical work. Forty years on, Sheppard was among those honoured in March 1992 at The Third James L. Waters Annual Symposium set up to Recognize Pioneers in the Development of Analytical Instrumentation which at Pittcon ’92 ‘Saluted Infrared Spectroscopy’. The Analytical Division has only recently had a Molecular Spectroscopy Group! Today’s trend for having modular schemes of study and the changes that will inevitably result from the Higher and Further Education Bill provide excellent flexibility for bridging con- cepts through analytical chemistry and thus to portray a relevant perspective of chemistry.However, with pressure on curriculum time it will be necessary to be on the offensive (the best line for progressive action) in order to ensure proper regard for analytical chemistry. Education and training has to be aimed at placing the new graduate in a position to be able to perform adequately, not just as an analytical chemist, but also for graduates in chemistry to be effective in diverse ways, for example, as better synthetic chemists by more intelligent use of essentially analytical type approaches, such as gel permeation chromatography for purifying products, and sampling pro- cedures and quantification methods for purity assessment.Academic-Industrial Bridges There can be no doubt that in order to establish methods of analysis much fundamental research or investigation work of an academic nature must be carried out. Therefore, having regard to the front-line role of analysis in manufacturing and commerce, bridging links between academic institutions and industry are paramount in the development of analytical chemistry. There are various ways for effecting these, includ- ing: (i) encounters at scientific meetings, such as those of the Analytical Division of The Royal Society of Chemistry; (ii) interactions through industrial training of students on sandwich courses; (iii) interactions through vacation employment, day release of students, short courses and graduate recruitment; (iv) job changes (industry to academic life and the reverse); (v) research co-operation through funding by industry or in co- operation with funding agencies, such as the Science and Engineering Research Council (SERC) and the Analytical Chemistry Trust; and ( i v ) consultancies, industrial professor- ships and lectureships.The first three are relatively informal, but they are essential ingredients in establishing rapport between the two sectors. They have the benefits of cultivating expert contacts for quick advice in problem solving. Regarding (i), it was in the 1986 Presidential Address of the Analytical Division that the view was expressed that it might be to the benefit of employers if credit in promotion and salary progression were given to employees attending appropriate scientific meetings.This certainly now figures in staff appraisal schemes of some universities. The matter might be on a points system, such as that detailed in a note on page 51 of the PITTCON ’92 programme relating to accreditation: ‘The American Board of Industrial Hygiene awards f point for each t day attendance at the Pittsburgh Conference’. Since the originator of the suggestion is involved with the Society’s Register of Analytical Chemists maybe we shall in due course see similar points awards for the Register. From personal experience, I consider the benefits of job transition from industry to academic life and vice versa to be invaluable. They serve to develop the ability to recognize problems, to determine priorities and to inspire ideas.The remainder of the above bridging links frequently derive from the others. However, the origins of research links may be more discrete, as I found from research on peat constitution. In Fig. 3 Preparing the Presidcntial Address in 1992 that work, the ion-exchange separation following a solvent extraction stage had resolved phenolic materials, aromatic and aliphatic esters. Further work on the ion-exchange behaviour of phenols, presented at a meeting of the Chromatography and Electrophoresis Group of this Division, attracted attention from industry and led to consultancies and a lengthy 15 year period of sponsored research.The sponsored research yielded a polystyrene-based support and mixed solvent electrolyte system for the electrophoresis of oil-soluble materials, insight into bacteriological initiation of corrosion, a computer-based monitoring system for process streams, and some insight into changes in oil additives during engine wear. There were also incidental rewards in the field of potentiometric ion sensors as in the polarography of imida- zoles. This was a rich harvest. Certainly, academic-industrial bridges can result from a lengthier list to include: (i) computerized file records by industry to keep track of academic research; (ii) lectures by academics and industrialists to indicate mutuality of interests; (iii) pressure by grant-awarding bodies (SERC-CASE awards, SAC Studentships, DTI LINK schemes, etc.); and (iv) the old- boy network.The third method of this secondary list extends to the broader role of the Science and Engineering Research Council (SERC) in the funding of academic research. This is aimed at exploitation of academically based research, and is also designed to promote a wider base for the funding of research. Personal experience has demonstrated this to be a superb catalyst for building strong bridges with various industries. It has also served to bridge multidisciplinary groups, that is, by linking groups of sophisticated organic systhesis and structural determination with our expertise in potentiometric and piezo- electric quartz crystal types of chemical sensing. Of special interest to our Symposium on Pesticide Analysis is the role of crown ethers, particularly dibenzo-30-crown-10 (DB30C10) and its derivatives as ion-selective electrode (ISE) sensors for diquat (DQT) and paraquat (PQT), the best ISE membrane being based on DB30C10 with DQT tetraphenylborate and 2- nitrophenyl phenyl ether in PVC.Of the DB3nCn derivatives, the maximum stability with DQT occurs when n = 10, that is for DB30C10. An X-ray structure shows the plan of the DQT molecule to be enclosed in a U-shaped cavity formed by DB30C10, facilitated by three DB30C10 (host)-DQT (guest) interactions. (i) DB30C10 catechol-oxygen electrostatic interaction with the positively charged nitrogen atoms in DQT. For this, the crown ether catechol 0-0 separation (2.6 A) and N-N separation in DQT (2.8 A) are similar, so that the former are nearly directly above and below the latter in the [DQT.DB30ClO]’+ complex.(ii) DB30C10 benzene ring n-electron charge transfer to the electron deficient DQT’+. (iii) Hydrogen bonding between H38 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 atoms on the carbons next to the nitrogens in DQT with oxygen atoms in the DB30C10 framework. International Bridges Analytical chemistry, as well as several other subject disci- plines in Britain, and indeed in most developed countries, has benefited from the influx of researchers from foreign countries. In meeting their desires to study and gain experience in many areas, such people have provided seed-corn for subsequent formal funding by their work on essentially speculative projects. This feature is well illustrated by the studies of an overseas student on the ion-sensing qualities of electrode membranes based on metal complexes with polyalkoxylates.Such systems also demonstrated electrochemical sensing quali- ties for polyalkoxylates themselves, and led to several funded projects and to sensors for alkoxylate type non-ionic surfactants. Other examples of our experience of the helpful roles of overseas research workers consist of initiating work on enzyme-based biosensors, subsequently funded by the Depart- ment of Trade and Industry and by industry itself; studies of the stability of ion-exchange resins in non-aqueous media which led on to the ion-exchange separation of phenols and then to industrial sponsorship of other projects; polyurethane foam chromatographic and extraction studies; and piezoelectric quartz crystal detection of gases and vapours.Particularly exciting has been the demonstration of the role of ring type epoxyoctahydro[ 12lcyclacene and of certain chemically modi- fied cyclodextrin derivatives as piezoelectric quartz crystal coating sensors for aromatic vapours. This last example is like a multi-arch viaduct or aqueduct, by also involving SERC and academic-academic links. As a result of research in Britain, trained personnel have returned to foreign countries with a wealth of expertise for embarking on their own programmes. Apart from the obvious electrochemical ion-sensing, our own research degree gradu- ates have gone forth with experience of nuclear magnetic resonance and other spectroscopic approaches, immobilization of enzymes and their characterization, chemometric principles, microbiological approaches, dielectric and thermal measure- ment expertise, etc.It is gratifying that the Analytical Chemistry Trust, through the SAC Studentship Scheme, has participated indirectly in this by coincidentally supporting an oveseas student graduate of a British university (helped also by the Overseas Research Studentship Scheme of the Committee of Vice-Chancellors and Principals). In this way, there has been high-level support of developing countries to complement the salutary initiative of the Analytical Chemistry Trust in supporting VSO (Voluntary Service Overseas) teachers. Presentation of research at conferences and during consul- tations and other activities in foreign countries, and in reverse by listening to visitors to Britain, provides forums for learning of interests of prospective research collaborators. The present day ease of travel and communication, compared with even a quarter of a century ago, makes for rapid bridge building, to the benefit of analytical chemistry.However, the closeness with the far reaches of the world, created by air travel and satellite communication, whilst promoting rapid progress and development, can hinder the generation of completely novel ideas. This is because of a natural desire to follow fads and fashion rather than undertake brainstorming sessions. . On a broader front, methods of analysis and the interpre- tation of numerical data and figures thereby obtained have to be international. It is not sufficient for duplicates of an analysis to agree, satisfying though that maybe.Replicates must also agree, and these conducted not by one individual worker or at one laboratory, but by several. It is on the basis of such agreements that contracts of national and international trade and commerce can be properly conducted. The imminence of the European single market has brought the need for confidence in the uniform definition of chemical ingredients through analysis into stark perspective. Hence, bodies like EURACHEM, advised in this country by CHEMAC (the Chemical Measurement Advisory Committee run by the Laboratory of the Government Chemist) have come into being. Of course, such bodies are complementary to The International Standards Institution (ISO) and its national adhering body, The British Standards Institution (BSI). Our own Analytical Methods Committee of the Analytical Division, apart from bringing together and promoting co- operation between people of diverse interests, has bridged the Atlantic Ocean in an understanding with the Association of Official Analytical Chemists.On a stronger footing is the affiliation of the Analytical Division to the Federation of Analytical Chemistry and Spectroscopy Societies (FACSS), which in the autumn of 1991 resulted in a Division-sponsored symposium on atomic spectrometry at the Anaheim FACSS Meeting. Capillary zone electrophoresis is planned to be Division-sponsored at the FACSS Meeting of 1993. Finally, in international bridging, there is the International Union of Pure and Applied Chemistry which emphasizes international agreement, and significantly has a Division of Analytical Chemistry. Similarly, the Federation of European Chemical Societies (FECS) has its Working Party on Analyti- cal Chemistry, which is very active in the European scene of bridging in analytical chemistry. To mark the Sesquicentenary in 1991 of the founding of the Chemical Society in 1841, the Analytical Division through the Analytical Chemistry Trust sponsored The Robert Boyle Anniversary Fellowship in Analytical Chemistry of The Royal Society of Chemistry. The Fellowship was held by Professor Alan M. Bond of La Trobe University, Australia, during 1991 at the University of Oxford. Conclusion Analytical chemistry is an advancing science, and because of its many implications in daily life and work, it has to be an organized science. In such roles it has to meet challenges, as do all who are engaged in it. Bridges help to overcome these challenges and to promote progress and understanding.

