variables in the high-performance anion-exchange chromatography of proteins

ANAL.YTICAL BIOCHEMISTRY 109. 345-353 ( 1980)

Variables in the High-Performance Anion-Exchange Chromatography of Proteins’

GEORGE VANECEK AND FRED E. REGNIER

The effect of mobile phase velocity, separation time, support pore diameter, column length, and temperature on resolution and loading capacity of a new commercially avail- able high-performance anion-exchange support. SynChropak AX-300. has been examined. This material is a macroporous spherical silica of IO grn particle size with a bonded poly- meric amine layer. It was found that the heterogeneity of ovalbumin samples. combined with bovine serum albumin, make them useful probes in evaluation of anion-exchange supports. In the columns of 4.1 mm i.d., the highest resolutions of proteins were achieved at a flow rate of 0.25 mlimin. Up to IO mg of protein per injection could be applied on a 4. I x 250 mm AX-300 column with good resolution. Columns of 50 mm length had one-tenth the protein load capacity ofa 250.mm column. retaining approximately 75% ofthe resolution.

Anion-exchange chromatography has proven to be a valuable and widely used technique for the fractionation of proteins. Chromatographic materials used in these separations are usually some type of car- bohydrate gel matrix with a covalently bonded diethylaminoethyl (DEAE) ion- exchange group. Although these materials have been very useful in protein purifica- tion, they have inherent problems such as poor mechanical stability under pressure and the tendency to change volume during the course of gradient elution and recycling.

In 1976. Chang published a series of papers (l-3) in which he reported the preparation of weak anion-exchange supports for protein fractionation that were porous inorganic materials with weak organic ion-exchange groups bonded to their surface. These supports were able to withstand several thousand pounds of pressure and allowed liquid to be forced through columns in minutes. Through the use of a column packed with controlled porosity silica hav-

I This is Journal Paper No. 8148 from the Purdue University Agricultural Experiment Station.

ing a DEAE-bonded phase, it was possible to fractionate proteins in 30 min or less in a high-performance liquid chromatograph and still achieve high recovery of biological activity. These supports have also been used by Toren and his associates in the fractionation of lactate dehydrogenase and creatine kinase (4-7).

More recently, Schlabach (8,9) and Alpert t 10) have developed alternate procedures for the preparation of high-performance anion-exchange support materials. In the first of these papers (8), diethylamine was bonded to the surface of the inorganic support through an epoxy monomer, while in the other (IO), an adsorbed and cross- linked polyethylenimine coating was used. On the basis of elution profiles obtained with lactate dehydrogenase and creatine kinase isoenzymes, it may be concluded that all three of these different bonded phases are similar in terms of their chromatographic selectivity with proteins. Basically the same buffers used in the elution of DEAE-cellulose and DEAE- Sephadex columns were used with the high-performance supports and gave the

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346 VANECEK AND REGNIER

same elution profiles. Recovery of lactate dehydrogenase-5 from the inorganic DEAE supports has been reported to be greater than 95% (9) while recovery of lactate dehydrogenase-1, lactate dehydrogenase-5. trypsin, and DAHP synthetase from the inorganic polyamine support was 100,95, 100, and 89%, respectively (10). The polyamine supports were used exclusively in the work presented in this paper.

On the basis of the resolution, speed, convenience, and enzyme recovery, these high-performance ion-exchange systems should be of value to many biochemical researchers. However. broad usage depends on a knowledge of their chromatographic characteristics. It is the purpose of this paper to examine the effect of mobile phase velocity, separation time. support pore diameter, column length. and temperature on resolution and loading capacity in high-performance anion-exchange chromatography of proteins.

EXPERIMENTAL Apparatus. All chromatographic runs

were performed on anion-exchange columns obtained from SynChrom Inc., Lin- den, Indiana. Column dimensions were 4. I mm i.d. and a length of either 5, 10, 15. 20, or 25 cm. The pumping system consisted of a Constametric solvent delivery system (Laboratory Data Control, Riviera Beach. Fla.) with Constametric I, II, and IIG pumps, a Gradient master, and a mixing device. The detector was either an Altex Analytical uv detector Model 153 (Altex Scientific, Berkeley. Calif.) or a Model HM uv-Vis holochrome spectrophotometer (Gilson Medical Electronics, Inc., Middle- ton, Wise.). The recorder was a Fischer Recordall Series 5000. Sample injection was achieved with a Model 7125 syringe loading sample injector (Rheodyne, Berkeley. Calif.) using 20-, lOO-. and 400-~1 sample loops, respectively.

