January, 1975 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY 25 High-performance Liquid Chromatography (HPLC) The following are summaries of three of the papers presented at a meeting of the Midlands and East Anglia Regions and the Special Techniques and Chromatography and Electro- phoresis Groups held on May 23rd, 1974, and reported in the June issue of Proceedings (p. 137). Developments in H ig h-performance Liquid Chromatography Packings and Columns R.E. Majors The renaissance in liquid chromatography (LC) is due, in part, to developments in column packing materials. Classical column chromatography utilised large-diameter porous packings such as silica gel or alumina, with particle diameters of 100 pm or greater. These particles are undesirable for high-performance liquid chromatography, particularly at high flow- rates, because of band broadening due to slow mass transfer of sample components, which is caused by the deep pores and convective mixing within interparticle channels.On the other hand, these porous particles, due to their high surface area, typically 200 to 400 me g-1, have large sample capacity. Sample capacity is particularly important for preparative chromatography or when larger samples are required for detectors of low sensitivity.Theoreticians predicted that there were at least two ways to reduce these band broadening phenomena. One way was to use the so-called porous layer beads (PLB) developed in the late 1960s. These packings consist of a solid non-porous core (usually glass) of approxi- mately 40pm and a thin porous outer shell approximately 1 to 2 pm thick.The outer layer usually consists of silica gel, alumina or an ion-exchange resin (referred to as a pellicular). Owing to the thin coating, solute mass transfer in the stationary phase is improved and high flow-rates can be used with little loss in efficiency. Most important, the thin coating also means that relative to the totally porous packings, sample capacity is reduced.An alternative way to reduce band broadening due to long diffusion paths and convective mixing is to reduce the particle diameter of the packing. This fact had been known for some time but techniques of sizing and packing such small particles were unavailable. During the last 2 years, these problems have been overcome and a renewed interest in porous particles has taken place.When properly packed, small porous particles (SPP) not only exhibit high efficiency but still retain the advantage of the large porous particles-large sample capacity. A large number of both PLB and small porous particles, with diameters of less than 15 pm, have become commercially available. Comparative performance of small porous particles and PLB has both theoretical and practical interest.Table I compares the relative efficiency (HETP) for various packings at a linear velocity of 1 cm s-l. Although not an exact comparison, the data indicate that, for best efficiency, columns packed with the Varian A ssociates Limited, Russell House, Molesey Road, Walton-on-Thames, Surrey, KT12 3P J EFFICIENCY Packing Durapak-OPN Merckosorb Si-60 Corasil I (1% BOP) Corasil I1 Zipax (0.6% BOP) Spherisorb silica Merckosorb Si-60 TABLE I AS A FUNCTION OF PARTICLE SIZE* Average particle P 55 3.30 P 45 2.50 PLB 44 1.40 PLB 44 0.80 PLB 30 0.66 P 20 0-66 P 6 0.07 T v e t diameter/ pm HETP/mm Packing technique Dry or slurry, Dry Dry Dry Dry b.d.+, Dry or slurry.Slurry, b.d. b.d. * For test solute, capacity factor (R') = 1 to 2, and linear velocity (V) = 1 cm s-l.P = porous; PLB = porous layer bead. b.d. = balanced density.26 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY Proc. Analyt. Div. Chem. SOC. smallest diameter porous packing should be used. However, as the particle size is decreased, the back-pressure increases. Then, the pressure output of the LC pump and/or system may become the limiting factor.Roughly speaking, for a given column length and set of con- ditions, if the particle diameter is reduced by one half, the pressure drop across the column is raised by a factor of 4. However, due to the greatly increased efficiency of SPP, short columns may generate many theoretical plates. These short columns require only moderate pressures. For example, at normal flow-rates (2 ml min-l) a 15-cm column of 10-pm silica gel may generate one thousand or more theoretical plates but require a pressure of only several hundred pounds per square inch for low-viscosity mobile phases.Thus, to accomplish the same separation in the same analysis time, SPP may require lower pressures than PLB.1 However, for difficult separations