ISSN:0144-557X    

DOI:10.1039/AP9933000035

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 OH 39 Short Papers in Pharmaceutical Analysis The following are summaries of five of the papers presented at a Meeting of the Joint Pharmaceutical Analysis Group held on' October 17th, 1991, in the Royal Pharmaceutical Society of Great Britain. Development and Application of an Assay Procedure for Hyoscine in Urine Using Solid-phase Extraction and High-performance Liquid Chromatography with Electrochemical Detection Peter R. Hurst British Pharmacopoeia Commission Secretariat, Market Towers, I Nine Elms Lane, London SW8 5NQ Hyoscine (scopolamine) is a potent anti-cholinergic alkaloid (Fig. 1). It is used as a pre-medicant and also to counteract motion sickness, for which purpose it may be administered orally or via a transdermal patch.A transdermal patch delivers an average of 500 pg of hyoscine over 72 h, producing an estimated steady-state plasma concentration of around 100 pg ml-'.' The compound is unstable; it decomposes to apohyoscine on gas chromatography columns and at high pH values it hydrolyses to scopoline and tropic acid (Fig. 1). This instability together with the low concentrations expected poses severe analytical problems. Analytical approaches to date include a gas chromatography-mass s ectrometry (GC-MS) method' and radioreceptor assays.'-' A high-performance liquid chromatographic (HPLC) method with electrochemical detection, suitable for the measurement of hyoscine in urine after oral doses o r administration of a transdermal patch, is presented in this paper. Experimental Transdermal patches (Scopoderm TTS, Ciba Laboratories, Horsham, Sussex) were applied to volunteers on two occasions one week apart on a double blind cross-over basis.Urine samples collected were assayed before and after incubation with P-glucuronidase-aryl sulfatase solution (Boehringer, Lewes, Sussex). Chromatography Compounds were chromatographed using a straight-phase Spherisorb S5W silica column 150 x 4.6mm (Phase Sepa- Scopoline H<p--> 0 I co I Hyoscine H C-CH 2 0 H O-H 0 I co I C=C H 2 Apohyoscine Fig. 1 Decomposition of hyoscine40 75 50 25 a m 3 0 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 (a) (b) - - 80 Yo 50 % - - - - Lc -L rations, Queensferry, Clwyd) with an eluent consisting of acetonitrile, methanol and 0.1 mol I-' ammonium nitrate buffer pH 9.0 in the proportions (7 + 2 + 1 v/v/v).Detection was via an Environmental Science Associates Model 5100A Coulochem detector. The compounds were oxidized at +0.9 V. & 75 a 50 25 0 Solid-phase Extraction Method Development Tritiated hyoscine and apohyoscine were employed to deter- mine the most suitable solid-phase extraction columns for the assay and also to develop a method capable of specifically extracting hyoscine but not apohyoscine. Urine samples spiked with the tritiated compounds were extracted on to a variety of solid-phase extraction columns and then eluted from them using successive 100 pl aliquots of eluent. Measurement of the radioactivity in each eluted fraction allowed an elution 'profile' to be produced for each sorbent type, allowing subtle yet significant differences in recovery and elution characteristics to be revealed. This approach has also been successfully applied to the development of an assay procedure for physostigmine.' Two-column Solid-phase Extraction Method Urine samples ( 5 ml pre-incubation and 1 ml post-incubation) were adjusted to pH 9 by addition of an equivalent volume of 0.1 mol 1-' borax.Internal standard (250 ng N-ethyl norhyo- scine in 50 p1 of water) was added and samples centrifuged. The supernatants were extracted via alkali-prepared large reservoir 100 mg CI8 columns. The columns were washed with 0.1 mol I-' borax (3 ml), water (3 ml) and 20% methanol- water (1 ml) before eluting the compounds with 1 ml of 50% methanol-water directly into 9 ml of water which had been placed in the reservoirs of acid-prepared 100 mg CN columns.The water-eluate in each reservoir was mixed, extracted via the CN column and the column washed with water (3 ml). The compounds were eluted with 500 pl of HPLC eluent, 200 pl of which was transferred to autosampler vials and centrifuged before injecting on to the chromatogram. (d) (el ( f ) - - - 80% 50 % 40 % c - - - - - 1 1 1 1 1 1 l ~ - Results and Discussion Using the chromatographic system above the resolution of the internal standard (N-ethyl norhyoscine) and apohyoscine was incomplete. However, the possible interference of apohyos- cine was avoided at the extraction stage by judicious choice of elution solvent. The use of 1 ml of 50% methanol-water to elute the C18 column meant that almost all of the hyoscine was eluted whilst apohyoscine remained on the column (Fig.2). The use of 2 x 1 ml of 20% methanol-water allowed many of the urine pigments to be washed from the column without any loss of the compounds of interest. However, some components of the extracted urine sample remained on the column, were co-eluted in the eluate and subsequently interfered with the 'binding' of the compounds of interest with the CN column. Consequently, it was essential that the eluate eluted from the CI8 column was diluted with water before extraction. For this assay diluting the eluate to 5% of its original concentration was sufficient to ensure that all the compounds of interest were extracted. As a consequence of the large number of sample manipula- tions this method has a lower recovery (64%) than alternative approaches (for example a toluene extraction followed by solid-phase extraction gives a recovery of about 85%).This was not considered a problem as the assay was sufficiently sensitive (lower limit of quantitation 2 ng mI-'), precise (coefficients of variation of 4.7, 4.1 and 11 at 100, 10 and 2 ngml-', respectively), linear over the range 1-100 ng ml-' (Y = 0.9998) and gave much cleaner chromatographic traces than any produced by techniques involving liquid-liquid extraction steps. Indeed it is worth noting that with regard to the assay of Table 1 Concentrations of hyoscine in the urine of volunteers after administration of a transdermal patch Subject 1 2 3 4 5 6 7 8 9 10 11 12 Hyoscine concentrationhg ml-' ~ Total? Free* 7 122 13 55 8 67 5 35 12 121 7 72 4 31 9 69 19 128 9 99 19 118 9 94 * Free = concentration before incubation with (3-glucuronidase-aryl t Total = concentration after incubation with P-glucuronidase-aryl sulfatase at 37 "C for 24 h.sulfatase at 37 "C for 24 h. 10 20 20 10 20 Fraction number (0.1 rnl) Fig. 2 extraction of each compound from 5 rnl urine on to the alkali prepared C18 column) Elution of hyoscine [(a)-(c)] and apohyoscine [ ( d ) - ( f ) ] from a 100 mg C18 column using varying percentages of methanol-water (afterANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 41 drugs from biological matrices (e.g., blood, plasma, urine) a References sample preparation step which results in a decreased recovery of the analyte may be accompanied by an increase in sensitivity of the assay.Assay results (Table 1) indicate that a large proportion of the hyoscine present in urine is present in a conjugated form, only about 10% of the total being present as hyoscine. The presence of apohyoscine (which was shown to be present in some urine samples) was significant as this casts some doubt upon the results of workers who assayed hyoscine indirectly by measur- ing its hydrolysis product scopoline.’ Scopoline would also be formed by hydrolysis of apohyoscine (Fig. 1). 1 Walker, G., and Massam, D., ABPl Data Sheet Compendium IYYl-92, Datapharm Publications Ltd., London, p. 313. 2 Bayne. W. F., Tao, F. T., and Crisologo, N., J. Phurm. Sci., 1975, 64, 288. 3 Metcalfe, R. F.. Biochem. Pharmacol.. 1981, 30, 209. 4 Cintron, N.M.. and Chen, Y.-M., J. Phurm. Sci., 1987, 76, 328. 5 Kentala, E., Kaila, T., Ali-Melkkila, T., and Kanto, J., lnt. J . Clin. Pharmacol. Ther. Toxicol., 1990, 28, 487. 6 Hurst, P. R., and Whelpton, R.. Biomed. Chromatogr., 1989.3, 226. Direct Non-destructive Colour Measurement of Pharmaceuticals Nicola Stock Pharmaceutical Analysis Department, Glaxo Group Research Ltd., Ware, Hertfordshire SG 72 ODP This paper briefly discusses colour measurement techniques with emphasis on tristimulus measurements. Recent work to modify a commercially available colour measurement system to accommodate the measurement of new pharmaceutical preparations in a direct, non-destructive manner is described. Colour is a part of our everyday lives and is a phenomenon that is taken for granted.Measurement of colour is undertaken for various reasons. In paint and cosmetics industries, for example, the final colour and consistency of colour of the product are vital, whereas in the pharmaceutical industry colour measurement can provide information about the formation of coloured impurities or can be used to control a manufacturing process. Colour is not an absolute measurement; people have different perceptions of colour and perceived colour is dependent on such parameters as particle size, texture, shape, angle of observation and distance. Colour measurement can be divided into two categories: operator observation and instru- mental techniques. Operator observation can be as simple as a description, t?.g., a yellow liquid, or can be comparative such as in pharmaco- poeial techniques, where a sample can be compared against a range of colour standards (for instance BY, GY solution series found in the British Pharmacopoeia).Instrumental techniques include spectrophometric and colorimetric methods. APHA measurement is an example of a spectrophometric technique where an inorganic colour standard at different strengths is measured at a particular wavelength, a calibration graph constructed and the sample then measured against that graph. Tristimulus measurements are derived from the amount of light transmitted or reflected from a sample in the visible range. Instrumental techniques are to be preferred because they are objective and do not rely on visual comparison of inorganic standards with organic samples. Tristimulus measurements are the most convenient and reliable way of measuring colour because they can be manipulated into readily understandable scales. , Tristimulus Colour The eye can detect three types of colour variation via three receptors: hue, brightness and saturation.The response of the eye has limiting wavelengths at 380 nm (deep violet) and 780 nm (deep red). Beyond these limiting wavelengths the invisible part of the electromagnetic spectrum is reached. Colour can be broken down into thrce radiant stimuli which excite the three receptors in the eye. It is possible to mix the three colour primaries red, green and blue to match light of any given colour. In practice this results in a little of each primary being added to the colour under test to obtain a match.This can be represented thus: Colour = Y(R) + g(G) + b(B) (1) where Y, g and b are the equivalent amounts of red, green and blue primaries. The measurement of these amounts corres- ponds to the tristimulus value. Transcription of these values into co-ordinate form causes practical problems which led the Commission Internationale de I’Eclairage (CIE), the body who are responsible for setting colour standards, to carry out some colour matching experi- ments with volunteers of normal colour