There s Great Chemistry Between Us.

There s Great Chemistry Between Us.

Introduction HPLC Columns Theory, Technology and Practice Uwe D. Neue, Waters Corporation, Milford, MA published by John Wiley & Sons High performance liquid chromatography and its derivatives techniques have become the dominant analytical separation tools in the pharmaceutical, chemical, and food industries, environmental laboratories, and therapeutic drug monitoring. Although the column is the heart of the HPLC instrument and essential to its success, until now no single book has focused on the theory and practice of column technology. HPLC Columns provides thorough, state-of-the-art coverage of HPLC column technology for the practicing technician and academician alike. Along with a comprehensive discussion of the chemical and physical processes of the HPLC column, it includes fundamental principles, separation mechanisms, available technologies, column selection criteria, and special techniques Special features include: Explanation of the underlying principles of HPLC columns Methods for selecting columns Practical advice on using and applying columns, including examples Section by M. Zoubair El Fallah on methods development Special techniques, including preparative chromatography, continuous chromatography, and the simulated moving bed Troubleshooting section Description Part No. Price HPLC Columns Theory, Technology and Practice WAT038216 89.95 Choice of HPLC Material Base Material Packings for high-performance liquid chromatography (HPLC) can be based on either an inorganic ceramic or an organic polymeric substrate. The inorganic ceramics used are predominantly silica and alumina. Inorganic packings have a high rigidity and do not swell in any solvent. Polymeric HPLC-grade packings are based on crosslinked styrene-divinylbenzene or methacrylates. Polymeric packings do not have the rigidity of inorganic packings, and are more compressible. Solvents and analytes can penetrate the polymer matrix, which can cause swelling of the particles and results in low column efficiency due to reduced transfer. lica lica is the most popular base material for HPLC packings. In addition to the high physical strength that it shares with other inorganic packings, silica provides a surface to which a broad range of ligands can be attached using well-established silanization technology. With this surface modification technique, packings for reversed-phase, ion-exchange, hydrophilic-interaction, hydrophobic-interaction or size-exclusion chromatography can be prepared. lica-based packings are compatible with a broad range of solvents from polar to non-polar. Their weakness is the limited stability in aqueous alkaline mobile phases. In general, a ph range of 2 to 8 is recommended for the routine use of silicabased packings. Polymeric Packings Highly crosslinked styrene divinylbenzene-based and methacrylate-based packings are compatible with the typical pressures of HPLC. However, their pressure limit is lower than the limit of most inorganic packings. The styrene-divinylbenzene matrix is very hydrophobic. It is compatible with all mobile phases, including the entire aqueous ph range. The compatibility with strongly acidic and strongly basic eluents allows thorough cleaning of the packing with NaH or strong acids. The methacrylate matrix is intrinsically more hydrophilic than the styrene-divinylbenzene resin, and it can be made very hydrophilic with appropriate modification of the functional groups. This resin is not quite as stable as the styrene-divinylbenzene resin at both extremes of the ph scale, but it is still stable enough to allow repeated cleaning of a column at ph 13. All polymeric packings exhibit swelling and shrinking with a change of the mobile phase. For the highly crosslinked packings useful for HPLC, the swelling and shrinking is limited. Due to the penetration of solvents and small molecules into the polymeric matrix, the mass transfer of small molecules is slower in polymeric supports than in ceramic packings. This leads to lower separation efficiencies for small molecules. For large molecules like proteins or synthetic polymers, the performance of polymeric packings is comparable to ceramic-based packings. Therefore, polymeric packings are used extensively in the separation of natural and synthetic polymers. As with silica-based packings, a broad range of surface chemistries is available for reversed-phase, ion-exchange, hydrophilicinteraction, hydrophobic-interaction or size-exclusion chromatography. 44

Alumina Spherical Alumina has the same favorable physical properties as silica with the added compatibility of an extended ph range. Like silica, it is very rigid and does not swell or shrink in any solvents. Unlike silica, bonded phases on alumina are not stable in aqueous mobile phases, however, coated alumines have been produced that show excellent ph stability in aqueous systems. Choosing the ight Base Material lica Alumina Styrene- Methacrylate divinylbenzene rganic Solvents +++ +++ ++ ++ ph ange + ++ +++ ++ Swelling/Shrinking +++ +++ + + Pressure Capability +++ +++ ++ + Surface Chemistries +++ + ++ +++ Efficiency +++ ++ + + +++ Good ++ Average + Poor lica-based packings are used for most HPLC analyses, especially for low-molecular-weight analytes. Polymers are the packings of choice for the analysis of high-molecular-weight compounds, with size-exclusion and ion-exchange chromatography as primary application areas. Particle Shape Chromatographic packings are available in two particle shapes: irregularshaped particles and spherical particles. Irregular The majority of currently available analytical packings are spherical. They are easier to pack than irregular packings. High performance, good column stability and low backpressure can be achieved reproducibly. Spherical materials are available with particle sizes from 3 µm to over 20 µm. The most popular particle size for analytical columns is 5 µm, but the use of short 3 µm columns is increasing. This is due to the fact that the technology for the preparation of 3 µm columns has improved over the last few years. Today, short 3 µm columns achieve the same column life time and performance as 5 µm columns, but provide the benefit of shorter analysis times. Particle Shapes of Various Packings Material Shape Material Shape Bondapak Irregular esolve Spherical µbondapak Irregular Waters Spherisorb Spherical Delta-Pak Spherical Symmetry Spherical Nova-Pak Spherical For reproducibility and high stability, spherical packings offer the best choice for analytical applications. The availability of larger particle sizes at reduced cost makes irregular-shaped packings attractive for preparative applications. Comparison Irregular Spherical Stability + +++ Particle size range +++ ++ Analytical + +++ Preparative +++ + Analytical Chromatography Irregular-shaped particles are obtained by milling and sizing of large particles. The original high-performance chromatographic packings were prepared by this technology. Many standard analytical methods are based on these materials. An example is µbondapak C 18 which is specified in about 50% of the assays in the US Pharmacopoeia. Irregular-shaped packings are available in larger sizes than spherical packings, but are not usually available less than 5 micron in diameter. A few have higher specific surface areas than those found in spherical particles. In general, irregular particles are more difficult to pack than spherical particles. Columns packed with small irregular particles may exhibit poorer packed bed stability than those prepared from spherical particles of the same size. However, the range of larger particle sizes and surface areas as well as their lower price makes irregular particles more attractive for larger scale preparative applications. +++ Good ++ Average + Poor 45

