Rodney R. Walters Department of Chemistry Iowa Stale 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 hy 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 he used for the assay of many dif-
0003-2700/85/A357-1099$0 1.50/0 @ 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 he as little as one minute. Clearly, the latter approach is particularly useful for repetitive types of separations such as industrial and lahoratorv. Durification and clinical . analysis. Although affinity chromatography was used as far back as 1910, modern affinity chromatomaDhs. heaan - with the development the cyanogen hromide method for the immobilization of ligands on agarose supports by Axen et al. in 1967 ( I ) . The use of rigid, microparticulate supports led to the development of high-performance affinity chromatography (HPAC) by
of
Ohlson et al. in 1978 (2).Other related methods that will not he discussed here include affinity partitioning, affinity filtration, and affinity targeting of drugs. AHhlty 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 HPAC separations in less than a minute (3). General ligands (or group-specific ligands), such as nucleotide analogues and lectins, bind to certain groups of
ANALYTICAL CHEMISTRY, VOL. 57. NO. 11. SEPTEMBER 1985
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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 Chroi..,., The basis of the selectivity of affinity :hromatography is the Jse ot immobilized iiochemicals as the stationary phase. These iiochemicals, which are :aPed affinity ligands, a n be antibodies, anzyme inhibitors, ectins, mother mole:ules that reversibly and bimelectively bind ,o the complementary analyte molecules in he sample. The sepa'atiins exploit the "lock nnd key" binding that is irevalent in biolooical " ;ystems. A typical affinity chre natographic separation ifamixtureotenzvmes s illustrated in the' accompanying figure. A %ample containing several enzymes (large Jircles) is applied to a mlumn containing an inhibitor (small circles) that has been wvaently bonded to an nert suppwt material. 4s the aample is Nashed through the
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Regenerate
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11OOA* ANALYTICAL CHEMISTRY, VOL. 57, NO. 11, SEPTEMBER 1985
lustrated by the separations of immuuoglohulins 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 (IgG1 and IgG,), 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 hecause 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 chowing 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. suppals 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-g&CtWe and 3,6-anhydro-~-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% agarwe and have pore sizes of approximately 300 and 150 A, respectively. A typical particle size is 50-150 pm. The recommended operating conditions for Sepharose 4B include a pH range of P 9 and a maximum pressure of 1psi. The agaroses can be cross-linked with 2,3-dibromopropanol to provide better chemical and mechanical
Table 1.
General ligands and their speclRdtles uond
1
Cibacron Blue F3GA dve. derivatives Of AMP, NADH, and NADPH
sp.*IlCl*
Certain dehvdroaenases via bindina- at . ihe nucleotide binding site .. .
streneth. For examnle. Senharose CL6B cin tolerate pH's12 i n d 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 LKBs 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 am or larger in diameter. Pierce sells Fraetogel 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. Trisacry1 can tolerate pH 1-13 and 40 psi. Bonded-phase silicas are available from Serva (pore size 500 A) and J.T. Baker (pore size 60-90 A). Bondedphase controlled porosity glass is available from Pierce (pore sizes 500 and 200 A). A typical bonded phase is 3-(glycidoxrpropyl)trimethoxysilane. The epoxide groups can be used directly for immobilization or hydrolyzed to form diol groups that can he activated with a variety of reagents. Aminopropyl phases are also common. These materials are stable at pH 2-8 and can withstand over lo00 psi. Only the Serva support is available with ligands already immobilized. The only commercially available 1102A
.. .. ...
..-. ......,
HPAC support is the Beckman Ultraffinity-EP column, which contains 10-pm bonded-phase silica of 300 A 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 (0.7and 1.5 am), which greatly improve diffusional mass transfer rates and have moderate surface areas cross-linked agaroses of 3-10-pm particle size that can withstand several hundred psi (8),and Superose, a 13pm size-exclusion support from Pharmacia that can withstand 200 psi.
