|
Thin-layer chromatography (TLC) is a simple and inexpensive technique that is used routinely in labs. when optimum separation efficiency is not a prerequisite. It is one of the simpler chromatographic techniques, operating in a planar format on an open bed of stationary phase. The mobile phase is transported across the TLC plate by capillary action and does not require any outside help. TLC offers a key advantage over HPLC in that it is possible to conduct parallel separations on the same plate. In contrast, HPLC is a high-resolution separation technique operating in a closed system but is relatively expensive to operate compared with TLC, the main costly components being the solvent delivery system and the detector. Nevertheless, HPLC continues to advance with the development of new stationary phase materials and new techniques such as hydrophilic interaction chromatography. One of the principal reasons for the difference in separation efficiency between HPLC and TLC is the stationary phase particle size. Those used to prepare HPLC columns are generally uniform in size with typical diameters of 5 µm but in TLC, the particle sizes are non-uniform and typically range from 5-25 or 50-200 µm. The simplicity and low cost of TLC and the power of HPLC are both attractive properties and scientists in the UK decided to see if they could be combined. This has led to a new technique, named capillary action liquid chromatography (caLC), which relies on capillary action of the mobile phase to separate analytes in a capillary column packed with HPLC-type stationary phase. It was devised by David Goodall, Bo Zhang and Edmund Bergstrom from the University of York, and Peter Myers who is jointly affiliated to the Universities of York and Liverpool. In the first instance, the capillaries were prepared from glass micropipettes, with one end tapered by heating to leave a narrow orifice. Since the analytes to be separated will remain in the capillary column and the solvent does not flow beyond the end of the column, the columns are, by definition, disposable. So, the researchers developed a novel way of dry packing them which was rapid and efficient. Each capillary was inserted into a pipette tip that was loaded with the packing material and the assembly was fitted into a plastic centrifuge tube that was cut away at the bottom. Ten of these were fitted into two centrifuge cartridges and they were spun for 3 minutes at 4000 rpm. This packing method enabled particles of varying sizes (1-40 µm) and chemistries to be packed in capillaries of different volumes (2, 5 and 10 µL). The caLC setup was demonstrated in normal-phase mode with a selection of bare silica stationary phases of different particle size. The test analytes were a mixture of 5 dyes: Fat red 7B, Solvent Green 3, Sudan Orange G, Sudan II and solvent Blue 35, which were loaded by dipping the open end of the capillary column briefly in the solution. A sample plug about 0.5-1 mm long was obtained each time. The capillary was then laid horizontally with the open end inserted into the side of a reservoir containing the mobile phase, as shown in the uppermost figure opposite. The whole assembly was positioned on the object plate of a digital optical microscope as detector. With toluene as the mobile phase, the analytes migrated along the column by capillary action alone, becoming separated along the way. The separation was monitored with snapshots taken at intervals and recorded in real time by a PC connected to the microscope. The results are shown in the second figure opposite, which also illustrates the good reproducibility between columns, confirming the success of the dry packing procedure. In every case, the speed of the solvent front decreased with increasing migration time. Goodall showed that this behaviour was the same as in TLC, with the movement of the solvent front quadratically related to time. So, the transport behaviour in caLC can be understood using the fundamental theories of TLC. Using the van Deemter equation to describe the band broadening behaviour in caLC, molecular diffusion appeared to be the dominant contribution to peak dispersion. The best plate height was achieved with a 2.6-µm silica, the value of 8.8 µm being comparable to those achieved by HPLC and close to the lowest possible value of 5.2 µm with these particles. The researchers declared that approaching this value "with an extremely simple and low cost caLC setup was an excellent outcome of this study." All data so far were measured manually but the team showed how computer-assisted image analysis could be used to interpret the data and convert it into a conventional chromatogram, as illustrated in the lowest figure opposite. The colour bitmap of the snapshot at the top was separated into the red, green and blue components and the pixel values were integrated across the column width. This led to the single chromatogram at the bottom of the figure. The average peak height of the starred peak (for Solvent Blue 35) was 35 µm, in reasonable agreement with the value of 41 µm calculated from the manual bandwidth measurement. The early success of this nascent technique has demonstrated its feasibility and the suitability of the dry column packing procedure. Goodall noted that the sample loading procedure would need to be improved and also suggested that CCD detectors and active pixel sensor area detectors could be adopted for real-time, on-column UV detection. Related links:
The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd. |
The caLC set up
caLC separation of the same mixture of dyes on five columns
All images courtesy Journal of Separation Science |
|
|
|
|