Specific Surface Area Measurement and eporting The specific surface area of chromatographic packings can be measured by nitrogen adsorption or mercury intrusion methods. Different methods may give slightly different results. Manufacturers usually report nominal values. The dimension of the specific surface area is m 2 /g. Surface Area and Pore ze The specific surface area of a packing is determined by the size of the structural elements that form its skeleton. This size is in turn closely linked to the pore size of the packing. Therefore there is a fundamental relationship between the specific surface area of a packing and the size of its pores, especially at constant specific pore volume. Packings with a small pore size have a large specific surface area, packings with a larger pore size have a small specific surface area. For example, packings with a specific pore volume of about 1 ml/g and a pore size of 10 nm (100Å) have a specific surface area of 300 m 2 /g, while a packing with the same specific pore volume and a pore size of 30 nm (300Å) has a specific surface area of 100 m 2 /g. You can use the following rule of thumb for the relationship between specific surface area, pore size and specific pore volume: SA 3 SV PD A convenient simplified measure for the retentivity and capacity of a packing is the ratio of the specific surface area to the specific pore volume, called the particle phase ratio. As one can see from the first equation above, the particle phase ratio, and therefore the retentivity and capacity, are exclusively determined by the pore diameter of the packing. Generally, packings with a smaller pore size have a higher capacity than packings with a larger pore size, if the sample molecules are small enough to penetrate the smaller pores. The exact same relationship holds for bonded phases. You should select the packing primarily based on the pore diameter and the size of your analyte molecules. Pore ze Measurement and eporting The pore-size distribution and the specific surface area can be determined by the same methods, that is, nitrogen adsorption and mercury porosimetry. The average pore size can also be determined by an inverted sizeexclusion method, but a true pore-size distribution can not be established by this method. However, due to problems with the other methods, the inverted size-exclusion method might nevertheless be the method of choice for polymeric packings. The pore size is given either in nm or in Å. The values for the average pore size are nominal, and different manufacturers make different assignments. Therefore, one manufacturer s designation of 10 nm (100Å) might be identical to another manufacturer s designation of 8 nm (80Å) or 12 nm (120Å). SA is the specific surface area, SV is the specific pore volume and PD is the pore diameter. Surface Area and etention Surface area is often used as a measure for the retentivity or the capacity of a packing. However, it needs to be pointed out that this leads to erroneous conclusions, since both are proportional to the surface area per volume of packed bed rather than per gram of packing. The surface area per packed bed volume, which is also called the phase ratio ß, depends on the packing density. The packing density in turn is primarily a function of the specific pore volume. For a normal packed bed of silica particles, the phase ratio can be estimated as follows: SA ß = 0.6 SV + 0.45 The factor 0.6 stems from the fact that the particles occupy only 60% of the column volume, and 0.45 is the volume of the silica skeleton of 1 g of packing. Pore ze and etention As pointed out in the last section, the pore size is the fundamental factor for the physical properties of the packing and determines the particle phase ratio. The particle phase ratio in turn determines the retentivity and the capacity of a packing. 46

Pore ze and Molecular Weight Specific Pore Volume and Particle Strength The size of analyte molecules increases with molecular weight. If the size of the molecule is about one third of the pore size or larger, mass transport in the pores is severely hindered and low efficiency and peak broadening result. Under these circumstances, packings with a larger pore size should be used. For all standard analytical problems, packings with pore sizes between about 6 nm (60Å) and 13 nm (130Å) are typically used. The larger pore size packings can also be used for peptide separations. For analytes with a larger molecular weight, for example proteins, packings with a 30 nm (300Å) pore size are more suitable. But these packings do not work well with smaller molecules due to the reduced surface area. For analytes with a molecular weight of less than 3,000, the smaller pore size should be used. If the molecular weight is larger than 10,000, packings with a pore size of 30 nm should be used. For analytes with a molecular weight between 3,000 and 10,000, both packings may give satisfactory results. Pore ze Distribution Most HPLC packings have a pore-size distribution that spans about an order of magnitude. Such a distribution is sufficiently narrow for most practical purposes. However, the performance of a packing improves when micropores are absent. This may give an advantage to packings with a narrow pore-size distribution such as Waters Spherisorb packing. Nonporous Packings Nonporous packings can be used for analytes with a very high molecular weight that exhibit restricted diffusion in large-pore packings. However, only a limited surface area is available. The specific pore volume is a measure of the empty space in a particle. The larger the specific pore volume, the smaller is the fraction of the particle that is occupied by the particle skeleton. Therefore, particles with a large specific pore volume are less strong than particles with a small specific pore volume. licas with a specific pore volume of 0.5 ml/g or less, such as Waters Spherisorb or Nova-Pak, are mechanically extremely rugged. Many HPLC-grade silicas have a specific pore volume of 1 ml/g, which is generally sufficient for most applications. Choosing a ugged Packing Specific Pore Volume Strength/uggedness 1.0 ml/g ++ 0.5 ml/g ++++ ++++ Excellent ++ Average Specific Pore Volume and ze Exclusion Chromatography In size-exclusion chromatography, the particle porosity is one of the key parameters that determines the usefulness of a packing. The particle porosity increases with increasing specific pore volume. If silica-based packings are used for size-exclusion chromatography, packings with a specific pore volume of about 1 ml/g or higher are preferred. Choice Analytical Chromatography Choosing the ight Pore ze Analyte Molecular Weight ecommended Pore ze of Packing < 3,000 6 nm (60Å) - 13 nm (130Å) 3,000-10,000 10 nm (100Å) > 10,000 (for example, proteins) 30 nm (300Å) - 100 nm (1000Å) very large molecules non-porous Specific Pore Volume Measurement and eporting The same techniques used for the measurement of the specific surface area and the pore size distribution yield results for the specific pore volume of a packing. However, it can be determined by other techniques as well, for example size exclusion chromatography or titration. It is reported in ml/g. Product Specific Specific Pore Surface Pore ze Area Volume [Å] [m 2 /g] [ml/g] µbondapak 330 1.00 125 Delta-Pak 300 1.00 100 Delta-Pak 300 125 1.00 300 Nova-Pak 120 0.30 60 esolve 200 0.50 90 Symmetry 335 0.90 100 Waters Spherisorb 200 0.50 80 47