requiring thousands of theoretical plates, long columns packed with small particles (5 pm) and high pressures may still be required.Table I1 compares PLB and SPP on the basis of several general criteria. Column efficiency is shown as the best values obtainable at reasonable flow velocities. The capacity of PLB is lower than that of SPP on a milligram of solute per gram of packing basis but when compared on a column volume basis (PLB are more dense than porous packings) the difference is reduced.Pressure requirements are compared for columns of equal diameter and length operated under the same conditions (i.e., the same linear velocity, mobile phase, etc.). The performance factorg compares columns in terms of their effective plates normalised for pressure drop and separation time.In other words, if we compare effective plates per unit pressure drop for the same analysis time, SPP give higher values than PLB. On a practical basis, PLB can be successfully dry-packed using packing techniques not unlike those used for gas-chromatographic columns. On the other hand, SPP, especially those below 20 pm, require special packing procedures. Slurry packing procedures are the easiest to use but dry packing can be mastered by those with patience and/or a “soft touch.” On a cost basis, SPP purchased in bulk are generally less expensive.Prices for pre-packed columns of both types are in the same range. However, due to the higher number of plates for an SPP column, more than one PLB column may be required to equal a single SPP column.COMPARISON OF Factor Efficiency (best values) Capacity TABLE I1 POROUS LAYER BEADS AND SMALL PARTICLE SIZE POROUS PACKINGS Pressure (50 cm x 2.2 mm column) Performance factor (N/ AP) Packing ability Cost : Packing Prepacked columns Fig. 1 presents the results of Porous layer beads (40 pm) Reasonable (H = 0.2 to 0.3 mm) Low (0.1 mg g-1) Low (300 to 500 lb i r S ) 10 to 1s Easy, dry pack Difficult (slurry pack or pre- High: L2 to 3/g flS0 to 90 Small porous packings (5 to 10 pm) High (H = 0-01 to 0.03 mm) High (5 mg g-l) Higher (greater than 2000 lb in-2) 50 to 100 packed columns) Lower: fl0-5 to l.O/g L60 to 110 a separation run under comparable conditions on a PLB (40-im) column and an SPP (5-pmj silica gel column.Although selectivity (relative re- tention) for the two columns is slightly different, a number of useful comparisons can be made.To achieve base-line separation of all four components on the PLB column, a I-m column was required. A better separation was achieved on a 15-cm column of 5-pm silica. This improved separation was a result of both increased efficiency (lower HETP) and increased selectivity. For N-ethyl-9-phenylazoaniline, an impurity was resolved, which remained unresolved on the PLB column.With the SPP column, analysis time could have been shortened considerably by (a) increasing the flow-rate but at the expense of pressure; (b) increasing the mobile phase strength; and/or (c) decreasing the column length. For the PLB column, the pressure drop was only 180 Ib in-2 compared to 1350 Ib in-2 for the 5-pm SPP.Through the chemical bonding of stable siloxane (5-0-Si) phases on to both PLB and SPP, unique separation selectivity may be obtained. These phases do not “bleed,” are stable in aqueous and non-aqueous media and, most important, may be used with solvent programming (gradient elution). Bonded phases on SPP have the advantage of higherJanuary, I975 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY la) e N = N e N E t 2 I I I I I I 1 I I I 0 2 4 6 8 1 Time/mi nutes 27 JYmpurity I I 3.8 4.8 ! 3 0.5 1 1 *5 1 -8 Time/minu tes Fig.1. Comparative separation of azo compounds on porous layer beads and on small porous particles. (a) Column dimensions, 1000 mm x 2-2 mm; average particle diameter of porous layer beads, 37 to 50 pm; pressure drop, 180 lb in-2; linear velocity, 1 cm s-l; mobile phase, 1 per cent.methylene chloride in hexane. (b) Column dimensions, 150 mm x 2-2 mm; average particle diameter of porous silica, 5 pm; pressure drop, 1350 lb i r 2 ; linear velocity, 1 cm s-l; mobile phase, 10 per cent. methylene chloride in hexane. 3 loading than the PLB ones. Both non-polar phases, such as those containing octadecylsilane groups, and polar phases, such as alkylnitrile and alkylamine, have been permanently bonded to 10-pm silica gel.3 Compared with silica gel, which has partially acidic hydroxyl groups on its surface, the -NH, phase has basic properties and, in acidic media, may function as a weak anion exchanger.Owing to their obvious advantages such bonded phase packings should eventually replace conventionally coated liquid - liquid chromatographic materials.28 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY R o c .Artalyt. Div. Chem. SOC. References 1. 