Chapter 11 Chromatographic Characterization of Acrylic Polyampholytes
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C. S. Patrickios, S. D. Gadam, S. M. Cramer, W. R. Hertler, and T. A. Hatton Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139
The adsorption properties of synthetic polyampholytes based on methacrylic acid were investigated by gradient and frontal techniques on a strong anion-exchange column. These polyampholytes are of well-defined size (4,000 Da) and composition, contain low levels of impurities, and include random, diblock and triblock copolymers. While the random copolymers show no retention in the gradient elution and low adsorption in the frontal experiments, the block copolymers are strongly retained and adsorbed at large amounts. The diblock polyampholyte, which does not form micelles, exhibits similar adsorption properties as the triblock polyampholytes which form micelles. This suggests that the micellar character of the triblocks does not inhibit or reduce adsorption; on the contrary, the higher (than the single chain) micellar charge may provide a greater driving force for polymer adsorption.
Ion-exchange displacement chromatography of proteins is a separation technique i n which a mixture of proteins is adsorbed on the column and subsequently displaced by a polyelectrolyte of higher column affinity, the displacer. This technique results i n concentrated protein fractions and is therefore particularly suitable for separations of mixtures i n which the desired proteins occur in very l o w concentrations. A s displacement chromatography is gaining popularity, the quest for more efficient displacers is becoming necessary (7). W e recently synthesized low-molecular-weight block polyampholytes based on methacrylic acid (Patrickios et al., M I T , submitted for publication). Initial experiments showed that these polymers can successfully displace and separate protein mixtures in ion-exchange columns. M o s t importantly these polymers possess two novel features: the column regeneration can be accomplished either by p H change or salinity increase, and any polymer contaminating the last protein fraction can be precipitated at the isoelectric point of the polymer. These polyampholytes contain up to four different methacrylic residues presented in Figure 1 : methacrylic acid (Ac) which can be negatively charged and has a p K of 5.4 (2), dimethylaminoethyl methacrylate (B) which can be positively charged and has a p K of 8.0 (2), methyl methacrylate (M) which is neutral and hydrophobic, and phenylethyl methacrylate (P) which is neutral and more hydrophobic than methyl methacrylate. The Group Transfer Polymerization (GTP) technique used for the
0097-6156/94/0548-0144$06.00/0 © 1994 American Chemical Society
Schmitz; Macro-ion Characterization ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date: December 13, 1993 | doi: 10.1021/bk-1994-0548.ch011
11.
PATRICKIOS ET AL.
Characterization of Acrylic Polyampholytes
145
synthesis resulted i n polymers with polydispersities as l o w as 1.3 and high composition homogeneity. The molecular weight of the polyampholytes is approximately 4,000 Da, with the exception of one polymer which is 15,000 D a . One neutral and one base-rich random polyampholytes were also synthesized. One polyampholyte is a neutral diblock ( M W = 2,500 Da) and the rest are A B C triblocks with different acid/base ratio, hydrophobicity, and block sequence. Table I, i n the Results and Discussion section, gives the composition and sequence of most of our copolymers. Both the diblock and the triblock copolymers precipitate around the isoelectric point. Light scattering studies revealed that at intermediate p H (=3-10) the triblocks, not the diblock, form micelles with hydrodynamic size larger than 10 nm. A steady-state pyrene fluorescence study on Polymer 6 (Table I) at p H 4.5 indicated a very l o w critical micellar concentration (below 0.1mg/mL) suggesting that, at the polymer concentrations employed in this study, typically 10 m g / m L , the triblock polyampholytes occur as micellar aggregates rather than free chains. The aim of this study is to investigate the chromatographic behavior of these novel polyampholytic displacers in the absence of proteins. M o s t of the experiments were performed at p H 8.5 at which all of the polymers are soluble. The polymer parameters determined are the adsorptive capacity, the characteristic charge, and the steric factor (3-6). The characteristic charge is determined as the number of ionic bonds that a polymer forms with the stationary phase. The steric factor is the number of inaccessible column sites per polymer molecule at m a x i m u m (lowest salt concentration) column saturation.
Experimental A n analytical Waters Ion Exchange column of internal diameter 5 mm was packed to a length of 39 mm with 8 μηι strong anion exchange (quaternary methylamine) beads of 100 nm average pore size. The same column was used for both the gradient elution and the frontal experiments. The equilibration buffer was Tris, typically at p H 8.5 and containing 50 m M C l " . A Waters M a x i m a 820 workstation was used for data acquisition.
Gradient Elution.
A linear 10 min-gradient from 0.2 to 1.0M N a C l was applied at a flow rate of 0.5 m L / m i n using a Waters 600 Multisolvent Delivery System. The gradient delay was 9 min (due to the volume of the mixing chamber) and the dead volume of the column was 0.6 m L . A Waters 481 Lamda-Max L C Spectrophotometer was used to monitor the column effluent at 240 nm. It was not convenient to employ the wavelength of 310 nm used in the frontal experiments because the signal-to-noise ratio was very low. 20 of 10 mg/mL polymer samples prepared in Tris of p H 8.5 and 50 m M CI" were injected using a Rheodyne manual injector.
Frontal Experiments.
Five frontal experiments (five steps) were performed for the characterization of each polymer at column saturation (5). A n L K B 2150 H P L C pump was used for solvent delivery and a Spectroflow 757 detector was used to measure the absorbance of the effluent at 310 nm. A ten-port Valco manual injector with a 10 m L loop was used to inject the polymer, the nitrate, and the régénérant solutions. In the first frontal experiment (step 1), the column capacity in small anions was calculated by passing a front of 100 m M sodium nitrate at 0.5 m L / m i n through the equilibrated column and determining the nitrate breakthrough time. Second, after reequilibrating the column with buffer, a front of 10 mg/mL (2.5 m M ) polyampholyte solution was passed at 0.2 m L / m i n , the polymer breakthrough time was determined and the amount of adsorbed polymer was calculated (step 2). The lower flow rate i n step 2 secures low levels of pressure drop and adequate time for polymer adsorption. The nonadsorbed polymer i n the dead volume was washed with buffer for 10 column volumes. Third, with the polymer adsorbed, a 30 m M sodium nitrate front was
Schmitz; Macro-ion Characterization ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
146
MACRO-ION CHARACTERIZATION
CH I CH2 — C— I 3
dissociated methacrylic acid residue
coo-
CH I protonated — CH9 — C — dimethylaminoethyl methacrylate residue I COOC2H NH (CH3)2
Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date: December 13, 1993 | doi: 10.1021/bk-1994-0548.ch011
3
+
4
CH I — CH2 — C — I 3
methyl methacrylate residue
COOCH3
CH I — CH2 — C — 3
1
2-phenylethyl methacrylate residue
COOC2H4C6H5 Figure 1. Chemical formulas of the polyampholyte residues.
Table I. Salt concentration required for polymer elution at pH=8.5 Polymer
2
l 2 3 4 5 6 7 8 9 10 11 12 2
3
Sequence
Composition
[NaCl] (mM)
B-M-Ac B-M-Ac M/Ac/B B/Ac M/Ac/B B/M/Ac B/M/Ac B/M/P/M/Ac P/B/M/Ac B/M/Ac B/M/Ac B/P/Ac
16-12-8 12-12-12 12/12/12 10/10 36/36/36 12/12/12 8/12/16 12/6/1/6/12 1/12/12/12 16/12/8 10/20/10 10/10/10