Comparison Choosing a Normal Phase Packing Pore ze etentivity small - Capacity small - Strength small small Mass Transfer large - ze Exclusion - large Large Analytes large - Specific Pore Volume Packing lica Alumina Diol CN NH 2 Application general purpose sorbent general purpose sorbent, more retentive than silica; group separation of aromatic hydrocarbons less polar than silica, equilibrates quickly least retentive normal phase sorbent most polar bonded phase; different selectivity than silica; group separation of aromatic hydrocarbons For retentivity, capacity and strength, packings with a small pore size should be selected. The lower limit is determined by mass transfer in the pores. For analytes with a larger molecular weight, packings with a larger pore size should be selected. For size-exclusion chromatography, packings with a large specific pore volume should be chosen. Bonded Phase and Mode of Separation Most analytical chromatography is carried out using packings whose sorption properties have been modified by attaching a covalently bonded phase to the surface of silica. Alternatively, the surface of a packing can be modified by coating it with a chemically stable adsorptive layer. Chemically stable bonds between the packing and the ligands that are responsible for retention are only available for silica and polymeric packings. The surface of the silica can easily be derivatized through silanization. The most commonly used HPLC packing is obtained by derivatization of the surface of a silica sorbent with a long-chain aliphatic silane. The aliphatic chain is 18 carbons long, and the packing is called C 18 or DS (for octadecyl silane). However, several other surface derivatizations of silica are available which result in packings with widely varying properties. Also polymeric packings with a similar range of surface properties are used in HPLC. In the following, we will briefly discuss the available packings based on the mode of separation. Normal Phase Chromatography Normal-phase chromatography is the classical form of chromatography using polar stationary phases and non-polar mobile phases. The solute is retained by the interaction of its polar functional groups with the polar groups on the surface of the packing. Classically, unbonded silica and alumina have been used for this application, but today polar bonded phases can be used with the following advantages: bonded phases equilibrate faster, are less sensitive to minute concentrations of water in the mobile phase, and yield different selectivities. eversed-phase Chromatography eversed-phase chromatography has become the most popular mode of chromatography. In reversed-phase chromatography, the stationary phase is non-polar and the mobile phase is polar. Typical mobile phases are mixtures of water or aqueous buffer with methanol, acetonitrile or tetrahydrofuran. Typical stationary phases are silica-based bonded phases with aliphatic hydrocarbons as ligands. ther packings for reversed-phase chromatography are graphitized carbon and styrenedivinylbenzene packings. The performance of reversed-phase bonded phases depends also on the activity of residual silanols. lanols interact with the polar functional group of the solutes. Therefore, packings exhibit different selectivities depending on the activity of the silanols. Also, tailing peaks are often observed for basic compounds on packings with a high level of silanol activity. ne way of modifying silanol activity is by endcapping, that is reaction with a silanization reagent that converts the silanols to trimethylsilyl groups. Nevertheless, the surface concentration of residual silanols is always higher than the total concentration of bonded ligand including the endcapping ligand. lanol activity also depends on the pretreatment of the silica ( base-deactivation ) and the purity of the silica. Fully endcapped bonded phases based on high-purity silicas are recommended for the chromatography of basic analytes. Non-endcapped packings can be used with advantage in many other applications to obtain a different selectivity. Surface of a Typical eversed-phase Packing lanol H H3C H3C CH3 H H3C C 8 Ligand CH3 CH3 H3C H H3C CH3 H H3C H3C H3C CH3 H H3C CH3 H Endcapping H3C H CH3 CH3 eversed-phase Packings C 18 (DS) P (Phenyl) C 2 (Ethyl) (P 2 ) Shielded eversed-phase C 8 (ctyl) (MS) (P8) C 4 (Butyl) C 1 (Methyl) (SAS) Polymer P C 6 (Hexyl) C 3 (Propyl) CN (Nitrile) (Cyano) 48

Ion-Exchange Chromatography Packings for Hydrophilic Interaction Chromatography The separation of solutes by their charge is possible by using phases which contain fixed ionic charges. In the case of silica-based ionexchangers, the ionic species are attached to the surface using standard silanization techniques. In the case of polymer-based ion-exchangers, the ion-exchange groups are distributed throughout the matrix. There are four categories of ion-exchangers: strong and weak cation exchangers and strong and weak anion exchangers. Weak ion-exchangers are characterized by the fact that the charge is a function of the ph. Exchangers with carboxylic acids as functional groups are an example of weak cation exchangers. Weak anion exchangers comprise primary, secondary and tertiary amines. The charge of strong ion-exchangers is for the most part independent of ph. Quaternary amines form strong anion exchangers, and sulfonic acids are classified as strong cation exchangers. All these functional groups are available on polymeric packings, primarily for the separation of large biomolecules. All but the weak cation exchanger are available on silica. Also, special ion-exchangers are available for ion chromatography. The latter are characterized by a low ion-exchange capacity, which makes it possible to use them with low-ionic-strength mobile phase, which is a requirement for ion chromatography with conductivity detection. Designations of Ion-Exchangers Strong Anion Weak Anion Strong Cation Weak Cation Exchanger Exchangers Exchangers Exchangers SAX NH 2 SCX CM DEAE Ion-Suppression Chromatography This technique is widely used for the analysis of organic acids in eluents at low ph. It is simply a subcategory of reversed-phase chromatography. Special polymer phases are available for this application. In addition silica phases with C 18, C 8 or C 6 ligands may also be used with ph 2 eluents. Hydrophilic-Interaction Chromatography Hydrophilic-interaction chromatography is the extension of normal-phase chromatography to aqueous eluents. Polar stationary phases are used in conjunction with aqueous-organic mobile phases. Contrary to reversed-phase chromatography, retention increases with increasing organic content. The most popular stationary phase for this application is a aminopropyl bonded phase, however, native silica, diol phases or other polar phases can also be used. The most common application is the separation of carbohydrates using the aminopropyl phase. Columns specially prepared for this application are available under the name of carbohydrate columns. NH 2 (Amino) Diol lica Alumina Carbohydrate Polar Polymeric Packings The Nature of Bonded Phases Carbon Load and Ligand Density Measurement and eporting The carbon content of a packing is determined by elemental analysis. ften the results are reported directly as the carbon load of a packing. From this value and with the knowledge of the specific surface area of the packing and the molecular weight of the bonded ligand, the ligand density can be calculated. The ligand density is usually expressed in µmol/m 2. It is also sometimes called the surface coverage. The ligand density is a very important measure of the composition of the surface of the bonded phase, and is one of the important parameters determining the selectivity of a packing. Carbon Load At equal ligand density on the same base silica, packings with larger ligands have a higher carbon load that is, C 18 phases have a higher carbon load than C 1 phases. For the same ligand on the same silica, the carbon load increases with surface coverage. For the same ligand at equal surface coverage on packings with different pore sizes, the carbon load decreases with increasing pore size, such as decreasing specific surface area. From this discussion, we can see that carbon load alone is not a good measure for the retentivity or the selectivity of a packing. Ligand Density Ligand density, also called surface coverage, is a much better measure of the characteristic properties of a packing. It is calculated using the following equation: χ = 100 SA 1 - %C 100 SA is the specific surface area, %C is the carbon load, MW is the molecular weight of the ligand and nc is the number of carbon atoms in the molecule. %C MW - 1 nc 12 Analytical Chromatography 49