Reagents. Tris(hydroxymethyl)amino- methane (Trizma Base. reagent grade),

glycyl-L-tyrosine (GY)” and sodium salts of adenosine-5’-mono, -di-. and -triphosphoric acid (AMP, ADP, ATP) were supplied by Sigma Chemical Company (St. Louis, MO.) and anhydrous sodium acetate AR by Mallinckrodt, Inc. (St. Louis, MO.).

For the study of protein resolution, ovalbumin (OV) samples from the follow- ing companies were used: (a) Boehringer- Mannheim GmbH (BMC) (W. Germany), protein calibration kit Combithek: (b) Phar- macia Fine Chemicals (Piscataway. N. Y.). calibration kit No. 1 IA; (c) Schwarzl Mann (Orangeburg, N. Y.), twice crystallized, Catalog No. 907338, Lot No. Y3872; (d) ICN Pharmaceuticals, Inc. (Cleveland, Ohio), twice crystallized, Lot Nos. 1319 and 4561; (e) Mann Research Laboratories, subsidiary of B-D Labo- ratories (New York, N. Y.), twice crystallized, U-2816. Crystallized and lyophilized bovine serum albumin (BSA), Cat. No. A- 4378. Lot 118C-8096 and bovine hemoglobin (Hb) as a crude powder Type II, Lot 78C-8045 were products of Sigma Chemical Company. Solutions of ovalbumin-ICN were filtered through a Millex disposable filter unit, 0.22 pm (Millipore Corp., Bedford, Mass.).

The linear salt gradient at pH 8.0 was formed from the initial starting buffer of 0.02 M Tris buffer (solution A) to a final buffer (solution B) consisting of solution A containing 0.5 N sodium acetate. Both solutions were adjusted to pH 8 with glacial acetic acid (10). The theoretical plate number N I= 16(r,lAf,)‘] and the height equivalent to a theoretical plate I-I l=L/Nl were determined for columns at several flow rates using glycyltyrosine (GT). GT was eluted from columns with buffer B. Resolu- tion was calculated by means of the equation

? Abbreviations used: BSA. bovine serum albumin; GY, glycyl-r-tyrosine; Hb. bovine hemoglobin; OV. ovalbumin; GT, glycyltyrosine: DAHP, 3-deoxy-I>- arabino-heptulosonate 7.phosphate: BMC, product of Boehringer-Mannheim GmbH: ICN, product of ICN Pharmaceuticals. Inc.

PROTEIN HIGH-PERFORMANCE ANION-EXCHANGE CHROMATOGRAPHY 347

& = wr2 - [,I)

At,, + At,? ’

where t,.l and tr2 are the retention times of the first and second components, respectively, and ht is the corresponding peak width in minutes. Relative retention time of any two components was expressed by the ratio of their retention times. trzlt,, .

Picric acid ion-pairing capacities of supports were determined according to the procedure of Alpert (IO). The anion-exchange capacities of supports for hemoglobin were determined according to the procedure of Chang (1) with the modification that 100 mg of support were used in- stead of a l-ml volume. The ion-exchange capacities for BSA and OV-BMC were determined in the same manner except that the concentration of the released protein was measured by absorbance at 280 nm and compared to a calibration curve.

Flow rates of 0.25, 0.5, 1.0. and 2.0 ml/ min and the linear gradient times of 10. 20. 40, and 80 min were used to examine the resolution of OV-BMC, the mixture of OV- ICN. and BSA, and the mixture of AMP. ADP, and ATP. The OV-BMC sample was used for the pilot loading study and later replaced by OV-ICN to which BSA was added.

During the chromatographic runs at 4 and 25”C, the chromatographic columns. the bottom half of the sample injector. and the buffer containers were inserted into a thermostatically controlled water bath.

