Summary
An interpretive optimization procedure in which pH can be one of the variables is presented with the emphasis on optimizing separations. When varying the pH in reversed-phase liquid chromatography the retention of ionogenic solutes will change. Thus, the selectivity between ionogenic and neutral solutes or between ionogenic solutes mutually can be optimized. However, pH also greatly affects the efficiency (plate count) and peak shape (asymmetry). Optimum selectivity (i.e. large differences in retention times) may be observed under conditions where peaks are broad and asymmetrical. Thus, it is essential to simultaneously consider retention, peak width and peak shape and their effects on separation (effective resolution) in pH-optimization studies. A procedure in which this is done is presented and applied to optimizing the separation of a synthetic mixture of selected pharmaceuticals. After initial experiments to establish the parameter space (boundaries for pH and binary methanol — water composition), twelve experiments are performed according to a 3×4 experimental design. At each loaction the retention, peak height, peak area and peak symmetry are recorded for each solute. These data are then used to build models for each of the four characteristics and for each solute. From this set of models the response surface, describing the quality of separation as a function of pH and composition, can be calculated. A variety of optimization criteria (quantifying quality of separation) can be used. The optimum corresponds to the highest point on the response surface.
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References
P. J. Schoenmakers, Optimization of Chromatographic Selectivity, A Guide to Method Development, Elsevier, Amsterdam, 1986.
J. C. Berridge, Techniques for the Automated Optimization of HPLC Separations, Wiley, Chichester, 1985.
T. Hamoir, M. de Smet, H. Pirijns, P. Conti, N. Vandendriesche, D. L. Massart, F. Maris, H. Hindriks, P. J. Schoenmakers, J. Chromatogr.589, 31 (1992).
P. J. Schoenmakers, S. van Molle, C. M. G. Hayes, L. G. M. Uunk, Anal. Chim. Acta250, 1 (1991).
R. M. Lopes Marques, P. J. Schoenmakers, J. Chromatogr.592, 157 (1992).
L. R. Snyder, J. Chromatogr.592, 183; 197 (1992).
A. Drouen, J. W. Dolan, L. R. Snyder, A. Poile, P. J. Schoenmakers, LC-GC9 (10), 714 (1991).
G. K. Low, Á. Bartha, H. A. H. Billiet, L. de Galan, J. Chromatogr.478, 21 (1989).
P. J. Schoenmakers, Á. Bartha, H. A. H. Billiet, J. Chromatogr.550, 425 (1991).
P. J. Schoenmakers, H. A. H. Billiet, L. de Galan, J. Chromatogr.205, 13 (1981).
P. J. Naish, R. J. Lynch, T. Blaffert, Chromatographia27, 343 (1989).
P. J. Naish, R. J. Lynch, Chromatographia29, 79 (1990).
M. J. P. Gerritsen, On-line curve resolution in HPLC using diode-array detection, Ph. D. thesis, University of Nijmegen, 1992.
R. J. Lynch, C. Measures, Pittsburg Conference, New Orleans, March 9–13, 1992, Paper no. 463 P.
R. M. Lopes Marques, P. J. Schoenmakers, C. B. Lucasius, G. Kateman, 19th International Symposium on Chromatography, Aix-en-Provence, September 1992, submitted for publication in Chromatographia
S. Sekulic, P. R. Haddad, J. Chromatogr.459, 65 (1988).
P. R. Haddad, S. Sekulic, J. Chromatogr.459, 79 (1988).
P. J. Schoenmakers, J. K. Strasters, Á. Bartha, J. Chromatogr.458, 355 (1987).
A. Peeters, L. Buydens, D. L. Massart, P. J. Schoenmakers, Chromatographia26, 101 (1988).
P. J. Schoenmakers, N. Dunand, A. C. Cleland, G. Musch, T. Blaffert, Chromatographia26, 37 (1988).
P. J. Schoenmakers, N. Dunand, J. Chromatogr.486, 219 (1989).
P. J. Schoenmakers, J. Liq. Chromatogr.10, 1865 (1987).
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Schoenmakers, P.J., Mackie, N. & Marques, R.M.L. Optimizing separations in reversed-phase liquid chromatography by varying pH and solvent composition. Chromatographia 35, 18–32 (1993). https://doi.org/10.1007/BF02278552
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DOI: https://doi.org/10.1007/BF02278552