Curtiss N. Renn

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.

Pointing-Stability-Limited Dual-Beam Absorbance Measurements by Position-Sensitive Detection

A HeCd laser was examined as a light source for dual-beam absorbance neasurements by position-sensitive detection. The measurement was limited by the laser point stability and, also, unspecified laser radiation that was effectively reduced by a spatial and spectral filter combination. An absorbance detection limit of 5 × 10−4 AU (3 × rms baseline noise) was predicted from an experimental optimization procedure. The detection limit was supported by microbore high-performance liquid chronatography of a B-2 vitamin sample, detecting riboflavin absorbance at 325 nm. Issues regarding the improvement of dual-beam absorbance measurements by position-sensitive detection are addressed.

Novel approach for the refractive index gradient measurement in microliter volumes using fiber-optic technology

The refractive index gradient (RIG) of hydrodynamically controlled profiles can be universally, yet sensitively, measured by carefully probing the radial RIG passing through a z-configuration flow cell. Fiber optic technology is applied in order to provide a narrow, collimated probe beam (100 micrometers diameter) that is deflected by a RIG and measured by a position sensitive detector. The fiber optic construction allows one to probe very small volumes (1 (mu) L to 3 (mu) L) amenable to microbore liquid chromatography ((mu) LC). The combination of (mu) LC and RIG detection is very useful for the analysis of trace quantities (ng injected amounts) of chemical species that are generally difficult to measure, i.e., species that are not amenable to absorbance detection or related techniques. Furthermore, the RIG detector is compatible with conventional mobile phase gradient and thermal gradient (mu) LC, unlike traditional RI detectors. A description of the RIG detector coupled with (mu) LC for the analysis of complex polymer samples is reported. Also, exploration into using the RIG detector for supercritical fluid chromatography is addressed.