Ind. Eng. Chem. Res. 1995,34, 2685-2691
2685
Nonlinear Chromatography of p-LactoglobulinsA and B: Non-Langmuirian Behavior? S.-C. David Jen* and Neville G. Pinto Department of Chemical Engineering, University of Cincinnati, Cincinnati, Ohio 45221-0171
The chromatographic retention and wave characteristics of P-lactoglobulins A and B were studied experimentally for various nonlinear chromatographic modes of operation, including isocratic and gradient elution and frontal and displacement chromatography. I n contrast to typical Langmuirian behavior, B-lactoglobulins A and B show increasing retention times with increasing column loading and diffuse fronts and sharp rears at moderate injection volumes. At higher injection volumes, the retention times increase initially and then drop. Significant tailing fronts are found in frontal chromatography, and the displacement effect is observed at high feed concentrations. The unusual behavior for both modes of overloaded chromatography has been explained consistently with a n S-shaped isotherm model. The displacement chromatography of these non-Langmuirian adsorbates has also been analyzed. Serious tailing of displacement trains is observed, and, consequently, resolution is severely affected. The cause of nonLangmuirian behavior is postulated to be aggregation of adsorbed proteins on the stationary phase.
Introduction Nonlinear chromatography with adsorbates obeying the Langmuir adsorption isotherm is frequently observed and gives patterns that are well-known in terms of retention times and wave characteristics. In overload elution, the retention times are decreased with increasing column loading, as a result of a finite monolayer adsorption capacity of the Langmuir isotherm. The overload elution peak shapes are thus sharp fronts and diffuse rears. In frontal chromatography, a sharp front is obtained, while for displacement development the fully developed pattern is made up of a series of sharp waves migrating at the same velocity (Helfferich and Klein, 1970). These responses are obtained because for a Langmuir isotherm concentration velocities increase with increasing concentration (DeVault, 1943). In contrast, non-Langmuirian adsorption behavior results in increased retention time with increased column loading in overload elution chromatography (Svoboda, 1990; Diack and Guiochon, 1992). Diffuse fronts are also generated at some overload concentrations. In frontal chromatography, diffuse, instead of sharp, fronts can be obtained (Blanco et al., 1989). These responses can mostly be accounted for by an S-shaped adsorption isotherm. Displacement chromatography for non-Langmuirian adsorbates has not been clearly identified. However, it is t o be expected that a displacement train with sharp fronts may not be obtained. There has not been a general experimental study to include various modes of nonlinear chromatography for specific proteins in a multicomponent system. In this paper, we report the results of a study on the nonLangmuirian, nonlinear chromatographic behavior of P-lactoglobulins. The behavior of these proteins in the elution, frontal, and displacement modes has been examined. The chromatographic patterns and wave characteristics of the proteins have been analyzed and contrasted with the Langmuirian behavior of other This work was presented a t the Annual National AIChE Meeting, Los Angeles, CA, November 1991. * Author t o whom correspondence should be addressed. Present address: SyStemix, Inc., 3155 Porter Dr., Palo Alto, CA 94304. 0888-588519512634-2685$09.00/0
proteins, such as lysozyme and bovine serum albumin. Possible causes for the non-Langmuirian behavior have been postulated. Finally, the implications of the nonLangmuirian behavior in optimizing large-scale chromatography are examined.