The higher this number, the higher is the density of the primary ligand on the surface of the packing. It is also a measure of the reduction of the density of silanols on the surface. n a fully hydroxylated silica, the surface concentration of silanols is around 8 µmol/m 2. Ligand densities of the primary ligand vary between 0.5 to 4 µmol/m 2 for different bonded phases based on monofunctional ligands (see below). The reproducibility of the ligand density is of utmost importance for the reproducibility of the retentivity and selectivity of a packing. In addition, packings with a high ligand density tend to be hydrolytically more stable than packings with a low ligand density. For applications in hydrolyzing conditions of high or low ph and high aqueous content, it is best to select a robust silica with trifunctional bonding. All silica-based ion-exchangers are trifunctional. If nitrile packings are being used for reversed-phase chromatography, the better packings are based on a robust silica (low pore volume) and a trifunctional silane. Some manufacturers produce a nitrile bonding specifically for use in reversed-phase chromatography. Monofunctional Ligands Trifunctional Ligands C 18 Packings Grouped by Ligand Density Low Medium High Symmetry C 18 Nova-Pak C 18 µbondapak C 18 Waters Spherisorb DS2 Waters Spherisorb CN µbondapak C 18 esolve C 18 Symmetry C 18 Waters Spherisorb DS1 Waters Spherisorb DS2 Nova-Pak C 18 Type of Bonded Phase and Endcapping Bonded phases are produced by the chemical reaction of an organosilane with the silica surface. The target is to achieve a uniform monomolecular layer. There are different types of silanes that are used for this bonding reaction. The silanes are characterized as mono-, di-, or trifunctional silanes depending on the number of groups on the silane that can react with the silica surface. With monofunctional silanes, the ligand density does not exceed 4 µmol/m 2 due to steric hindrance. With multifunctional silanes, higher ligand densities can be achieved. ften, packings with these higher ligand densities are referred to as polymeric packings, although there is little evidence for the formation of a polymer. Conversely, packings with a low ligand density are referred to as monomeric. The selectivity of a packing depends on the ligand density. Especially different shape selectivities have been demonstrated for polyaromatic hydrocarbons at different ligand densities. The majority of commercially available bonded phases have a ligand density under 4 µmol/m 2 and are therefore of the monomeric type. Monofunctional ligands give a more predictable coverage of the silica since complicating side reactions of the second reactive group are not possible. However, only one bond is made with the surface, which can result in an accelerated hydrolysis compared with phases prepared from multifunctional ligands. Trifunctional ligands are more difficult to bond reproducibly to the surface. n the average, two bonds are made with the surface, which increases the resistance to hydrolysis. Trifunctional ligands are preferred when low ph mobile phases are used. Manufacturers do not always reveal their bonding chemistries. However most will indicate if a C 18 phase is monofunctional. More recently introduced reversed-phase packings are usually of that type because bonded phases can be produced with a higher reproducibility using monofunctional silanes. Endcapping For many reversed-phase packings, a secondary bonding step is carried out to cover unreacted silanol sites on the silica surface. A small silane, usually trimethylchlorosilane (TMCS), is used to produce a maximum coverage. This process is called endcapping. Endcapping is applied to most bonded phases used in reversed-phase chromatography. Phases used in normal-phase chromatography or other modes of chromatography are not endcapped. eversed-phase packings that are not endcapped often exhibit a significantly different selectivity than endcapped packings. However, basic analytes tail on non-endcapped reversed-phase packings. Trimethylsilyl groups (endcapping groups) are subject to hydrolysis in acidic conditions. Therefore, endcapped packings should not be used at ph < 2. eversed-phase Packings without Endcapping Waters Spherisorb DS1 esolve C 18 and C 8 Characterization of eversed-phase Packings It is clear that the final properties of a reversed-phase packing depend on many different parameters of bonded-phase design and on the details of the manufacturing process of the different packings. To condense this large body of data into information that is useful to the chromatographer, it is important to focus on the most relevant properties of reversed-phase packings. There are two basic properties of a reversed-phase chromatographic packing. ne is the strength of the hydrophobic interaction which can easily be measured by the retention factor of a purely hydrophobic analyte. The second property is the strength of the silanophilic interaction. This can best be measured by using the relative retention between an analyte that interacts by hydrophobic and silanophilic interaction and an analyte that interacts by hydrophobic interaction only. This has been done for many of the C 8 and C 18 packings that can be found in this catalogue as well as for some others, for which the relevant information was available. 50

