CHAPTER 3 COLUMN SELECTION IN HIGH

CHAPTER 3 COLUMN SELECTION IN HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Roy Eksteen

1. Introduction

The term high-performance liquid chromatography (HPLC) was coined in the early 1970s. It correctly reflected the tremendous improvement in resolution that was achieved by reducing the particle size of the column packing materials that had been used traditionally in liquid chromatography. The large number of HPLC columns and column types that has since been introduced makes it difficult to choose the right column for the separation problem at hand. By some estimates one can select the most popular C18 (reversed phase) column from at least 100 manufacturers worldwide. At the same time, the pace of new column development has not yet slowed down. For example, thirty new columns for analyzing small molecular weight compounds were introduced at tl1e 1990 Pittsburgh Conference, in addition to twenty new columns just for biopolymer analyses [1]. This trend continued at the 1992 meeting, where, for the first year, more polymerbased than silica-based columns were introduced [2]. Five-micron packings have now been available for more than a decade, and the high performance in HPLC has become the rule rather than the exception. Although the technique is most often referred to as HPLC, it is known in biochemical laboratories by such abbreviations as FPLC (Fast Protein), HRLC (High Resolution), and FFLC (Fast Flow).

96

Of course, HPLC is not the only qualitative and quantitative analytical separation technique, and a brief overview of methods available for characterizing biomolecules is given in Chapter 10. In this chapter it will be assumed, however, that the analyst has good reasons to pursue HPLC for the sample to be analyzed. It will further be assumed that the liquid chromatograph to be utilized has been properly adapted to take full advantage of the separation capability of the column. In other words, column efficiency is not compromised by so-called extra-column band broadening effects. Refer to Chapter 2 for a discussion of extra-column band broadening effects. This discussion of column selection will focus on the properties of column packing materials. Besides the popular silica-based packings, recent advances in resin-based packings as well as other pH-stable materials will be reviewed with respect to their application to the analysis of proteins, peptides, nucleic acid fragments and carbohydrates. In writing this chapter the emphasis has been to include common and accepted practices of highperformance liquid chromatography in an attempt to clarify the basis upon which the technology rests. Hopefully this will set the stage for further study of the literature. Developing an analysis is a complex process that consists of several interacting steps, one of which is column selection. A column is chosen based on the knowledge of the sample and on the expectation of how its components will physically and chemically interact with the packing material. After establishing that the developed procedure is satisfactory, optimization of the design of the column (particle size and column dimensions) should be considered, as discussed in Chapter 2. Optimization of column parameters and, to a lesser extent, of other operating conditions such as mobile phase composition can be omitted when one or a few samples have to be analyzed. Mobile phase and column optimization procedures, although of great importance when developing a method for a routine analysis, will not be discussed, whereas differences in column hardware will be described and guidelines will be provided on bow to protect the column once it has been selected.

97

This chapter concludes with a guide for column selection for molecules having molecular weights below and above 2000 (Tables 1 and 2), as an aid in selecting the best column for a particular analysis. Columns for analyzing biopolymers by HPLC are organized for each of the major separation modes.

2. HPLC Column Hardware

2.1. Column Blank Material Because separation takes place on the column, the column is often referred to as the heart of the chromatographic system. It usually consists of a piece of tubing that contains the packing with closures that are designed to connect it to the chromatograph and allow leak-free operation at pressures varying from 0 to 40 MPa (400 kg/cm2 or 6,000 p.s.i). It should be noted that while the majority of silica-based columns can indeed be operated under such pressures, in practice the column inlet pressure, even for silicabased columns, does not often exceed 20 MPa. Traditionally, stainless steel has been the tubing material of choice in HPLC, while glass has been the preferred column material in open column (low pressure) liquid chromatography, a technique widely practiced in the biochemical laboratory. Initially, high efficiency columns for biopolymer separations were predominantly made from stainless steel. In recent years the use of other column materials, such as glass and PEEK (polyetheretherketone) has been advocated to reduce the irreversible adsorption of some proteins to stainless steel [3,4]. Several instrument manufacturers have promoted the use of titanium in system components, particularly pump heads, since stainless steel is known to corrode under high salt concentrations, particularly in the presence of chloride ions. Denner et a!., using size-exclusion and ion exchange chromatography, measured 75-95% activity recovery of [3H]progesterone-labeled receptor on glass columns versus only 10-45% in stainless steel columns [5]. In contrast, Herold et a!. found that the residence time of metal-sensitive enzymes in the (stainless steel) column and connecting tubing was too short to have a negative effect on activity recovery [6]. On the other hand, the