vision. The distributing coefficients for each visible wavelength (400-700 nm) giving relative amounts of stimulation for each receptor caused by light of that wavelength were measured. The resulting curves (Fig. 1) (1931 CIE system for a two-degree field of view) give the spectral distribution and amounts of idealized primaries required to match any colour in the visible range.The y curve is the spectral luminous efficiency curve for the photopic eye and therefore gives lightness information, while the x and z curves give colour information only. The amount of each primary required to colour match is the tristimulus value. Tristimulus measurements can be reduced to a two-dimen- sional chromaticity plot (Fig. 2), where x and y are the nor- malizcd tristimulus [x = X/(X+Y+Z), y = Y/(X+Y+Z)]. The 350 42 5 500 575 650 725 Wavelengthlnm Blue Green Red Fig. 1 Spectral distribution coefficients42 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 Y Magenta 0 1 4 0 6 2 3 4 5 6 7 8 X Fig. 2 CIE chromaticity diagram enclosed area contains all the colours which can be detected by the human eye.For a perceived red colour a positive amount of red and a negative amount of blue and green radiation are seen, producing a redder than red imaginary primary. Chroma- ticity mesurements have disadvantages in that they do not represent equal colour differences as equal distances in space. The CIE have created more simple scales, the CIELUX (for television and graphic arts) and the CIELAB (e.g., cosmetics, paint, pharmaceuticals). The LAB values are a mathematical transformation of tristimulus data defined by three axes (Fig. 3). The L axis is the third dimension lightness scale derived from the tristimulus Y by a non-linear transformation, A and B are Cartesian coordinates where a positive A value represents redness and a negative value greenness, a positive B value represents yellow and a negative one blue. The understanding of colour can be further clarified by expressing the values as polar coordinates (Fig.3). The distance of the colour from the origin is the chroma or strength of colour and the actual colour is the hue angle in the range 0- 360" counter-clockwise, where 0" is red, 90" yellow, 180" green, 270" blue, back to red at 360". LAB values can be expressed using the derived value DE: A E = (AL? + AA: + AB?); (2) where A L , A A and AB are the differences between colour coordinates of two samples being compared, e.g., a sample and its recognized standard or a stressed stability sample with the controlled temperature sample. The eye can detect a nE value T White L = 100 Light Fig. 3 LAB and LCH colour coordinates of about 4.This value will demonstrate a difference between samples but will give no information about what that difference is. Tristimulus Instrumentation Instruments commonly available are the Trivector colour measurement systems, the most recent model being the CL6000 which is available in both reflectancc and transmission modes. The pharmaceutical industry uses the reflectance mode for solid colour assessment such as tablets, whereas the trans- mission mode is used for liquids such as injectable prep- arations. Both types of measurements are performed using transmission or reflectance measuring heads which are con- nected to a control unit which can be linked to a personal computer with appropriate software (Fig.4). The transmission head measures the liquid sample placed in either a 1 cm o r 5 cm UV cell, whereas the reflectance head usually measures the sample directly. Experimental The development of a new formulation at Glaxo Group Research involving very small volumes (125 PI) of liquid in unit dose devices presented an interesting problem for conventional instrumentation. The use of a 1 cm cell would involve bulking a large number of sample devices, which is not only wasteful but gives no single device information. Small volume flow cells were also investigated but problems occurred with the width of the light beam in the CL6000 transmission head and more than one sample device was still necessary. Therefore, a direct method of measuring colour was required, preferably in a non- destructive manner so that a single sample device could be used to gain colour information and then be used for other analyses such as assay and impurities, thereby enabling correlation of colour with possible degradation profiles.Development Early work suggested the use of fibre optic cables to pass light directly through a sample. A prototype system was developed (Fig. 5 ) which directed light from an independent light source via fibre optic cables through a sample device into a CL6000 transmission head. Initial sample repositioning problems were overcome by design of a crude sample holder to keep the device rigid, in order to minimize optical effects from movement of the glass sample container. Tables 1 and 2 compare results with and without the sample stand and demonstrate that excellent reproducibility can be obtained with the stand.Some varia- bility was seen between sample devices which can be attributed to the nature of the sample. The success of this prototype system led to the commercial manufacture of a modified Trivector CL6000 transmission DL-,n Optional PC 7 1 cm or 5 cm cells I I Control unit Transmission head Fig. 4 Schcmatic diagram of a conventional Trivector CL6000 transmission systcmANALYTICAL PROCEEDINGS. JANUARY 1993. VOL 30 43 Transmission Sample Fig. 5 guides Schematic diagram of prototype system with fibre optic light Table 1 Tristimulus measurements without sample stand Chromaticity [x = ( X x lOOO)l(X + Y + Z ) ] Tristimulus Position X Y 2 X Y I 381 327 73 488 419 2 285 251 54 483 426 3 280 244 53 485 423 4 372 320 69 488 420 5 296 252 54 492 419 6 219 192 42 483 424 7 244 195 44 484 42 1 8 243 213 46 484 424 9 244 215 49 480 423 10 394 341 72 488 423 Mean 294 255 56 486 422 RSD(%) 66 56 12 3 2 Table 2 Tristimulus measurements with sample stand Chromaticity [x = ( X X 1000)l(X + Y + Z ) ] Tristimulus Position X Y Z X Y I 2 3 4 5 6 7 8 9 10 261 228 46 263 229 46 265 231 47 264 230 47 265 231 47 262 229 46 265 231 47 261 228 46 263 230 47 263 230 47 488 489 488 488 488 488 488 488 487 487 426 426 225 425 425 426 425 426 426 426 Mean 263 230 47 488 426 RSD(Yo) 1 1 1 1 0 Transmission head Sample holder Optional PC Fig.6 Schematic diagram of modified CL6000 transmission system Table 3 Type 1 sample device repositioned ten times using the modified system Chromaticity [x = ( X x 100O)l(X + Y + Z ) ] Tristimulus Position X Y 2 X Y 1 571 534 76 483 452 2 555 519 75 483 452 3 554 518 75 483 452 4 553 518 75 483 452 5 557 520 75 484 45 1 6 553 517 75 483 452 7 556 520 75 483 452 8 560 523 75 484 452 9 555 519 75 483 452 10 556 519 75 483 45 1 Mean 555 521 72 483 452 RSD (Yo) 1.0 0.9 0.4 0.1 0.1 Table 4 Type 2 sample device repositioned ten times using the modified system Chromaticity [x = ( X X 100O)l(X + Y + Z ) ] Tristimulus Position X Y Z X Y 1 956 964 851 345 348 2 954 963 849 345 348 3 957 966 854 345 348 4 960 968 855 345 348 5 960 969 856 345 348 6 833 841 743 345 348 7 954 963 849 345 348 8 959 967 855 345 348 9 958 967 853 345 348 10 945 954 842 345 348 Mean 944 952 840 345 348 RSD (Yo) 4.1 4.1 4.1 - - system (Fig.6). This differs from the prototype system in that there is n o independent light source and the sample holders have been optically designed to minimize effects from the glass sample container. The validation of the instrument (see Tables 3 and 4) showed excellent reproducibility of measure- ment and comparable results, allowing for differences in path- length between samples measured on the modified system and on an independent conventional system. Different sample holders can be interchanged within the system and the modified optics in the transmission head can be returned to the conventional mode, using 1 or 5 cm cells, in approximately 20 min. The modified system is now in routine use within Glaxo Group Research using two types of sample holders for different pharmaceutical preparations and has proven to be a valuable addition to routine colour measure- ment instrumentation. In conclusion this paper demonstrates that a Trivector CL6000 colour measurement system can be modified for use with fibre optic light guides with the advantages of: non- destructive colour assessment; detection of device-to-device variation for pharmaceutical preparations; correlation of colour and assayhmpurity profiles; rapid, clean technique; possible design of other sample holders; possible conversion back to a conventional instrument; and linkage to a personal computer for easy data manipulation and convenient print-outs.The author is grateful to E. Scorer, Glaxo Manufacturing Services, County Durham and to M.Petty, Cosense Ltd., Cambridge. References Throughout the section on Tristimulus Colour reference has been made to the following: 1 2 Trivector CL6000 System Manual. Unired Srares Phul-mucopeia. 1990, XXIl(1061). 1627.44 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 Trace Analysis of Acyclovir Esters David S. Ashton and Andrew Ray Department of Ph ysical Sciences, Wellcom e Research Laboratories, L ang le y Court, Becken ham, Kent BR3 3BS Acyclovir, 9-(2-hydroxyethoxymethyl)guanine, is an anti-viral drug manufactured by The Wellcome Foundation Ltd. It was the first non-toxic drug to be developed for systemic use against herpes roup viral infections such as cold sores and genital herpes. 'The drug is available for parenteral, oral and topical administration using different formulations.Acyclovir has recently been used to treat shingle^^.^ and a New Drug Application (NDA) has been filed in the USA for the use of acyclovir in the treatment of chickenpox. An analytical method has been established to extract and confirm the presence of esters which are route indicative impurities found during the synthesis of acyclovir. These esters can be important in enforcing patent rights. The method involves liquid extraction of the sample followed by high- performance liquid chromatography (HPLC) in which frac- tions are collected and then further analysed by mass spectrometry. The samples analysed are generally from one of three types: bulk acyclovir powder, tablets and creams (or ointments). The composition of the samples may not be known so a universally applicable method must be used.Experimental Extraction To approximately 1 g of bulk acyclovir powder was added 20 ml of methanol-water (2 + 8). The mixture was shaken for 30 min and then centrifuged for 30 min at 2500 r.p.m. The supernatant liquid was removed by Pasteur pipette and blown down to approximately 2 ml under nitrogen. The sample was then filtered through a 0.45 pm Millex HA filter. An extraction blank was prepared prior to the analysis using the procedure described above to ensure that there was no contamination. HPLC HPLC separations were performed on a Waters 600ms System with a Waters WISP 715 Sample Processor. Separation of the 0 Acyclovir 9-(2-hydroxyethoxymethyl)guanine 0 0-Acetylguanine compound 9-(2-acetoxyethoxymethyl)guanine esters was achieved with a Dupont Zorbax-CS (25 x 0.46 cm) column.A Waters 991 Photo Diode Array detector was used for detection. Data collection and reduction was performed with a VG Multichrom chromatography data system. Water was obtained from a Scorah triple distillation system and HPLC grade methanol was obtained from Rathburns. The HPLC conditions were: methanol-water (2 + 8); flow rate, 1 ml min-'; temperature, 30°C. Detection was by UV moni- toring at 254 nm. Injection size was 100 PI. Mass Spectrometry The fraction corresponding to the compound of interest was analysed by high resolution mass spectrometry. Ions with the mass to charge ratio ('rnlz') of the singly charged molecular ion of the compound of interest were detected by means of high resolution mass spectrometry, using a peak matching tech- nique.The mass spectrometer was a Kratos Concept 1s with a maximum resolving power of 80000 and an accuracy of m/z measurement of 0.0006 a.m.u. at 10 000 resolution in the region of 300 a.m.u. The probe was a direct insertion type. A Sun computer system with Kratos Mach-3 software was used to record the series of scans and to provide averaged results in graphical form. Method Extraction and HPLC Standards of each ester were injected, then a blank injection was made (machine blank) to ensure no residual contamina- tion. This was followed by the extraction blank and finally the sample. Fractions were collected for the two blanks and the sample at the appropriate retention times of the standards.The sample injection was repeated six times and fractions collected. The fractions corresponding to the standard peaks were blown down under nitrogen to approximately 300 pl. These fractions were reinjected on to the HPLC using the same conditions as above. Fractions were again collected and analysed by mass spectrometry. 