Determining molar mass averages in GPC. By Amandaa K. Brewer & Regina Roemling
Gel permeation chromatography (GPC) coupled to a differential refractive index (RI) detector is widely used for obtaining molar mass averages and distributions of polymers. Here, we have studied the repeatability, reproducibility and baseline stability of dual-flow RI detectors for the determination of molar mass averages.
GPC is currently the most widely accepted and used analytical method for obtaining molar mass averages and distributions of both synthetic and biopolymers. Traditionally, molar mass averages and distributions are obtained via a peak position calibration involving a series of standards of known molar mass and chemistry analysed by GPC coupled to a differential refractive index (RI) detector. In the context of GPC, GPC/RI continues to be heavily employed as it provides excellent day-to-day reproducibility and is ideal for quality control procedures.
One caveat to single detector GPC is the baseline stability of the RI detector. A conventional RI detector is constructed in such a way that there are two sides: the reference side consisting of stagnant pure solvent; and the sample side, containing a flowing stream of analyte in the same solvent as in the reference side. Over time, the reference side slowly changes, resulting in baseline drift. For peak position calibration a drift in the RI baseline has been shown to drastically affect the accuracy and precision of molar mass averages and distributions with errors up 25%.
Here, we have studied the repeatability, reproducibility and baseline stability of a dual-flow RI detector in the EcoSEC GPC system for the determination of molar mass averages via peak position calibration. The dual-flow design of the RI detector is constructed in such a way that the reference side of the RI flow cell contains a flowing stream of pure solvent. The dual-flow design is shown to compensate for the changes in the refractive index of the solvent over time by continuously flowing pure solvent through the reference side of the flow cell.
GPC analysis was performed on a system consisting of either an all-in-one EcoSEC GPC system equipped with a dual-flow refractive index detector or a modular HPLC system with an external conventional refractive index detector. Separation of polystyrene standards (PS) occurred over a column bank consisting of TSKgel SuperMultiporeHZ-M columns, with THF as the mobile phase.
To demonstrate the repeatability, reproducibility and baseline stability of a dual-flow RI detector compared to a conventional RI detector a series of identical experiments was performed on the EcoSEC GPC system (Fig. 1) and two conventional HPLC systems. As shown in Fig. 2, five consecutive injections of polystyrene standards with run times of one hour without auto zeroing the detector between injections for a total of five hours, resulted in an extremely stable baseline with low baseline drift for the dual-flow RI detector and a significantly drifting baseline on the two conventional RI detectors.
The repeatability and reproducibility of the molar mass averages as obtained via the dual-flow and conventional RI detectors were also compared. The reproducibility of the weight-average molar mass, Mw, of the dual-flow RI detector was determined to be superior by a factor of 3 to that of a conventional RI detector. Additionally, the day-to-day reproducibility and repeatability for the determination of molar mass averages was shown to vary less than 0.5% for the dual-flow RI detector, while the conventional RI detector produced day-to-day variations in molar mass averages between 1% and 3%.
A stable RI detector baseline is required for successful experiments, and repeatable and reproducible molar mass averages. Extreme care must be taken when molar mass averages and distributions are determined via peak position calibration as uncertainties and instabilities in the RI baseline can result in relatively large errors, inconsistencies, and deviations in molar mass averages. The repeatability and reproducibility of the molar mass averages were shown to increase greatly when a conventional RI detector was replaced with a dual-flow RI detector.
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Amandaa K. Brewer & Regina Roemling are with Tosoh Bioscience.