Experimental Section Materials. Crude P-lactoglobulins (containing both A and B form), pure ,&lactoglobulin A and B, bovine serum albumin (BSA),lysozyme, and dextran sulfates (from dextran molecular weight of 5000) were obtained from Sigma (St. Louis, MO). Tris(hydroxymethy1)aminomethane, imidazole, histidine, and sodium chloride were purchased from Aldrich (Milwaukee, WI). Matrex PAE-300, a polyethylenimine-coated, silicabased, weak anion-exchanger (10 pm diameter, 300500 A pore size from Amicon, Danvers, MA), was used as the stationary phase. Apparatus and Column Packing. The chromatograph apparatus consisted of a SP8800 ternary gradient HPLC pump (Spectra Physics, San Jose, CAI connected to a 25 cm x 0.46 cm i.d. chromatographic column via a Model CST4UW multiposition valve (Valco, Houston, TX). The column effluent was monitored with a Model 2550 UV/vis detector (Varian Instruments, Sunnyvale, CAI. The detector response was recorded and analyzed with an HP 3396A integrator (Hewlett Packard, Palo Alto, CA). Samples to be analyzed were injected automatically by a Model 655A-40 autosampler (Hitachi, Danbury, CT). A 100pL polypropylene sample vial with a Teflon-faced silicone septum (Hitachi, Danbury, CT) was used for holding the fraction collected. A manual 10-port sampling valve (Supelco, Bellefonte, PA) was used for calibration, as well as for isocratic elution experiments. All columns were packed with the ion exchange PAE300, using the conventional upward slurry procedure. A Haskel air driven liquid pump purchased from Alltech (Deerfield,IL) was used for this purpose. The resin was first equilibrated and slurried in methanol in an ultrasonic bath. The slurry was then poured into a slurry holding tube, and the column was immediately attached to the tube. A packing pressure of 6000 psi was applied.
0 1995 American Chemical Society
2686 Ind. Eng. Chem. Res., Vol. 34, No. 8, 1995
About 20 min was required to pack a 0.46 x 25 cm column. The packing density obtained, as per the manufacturer's specifications, was approximately 0.45 g/ml (Amicon, 1989). The dead volume for the system (containing a filter, a guard column and the 0.46 x 25 cm column) was determined to be 3.3 mL. Ten microliters of 0.1 mg/mL myoglobin at a flow rate of 0.2 m u min and mobile phase of 0.3 M Tris, pH 8, was used for this determination; under this condition the myoglobin was considered to be a nonretained species. The porosity of the column calculated from these measurements was close t o 79%.
Lysozyme
Procedures
A 25 cm x 0.46 cm i.d. PAE-300 column was used for all the chromatographic experiments except gradient elution, which used a 10 cm x 0.21 cm i.d. PAE-300 column. For all experiments requiring a constant composition of mobile phase, 0.3 M Tris, pH 8, was used, except where indicated. Effluent from the column was monitored at 280 nm for all the elution experiments and one frontal experiment at low feed concentration (0.2 mg/mL of crude P-lactoglobulins). For two frontal experiments at higher feed concentrations (1and 5 mg/ mL) and all displacement experiments, 310 nm was used to avoid detector overload. Overload Elution Chromatography. Except where indicated, all the mass overload experiments with /3-lactoglobulins were carried out at a flow rate of 0.2 mumin and sample volume of 100 yL. For volume overload experiments, crude P-lactoglobulin concentrations of 10 mg/mL were used at a flow rate of 0.2 mL/ min, except where indicated. The composition of crude P-lactoglobulin was determined by analytical gradient elution (see Analytical Procedure). The analytical gradient elution conditions, other than sample load, were also used for overload gradient elution chromatography. Frontal Chromatography. Frontal chromatography was carried out at three different concentrations of crude ,!%lactoglobulins:0.2, 1, and 5 mg/mL. A flow rate of 0.5 mumin was used. Samples were filtered before being loaded onto the column. Displacement Chromatography. Displacement chromatography of /?