Efficiency of packed microcolumns compared with large‐bore packed columns in size‐exclusion chromatography

Abstract

Abstract.Columns with inner diameters of 50 and 28 pm were slurry-packed with 4-5 pm size-exclusion chromatography particles (Du Pont GF 250 Zorbax Bioseries) and compared with a commercially prepared 9.4-mm i.d. column that was packed with the Same particles. The columns were compared on the basis of efficiencies, peak shapes, and van Deemter coefficients obtained for three proteins used as test analytes. The microcolumns provided more symmetrical peak shapes and lower plate heighls than those provided by the commercial column if the peaks were analyzed using statistical moments. For example, h,,, for bovine serum albumin was 5.6 in the commercial column and 2.1 in the microcolumn. This difference is attributed to the quality of the packing process, based on the fact that the van Deemter A term was significantly lower for the microcolumn than for the conventional column. The van Deemter data demonstrate that the A term was the major source of band broadening in the commercial column throughout the range of velocities used, and both theA and C terms were significant for the microcolumn. A 90 cm X 50 pm column was prepared and showed approximately 80% of the expected increase in efficiency.

Microcolumn size exclusion chromatography with polymeric stationary phases

“…fused-silica packed with PL-QEL, 5 /im, 1000 A; conditions as In Figure 7. Peaks: (1) polystyrene (PS) (MW 2.2 million), (2) PS 485 000, (3) PS 68 000, (4) PS 22 000, (5) PS 12 500, (6) PS 5050, (7) Irganox 1010 (MW 1178), (8) Irganox 1076 (MW 530), (9) Cyanosorb UV-531 (MW 326), ( 10) trlphenylmethane (MW 244), (11) Impurity, ( 12) biphenyl (MW 154), and ( 13) toluene (MW 92). Conditions as In Figure 7.…”

Microcolumn size exclusion chromatography with polymeric stationary phases

“…fused-silica packed with PL-QEL, 5 /im, 1000 A; conditions as In Figure 7. Peaks: (1) polystyrene (PS) (MW 2.2 million), (2) PS 485 000, (3) PS 68 000, (4) PS 22 000, (5) PS 12 500, (6) PS 5050, (7) Irganox 1010 (MW 1178), (8) Irganox 1076 (MW 530), (9) Cyanosorb UV-531 (MW 326), ( 10) trlphenylmethane (MW 244), (11) Impurity, ( 12) biphenyl (MW 154), and ( 13) toluene (MW 92). Conditions as In Figure 7.…”

Instrumental Requirements for Nanoscale Liquid Chromatography

“…The first attempts at using packed capillary columns with very small inner diameters, ranging from 20 to 70 µm, and flow rates on the order of 50-200 nl/min were reported several years ago by Novotny 15,16 and Jorgenson. 17,18 The direct coupling of packed microcapillary columns with ESI/MS was shown recently by Hunt et al 19,20 In this paper, instrumental requirements such as highly reproducible delivery of nanoliter flows under isocratic and gradient conditions, highly sensitive UV detection using Z-or U-shaped nanoliter UV flow cells, and the use of zero dead volume connections are presented, in order to realize the potential of…”

Preparation of Polymer Monoliths That Exhibit Size Exclusion Properties for Proteins and Peptides

“…In addition, they have achieved more efficient separations than conventional columns after optimization. 10 However, care must be taken to minimize dead volume in the capillary LC system, since the capillary column bed is only a few microliters in total volume. 11 A monolithic column structure would be advantageous as an SEC stationary phase in view of its ease of preparation and low cost, as well as good stability.…”

“…Even capillary SEC was demonstrated in the early years of microcolumn separations. , Compared to conventional SEC, capillary SEC offers several advantages, such as low eluent consumption, ease of interfacing with a mass spectrometer, and ability to prepare long columns. In addition, they have achieved more efficient separations than conventional columns after optimization . However, care must be taken to minimize dead volume in the capillary LC system, since the capillary column bed is only a few microliters in total volume …”