RESULTS

Chromatographic evaluation of any system requires specific methods and probe molecules. After the analysis of a number of samples, it was found that a few stable inexpensive proteins functioned well as probes of both column resolution and loading capacity. Gradient elution chromatograms of a series of commercial oval-

FIG. 1. Comparison of the chromatographic profiles of ovalbumin samples of different commercial sources. Column. SynChropak AX-300, 250 x 4.1 mm i.d.: protein samples, 100 ~1 of 0.5% solution in the buffer A; 20-min linear gradients between buffers A and B: flow rate, 2.0 mlimin.

bumins on an AX-300 column are seen in Fig. 1. These chromatograms establish both that the commercial ovalbumins are not chromatographically homogeneous and that ion-exchange analysis of a sample may be carried out in 15 min or less. The complexity of the OV-BMC sample with its distribution of components throughout the elution gradient made it a sensitive indicator of both resolution and column loading capacity. The resolution of the BMC material was reproducible between lots, Addition of BSA to the OV-ICN sample produces a convenient mixture of still greater complexity. These protein samples were used in all further resolution and loading studies.

Chromatograms of the ovalbumin-BMC sample at different mobile phase velocities are shown in Figs. 2 (a, b, and c). Through adjustments in the gradient slope as a func- tion of time. total elution volumes of


b

TIME Im~n,

TIME Imml

approximately 40 ml were maintained in all cases. It is seen that decreasing the flow rate from 2 mlimin (Fig. 2a) to 0.5 mlimin (Fig. 2b) substantially increases the resolution. The resolution equation outlined above indicates that this fourfold decrease in mobile phase velocity results in a 15X% increase in resolution. A further decrease in flow rate from 0.5 mlimin to 0.2 ml/min (Fig. 2~) produced only a S% increase in resolution. The IO-fold variation in mobile phase velocity between Figs. 2a and c resulted in a 160% increase in resolution.

Thus, it is seen that there is an inverse relationship between resolution and separation time. Due to the inaccuracy of commercial liquid chromatographic pumping systems below 0.2 ml/min, it was not possible to extend this study to still lower flow rates. It is assumed that there is a point at which further decrease in mobile phase velocity will no longer produce sufficient increases in resolution to be worthwhile.

The influence of column length on resolution of proteins was investigated. To establish that all of the columns used in the study were of comparable efficiency, plate heights (HI and plate numbers (N) were determined using the small peptide, glycyltyrosine. as shown in Table 1. The N and H values of columns were averaged from three to six chromatographic runs. When columns varying in length from 5 to 20 cm were eluted in a 40-min linear gradient at flow rates of 0.5 and 2.0 ml/min. the BMC ovalbumin sample gave the results shown in Fig. 3. At both flow rates each

FIG. 2. Chromatographic profiles ofovalbumin-BMC

at different flow rates and times of the linear gradient

between buffers A and B. Column. SynChropak

AX-300, 250 x 4.1 mm i.d.: samples. IO0 ~1 of 0.5%’

solution in the buffer A. (a) 20-min gradient; flow rate.

2.0 mlimin. (b) 75.min gradient: flow rate, 0.5 mlimin.

(c) 200.min gradient: flow rate, 0.2 mlimin.


TABLE I

EFFICIENCIES OF SYNCHROPAK COLUMNS USED IN THESE STUDIES

Columns (cm)

Flow rate (mUmin)

0.25 0.5 I .o 2.0

N H N H N H N H

AX-300 (5) 386 0.013 274 0.018 214 0.024 148 0.034 AX-300 (IO) 482 0.021 355 0.028 261 0.038 158 0.063 AX-300 (15) 898 0.017 799 0.019 596 0.03 318 0.047 AX-300 (20) I.571 0.013 1219 0.016 801 0.025 499 0.040 AX-300 (25) 1949 0.013 1555 0.016 1082 0.023 691 0.036 AX-100 (5) 262 0.020 210 0.024 156 0.032 103 0.048 AX-500 (5) 215 0.023 149 0.034 96 0.052 62 0.080

5-cm incremental increase in length produced approximately 11% increase in resolution.

Resolution of the ovalbumin-BMC sample on a 4.1 x 50 mm AX-300 column operated at 0.25 ml/min with a IO-min gradient is illustrated in Fig. 4. Although resolution is lower than that shown in Fig. 2 above, it is surprisingly good for such a short column and short gradient time. Resolution (R,) of the two major peaks in Fig. 4 was 2.03. Resolution could be increased to 2.32 with this system by simply extending the gradient time to 40

COLUMN LENGTH lcm)

FIG. 3. Resolution of ovalbumin-BMC on Syn- Chropak AX-300 columns of varying length. (0) Flow rate 0.5 mlimin, (0) flow rate 2.0 mlimin.

min while the other variables were held constant. As noted above, when the flow rates were increased to 0.5 and 2.0 ml/min with a lo-min gradient, R, decreased to 1.87 and 1.53, respectively.