-lactoglobulinswas carried out with dextran sulfate as the displacer. A sample loop of 5 mL was used to inject 5 mg/mL of crude P-lactoglobulins at a flow rate of 0.5 mumin. Three experiments at pH 8 (0.3 M Tris) with displacer concentrations of 15, 10, and 2 mg/mL were performed at a flow rate of 0.2 mumin. Additional experiments without the displacer were also performed for comparison. The column was regenerated with 0.5 M NaCl(30 min) followed by 2 M NaCl(30 min) in 0.05 M Tris, pH 8, at a flow rate of 0.5 mumin. Analytical Procedure. Fractions collected from the frontal and displacement runs were analyzed by gradient elution chromatography on a 10 cm x 0.21 cm i.d. PAE-300 column. A 30 min linear gradient of 0.0-0.5 M NaCl in 10 mM Tris, pH 8, was used, with a flow rate of 0.7 mumin. Five microliter samples were injected by the autosampler. The column effluent was monitored at 280 nm. The concentrations of /?-lactoglobulins A and B were calibrated using pure components in gradient elution. Results and Discussion Overload Elution Chromatography of p-Lactoglobulins A and B. Mass overload elution is obtained
l-r
m
200
E f f l u e n t T i m e (min)
Figure 1. Mass overload elution chromatograms showing Langmuirian behavior of lysozyme; sample volume, 100 pL; mobile phase, 0.05 M NaCl in 0.05 M Tris-HC1, pH 8; flow rate, 0.5 m u min. 6-Lactoglobulin B
$8
/
i
6 -Lac A t o g lo b u l i n
A
"
250
Figure 2. Mass overload elution chromatograms of P-lactoglobulins: sample volume,100 p L ; flow rate, 0.2 mumin; numbers on the curves are total crude P-lactoglobulin concentrations.
by keeping the injection volume constant while increasing the sample concentration. Typical mass overload elution profiles, i.e., profiles for Langmuirian systems, show reduced retention times with increased column loading, as shown in Figure 1 for lysozyme elution on PAE-300. However, for mass overload elutions of P-lactoglobulins A and B on the same stationary phase, the retention time, as shown in Figure 2, increases with increasing column loading. Also, the peaks of both ,&lactoglobulin A and B have gradually ascending fronts and sharply descending rears. This is unusual, since typical (Langmuirian) mass overload peaks (e.g., Figure 1)have the opposite peak shapes (Helfferich and Klein, 1970; Katti et al., 1990). To establish if the nontypical behavior observed is due t o interactions between the A and B variants of the protein, mass overload elution with pure fractions was performed. The pure samples were obtained from displacement chromatography of the mixture, as described later. Figure 3 shows the behavior of pure P-lactoglobulin B. A similar result was obtained for @-lactoglobulinA. The dashed line in Figure 3 is for the mixture. It is clear that the general characteristics of the peak for the pure protein are the same as those obtained when the protein is in the mixture. This indicates that the cause of the non-Langmuirian behavior is rooted in the adsorption characteristics of the pure
Ind. Eng. Chem. Res., Vol. 34, No. 8, 1995 2687 ,,@-Lac t o g lo bulin B
@-Lactoglobulin B
Figure 3. Mass overload elution chromatograms of /?-lactoglobulin B: sample volume, 100 pL; flow rate, 0.2 mumin. Numbers on the curves are concentrations determined by analytical gradient elution.
1
Figure 5. Heavily mass overload elution chromatograms of /?-lactoglobulins: sample volume, 2000 pL; flow rate, 0.2 mumin. Numbers on the curves are total crude /?-lactoglobulin concentrations.
we
n@-LactogZobulin B
10
\ :
0
1
5
10
15
20
Figure 6. First derivative of S-shaped isotherm.
It should also be noted that the rectangular bands m 300
Figure 4. Volume overload elution chromatograms of /?lactoglobulins: total crude /?-lactoglobulin concentration, 10 mgl mL; flow rate, 0.2 mumin. Numbers on the curves are injected volumes.
