chromatography • spectroscopy 25
Differential refractive index detector
Jens Reichenberger, Marina Urmann and Amandaa K Brewer outline the benefits of a dual-flow differential refractive index detector and semi micro columns for calculation of molar mass averages in size exclusion chromatography.
S
ize exclusion chromatography (SEC) is the most widely accepted
and used analytical method for obtaining molar mass averages and distributions of both synthetic and biopolymers1
. Traditionally, molar
mass averages and distributions are obtained via a peak position (calibrant-relative) calibration involving a series of linear, narrow polydisperse standards of known molar mass and chemistry analysed by SEC coupled to a differential refractive index (RI) detector. Te RI baseline drift has been shown to drastically affect the accuracy and precision of molar mass averages and distributions2-4
which means
a restriction for the use of single detector SEC.
In the most common type of differential RI detector, a deflection- type detector employing the principles of Snell’s law of refraction, light emitted from a source is transmitted through the flow cell of the RI detector and strikes a detector element.
Fig. 1. Construction of a conventional RI detector.
A
A conventional RI detector is constructed in such a way that the flow cell consists of two sides (A): Te reference side filled with stagnant pure solvent and a sample
side containing a flowing stream of analyte in the same solvent as in the reference side. Over time the refractive index of the stagnant solvent in the reference cell will slowly change due to solvent degradation resulting in a baseline drift (B). See Fig. 1.
A dual-flow RI detector, such as that in the EcoSEC GPC System also consists of a flow cell with a reference and a sample side (A). Te reference side of the dual-flow RI detector contains a flowing stream of pure solvent and the sample side contains a flowing stream of analyte in the same solvent as in the reference side. Te unique flow design of the EcoSEC GPC System results in reduced baseline drift (B) and superb RI baseline stability. See Fig. 2.
For equal comparison between the dual flow and conventional RI detectors, all experiments were performed on both semi-micro and conventional SEC columns. Two conventional RI detectors were connected to a modular HPLC or SEC system optimized for the use of conventional SEC columns while the dual-flow RI detector is housed within the EcoSEC GPC System, an all-in-one system engineered to minimize extra column dead volume
B
by reduced tubing lengths, low dead volume flow cells and small stroke pumps allowing the system to maintain the efficiency of semi-micro (4.6mm ID × 15mL) as well as conventional (7.8mm ID × 30cmL) SEC columns.
Fig. 3a and b show five consecutive injections of polystyrene standards on semi-micro (0.35mL/min) and on conventional SEC columns (1.0mL/min) with a total runtime of five hours (one hour per sample) without auto zeroing the detector between the injections. For both semi-micro and conventional SEC columns the dual-flow RI detector shows a very stable baseline. With the semi-micro SEC columns a significant baseline drift was observed for the conventional detector A and a slight drift for detector B. Both conventional detectors show a significant drift and an inconsistent baseline for the conventional SEC columns.
Te comparison of the superposition of five sequential injections of dicyclohexylpthalate (DCHP) as obtained using a dual-flow and two conventional RI detectors with semi-micro and conventional SEC columns, shows a negligible or significantly less baseline drift
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