(n,
lmmObilization Affinity ligands are usually attached to supports by covalent bond formation. Ideally, a dense, stable coverage of the support with fully active ligand is desired. It must he remembered that many of the ligands are large, fragile molecules whose three-dimensional structure and orientation are important for their activity. Hence,
ANALYTICAL CHEMISTRY, VOL. 57, NO. 11, SEPTEMBER 1985
optimum immobilization conditions often must he found empirically. Immobilization is usually a two-step process. In the activation step, the support is treated with a reactive compound 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 matrix and ligand is performed in aqueous solution. Sometimes the two steps can be combined, but only if the ligand does not react with the activating agent in a bifunctional manner, thereby causing polymerization. Some of the factors to consider in choosing immobilization conditions are listed below. Functional groups on t h e ligand. Most immobilization methods use free amino groups on the ligand for coupling. Other functional groups, such as carboxylic acid, phenol, and thiol groups, can also he 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 effects. Shaltiel et al. (9) and Hofstee (10) were the first to notice that spacer arms alone caused adsorption. These effects are used in hydrophobic interaction chromatography and reversed-phase chromatography. In general, neutral, hydrophilic spacer arms minimize such effects. pH. The reactivity of.the functional groups on the ligand and the reactivity or hydrolysis of active groups on the support may he affected by pH. In addition, the ligand or the support can be irreversibly harmed by extremes of PH. Density of active groups. A monolayer of small molecules on the surface of a support has a surface concentration of a few pmol/m2. A monolayer of a moderately large protein is approximately 0.01 amol/m2 if the protein covers a 100 A x 100 A 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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gure 1. Effects of immobilization of a macromolecule
, idaal case: (b) altered thee-dlmenalanal structure: (c)lwropw MientationOT SFmclng iecific activity and amount attached. m e immobilization methods exhibit uch better control of this variable ian others, and some ligands are ore sensitive to this variable than .hers. Many activated supports are stable #definitely,so many preactivated ipports are commercially available. m e of the coupling reactions heween activated supports and ligands .e shown in Table 11. Preactivated :arose supports are available from all 'the companies listed previously. upports containing spacer arms with !minal carboxylic acid or amine .oups are also available. Bio-Rad emhasizes the active ester method in its roducts, whereas Pierce primarily 38s 1,l'-carbonyldiimidaole.With lica-based supports, epoxide, diazoium, active ester, carbonyldiimidade, and glutaraldehyde methods ominate. The availability of preactivated ipports has greatly increased the use
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of affinity Chromatography. Cyanogen bromide is an inconvenient and potentially hazardous activating agent, but the commercial availability of cyanogen bromide-activated agarose has made it the most popular immobilization reagent in spite of well-known problems of ligand leakage and nonspecific adsorption on charged isourea groups. Whereas medium- and low-performance supports are generally packed into columns by the user after immobilization, the new HPAC support from Beckman is supplied in a column. Coupling takes place while a ligand solution is slowly pumped through the column. This presents a problem in that trial and error adjustment of pH or other conditions could become quite expensive. Beckman has solved this by supplying an Ultraffinity Introduction Kit that contains activated support in a vial which can be used for initial optimization of coupling conditions.
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Table II. Reactkns of activated Buppats Cyanogen bromide Su--O--CtN
,
+ RNH,
-
NH S u d - N H R
Xlde
p\+H* + R", (ROH. RSH)
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:Mknmelh0ds Elution methods can be divided into iospecific and nonspecific methods. I biospecific elution, the mobile hase modifier (called the inhibitor) is free ligand similar or identical to the nmobiliied affinity ligand or the solte.The inhibitor competes for sitea n the ligand or solute and thus dereases the capacity factor (k') of the )lute. Biospecific elution is most mmonly used when a lowmolecular,eight inhibitor is available. For exmple, if immobilized glucosamine 'ere used to purify a lectin, glucose or I-acetyl-D-glucosamine might be sed as the inhibitor. In this case, the Ffinity ligand and inhibitor compete ir the binding sites on the analyte. If n immobilized lectin were used to puify a glycoprotein, the inhibitor again light be glucose. In this case, the anarte and inhibitor compete for sites on he affinity ligand. This is sometimes alled reuersed-role afflmity chroma3graphy. Biospecific elution in norla1 and reversed-role affinity chroma3graphy is illustrated in Figure 2. Nonspecific elution involves denauration of either the ligand or analyte ~ymeans of pH, chaotropic agents e.g., KSCN, urea), organic solvents, i ionic strength. Typically, these con-
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-l, leading to diss to many sociation half-lives of hours. Clearly the longer half-lives (> 1 s) can lead to excessive band broadening. In the ca%eof 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 ismatic elution, in which the analyte must adsorb and desorb many times as it passes down the column. Step elution helps to minimize this
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Flgure 2. BioSpecificelution In (a)n m l and (b) reversed-role affinity chromatography The 801ut0 is in red and the lnhlbnw In -n.