proteins. It should be noted that there is a difference between the retention times of the P-lactoglobulin B peaks (at the same conditions) in Figures 2 and 3. This small difference is attributed t o irreversible blockage of a small number of active sites after extensive use of the column; the data in Figure 2 were obtained on a freshly packed column, and numerous frontal and displacement runs were performed on this column before the data in Figure 3 were obtained. Volume overload is a mode of operation in which the sample concentration is kept constant and injection volume is increased. This mode is normally used for identifying the maximum column loading in the optimization of large-scale liquid chromatography. Figure 4 shows column behavior as the sample volume changes. It was observed that the retention times increased initially and then decreased with increasing sample volume. Figure 5 shows the effects of feed concentration on volume overload. All the responses in this figure are for an injection volume of 2000 pL. At low concentrations, the retention time increases with increased concentration, while at higher concentrations an opposite effect is obtained. This behavior is remarkably similar to that obtained for the effect of sample volume (Figure 4). In fact, for both cases about 10 mg is the loading at which the retention time starts to decrease.
conventionally expected for volume overload (Eisenbeiss et al., 1985) were never obtained, even at very low concentrations of the protein. The elution behavior of /?-lactoglobulins (Figures 2, 4, and 5) suggests that the adsorption isotherm is S-shaped. This can be inferred from DeVault’s equation. According to this equation, the concentration velocity is inversely proportional to the slope of the isotherm (dq/dc): II
At low column loadings, corresponding to the low concentration region of the isotherms, the concentration velocity decreases with increasing concentration (Figure 2). From DeVault’s equation, this implies increasing isotherm slope with increasing concentration. At even lower sample concentrations ( ~ 0 . 2 mg/mL, data not shown), it was observed that the velocities of both proteins are independent of concentration, indicating the existence of a Henry’s law (linear) region. In the high-concentration region of the isotherm (e.g., high column loading case with 2000 p L injection, Figure 51, peak retention time decreases with increasing concentration. From DeVault’s equation, this corresponds to a decrease in the slope of the isotherm with increasing concentration and further implies the existence of a maximum at some intermediate concentration. Figure 6 summarizes the deduced characteristics of the slope of the isotherms for the P-lactoglobulins. This slope behavior translates into the S-shaped isotherm shown in Figure 7. Note that the isotherm information shown
2688 Ind. Eng. Chem. Res., Vol. 34, No. 8, 1995 a
1
I 2
0
4
8
6
10
12
14
16
18
EOlnent Volume (ml)
20
Figure 9. Frontal chromatograms showing anti-Langmuirian behavior of ,%lactoglobulinA and B. Numbers on each curve show the feed concentrations of @-lactoglobulinand W wavelength (in parentheses).
Mobile Phase Concentration
Figure 7. Hypothetical S-shaped isotherm. 2500 I
,
I
Emneat c a ” m t l o n (“l)
Emuent Volume (ml) 0
0
5
10
15
20
25
30
Effluenl Time (min)
Figure 8. Mass overload gradient elution chromatograms of @-lactoglobulinB: sample volume, 5 pL; lactoglobulin B concentrations (mg/ml); see figure legend.
in both Figures 6 and 7 is deduced qualitatively from the band profiles of the elution chromatography at low and high column loadings. As will be shown later, further support for this type of isotherm is provided by frontal and displacement data. No attempt was made to measure the isotherms due to the very high cost of pure /?-lactoglobulins.Also, significant amounts of pure protein are required for measurement by either the batch or frontal methods. The overload elution behavior under salt gradient conditions was also investigated. Shown in Figure 8 are the chromatograms obtained with /3-lactoglobulinB; the behavior is also typical of the behavior observed with pure P-lactoglobulinA and P-lactoglobulinmixtures. As expected, the peaks are sharper than corresponding isocratic peaks. More importantly, peak retention times were once again found t o increase with column loading. This indicates that the non-Langmuirian adsorption behavior occurs over a range of modulator (NaC1) concentration. Frontal Chromatography of /$Lactoglobulins A and B. For species following Langmuirian behavior it is well-known that the waves generated by a step increase in concentration are always self-sharpening (Helfferich and Klein, 1970) and roll-up increases with increased feed concentration. For ,&lactoglobulins, the frontal responses obtained are shown in Figure 9 for three feed concentrations; the responses are in terms of the detector signal. At low feed concentrations, 0.2 and 1 mg/mL, the fronts for both /3-lactoglobulins are diffuse (proportionate). At the higher concentration, 5
Figure 10. Analysis of frontal chromatogram of 1 mg/mL /Mactoglobulin A and B in Figure 9. mumt coocenlTatton (“l)
o
5 1 0 1 ~ 2 4 1 ~ 3 0 3 5 1 0 4 5 5 0
Emueat Voltme (ml)
Figure 11. Analysis of Frontal Chromatogram of 5 mg/mL @-lactoglobulinA and B in Figure 9.
