doi:10.1016/j.chroma.2005.08.071 
Copyright © 2005 Elsevier B.V. All rights reserved.
Fast size-exclusion chromatography—Theoretical and practical considerations
Simona T. Popovicia and Peter J. Schoenmakersa, b, 
, 
avan’t Hoff Institute for Molecular Sciencies (HIMS), University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands
bDutch Polymer Institute, P.O. Box. 902, 5600 AX Eindhoven, The Netherlands
Received 2 July 2004;
revised 22 August 2005;
accepted 24 August 2005.
Available online 9 September 2005.
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Abstract
Fast SEC is a very interesting modification of conventional SEC. The need for it emerges from combinatorial chemistry and high-throughput experimentation, where high-speed analyses are required. The different approaches to change the speed of analysis are extensively described in this paper. Special attention is paid to the trade-off between analysis time and resolution and to the selection of optimal column lengths and flow rates. Simulations are used to design and to understand experiments. Integrity plots are constructed to judge the quality of various SEC systems. Fast separations in size-exclusion chromatography are found to be more favorable than suggested by conventional theory. The results are based on experimental data obtained for polystyrene using THF as mobile phase.
Keywords: Size-exclusion chromatography; Fast-SEC; High-throughput screening; Band broadening; Polydispersity
Fig. 1. Reduced plate height vs. reduced velocity plot according to conventional theory (Eq. (4)) and according to Eq. (4a).
Fig. 2. Dimensionless plate-height (h) vs. reduced velocity (ν) plotted on a logarithmic scale for polystyrene standards (Table 3) on the PL-Gel-Mixed-C 50 mm × 7.5 mm i.d. column. See text for description of experimental data.
Fig. 3. Simulated chromatogram for a PS standard of 117,000 Da on a 300 mm × 7.8 mm i.d. column, flow rate 1.0 ml/min, comparison between PDI = 1.00 (- - -) and (a) PDI = 1.01, (b) PDI = 1.03, (c) PDI = 1.06 and PDI = 1.2.
Fig. 4. Simulated chromatogram for a PS standard 117,000 Da on a 50 mm × 7.5 mm i.d. column, flow rate 1.0 ml/min, comparison between PDI = 1.00 (- - -) and (a) PDI = 1.01, (b) PDI = 1.03, (c) PDI = 1.06 and PDI = 1.2.
Fig. 5. Comparison between simulation and experimental chromatograms (- - -) PDI = 1 and (—) PDI values as specified by the manufacturer (see Table 3). Experimental conditions: column 50 mm × 7.5 mm i.d. at 1 ml/min and polystyrene M: 1700; 10,900; 117,000; and 2,200,000 Da.
Fig. 6. Separation of a mixture of 1700, 10,900, 117,000, and 2,200,000 Da polystyrene standards at different flow rates (conditions see text).
Fig. 7. (a) Calibration curves at different flow rates (0.3, 0.5, 0.7, 1.0, 1.2, 1.4, 1.6, and 1.8 ml/min) on the 7.5 mm × 50 mm i.d. column (b) Enlarged region of part (a) (flow rates increase from left to right).
Fig. 8. Integrity plots for the fast-SEC column 7.5 mm × 50 mm i.d. at different flow rates (a) 0.5 ml/min, (b) 1.0 ml/min (c) 1.2 ml/min, (d) 1.4 ml/min, and (e) 1.6 ml/min.
Fig. 9. Integrity plot for the Plgel 103 Å column at 0.7 ml/min flow rate.
Fig. 10. Effect of concomitant changes in the column length and flow rate on the resolving power of Fast-SEC columns (a) 4.6 mm × 50 mm i.d. at 0.3 ml/min; (b) 4.6 mm × 100 mm i.d. at 0.6 ml/min; (c) 4.6 mm × 150 mm i.d. at 0.9 ml/min.
Fig. 11. Integrity plots of 4.6 mm × 50 mm i.d. (at 0.3 ml/min) compared with 4.6 mm × 100 mm i.d. at 0.9 ml/min and 4.6 mm × 150 mm i.d. at 0.9 ml/min flow rate.
Table 1.
Different approaches to change the speed of analysis in SEC
dp is the particle diameter, L the column length, F the flow rate, N the plate number, tR the retention time and Δp is the pressure drop across the separation column. Potential approaches to fast SEC are printed in bold.
a The symbol “>” indicates “better than”; The symbol “<” indicates “worse than”. Effects are moderated, because the plate height varies less than proportionally with the flow rate.
Table 2a.
Dimensions of Fast-SEC columns (dc is column diameter, L is column length, dp is particle diameter, Vcol is column volume)
a Assuming 100 second-dimension runs.
Table 2b.
Suggested flow rates and column diameters in LC × SEC
Table 3.
Polystyrene standards obtained from different manufacturers with their specified PDI values
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