Report Rodney R. Walters Department of Chemistry Iowa State University Ames, Iowa 50011
Affinity Chromatography One can imagine two extremes in the chromatographic separation of a complex sample. At one extreme, typified by capillary gas chromatography, a long, highly efficient column of low to moderate selectivity is used to separate a sample into hundreds of peaks. At the other extreme, typified by affinity chromatography, a short, inefficient column of high selectivity is used to separate one or a few solutes from hundreds of unretained solutes. Both of these approaches have obvious advantages and disadvantages. In the former, long analysis times are typical and overlap of critical peaks may occur, but a great deal of analytical information is obtained and the columns can be used for the assay of many dif-
0003-2700/85/A357-1099$01.50/0 © 1985 American Chemical Society
ferent solutes. In the latter approach, the column may be useful for the assay of just one solute, but the separation time may be as little as one minute. Clearly, the latter approach is particularly useful for repetitive types of separations such as industrial and laboratory purification and clinical analysis. Although affinity chromatography was used as far back as 1910, modern affinity chromatography began with the development of the cyanogen bromide method for the immobilization of ligands on agarose supports by Axen et al. in 1967 (2). The use of rigid, microparticulate supports led to the development of high-performance affinity chromatography (HPAC) by
Ohlson et al. in 1978 (2). Other related methods that will not be discussed here include affinity partitioning, affinity filtration, and affinity targeting of drugs. Affinity ligands Affinity ligands are divided into two groups: specific and general. Specific ligands, such as antibodies, presumably bind to only one particular solute. One can take advantage of this high selectivity by using short columns (~1 cm) and step elution methods to perform HP AC separations in less than a minute (3). General ligands (or group-specific ligands), such as nucleotide analogues and lectins, bind to certain groups of
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solutes. For example, the lectin concanavalin A binds to solutes containing terminal glucosyl and mannosyl residues. These solutes can be separated as a group using step elution techniques or separated from each other
using isocratic or gradient elution techniques. In the latter case, the selectivities of the solutes may be similar, so that longer, more efficient columns must be used. These two modes of operation are il-
Affinity Chromatography—Basic Principles The basis of the selectivity of affinity chromatography is the use of immobilized biochemicals as the stationary phase. These biochemicals, which are called affinity ligands, can be antibodies, enzyme inhibitors, lectins, or other mole cules that reversibly and bioselectively bind to the complementary analyte molecules in the sample. The sepa rations exploit the "lock and key" binding that is prevalent in biological systems. A typical affinity chro matographic separation of a mixture of enzymes is illustrated in the accompanying figure. A sample containing several enzymes (large circles) is applied to a column containing an inhibitor (small circles) that has been covalently bonded to an inert support material. As the sample is washed through the column, the enzyme molecules complemen tary to the affinity ligand molecules are adsorbed while the other compo nents elute without retention. The adsorbed enzyme molecules are then eluted by means of a change in the mobile phase composition. For example, a pH change will often dissociate the enzyme-inhibitor com plex because of changes in the ioniza tion of acidic or basic functional groups on the molecules. The column is then ready for another run after returning to the initial mobile phase composition. A typical chromatogram, as shown in the figure, has a nonretained peak contain ing many components and a retained peak that ideally contains only the one biospecifically adsorbed enzyme. The term affinity chromatography is somewhat vague and encompasses not only the above types of separations but also biomacromolecule separations using immobilized metal chelates, chiral separations of drugs using immobi lized albumin, and even adsorption via covalent disulfide bond formation be tween biomacromolecules and immobilized thiol groups.