0 CO I CH3 N-Acetylguanine compound Nz-acetyl-9-(2-hydroxyethoxymethyl)guanine 0 Diacetylguanine compound 9-(2-acetoxyethoxymethyl)-W-acetylguanineANALYTICAL PROCEEDINGS, JANUARY 1993. VOL 30 90 80 45 Peak match + El Scans 87-121 100% = 147 mV - - Sufficient tablets were used to give 1 g of acyclovir. The tablets were crushed using a pestle and mortar and the sample washed into a scintillation vial using 20 ml of methanol-water (2 + 8); the vial was shaken and centrifuged as described previously.The sample was then blown down under nitrogen to approximately 2 ml. For cream or ointment samples a method that removed detergents which might affect the HPLC and mass spec- trometry of the sample was necessary. The method involved dispersing 1 g of cream thoroughly in 20 ml of methanol-water (2 + 8). The sample was shaken for 30 min and centrifuged for 30 min. The centrifuging produced three separate layers, the depth of each being dependent on the composition of the cream. The top layer consisted of any detergents present in the formulation which had flocculated together. This layer was normally a milky white froth. The middle layer was the aqueous phase and was the largest layer, generally a cloudy solution.The third layer was a precipitate on the bottom of the vial containing any heavy organics used in the formulation which were not soluble in the aqueous phase. The middle aqueous layer contained the acyclovir and its related esters. This was carefully removed by pipette to ensure that the other two phases were left behind. This layer was then blown down under nitrogen to 2 ml, filtered through a 0.45 pm filter and injected on to the HPLC as described previously. Mass Spectrometry The mass spectrometer was tuned to an indicated resolution of 10 000 and to its maximum sensitivity for that resolution using an ion of perfluorokerosene with an mlz value of 268.9824 as the reference peak. The instrument was then set to peak matching mode and calibrated against another ion of perfluor- okerosene with an rnlz value of 266.9856.The molecular ion of the O-acetylguanine compound at rnlz 267.0968 was then monitored. A capillary tube was filled with part of the appropriate fraction from the extraction blank and placed in the probe tube. The solvent was removed under vacuum. The probe was then inserted into the mass spectrometer through the vacuum lock and heated gently to a temperature of up to 300°C. The procedure was then repeated with the appropriate fraction collected from the HPLC of the extracted sample. The data acquired was processed by the computer and a graphical output displayed and recorded for each fraction. Results The relatively large amount of acyclovir injected can swamp trace amounts of ester, particularly the N-acetyl ester.It was often necessary to collect fractions about the retention times of the esters, then reduce the volume of the fraction under nitrogen before reinjecting. The re-injected fraction produced a much cleaner chromatogram with more consistent retention times. Fractions from the re-injected run were collected and further analysed by mass spectrometry. This HPLC method was applied to all three esters, whether looking for all three or just one. The only time that the method was changed was when the fraction containing the diacetylguanine compound was re- injected; here the methanol percentage of the mobile phase was increased to shorten the run time and to lower the peak width. A typical analysis of acyclovir cream which attempted to confirm the presence of the O-acetylguanine compound produced Fig.1. The fractions collected on this run and five additional runs were concentrated under nitrogen and re- injected. This fraction, corresponding to the O-acetylguanine compound, was run with a mobile phase of methanol-water (2 + 8) and produced Fig. 2. The fraction from the re-injected run was blown down for analysis by mass spectrometry. Fig. 3 shows the extraction blank monitored for the molecular ion of the O-acetylguanine compound at mlz 267.0968 and Fig. 4 shows the molecular ion of the O-acetylguanine compound at rnlz 267.0968 for the fraction collected from the re-injected HPLC fraction. Discussion Several methods for the analysis of acyclovir have been reported.- These methods are not applicable here due to the instability of the ‘acetyl esters’ at acid pH.The esters undergo a predictable hydrolysis in solution, slowly breaking down to give acyclovir (the diacetyl ester also produces both the mono- acetyl esters). The method described is difficult to quantitate. The overall extraction efficiency is approximately 60%. HPLC can detect the appropriate ester at approximately 0.005 ppm, but this can often be obscured by other components from the preparation. High resolution mass spectrometry detects the accurate mass of the molecular ion of the appropriate ester, demonstrating unequivocally the presence of the ester. Although more sensitive detection can be achieved by both flow liquid secondary ion mass spectroscopy (LSIMS) techniques and thermospray mass spectrometry, these methods do not give the I 1000 1 900 > 800 & 700 $600 500 E 400 300 200 100 - o c a I I I I I 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 Ti me/m i n Fig.1 Extract from a cream m 0 500 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 Time/m i n Fig. 2 Reinjected O-acetylguanine fraction I 0 10 20 30 40 50 60 70 80 90 100 110 120 Channel Fig. 3 fraction rnlz 267.0968 Mass spectrum of extraction blank of O-acetylguanine HPLC46 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 100 90 80 70 60 E $ 50 40 30 20 10 0 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 Channel Fig. 4 from extract mlz 267.0968 Mass spectrum of O-acetylguanine HPLC fraction collected confirmatory accuracy of a high resolution mass spectrometric measurement. References Schaeffer, H.J . , Beauchamp, L . , de Miranda, P., Elion, G. B., Bauer. D. J . , and Collins, P . , Nature, 1978, 272, 583. McKendrick, M. W., McGill, J . I., White, J. E., and Wood, M. J., Br. Med. J . , 1986, 293, 1529. Huff, J . C., Bean, B., Balfour, H. H., Laskin, 0. L., Conner, J . D.. Corey, L., Bryson, Y. J . , and McGuirt, P., Am. J. Med., 1988, 85 (Suppl 2A), 84. Molokhia, A. M., Niazy, E. M., El-Hoofy. S. A., and El- Dardari, M. E.. J . Liq. Chromatogr., 1985, 13, 981. Smith. R. L . , and Walker, D., J . Chromutogr.. 1985, 343, 203. Bouquet. J . , Regnier, B., Quehen, S . . Brisson, A. M., Courtois, P., and Fourtillan, J. B. N., J . Liq. Chromatoer., 1985, 8 , 1663. Investigation into the Reversed Phase High-performance Liquid Chromatographic Behaviour of Tipredane Metabolites and Related Substances Melvin R.Euerby, Christopher M. Johnson* and Steven C. Nichols Analytical Chemistry, Research and Development Laboratories, Fisons Pharmaceuticals plc, Bakewell Road, Loughborough, Leicestershire LEI 1 ORH Tipredane (INN, I), a novel corticosteroid, undergoes oxida- tive metabolism in man to yield the 6~-hydroxy-17-methylsulfi- nyl diastereoisomers (FPL 66365XX and FPL 66364XX) and the corresponding sulfone (FPL 66366XX) derivatives. Syn- thesis of gram quantities of these compounds was required, therefore it was essential that a high-performance liquid chromatographic (HPLC) purity screen was developed which separated the sulfoxide diastereoisomers (FPL 66365XX and FPL 66364XX) from each other and from FPL 66366XX and the related synthesis impurities (FPL 67526XX, and com- pounds I1 and 111).This paper describes the complex chromatographic behav- iour of the tipredane metabolites and related substances, and the subsequent development, using a systematic modular approach, of a selective multisolvent gradient elution HPLC method for the separation of the desired compounds, where solvent strength, selectivity and composition were simulta- neously varied during the analysis. This technique has the advantage that it does not require the use of complex optimization software and can be performed using a binary HPLC delivery system. With this technique the effect of changes in various conditions on the separation of critical pairs of compounds of similar polarities was sequentially investigated.Parameters such as organic modifier type and concentration, pH, tempera- ture and column type were studied. Experimental Reagents Tipredane metabolites and related substances were supplied by Fisons Pharmaceuticals Division (Loughborough) . Methanol (MeOH) (gradient HPLC grade) , acetonitrile (MeCN) (Far UV HPLC grade) and tetrahydrofuran (THF) (HPLC grade) used as the mobile phase modifiers and potassium dihydrogen phosphate (AnalaR grade) and orthophosphoric acid (85 .O%) * To whom correspondence should be addressed. (AnalaR grade) used to prepare pH buffers were supplied by Fisons Scientific Equipment Division (Loughborough). Water was purified by an Elgastat spectrum RO system supplied by Elga (High Wycombe). Chromatography HPLC analyses were performed on a HP1090M supplied by Hewlett-Packard (Stockport) and a Beckman Gold supplied by Beckman (High Wycombe).Both instruments possess diode array UV detection, column ovens and binary solvent delivery systems with solvent switching which allowed the use of two further solvents. General Chromatographic Conditions Unless otherwise stated chromatography was performed with a Waters Nova-Pak C18 (5 pm, 150 x 3.9 mm) column. Detec- tion was at 240 nm, based on the h,,, of tipredane. The flow rate was 1.5 ml min-' and the oven temperature held at 40 "C. Isocratic chromatography was performed unless otherwise stated. The suppliers of the columns used are shown in Table 1; all columns possessed stationary-phase packing of 5 pm except for the Hypersil column which possessed 3 pm material.The identity of the peaks in the chromatograms was established by