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


(a) Immobilised polysaccharide-based phases Columns+: 1. CHIRALPAK®


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.


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


. 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® 2.CHIRALCEL® OD®


(amylose tris(3,5-dimethylphenylcarbamate)) (cellulose tris(3,5-dimethylphenylcarbamate))


3.CHIRALPAK AS® (amylose tris(S-α-methylbenzylcarbamate)) 4.CHIRALCEL OJ®


(cellulose tris(4-methylbenzoate))


Mobile Phases 1. 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


. 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.


For SMB processes (see below) this value should be reduced for maximum production rate 2


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


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