DYE ASSISTED HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC).

Abstract

During the course of study of reversed phase ion partition chromatography, it was observed that non-ionic substances such as alcohols, ester, and ketones undergo chromatographic separation in some as yet undefined association with cationic dyes (methylene blue chloride or brilliant green). Detection of non-chromophore trace level compounds has been a major problem in liquid chromatography due to the lack of a universal detector. Refractive index detector, although a universal detector lacks sensitivity. The U.V. absorbance detector is the work horse of liquid chromatography. Its major drawback is its lack of universality. It is often desirable to extend the utility of this detector to compounds toward which they are normally insensitive. This research was directed to developing the U.V.-Visible absorbance detector into a 'universal detector'. Using a mixture of methanol/water or dioxane/water solvent containing 1 x 10⁻⁴ M cationic dye such as the mobile phase it was shown to separate on Partisil ODS or on Zorbax ODS a series of C₂-C₅ alcohols and other neutrals at submicrogram levels with good base line separation. The alcohols carry with them some dye which must come from the presaturated dye ODS column. The dye peaks are detected spectrophotometrically at λ maximum of dye and becomes a measure of the eluting alcohol concentration. Normally, these aliphatic alcohols have a poor sensitivity either with refractive index or U.V. absorbance detector. This novel mode of detection and separation of trace quantities of alcohols and other neutral species represents a significant increase in sensitivity and should be widely applicable. The experimental data on which these separations are based, poses very interesting questions. For example, is there a specific kind of interaction between the alcohol and the dye or do the alcohols distribute between the Partisil ODS and mobile phase in their customary fashion and dissolve some of the immobilized dye because of their local excess concentration? Furthermore why do the slopes of the linear calibration curves obtained for each of the alcohols vary with the particular alcohol? Retention model based on dye alcohol complex formation and equilibrium partitioning of these species is advanced.