The circles repreMmt macmmolecdes.and l h squares ~ represerd 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 constant. For example, it has been estimated that the dissociation rate constant 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 denaturing agent can help to bypass the slow dissociation rate of the native complex. Narsped(kadsaptkn Perhaps the greatest limitation to the selectivity and detection limits of affinity chromatography is the problem of nonspecific adsorption. This term refers to the retention of solutes that do not adsorb in the usual biospecific manner. Nonspecific adsorption is not unexpected because the affinity ligands and supports frequently contain ionic and hydrophobic sites that may retain solutes by the same mechanisms as in ion-exchange and reversed-phase chromatography. Nonspecific adsorption can be minimized by the careful choice of support, spacer arm, immobilization method, and mobile phase ionic strength. Examination of Table I1 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 methods,or whether these groups can be removed in a way that leaves uncharged sites, e.g., by hydrolysis to hydroxyl groups. IIIOA
The amount of nonsnecific adsomtion is usually small, b;t it can have a significant effect if the analyte is present in trace amounts. A consequence is that affinity chromatography is seldom used as a single-step purification method. Precipitation, sizeexclusion chromatography, or ionexchange chromatography is frequently 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.
&P-I Glycosylated hemoglobin away. In spite of the great selectivity of affinity chromatography, there have been relatively few clinical applications. 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 chromatopraphy should he competitive in many instances. One example of the use of affinity chromatography for clinical diagnosis is the glycosylated hemoglobin assay. The concentration of glycosylated hemoglobins (HbA1) in blood depends on the long-term (several weeks) glucose 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 detection of hemoglobins at 414 nm. The procedure involves hemolysis of the erythrocytes, separation of nonre-
ANALYTICAL CHEMISTRY, VOL. 57. NO. 11. SEPTEMBER 1985
tained hemoelohins and retained hemoglohins ( e h e d with sorbitol) by affinity 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 superior with respect to precision and interferences compared with ion-exchange and other methods. Commercial kits that use agarose supports are available from Pierce Chemical Company, Isolah, and Helena Laboratories. Purification of allergens. Affinity chromatography has considerable potential as a therapeutic tool. One interesting application is in the purification 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 hind to the patient’s immunoglobulin E (IgE). To achieve this, Sondergaard and Weeke (14) used an affinity chromatography column containing immobilized 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 possible to obtain microgram quantities of the allergens. Biotechnology. A recent REPORT (15) emphasized the importance of affinity chromatography in the purification of the products of genetic engineering. The crude products are typically very dilute and may represent only a tiny fraction of the soluble material. Excellent purification of such
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products is often obtained using immobilized 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 increases in specific activity of the interferon in a single step. Preliminary steps consisted of cell disruption, clarification, concentration, and precipitation of nucleic acids. Two milligrams of interferon were obtained from several liters of cells with a yield of 65%. A less obvious application is the selection of bacterial mutants by affinity chromatography. If such mutants have altered cell surface markers, it is possible to separate them using appropriate immobilized ligands. For example, mutant E. coli strains lacking affinity to maltodextrins have been isolated using immobilized starch (17). Two-dimensional separations. Although 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 limitations 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 column (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 gradient. As many as 14 ribonucleosides can be separated in 40 min with relatively few interferences from other sample components. Fundamental studies. As in all types of chromatography, slow diffusion is an important cause of band broadening in affinity chromatography. More specific to affinity chromatography is the sometimes large contribution 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 dissociation of biochemical complexes could be measured chromatographically or, conversely, that chromato-
graphic band-broadening theories could be proven using model biochemical 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 A-solute sugar interaction. If the ligand is a sugar and concanavalin A is the solute, then the band broadening depends on both the concanavalin A-ligand and concanavalin A-competing sugar kinetics. The former case was examined by Muller and Carr (20).In contradiction of theory, they found that the rate constant was a function of Fz’. Further work is needed to show whether the rate constants 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 columns (12).Under these conditions, a pure analyte may elute as two peaks: a
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nonretained peak and a strongly adsorbed peak. The 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 constant of the analyte. It is particularly useful for strongly adsorbed solutes for which competing inhibitors are not available, e.g., antigen-immobilized antibody systems. I t is likely that unusual methods such as this will prove to be more useful for studying chromatographic rate processes of macromolecules than conventional isocratic plate height methods.
