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.
Size 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. The 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.
A conventional RI detector is constructed in such a way that the flow cell consists of two sides (A): The 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). The 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. The 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 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 x 15mL) as well as conventional (7.8mm ID x 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.
The 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 occurs using a dual-flow RI detector compared to that of a conventional RI detector.
The reproducibility of the weight-average molar mass, Mw, for the dual-flow detector was superior by a factor of three than the reproducibility for the conventional RI detector.
In addition the day-to-day reproducibility and repeatability for the determination of Mw varies by less than 0.5 per cent for the dual-flow RI detector, while the conventional RI detector shows a variation of 1-3 per cent.
A stable RI detector baseline is required for successful experiments, in particular for repeatable and reproducible molar mass averages. The repeatability and reproducibility of the molar mass averages have been shown to significantly increase by replacing a conventional RI detector with a dual-flow RI detector.
The dual-flow RI detector shows unmatched baseline stability, excellent retention time reproducibility and day-to-day consistency compared to conventional RI detectors. Due to these features the dual-flow RI detector is an ideal tool for both single detector SEC and multi-detector SEC.
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Jens Reichenberger and Marina Urmann are with Tosoh Bioscience GmbH, Stuttgart, Germany. Amandaa K Brewer is with Tosoh Bioscience LLC, King of Prussia, PA, USA. www.tosohbioscience.com [Page Break]
Samples: Polystyrene standards, molar mass from 266 to 7.06 x 105g/mol, Mw/Mn = 1.01 from Tosoh Bioscience; Dicyclohexylphthalate, 99 per cent pure; THF
Columns: TSKgel SuperMultiporeHZ-M, 4µm, 4.6 mm ID x 15cm L x 2 + guard column TSKgel GMHXL-L, 6µm, 7.8mm ID x 30cmL + guard column
Solvent/mobile phase: THF; Flow rate: 0.35 and 1.0mL/min
Temperature: 40°C (pump and column ovens and RI detector in the EcoSEC GPC System; column oven and RI detector in the modular system)
References:
1 Striegel, A M; Yau, W W; Kirkland, J J ; Bly, D D Modern Size-Exclusion Liquid Chromatography, 2nd edition; Wiley: New York, 2009;
2 Goetz, H; Schulenberg-Schell, H Int J Polym Anal Charact, 2001, 6, 565;
3 Tchir, W J ; Rudin, A ; Fyfe, C A J Polym Sci, 1982, 20, 1443;
4 Ritter, A; Schmid, M; Affolter, S Polym Test, 2010, 29, 945;
5 Trathnigg, B; Jorde, Ch. J Liq Chromatogr, 1984, 9, 1789.