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lustrated by the separations of immunoglobulins using immobilized protein A, a protein from the cell wall of Staphylococcus aureus. A 5-cm column, operated with step elution, separates the retained immunoglobulins in blood serum in 4 min (4). A 30-cm column, operated with gradient elution, is able to resolve two immunoglobulin G subclasses (IgGi and IgG2), but the separation requires one hour (5). Table I lists some of the more common general ligands and their specificities. More extensive lists can be found in the suggested readings at the end of this REPORT. Although less selective, general ligands are widely used because of their greater convenience. Specific ligands are often more difficult to obtain but are more suitable for repetitive separations. Of course, there is really a continuum between specific and general ligands. Some ligands, like concanavalin A, bind to dozens of substances, while others, like soybean trypsin inhibitor, bind to only a few. Clearly, the suitability of a general ligand will also depend on what other components are in the sample. Other considerations in choosing the ligand include the strength and kinetics of binding and the conditions required for elution. Among the more unusual ligands are the triazine dyes, such as Cibacron Blue. These dyes bind at the nucleotide-binding sites of many enzymes but also bind hydrophobically to many other proteins including albumin. They have been used for the purification of more than 500 different proteins. Supports
The ideal support for affinity chromatography would be a rigid, stable, high surface area material that does not adsorb anything itself. Existing supports do not quite measure up to these standards. For conventional affinity chromatography, organic gels such as agarose, cellulose, dextran, polyacrylamide, and combinations of these polymers have been used. The most popular is the agarose gel sold by Pharmacia under the trade name Sepharose. Agarose is a polymer of D-galactose and 3,6-anhydro-L-galactose. Agaroses are available in a variety of pore sizes based on the percentage agarose used to prepare the gel. For example, Sepharose 4B and 6B are 4% and 6% agarose and have pore sizes of approximately 300 and 150 À, respectively. A typical particle size is 50-150 μνα. The recommended operating conditions for Sepharose 4B include a pH range of 4-9 and a maximum pressure of 1 psi. The agaroses can be cross-linked with 2,3-dibromopropanol to provide better chemical and mechanical
Table 1.
General ligands and their specificities Ligand
Specificity
Cibacron Blue F3G-A dye, derivatives Of AMP, NADH, and NADPH
Certain dehydrogenases via binding at the nucleotide binding site
Concanavalin A, lentil lectin, wheat germ lectin
Polysaccharides, glycoproteins, glycolipids, and membrane proteins containing sugar residues of certain configurations
Soybean trypsin inhibitor, methyl esters of various amino acids, D-amino acids
Various proteases
Phenylboronic acid
Glycosylated hemoglobins, sugars, nucleic acids, and other c/s-diolcontaining substances
Protein A
Many immunoglobulin classes and subclasses via binding to the Fc region
DNA, RNA, nucleosides, nucleotides
Nucleases, polymerases, nucleic acids
strength. For example, Sepharose CL6B can tolerate pH 2-12 and up to 6 psi. Agarose gels are available from many companies including Pharmacia, Bio-Rad, Pierce, LKB, Sigma, and United States Biochemicals; some companies sell as many as 200 different ligands already immobilized. One particularly interesting support is LKB's Magnogel, which contains iron oxide as well as agarose and polyacrylamide. Because it is magnetic, this support can be quickly collected, washed, etc., without the use of centrifugation or filtration. Several medium-performance supports are available that can withstand higher pressures and are typically 40 (im or larger in diameter. Pierce sells Fractogel TSK (a hydrophilic vinyl polymer) and Trisacryl GF (a hydrophilic polyacrylamide) with several ligands attached. The Fractogel can tolerate pH 1-14 and 100 psi. Trisacryl can tolerate pH 1-13 and 40 psi. Bonded-phase silicas are available from Serva (pore size 500 Â) and J.T. Baker (pore size 60-90 Â). Bondedphase controlled porosity glass is available from Pierce (pore sizes 500 and 200 À). A typical bonded phase is 3-(glycidoxypropyl)trimethoxysilane. The epoxide groups can be used directly for immobilization or hydrolyzed to form diol groups that can be activated with a variety of reagents. Aminopropyl phases are also common. These materials are stable at pH 2-8 and can withstand over 1000 psi. Only the Serva support is available with ligands already immobilized. The only commercially available