98

adsorption of proteins on silanol groups on glass surfaces is well known in biochemistry [7] and references therein). PEEK has not been used long enough to conclude that it is less adsorptive than either stainless steel or glass. The first high efficiency glass columns for protein separations by ion-exchange chromatography were packed with monodisperse spherical 9.8 micron particles [8]). Their narrow particle size distribution allowed them to be employed at pressures below 3.5 MPa (500 p.s.i.). Larger bore glass columns (8 mm ID) are available packed with spherical 5 micron particles for separating biopolymers, e.g. by size-exclusion chromatography. At a flow rate of 1-1.5 mi/min, i.e. by using a low linear velocity, such short columns provide high efficiency while generating less than 4 MPa back pressure. 5 25

"'0'

....

20

2

-

~ !j

a 15-

3

1

-" ...

4

0

~

-"


Zr-0-Si-R > Ti-0-Si-R >> Al-0Si-R [68]. Trudinger et at. [69] prepared porous zirconia and titania, and modified these packings with octadecyltrimethoxysilane to obtain polymeric CIS bonded phases. They found that titania-based CIS packings are stable up to pH I! and that zirconia-based CIS packings are stable up to pH 12. The same authors also concluded that titanium and zirconium oxides are not attacked by strongly alkaline solutions. Using CIS-bonded zirconia, Yu and El Rassi noticed a 30-75% decrease in retention of non-polar compounds during the first 4000 column volumes when flushing the column with a pH 2 or a pH I2 mobile phase. The stability of polymeric CIS-zirconia packings was better than for endcapped monomeric CISzirconia [70].

124

Although, all alumina- and zirconia-based C 18 packing materials showed typical reversed phase retention behavior for alkyl and aryl compounds, more information regarding the long-term stability of retention and selectivity, particularly at high pH, is needed in order to determine the long-term potential of these packing materials as alternatives to silica-based C18 columns. 5.3.3. Hydroxylapatite

Since its first use in classical open column chromatography (71), hydroxyapatite, Ca5(P04) 30H, has been known for its excellent selectivity for separating nucleic acids [72] and medium and high molecular weight proteins [73]. Separation on hydroxylapatite involves ion exchange via the calcium ion in the adsorbent, while steric exclusion also plays a role, particularly in the retention of polynucleotides. Elution of the sample is usually accomplished by increasing the phosphate concentration in the mobile phase. The presence of salts commonly used in the purification of proteins, such as sodium chloride (ion exchange) and ammonium sulfate (hydrophobic interaction), does not seem to influence the elution process.

5

10

20 15 Minutes

25

30

Column, TSKgel HA-1000, 5 J.lm, 7.5 em x 7.5 mm; mobile phase, from 10 to 500 nM sodium phosphate, pH 6.8, over 30 min.; flow rate, 1.0 mllmin; detection, 260 nm UV; injection, plasmid mixture of 12 J.lg Ml4mp8 and 11 J.lg of pBR322.

Figure 10 Hydroxylapatite Separation of Single and Double Stranded DNA

125

The traditional support material has suffered, however, from poor flow properties and instability under separation conditions. Several spherical hydroxylapatite packing materials have been developed in recent years, including 5 IJ.m particles with 1000 A pores showing improved flow characteristics [74]. Hydroxylapatite is primarily used for the separation of nucleic acids, particularly single and double stranded DNA, and proteins. The addition of 0.1mM CaC1 2 to the mobile phase increases column lifetime without affecting retention and selectivity. Figure 10 shows the separation of single and double stranded DNA on a TSKgel HA-1000 column [75]. The properties of commercial hydroxylapatite columns are listed in Table 10 at the end of this chapter. 5.3.4. Carbon

Carbon adsorbents are an intermediate between inorganic and organic LC packings. Efficient carbon-based HPLC columns capable of withstanding high back pressure were first described by Knox and Gilbert [76]. The particles are produced by impregnating a porous silica with a phenol/ hexamine resin mixture, polymerizing this mixture within the pores of the silica gel, pyrolysing the resin in nitrogen, dissolving out the silica template in hot potassium hydroxide, and heating the remaining porous carbon to temperatures in excess of 2000 'C [77]. The particles are free of micropores, which cause poor chromatographic efficiency, since their pore structure is the inverse of the silica template. Hypercarb (Shandon), a porous graphitized carbon prepared by this procedure, required a higher percentage of organic modifier in the mobile phase relative to CIS-silica, typically 95% methanol compared to 50% methanol [77]. Other properties of this spherical carbon support include a 7 IJ.m particle size, 95-125m2/g surface area, while the particles have a porosity of 60% with 250 A pores, and are stable to pressures up to 40 MPa. An alternative support, TSKgel Carbon500 (Tosoh), contains spherical5 ~J.m particles with 500 Apores. Compared to a silica-based C18 column with 80 A pores and 350 m2fg surface area, the Carbon-500 column showed less retention for alkylbenzenes, similar retention for alkyl benzoates, and was more retentive for p-hydroxyalkylbenzoates [78].

