Determination of UV-transparent compounds by liquid chromatography using the UV detector

An indirect UV photometric detection technique is described in which a low concentration of a UV-absorbing compound (UVAC) is added to the mobile phase in reversed phase liquid chromatography, thereby making it possible for non UV-absorbing compounds such as the lower alcohols to be detected by the UV detector. This happens because the injected analyte may extract a portion of the UV absorbing compound from the mobile and/or stationary phase and the complex is co-eluted as a positive peak at the retention time of the analyte. Alternatively, the injected analyte may appear as a negative peak if the UV-absorbing compound is transferred to the mobile and stationary phases. In any case, the injected compound appears either as a positive or negative peak depending on the relative polarities and concentrations of all the compounds in the system. In addition, the resulting excess or deficiency of detection agent in the stationary phase is eluted separately as a positive or negative peak, indicating that the system has returned to equilibrium. In the work described herein, the chromatographic conditions and variables of the indirect photometric technique were studied to develop a quantitative HPLC method for UV-transparent compounds. It was found that under optimal conditions it is possible to determine some analytes quantitatively at concentrations as low as 0.05%.