Microcolumn size exclusion chromatography with polymeric stationary phases

“…fused-silica packed with PL-QEL, 5 /im, 1000 A; conditions as In Figure 7. Peaks: (1) polystyrene (PS) (MW 2.2 million), (2) PS 485 000, (3) PS 68 000, (4) PS 22 000, (5) PS 12 500, (6) PS 5050, (7) Irganox 1010 (MW 1178), (8) Irganox 1076 (MW 530), (9) Cyanosorb UV-531 (MW 326), ( 10) trlphenylmethane (MW 244), (11) Impurity, ( 12) biphenyl (MW 154), and ( 13) toluene (MW 92). Conditions as In Figure 7.…”

Instrumental Requirements for Nanoscale Liquid Chromatography

“…The first attempts at using packed capillary columns with very small inner diameters, ranging from 20 to 70 µm, and flow rates on the order of 50-200 nl/min were reported several years ago by Novotny 15,16 and Jorgenson. 17,18 The direct coupling of packed microcapillary columns with ESI/MS was shown recently by Hunt et al 19,20 In this paper, instrumental requirements such as highly reproducible delivery of nanoliter flows under isocratic and gradient conditions, highly sensitive UV detection using Z-or U-shaped nanoliter UV flow cells, and the use of zero dead volume connections are presented, in order to realize the potential of…”

Preparation of Polymer Monoliths That Exhibit Size Exclusion Properties for Proteins and Peptides

“…In addition, they have achieved more efficient separations than conventional columns after optimization. 10 However, care must be taken to minimize dead volume in the capillary LC system, since the capillary column bed is only a few microliters in total volume. 11 A monolithic column structure would be advantageous as an SEC stationary phase in view of its ease of preparation and low cost, as well as good stability.…”

“…Even capillary SEC was demonstrated in the early years of microcolumn separations. , Compared to conventional SEC, capillary SEC offers several advantages, such as low eluent consumption, ease of interfacing with a mass spectrometer, and ability to prepare long columns. In addition, they have achieved more efficient separations than conventional columns after optimization . However, care must be taken to minimize dead volume in the capillary LC system, since the capillary column bed is only a few microliters in total volume …”

Microcolumn size exclusion chromatography with polymeric stationary phases

“…fused-silica packed with PL-QEL, 5 /im, 1000 A; conditions as In Figure 7. Peaks: (1) polystyrene (PS) (MW 2.2 million), (2) PS 485 000, (3) PS 68 000, (4) PS 22 000, (5) PS 12 500, (6) PS 5050, (7) Irganox 1010 (MW 1178), (8) Irganox 1076 (MW 530), (9) Cyanosorb UV-531 (MW 326), ( 10) trlphenylmethane (MW 244), (11) Impurity, ( 12) biphenyl (MW 154), and ( 13) toluene (MW 92). Conditions as In Figure 7.…”

Instrumental Requirements for Nanoscale Liquid Chromatography

“…The first attempts at using packed capillary columns with very small inner diameters, ranging from 20 to 70 µm, and flow rates on the order of 50-200 nl/min were reported several years ago by Novotny 15,16 and Jorgenson. 17,18 The direct coupling of packed microcapillary columns with ESI/MS was shown recently by Hunt et al 19,20 In this paper, instrumental requirements such as highly reproducible delivery of nanoliter flows under isocratic and gradient conditions, highly sensitive UV detection using Z-or U-shaped nanoliter UV flow cells, and the use of zero dead volume connections are presented, in order to realize the potential of…”

Preparation of Polymer Monoliths That Exhibit Size Exclusion Properties for Proteins and Peptides

“…In addition, they have achieved more efficient separations than conventional columns after optimization. 10 However, care must be taken to minimize dead volume in the capillary LC system, since the capillary column bed is only a few microliters in total volume. 11 A monolithic column structure would be advantageous as an SEC stationary phase in view of its ease of preparation and low cost, as well as good stability.…”

“…Even capillary SEC was demonstrated in the early years of microcolumn separations. , Compared to conventional SEC, capillary SEC offers several advantages, such as low eluent consumption, ease of interfacing with a mass spectrometer, and ability to prepare long columns. In addition, they have achieved more efficient separations than conventional columns after optimization . However, care must be taken to minimize dead volume in the capillary LC system, since the capillary column bed is only a few microliters in total volume …”