170 CHROMATOGRAPHY [8] [8] High-Performance Ion-Exchange Chromatography By FRED E. REGNIER Liquid chromatography of proteins is a separation process based on differential rates of molecular migration through a bed of particles. When purifying a single substance by this process, the objective is to choose conditions and materials that maximize the difference between the migra- tion of this substance and all others in the sample. The difference in migration between the substance being purified and that of any other component in the sample depends on a number of variables, including the resolving power of the system. Resolution (R0 of a particular component from any other is expressed by Eq. (1) R~ = 2(Ve2 - Vel)/(AVe I + AVe 2) (1) in which Vel and Ve2 refer to the elution volumes of the first and second components, respectively, to elute from the column. Peak width for each of these components is designated, with the appropriate subscript, as AVe. The discussion here will deal with the rationale used in column selection, operation of ion-exchange columns, and maximization of reso- lution through manipulation of Ve and AVe values. Obtaining successful ion-exchange separations of proteins from a se- ries of different samples has two requirements: a good column and an understanding of the ion-exchange process. The first requirement is gen- erally satisfied by the purchase of a commercial column, but the second requires an intellectual commitment from the investigator. Actually, many individuals fail to see the need for the second requirement since they view chromatography as a procedure that can be accomplished by a recipe. Although much can be done by recipe, that approach is as restric- tive as an attempt to paint a landscape in monochrome. Each protein is unique and, therefore, offers unique opportunities for its purification. The chromatographer must be aware of the means for finding and exploiting these opportunities. Retention Protein retention on an ion-exchange column is obviously the result of electrostatic interactions in which retention increases in proportion to the charge density on both the ion-exchange matrix and the protein. Since proteins are amphoteric, their net charge and charge density will vary Copyright © 1984 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 104 All rights of reproduction in any form reserved. ISBN 0-12-182004-1
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`KASHIV EXHIBIT 1027
`IPR2019-00791
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`[8] HPLC: ION EXCHANGE 171 with the pH of the solution. Two additional variables that may influence retention are the nature of the ion-exchange support and the distribution of charge in the protein. Some ion-exchange materials have broad titra- tion curves that will cause ion-exchange ligand density to vary with the solution pH. The ionic properties of the ion-exchange material and of proteins may vary independently with pH. That all the charges on the surface of a protein may not be able to interact with the ion-exchange matrix simultaneously is an important consideration. That is, two proteins of identical charge can interact differently with an ion-exchange support because of differences in the distribution of charges rather than the net charge alone. In order to control the retention process in ion-exchange chromatogra- phy and to manipulate elution volumes of the various protein compo- nents, it is necessary to examine the ionic properties of proteins. Proteins are amphoteric species that bear a net positive charge under acidic condi- tions and a net negative charge under basic conditions and are isoelectric at a specific intermediate pH designated as the isoelectric point (pI). Titration curves indicate that the amount of charge on proteins increases steadily as the pH of the enveloping solution deviates from the pI and that each protein has a unique titration curve. This implies that a pH could probably be found at which the difference in charge between the protein being purified and all other proteins will be at a maximum. That pH would obviously be the one at which the protein should be chromatographed on an ion-exchange column. Two factors complicate this rationale: all the charged groups within a protein will not be able to interact simultaneously with the ion-exchange matrix, and charge distribution on the surface of all proteins may not be uniform. Ion-exchange characteristics are based only on those groups of the protein that are capable of interacting with the ion- exchange matrix. Ion-exchange surfaces can recognize the difference be- tween two proteins that are identical in every respect except that one has uniform charge distribution and the other is asymmetric. (Significantly, electrophoretic systems will not distinguish between these two species because both have the same net charge and pI.) In contrast, two mole- cules having a different pI and net charge could possibly cochromato- graph if they have the same surface characteristics. From these examples, it can be seen that any comparison between electrophoresis and ion- exchange chromatography has the inherent flaw that the two techniques are effective by different mechanisms using charge differences as the basis for separation. The difference in separation mechanism between ion-exchange and electrophoretic techniques is most prominent at the pI where the net charge of a protein is zero and where it does not migrate in an electric