mg/mL, the composition fronts are composite waves made up of a diffuse front and a sharp rear. The difference between the two responses is strikingly clear when concentrations are compared, rather than detector responses. Figures 10 and 11 show the effluent composition history for feed concentrations of 1 and 5 mg/ mL, respectively. At the higher concentration (Figure ll), the rears of the both the first and the second composition waves are very sharp, in marked contrast to the waves in Figure 10. Also, the leading edges of both waves in Figure 11are diffuse, and there is a clear transition to the sharp rear. The observed frontal behavior can be explained consistently with the S-shaped adsorption isotherm proposed earlier. The low concentration chromatograms of Figure 9 are restricted to the region of the isotherm that corresponds to the left of the maximum in Figure 6. In this region, with the exception of Henry’s law concentrations, according t o DeVault’s equation, con-
Ind. Eng. Chem. Res., Vol. 34, No. 8, 1995 2689 centration velocities will decrease with increasing liquid concentration. Thus, lower concentrations will migrate through the column faster, leading t o diffise frontal waves as has been observed in Figures 9 and 10. At frontal feed concentrations higher than the concentration corresponding to the (dqldc),, in Figure 6, since higher concentrations will have higher migration velocities than some lower concentrations, the rear of the front will self-sharpen. This sharpening process will continue as the wave propagates, leading to a gradual increase in the size of the sharp segment and a corresponding decrease in the size of the diffuse downstream segment. Eventually, the sharp upstream segment will have a wave velocity equal t o that of the tailing end of the diffuse segment. At this time, the sharp segment will cease to grow further, and will propagate as a constant pattern. The diffise downstream segment will continue to spread proportionately. This composite wave behavior is what is observed for the 5 mg/mL case (Figure 11). These findings are also supported by data reported in the literature on the single component behavior of P-lactoglobulin A on a weakly hydrophobic resin (Blanco et al., 1989). In this case also a diffuse front was observed, and the isotherm was found to be S-shaped. The retention volumes of both fronts for the feed concentration of 0.2 mg/mL concentration (i.e., 12.5 and 22.8 mL) are very close to those of elution peaks obtained in isocratic elution with 0.2 mg/mL (Figure 2) (i.e., 13.4 and 23.4 mL). This indicates that a t this feed concentration the frontal experiment is close to the linear condition. Thus, there should be no competition for adsorption sites, and no roll-up should be observed in the effluent profile. In fact, this appears to be the case even at 1mg/mL @-lactoglobulins,since in Figure 10 both species have plateau concentrations that are identical to the feed concentrations. At a higher column loading, significant roll-up of the more weakly retained species is observed (Figure 11). That is, the concentration of /?-lactoglobulinB is raised from ca. 2.5 to 4 mg/ mL, suggesting that the feed concentrations are high enough such that there is competition between the proteins for a limited number of adsorption sites. This result also demonstrates that the displacement effect can occur in non-Langmurian systems, and this effect may be used to enhance separation in other modes of chromatography. Displacement Chromatography of p-Lactoglobulins A and B. To further investigate the displacement effect between P-lactoglobulins,displacement chromatography was performed using low molecular weight dextran sulfate (Jen and Pinto, 19911, an anionic polyelectrolyte, as the displacer. Figure 12 shows four displacement chromatograms, each at a different displacer concentration, The front of the dextran sulfate displacer is not shown, because the location is difficult to determine at 310 nm. The positions of the displacer fronts were, however, measured in independent frontal experiments with only the displacer. It was found that the displacer behavior was Langmuirian, and for displacer concentrations of 15,10,and 2 mg/mL, the fronts were observed at 13.4, 16.2, and 30.5 mL, respectively. These results are consistent with the end points of protein zones in Figure 12. For the chromatogram with 2 mg/mL dextran sulfate, there is effectively no displacement of the proteins by the displacer. Consequently, the chromatogram is virtually identical to that in the absence of the displacer (0 mg/mL dextran sulfate). Also, the proteins are not concentrated, and the tailing is very long. This implies
16000
C
d
8
0 5
3.E 5
io000 .8000
--
6000 .4000 ..
