A Method of Correcting for Flow-Rate Fluctuations in Size Exclusion Chromatography Calculations: Applications to Methylene Chloride/Hexafluoroisopropanol Solvent System

Abstract

The mixture of methylene chloride/hexafluoroisopropanol (70/30, v/v) is an excellent polyester solvent, but its low boiling point causes unstable flow when it is used for size exclusion chromatography (SEC). In high-performance SEC experiments, retention time is normally used to measure elution volume; however, unstable flow makes it difficult to calibrate an SEC column set or calculate molecular weight parameters from a chromatogram. We have devised a simple and inexpensive method to compensate for the effect of unstable flow in SEC calculations. A calibration marker injected along with each sample is used to indicate flow-rate variations. The ratio of the sample retention time to the marker retention time is invariant to flow-rate changes and is used in place of retention time as a measure of elution volume in the universal calibration technique. Calibrating a column set and analyzing chromatograms by this method result in a large improvement in the accuracy and precision of calculated molecular weight parameters.