Positive peak identification in liquid chromatography using absorbance ratioing with a variable-wavelength spectrophotometric detector.

It is frequently necessary to confirm the identification of component peaks isolated in high-performance liquid chromatograms. The UV spectrum of the pure compound is usually a very powerful signature for most compounds. For liquid chromatographic instruments that permit stop-flow operation, a simple approximation is obtained with a variable wavelength UV detector by determining the absorbance ratios at several specific wavelengths to characterize the compound. If the wavelengths are well chosen, these absorbance ratios provide a very specific and a very reliable technique for peak identification. The absorbance ratios are independent of the concentration of the compounds in the detector flow cell. Identity or purity of individual peaks can be confirmed when absorbance ratios are taken from the peaks of individually injected pure standards. Flow can be stopped several times on the leading and trailing edges of broad peaks, and ratios calculated. If the ratios are identical, the peak can be considered to be pure; if ratios differ, the presence of unresolved components can be suspected. Stop-flow ratioing can be performed during gradient elution: standards are injected under rapid elution conditions and the resulting ratios are compared with those of the peaks from the gradient run. Ratios are found to be identical for similar components, regardless of the differences in retention time, thus providing rapid identification of the compounds in the gradient chromatogram. Factors that influence the overall precision of the method are: ability to stop and re-start eluent flow without loss of chromagraphic efficiency, ability to obtain numerical absorbance data from the detector, the wavelength repeatability of the detector, and the requirement that the detector cell design allow slow interruptions without baseline upset.