HP AC support is the Beckman Ultraffinity-EP column, which contains lO-^m bonded-phase silica of 300 À pore size and is prepacked in 5 X 0.46cm and 10 X 1-cm columns. The support contains epoxide groups for immobilization of ligands. No preimmobilized ligands are currently available. Of course, almost any silica or aqueous size-exclusion chromatographic support can be adapted for use in HPAC. Wide-pore supports such as Hypersil (Shandon), LiChrospher (EM Science), and Separon (Laboratory Instrument Works) have been widely used. High-pressure slurry packing of microparticulate supports is often unnecessary if short columns are used. A simple aspirator-vacuum method has been developed for such columns (6). Other promising new supports include very small nonporous particles (0.7 and 1.5 μηι), which greatly im prove diffusional mass transfer rates and have moderate surface areas (7), cross-linked agaroses of 3-10-/xm par ticle size that can withstand several hundred psi (β), and Superose, a 13μτη size-exclusion support from Phar macia that can withstand 200 psi. Immobilization Affinity ligands are usually attached to supports by covalent bond forma tion. Ideally, a dense, stable coverage of the support with fully active ligand is desired. It must be remembered that many of the ligands are large, fragile molecules whose three-dimen sional structure and orientation are important for their activity. Hence,
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optimum immobilization conditions often must be found empirically. Immobilization is usually a two-step process. In the activation step, the support is treated with a reactive com pound in aqueous or organic solution to produce an activated matrix. Most commonly, the reagent reacts with hydroxyl groups on the support. After removal of excess activating agent, the coupling step between activated ma trix and ligand is performed in aque ous solution. Sometimes the two steps can be combined, but only if the lig and does not react with the activating agent in a bifunctional manner, there by causing polymerization. Some of the factors to consider in choosing immobilization conditions are listed below. Functional groups on the ligand. Most immobilization methods use free amino groups on the ligand for cou pling. Other functional groups, such as carboxylic acid, phenol, and thiol groups, can also be used. Spacer arms. When small solutes are immobilized, a spacer arm is often needed so that the ligand will be able to reach into the binding site of the analyte macromolecule. A common spacer arm is 6-aminohexanoic acid. However, the spacer arm may alter the strength of interaction with analyte because of hydrophobic or ionic ef fects. Shaltiel et al. (9) and Hofstee (10) were the first to notice that spac er arms alone caused adsorption. These effects are used in hydrophobic interaction chromatography and reversed-phase chromatography. In gen eral, neutral, hydrophilic spacer arms minimize such effects. pH. The reactivity of,the functional groups on the ligand and the reactiyity or hydrolysis of active groups on the support may be affected by pH. In ad dition, the ligand or the support can be irreversibly harmed by extremes of pH. Density of active groups. A mono layer of small molecules on the surface of a support has a surface concentra tion of a few fimol/m2. A monolayer of a moderately large protein is approxi mately 0.01 umol/m 2 if the protein covers a 100 À X 100 À area. Thus, activation of all the functional groups on the surface of the support could lead to the immobilization of the ligand via 100 bonds per ligand molecule (however, the ligand may not have that many available functional groups). This process of multipoint attachment would lead to a very stable stationary phase, but with little biochemical activity due to distortion of the ligand, steric hindrance of its binding site, or other undesirable effects. This is illustrated in Figure 1. Intermediate levels of activation lead to optimum coupling in terms of both
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Figure 1. Effects of immobilization of a macromolecule (a) ideal case; (b) altered three-dimensional structure; (c) improper orientation or spacing
specific activity and amount attached. Some immobilization methods exhibit much better control of this variable than others, and some ligands are more sensitive to this variable than others. Many activated supports are stable indefinitely, so many preactivated supports are commercially available. Some of the coupling reactions be tween activated supports and ligands are shown in Table II. Preactivated agarose supports are available from all of the companies listed previously. Supports containing spacer arms with terminal carboxylic acid or amine groups are also available. Bio-Rad em phasizes the active ester method in its products, whereas Pierce primarily uses Ι,Γ-carbonyldiimidazole. With silica-based supports, epoxide, diazonium, active ester, carbonyldiimidazole, and glutaraldehyde methods dominate. The availability of preactivated supports has greatly increased the use
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of affinity chromatography. Cyanogen bromide is an inconvenient and poten tially hazardous activating agent, but the commercial availability of cyano gen bromide-activated agarose has made it the most popular immobiliza tion reagent in spite of well-known problems of ligand leakage and non specific adsorption on charged isourea groups. Whereas medium- and low-perfor mance supports are generally packed into columns by the user after immo bilization, the new HPAC support from Beckman is supplied in a col umn. Coupling takes place while a ligand solution is slowly pumped through the column. This presents a problem in that trial and error adjust ment of pH or other conditions could become quite expensive. Beckman has solved this by supplying an Ultraffinity Introduction Kit that contains ac tivated support in a vial which can be used for initial optimization of cou pling conditions.