comparison of retention times with those of individually chromatographed authentic samples. Results and Discussion Choice of Mobile Phase Initial selection of solvent strength In order to allow mapping of the resolution surface it was necessary to establish organic modifier-water mixtures for each organic modifier which gave suitable capacity factors of between 4 and 10 for all components being studied. As the elution order of the tipredane metabolites and related sub- stances could only be partially predicted by lipophilicityANALYTICAL PROCEEDINGS, JANUARY 1993. VOL 30 47 FPL 66365XX and FPL 66364XX OH OH e0 / - - - - 0 OH &yo / - - 0 FPL 67526XX 111 Table 1 Manufacturers and suppliers of columns studied Column and dimension/mm Manufacturer Supplier Hypersil Excel ODS Shandon Nova-Pak C l x Waters Ultrasphere C18 (150 Beckman Sphcrisorb ODs2 Phase Spherisorb ODS 1 Phase Resolve Clx Waters p-Bondapak C l x Waters Suplex pKb 100 Supelco Lichrosorb RP E. Merck (150 x 4.9) Scientific (150 x 3.9) x 4.6) (150 x 4.6) Separations (150 x 4.6) Separations (150 x 3.9) (300 x 3.9) (250 x 4.6) (125 x 4.0) select B Hichrom, Reading Fisons Scientific Equipment, Beckman, High Wycombe Loughborough Fisons Scientific Equipment, Fisons Scientific Equipment, Fisons Scientific Equipment, Fisons Scientific Equipment.Supelchem UK. Saffron Fisons Scientific Equipment. Loughborough Loughborough Loughborough Loughborough Walden Loughborough considerations and it has been demonstrated by Snyder et al.that suitable conditions for isocratic separation can be calcu- lated from the elution of the compounds under gradient conditions, initial chromatographic data for the compounds was established by chromatography using the tipredane related substances screen.2 The chromatogram obtained is shown in Fig. 1 and the identities and retention times of the peaks are given in Table 2. Only partial separation was achieved by this method with the compounds eluting as three pairs of poorly separated peaks. The compounds fall into three classes by polarity, the relatively polar sulfoxide diastereoisomers FPL 66365XX and FPL 66364XX, the moderately polar FPL 67526XX and FPL 66366XX and the relatively non-polar compounds I1 and 111.In general, chromatographic separations can be categorized into three classes as follows: (1) compounds which have very similar lipophilicities for which resolution is most difficult; (2) compounds with widely different lipophilicities which are easily resolved but will require gradient chromatography to obtain elution within a reasonable time scale; (3) compounds with moderately different lipophilicities which are readily separated by isocratic chromatography. The critical sepa- rations are those from categories (1) and (2) as these will determine the over-all nature of the method which is developed. From the results using the tipredane related substances screen it can be seen that the separation of FPL 66365XX from FPL 66364XX, FPL 67526XX from FPL 66366XX and of compounds I1 from 111 all fall into category (1).That of the first pair from the third pair falls into category (2), whereas the separation of the first pair from the second pair falls into the category (3). Approximate acetonitrile-water-buffer mixtures were iden- tified for each group of compounds in category (1) by reference to retention behaviour on the tipredane related substances 400 t FPL 66366XX I 0 1 I I I 5 10 15 20 25 Time/m i n Fig. 1 Chromatogram of tipredane metabolites and related sub- stances using tipredane related substances screen.’ Chromatographic conditions as described in the experimental section. Mobile phases A (0.025 mol dmp3 KH2POI) and B (0.025 mol dm-’ KH2POI in 65% v/v acetonitrile); a linear gradient was run over 20 min from 10 to 95% mobile phase B; then the eluent composition was held for a further 10 min. Each peak corresponds to approximately 1-2 pg loaded on to the column Table 2 Retention times (R.T.) of analytes on the tipredane related substances HPLC screen,’ calculated and actual acetonitrile contents of mobile phase used for isocratic elution MeCN R .T. /min Compound typical calculated(%) I FPL 66365XX 5.86 12.1 FPL 66364XX 6.01 12.6 FPL 67526XX 6.95 15.3 FPL 66366XX 7.03 15.5 I1 11.04 27.3 I11 11.17 27.6 MeCN used( % ) 12.5 12.5 12.5 12.5 25.0 25.048 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 gradient',2 (Table 2). The pH of the mobile phase was found to have no effect on the capacity factor, therefore water was used to adjust the elutropic strength instead of a suitable buffer. Solvent type The selectivity of reversed-phase systems is highly dependent on the mobile-phase modifier chosen.From the work of Snyder,3 methanol, acetonitrile and tetrahydrofuran were chosen as the most appropriate solvents for further studies due to their different selectivities. As will be shown later, the resolution of the tipredane metabolites and related substances requires the use of modifiers with different selectivities. Initially, the proportions of organic modifier necessary to obtain similar retention times with methanol and tetrahydro- furan mobile phases (isoelutropic mobile phases) were estab- lished by reference to literature values for solvent strength.4 Where these mobile phases did not elute the compounds in the required capacity factor window (4-10) the mobile phases were adjusted until suitable capacity factors were obtained (Table 3).Isocratic optimization f o r category ( I ) separations Category (1) separations were studied using the statistical design for selecting an optimum mixture of three solvents based on that suggested by Glajch et al.s using mobile phases which gave suitable capacity factors (see Table 3). The statistical design takes the form of a two-dimensional lattice search (Fig. 2). From these seven experiments it was possible to conclude which organic modifier gave the best resolution, as determined by the critical response factor (CRF)' for each of the category (1) separations, and whether ternary or quaternary mobile phases would give any improvement in resolution.From the CRF values shown in Table 4 (values of 0 and ~0 indicate baseline separation and co-elution, respectively) it was deduced that for compounds I1 and I11 methanol was required for resolution, whereas for the diastereoisomeric sulfoxides FPL 66365XX and FPL 66364XX acetonitrile gave the best resolution under the conditions employed. Binary solvent mixtures were chosen as ternary and quaternary systems offered no advantage. As the lipophilicities of FPL 67526XX and FPL 66366XX are similar to those of FPL 66365XX and FPL 66364XX, aceto- Table 3 Calculated and actual tetrahydrofuran and methanol concentrations for mobile phases with similar capacity factors to acetonitrile based mobile phase THF( "/o) MeOH(%) Compound MeCN(%) Calc. Used Calc. Used FPL 66365XX 12.5 8.9 7.5 15.4 40.0 FPL 66364XX 12.5 8.9 7.5 15.4 40.0 I1 25.0 17.8 15.0 30.8 40.0 III 25.0 17.8 15.0 30.8 40.0 Fig.2 Two-dimensional lattice search for three solvents A, B and C. The position of the numbers represent the trilinear coordinates of A, B and C for the seven mobile phases investigated Table 4 CRF values from two-dimensional lattice search FPL 66365XX FPL 66364XX and I11 and Compounds I1 Organic modifier Organic(%) CRF Organic(%) CRF Acetonitrile 12.5 0.02 25.0 0.9 Methanol 40.0 1.3 40.0 0.0 THF 7.5 Co 15.0 1.4 Acetonitrile-Methanol 6.3-20.0 0.08 12.5-20.0 0.0 Acetonitrile-THF 6.3-3.8 ~0 12.5-7.5 0.2 Methanol-THF 20.0-3.8 20.0-7.5 0.0 Acetonitrile-Methanol- THF 4.2-13.3-2.5 ~0 8.3-13.3-5.0 0.2 nitrile at the concentration used for the separation of FPL 66365XX from FPL 66364XX was the organic modifier of choice on practical grounds.This would avoid a rapid change in solvent strength or selectivity which would give rise to baseline fluctuations. Baseline separation of FPL 67526XX from FPL 66366XX was achieved with this mobile phase so no further optimization was performed. Tetrahydrofuran did not possess any advantages over the other organic modifiers and, given its incompatibility with the PTFE HPLC tubing and its greater toxicity, no further work was performed with this solvent. Optimization of solvent strength In order to gain more detailed information before moving from these isocratic methods to a gradient method further investi- gations into the chromatographic behaviour of the compounds were made by studying the relationship between organic modifier strength and capacity factor (k').The effect of methanol concentration on Ink' for com- pounds I1 and I11 is shown in Fig. 3. Unexpectedly the separation improved with increasing methanol concentration. This phenomenon was investigated further by studying the effect of acetonitrile concentration on the separation and the results are shown in Fig. 4. Here, elution crossover can be seen at about 22% acetonitrile: below this level compound I11 eluted before compound 11, whereas above 22% the elution order was reversed. Elution crossover was a direct result of differences in the gradient (S) of the line, in the graph of In k' versus organic modifier concentration, for each compound. A number of factors may lead to this difference in S .The difference in chemical nature of the analytes may be one factor; for example, the sulfone group of compound I1 may participate in dipole-dipole interactions with the mobile phases, whereas such interaction may be absent for the keto group of compound 111. In addition, the decrease in molecular size of compound 111 4.5 I I I I I I i I l l I 24 26 28 30 32 34 36 38 40 42 44 46 MeOH (%) Fig. 3 Effect of methanol concentration on In k' for compounds I1 and 111. Chromatographic conditions as described in experimental section. Compound I1 (A) and compound 111 ( A )ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 49 19.5 24.5 29.5 34.5 MeCN (%) Fig. 4 Effect of acetonitrile concentration on In k’ for compounds I1 and 111. Chromatographic conditions as described in experimental section.Key as in Fig. 3 compared with compound I1 may be another contributing factor: similar effects have previously been reported in studies of an homologous series of alkanes and carboxylic acids on p- Bondapak CI8 columns.6 In an analogous manner FPL 66366XX and FPL 67526XX exhibited differing gradients of In k’ versus acetonitrile concen- tration (Fig. 5 ) . In the light of our experience with the non-C6 hydroxy analogues I1 and 111, the effect of methanol concen- tration on In k’ was also studied and the results shown in Fig. 6. It can be postulated that if it had been possible to use higher percentages of acetonitrile without elution on the solvent front or lower concentrations of methanol without excessively long retention times elution crossover would have been seen in these cases as well.When acetonitrile was employed the elution order of the C6 3.5 3 2.5 & 2 1.5 1 0.5 0 C - 9.5 15.5 MeCN (Yo) 21.5 Fig. 5 Effect of acetonitrile concentration on In k’ for FPL 66365XX, FPL 66364XX, FPL 67526XX and FPL 66366XX. Chromatographic conditions as described in experimental section, with the exception that a Hypersil Excel ODS column was used. FPL 66365XX (0), FPL 66364XX (O), FPL 67526XX (0) and FPL 66366XX (H) 3.5 1 I 18 20 22 24 26 28 30 32 34 36 38 40 42 MeOH (Yo) Fig. 6 Effect of methanol concentration on In k’ for FPL 66365XX, FPL 66364XX. FPL 67526XX and FPL 