Although a mobile phase velocity of 0.25 ml/min produced higher resolution, re- producibility became erratic. This problem may be attributed to the very small volume of liquid used to develop these short columns and the inherent problems of the pumping system in producing gradients of 5 to lo-ml total elution volume.

It was also noted that detection sensitivity could be enhanced with short columns when they are operated at low Ilow rates

FIG. 4. Chromatographic profile of ovalbumin- BMC on a SynChropak AX-300 column, 50 x 4. I mm i.d. Sample. IS ~1 1% solution in buffer A: lo-min linear gradient of buffer A to 50% buffer B; flow rate. 0.25 ml/mint chart speed. 0.S in./min.


TIME imn,

FIG. 5. Loading capacity effect on the resolution of ovalbumin (Mann Res. Lab.). Column, SynChropak AX-300, 250 x 4.1 mm i.d.: SO-min linear gradient of buffer A vs buffer B: flow rate. 0.5 mlimin: chart speed. 0.1 in./min. Arrow identifies the point of peak fusion.

(0.25 ml/mm) with short gradient times. The entire sample could be eluted from the column in less than 20 min with a total elution volume of 5 ml or less. This results in a higher protein concentration per unit volume of eluant. Sensitivity could be increased as much as sixfold by this technique.

Some of the attractive features of these short columns are that they are easier to slurry pack, may be operated at 100 psi or less, they are less expensive and have a longer life, sample detection limits may be lower, and resolution is only slightly less than that achieved on 25cm columns. There are, however. disadvantages. The loading capacity of 5-cm columns is at least IO times less than that seen with the 25cm column as will be shown later. Serious overload- ing problems were seen when more than I-mg samples of protein were applied to a S-cm column. A second potential problem with 5-cm columns is the very small elution volume of the column relative to the “dead- volumes” of the pumping system and de-

tector cell in the instrument used to operate the column. The volume within the tubing connecting the pumps, mixing chamber, and valve may be 3-5 ml on some instru- ments. Since the total elution volume of a 5-cm column might be 5 ml, some instru- ments are not able to provide the selected gradient shape within the desired time. Deviations in elution profiles from those obtained on 25-cm columns are often due to differences in gradient shape and slope.

Protein loading capacity of chromatographic supports was established by a dynamic and a static method. The results of the dynamic test of loading are illustrated in Fig. 5, showing the elution pat- terns of ovalbumin samples (Mann Res. Lab.). The major protein peak and the component, which just precedes it, are clearly resolved at loads up to 2 mg on a 4.1 X 250-mm column. At 5 mg the preced- ing component still appears as a shoulder but at 12 mg the two components are no longer distinguishable. The arrow in Fig. 5 identifies a point at which peak fusion occurs at high loading. Results of the chromatography of a mixture of OV-ICN and BSA on a 5-cm-long column of SynChropak AX-300 applied in increasing loads from 0.2 mg of each protein to 3.2 mg are illustrated in Table 2. As mentioned earlier, the short column resolves well at loads of 0.2 mg but loses efficiency as loading increases.

Picric acid, ion-pairing and ion-exchange capacities of SynChropak AX-300, and a series of other supports in static tests with hemoglobin, bovine serum albumin, and ovalbumin are seen in Table 3. Other supports were prepared by a modification of the Alpert procedure (10) to be described in a forthcoming paper ( 11). It is seen in Table 3 that the 300-A pore diameter support has the highest ion-exchange capacity for proteins in this static test. This observation is in agreement with that of Chang (l), who reported that on controlled porosity glass


TABLE 2 TABLE 3

R, VALUES OF OV-1CN AND BSA CHROMATOGRAPHED

ON A S-cm SYNCHROPAK AX-300 COLUMN WITH

INTREASING LOADS”

COMPARISON OF PICRIC ACID ION-PAIRING ANII

PROTEIN ION-EXTHANGF CAPACITIES OF SYN CHROPAK

AX-300 AND VARIOUS PORL DIAMFTER SILICA SLIPPORl S

Milligram amounts

0.2 0.4 0.8 1.6 3.2

R.l.2 R,,.:, Rir.:i

1.50 1.73 0.90 1.29 1.71 0.81 1.13 1.31 0.79 0.77 - 0.37 -

Note. Flow rate I.0 mlimin: 40 min linear gradient of buffer A to buffer B.