8
13
18 EHluenl Volume (mL)
23
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Figure 12. Effect of displacer concentrations on displacement chromatography. Displacer concentrations: see figure legend. Efluent volume includes sample volume.
that at 2 mg/mL the velocity of the displacer front is too small to catch the migrating proteins. Therefore, the separation pattern is achieved mainly by elution development. In contrast, displacement chromatography with 10 and 15 mg/mL dextran sulfate shows faster breakthrough and significantly concentrated proteins, signifying a strong displacement effect exerted by dextran sulfate at these higher concentrations. The fact that dextran sulfate, a Langmuirian adsorbate, displaces the non-Langmuirian P-lactoglobulins indicates that both the displacer and the proteins compete for the same adsorption sites. A significant feature of the protein bands in Figure 12 is that they exhibit behavior that is typical of Langmuirian species, i.e., sharp fronts and diffise rears. This is also the case for chromatograms where there is effectively no displacement (chromatograms with 2 and 0 mg/mL dextran sulfate). This appears to be inconsistent with the overload elutions shown earlier (Figures 2 and 5). However, when examined closely, the behavior is generally consistent with an S-shaped isotherm. The displacement experiments of Figure 12 were performed under extremely overloaded conditions (25 mg), larger than the highest loading (20 mg) in Figure 4. On the basis of earlier arguments (Figure 6), the pattern obtained for P-lactoglobulin B (more weakly retained protein) should consist of a leading sharp front under heavily overload condition for which the concentration velocity is monotonically increasing. This is exactly what is obtained for all the chromatograms in Figure 12. Note that the minor diffuse fronts of the /3-lactoglobulin B zones a t 10 and 15 mg/mL displacer are related to nonretained contaminants. Also, based on the frontal experiments a t higher feed concentrations, the displacement effect should sharpen the rear of the P-lactoglobulinB zone at higher displacer concentrations (10 and 15 mg/mL). Shown in Figure 13 are the concentration profiles of the displaced ,&lactoglobulins for the 15 mg/mL displacer case. While most of the rear of ,!?-lactoglobulinB band has been sharpened by the displacement effect, there is a significant diffuse tail. This type of behavior has also been observed previously with 0.24 M Tris as the buffer (Jen and Pinto, 1990). It should be noted that the tail of the ,&lactoglobulin B zone in Figure 13 corresponds to the diffuse leading front of the ,!?-lactoglobulinA zone. This correspondence suggests that for proteins with S-shaped isotherms the
2690 Ind. Eng. Chem. Res., Vol. 34, No. 8, 1995
Emmad v0l.u (d)
Figure 13. Analysis of displacement chromatogram with displacer of 15 mg/mL dextran sulfate in Figure 12.EMuent volume includes the volume for sample injection.