66366XX. Chromatographic conditions as described in experimental section, with the exception that a Hypersil Excel ODS column was used.Key as in Fig. 5 hydroxy derivatives was observed to be FPL 66365XX followed by FPL 66364XX, FPL 67256XX and FPL 66366XX (see Fig. 5 ) , whereas with methanol there was a change in selectivity; the sulfone derivative FPL 66366XX now co-eluting with the sulfoxide FPL 66365XX (see Fig. 6). The diastereoiso- meric sulfoxides FPL 66365XX, FPL 66364XX and sulfone FPL 66366XX exhibited comparable gradients for the plots of In k’ versus methanol or acetonitrile concentration (see Figs. 5 and 6) and hence for these compounds no change in selectivity was observed over the organic modifier concentration range examined. However, the gradient for the keto derivative FPL 67526XX was consistently smaller, which was also the case for the corresponding non-C6 hydroxy keto derivative (compound 111).This suggested that molecular size may be a vital parameter in determining the magnitude of the gradient. PH There was no significant change in retention time or selectivity of either the sulfoxide diastereoisomers (which possessed an ionic character) or the compounds FPL 67526XX and FPL 66366XX (which lacked ionic character) over a mobile-phase pH range of 2.5-7.5 compared with that of unbuffered mobile phases. Column Temperature The capacity factors of FPL 66365XX and FPL 66364XX were measured at different temperatures by the use of a thermostati- cally controlled column oven. The expected linear reduction in Ink’ was found.7 There was no significant change in the selectivity within the temperature range examined (30-70 “C). Column Type The chromatographic behaviour of the sulfoxide diastereo- isomers (FPL 66365XX and FPL 66364XX) was investigated on columns of varying silica pre-treatment and degree of endcapping (Table 5 ) .Standard isocratic conditions of 12.5% acetonitrile in water were used for all the investigations, a flow rate of 1.5 ml min-’ was employed for columns of 150 mm length and the flow rate of column of other dimensions was adjusted to maintain comparable capacity factors. The retention times of FPL 66365XX, FPL 66364XX and FPL 66366XX relative to the retention time of FPL 67526XX on various columns are shown in Table 6. (Compounds FPL 67526XX and FPL 66366XX acted as markers and proved that the columns investigated had similar efficiencies.) Factors such as YO carbon load and trace metals content of the stationary phases were considered, but the only parameter which appeared to be closely related to the resolution of the sulfoxide diastereoisomers was the degree of ‘endcapping’ or deactiva- tion of the silica surface.Silica based columns, primarily designed for use with basic compounds (Tables 5 and 6, rows 7-9), which had been deactivated to yield a stationary phase with a very low numbers of active sites, gave no resolution of the sulfoxide diastereo- isomers. In addition, silica based columns which had not been deactivated (Tables5 and 6, row 6) and therefore possessed Table 5 Manufacturers information on columns used in study Silica pre- Column number Column Endcapped treatment 1 Hypersil Excel ODS Fully No 2 Nova-Pak C18 Fully No 3 Ultrasphere C18 Fully No 4 Spherisorb ODs2 Fully No 5 Spherisorb ODSl Partially No 6 Resolve CI8 No No 7 p-Bondapack CI8 Fully Yes 8 Suplex pKb 100 No Yes 9 Lichrosorb RP select B No Yes50 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 Table 6 Effect of column selection on relative retention time and selectivity Column number 1 2 3 4 5 6 7 8 9 Column Hypersil Excel ODS Nova-Pak CIS Ultrasphere C18 Spherisorb ODs2 Spherisorb ODS 1 Resolve C18 y-Bondapak C18 Suplex pKb 100 Lichrosorb RP select B Selectivity for FPL 6636SXW FPL 66364XX 1.13 1.10 1.11 1.09 1 .00 1.03 1 .OO 1 .OO 1 .00 Relative retention times FPL 6636SXX 0.63 0.69 0.64 0.69 0.97 0.91 0.85 0.76 0.87 FPL 66364XX 0.71 0.76 0.71 0.75 0.97 0.94 0.85 0.76 0.87 FPL 67526XX 1 .oo 1 .OO 1 .OO I .00 1 .OO 1 .OO 1 .OO I .OO 1 .OO FPL 66366XX 1.11 1.14 1.09 1.10 1.10 1.16 1.12 1.09 1.18 large numbers of active sites or were only partially endcapped (Tables 5 and 6, row 5 ) and contained a substantial number of active sites gave longer retention times and little or no resolution of the sulfoxide diastereoisomers.However, the sulfoxide diastereoisomers were resolved using silica based columns which were classified as ‘fully’ endcapped, yet without silica pre-treatment, by the manufacturers (Tables 5 and 6 , rows 1-4). The method of deactivation of these types of stationary phases gives rise to a small number of isolated active sites which are probably responsible for the selectivity observed. It is possible that the selectivity of the ‘fully’ endcapped columns arose from interaction of the sterically hindered active sites at the column surface with the sulfoxide moiety.Silanophilic interactions have previously been reported to explain the unusual chromatographic behaviour of antibiotics8 and of dibenz0-18-crown-6-ethers.~ However, this is the first reported example of selectivity differences for diastereo- isomers being attributed to these silanophilic interactions. Further investigations into these types of interactions are in progress. As a result of the above findings baseline resolution of the sulfoxide diastereoisomers could be achieved by the use of a 3 pm Hypersil Excel ODS column, which possessed the required characteristics, i.e. , ‘fully’ encapped and no silica pre- treatment, to provide the selectivity and give sharper peaks due to its higher efficiency compared with the other columns investigated.Selective Multisolvent Gradient Elution Method The final stage in the method development for the analysis was linking the conditions appropriate for each of the category ( 1 ) separations (compounds with very similar capacity factors) by a gradient to facilitate the category (2) separation (compounds with very different capacity factors) which was achieved by a simple linear gradient between the two sets of conditions. The resultant chromatogram is shown in Fig. 7. By definition the category ( 3 ) separation in acetonitrile based mobile phase (compounds with moderately different capacity factors) was readily achieved utilizing these conditions.Baseline separation of all six components was achieved within a satisfactory run time of 36 min. Conclusions Several interesting phenomena were observed during this investigation which re-emphasize the complex nature of the interactions which occur in reversed-phase HPLC. Firstly, the interaction of the sulfoxides with the stationary phase high- lighted the importance of column endcapping and silica pre- treatment in determining selectivity; secondly, the elution crossover which occurred showcd the importance of solvent strength in determining selectivity; thirdly, the literature values used to obtain isoelutropic mixtures did not give results in good agreement with experimental data, due to the importance of specific solute-solvent interactions in deter- 300 m z X $ 200 + :: 2 100 C m 0 Fig.7 10 20 30 Tirne/rn i n Chromatogram of tiorcdane metabolites and related sub- - stances on the selective multisolvent gradient elution method. Chro- matographic conditions as described in the experimental section. Hypersil Excel ODS 3 ym (150 x 4.9 mm) column was used. Mobile phases A (12.5% v/v acetonitrile in water) and B (95% v/v methanol in water), the mobile composition was kept at 0% B for 20 min then a linear gradient was run over 30min up to 100% B; then the composition was held for a further 5 min. Each peak corresponds to approximately 1-2 pg loaded onto the column mining retention times. Given the latter two observations, the relationship between In k’ and organic modifier concentration for each solvent and for each solute should be determined before selecting the mobile phases in the two-dimensional lattice optimization.This relationship can be more rapidly estimated for each organic modifier by comparison of capacity factors using two or more isoselective gradient elution methods with different rates of change of solvent strength. ‘O A selective multisolvent gradient elution method, where solvent strength, selectivity and composition were simulta- neously varied during the analysis, was developed using a systematic modular approach which relied to a greater extent upon analytical knowledge and interactive decision making than on fully automated optimization techniques. This resulted in baseline separation of the analytes of interest. The transfer from isocratic to gradient analysis was made easier by the greater over-all understanding of the separation obtained and because specific groups of compounds which were difficult to separate were identified.No optimization software was used and only a binary solvent delivery system was required. The method used requires the availability of individual groups of the compounds of similar capacity factor to allow isoelutropic investigation of areas of the solvent strength/ selectivity domain. In this particular application the individual compounds were also required for identification purposes, as the UV spectra of the individual compounds did not differ sufficiently for positive identification. The authors thank C. Thomson, who synthesized the com- pounds used in this investigation; V. Gerdelat and P.C. Downing for technical assistance; and R. J. Lewis for valuable discussions.ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 51 References 6 Snyder, L. R., Dolan, J. W . , and Gant, J. R., J . Chromatogr.. 1979, 165, 3. 7 Eucrby, M. R., Hare, J . , and Nichols, S. C., J . Pharm. Hiomed. Anal.. 1992, 10, 269. Snyder, L. R., J. Chromatogr. Sci.. 1978, 16, 223. 8 Intcrscience, New York, 1988, p. 136. togr., 1980, 199, 57. 1 2 3 4 Meyer, V. R., Practical HPLC Method Dcvetopment, Wiley- 9 5 Glajch, J . I,., Kirkland, J . J . , and Squire, K. M., J . Chroma- 10 Tanaka. N., andThornton. E. R., J. A m . Chem. Soc., 1977.99, 7300. Horvath, C., Melander, W . , and Molnar. I . , J. Chromatogr., 1976, 125, 129. Moats. W. A., J . Chromatogr., 1986. 366. 69. Nahum, A., and Horvath, C., J .Chromatogr.. 1981, 203, 53. Quarry, M. A . , Grob, R. L., and Snyder, L. R., Anal. Chem., 1986, 58, 907. Studies on the Use of Base-deactivated and Polymeric Stationary Phases in Reversed-phase High-performance Liquid Chromatography of Anthracyclines and Their Metabolites Glynis Nicholls, Brian J. Clark and J. E. Brown Pha rma ce u tica I Ch em is try, Sc h o o I o f Pharmacy, University o f Brad fo rd, Brad ford, West Yo rks h ire BD7 7DP The anthracycline group of antibiotics are amongst the most clinically important antitumour agents in current cancer chemotherapy; doxorubicin in particular has the broadest spectrum of activity of any known antineoplastic agent.’ The 4’-epimer epirubicin has recently been introduced into clinical medicine,* and has been reported to have equivalent antitu- mour activity to doxorubicin but with lower cardiac and systemic toxicity.We have recently described a method for the optimal separation of doxorubicin, epirubicin and their clinic- ally important metabolites using reversed-phase high-perfor- mance liquid chromatography (HPLC).3 In method develop- ment , formal simultaneous optimization strategies (the solvent selectivity triangle and factorial design approach) were used to define an area of factor space, within which the global optimal mobile phase conditions could be located using the modified simplex sequential optimization method. During this work, a complex mechanism of interaction between the stationary phase , mobile phase and anthracycline drugs and metabolites was observed. This included consider- able peak tailing when ionic forms of the drugs and metabolites were present and triethylamine was incorporated into the mobile phase as an endcapping agent to improve peak shape.During method development, complete resolution of all components was attained; however, only minor changes in mobile phase composition had significant effects on both resolution and retention times. Consequently, the robustness of the assay required further investigation, and in this work the function of the stationary phase in the separation was closely examined. This related in particular to the effect of the interaction of the anthracyclines with residual silanol groups present on the previously used silica-based stationary phase.Recent commercial developments in column technology have attempted to address the effect of residual silanols in reversed-phase HPLC separations, and alternatives to the traditional C18-bonded silicas have been introduced, especially for basic compounds. Of particular interest are the base- deactivated reversed-phase bonded silicas and polymeric column packings. In both types of stationary phase the effect of residual silanols on the chromatographic separation is neglig- ible, either because of the type of process used to manufacture the silica backbone, or through the use of non-silica materials. Therefore, these alternative stationary phases were used to investigate whether the over-all dependence of the assay method to changes in mobile phase composition could be improved by altering stationary-phase composition, whilst maintaining good resolution of the anthracyclines and their metabolites.Experimental The anthracycline drugs and metabolites assayed were doxoru- bicin, epirubicin, doxorubicinol, epirubicinol, 7-hydroxydox- orubicinol aglycone, 7-hydroxydoxorubicin aglycone, 7-deoxy- doxorubicin aglycone and daunorubicin (internal standard). The HPLC system consisted of: LKB 2150 dual reciprocating pump (LKB-Pharmacia, Uppsala, Sweden) , Hewlett-Packard 1046A fluorescence detector (Hewlett-Packard, Waldbronn, Germany) and Hewlett-Packard HP3394A computing integra- tor. For sensitive and selective detection fluorescence was used (detection limit for doxorubicin 1 ng ml-I), with excitation wavelength set at 480 nm and emission wavelength at 560 nm.Columns and Mobile-phase Systems Used (a) Five micrometre Hi-Chrom Spherisorb ODS 1, 250 X 4.6 mm i.d., with an optimized mobile phase: acetonitrile- 0.06 mol 1-’ citrate buffer (35 + 65 v/v), containing 0.05% (v/v) triethylamine, pH 4.6. ( b ) Five micrometre Hi-Chrom RPB Spherisorb, 250 x 4.6 mm i.d. Initial mobile phase was acetonitrile-0.06 mol I-’ citrate buffer (35 + 65 v/v), pH 4.6; this was modified by decreasing the acetonitrile content from 35% (v/v) to 30% (v/v) to improve chromatographic peak resolution. (c) Five micrometre Regis ‘Rexchrom’, 250 X 4.6 mm i.d. Initial mobile phase: acetonitrile-0.06 mol I-’ citrate buffer (35 + 65 v/v), pH 4.6. The optimized mobile phase as stated in ( a ) was also used. ( d ) Five micrometre Polymer Laboratories PLRP-S, 250 x 4.6mm i.d.Mobile phase: acetonitrile-0.06 mol I-’ citrate buffer (35 + 65 v/v), pH 4.6. Chromatographic peak efficiency was considerably improved in this experimental programme over the previous method3 by using 250 mm length columns instead of 150 mm. Peak efficiency values (theoretical plates) for all columns were calculated, relative to the parent drug doxorubicin. Resolution values were also calculated for all incompletely resolved peaks. Results and Discussion Conventional C18 reversed-phase packing materials often have residual silanol groups on the silica surface; these may interfere with the chromatographic separation of basic solutes and cause peak trailing and low efficiency. The glycosidic anthracyclines (Fig.1) contain an amine group which is protonated at all pH values at which the best conditions for resolution occur (pH (7). Interactions between the amine group and residual52 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 0 OH 'n Table 1 Stationary phases used and comparison of their effects on the separation of the anthracyclines and their metabolites. Mobile phase conditions as described in Figs. 2 and 3 Particle Nlplates Peak Resolu- Column size/nm m-'* shape tion RT Spherisorb ODS 1 5 26000 Good >1.5 RPB Spherisorb 5 31 000 Excellent 0.684 Regis 'Rexchrom' 5 6800 Poor 0.99 PLRP-S (polymer) 5 1000 Poor 0.77 drug, doxorubicin. separated pair of peaks in the chromatogram. modifier. * Peak efficiency ( N ) was calculated from the value for the parent T Resolution values were based on the resolution for the worst 4 Resolution was increased to R = 1.3 using 32% (v/v) organic Anthracyclinone ring system Daunosamine sugar residue (D) R1 R2 R3 R4 Doxoru bicin Epirubicin Doxoru bicinol Epirubicinol 7-OH Doxorubicinol aglycone 7-OH Doxorubicin ag lycone 7-Deoxy doxorubicin aglycone Daunorubicin D D D D OH OH H D -COCH20H H OH -CHOHCH2OH H OH -COCH20H OH H -CHOHCH20H OH H -CHOHCH20H - - -COCH20H - - -COCH20H - - -COCH3 H OH Fig.1 Structures of the anthracyclines 6 8 10 12 14 16 silanols were minimized in the original method (which used a partially endcapped Spherisorb ODS 1 stationary phase, with 7% carbon loading) by the addition of a short alkyl amine (triethylamine) to the mobile phase. An alternative approach is to use a stationary phase with a higher carbon loading (e.g., Spherisorb ODS 2, 12% carbon loading), although an endcap- ping mobile-phase additive is still required and the optimal component resolution conditions remain in a very narrow operating range.In addition, considerable variation in perfor- mance was found between different batches of Spherisorb ODS 1 stationary phases. Two alternative types of column packings were therefore investigated, which did not have the silanol effect problem and consequently may have a broader range of operating conditions. Base-deactivated Stationary Phases This type of stationary phase was developed primarily for the chromatographic assay of basic compounds, and is manufac- tured selectively to remove residual silanols. The packing material is not deactivated with a basic coating as the name implies, but is deactivated towards basic compounds.Hence, there is often no need for the inclusion of an endcapping agent in the mobile phase and good peak shape can be achieved, even with ionized basic compounds. Two different base-deactivated column packings were investigated, initially using the mobile phase conditions outlined in the previous paper3 but without the inclusion of triethylamine. Peak shape, efficiency and resolution were directly compared with those obtained using a 250 mm length Spherisorb ODS 1 column. 6 I I 1 1 I I I I 2 4 6 8 10 12 14 16 1 I 2 6 Hi-Chrom RPB Spherisorb By using the Hi-Chrom base-deactivated stationary phase, peak shape was improved for all of the anthracycline drugs and metabolites, and therefore peak efficiency was higher than that obtained using the Spherisorb ODS 1 stationary phase (Table 1).However, only partial resolution was obtained for two of the more polar metabolites (doxorubicinol and epirubi- cinol) compared with the baseline resolution attained pre- viously [Fig. 2(a), ( b ) ] . An improvement in resolution could, however, be achieved by decreasing the organic modifier content of the mobile phase from 35% (v/v) to 32% (v/v), but this resulted in an over-all retention time increase from 15.8 min to 18.2 min [Fig. 2(c)]. Baseline resolution of all peaks could only be achieved by reducing the acetonitrile content to 30% (v/v) of mobile phase (total run time 26 min). I I I I I 1 I I I 2 4 6 8 10 12 14 16 18 20 E I uti o n ti me/m i n Fig.2 HPLC separation of the anthracyclines and their metabolites using Spherisorb stationary phases: ( a ) Hi-Chrom Spherisorb ODS 1, with mobile phase 35% (v/v) acetonitrile-0.06 mol I-' disodium hydrogen phosphate-0.03 mol I-' citric acid containing 0.05% (vlv) triethylamine, pH 4.6; (b) Hi-Chrom RPB Spherisorb, with mobile phase as ( a ) ; ( c ) Hi-Chrom RPB Spherisorb, with mobile phase 32% (v/v) acetonitrile-0.06mol I-' disodium hydrogen phosphate- 0.03 mol I-' citric acid. pH 4.6. All columns were 250 x 4.6 mm i.d. stainless steel, packed with 5 pm size material. Flow rate was 1 ml min-', excitation wavelength was 480 nm, and emission wave- length was 560 nm. Key to peaks: 1, doxorubicinol; 2, epirubicinol; 3, 7-OH doxorubicinol aglycone; 4, doxorubicin; 5 , epirubicin; 6 , 7-OH doxorubicin aglycone; 7, daunorubicin; 8, 7-deoxy doxorubicin agl yconeANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 53 Although over-all resolution was not obtained without modification of mobile phase composition, the peak profile was considerably improved, which may significantly enhance sensitivity of detection in biological samples.However, it appears that the separation of the more polar anthracycline metabolites on Spherisorb ODS 1 packing material is partially dependent on secondary interactions with the residual silanol groups. Hence the ‘total’ endcapping found in this base- deactivated column resulted in poorer resolution of doxorubi- cinol and epirubicinol. This can be overcome by decreasing organic modifier content, but the over-all run time is increased, primarily because of the longer residence time in the column of 7-deoxy doxorubicin aglycone, the most lipophilic solute.Regis ‘Rexchrom ’ This base-deactivated stationary phase gave extended reten- tion times for all the anthracyclines when compared with the Spherisorb ODS 1 or the Hi-Chrom RPB Spherisorb packings. Peak shape was generally poorer and therefore efficiency was low (Table 1). However, with the exception of epirubicin and 7-hydroxy doxorubicin aglycone, all components were well separated [Fig. 3(a)]. In both of these base-deactivated stationary phases there is a possibility of some residual silanol effects. This was investi- gated by addition of 0.05% (v/v) triethylamine to the mobile phase, but in each case neither peak shape nor resolution were improved.Further optimization of the mobile phase was not attempted at this stage, but this may lead to some improve- ments in the separation. Thus the Regis column appears to be unsuitable for this particular application, as interaction between the anthracyc- lines and the stationary phase caused considerable peak broadening. Overall, the differences in resolution and efficiency observed between the Regis and Hi-Chrom station- ary phases probably reflect differences in manufacture or type of packing material used. Polymer based stationary phase (Polymer Laboratories Polymeric packings, recently developed for reversed-phase chromatography, have been promoted as a suitable alternative PL R P-S) to bonded-phase silica because of their stability over a wider pH range.They can be used ‘bare’ (e.g., polystyrene divinyl- benzene copolymers or PS-DVB) or derivatized with alkyl functional groups (e.g., Clx), and are reported to have different selectivity to silica-based