‘I In defining resolution R,. the subscripts I. 2, and 3 represent the first major ovalbumin peak. the second major ovalbumin peak, and BSA. respectively.

Bovine Picric Hem- \er-urn OV& acid glohin alhurnin humrn

support (@INdig) t~mol.‘gl ImEg) tinpig)

SynChropak AX-300 6% 39 105 9x AX-IOOA 1415 1’) 64 59 AX-300A 871 38 93 91 AX-500A 308 13 59 76

rli,jttz. Supports with the A designation were synthesized ( I I).

(CPG) of 100, 250, and 500 A pore diameter with a DEAE-bonded phase, 23, 40, and 18 mg/g of hemoglobin were bound, respectively. On the basis of these data, it may be concluded that AX-300 has as high an ion-exchange capacity as any inorganic support prepared for these studies or reported in the literature and that its pore diameter is in the region of 300 A.

creases. This should result in shorter retention times for solutes. It is interesting to note in Fig. 6 that there is a differential variation in retention times between the low- molecular-weight nucleotides and high- molecular-weight proteins on different pore diameter supports. For example. ADP and the protein OV, virtually coelute on the AX-loo-A column but are widely separated on both the AX-300- and the AX-500-A columns. The reason for this phenomenon is unknown.

The influence of support pore diameter on both resolution and retention was examined by using both low- and high- molecular-weight probes. It will be seen in Table 4 that resolution of both the AMP- ADP and ADP-ATP pairs decrease with increasing pore diameter. However, in the case of the macromolecular ovalbumin components a different pattern is observed. The 300-A pore diameter support showed the highest resolution. Since ovalbumin is capable of penetrating the pores of all of these supports (12) and 100-A pore diameter supports are routinely used in the gel permeation chromatography of ovalbumin. these results were unexpected.

Tcmperntnrc ,!zIjfkct

It is general practice in the low-pressure column chromatography of proteins to use

TABLE 4

RESOLUTION R, OF OV.AI.BUMIN (BMC) AND SEV~RAI

NUCLEOTIDES ON S-cm COLUMNS OF SYNCHROPAK

ANION-EXCHANCF SUPPORTS WITH DIFFFRENT PORE

DIAMETERS

Pore size should also influence retention of solutes on supports. It is known that as pore diameter increases, surface area de-

R, Support pore

diameter ,4MP/ADP ADPIATP ov, /ov2

AX- 100 9.04 6.03 I .s4 AX-300 5.15 3.42 2.1 I AX-500 5.40 3.36 I.14

Note. Flow rate 1.0 mlimin: 40 min linear gradient from buffer A to buffer B.


proteins for anion-exchange supports. In addition to being useful in comparing columns, they may be used in optimizing chromatographic operating conditions. Adop- tion of some uniform evaluation techniques for ion-exchange chromatography columns used in protein fractionation is strongly urged.

I I I I As expected, it was found that decreasing IO 20 30

RETENTION TIME (mm) the mobile phase velocity increased resolu-

FIG. 6. Effect of support pore diameter on retention. The very-low-diffusion coefficients of tion time of low- and high-molecular-weight probes. macromolecules require that the mobile Columns. 5-cm-long SynChropak AX-100. AX-300. phase velocity be decreased up to IO-fold AX-500; flow rate, 1.0 mlimin; 40-min linear gradient between buffers A and B. Nucleotides (- - -). pro-

to achieve the resolution obtained with

teins (-). AMP (Cl), ADP (A), ATP (O), OV first small molecules (13). peak (0). OV second peak (A), BSA (W). The loading capacity of the 4.1 x 250 mm

SynChropak AX-300 column is 10 times higher than high-performance gel per-

subambient elution conditions. Although meation chromatography columns of the

elution times in high-performance liquid same dimensions (14). As noted above, the chromatography are much shorter and pro- greater loading capacity of the intermediate tein stability less of a problem, it could still (300 A) pore diameter supports has been be useful to separate some sensitive pro- observed previously (1). teins in refrigerated high-performance col- The relationship of resolution to the columns. Resolution of the BMC ovalbumin umn length observed was unexpected and sample was found to decrease 40% as the difficult to explain. Since resolution is pro- temperature was lowered from 25 to 4°C. portional to the square root of column length, Accompanying this change in resolution a fourfold decrease in column length was a twofold increase in column head pres- would be expected to produce a 50% de- sure. Although lowering column tempera- crease in resolution. This was not the case. ture might be necessary to protect sensi- In the study using 2.0 ml/min only a 23% tive proteins, these results discourage the decrease was observed for a fourfold change routine use of subambient elution. in column length. Further studies are re-

quired to resolve this lack of correlation. DISCUSSION The 300-A pore diameter support pro-