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`172 CHROMATOGRAPHY [8] 35 30 25 T I I 20 o 1'5 IN 10 0 2 - Lactoglobulin I i I i I ~ I l I I I l / // s ,x 4 6 8 I0 pH ao i-- ~z I0 0 I- Z bJ I- UJ I-- U 0 .J W FIG. I. Chromatographic retention as compared to titration and electrophoretic mobility curves for fl-lactoglobulin, pl = 5.1. SAX refers to the Pharmacia Mono Q column; SCX, to the Pharmacia Mono S column. A 40-min linear gradient to 1.0 M NaC1 was used at eluent pH 3.0 on the SCX column. field. The absence of electrophoretic migration does not mean that the protein is devoid of charge. There will still be cationic and anionic groups within the molecule at its pl, and if their distribution is sufficiently asym- metric, i.e., if some of the charges are clustered, they will cause ionic interactions with an ionic surface. Thus, it is not surprising that 75% of the proteins examined by Kopaciewicz ~ were retained on both anion- and cation-exchange columns at their pI. The term "ion-exchange retention," as used here, refers to either the retention time or the elution volume of a protein from a column that has been eluted with a gradient. Two major modes of gradient elution are used in protein separations: ionic strength gradients, and pH gradients. Ionic strength gradients are by far the most common and the easiest to gener- ate. Chromatofocusing provides a form of pH gradient elution and will be discussed separately. The reason for separating most proteins by gradient elution will also be discussed. To examine the difference between electro- phoretic and ion-exchange separations, Kopaciewicz ~ compared the ] W. Kopaciewicz, M. A. Rounds, J. Fausnaugh, and F. E. Regnier, J. Chrornatogr. 2,66, 3 (1983).
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`[8] HPLC: ION EXCHANGE 173 charge, electrophoretic mobility, and chromatographic retention of/3- lactoglobulin at a number of pH values. The results, shown in Fig. 1, indicate that retention on both strong cation- and anion-exchange columns occurs at the pI at which net charge and electrophoretic mobility are zero. This would indicate that/3-1actoglobulin represents a protein in which the charge distribution is asymmetric. The figure also shows that chromatographic retention begins to plateau several pH units above the p! of/3-1actoglobulin whereas its charge and electrophoretic mobility con- tinue to increase. Further analysis of the titration and retention data indi- cates that the protein had a total of 17 negatively charged residues at pH 10 but that only 6 were involved in the ion-exchange process. Plots of chromatographic retention versus pH are referred to as reten- tion maps ~ (see also this volume [11]). Comparison of the retention maps (Fig. 2) of several proteins indicates that many proteins are retained on both strong anion- and cation-exchange columns near their pI; that reten- tion on both types of columns increases as mobile-phase pH is moved away from the pl; that each protein has a unique retention map, just as it has a unique titration curve; and that there is an optimum pH that pro- duces the largest difference in retention of a specific component from others in a mixture. Questions relating to properties of the ion-exchange support and oper- ating parameters to resolution are discussed separately. Macromolecules have been separated chromatographically on the ba- sis of charge, hydrophobicity, size, and bioaffinity. Obviously, charge characteristics are the most important in ion-exchange separations, but hydrophobicity and molecular size also play a role. For example, mole- cules that are of limited water solubility may interact with an ionic matrix by a hydrophobic mechanism in addition to coulombic forces. In extreme cases, a protein may be so hydrophobic that it lacks water solubility. Separation of proteins of limited water solubility, such as membrane pro- teins, will also be discussed (see also this volume [16] and [18]). The contributions of molecular size to ion-exchange chromatography, in con- trast to hydrophobic effects, are usually evident in the capacity of an ion- exchange medium. The pore diameter of a support must be matched to the molecular size to obtain maximum ion-exchange capacity. The role of these variables should become clearer from the discussion that follows. Column Selection The most important question confronting the neophyte chromatog- rapher is that of the nature of the column to be used. For a rational choice, the chromatographer should have some knowledge of the protein