126

Since carbon is stable at any pH from 0 to 14, carbon columns are an alternative to silica-based reversed phase columns, particularly when there is a need to work at alkaline pH, such as for the analysis of basic compounds. More important, however, carbon-based columns have better selectivity for separating diastereoisomers and geometric isomers than silica-based reversed phase columns [79]. Carbon columns are, in general, more retentive and have higher hydrophobic selectivity than silica-based reversed phase columns, but efficiencies are lower and peak shape is less symmetrical.

6.

Column Selection

This chapter concludes with 10 tables, the first two of which give an overview of the HPLC techniques available for various sample types. Table 1 is for solutes with molecular weight below 2000, and Table 2 for compounds with molecular weight above 2000. Besides molecular weight, it is further assumed that the polarity of the sample is known. Charts similar to Tables I and 2 can be found in column manufacturers' catalogs, some more detailed than others depending on how broad the supplier's column line is. Table 3 tabulates commercially available columns for the analysis of small molecular weight basic compounds, while Tables 4 through 10 list columns for analyzing biopolymers by size-exclusion chromatography (Table 4), anion and cation exchange chromatography (Tables 5 and 6), reversed phase chromatography of peptides, proteins, and polynucleotides (Table 7), hydrophobic-interaction chromatography (Table 8), affinity chromatography (Table 9), and hydroxylapatite chromatography (Table 10). Since there are hundreds of manufacturers and suppliers of columns for the analysis of low molecular weight compounds, it was decided not to include these columns in the tables. As noted, an exception was made for the analysis of basic compounds (Table 3). Also note that no effort was made to obtain a complete list of all available columns for any of the techniques. Those columns listed represent most of the major column brands, although it is likely that several important column types have been overlooked. The

127

presence of a product in any of the tables does not imply a recommendation of that product nor its manufacturer or supplier. Each table is organized alphabetically by manufacturer, starting with the packing material that, according to this author, is most often employed for each individual technique. For example, silica-based columns are most widely used for analyzing basic compounds by reversed phase (see Table 3), followed by resin-based columns, followed by other packing materials such as alumina. This section continues with a short discussion of issues involved in selecting a column for low molecular weight solutes, followed by a similar discussion for higher molecular weight solutes, and concludes with brief comments about the analysis and purification of peptides and proteins. Refer to Chapter 8 (carbohydrates) and Chapter 9 (proteins) for more detailed discussions about these sample types. A separate section on column selection for nucleic acids and nucleic acid fragments completes this chapter. 6.1 Samples with MW below 2000 Table I shows the types of columns involved in analyzing a sample with a molecular weight of less than 2000 Daltons. Often, the solvent(s) in which the sample dissolves is (are) known. If unknown, a proper solvent needs to be determined, taking into consideration that it is desirable that both the solute and the solvent are soluble in the mobile phase. If this is all that is known about the sample, it is recommended to select a silica-based C18 reversed phase or a polar bonded phase column, depending on whether the sample is soluble in a typical RPLC mobile phase or in an organic solvent, respectively. Note that RPLC can still be used if the sample is only sparsely soluble in RPLC solvents. Assuming that an RPLC column was selected, a buffer is chosen for the aqueous portion of the mobile phase and acetonitrile for the organic solvent. The choice of the buffer would, of course, be influenced by knowledge of the charge of the solute in the pH range 2-7 for silica-based columns, although a buffer would still be selected if the ionic form of the