leading diffuse regions are ineffective in the displacement process. This has important practical implications, since it exacerbates cross-contaminationof protein zones. Thus, displacement as a separation technique is not as effective for proteins that have S-shaped isotherms as it is for Langmuirian proteins; for the latter, equilibrium characteristics lead to sharp front and rear waves, minimizing the contamination between zones (Helfferich and Klein, 1970). The rear wave of the @-lactoglobulinA zone represents the displacement of this non-Langmuirian protein with a Langmuirian displacer. Clearly, there is a displacement effect, as evidenced by the high concentration of the protein. However, the wave is unexpectedly diffuse, though not as diffuse as in the absence of the displacement effect (Figure 12,O and 2 mg/mL). This behavior once again suggests that the displacement of nonLangmuirian proteins is less efficient than Langmuirian proteins, at least when a Langmuirian displacer is used. Possible Causes for Non-LangsluirianBehavior. The S-shaped isotherm behavior of the p-lactoglobulins suggests that there are a t least two steps in the adsorption process (Svoboda, 1990). It is postulated that the first step involves mainly electrostatic interactions. This is supported by experimental evidence in an earlier study that the retention behavior of both proteins on the same ion exchanger at low column loading can be described by the stoichiometric displacement model with chloride ion as the modulator (Jen and Pinto, 1991). Furthermore, the proteins are not bound to the anion exchanger when high ionic strength solutions of 0.5 M and 1.0 M NaCl in 0.05 Tris buffer, pH 8, are used as the mobile phases. The second step of the process is postulated t o be the adsorption of proteins onto already adsorbed proteins, i.e., protein aggregation on the stationary phase. @-Lactoglobulins are well-known for their aggregation behavior (McKenzie, 1971), and protein aggregation in the mobile phase (Grinberg et al., 1989)or on the stationary phase (Nygren and Stenberg, 1990) are known to occur. However, the S-shaped isotherm cannot be rationalized on the basis of protein aggregation in the mobile phase (Blanc0 et al., 1989) but is consistent with protein aggregation or other reactions on the stationary phase (Svoboda, 1990). The second step of adsorption is insignificant at low column loadings, due to the stronger attraction of the proteins to ionic sites on the stationary phase, consistent with the finite initial slope of the isotherm. At higher surface coverage, the adsorbed proteins provide thermodynamically competitive adsorption sites, leading to protein aggregation. It is important to note the difference between this work and the work by Grinberg et al., 1989. In contrast
to their work, which focused on the formation of @-lactoglobulin A aggregates at acidic pH in hydrophobic interaction chromatography (HIC), the formation of multiple peaks for /3-lactoglobulins A and B in linear chromatography has not been observed in this work. Several forms of protein aggregates have been shown by Grinberg et al. to be present in the mobile phase at pH 4.5, based on the formation of multiple peaks in HIC and low-angle light scattering data for @-lactoglobulin A. However, the formation of more than one aggregate in the mobile phase may not occur under different pH conditions. For example, Grinberg et al. (1989) showed that only one form of ,&lactoglobulinA aggregate exists at pH 6. Single peaks were observed in ion exchange HPLC for @-lactoglobulinsA and B at pH 7 (Liao et al., 1987). A pH of 8 has been used in this study, and single peaks were observed for ,+lactoglobulins A and B. Therefore, it is less likely that the mechanism of mobile phase aggregation is responsible for the unusual chromatography observed in this study. Liao et al. (1987)have reported dramatically different results in a study with the same proteins but with different stationary and mobile phases. Behavior consistent with the Langmuirian isotherm was observed in isocratic, gradient, and displacement chromatography of @-lactoglobulinsA and B. For example, in displacement chromatography, sharp displacement trains with high recovery of pure proteins were reported, in contrast to the diffuse displacement trains and low recovery observed in this study. These differences can be attributed to either the different mobile phase (i.e., 25 mM phosphate, pH 7 in displacement chromatography) or the different stationary phase (i.e., TSK DEAE-5PW) used. Both can affect the