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`174 CHROMATOGRAPHY [8] 20 16 12 8 4 ._~ E 20 16 12 8 4 • I I l ! I I ANION EXCHANGE pI<7 CATION EXCHANGE pI<:7 | 45678910 pH ! I [ I I I I ANION EXCHANGE pI:>7 ~L -- GE I I I I I I l 345678910 pH Flo. 2. Retention maps of some common proteins. Five acidic and five basic proteins were chromatographed on both a strong anion (Mono Q) and strong cation (Mono S) ex- change column. The symbols represent proteins within the vertical column. A suitable buffering ion (10 mM) was chosen for each pH. Proteins were eluted with a 20-rain linear gradient from 0 to I = 0.5 M NaCI at a flow rate of I rnl/min. Anion exchange: ©, lysozyme; A, cytochrome c; [3, ribonuclease; O, chymotrypsin; A, carbonic anhydrase; cation ex- change: (D, /3-1actoglobulin; A, soybean trypsin inhibitor; G, ovalbumin; A, a-amylase; 0, conalbumin. that is to be separated in terms of its pI, size, and even hydrophobicity. The retention maps of proteins in Fig. 2 indicate that greatest retention on cation-exchange columns occurs below the isoelectric point, where the protein has a net positive charge, whereas the largest retention on anion- exchange columns occurs above the isoelectric point, where proteins are negatively charged. For example, a protein with a pl of 7 would be retained on a cation-exchange column below pH 7 and on an anion-
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`[8] HPLC: ION EXCHANGE 175 exchange column above pH 7. Less than 5% of the proteins examined are an exception to this statement. 1 In the event that the chromatographer does not know the pl of the protein he is trying to separate, the informa- tion may be obtained either chromatographically or electrophoretically. A simple technique for approximating pI is to chromatograph the protein on both anion- and cation-exchange columns over a range of 3 pH units at near-physiological conditions. Retention properties of the protein may be mapped and the most suitable column selected by this simple process. A superior method for determining pI is the isoelectric focusing titration curve of Rhigetti. 2 In addition to giving the isoelectric points of all pro- teins in a sample, information on the charge characteristics of all sample proteins at any pH from 3 to 10 is also provided. Although there is not a direct correlation between the charge characteristics as observed in elec- trophoresis and chromatographic retention, electrophoresis data suggest the most useful pH at which to begin ion-exchange separations. A number of instances have been observed in which electrophoresis was used to predict optimum pH for ion-exchange chromatography. 3 Once it has been established that either an anion- or cation-exchange column is the more likely to provide the desired separation, the experimenter may proceed with column selection. Four basic types of high-performance ion-exchange chromatography (HPIEC) columns are available: (I) weak anion-exchange (WAX), (2) strong anion-exchange (SAX), (3) weak cation-exchange (WCX), and (4) strong cation-exchange (SCX) materials. Unfortunately, the terms "strong" and "weak" are often misunderstood in reference to ion-ex- change chromatography. They are not intended to suggest the strength with which a substance is retained on the ion-exchange matrix, as the name would imply. Rather, they indicate the degree of stationary phase ionization at various extremes of pH. Strong cation- and anion-exchange materials are usually sulfonates and quaternary amines, respectively; both remain fully ionized within the normal operating range of high-per- formance ion-exchange chromatography. In contrast, weak cation- and anion-exchange supports usually contain carboxyl groups or primary and secondary amines, respectively, as the ionizable species. The pKa of the average weak cation-exchange support is roughly 4, and that of a weak anion-exchange material is in the range of 8 to 10. Some ion-exchange materials even have very broad titration curves that span 5 pH units. 4 ~" P. G. Righetti and J. W. Drysdale, in "Isoelectric Focusing," p. 341. North-Holland Publ., Amsterdam, 1976. Technical Bulletin, "The Pharmacia HPLC System." Pharmacia Fine Chemicals AB, Uppsala, Sweden. 4 A. J. AIpert and F. E. Regnier, J. Chromatogr. 185, 375 (1978).