128

sample is unknown. Next, a linear gradient is run from low to high percent of acetonitrile. It is possible that the solute of interest elutes unretained, either because it is very polar and is not even retained by the column in a 100% aqueous mobile phase, or because it is a strong base, a very strong acid, or a salt of a strong base or acid, and as such is still ionized in the entire acceptable pH range. Although this possibility is not uncommon, strong bases and acids are often retained in RPLC due to sufficient hydrophobic character. Continuing with the example, an oppositely charged ion pair reagent can be added to the mobile phase when a basic or strongly acidic solute is insufficiently retained. Alternatively, an ion exchange column can be used, or, in the case of strong base, one can opt for a column filled with a polymeric packing material. Rather than providing additional examples, it is suggested to consult the literature for a specific application. Others may want to learn more about how an HPLC method is developed [80]. In a recent survey of LC users it was reported that 57% of the respondents used reversed phase columns [81]. As a result, at least several hundred different brands of reversed phase columns are commercially available, making it impossible to list all or a majority of them in a table dedicated to columns for analyzing small molecular weight compounds. Instead, Table 3 lists columns that were specially developed for the analysis of basic compounds, which is still an active research topic in industry and academia. Other current research in packing materials is focused on sample preparation and chiral separations. Finally, Table 1 concludes with a short list of specialty columns. These columns are marketed for a particular separation, such as for the separation of basic drugs, tricyclic antidepressants, poly aromatic hydrocarbons, amino acids etc. Often the separation of the particular class of compounds was difficult to reproduce from column to column because of sample complexity, and/or Jack of control in silica or bonded phase preparation. In some cases, the compounds of interest could not be completely separated on a conventional (RPLC) column but could be separated on a slightly different stationary phase or by changing the coverage of the bonded phase on the

129

surface. The number of specialty columns has increased dramatically in recent years. 6.2. Samples with MW above 2000 As shown in Table 2, a variety of high performance techniques are available for biopolymer analysis. Most of the options are high-performance extensions of low or medium performance supports, with reversed phase liquid chromatography being the notable exception. The reader is advised to consult one or more of the many textbooks for more detail about individual separation techniques and applications thereof. References [82-84] examine the theory and application of size-exclusion chromatography similarly, [16] and [85-87] are devoted to affinity chromatography, references [37] and [88-93] are recent general texts about protein purification and analysis, and reference [94] provides greater detail about the various HPLC packing materials. A similar approach to that described above can be devised for each of the sample types listed in Table 2. In general, sample solubility will be known or can quickly be determined. Some additional information about the sample (charge, prevalent functional groups, hydrophobicity) will usually be known to allow a decision to be made about the first step in an analysis or purification scheme. This section concludes with brief remarks about each of the HPLC modes employed in the separation and purification of proteins, and a short discussion about the role of HPLC in the analysis of nucleic acid fragments. The reader is referred to the monographs about affinity chromatography (14, 85-87) or to the appropriate sections in the general textbooks [34, 8590] for a detailed account of the theory and applications of high performance affinity chromatography. Commercially available high performance affinity columns are listed in Table 9. 6.3. Proteins Gel filtration chromatography is often a useful first, and simple, step in the purification or characterization of the sample. In addition, GFC allows a

130

confirmation of the molecular weight that has often earlier been determined by gel electrophoresis. Whether working with a biopolymer or a watersoluble industrial polymer, it is recommended to vary the mobile phase composition (buffer concentration, type and concentration of salt, pH, and/or organic modifier) to prevent sample adsorption, and to ensure that the solute elutes within the pore volume of the column. Because most proteins are selectively excluded from a packing material with pores of 500 A or less, several commercial column lines for protein gel filtration chromatography consist of silica-based columns with 125 A, 250 A, and 450 A pores. Although these pore sizes are also available for hydrophilic resin-based columns, the silica-based supports have often larger pore volumes and thus better MW selectivity [95]. However, excellent results have recently been achieved on columns packed with cross-linked polysaccharide particles, such as those shown in Figure I. Hydrophilic resin-based columns are preferred for synthetic water-soluble polymers and nucleic acids [96], as these samples require very large -- and/or a large variety of pore sizes. Table 4 lists commercially available columns for gel filtration chromatography. Although size-exclusion chromatography is a reasonable choice as a first step in the purification of an 'unknown' sample, the low peak capacity of this technique is often insufficient to obtain the required sample purity. Depending on the stability of the sample, as well as its charge, hydrophobicity, shape or function, ion exchange, reversed phase, hydrophobic interaction or affinity chromatography would be selected for the next step in the purification. It has been estimated that ion-exchange chromatography is included in 75% of purification protocols [followed by affinity chromatography (60%) and gel filtration (50%)], because the technique is simple yet versatile and has high resolvicg power and capacity [97,98]. In recent years, the efficiency of the technique has been upgraded by incorporating ion exchange groups in small particle supports prepared from hydrophilic or hydrophobic polymers and silica. For scale-up of the separation from analytical to preparative or process size columns, several manufacturers offer the same support in particle sizes ranging from 5-10 iJ.m to as large as 100 iJ.m.

