TEMPERATURE PROGRAMMING AND GRADIENT ELUTION IN REVERSED-PHASE CHROMATOGRAPHY WITH PACKED CAPILLARY COLUMNS

The two major anisocratic elution modes were compared in reversed-phas e chromatography with 180 mu m I.D. fused-silica capillary columns pac ked with 6 mu m Zorbax SE ODS-silica. By evaluating the retention fact ors of alkylbenzenes at acetonitrile concentrations varying from 60 to 80% (v/v) in the aqueous eluent and in the temperature range of 30-80 degrees C, it was found that a 5 degrees C change in column temperatu re and a 1% change in acetonitrile concentration have almost the same effect on retention. This is illustrated by the almost identical chrom atograms of an alkylbenzene sample obtained by temperature programming and by gradient elution under the same conditions otherwise and by si mulation of the trajectories of the eluent peaks moving down the colum n. The results suggest that in reversed-phase HPLC with packed capilla ry columns temperature programming offers an alternative to gradient e lution in a relatively narrow range of the required elution strength. Thermodynamic data from isocratic chromatographic measurements were us ed to predict the retention times of alkylbenzenes in reversed-phase c hromatography with temperature programming at different heating rates and column inlet pressures. Temperature programming was used to separa te beta-lactoglobulins A and B by reversed-phase chromatography. It wa s also employed concomitantly with gradient elution to enhance the sep aration of a mixture of four standard proteins. The results indicate t hat temperature programming could serve as an adjunct to gradient elut ion by means of fine retention tuning to bring about or increase the r esolution of closely related macromolecules. (C) 1997 Elsevier Science B.V.