The separation of high-molecular compounds under isocratic conditions is very difficult, if possible at all, and thus gradient elution is needed. The theory of gradient elution for small molecules is well established; however, its applications to reversed-phase gradient separations of biopolymers are not straightforward because of specific problems, such as slow diffusion, limited accessibility of the stationary phase for larger molecules, or possible sample conformation changes during the elution.
The first step of our study was the determination of the experimental data, and then these data were used to predict gradient retention times. High performance liquid chromatography was used to investigate the reversed-phase chromatographic behaviour of four proteins. The influence of experimental parameters was examined using a water/organic solvent/trifluoroacetic acid system. Chromatographic results from four Zorbax stationary phases supports were comparable.
1. Aguilar, M. I. and Hearns, M. T. W. High resolution reversed phase high performance liquid chromatography of peptides and proteins. Meth. Enzymol., 1996, 270, 3–26.
http://dx.doi.org/10.1016/S0076-6879(96)70003-4
2. Mant, C. T. and Hodges, R. S. Analysis of peptides by high performance liquid chromatography. Meth. Enzymol., 1996, 271, 3–50.
http://dx.doi.org/10.1016/S0076-6879(96)71003-0
3. Fekete, S., Veuthey, J. L., and Guillarme, D. New trends in reversed-phase liquid chromatographic separations of therapeutic peptides and proteins: theory and applications. J. Pharm. Biomed., 2012, 69, 9–27.
http://dx.doi.org/10.1016/j.jpba.2012.03.024
4. Hancock, W. S., Bishop, C. A., Prestidge, R. L., Harding, D. R., and Hearn, M. T. W. Reversed phase, high-pressure liquid chromatography of peptides and proteins with ion-pairing reagents. J. Chromatogr. Sci., 1978, 200, 1168–1170.
http://dx.doi.org/10.1126/science.206966
5. Dolan, J. W., Lommen, D. C., and Snyder, L. R. DryLab computer simulation for high-performance liquid chromatographic method development. II. Gradient elution. J. Chromatogr. A, 1989, 485, 91–112.
http://dx.doi.org/10.1016/S0021-9673(01)89134-2
6. Ghrist, B. F. D. and Snyder, L. R. Design of optimized high-performance liquid chromatographic gradients for the separation of either small or large molecules. II. Background and theory. J. Chromatogr. A, 1988, 459, 25–41.
http://dx.doi.org/10.1016/S0021-9673(01)82016-1
7. Ghrist, B. F. D., Coopermann, B. S., and Snyder, L. R. Design of optimized high-performance liquid chromatographic gradients for the separation of either small or large molecules: II. Minimising errors in computer simulations. J. Chromatogr. A, 1988, 459, 1–23.
http://dx.doi.org/10.1016/S0021-9673(01)82016-1
8. Dolan, J. W., Snyder. L. R. and Quarry, M. A. Computer simulation as a means of developing an optimized reversed-phase gradient-elution separation. Chromatographia, 1987, 24, 261–276.
http://dx.doi.org/10.1007/BF02688488
9. Dolan, J. W., Snyder, L. R., Djordjevic, N. M., Hill, D. W., and Saunders, D. L. Simultaneous variation of temperature and gradient steepness for reversed-phase high-performance liquid chromatography method development: I. Application to 14 different samples using computer simulation. J. Chromatogr. A, 1998, 803,
1–31.
http://dx.doi.org/10.1016/S0021-9673(97)01293-4
10. Gritti, F. and Guiochon, G. Peak compression factor of proteins. J. Chromatogr. A, 2009, 1216, 6124–6133.
http://dx.doi.org/10.1016/j.chroma.2009.06.063
