Temperature programming and gradient elution in reversed-phase chromatography with packed capillary columns

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The two major anisocratic elution modes were compared in reversed-phase chromagraphy with 180 μm I.D. fused-silica capillary columns acked with 6 μm Zorbax SB ODS-silica. By evaluating the retention factors of alkylbenzenes at acetonitrile concentrations varying from 60 to 80% (v/v) in the aqueous eluent and in the temperature range of 30–80°C, it was found that a 5°C change in column temperature and a 1% change in acetonitrile concentration have almost the same effect on retention. This is illustrated by the almost identical chromatograms of an alkylbenzene sample obtained by temperature programming and by gradient elution under the same conditions otherwise and by simulation of the trajectories of the eluent peaks moving down the column. The results suggest that in reversed-phase HPLC with packed capillary columns temperature programming offers an alternative to gradient elution in a relatively narrow range of the required elution strength. Thermodynamic data from isocratic chromatographic measurements were used to predict the retention times of alkylbenzenes in reversed-phase chromatography with temperature programming at different heating rates and column inlet pressures. Temperature programming was used to separate β-lactoglobulins A and B by reversed-phase chromatography. It was also employed concomitantly with gradient elution to enhance the separation of a mixture of four standard proteins. The results indicate that temperature programming could serve as an adjunct to gradient elution by means of fine retention tuning to bring about or increase the resolution of closely related macromolecules.