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`176 CHROMATOGRAPHY [8] Obviously, as the operating pH of a column approaches within a few pH units of the pK~, charge on the ion exchange matrix decreases along with ligand density. Since the charge density of both the support and the pro- tein vary with the pH of the mobile phase in an unrelated manner, reten- tion characteristics of proteins on weak ion-exchange columns are less predictable than with strong ion exchangers. There are instances in which the retention of a protein will be greatest at an intermediate pH and will diminish toward the pH extremes as ionization of the WAX or WCX support collapses. As a general rule, it is best to carry out the initial separations of a protein on a strong ion-exchange material, although this is not meant to suggest that the strong ion-exchange support will neces- sarily be superior to a weak ion-exchange column in resolution. The sub- ject of resolution is discussed below. A number of ion-exchange columns are now available (Table I). There are two broad classes of ion-exchange supports in terms of support matri- ces: those totally organic and those that are inorganic with an organic surface coating. The inorganic supports are available in a series of pore diameters ranging from 100 to 4000 ~ and particle sizes from 3 to 30/zm. The only microparticulate organic-based support currently available for proteins is the Pharmacia MonoBead material; it is available in a strong cation- and anion-exchange form in addition to a weak anion-exchange material for chromatofocusing (see also this volume [11]). Support pore diameter in the MonoBeads is reported 3 to be approximately 800/~, with the matrix chemically stable between the limits of pH 2 and 12. This is considerably broader than the pH 2 to 8 limit of silica-based materials. Present silica supports have an upper pH limit of 8 or 9 because of the increased erosion of the silica matrix at basic pH. An important question centers about the pH to be used in protein separations. Chromatographic band broadening, the tendency of enzymes to denature, and the amount of retained material at the inlet of ion-ex- change columns increase substantially when columns are operated more than 2 pH units from physiological pH. This does not mean that ion- exchange separations are limited to the limited pH 5 to 9 range, but rather that most separations can be achieved within this range. Pressure limits and expected column life are generally not available from manufacturers (see also this volume [6]). Since most separations may be achieved at less than 30 atmospheres with properly designed columns, mechanical stability is probably not a problem with any of the current semirigid and rigid packing materials. Expected column life, in contrast, is more difficult to quantitate since it depends on operating conditions, type of sample applied, and column care. There is now evi- dence that many columns will survive the analysis of 500 or more serum samples at pH 7 if routinely cleaned when resolution begins to fail.
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`[8] HPLC: ION EXCHANGE 177 u~ la. O r~ O r.~ m Z < 0 u~ o g e~ z .~ r/l oc5- <<< ZZZ ZZ ~ © ×××x~ <<<< << ~zz~ ~~~~ d AAAA <<<< ZZZZ ZZZZ 0 0 o o o e. 0 la0 < "~ o
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`178 CHROMATOGRAPHY [8] Loading Capacity An additional parameter in column selection is that of the amount of protein that must be separated. In current practice, a 4.6 x 250 mm column is considered to be one for analytical use. Most ion-exchange columns of this size will have a loading capacity ranging up to 10 mg of protein without loss of resolution. As sample loading is increased, resolu- tion decreases steadily up to loads of 40 or 50 mg, at which point protein often saturates most of the length of the column and normally retained proteins begin to break through with other nonretained material. The relative degree of separation between the proteins of interest determines to a large extent whether such a large sample may be accommodated. Loads of 50 mg may give very acceptable separation of proteins that separated widely in an analytical sample, whereas a 15-mg load may produce unacceptable resolution between peaks that are immediately ad- jacent. The loading capacity of any support is due to a combination of variables that includes the ratio of support pore diameter to solute diame- ter, support surface area, and ligand density. The loading capacities pre- sented here are in terms of what might be expected with a protein (Mr = 50,000) on a 300-/~ pore diameter support. Proteins of higher molecular weight would be expected to exhibit lower loading because they cannot penetrate the support matrix. Smaller proteins might load more heavily