Table II.
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Reactions of activated supports
Elution methods Elution methods can be divided into biospecific and nonspecific methods. In biospecific elution, the mobile phase modifier (called the inhibitor) is a free ligand similar or identical to the immobilized affinity ligand or the solute. The inhibitor competes for sites on the ligand or solute and thus decreases the capacity factor (k') of the solute. Biospecific elution is most commonly used when a low-molecularweight inhibitor is available. For example, if immobilized glucosamine were used to purify a lectin, glucose or N-acetyl-D-glucosamine might be used as the inhibitor. In this case, the affinity ligand and inhibitor compete for the binding sites on the analyte. If an immobilized lectin were used to purify a glycoprotein, the inhibitor again might be glucose. In this case, the analyte and inhibitor compete for sites on the affinity ligand. This is sometimes called reversed-role affinity chromatography. Biospecific elution in normal and reversed-role affinity chromatography is illustrated in Figure 2. Nonspecific elution involves denaturation of either the ligand or analyte by means of pH, chaotropic agents (e.g., KSCN, urea), organic solvents, or ionic strength. Typically, these con-
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ditions are empirically determined, although workers such as van Oss have examined more fundamental and general ways to disrupt the hydrogen bonds, hydrophobic interactions, and coulombic attractions that exist in the binding site between ligand and analyte (11). The conditions chosen for nonspecific elution should be mild enough that the support and ligand are not irreversibly damaged. If the analyte is being purified for further use, it must not be irreversibly denatured. One subtle difference between biospecific and nonspecific elution is related to the kinetics of elution. The dissociation rate constant of many ligand-analyte complexes is in the range of 103 to 10 - 5 s _1 , leading to dissociation half-lives of 10 - 3 s to many hours. Clearly the longer half-lives (> 1 s) can lead to excessive band broadening. In the case of biospecific elution, it is generally thought that the competing inhibitor does not alter the dissociation rate constant of the analyte-ligand complex. Therefore, slow dissociation will lead directly to excessive band broadening. The worst case is isocratic elution, in which the analyte must adsorb and desorb many times as it passes down the column. Step elution helps to minimize this
Figure 2. Biospecific elution in (a) normal and (b) reversed-role affinity chromatography The solute is in red and the inhibitor in green. The circles represent macromolecules, and the squares represent small molecules
problem because a high concentration of inhibitor can reduce the number of adsorptions and desorptions to one. However, nonspecific elution may be even better because the denaturation may alter the dissociation rate con stant. For example, it has been esti mated that the dissociation rate con stant of the protein A-immunoglobulin complex is 3 X 10" 4 s _ 1 (half-life = 36 min), yet a pH change to 3 elutes the immunoglobulins within a minute (4, 12). Clearly, elution using a dena turing agent can help to bypass the slow dissociation rate of the native complex. Nonspecific adsorption Perhaps the greatest limitation to the selectivity and detection limits of affinity chromatography is the prob lem of nonspecific adsorption. This term refers to the retention of solutes that do not adsorb in the usual biospe cific manner. Nonspecific adsorption is not unexpected because the affinity ligands and supports frequently con tain ionic and hydrophobic sites that may retain solutes by the same mecha nisms as in ion-exchange and reversed-phase chromatography. Non specific adsorption can be minimized by the careful choice of support, spac er arm, immobilization method, and mobile phase ionic strength. Examina tion of Table II indicates that many immobilization methods introduce anion-exchange sites. The extent of the problem also depends on whether excess reactive groups are treated with a reagent such as ethanolamine that will yield a charged site in the epoxide, tresyl chloride, and some other meth ods, or whether these groups can be removed in a way that leaves un charged sites, e.g., by hydrolysis to hydroxyl groups.
