Refractive index gradient detection of biopolymers separated by high-temperature liquid chromatography

Abstract

A refractive index gradient (RIG) detector has been successfully applied in both mobile phase gradient (MPG) and thermal gradient (TG) microbore liquid chromatography (μLC). The RIG detector is based upon probing the radial RIG of the material passing through a small-volume z-configuration flow cell with a 390-μm internal radius. The optimum RIG sensitivity was at a position of 225μm offset parallel to the flow cell center axis. This optimum position was probed by a 200μm diameter collimated laser beam produced by fiber optic techniques. The performance of MPG-μLC and TG-μLC with RIG detection was evaluated using mixtures of n-alkanes and 1,2-diacylphosphatidylcholines (a class of phospholipid biopolymers). Baseline drift for the gradient separations was found to be quite small, in contrast to the performance one would obtain using conventional RI detection. Furthermore, the detection limit for the integrated RIG signal was routinely 2.10 −8 RI units (3 × baseline root mean square noise). The technique of TG-μLC with RIG detection was found to dramatically reduce the analysis time for the biopolymer separation, while providing detection limits below 100 ng injected phospholipid.