2 . 3. Majors, R. E., and MacDonald, F. R., J . Chromat., 1973, 83, 169. Snyder, L. R., in Stock, R., and Perry, S. G.. Editors, “Gas Chromatography 1970,” Institute of Majors, R. E., in Grushka, E., Editor, “Bonded Stationary Phases in Chromatography,’’ Ann Petroleum, London, 1971, pp.81 to 111. Arbor Science Publishers, Ann Arbor, Michigan, 1974, Chapter 8, pp. 139-172. Large-scale Moving- bed Liquid Chromatography P. J. Carr Unilever Research, Port Sunlight Laboratories, Wirral, Cheshire, L62 4XN In recent years resurgence of interest in liquid chromatography in columns has led to the optimisation of materials and operating parameters, giving vast improvements in speed and resolution.As a research technique, high-performance liquid chromatography has narrowed the gap considerably, between itself and gas chromatography in terms of speed, resolution and convenience of operation. The coupling of liquid chromatography with highly sensitive spectroscopic methods has meant that individual components can be isolated from admixtures in microgram to milligram amounts for subsequent identification by spectroscopic methods such as infrared spectroscopy, mass spectrometry and nuclear magnetic resonance spectroscopy. However, we have found that this type of chromatographic service has, by itself, been unable to satisfy completely the demands of many research workers in product development chemistry.There is now a growing need for the isolation of preparative amounts of pure compounds, or narrow fractions from commercial and reaction products in order to evaluate their behaviour in user property tests. Preparative amounts are normally defined as any level of material that is isolated for subsequent testing; this paper deals with tens of grams amounts sufficient for the testing of user properties, e.g., which component in a mixture gives improved deter- gency or foaming.We have found that simple scale-up of conventional columns has enabled us to achieve separations of up to 0-5 to 1.0 g of material into narrow fractions (or values covering the range 1.05 to 1.50) on l-inch diameter columns without any loss of resolution; with 2-inch columns a marked loss in resolution occurred.However, the demand for increased scale- up persuaded us to look at the area of moving-bed continuous chromatography by means of an instrument known as a sequential separator. The instrument is now manufactured by Precision Engineering Produds (Suffolk) Ltd.* There are two basic approaches to moving- bed chromatography : (i) A free-falling system in which the adsorbent is allowed to fall freely down a vertical column up which the mobile phase containing the feed is passed; (ii) A closed-column system in which a number of columns are flow programmed to operate in sequence as (a), a series of independent columns or (b), a continuous column.The liquid sequential separator is based on the second of these concepts and can be used to operate under overload conditions or as a continuous analytical instrument. Principle Sequential separation is a form of moving-bed chromatography which can be used to separate continuously or to concentrate a feed solution.Up to twenty independently controlled chromatographic columns may be used. When the columns are set to operate in sequence the mobile phase and sample are programmed to move along the bed in a pre- determined manner ; take-off points for separated components are similarly programmed.The process of sequential separation is made possible by means of a multi-port rotary valve that consists of a “stator” (which is fixed and into which sample and solvents are fed and individual components are eluted from the system), a graphite-loaded PTFE programmable disc and a “rotor” (which can move and from which all the columns are suspended). When the rotor is moved between positions (indexed) the columns are moved opposite to the mobile phase flow.Thus, slow-moving components are moved counter-current to the mobile phase * Atlas Works, Cullum Road, Bury St. Edmunds, Suffolk.January, 1975 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY 29 flow (upstream) while faster moving components leave the downstream end of the system, together with the mobile phase.Also, by matching the index time to the rate of movement of one component in a mixture, it is possible to concentrate that component at or near the central feed-point. Applications The equipment is capable of separating