5 ignored. There may be advantages in an HPLC process - better selectivity or solubility, for example - which allow faster purification despite the lower flow rates typically used. Sometimes separations can be achieved using one of the techniques and not the other; thus it is worth screening both, especially where the scale of separation may be increased at some time in the near future and the most effective separation will be required. Method Development. Whether one is developing an HPLC or an SFC separation, the procedure is very similar. As there is currently no way to predict which column - mobile phase combination will give a separation of the desired product ( and it is probable that such a prediction will continue to elude scientists in this field for some years to come!) the method development process generally involves screening a number of chiral stationary phases and potential mobile phases in a systematic scheme. This is aided by statistical information which tells us that for past separations there are sets of chiral phases which will give at least an 85 to 95% chance that such a set will provide conditions suitable for the preparative separation. This is not, of course, a guarantee, especially when new molecular structures are in development. Typical sets of columns and mobile phases for primary HPLC screening are shown in Table 1. If this initial screening is not successful, typically one moves to a secondary screen, where the lesser used columns and solvents are employed, again in a similar process. Usually the column sets are mounted on switching valves in the chromatograph and the whole is operated automatically, allowing much of the screening process to be run overnight in an unattended fashion. A typical screening result is shown in Figure 1. For larger scale separations it is often most convenient to run a full screen of all available columns and mobile phases for the separation since at this point the best rather than a merely adequate separation is often required. Screening in this case can be an involved process. At Chiral Technologies, for example, a full screen involves more than 100 solvent - column combinations while a screen for an industrial process in which at least 70 to 80 additional chiral phases are investigated involves even more. Such a full screen can take a long time to complete and ways to reduce this are continuously researched. Besides the use of SFC, which as noted above reduces the analysis time by a factor of around 4 from HPLC, screening can be accelerated by use of smaller particle size columns. A column 5 cm in length packed with 3 micron particles will have higher efficiency than the 15 cm column packed with the 20 micron CSP often used for larger scale separations and can give selectivity and retention data in an order of magnitude less time. It is essential, of course, that the small particles have chromatographic properties identical with the larger particles that will be used for the separation project. Parallel chromatography systems have been developed as another approach to rapid screening. These typically use 8 channels with either conventional columns ( Sepiatech, both HPLC and SFC) or microflow columns of 0.3 mm id ( Eksigent). Such parallel systems allow a screen of 8 columns in the same time as conventionally used in screening just one. Coupled with solvent switching to allow fully automated screening gives these systems an 8- fold time advantage over the conventional single channel units. Optimisation. Once screening is complete, the separation is generally optimized to maximize the selectivity and to bring retention times into an acceptable window. This process can be more time consuming than the screening, especially as this step relies on the expertise of the chromatographer to develop the most effective procedure. For HPLC processes, it has been calculated that the optimum retention factor for the first peak in the chromatogram should have a value around 11. For SMB processes ( see below) this value should be reduced for maximum production rate 2. Optimisation also may include investigation of the sample solubility; if a solubility of only a few g/ l is attained, the preparative method will always be slow and expensive. In this respect, the use of a combination of immobilized chiral phases and mid- polarity range solvents such as dichloromethane, ethyl acetate and THF ( see Table 1) have been found to be extremely useful; many drug candidates are not especially soluble in the more conventional hexane - alcohol mobile phases employed in chiral chromatography . The method development process is completed by a loading study in which increasing quantities of the racemic compound are injected to the point where the two enantiomer peaks overlap. For small scale separations this process is stopped at the point at which the two chromatographic bands just touch. As the scale increases it may be better to sacrifice some recovery in favour of increasing the production rate of the separation by increasing load further, allowing the bands to overlap and taking the appropriate fractions which give the desired combination of purity and product yield. Particle size and column technology. At this point it is also necessary to make decisions on the particle size of the media that will be employed in the larger scale separations. Small particles, while they give high separation efficiency and allow difficult separations, produce high operating pressures. This is not an issue in small scale operations ( up to ~ 5 cm id columns) for many ( a) Immobilised polysaccharide- based phases Columns+: 1. CHIRALPAK ® IATM ( immobilized amylose tris( 3,5- dimethylphenylcarbamate)) 2. CHIRALPAK IBTM ( immobilized cellulose tris( 3,5- dimethylphenylcarbamate)) 3. CHIRALPAK ICTM ( immobilized cellulose tris( 3,5- dichlorophenylcarbamate)) + Other solvent- stable chiral columns such as Whelk- O 1 ( etc) may be included in the set. Mobile phases: 1. Hexane - 2- Propanol( 80: 20) 2 Hexane - Ethanol ( 80: 20) 3. Methyl tert- Butyl Ether - Methanol ( 98: 2) 4. Hexane - Dichloromethane - Methanol* ( 49: 49: 2) * Alternatively Hexane - THF - methanol may be used in place of the chlorinated solvent. ( b) Coated Polysaccharide- based Phases. Columns: 1. CHIRALPAK AD ® ( amylose tris( 3,5- dimethylphenylcarbamate)) 2. CHIRALCEL ® OD ® ( cellulose tris( 3,5- dimethylphenylcarbamate)) 3. CHIRALPAK AS ® ( amylose tris( S- a- methylbenzylcarbamate)) 4. CHIRALCEL OJ ® ( cellulose tris( 4- methylbenzoate)) Mobile Phases1. Hexane - 2- Propanol ( 85: 15) 2. Hexane - Ethanol ( 80: 20) 3. Methanol ( 100%) 4. Acetonitrile( 100%) The solvent strength of the mobile phases used in screening should be adjusted to obtain reasonable elution times by changing the proportion of the polar ( alcohol) modifier. ( CHIRALPAK, CHIRALCEL, AD, OD, OJ and AS are registered trademarks of Daicel Chemical Industries, Ltd.) Table 1. Screening conditions for HPLC Method Development Figure 1. Screening results for benzoin ethyl ether. Columns 250 x 4.6 mm. Mobile phase hexane : 2- propanol ( 85: 15), flow rate 1 ml/ min. Columns: 1: CHIRALCEL OD; 2: CHIRALPAK AD; 3: CHIRALPAK AS; 4: CHIRALCEL OF; 5: CHIRALCEL OB; 6: CHIRALCEL OG; 7: CHIRALCEL OJ.

6November/ 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. 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. CO2is 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 CO2by 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 CO2to 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 CO2is a gas, the solubility of both the samples and the mobile 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 CO2is condensed by cooling the stream. 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 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. 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 Figure 2. Schematic of a Preparative SFC unit. Figure 3. Result of Inlet Frit Blockage and Consequent Over- pressure in an SFC Column. CHIRALPAK AD- H, 250 x 50 mm.