Reterences (1) Aaen. R.; Porath. J.; Ernback. S. Na-
ture (London) 1967.214,13024. (2)Ohlson, S.;Hansson, L.; Larsson, P.-0.; Mosbaeh. K. FEES Lett. 1978.93.5-9, (3) Walters, R. R. Anal. Chem. 1983.55, 1395-99. (4) Crowley. S. C.; Walters. R. R. J . Chromntogr. i983.266.15762. (5) Ohlaon. S . In “Affinity Chromato ra phy and Biological Recognition”; &ai: ken. I. M.: Wilehek. M.: Parikh. I.. Eds.: Academic’Press: Orlando, Fla., ‘1983; pp. 255-56. (6) Moore, R. M.; Walters. R. R. J . Chromotogr. 1984,317,119-28. (7) Anspach, B.; Un er, K.; Giesche, H.; Hearn. M.T.W. 4 t f International Symposium on HPLC of Proteins, Peptides, and Polynucleotides. Baltimore, Md.,
1984; paper No. 103. (8)Hjerten. S.; Yang. D. J. Chromatogr. 1984,316,301-9. (9) Er-el. 2.; Zaidenzaig, Y.; Shaltiel, S. Biockem. Biopkys. Res. Commun. 1972. 49,383-90. (10) Hofstee. B.H.J. Anal. Bioehem. 1973. 52,430-48. (11) Van Oss, C. J.; Absolom, D. R.; Neumann. A. W. Colloids Surf. 1980, I, 45-56. (12) Hage, D.S.; Walters. R. R.;Hethcote. H.W.. submitted for publication in Anal. Ckem. (13) Mallia, A. K.; Hermanson,G. T.; Krohn, R. 1.; Fujimoto, E. K.; 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) Vsks. B.; Mory, Y.; Pederson, J. U.; Horovitz. 0.Biotechnol. Lett. 1984.6, 621-26. (17) Ferenci, T.Trends Bioehem. Sci. 1984,9,44-48. (18) Hagemeier, E.;Kemper, K.; Bws, K.-S.; Schlimme, E. J . Chromatogr. 1983,282,66349, (19) Hethcote, H. W.; DeLisi. C. J . Chromatogr. 1982,248,183-202. (20) Muller, A. J.;Carr, P. W. J . Chromotogr. 1984.284,33-51.
syleestdreaangs (1) Turkova. J. “Affinitv Chromatoeraphy”; Elsevier: Amsteidam, 1978.” (2) Scouten, W. H., Ed. “Solid Phase Bia-
chemistry”; Wiley: New York, N.Y., 10Q1 IO”“.
(3) Dean, P.D.G.; Johnson, W.S.; Middle, F. A., Eds. “AffinityChromatography”; IRL Press: Oxford, U.K., 1985.
INDICATING DESICCANT changes from blue when dry to rose-red when exhausted Indicating DRIERITE is a chemical d r y i n g agent for the efficient a n d rapid drying of air and gases in systems where visual indication of active desiccant is desired. In use, the color changes sharply to a rose-red as the margin between t h e exhausted and active desiccant progresses through t h e t u b e or column. It is available in 8 Mesh, t h e s t a n d a r d granule size, and in 20-40. 10-20.6 and 4 M e s h . Impregnated with cobalt chloride, Indicating DRIERITE retains t h e high efficiency of R e g u l a r DRIERITE, anhydrous calcium sulfate, plus t h e added a d v a n t a g e of t h e color c h a n g e for indication. I t dries air and gases to a terminal dryness o f 0.005 milligram per liter of gas. Regeneration reverses t h e color change and makes possible repeated use after heating at 375-450°F while spread o n e granule deep for one hour. Regular white DRIERITE (non-indicating) is available in t h e above mentioned sizes and also in 200 Mesh.
DRIERITE is a product of W.A. H a m m o n d Drierite Co., Xenia, Ohio 45385.
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(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.-0. Methods Enzymol. 1984,104,212-23.
Rodney Walters received a B.S.in chemistry a n d biology from Iowa State University in 1975 a n d a Ph.D. in analytical chemistry from the University of North Carolina at Chapel Hill in 1980. Since then he has been a n assistant professor of analytical chemistry a t Iowa State Uniuersity. His research interests include highperformance affinity chromatography, fundamental chromatographic studies, computer modeling, and diffusion coefficient measurements.