because they have greater access to the support surface inside pores. At submicrogram loadings, sample recovery may decline because the surface area-to-solute mass ratio in the column is very large; the column is undefloaded. The small number of imperfections in a support that irre- versibly adsorb or denature protein can dominate the separation in an underloaded column. It is better to use a smaller column for micropre- parative work. Submicrogram samples also begin to exceed the detection limits of the system unless such sensitive indicators as radioactivity, en- zyme activity, or fluorescence are being monitored. Columns of 50-ram length or less are becoming common in analytical separations because column length contributes little in the resolution of proteins. The loading capacity of a 4.2 × 50 mm column is in the range of 1-2 mg of protein. The principal advantages of short columns are that they are less expensive, mechanical stress on the packing bed is reduced, they may be eluted more quickly, they are easier to pack, and solutes may be eluted in smaller volumes of mobile phase thereby de- creasing sample dilution and increasing detector sensitivity. When the intent is to separate very large quantities of protein, the chromatographer must go to the true preparative-scale column. It is to be expected that the loading capacity of ion-exchange columns will increase with the square of
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`[8] HPLC: ion EXCrtANGE 179 the column radius. Thus, by increasing column dimensions from 4 mm (analytical dimensions) to 10 mm (preparative dimensions), loading capac- ity is expected to increase sixfold. The design and application of prepara- tive ion-exchange columns to protein separations remains in an early stage, but there is sufficient knowledge available to begin a discussion of the subject. Two types of ion-exchange users are anticipated for the future: those scaling up an analytioal procedure to produce a few grams or less of protein, and the industrial user who intends to produce tens of grams to kilograms of protein per hour. Their needs and their approaches will be completely different. In the first case, simply scaling up the analytical separation by using the same packing material and a two- to fourfold larger column would probably provide the best solution. The need for redesigning a separation system and elution protocol is thereby avoided for occasional use. The cost per unit mass of protein isolated may be much more expensive by this approach than with the large, preparative system required in the commercial production of proteins, but it requires much less development. With the advent of genetic engineering, the ability to prepare kilogram quantities of protein is in demand. The preparative systems used in the purification of these proteins will be designed for specific separations. Even the support materials may be optimized for loading capacity and the resolution of a single protein species. Elution A major initial concern of a chromatographer attempting a separation on a new type of column is "how to get a peak." Since ion-exchange chromatography of proteins has been in use for the past 25 years and is quite similar to HPIEC, a large body of information is available on ion- exchange elution protocols. As a first suggestion, it is recommended that the chromatographer draw upon his own experience or search the litera- ture for separations similar to the one he is undertaking. Conditions simi- lar to those reported with conventional gel-type ion-exchange columns usually work well on HPIEC columns. The most widely used technique for eluting ion-exchange columns over the last 25 years has been with ionic-strength gradients at a fixed pH. Although pH gradients have been successful, they are more difficult to produce and generally give poorer resolution than ionic-strength gradients. The possible exception would be in chromatofocusing columns. The initial step in gradient elution of increasing the ionic strength of an ion-exchange column is to load the sample on the column in a buffer at a
