Summary
β-Lactoglobulin A and B (β-LACT) were separated by displacement chromatography (DSC) on an ionexchange column using dextran sulfate as the displacer. A LALLS photometer and a UV detector, in series, were used to determine the molecular weight (MW) of the proteins, on-line. The results indicate that both, β-LACT A and B, were present as dimers in the buffer used for the mobile phase. The MWs of the proteins were about 6–8% higher than the theoretical MW of a dimer (∼37,000). Additional control studies have shown the presence of a high molecular weight species in both the proteins, which could possibly be an aggregate. This species was observed in the LALLS signal but was nearly absent in the UV signal. Our work has demonstrated the feasibility of interfacing LALLS with displacement chromatography for detecting impurities or aggregates which may be difficult to detect by conventional detectors used for chromatography.
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Abbreviations
- DSC:
-
displacement chromatography
- DSC-LALLS/UV:
-
DSC with LALLS and UV detection
- dn/dc:
-
differential refractive index of the protein
- FIA:
-
flow injection analysis (no column, direct injection into the detector)
- FIA-LALLS/UV:
-
flow injection analysis with LALLS and UV detection
- FIA-UV:
-
flow injection analysis with UV detection
- β-LACT:
-
β-lactoglobulin
- LALLS:
-
Low angle laser light scattering
- MPB:
-
modified protein buffer (composition in the experimental section)
- n:
-
refractive index
- SEC:
-
size exclusion chromatography
References
G. Subraman an, M. W. Phillips, S. M. Cramer, J. Chromatogr.439, 341 (1988).
J. Frenz, Cs. Horvath, in High Performance Liquid Chromatography. Advances and Perspectives. Cs. Horvath, Ed., Academic Press, New York, 1988, Vol. 5, p. 211.
S. M. Cramer, Nature351, 251 (1991).
Cs. Horvath, in The Science of Chromatography, F. Bruner, Ed., Elsevier, Amsterdam, 1985, p. 185.
J. Frenz, Cs. Horvath, AiChE J.31, 400 (1985).
D. Devault, J. Anal. Chem. Soc.65, 532 (1946).
E. Glueckuf, Proc. Roy. Soc. A186, 35 (1946).
E. A. Peterson, Anal. Biochem.90, 767 (1978).
E. A. Peterson, A. R. Torres, Anal. Biochem.130, 271 (1983).
A. R. Torres, B. E. Dunn, S. C. Edberg, E. A. Peterson, J. Chromatogr.316, 125 (1984).
A. R. Torres, G. G. Krueger, E. A. Peterson, Anal. Biochem.144, 469 (1985).
A. R. Torres, S. C. Edberg, E. A. Peterson, J. Chromatogr.389, 177 (1987).
B. E. Dunn, S. C. Edberg, A. R. Torres, Anal. Biochem.168, 25 (1988).
A. W. Liao, Cs. Horvath, in Biochemical Engineering VI, Annals of the New York Academy of Sciences. W. E. Goldstein, D. Dibisia, H. Pederson, Eds., New York Academy of Sciences, 1990, Vol. 589, p. 182.
A. W. Liao, Z. El Rassi, D. M. LeMaster, Cs. Horvath, Chromatographia24, 881 (1987).
A. L. Lee, A. W. Liao, Cs. Horvath, J. Chromatogr.443, 31 (1988).
S.-C. D. Jen, N. G. Pinto, J. Chromatogr.519, 87 (1990).
S.-C. D. Jen, N. G. Pinto, J. Chrom. Sci.29, 478 (1991).
S. Ghose, B. Mattiason, J. Chromatogr.547, 145 (1991).
J. A. Gerstner, S. M. Cramer, Biopharm.5, 42 (1992).
J. A. Gerstner, S. M. Cramer, Biotechnol. Prog.8, 540 (1992).
S. D. Gadam, G. Jayraman, S. M. Cramer, J. Chromatogr.630, 37 (1993).
G. Jayaraman, S. Gadam, S. M. Cramer, J. Chromatogr.630, 53 (1993).
N. Grinberg, R. Blanco, D. M. Yarmush, B. L. Karger, Anal. Chem.616, 514 (1989).
H. H. Stuting, I. S. Krull, Anal. Chem.62, 2107 (1990).
R. Mhatre, I. S. Krull, H. H. Stuting, J. Chromatogr.502, 21 (1990).
D. J. Magiera, I. S. Krull, J. Chromatogr.606, 264 (1992).
H. H. Stuting, I. S. Krull, J. Chromatogr.539, 91 (1991).
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Mhatre, R., Qian, R., Krull, I.S. et al. Characterization of proteins separated by displacement chromatography using low angle laser light scattering photometry. Chromatographia 38, 349–354 (1994). https://doi.org/10.1007/BF02269779
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DOI: https://doi.org/10.1007/BF02269779