131

Janzen and co-workers developed novel ion exchange stationary phases by anchoring flexible polymeric or oligomeric ligands to resin and silica-based supports [99]. They showed that these multi-functional 'tentacle' supports have several-fold higher capacity than the same suppmt with short-chain functional groups, and allow long-term use of silica-based ion exchangers at alkaline pH. A typical application of ion exchange chromatography is the purification of isoenzymes (multiple forms of an enzyme within a species or a cell), which catalyze the same reaction but differ slightly in amino acid composition and pi values. Since their pi values are often greater than 8, resin-based columns are preferred over silica-based columns [100].

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70

90

Volume (ml)

Column, Progel-TSK DEAE-5PW, 10 !liD, 7.5 em x 7.5 mm; mobile phase, 200 mllinear gradient from 0 to 200 mM KCI in IOmM Tris-HCl containing 8 % glycerol, 3mM 2 mercaptoethanol, pH 7.5; flow rate, I ml!min; detection, fractions of 1 ml were collected and assayed for PNPase activity; recovery of enzyme activity was 86%; injection, 500 Jll

hemolysate.

Figure 11 Separation of Multiple Forms of Purine Nucleoside Phosphorylase from Human Blood Cells

132

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Column, Mono P, 10 ~m, 5 em X 5 mm; mobile phase, pH gradient developed with 30 ml of 1/10 diluted polybuffer 74 (pH 4.0) containing 6 M urea; detection, 280 nm UV; sample, apoliporoproteins (approximately 8 mg protein) injected in 6 M urea (pH 6.3);

seven fractions were collected and isolated.

Figure 12 Fractionation of Soluble VLDL

Stocchi et al. recently reported on the optimization of the separation of red blood cell isoenzymes using columns packed with analytical (10 ~m ProgelTSK DEAE-5PW) and preparative (35 ~m Toyopearl DEAE-650S) supports, which have very similar chemical composition [101]. Figure II shows the separation of isozymes of purine nucleoside phosphorylase on an analytical column. Slightly improved resolution was shown on the preparative column at the expense of a 10-fold longer analysis time. Another example of high-performance ion exchange chromatography is displayed in Figure 12, where human very-low-density apolipoproteins (VLDL) are separated by chromatofocusing using a decreasing pH gradient on a weak anion exchange column [102]. Finally, anion ·as well as cation exchange chromatography, sometimes in combination with hydrophobic-interaction

133

chromatography, play an important role in the purification of monoclonal antibodies [103]. Tables 5 and 6 lists commercially available columns for anion- and cation-exchange chromatography respectively. Recognized as a major tool in peptide and protein analysis, reversed phase chromatography is versatile, has unique selectivity and high peak capacity [104]. In recent years, RPC has become the most prominent technique in the analysis of peptides and proteins, although the use of denaturing conditions has hampered its wide-scale application in industrial purification of proteins. Wide-pore (300 A) C4-silica is commonly used for reversed phase separations of proteins, and small-pore (120 A) to wide pore (300 A) Cl8-silica columns are the columns of choice for analyzing peptides. Alternatively, large pore resin-based columns have been shown to provide good recoveries for large proteins [105]. As discussed earlier and as shown in Figures 6 and 7, fast analysis of peptides (and proteins) has recently become possible using either very large pore size or non-porous resin- or silica-based columns. Many examples of how reversed phase HPLC is applied to the analysis and isolation of peptides and proteins can be found in ref. [93]. In contrast to reversed phase chromatography, the separation of biopolymers in hydrophobic-interaction chromatography is governed by the hydrophobicity of the amino acids positioned on the surface of the molecules, since the mobile and stationary phase conditions are mild enough to leave most macromolecules in their folded state. As mentioned above, sample molecules are retained in HIC in a (phosphate or acetate) buffered mobile phase containing a high concentration of salt and are eluted from the column by a decreasing salt gradient. Although different salts have slightly different effects on retention and selectivity of proteins (see, e.g., ref. [106]), ammonium sulfate is most commonly used because of its high solubility, UV transparency, and its availability in high purity grades. Gooding et al. showed that the length of the hydrophobic ligand on the stationary phase in me greatly affects retention and selectivity [107]. Miller and Karger developed an ether-bonded silica column for dual purpose in HIC and SEC, and demonstrated that protein elution closely followed Snyder's gradient model [108].