up to 100 g h-l of complex synthetic and natural organic mixtures into as many as twelve fractions.By using solvents of increasing strength around the system, separations can be achieved (via stepwise gradient elution) which are comparable in resolution to medium-resolution liquid chromatography. The separations obtained on 1 x 50 cm columns packed with 60-pm silica gel include: (i) separation of isooctane and toluene from admixture at a sample throughput rate of 100 g h-l; (ii) separation of an organic reaction product into hydrocarbon, monomer and dimer fractions at a sample throughput rate of 5 g h-l; (iii) fractionation of ethoxylated non-ionic compounds into thirteen narrow-cut fractions using a multi-step solvent gradient system; a sample throughput rate of 2 g h-l was obtained; (iv) isolation of a single ethoxylated non-ionic compound in high purity (greater than 98 per cent.) from a wide distribution mix at a sample throughput rate of approxi- mately 2 g h-l.The technique has also been applied to the separation of dyes and drugs and to the con- centration of trace components. SUMMARY AND CONCLUSIONS Organic reaction products have been separated by means of a sequential separator into individual components or narrow cuts at sample throughput rates of lOOg h-l on 1 x 50 cm columns; high resolution can be obtained when sample throughput is sacrificed (e.g., 2 g h-1 throughput).The instrument has obvious applications in the purification of fine chemicals and polymers and in the enrichment of trace components in pollution studies.Practical High-performance Liquid Chromatography Using Small Size Particles Brian H. Freeman James A . Jobling G- Co. Ltd., Laboratory Division, Stone, Staffordshire The survival of classical liquid chromatography has been due, to a great extent, to its great versatility in comparison with other forms of chromatography.In comparison with the inert gas generally used in gas chromatography, the mobile phase in liquid chromatography can play a central role in the separation process by selective interactions within the mobile phase. The recent advances in liquid column chromatography have been caused in part by im- proved packing materials. Both pellicular and microparticular materials are used to solve the problems caused by the slow rate of diffusion in liquids.Under pressure, high flow-rates can be obtained and resolution of compounds can take place in a few minutes. In order to obtain the efficiency necessary for high-performance liquid chromatography these particles must be packed into regular and homogeneous columns. The two methods of packing most uni- versally accepted are the Kirkland modified tap - fill methodl of dry packing and the high- pressure slurry packing method.2 Pellicular particles consist of a non-porous sphere coated with a thin layer of adsorbent material approximately 1 pm in thickness.They are generally 30 to 40 pm in diameter and can be adequately packed by the dry-packing procedure. They give reasonable efficiency but have low sample capacity and are relatively expensive.Microparticles are totally porous small size particles normally 5 to 10 pm in diameter. They are more difficult to pack, usually requiring a high-pressure slurry method, but they do have a high sample capacity, can give very high efficiencies and are relatively cheap. Unfortunately, at the moment, there is no universally accepted criterion by which the performance of different columns can be easily compared.Work on the packing of micro-30 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY Proc. Anatyt. Div. Chem. SOC. particles has shown that the efficiency of a column, expressed as the height of a theoretical plate, can vary with the flow-rate and the retention time of the solute. Another important variable is the pressure drop across the column.To compare fairly the performance of different types of packing both the flow-rate and the capacity factor should be virtually constant. A number of nicroparticulate silicas have been packed by the high-pressure slurry method at 6OOO lb in-2 in glass-lined stainless-steel columns (300 mm x 1.8 mm id.) and their performances compared (Table I). TABLE I PERFORMANCE OF MICROPARTICULATE SILICA COLUMNS Particle diameter/ Clm Sorbsil-0520 5-20 Merckosorb S 1-60 10 Partisil- 10 11 Partisil-5 7 Spherisorb Slow 10 Linear velocity/ cm s-l 0.26 0.30 0.30 0.32 0-24 Capacity factor Pressure HETP, (k‘) drop (AP) H/mm 2.0 800 0.16 1.7 280 0.07 1.6 260 0.09 1.7 210 0.12 1.9 800 0-04 The system consisted of a Jobling LD711 Eluent Delivery Unit using hexane modified with 1 per