Chapter 9
Packed Capillary Column Chromatography with Gas, Supercritical, and Liquid Mobile Phases 1
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Keith D. Bartle , Anthony A. Clifford , Peter Myers , Mark M. Robson , Katherine Seale , Daixin Tong , David N. Batchelder , and Suzanne Cooper 1
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School of Chemistry and to Department of Physics and Astronomy, University of Leeds, Leeds S2 9JT, United Kingdom
Introduction
The concept of unified chromatography was defined by Giddings over thirty years ago, when he pointed out (1) that there are no distinctions between chromatographic separation modes which are merely classified according to the physical state of the mobile phase (GC, SFC and HPLC). Recently, Chester has described (2) how a consideration of the phase diagram of the mobile phase shows that a one-phase region (Figure 1) is available for the setting of mobile phase parameters, and that the boundaries separating individual techniques are totally arbitrary. By varying pressure, temperature and composition, solute - mobile phase interactions can be varied so as to permit the chromatographic elution of analytes ranging from permanent gases to ionic compounds; the dependence of solute diffusion coefficient in the mobile phase on pressure, temperature and composition, (Figure 1) also influences mass-transfer characteristics and also has an important bearing on the choice of an appropriate mobile phase.
Towards the end of the 1980s, the concept arose of using a single chromatographic system to carry out separation in different modes; the principles and applications of unified chromatography have recently been reviewed (3). The purpose of this paper is to show how: capillary columns with i.d. in the range 50 to 500 μηι and packed with 3
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© 2000 American Chemical Society
Parcher and Chester; Unified Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1999.
143 (generally bonded) silica particles with supercritical C 0 carrier permit GC, SFC and 2
HPLC in the same chromatograph; (b) such columns can be used in capillary electrochromatography (CEC); and (c) that micro Raman spectroscopy is a promising detector for microchromatography. Packed capillary columns offer the substantial advantages of small volumetric flow 1
rates (1-20 μL min" ) which have environmental advantages, as well as permitting the use of 'exotic' or expensive mobile phases. Peak volumes are reduced, driven by the necessity of analysing very small (picomole) amounts of substance available, for example, in small volumes of body fluids, or in the products of single-bead combinational chemistry. Packing capillary columns
Capillary columns have most commonly been packed with 1 m) columns to be packed, and packing medium density and viscosity to be varied by changing the applied pressure and temperature (4). Packing material is well dispersed in liquid C 0 in a reservoir at room temperature, 2
and packed under supercritical conditions (above 32°), maintained by a restrictor at the column exit which can be changed to vary the packing velocity. The column is subjected to ultrasonication during packing.
Columns packed in this way, and tested in SFC are HPLC are highly efficient and show the classical van Deemter behaviour (Figure 2) with the minimum in the reduced plate height (h') shifted to faster carrier linear flow rates u and a flatter curve at high u because of a higher solute diffusion coefficient. An investigation of the dependence of column performance on packing variables revealed that: sonication is vital to avoid column voids; higher packing pressure gives better efficiency, with Parcher and Chester; Unified Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1999.
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Figure 1. Three dimensional two-component phase diagram. Shaded area is twophase region. After reference 2.
COMPARISON O F V A N D E E M T E R B E H A V I O U R O F P A C K E D C A P I L L A R Y C O L U M N S IN H P L C , SFC AND C E C
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(mm/e)
Figure 2. Van Deemter plots of reduced plate height, h, versus linear mobile phase velocity for packed capillary columns in HPLC, SFC and CEC. Columns: HPLC and SFC, 30 cm X 250 μιη packed with Water Spherisorb ODS-2 5 /xm; CEC 50 μιη X 25 cm packed with Waters Spherisorb PAH 3 μπι. Test solutes: HPLC, pyrene (k ' = 6.7); SFC, chrysene (k' = 5.4); CEC, phenanthrene (k' = 5.0). lower minimum reduced plate heights (h' ) ~2; but, and counter-intuitively, looser, min
more porous packing results from higher packing pressures. In fact, a comparison with literature h values for columns packed by liquid slurry methods (5) show the same trend as our results (Figure 3). For dry-packed (6)
Parcher and Chester; Unified Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1999.
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Figure 3. Plot of minimum reduced plate height, h versus total porosity (e ) for capillary columns packed with: ( · ) supercriticalfluidcarrier; (•) dry-packing (reference 6); and (•) liquid slurry (reference 5). min
Parcher and Chester; Unified Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1999.
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146 columns there is an opposite trend (Figure 3), presumably because frictional behaviour is quite different during this procedure. The observation of looser packing with increasing pressure can be accounted for by a model (7) in which a greater slurry velocity during packed bed formation in the column centre produces a 'dome' of particles which rearrange laterally in a process which is favoured by reduced packing fluid density. The better efficiency of loose packed columns can be explained by a wall effect in which, for a tightly packed column bed, flow near the wall differs more from flow in the bed core than in a loosely packed column where there is a greater similarity of flows in the wall and core regions. A unified chromatograph for GC. SFC and HPLC The elements of the unified chromatograph (Figure 4) are: a helium cylinder with two-stage pressure regulator; syringe pump; reciprocating pump; injection valve with pneumatic actuator and digital valve sequence programmer; packed capillary column located in an oven; flame ionisation detector; and UV/visible detector with small volume flow cell (