The amount of nonspecific adsorp tion is usually small, but it can have a significant effect if the analyte is present in trace amounts. A conse quence is that affinity chromatogra phy is seldom used as a single-step pu rification method. Precipitation, sizeexclusion chromatography, or ionexchange chromatography is fre quently used either before or after the affinity chromatographic purification. In spite of nonspecific adsorption, the purification factors of 100-fold or more commonly achieved are superior to most other methods. Applications Glycosylated hemoglobin assay. In spite of the great selectivity of af finity chromatography, there have been relatively few clinical applica tions. This is probably because of the greater sensitivity of immunoassay methods and the ability to run many assays in parallel. However, affinity chromatographic methods are faster (minutes vs. hours), and the columns are reusable, so affinity chromatogra phy should be competitive in many in stances. One example of the use of affinity chromatography for clinical diagnosis is the glycosylated hemoglobin assay. The concentration of glycosylated he moglobins (HbAi) in blood depends on the long-term (several weeks) glu cose concentration. Thus, this assay is a more reliable indicator of diabetic control than blood or urine glucose levels. Somewhat surprisingly, the affinity chromatographic assay uses a ligand of rather low selectivity, phenylboronic acid, combined with selective de tection of hemoglobins at 414 nm. The procedure involves hemolysis of the erythrocytes, separation of nonre-
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tained hemoglobins and retained he moglobins (eluted with sorbitol) by af finity chromatography, and absorbance measurement of the two fractions. This method was first developed by Mallia et al. in 1981 (13) and has been widely used since. Many studies have shown that the affinity method is su perior with respect to precision and interferences compared with ion-ex change and other methods. Commercial kits that use agarose supports are available from Pierce Chemical Company, Isolab, and Hele na Laboratories. Purification of allergens. Affinity chromatography has considerable po tential as a therapeutic tool. One in teresting application is in the purifica tion of allergens for immunotherapy of patients with severe allergies. For the therapy, it is desirable to use only the particular substances that the patient is allergic to, i.e., those that bind to the patient's immunoglobulin Ε (IgE). To achieve this, Sondergaard and Weeke (14) used an affinity chroma tography column containing immobi lized antibodies specific for human IgE. After adsorption of the patient's IgE, a mixture of possible allergens was passed through the column. The allergens that adsorbed to the IgE were then eluted, along with the IgE, by means of a pH and ionic strength change. With this approach it was pos sible to obtain microgram quantities of the allergens. Biotechnology. A recent REPORT (15) emphasized the importance of af finity chromatography in the purifica tion of the products of genetic engi neering. The crude products are typi cally very dilute and may represent only a tiny fraction of the soluble ma terial. Excellent purification of such
products is often obtained using im mobilized monoclonal antibodies. For example, human interferon can be produced by E. coli containing a plasmid with the interferon gene. Using a monoclonal antibody column specific for interferon, Vaks et al. (16) were able to obtain 750- to 4000-fold in creases in specific activity of the inter feron in a single step. Preliminary steps consisted of cell disruption, clar ification, concentration, and precipita tion of nucleic acids. Two milligrams of interferon were obtained from sev eral liters of cells with a yield of 65%. A less obvious application is the se lection of bacterial mutants by affinity chromatography. If such mutants have altered cell surface markers, it is pos sible to separate them using appropri ate immobilized ligands. For example, mutant E. coli strains lacking affinity to maltodextrins have been isolated using immobilized starch (17). Two-dimensional separations. Al though general ligands are readily used for the separation of a group of related components, resolution of the individual components may not be good because of slow kinetics or limi tations of column size and selectivity. Therefore, several authors have used affinity chromatography to prefractionate a sample, followed by a high-