adsorption behavior profoundly. In order to investigate this, the mobile phase used in Liao’s study for isocratic elution chromatography, i.e., 130 mM phosphate, pH 7, was used with PAE300 as the stationary phase. Non-Langmuirian behavior was still observed in overload elution, indicating that the mobile phase is not the primary influence for nonLangmuirian behavior. The major difference between the two stationary phases is that the ligand for PAE-300 is a cross-linked polymer (Unger, 1989) with significant hydrophobicity, which manifests itself at high salt concentrations in the mobile phase. In contrast, TSK DEAE has a hydrophilic monolayer coating. This difference in the stationary phases may lead t o the adsorption of @-lactoglobulins with different orientations, facilitating aggregation in the case of PAE-300. The aggregation interaction between the @-lactoglobulins on the surface should be hydrophobic. These proteins have significant hydrophobicity, attributed to an antiparallel ,&barrel that can bind hydrophobic molecules (Papiz et al., 1986). Thus, ,&lactoglobulins have a notable tendency to form aggregates and have been shown t o aggregate in hydrophobic interaction chromatography (Grinberg et al., 1989). Non-Langmuirian behavior on PAE-300 was also observed with mobile phases of different pH and ionic strength, including 0.24 M Tris-HC1, pH 8, 0.3 M imidazole, pH 6.9, and 160 mM phosphate, pH 5.7. This suggests that the aggregation force is not primarily governed by ionic interactions. Mixed Behavior and Implication on Separations. The above experiments focused on sample mixtures exhibiting only non-Langmuirian behavior. Practical separations may involve mixtures of Langmuirian and non-Langmuirian proteins, and these mixed characteristics may have implications on opti-
Ind. Eng. Chem. Res., Vol. 34, No. 8, 1995 2691 ian adsorption behavior affects chromatographic separation significantly. It is postulated that protein aggregation through hydrophobic interactions is responsible for this type of equilibrium behavior.
BSA
,
pLaotoglobulin B
Acknowledgment This work was supported in part by Grant No. CTS8909742 from the National Science Foundation. This support is gratefully acknowledged. Literature Cited
Figure 14. Mass overload elution chromatograms of proteins. Concentrations for crude P-lactoglobulin, 10,5, 2.5,1.0,0.2 and 0.1;for BSA, 2, 1, 0.5,0.2,0.04,and 0.02mg/mL; flow rate, 0.5 mumin; sample volume, 100 pL.
mization of the separation. Shown in Figure 14 are mass overload isocratic elution chromatograms of BSA and /?-lactoglobulin A and B with 100 pL sample injection. In contrast to the non-Langmuirian behavior of the P-lactoglobulins, BSA gives peaks with sharp fronts and diffuse rears and decreasing retention times with increasing column loading. These are consistent with Langmuirian behavior. A n interesting feature of the chromatograms in Figure 14 is that the separation distance between BSA and P-lactoglobulin B is increased with increased feed concentration, indicating an enhancement in selectivity, i.e., increased resolution with increased column loading. However, there is an upper limit to this loading, beyond which the separation distance was found t o decrease. For the system under consideration (Figure 14) with 2000 pL injections (data not shown), the P-lactoglobulin retentions begin to decrease at crude protein concentrations between 2.5 and 5 mg/ml for P-lactoglobulin B and between 5 and 10 mg/mL for P-lactoglobulin A, while the BSA retention continues t o decline. Based on these results, it is also to be expected that if a Langmuirian protein with an affinity stronger than the P-lactoglobulins had been selected, cross-contamination of the proteins would occur more quickly from column overload than if the proteins were all Langmuirian. Clearly, the different behavior of non-Langmuirian proteins can have a significant effect on the separation and must be considered in the optimization of preparative chromatography.
Conclusions The chromatographic behavior of @-lactoglobulinshas been studied in detail under overload conditions. Significant deviations from Langmuirian behavior were identified. Based on chromatographic data from frontal, elution, and displacement development, it was concluded that the behavior of these proteins is generally consistent with S-shaped isotherms and non-langmuir-
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Received for review November 4, 1994 Accepted March 20,1995 @
I39406456 @Abstract published in Advance A C S Abstracts, July 1, 1995.
