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November/December 2009 reasons. A major factor is one of time. In the


transition from small to large particles there is a finite redevelopment time where the separation is modified to account for the lower column efficiency. Where the selectivity is high, this is not important, but for the more difficult separations there can be a significant loss in production rate. As most HPLC and SFC systems can cope with the pressures required to run semi-preparative columns at a reasonable flow rate the simplest and fastest option is to use the same particle size for the preparative separation as for the analytical scale column used for development. For larger scale separations the particle size becomes important as the column diameter is necessarily increased. For columns 10 cm id and above it is necessary to limit the operating pressure to prevent damage to the silica base particles since wall support for the chromatographic bed is lost in such wide diameter columns. Just as importantly, the production rate needs to be maximized for these larger scale separations to minimize the project duration and the costs. Larger particles of 10 to 20 microns diameter allow higher flow rates (albeit at a loss in plate count, which for the higher selectivity separations is less important a parameter) which give higher production rates. Thus larger particle sizes are preferred as the scale of operation increases, with SMB processes optimally operating toward the 20 micron end of the range.


The column technologies available for preparative chromatography have changed little over recent years. Axial compression technology, introduced in the 1980s4


,


revolutionized the preparative technique by allowing stable, high performance columns of diameters greater than 5 cm to be prepared from the small particles used in HPLC separations. Several variations on this theme have appeared more recently, but all such columns perform similarly with the compression technique compensating for the inevitable voiding and channeling that plagues large diameter columns. For columns 5 cm and less, there are several techniques used to pack high performance columns, some relying on axial compression schemes, others using more traditional high pressure slurry processes. For these, the performance of columns packed by different technologies is closely similar; a well-packed column has the same performance characteristics and lifetime regardless of how it is prepared.


Supercritical Fluid Chromatography (SFC). As noted above, SFC has supplanted HPLC as a preparative technique in many companies which are concerned with small scale separations at the discovery level. The reasons usually cited for this change in processing are the faster separations, due to the low mobile phase viscosity, and the reduction in organic solvent consumption which results in easier product recovery. The technique is promoted


as being “green” in that it uses less solvent (the carbon dioxide used in the systems is usually a by-product of other processes; its use in SFC separations merely delays its arrival in the atmosphere) and as such can make a small difference to the overall carbon emissions from the industry. Although SFC saves costs in terms of the low price of CO2, it must be remembered that it is more expensive to operate, as the pumps required for the CO2 are considerably larger than those required for similar flow rates of organic solvents and there are several phase changes through the cycle (see below) which require energy input. Unlike the situation for HPLC, the mobile phase in SFC is a compressible fluid at high pressure which requires significant safety considerations to be taken into account in equipment design and operation.


phase modifier becomes extremely small and these components drop out of solution as a fine mist. Collection of the organic components is usually done in a cyclone collector which efficiently separates out the mist, condensing the product as a solution in the mobile phase modifier. The carbon dioxide is then either vented to the atmosphere or is recycled back to the pump through a stripper to remove remnants of modifier or solutes.


In


the latter case, the pressure downstream from the backpressure regulator is maintained at around 50 bar and the gaseous CO2 condensed by cooling the stream.


is


One aspect of SFC that is currently problematic lies in sample introduction. The sample is usually introduced into the mobile phase stream with a loop injector or a sample pump as a solution in the organic modifier. This results in band distortion when the sample volume is large because the pulse of strong solvent causes premature elution of the solute molecules within it as it mixes with the mobile phase. This distortion can limit the injection volume that can be used. An alternative, to introduce the sample into the modifier stream before mixing with the CO2


, results in broader Figure 2. Schematic of a Preparative SFC unit.


A schematic of a preparative supercritical fluid chromatographic system is shown in Figure 2. The key differences from HPLC systems lie in the use of carbon dioxide as the main component of the mobile phase. CO2


is non


polar and for almost all applications a mobile phase modifier has to be used to increase the overall solvent polarity to solubilise the sample and to allow elution from the column. The CO2 has to be in the supercritical fluid state (or close to it) for the chromatographic step which means it has to be pressurized to greater than 73 bar at a temperature of greater than 31.1°C. In order to bring it to the required pressure it has to pumped, which means it needs to be in a liquid form at this point. This is usually accomplished either by using a cylinder with a dip tube or by condensing gaseous CO2


by


maintaining the pressure at around 50 bar and reducing the temperature to a few degrees above 0°C. Once the operating pressure is reached, the temperature is raised to bring the CO2


to the supercritical state after which it is


mixed with the mobile phase modifier. The sample, dissolved in the modifier, is introduced from a separate pump or from a loop injector. After the separation and the components are detected, the pressure is reduced in the back pressure regulator (BPR) to bring the supercritical fluid to the gaseous state. This pressure reduction results in rapid cooling and the temperature has to be controlled to prevent the equipment from being encased in a block of ice. Once the CO2


is a gas, the solubility of both the samples and the mobile


injection bands, especially when the modifier concentration is low. Another problem that can arise is that of sample solubility. A not infrequent situation is where the sample, or a sample component, is less soluble in the supercritical mobile phase than it is in the modifier. As the mobile phase and injected sample mix, the sample – or the insoluble component – may precipitate prior to reaching the column inlet. This often results in pressure increases on injection and can result in blocked and distorted frits, which destroys the column (Figure 3). The ideal solution, to dissolve the sample in the supercritical mobile phase, is not easily implemented and is not offered in commercial systems.


Figure 3. Result of Inlet Frit Blockage and Consequent Over-pressure in an SFC Column. CHIRALPAK AD-H, 250 x 50 mm.


High Performance Liquid Chromatography. HPLC has been around for many years and although at the small scale end of preparative chromatography it is being supplanted by SFC, nevertheless it remains the more important technique at larger scale. This is partly due to the size, availability and cost of large scale SFC equipment, as well as the services and costs required to run it.


In labs at


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