resolution separation of the retained components on a conventional HPLC column. One example is the analysis of ribonucleosides in urine or deproteinized serum by means of a boronic acid col umn (3-cm length) in series with a conventional reversed-phase column (18). The sample is applied at pH 7, nonretained components are eluted to waste, and the retained components are eluted at pH 3.5 and concentrated on the reversed-phase column, which is then eluted with a methanol gradi ent. As many as 14 ribonucleosides can be separated in 40 min with rela tively few interferences from other sample components. Fundamental studies. As in all types of chromatography, slow diffu sion is an important cause of band broadening in affinity chromatogra phy. More specific to affinity chroma tography is the sometimes large con tribution by slow adsorption-desorption rates. This is due in part to the slow rate constants characteristic of macromolecular interactions and in part to the low ligand densities used. This has led to speculation that the rate constants for formation or disso ciation of biochemical complexes could be measured chromatographically or, conversely, that chromato
graphic band-broadening theories could be proven using model biochem ical systems. Hethcote and DeLisi have derived equations to predict the statistical moments of solutes eluted isocratically using competing inhibitors (19). These theories depend on the valency of the molecules and which molecule is immobilized. For example, if concanavalin A is immobilized the kinetic band broadening should depend only on the concanavalin Α-solute sugar in teraction. If the ligand is a sugar and concanavalin A is the solute, then the band broadening depends on both the concanavalin A-ligand and concanav alin Α-competing sugar kinetics. The former case was examined by Muller and Carr (20). In contradiction of the ory, they found that the rate constant was a function of k'. Further work is needed to show whether the rate con stants are different for immobilized ligands compared with soluble ligands or if chromatographic theory does not adequately describe the situation. Another method for obtaining rate constant data has been developed by Hage et al. based on the unusual "split-peak" behavior observed at high flow rates or with very short col umns (12). Under these conditions, a pure analyte may elute as two peaks: a
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nonretained peak and a strongly ad sorbed peak. T h e nonretained peak results from the very short residence time of the molecules in the column. Some molecules do not have time to adsorb even once. This method yields the diffusional or adsorption rate con stant of the analyte. It is particularly useful for strongly adsorbed solutes for which competing inhibitors are not available, e.g., antigen-immobilized antibody systems. It is likely that un usual methods such as this will prove to be more useful for studying chro matographic rate processes of macromolecules than conventional isocratic plate height methods.
References (1) Axen, R.; Porath, J.; Ernback, S. Na ture (London) 1967,214, 1302-4. (2) Ohlson, S.; Hansson, L.; Larsson, P.-O.; Mosbach, K. FEBS Lett. 1978, 93, 5-9. (3) Walters, R. R. Anal. Chem. 1983,55, 1S95—99 (4) Crowley, S. C; Walters, R. R. J. Chromatogr. 1983,266, 157-62. (5) Ohlson, S. In "Affinity Chromatogra phy and Biological Recognition"; Chaiken, I. M; Wilchek, M.; Parikh, L, Eds.; Academic Press: Orlando, Fla., 1983; pp. 255-56. (6) Moore, R. M.; Walters, R. R. J. Chromatogr. 1984,317, 119-28. (7) Anspach, B.; Unger, K.; Giesche, H.; Hearn, M.T.W. 4th International Sym posium on HPLC of Proteins, Peptides, and Polynucleotides, Baltimore, Md.,
1984; paper No. 103. (8) Hjerten, S.; Yang, D. J. Chromatogr. 1984,376,301-9. (9) Er-el, Z.; Zaidenzaig, Y.; Shaltiel, S. Biochem. Biophys. Res. Commun. 1972, 49, 383-90. (10) Hofstee, B.H.J. Anal. Biochem. 1973, 52, 430-48. (11) Van Oss, C. J.; Absolom, D. R.; Neu mann, A. W. Colloids Surf. 1980,1, 45-56. (12) Hage, D. S.; Walters, R. R.; Hethcote, H.W., submitted for publication in Anal. Chem. (13) Mallia, A. K.; Hermanson, G. T.; Krohn, R. I.; Fujimoto, Ε. Κ.; Smith, P. K. Anal. Lett. 1981,14, 649-61. (14) Sondergaard, I.; Weeke, B. Allergy 1984,39, 473-79. (15) Warren, D. C. Anal. Chem. 1984,56, 1528-44 A. (16) Vaks, B.; Mory, Y.; Pederson, J. U.; Horovitz, O. Biotechnol. Lett. 1984, 6, 621-26. (17) Ferenci, T. Trends Biochem. Sci. 1984, 9, 44-48. (18) Hagemeier, E.; Kemper, K.; Boos, K.-S.; Schlimme, E. J. Chromatogr. 1983,282, 663-69. (19) Hethcote, H. W.; DeLisi, C. J. Chro matogr. 1982,248, 183-202. (20) Muller, A. J.; Carr, P. W. J. Chroma togr. 1984,284,33-51.