The information is summarized clearly in the chart below. The x-axis represents the hydrophobicity of a packing, and the y-axis is the silanophilic interaction of the packing. The hydrophobicity increases fundamentally with the chain length. Therefore, you will find most C 8 packings on the left side of the chart, and most C 18 packings on the right side of the chart. The silanophilic interaction is measured at neutral ph. Packings based on high-purity silicas that are well endcapped show a low silanol group activity and are found on the lower side of the graph. These are the packings that are preferentially used for basic analytes. n the upper end of the y-axis, you find packings with a high silanol activity. These are typically based on either lower purity silica and/or are not endcapped sometimes. These packings can be used to provide a different selectivity than packings with a low silanol activity, but they usually exhibit tailing for basic analytes at neutral mobile phase ph. You can select the type of packing most suitable for your separation based on this graph. If your analytes are basic and exhibit strong interactions with surface silanols, select packings on the lower part of the graph. If you are looking for a significant difference in polar selectivity, you should select packings that are located in different sections of the chart. If you are looking simply for differences in hydrophobicity, use packings that are far away from each other on the hydrophobicity axis. Note: The actual value obtained from the chromatographic evaluation is shown by the diamond symbol. Selectivity Chart of eversed-phase Columns lanol Activity ln [(α) Amiitriptyline/Acenaphthene] 2.7 2.2 1.7 1.2 0.7 Symmetry C Waters Spherisorb DS1 esolve C 18 µbondapak C 18 Waters Spherisorb DS2 YMC J'Sphere DS-L80 Nucleosil C 18 LiChrosorb Select B Waters Spherisorb C 8 YMC J'Sphere DS-M80 LiChrospher Select B Hypersil DS YMC J'Sphere DS-H80 Nova-Pak C 8 Inertsil C 8 Kromasil C 8 YMCbasic Nova-Pak C 18 YMC DS-AQ YMC-Pack Pro C4 YMC-Pack Pro C8 Hypersil BDS C 8 8 YMC-Pack Pro C18 Inertsil DS3 Analytical Chromatography 0.2 Symmetry300 C 4 Hypersil BDS C 18 Inertsil DS2 Kromasil C 18 Symmetry C 18 SymmetryShield P 8-0.3 SymmetryShield P 18 1.2 1.7 2.2 2.7 3.2 Hydrophobicity ln [k] Acenaphthene 51

eproducibility Particle ze Distribution The test of reproducibility is time. f the packings produced by the major suppliers, many have shown acceptable reproducibility characteristics from batch-to-batch, and over a long period of time. The amount and sophistication of the analytical characterization of HPLC packings has increased with the goal to reduce the variability in the manufacturing and bonding processes. Extensive chromatographic and physical testing of the packings demonstrates the reproducibility from batch-to-batch and columnto-column, and certification of these results is becoming more prevalent. Extensive data on the physical characterisation of Waters Spherisorb packings are available, showing control of bonding parameters and physical silica properties for key materials. The documentation of Waters Symmetry C 18 and C 8 materials leaves no doubt that the specifications, and therefore the control of the synthesis, are extremely tight, resulting in excellent batch-to-batch and column-to-column reproducibility. Although subjective, the age of a well established HPLC packing shows assurance of continuing supply and market acceptance, while newer products show evidence of improved reproducibility due to the application of state-of-the-art know-how. We are constantly reviewing our products to improve reproducibility and we have achieved better batch-to-batch control now than 5 or 10 years ago. The following table lists the packings by the decade of their first introduction. lder packings have a proven track record and are used in many standardized applications (for example, Pharmacopoeia methods). Newer packings use state-of-the-art technology, often resulting in improved properties of the packing and improved reproducibility. Introduction 1970s 1980s 1990s Particle ze µbondapak esolve Symmetry Waters Spherisorb Nova-Pak eporting and Measurement Particle size is expressed in µm and refers to the average diameter of the particles of the packing. This measurement may be carried out by a number of techniques and reported in different ways. For irregular packings, it is not possible to define a diameter. The reported values are equivalent spherical diameter as defined by the measurement technique. Commonly used HPLC packings have a distribution of diameters. The reported nominal particle diameter values could be either volume-averaged or number-averaged values. It is therefore difficult to compare the particle sizes of different manufacturers. If the same measurement technique and definition is used, packings with a nominal particle size of 5 µm could yield actual values between 4.0 µm and 6.5 µm. The true measure of particle size is ultimately the permeability (=normalized backpressure) of a packing. Comparison of Materials The mean particle size of the packing material has an effect on two parameters. The first is the operating backpressure of the column. Backpressure increases with the inverse square of the particle diameter. The second is separation efficiency. Smaller particles give higher efficiencies at constant column length due to the shorter diffusion path. A well packed 3 µm column will give almost twice the separation efficiency of an equivalent 5 µm column, but at the cost of four times the operating pressure at the same column length and linear velocity. Currently, the most popular packing size is 5 µm. Particles of this size exhibit higher efficiencies than 10 µm packings at a given analysis time, and with reasonable backpressures. 3 µm packings in short columns are becoming more popular, since they give still higher efficiencies at a given analysis time than 5 µm packings. Columns packed with 3 µm particles formerly suffered from short column life, but today s technology has sufficiently improved such that high efficiency can be obtained with modern 3 to 3.5 µm packings without compromising column life. 3 µm The particle size for fast analysis. If you choose a state-of-the-art packing, you can obtain very fast analyses without loss of efficiency or sacrificing column life. 5 µm The particle size of choice for most routine analytical columns due to its combination of high separation performance and moderate operating pressures. 10 µm nce the standard particle size for analytical applications, 10 µm packings have the ability to give good peak resolution with moderate column efficiencies at low operating pressures, even with long columns. These packings are now mainly used in routine QA methods or as a scout column for upscaling of the analytical method to preparative applications. 52