The practice of evaluating resolution and duced the highest resolution of the oval-

exclusion characteristics of gel permeation bumin standards (see Table 4) and had the

chromatography columns with standard highest ion-exchange capacity for proteins

protein samples is widespread. This makes (see Table 3). However, care must be taken

it possible to compare both the quality of in interpreting these results. The ovalbumin supports between manufacturers and inter- sample was taken from a gel permeation lot variations from the same company. In- calibration kit and all of the components terestingly, the same thing has not been are of similar molecular weight. Resolution done in ion-exchange chromatography. of molecules of other sizes may be a dif- These studies have indicated that the ferent matter. From Fig. 6. it appears that heterogeneity within ovalbumin and bovine the optimum pore diameter depends on the serum albumin make them ideal calibration particular pair of solutes being separated.


CONCLUSIONS

From the studies outlined above, several conclusions may be reached concerning the effect of various operating parameters on resolution and loading capacity of column. These conclusions are as indicated below.

1. The selection and use of suitable proteins such as ovalbumin and bovine serum albumin as molecular probes of resolution and loading capacity in ion-exchange columns makes it possible to optimize operating conditions and compare columns.

2. Separation times of 1.5 min or less may be used in ion-exchange columns of 4.1 mm i.d. and 5-2.5 cm length but the resolution of proteins increases as flow rate decreases and separation time increases with the practical minimum flow rate being 0.25 mumin.

3. A maximum protein ion-exchange capacity of over 10 mg/injection will be obtained with 300-A pore diameter supports packed in a 4.1 x 250 mm.

4. Short columns ranging down to 50 mm in length show 5- to IO-fold decrease in protein loading capacity when compared to 250-mm columns but have approximately 75% of the resolution.

5. The 300-A pore diameter supports produced maximum resolution of the component proteins in commercial ovalbumin samples.

6. Subambient operation of columns re- duces the resolution of proteins and is only

recommended if the protein is insufficiently stable at room temperature.

ACKNOWLEDGMENT

The authors gratefully acknowledge the support of the National Institutes of Health. U. S. Public Health Service (GM 25431) in this research.

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14.

REFERENCES

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Chang, S. H., Noel. R.. and Regnier, F. E. (1976) Anal. Chem. 48, 1839-1845.

Chang, S. H.. Gooding, K. M.. and Regnier. F. E. (1976) j. Chromtrtoxr. 125, lO3- 114.

Schroeder, R. R.. Kudirka. P. J., and Toren. E. C., Jr. (1977) J. Chrornaro~~r. 134, 83-90.

Schlabach. T. D.. Fulton. J. A., Mockridge. P. B., and Toren, E. C.. Jr. (1979) Cliri. Cl~c~m. 25, 1600- 1607.

Fulton. J. A., Schlabach. T. D.. Kerl. J. E., and Toren. E. C.. Jr. (1979) .I. C/zr~~mtrto,~r. 175, 269-7-81. 283-291.

Schlabach, T. D.. Fulton. J. A.. Mockridge. P. B., and Toren, E. C.. Jr. (1980) And. Chrm. 52, 729-733.

Schlabach. T. D.. Alpert. A. J.. and Regnier, F. E. (1978) C/in. Chem. 24, 1351-1360.

Schlabach. T. D. (1978) Ph.D. Dissertation. Purdue University. Lafayette, Indiana.

Alpert, A. J.. and Regnier. F. E. (1978) J. Chrr,- marogr. 185. 37.5-392.

Vanecek. G.. and Regnier. F. E.. manuscript in preparation.

Regnier. F. E.. and Gooding. K. M. (19801 AHU/. Biochc,m 103, I-75.

Regnier. F. E.. Gooding. K. M. and Chang. S. H. (1977) Cont. Top. Clin. Anal. Chem. 1, l-48.

Gooding, K. M.. Lu. K. C.. Vanecek, G., and Regnier. F. E. (1979) 4th International Sym- posium on Column Liquid Chromatography.

variables in the high-performance anion-exchange chromatography of proteins

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