columns. Additionally, polymer-coated or polymer-bonded silica materials are avail- able. The polymeric stationary phase used in this study was a PS-DVB copolymer. Using a mobile phase composition of acetonitrile- 0.06 mol I-’ citrate buffer (35 + 65 v/v) at pH4.6, the chromatographic peaks were generally poorly shaped, with peak tailing and incomplete resolution of four of the eight anthracycline components [Fig. 3(b)]. Nevertheless, retention times (R,) were comparable to the Spherisorb ODS 1 column, with the exception of the least polar metabolite 7- deoxy doxorubicin aglycone (R, >30 min), but plate efficiency was low (Table 1).The high retentive properties of the column, especially for the lipophilic metabolite, suggests that considerable hydro- phobic interaction occurred between the anthracyclinone ring system of the anthracyclines and the stationary phase. From this brief study, it can be concluded that ‘bare’ polymer-based stationary phases may be of less value than silica-based stationary phases for this application. Conclusions The base-deactivated stationary phases enable good resolution and peak shape to be attained for the anthracyclines under optimal conditions, providing there are only minimal hydro- phobic interactions of the anthracyclinone nucleus with the stationary phase. In this respect, the Hi-Chrom RPB column was found to be preferable to the Regis column, and it could be used as an alternative to the Spherisorb ODS 1 column if slightly longer run times were acceptable. The polymer column investigated was unsuitable for this application under the conditions chosen due to its high retentive properties for the anthracyclinone ring system. This was especially evident for the most lipophilic metabolite 7-deoxy doxorubicin aglycone. However, other less hydrophobic polymeric packings (e.g., alkyl-derivatized polymers) may be of use for this assay. With all the column packings investigated, the dependence of the 1 3 Inj. II II I I I I I 1 I I I I 1 I I Q, E O 2 4 6 8 10 12 14 16 18 20 22 24 26 28 .- 2 (b) 3 2 a, v) 6 3 U - 8 / - 7 I I I 1 I I I I I I I I I I I I I 1 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 Elution time/min Fig. 3 HPLC separation of the anthracyclines and their metabolites using: ( a ) base-deactivated stationary phase (Regis ‘Rexchrom’), with mobile phase 35% (v/v) acetonitrile-0.06 mol I-’ disodium hydrogen phosphate-0.03 mol 1-’ citric acid, pH 4.6; (b) polymer stationary phase (Polymer Laboratories PLRP-S), with mobile phase as (a). For other chromatographic parameters see caption to Fig. 2. Key to peaks as for Fig. 254 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 assay method to changes in mobile phase composition was not significantly improved. This study has demonstrated the influence of residual silanols present on silica-based reversed-phase HPLC supports on the retention of the anthracyclines, and their role in achieving baseline resolution of all components on a Spheri- sorb ODS 1 column. However, in considering alternative stationary phases, it is necessary not only to take into account potential drug-silanol interactions, but also the effect of interactions between the anthracyclinone ring system of these drugs and the hydrophobic components of the stationary phase. The authors thank the Science and Engineering Research Council for their support of this work, Farmitalia Carlo-Erba (Italy) for the donation of drugs and metabolites, and Hichrom Ltd. (Reading, UK) and Regis Chemical Company (Illinois, USA) for their donations of base-deactivated columns. References 1 2 3 Black. D. J . , and Livingston. R . B . , Drugs, 1990, 39, 652. Mross. K . , Maessen, P . , van der Vijgh, W. J . F., Gall, H . , Boven, E., and Pinedo, H . M., J . Clin. Oncol., 1988, 6 , 517. Nicholls, G., Brown, J . E.. Clark, B. J . . and Crawford. S. M., Anal. Proc.. 1993, in the press.

ISSN:0144-557X    

DOI:10.1039/AP9933000039

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 55 Biological Applications of Scanning Tunnelling Microscopy Some time ago a meeting on this topic was held in the University of Nottingham, convened on behalf of the Biological Methods Group by Dr. S. Tendler. At the time it proved impossible to secure extended summaries of the papers for Analytical Proceedings but because of considerable interest in the technique it has been decided to publish very brief abstracts of the papers as a report. STM of Biopolymers: How Does it Work, and What do the Images Mean? Professor S. M. Lindsay (Department of Physics, Arizona State University, Tempe, A 2 85287, USA) opening by stating that scanning tunnelling microscope (STMs) (and more recently atomic force microscopes, AFMs) have produced some impressive pictures of uncoated biomolecules.However, the STM (and AFM) tips interact strongly with substrates, producing artifacts and sweeping away loosely absorbed molecules. Electrochemical methods can be used to bind molecules reliably, and to control the environment of the adsorbate during imaging. The STM can image large ‘insulating’ molecules. While the contrast mechanism is not understood generally, theoretical and experimental studies indicate that resonant tunnelling plays a role, illustrating the potential of STM for probing electronic properties of individual adsorbed molecules. The complexity of the contrast mechanism can make interpretation of STM images very difficult, although chemical identification appears to be possible in certain instances. However, both the STM and AFM show promise of yielding important biological information on a nanometer scale.One example is the sequence directed kinking and bending of nucleic acid polymers. STM Analysis of Polysaccharides and Peptides - Molecu- lar Conformation, Packing and Surface Ordering of Biomolecules Drs. T . J. McMaster and V. J . Morris (Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich NR4 7UA) pointed out that scanning tunnelling microscopy (STM) is of great potential for the direct imaging of biological and biomedical molecular systems to angstrom resolution, without the necessary recourse to the coating and in vacuo operation normally associated with other high resolution microscopical techniques. This will be demonstrated by reference to the results of STM studies on a range of biomolecules.Polysaccharide, peptide and protein molecules displaying a variety of structural forms have been imaged. Linear molecules of poly(y-benzyl-L-glutamate) (PBLG) have been induced to form two-dimensional periodic arrays on a flat surface, and the internal secondary structure has observed. Isolated molecules of the small cyclic polypeptide, valinomycin, have been imaged and the internal cavity has been resolved. Images of less ordered structures such as the zein protein from maize and immunoglobulin molecules have been obtained. These pro- teins are not crystallizable, and no complete X-ray structure is known for them. Hence, STM provides three-dimensional structural information not available from other biophysical techniques.Scanning Tunnelling Microscopy of Proteins Dr. M. Steadman (National Physical Laboratory, Teddington, Middlesex T W l l 0LW) discussed aspects of her work on the STM analysis of proteins which includes immunoglobulins. In addition, the need for accurate calibration of STM systems was highlighted. Calibration methods were discussed. Scanning Probe Microscopy Professor Andreas Engel (M. E. Muller Institute for High- Resolution Electron Microscopy at the Biocenter University of Basel, CH-4056 Basel, Switzerland) pointed out that scanning tunnelling microscopy exploits the tunnel effect to guide a tip at atomic distances over a conductor using a sensitive servo system. Notoriously low conductance of biomacromolecules requires a conducting metal coat for reproducible measure- ments of the surface topography.Alternatively, minute forces as well as temperature changes can be used to probe the surfaces of insulators. Such sensors can be used in aqueous media and may ultimately become tools to measure structure and function of biological systems in situ. Application of Atomic Force Microscopy to Biology Hans-Jurgen Butt (Max-Planck-Institut fur Biophysik, Kenne- dyallee 70, 6000 Frankfurt 70, Germany) said that the atomic force microscopy (AFM) is a scanning probe microscope capable of resolving atoms on hard materials. As the AFM does not require a conductive sample, it was soon applied to biological materials. A lysine crystal was resolved with molecular resolution and the clotting of fibrinogen was imaged in water in real time. Two important fields of investigation are sequencing DNA and-even more important -imaging of membrane structures.Although encouraging progress has been made, sequencing DNA with the AFM is not yet reliable. The main problem is that on flat surfaces like mica the DNA is pushed around by the tip. However, samples of membrane structures, organic monolayers and bilayers, proteins embed- ded in a lipid layer, and membrane proteins, have been imaged with molecular resolution. In addition, the surfaces of bacteria, plants, and red and white blood cells were imaged in air and water. A problem in atomic force microscopy is the force applied by the tip to the sample. Are these forces strong enough to deform or even destroy biological materials? This question will be addressed and perspectives of atomic force microscopy in biology will be discussed.Biological Applications of Scanning Tunnelling Micro- scopy: Integration with Computational Chemistry Drs. D. E. Jackson, M. C. Davies, C. D. Melia, S. J . B. Tendler and P. M. Williams (VG STM Laboratory for Biological Applications, Department of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD)56 ANALYTICAL PROCEEDINGS, JANUARY 1993, VOL 30 produced the final paper. Dr. Tendler stated that the ability of scanning tunnelling microscopy to provide fundamental infor- mation on biological molecules was now being realized, with several studies reporting good image data on a range of molecule types. The potential for topographic information at near-atomic resolution on single surface-adsorbed biomol- ecules indicated that STM was emerging as a new and very important complementary biophysical technique for the struc- tural analysis of biomolecules.However, in order to establish STM in this biophysical role, initial data sets needed to be interpreted in the light of structural parameters derived by other techniques (e.g., X-ray crystallography, high-field NMR computational chemistry). His group believed that the cost effective way to integrate such complementary 3-dimensional data sets was through the development of appropriate software tools for use on high-performance computer graphics systems. The presentation described the ongoing programme of work in their laboratories to establish STM as a valuable tool for the structural elucidation of biomolecules.Initial results for the analysis of several biological samples including DNA and proteins were presented. New software algorithms were described for the manipulation, interrogation and display of STM data sets. In addition, image analysis techniques utilizing Fast Fourier Transforms convolutions were presented. 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ISSN:0144-557X    

DOI:10.1039/AP9933000055

出版商:RSC    

年代:1993

数据来源: RSC