> 10 µm Particles larger than 10 µm are primarily used in preparative applications. An increasing range of spherical preparative materials are available today, but with a size limit of 20 µm. Above 20 µm only irregular packings are available. Choice of Particle ze Not all manufacturers produce a full range of particle sizes in all their products. The following materials are available in the particle sizes shown. maximum plate count. Also, all curves stop at the same short analysis time. (However, at a given analysis time, the columns packed with the smaller particles give a higher plate count, and the plate-count maximum shifts towards shorter analysis times with smaller particle size. This is why the popularity of short 3 µm columns is increasing). For columns with a larger ratio of column length to particle size, the pressure drop at a given analysis time increases with the square of this ratio. n the other hand, columns with a smaller ratio of column length to particle size can give much faster analyses, but at the sacrifice of maximum resolving power. At a given pressure drop, a 75 mm, 5 µm column is four times faster than a 150 mm, 5 µm column. lica Particle ze µbondapak 10 Delta-Pak 100Å 5, 15 Delta-Pak 300 Å 5, 15 Nova-Pak 4, 6 lica Particle ze esolve 5, 10 Symmetry 3.5, 5, 7 Waters Spherisorb 3, 5, 10 Plot of Plate Count versus Analysis Time for Several Columns with the Same atio of Column Length to Particle ze. Choice of Column Dimensions Column Length and Particle ze The performance of a column depends primarily on the choice of particle size and column length. As a matter of fact, column efficiency (also called plate count), analysis time and backpressure are intimately linked to the ratio of column length to particle size. The maximum column efficiency is determined by this ratio. For a well packed column, the following equation can be used to estimate the maximum column efficiency N max : L N max = 0.4 dp L is the column length and dp is the particle size. Also, at a given solvent viscosity η, the product of backpressure Dp and analysis time t is proportional to the square of the ratio of column length to particle size: 2 L p t η dp ( ) Choice 300 mm/10 µm = 30,000 150 mm/5 µm = 30,000 90 mm/3 µm = 30,000 You should select your column length based on the column length to particle size ratio and the performance expectation. Use a ratio of 30,000 for normal analysis, a ratio of about 15,000 or less for fast analysis, and a ratio of about 50,000 or more for difficult analytical problems. Type of Analysis atio of Column Length to Particle ze Analytical Chromatography Therefore, the shortest analysis time that is possible at a given pressure limit is given by the ratio of column length to particle size, and the pressure drop at a given analysis time is determined by this ratio. This is illustrated in the figure, where the plate count is plotted versus analysis time for several columns with the same ratio of column length to particle size. The column to the right is 300 mm long and is packed with 10 µm packings, representative of the type of columns that were popular in the 1970s. The column in the middle is representative of the most popular column choice today, a 150 mm long column packed with 5 µm particles, and the column on the left is a short 90 mm column packed with 3 µm particles. ne can see that all three columns have the same difficult > 50,000 normal ~ 30,000 fast < 15,000 53

Column Length, Column Diameter and Column Volume For a given analysis, the column volume, which is determined by column length and diameter, determines the volume of the peaks as they exit the column. The peak volume is an important factor for the sensitivity. At a given injected mass, columns with a smaller column volume give a higher sensitivity than columns with a larger column volume. Therefore, for applications where the sample mass is limited, columns with a small volume should be chosen. Volumes of Available adial Compression Cartridges (ml) Column Diameter Column Length 5 mm 8 mm 25 mm 40 mm 47 mm 100 mm 1.96 5.03 49.1 125.7-300 mm - - - - 520 Smaller volume columns also use less solvent per analysis. This too makes smaller volume columns more desirable. However, smaller volume columns may require special HPLC instruments for use. The extra-column bandspreading of this instrument should be minimized to achieve maximal resolution with smaller diameter columns. While columns down to an internal diameter of 3 mm can be used with no or little modification to a standard HPLC instrument, the HPLC equipment needs to be optimized to use columns with internal diameters under 3 mm. Because of this instrument optimization issue, the user has to weigh carefully, whether or not smaller diameter columns are the best choice. For example, if a large enough amount of sample is available, the same sensitivity can be obtained with standard columns as with smaller volume columns. All one needs to do is inject a larger amount of sample. Volumes of Available Column Dimensions (ml) Column Diameter Column Length 0.5 mm 1 mm 2.1 mm 3.0 mm 3.9 mm 4.0 mm 4.6 mm 7.8 mm 10 mm 19 mm 20 mm 30 mm - - - - - - 0.50 - - - - 50 mm - - 0.173 0.353 0.597-0.83 - - - - 75 mm - - - - 0.896-1.25 - - - - 100 mm - 0.079 0.346-1.194-1.66-7.85 - - 125 mm - - 0.433 0.883 1.493 1.571 2.08 - - - - 150 mm 0.029 0.118 0.519 1.060 1.791-2.49 7.16-42.5-250 mm 0.049 0.196 0.866 1.766-3.142 4.15-19.63-78.5 300 mm - - 1.039-3.582 - - 14.33-85.0-54

HPLC lica Bonded*-Phase Structures Amino, NH 2 ctyl, C 8 (CH 2 ) 3 NH 2 (CH 2 ) 7 CH 3 Butyl, C 4 DS, C 18 (CH 2 ) 3 CH 3 (CH 2 ) 17 CH 3 Cyano, CN, Nitrile (CH 2 ) 3 CN Phenyl (CH 2 ) 3 Analytical Chromatography Hexyl, C 6 SAX CH 3 (CH 2 ) 5 CH 3 (CH 2 ) 3 N + CH 3 CI - CH 3 Methyl, C 1 SCX CH 3 CH 3 (CH 2 ) 3 S - 3 H + CH 3 * Monofunctional where is nonreactive. Multi or polyfunctional where group is reactive. 55

L1 ctadecyl silane {DS or C 18} chemically bonded to porous silica or ceramic particles - 3 to 10 micron in diameter. L5 Alumina of controlled surface porosity bonded to a solid spherical core - 30 to 50 micron in diameter. µbondapak 10 Irr 85 Delta-Pak 5 Sph 87 Hypersil 3, 5, 10 Sph 218 Inertsil 3, 5 Sph 222 Kromasil 5, 10 Sph 225 LiChrosorb 5, 10 Irr 228 LiChrospher 5, 10 Sph 228 Nova-Pak 4, 6 Sph 80 Nucleosil 3, 5, 10 Sph 232 Partisil 5, 10 Irr 236 esolve 5, 10 Sph 90 Waters Spherisorb 3, 5, 10 Sph 98 Symmetry 3.5, 5, 7 Sph 74 XTerra MS 2.5, 3.5, 5, 7 Sph 71 YMC-Pack ProC18 3,5 Sph 284 YMC-Pack DS-A 5 Sph 287 YMC-Pack DS-AM 5 Sph 290 YMC-Pack DS-AL 5 Sph 295 YMC-Pack DS-AQ 4 Sph 291 YMC J Sphere DS series 4 Sph 299 L2 ctadecyl silane {DS or C 18} chemically bonded to silica gel of a controlled surface porosity bonded to a solid spherical core - 30 to 50 micron in diameter. L3 L4 Porous silica particles - 5 to 10 micron in diameter. Hypersil 5, 10 Sph 218 Inertsil 5 Sph 222 Kromasil 5, 10 Sph 225 LiChrosorb 5, 10 Irr 228 LiChrospher 5, 10 Sph 228 Nova-Pak 6 {4} Sph 80 Nucleosil 5, 10 Sph 232 Partisil 5, 10 Irr 236 Porasil 10 Irr 89 esolve 5, 10 Sph 90 Waters Spherisorb 5, 10 Sph 93 YMC-Pack lica 5, 10 Sph 317 lica gel of a controlled surface porosity bonded to a solid spherical core - 30 to 50 micron in diameter. L6 L7 ctyl silane {C 8} chemically bonded to porous silica or ceramic particles - 3 to 10 micron in diameter. Hypersil {MS} 3, 5, 10 Sph 218 Inertsil 5 Sph 228 Kromasil 5, 10 Sph 225 LiChrosorb 5, 10 Irr 228 LiChrospher 5, 10 Sph 228 Nova-Pak 4, 6 Sph 80 Nucleosil 3, 5, 10 Sph 232 Partisil 5, 10 Irr 236 esolve 5, 10 Sph 96 Waters Spherisorb 3, 5, 10 Sph 93 Symmetry 3.5, 5, 7 Sph 74 XTerra MS 2.5, 3.5, 5, 7 Sph 71 YMC-Pack C 8 (ctyl) 3, 5, 10 Sph 305 YMCbasic 3, 5, 10 Sph 303 YMC-Pack Pro C8 5 Sph 284 L8 An essentially monomolecular layer of aminopropylsilane {NH 2} chemically bonded to totally porous silica gel support - 10 micron in diameter. L9 Strong cation exchanger packing - sulfonated fluorocarbon polymer coated on a solid spherical core - 30 to 50 micron in diameter. µbondapak 10 Irr 85 Hypersil {APS} 10 {3}{5} Sph 218 Kromasil 10 {5} Sph 225 LiChrosorb 10 {5} Irr 228 LiChrospher 10 {5} Sph 228 Nucleosil 10 {5} Sph 232 Waters Spherisorb 10 {3}{5} Sph 93 YMC-Pack NH 2 (Amino) 10 {5}{15} SPH 323 10 micron irregular, totally porous silica gel having a chemically bonded strongly acidic cation exchanger coating {SCX}. Partisil 10 Irr 236 56