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`180 CHROMATOGRAPHY 18] low ionic strength and at a pH appropriate for retention to occur. Buffer concentrations of I0-20 mM are generally used, although up to 50 mM buffer is appropriate when proteins are strongly retained. Desorption and elution of proteins is achieved by gradually increasing the ionic strength of the mobile phase. Proteins are generally displaced from the column in the order of their increasing charge, although the actual number of charges in contact with the surface are more important in determining retention than is net charge. The next consideration is the selection of a mobile phase consisting of a buffer and displacing ions. Since the ionic strength of the buffer is low, the pKa of the buffer chosen should be within 1 pH unit of the operating pH of the system. Manufacturer literature supplied with the column should also be consulted, since specific buffers are occasionally recom- mended. If there is no information on which to base a selection of the displacing ion or salt, it is best to start with simple salts such as sodium chloride, sodium acetate, or, possibly, sodium bromide. Sodium chloride has been so widely successful in conventional gel-type columns that its use is rec- ommended in HPIEC as well. However, it should be recognized that halide ions erode stainless steel surfaces of pumping systems unless the metal is passivated. The problems of metal erosion and passivation tech- niques have been discussed in this series. 5 Selection and use of a variety of other salts will be discussed below in the section dealing with optimiza- tion. The strong or displacing solvent (solvent B) usually consists of 0.5- 1.0 M displacing ion added to the initial buffer (solvent A). Seldom will a protein be so strongly adsorbed to a column that more than 1.0 M salt is required for elution; when a case of strong retention is encountered, bringing the operating pH of the column closer to the pI of the protein will diminish retention. In the event that all protein is eluted from the column by the time the gradient has reached 50% solvent B, it would be desirable to dilute solvent B until the most strongly retained proteins elute near 90% B in gradient elution. Although step gradients are commonly used in conventional gel-type columns and, on occasion, in HPIEC columns, complex mixtures are usually best resolved by a continuous gradient. Analytical columns may be eluted at 1 ml/min with a linear gradient ranging from 0 to 100% B in 20-30 min. A rationale for using different mobile-phase velocities and gradient slopes will be presented in the dis- cussion of optimization. 5 This series, Vol. 91, p. 137.
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`[8] HPLC: ION EXCHANGE 181 A note of caution should be added with regard to particulate matter in buffers, since both the column frits and the column bed have the proper- ties of a filtration medium. Any particulate matter in the mobile phases will accumulate in the column and eventually plug it. Proper care and preparation is discussed in detail in this volume [6]. Chromatofocusing As noted, charged proteins may be adsorbed to an ion-exchange sup- port and eluted with either a pH or salt gradient. Gradients are normally generated externally and fed into the ion-exchange column to effect elu- tion. Sluyterman and his co-workers 6-8 have developed a new technique for generating a pH gradient: chromatofocusing. A pH gradient is formed by pumping a buffer with a large number of different charged species into a column that has a natural buffering capacity. For example, if the pH of a weak anion-exchange column is adjusted to 8 and polybuffcr--a term introduced by Pharmacia Fine Chemicals to indicate a buffer of many charged components--of pH 5 is pumped into the inlet of the weak anion- exchange column, the most acidic components of the buffer will be ad- sorbed and other components will migrate farther down the column be- fore being adsorbed. The net effect of this process on pH is that the ion-exchange groups at the head of the column will be titrated and the pH of the medium will gradually be raised to that of the inlet buffer. This titration process will be repeated in all segments of the column until the whole length of the column is, in the example, at pH 5. Because the titrating buffer enters the head of the column, titration will occur sequen- tially in column segments and a pH gradient will be established across the length of the column. If a protein is introduced into the column during this titration process, it will migrate along the pH gradient until it reaches a point in the column where the pH is sufficiently high that it will become negatively charged. This pH will be near, but not necessarily at, the pI of the protein because some proteins have intensely charged areas on their surface that bind to an ionic surface even at their pI. As the pH gradient moves down the column, a protein is alternately adsorbed and desorbed in such a manner as to have a focusing or concentrating effect on sample components. Proteins will move along the column at or near their pl. As in other types of ion-exchange chromatography, resolution may be influenced by mobile-phase velocity and gradient slope. Figure 3 shows L. A. E. Sluyterman and J. Wijdenes, J. Chromatogr. 150, 31 (1978). 7 L. A. E. Sluyterman and J. Wijdenes, J. Chromatogr. 206, 429 (1981). 8 L. A. E. Sluyterman and J. Wijdenes, J. Chromatogr. 206, 441 (1981).