Suggested readings (1) Turkova, J. "Affinity Chromatogra phy"; Elsevier: Amsterdam, 1978. (2) Scouten, W. H., Ed. "Solid Phase Bio chemistry"; Wiley: New York, N.Y., 1983. (3) Dean, P.D.G.; Johnson, W. S.; Middle, F. Α., Eds. "Affinity Chromatography"; IRL Press: Oxford, U.K., 1985.
(4) Schott, H. "Affinity Chromatography" Marcel Dekker: New York, N.Y., 1984. (5) Wilchek, M.; Miron, T.; Kohn, J. Methods Enzymol. 1984,104, 3-55. (6) Larsson, P.-O. Methods Enzymol. 1984,104, 212-23.
Rodney Walters received a B.S. in chemistry and biology from Iowa State University in 1975 and a Ph.D. in analytical chemistry from the Uni versity of North Carolina at Chapel Hill in 1980. Since then he has been an assistant professor of analytical chemistry at Iowa State University. His research interests include highperformance affinity chromatogra phy, fundamental chromatographic studies, computer modeling, and dif fusion coefficient measurements.
INDICATING DESICCANT changes from blue when dry to rose-red when exhausted I n d i c a t i n g D R I E R I T E is a c h e m i c a l d r y i n g a g e n t for t h e e f f i c i e n t a n d r a p i d d r y i n g o f a i r a n d g a s e s in s y s t e m s w h e r e v i s u a l i n d i c a t i o n o f a c t i v e d e s i c c a n t is d e s i r e d . In u s e , t h e c o l o r c h a n g e s s h a r p l y t o a r o s e - r e d a s the margin between the exhausted and active desiccant p r o g r e s s e s t h r o u g h t h e t u b e or c o l u m n . It is a v a i l a b l e in 8 M e s h , t h e s t a n d a r d g r a n u l e s i z e , a n d in 2 0 - 4 0 , 1 0 - 2 0 , 6 a n d 4 M e s h . Impregnated with cobalt chloride, Indicating D R I E R I T E retains the high efficiency of Regular D R I E R I T E , anhydrous calcium sulfate, plus the added advantage of t h e c o l o r c h a n g e for i n d i c a t i o n . It d r i e s a i r a n d g a s e s t o a terminal dryness of 0 . 0 0 5 m i l l i g r a m per liter of g a s .
Size:2%"x 1 1 % " Laboratory Gas and Air Drying Unit
Regeneration reverses the color change and makes possible repeated use after h e a t i n g at 3 7 5 - 4 5 0 ° F while spread o n e g r a n u l e d e e p for o n e h o u r . R e g u l a r w h i t e D R I E R I T E ( n o n - i n d i c a t i n g ) is a v a i l a b l e in t h e a b o v e m e n t i o n e d s i z e s a n d a l s o in 2 0 0 M e s h . D R I E R I T E is a p r o d u c t o f W . A . H a m m o n d Drierite Co., Xenia, Ohio 4 5 3 8 5 . S 0 L I O S LIQUIDS CASES SOLIDS L I 0 U I D S GASES SOLIDS
DRIERITE 9*>s j 3sv9-sai*no)>s«ioi-i 3jvo somon s«no CIRCLE 95 ON READER SERVICE CARD 1114 A · ANALYTICAL CHEMISTRY, VOL. 57, NO. 11, SEPTEMBER 1985
Available from your nearest LABORATORY SUPPLY HOUSE.