134

0

60 30 Elution Time (Minutes)

90

Column, TSKgel Phenyl-5PW, 20 11m, 20 em x 55 mm; mobile phase, 60 minute linear gradient from 1.5 to 0 M (NH4)2S04 in 0.1 M phosphate buffer, pH 7.0: flow rate, 40 rnllmin; detection, 280 nm UV; injection, 500 mg (20 mg/ml) crude phosphoglucose

isomerase.

Figure 13 Preparative HIC Separation of Phospho glucose Isomerase

Table 8 shows several commercially available HIC columns, several of which are based on hydrophilic media modified with alkyl or phenyl groups. The first HIC media were prepared from alkyl-derivatized crosslinked agarose. Modern versions of this material and also hydrophilic resin-based supports are widely employed in high-performance HIC, as are the many silica-based packing materials. Although the peak capacity in HIC is not as good as in RPC or even ionexchange, the benefits of retaining biological activity and the distinct selectivity of HIC have contributed to its increased acceptance, particularly in large scale purifications. For example, Figure 13 shows the chromatogram for a 500 mg injection of crude phosphoglucose isomerase on a 20 em X 55 mrn !.D. column packed with 20 J.lm TSKgel Phenyl-5PW [109].

6.4. Nucleic Acids and Nucleic Acid Fragments Nucleic acids and their fragments have been separated by a variety of HPLC techniques. The analysis of nucleic acid bases and nucleosides is

135

commonly performed by reversed phase HPLC using C 18 or deactivated C 18 columns. Simpson and Brown published an extensive review of the profiling of nucleic acid components in physiological fluids [110]. Gehrke and Kuo edited a three volume set on chromatographic and other analytical techniques in nucleic acids modification research [Ill]. An example from their work, shown in Figure 14, demonstrates a high performance separation of nucleosides in unfractionated calf liver tRNAs. The presence of mono-, di-, or triphosphate groups in nucleotides has made anion exchange chromatography the obvious choice for many authors, although reversed phase or ion pair reversed phase separations often result in excellent resolution while benefiting from the proven excellent chemical stability of alkyl-bonded phase columns. The purification of synthetic oligodeoxyribonucleotides was recently reviewed by Zon [37]. The separation of !RNA's by reversed phase was demonstrated by Pearson eta/. [112], while El Rassi and Horvath separated Gradient Progrtm

Time

m'G

%A 100 100

(mil'l) 0 12

m1G \ A m,Z PRP-3 POROS R PLRP-S 300A IOOOA

Supelco

Silica

SynChromf Micra

Silica

Tosoh

Silica

YMC

Silica

Hamilton Perseptive Biosystems Polymer Labs

TSK-GEL Phenyl-5PW RP Octadecyl-4PW Octadecyl-NPR

Tosoh

POLYP

Mac-Mod

Hydrophobic polymer Hydrophic polymer Hydrophobic polymer

Hydrophilic polymer Fluorocarbon

25

X

4.6

5

300

CIS, CS, C4

5 10

X X

4.6 4.6

6.5 6.5

300 100

Cl8 Cl8

15

X

4.6

5

300

Cl8

25 25

X X

4.6 4.6

5 5

120 200

C8 (CIS) CIS

3 5

X X

4.1 4.1

4 10

Non-porous 300

PS/DVB PS/DVS

JO

X

4.6

20

6000

PS/DVB

5 5

X

4.6 4.6

5

X

s

300 1000

PS/DVB PS/DVB

10 7 2.5

1000 500 Non-porous

phenyl CIS Cl8

20

300

7.5 X 4.6 15 X 4.6 3.5 X 4.6 8

X

6.2

Table 8. Columns for hydrophobic interaction chromatography

Column description

Company

Support

HEMA BI0-1000 11Analyzer MP7 HIC Superose Phenyl Alkyl Shodex PH-814 SigmaChrom RIC-Phenyl TSK-GEL Phenyi-5PW Ether-5PW Butyi-NPR

Alltech

Hydrophilic polymer Hydrophilic polymer Hydrophilic polymer

Bakerbond HI-Propyl Spherogel CAA-HIC

JT Baker

Silica

Beckman

Silica

Bio-Rad Pharmacia Showa Denko Supelco/ Sigma Tosoh

Hydrophilic polymer Hydrophilic polymer Hydrophilic polymer

Dimensions

Particle

Pores

Functional

em x mm

size (I'm)