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`182 CHROMATOGRAPHY [8] E cO.J. N 0. 4 t,- 0.3 IlI ~0.2 mO 0.1 0 o , i t ,BI I s ~ m5 TIME (rain} i t i c 5 "T 4 t L 3~ 2 J I I 20 2S 30 FIG. 3. Chromatofocusing profiles showing the effect of flow rate on resolution of peaks ofconalbumin. A 4% suspension of conaibumin (20/,l) was chromatographed at (A) 2, (B) l, and (C) 0.5 ml/min. The pH gradient (---) was formed using a 1 : 10 dilution of PB 96 polybuffer, pH 6.0, after the column was equilibrated in 0.25 M imidazole-acetic acid at pH 7.4. I, II, and III represent the major components of conalbumin. the relationship between mobile-phase velocity and resolution of compo- nents in a conalbumin sample at the same gradient slope (percentage of change per unit volume of mobile phase). Flow rates even lower than 0.5 ml/min are useful if the pumping system retains its accuracy at these low velocities. Gradient slope is controlled in chromatofocusing by the concentration of polybuffer being pumped into the column. As the buffer becomes more dilute, a greater volume will be required to titrate the support and the pH change of the eluent per unit volume of buffer added to the column will be smaller. It is apparent that the amount of buffer required to titrate the column will be a function of the charge density of the support. Since charge density varies considerably among commercial supports, the column manufacturer's literature should be consulted for guidance, or several dilutions of the polybuffer examined to achieve the desired gradi- ent slope. Only two types of columns have currently been reported as useful: Pharmacia Mono P and the Synchrom AX series. Both are weak anion- exchange supports. Mono P is a proprietary material of undisclosed pore diameter; presumably, the pore diameter is 800 ~ as in the Mono Q support. The Synchropak AX materials have a polyethylenimine phase bonded to silica and are available on 100-, 300-, 500-, or 1000-~ diameter porosity. It has been reported s that polyethylenimine is useful in chroma-
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`[8] HPLC: ION EXCHANGE 183 tofocusing because it has a very broad titration curve and that the pore diameter contributes to resolution in chromatofocusing. Substantially bet- ter resolution of estrogen receptor proteins were obtained on 500-.~ pore diameter supports than 300 A.9 Although it is possible to prepare polybuffers in the laboratory, they are generally inferior to the products of Pharmacia. l° Pharmacia recom- mends that only a polybuffer be used over an interval of 3 pH units. To cover the full range of pH within which one must work, they offer three polybuffers: polybuffer 74 for the pH 7 to 4 range; polybuffer 96 for the pH 9 to 6 range; and Pharmalyte for the pH 10.5 to 8 range. Pharmacia polybuffers are supplied as sterile filtered solutions at 0.075 mmol per pH unit per milliliter. Before reusing a chromatofocusing column, all residual components from the previous sample should be removed by adjusting to pH 3 or less, using 1 M salt, or by a combination of low pH and high ionic strength. Two to five column volumes should be sufficient for recycling, depending on the nature of the sample. After this step, the column may be brought back to the initial pH required for chromatofocusing. The advantages of chromatofocusing are in provision of another method of ion-exchange chromatography that has a selectivity different from that of ionic strength gradient elution and requires only a single pump to form a gradient. The principal disadvantage is that fractionation is achieved near the isoelectric point of a protein at low ionic strength, both of which favor denaturation. As yet, there are relatively few reports of high-performance chromatofocusing. The true utility of the technique compared to ionic strength gradient elution remains largely undeter- mined. Optimization One of the questions regarding any separation is whether it is the best that can be achieved. This is particularly pertinent when trying to purify a protein with the minimum number of steps or when a coeluting substance prevents analytical quantitation. The answer to the question requires a more detailed discussion of resolution. Unfortunately, there is no single means by which it may be determined that a column is operating at maxi- mum resolution or to optimize resolution. Two sets of variables control resolution in a chromatographic system: those that are inherent to the c