L10 Nitrile groups {CN} chemically bonded to porous silica particles - 3 to 10 micron in diameter. L15 Hexylsilane {C 6} chemically bonded to a totally porous silica particle - 3 to 10 micron in diameter. L11 µbondapak 10 Irr 85 Hypersil {CPS} 3, 5, 10 Sph 218 LiChrosorb 5, 10 Irr 228 LiChrospher 5, 10 Sph 228 Nova-Pak 3, 6 Sph 80 Nucleosil 5, 10 Sph 232 esolve 10 Sph 90 Waters Spherisorb 3, 5, 10 Sph 93 YMC-Pack CN (Cyano/Nitrile) 3, 5, 10 Sph 315 Phenyl groups chemically bonded to porous silica particles - 5 to 10 micron in diameter. L16 Dimethylsilane {C 2} chemically bonded to a totally porous silica particles - 5 to 10 micron in diameter. L17 Waters Spherisorb 3, 5, 10 Sph 93 Strong cation exchange resin consisting of sulfonated, cross-linked styrene divinylbenzene copolymer in the hydrogen form, 7 to 11 micron in diameter. L12 L13 µbondapak 10 Irr 85 Hypersil 5, 10 {3} Sph 218 Inertsil 5 Sph 222 Nova-Pak {4} Sph 80 Nucleosil 7 Sph 232 Waters Spherisorb 5, 10 {3} Sph 93 YMC-Pack TMS 3, 5, 10 Sph 313 A strong anion exchanger packing made by chemically bonding a quaternary amine to a solid silica spherical core - 30 to 50 micron in diameter. Trimethylsilane {C1} chemically bonded to porous silica particles - 3 to 10 micron in diameter IC-Pak Ion Exclusion 7 Sph 181 PP -X200 10 Sph 215 Spak {6} Sph 91 L18 Amino {NH 2} and Cyano {CN} groups chemically bonded to porous silica particles - 3 to 10 micron in diameter. L19 Partisil {PAC} 5, 10 Irr 236 Strong cation exchange resin consisting of sulfonated, cross-linked styrene divinylbenzene copolymer in the calcium form - about 9 micron in diameter. Sugar-Pak 1 9 Sph 176 SC-1011 {7} Sph 176 Analytical Chromatography L14 Hypersil {SAS} 3, 5, 10 Sph 218 Kromasil 5, 10 Sph 225 Waters Spherisorb 3, 5, 10 Sph 93 YMC-Pack TMS 3, 5, 10 Sph 313 lica gel, 10 micron in diameter having a chemically bonded, strongly basic quaternary ammonium anion exchanger {SAX} coating. Nucleosil {SB} 10 {5} Sph 232 Partisil 10 Irr 236 Waters Spherisorb 10 {5} Sph 93 L20 Dihydroxypropane groups chemically bonded to porous silica particles - 5 to 10 micron in diameter. Protein-Pak 60 10 Irr 153 Protein Pak 125 10 Irr 153 Protein-Pak 300SW 10 Irr 153 Protein-Pak KW -802.5 7 Irr 154 Protein-Pak KW -803 7 Irr 154 Protein-Pak KW -804 7 Irr 154 YMC-Pack Diol 5, 10 Sph 321 57