cent.of acetonitrile as solvent. A specially designed syringe injection head allowed introduction of the sample, which was a mixture of standard solutions of pentane (unretained), benzene, phenetole, ethyl benzoate, nitrobenzene and benzophenone dissolved in the eluting solvent. Detection was effected by the Jobling LD1205 Ultraviolet detector operating at 254 nm.At similar flow-rates and capacity factors the efficiency obtained must be viewed in conjunction with the pressure drop across the column. The irregularly shaped silicas in this study, both with narrow particle diameter ranges, namely Merckosorb S1-60 and Partisil-10, both gave good, efficient columns with a reasonable pressure drop across the column. Partisil- 5 gave an improvement in efficiency but also a large pressure increase.Sorbsil-0520, with its wide range of particle size, gave only moderate efficiency and a high pressure drop and so fares very badly in this comparison. Spherisorb, although giving the lowest pressure drop, as expected from its narrow particle diameter range and its spherical shape, was gene- rally disappointing and is probably better packed by other methods.Extra-column effects become very much more important with microparticulate materials, the high efficiency obtained meaning that fairly short columns are used. Thus, design of injection head, detector and connectors are critical for good performance. The injection of sample should be as near on-column as practical aspects allow, connectors between the column and detector should be of minimum dead volume and the detector cell should be as small as practicable and have good flow characteristics.On-column injection quickly destroys the top of the column packing and gives rise to rapid deterioration of column performance so, in practice, it is necessary to have some kind of porous inlet filter at the top of the column although this can reduce efficiency by a factor of 2 to 3.3 Large variations in efficiency can be obtained with microparticulate packings, depending on the capacity factor of a particular solute.Efficiency is usually at a maximum when k‘w2 and the optimum range occurs when the solutes in question have capacity factors between about 1.5 and 4. For all types of column packings an increase in flow-rate decreases the time of analysis but also decreases the efficiency of the column and a higher pressure is required.For a particular analysis a compromise must be made between these conflicting effects. With the larger size pellicular particles longer columns are generally used and extra-column effects become less important. This means that less care is needed to optimise conditions for routine operation and the pellicular columns are very useful for “scouting” for conditions.Dry-packing procedures are generally accepted for particles of more than 20 pm, with the modified tap - fill method being the most generally used. However, dry-packing pro- cedures are difficult to reproduce with respect to column performance and are time consuming and tedious.High-pressure slurry packing is the method of choice for particles of less than 20 pm. It is comparatively reproducible but does require high-pressure apparatus. The slurryJanuary, 1975 R AND D TOPICS IN ANALYTICAL CHEMISTRY 31 can be prepared as a balanced-density or an ammonia-stabilised suspension; both give good results. Ultrasonic mixing is not essential provided the suspension is well shaken. The Suspension is then packed into a column at a high flow-rate and at 5000 lb in-2, to give regular and homogeneous packing. Some small size particles have been packed by dry-packing procedures, notably by tamping between additions and in published cases4 this method can give good results. How- ever, it remains tedious and lacks reproducibility. Spherisorb is normally packed using vibration and is reported to give very good results. However, in this case, the prepared column suffers badly from peak asymmetry, probably caused by flow-path inequalities due to disturbed packing at the column walls. When difficult separations are required, or very small amounts of solute need to be detected, the high-efficiency columns of microparticles are very useful. Examples include the concentration of aflatoxins in groundnuts, the separation of acetoxycholanates and the analysis of vitamins in foodstuffs. References Lower pressures do not give such good results. 1. 2. 3. 4. Kirkland, J. J., J . Chromat. Sci., 1972, 10, 129. Majors, R. E., Analyt. Chem., 1972, 44, 1722. Kirkland, J. J., J . Chronzat., 1973, 83, 149. Huber, J. F. K., Chirnia, Aarau, Supplement, 1970, 24.