L21 L22 A rigid, spherical styrene-divinylbenzene copolymer - 5 to 10 micron in diameter. PP -1 5 Sph 215 PP -1 10 Sph 215 Spak 613 6 Sph 91 A cation exchange resin made of porous polystyrene with sulfonic acid groups - about 10 micron in size. L28 L29 A multifunctional support which consists of a high purity, 100Å, spherical silica substrate that has been bonded with anionic (amine) functionality in addition to a conventional reversed phase C 8 functionality. Gamma alumina, reversed-phase, low carbon percentage by weight alumina-based polybutadiene spherical particles - 5 micron in diameter with a pore diameter of 80Å. L23 IC-Pak Ion Exclusion 7 Sph 181 Spak DC 613 6 Sph 91 SP-0810 8 Sph 176 An anion exchange resin made of porous polymethacrylate or polyacrylate gel with quaternary ammonium groups - about 10 micron in size. L30 Ethyl silane chemically bonded to a totally porous silica particle - 3 to 10 micron in diameter. L24 Protein-Pak Q 8H 8 Sph 147 A semi-rigid hydrophilic gel consisting of vinyl polymers with numerous hydroxyl groups on the matrix surface - 32 to 63 microns in diameter. L31 A strong anion-exchange resin-quaternary amine bonded on latex particles attached to a core of 8.5 micron macroporous particles having a pore size of 2,000Å and consisting of ethylvinylbenzene cross-linked with 55% divinyl benzene. L25 Packing having the capacity to separate compounds with a molecular weight range from 100 to 5,000 (as determined by polyethylene oxide), applied to neutral, anionic and cationic water-soluble polymers. A polymethacrylate resin base, cross-linked with polyhydroxylated ether, (surface contained some residual carboxyl groups) was found suitable. L32 A chiral-ligand exchange packing - L proline copper complex covalently bonded to an irregularly shaped silica particles - 5 to 10 micron in diameter. L26 Ultrahydrogel 120 10 Sph 176 Butyl silane {C4} chemically bonded to porous silica particles - 5 to 10 micron in diameter. L33 Packing having the capacity to separate proteins of 4,000 to 400,000 daltons. It is spherical, silica-based and processed to provide ph stability. YMC-Pack 200Å Diol 5-150 Sph 321 Delta-Pak C4 5 Sph {100+300Å} 87 Kromasil 5, 10 Sph 225 YMC-Pack Protein-P 5 Sph 328 YMC C 4 (Butyl) 5, 10 Sph 310 YMC-Pack Pro C4 5 Sph 284 L34 Strong cation-exchange resin consisting of sulfonated cross-linked styrene-divinylbenzene copolymer in the lead form, about 9 micron in diameter. L27 Porous silica particles, 30-50 µm in diameter. Hamilton HC-75 Pb 10 Sph 215 Porasil 37-55 Irr 89 YMC-Pack lica (S30/50) Irr 317 Source: Pharmacopeial Form, Vol 25(2), 1999 58

Physical Characteristics of HPLC Packings Brand Chemistry Particle Shape Particle ze(s) Pore ze Surface Area [m 2 /g] Pore Volume [cc/g] % Carbon Load Endcapped µbondapak C 18 Irregular 10 µm 125Å 330 1.00 9.8 yes µbondapak Phenyl Irregular 10 µm 125Å 330 1.00 9.3 yes µbondapak CN Irregular 10 µm 125Å 330 1.00 6.0 yes µbondapak NH 2 Irregular 10 µm 125Å 330 1.00 4.0 no Bondapak C 18 Irregular 15-20 µm 125Å 330 1.00 10.0 yes Bondapak C 8 Irregular 15-20 µm 300Å 100 1.00 6.0 yes Bondapak C 18 Irregular 15-20 µm 300Å 100 1.00 3.5 yes Delta-Pak C 4 Spherical 5, 15 µm 100Å 300 1.00 7.3 yes Delta-Pak C 18 Spherical 5, 15 µm 100Å 300 1.00 17.0 yes Delta-Pak C 4 Spherical 5, 15 µm 300Å 125 1.00 2.6 yes Delta-Pak C 18 Spherical 5, 15 µm 300Å 125 1.00 6.8 yes Nova-Pak C 18 Spherical 4, 6 µm 60Å 120 0.30 7.3 yes Nova-Pak C 8 Spherical 4 µm 60Å 120 0.30 4.0 yes Nova-Pak Phenyl Spherical 4 µm 60Å 120 0.30 4.6 yes Nova-Pak CN HP Spherical 4 µm 60Å 120 0.30 3.0 yes Nova-Pak lica Spherical 4, 6 µm 60Å 120 0.30 n/a n/a µporasil lica Irregular 10 µm 125Å 330 1.00 n/a n/a Porasil lica Irregular 15-20 µm 125Å 330 - n/a n/a esolve C 18 Spherical 5, 10 µm 90Å 200 0.50 10.2 no esolve C 8 Spherical 5 µm 90Å 200 0.50 5.1 no esolve CN Spherical 10 µm 90Å 200 0.50 3.0 no esolve lica Spherical 5, 10 µm 90Å 200 0.50 n/a n/a Symmetry C 18 Spherical 3.5, 5 µm 100Å 335 0.90 19.1 yes Symmetry C 8 Spherical 3.5, 5 µm 100Å 335 0.90 11.7 yes Analytical Chromatography SymmetryShield P 8 Spherical 3.5, 5 µm 100Å 335 0.90 15.0 yes SymmetryShield P 18 Spherical 5 µm 100Å 335 0.90 17.0 yes SymmetryPrep C 18 Spherical 7 µm 100Å 335 0.90 19.1 yes SymmetryPrep C 8 Spherical 7 µm 100Å 335 0.90 11.7 yes Symmetry300 C 18 Spherical 3.5, 5 µm 300Å 110 0.80 8.5 yes Symmetry300 C 4 Spherical 3.5, 5 µm 300Å 110 0.80 2.8 yes Waters Spherisorb DS 2 Spherical 3, 5, 10 µm 80Å 220 0.50 11.5 yes Waters Spherisorb DS Spherical 3, 5, 10 µm 80Å 220 0.50 6.2 no Waters Spherisorb DSB Spherical 5 µm 80Å 220 0.50 11.5 yes Waters Spherisorb C 8 Spherical 3, 5, 10 µm 80Å 220 0.50 5.8 yes Waters Spherisorb C 6 Spherical 3, 5, 10 µm 80Å 220 0.50 4.7 yes Waters Spherisorb C 1 Spherical 3, 5, 10 µm 80Å 220 0.50 2.2 no Waters Spherisorb Nitrile Spherical 3, 5, 10 µm 80Å 220 0.50 3.1 no Waters Spherisorb Amino Spherical 3, 5, 10 µm 80Å 220 0.50 1.9 no Waters Spherisorb Phenyl Spherical 3, 5, 10 µm 80Å 220 0.50 2.5 no Waters Spherisorb D/CN Spherical 5 µm 80Å 220 0.50 5.0 yes Waters Spherisorb SAX, SCX Spherical 5, 10 µm 80Å 220 0.50 4.0 no XTerra P P 18 Spherical 2.5, 3.5, 5, 7 µm 125Å 175 0.70 15.0 yes XTerra P P 8 Spherical 2.5, 3.5, 5, 7 µm 125Å 175 0.70 13.5 yes XTerra MS C 18 Spherical 2.5, 3.5, 5, 7 µm 125Å 175 0.70 15.5 yes XTerra MS C 8 Spherical 2.5, 3.5, 5, 7 µm 125Å 175 0.70 12.0 yes 59