Two measurements of success were used. The first was the number of separations with resolution >1.5 (i.e., baseline separations).
The second was the number of separations for which at least a partial separation was attained. Although neither metric is
perfect, they can be used to estimate the relative success of separation approaches. Table I shows the results from the first
set of data using the three immobilized CSPs available (8). Solvents were the conventional hexane–ethanol and hexane–2-propanol
mixtures together with mixtures based on dichloromethane (DCM), methyl t-butyl ether (MTBE) and tetrahydrofuran (THF). These latter solvents were used either pure or with an admixture of hexane
and/or alcohols to adjust the solvent strength to allow elution in a reasonable time.
Table I: Success rates using various screening mobile phases.*
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Although no one solvent system showed selectivity for all compounds, it is clear that a suitable combination of them can maximize
the success rate (see Table I). Based on these data, one can make recommendations on a set of initial screening solvents that
would give a high probability of finding separation conditions (see Table II).
Table II: Proposed screening solvents*
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In the second study, the separations were not optimized but were simply screened with a range of solvents. In this study,
again with 70 solutes, 61.4% were baseline separated, and 94.3% of all chromatograms showed a selectivity greater than 1.
Fourteen percent of the baseline separations required use of the extended range solvents (MTBE, DCM, THF etc.).
Gradients
Chiral separations are generally simple, involving the separation of a pair of enantiomers. For this reason, elution gradients
are rarely used for enantioselective chromatography. This approach is somewhat different from achiral separations where elution
gradients are frequently used.
Figure 4: Overlay of gradient runs. Column: Chiralpak IC, 250 × 4.6 mm. Mobile phase: A = 30% dichloromethane in hexane;
B = 10% methanol in dichloromethane. Both A and B contained 0.1 % diethylamine. Gradient: 0–100% B in 30 min; hold at 100%
B 10 min. Flow rate = 1.5 mL/min. UV detection at 250 nm. Blue = Mianserin, Red = Methocarbamol, Green = Sulpiride.
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There are, however, some solvent strength issues, especially for some extended range solvents. Although for many compounds
the solvent composition of Table II will suffice, some solutes will either not be retained or will not elute under these basic
screening conditions. Thus, before starting the screening, one should establish a suitable solvent composition using several
isocratic experiments involving various mixtures of solvents or with a gradient.
Figure 5: Isocratic separations arising from the separations in Figure 3. Top is mianserin, mobile phase is 20% dichloromethane
in hexane. Middle is methocarbamol, mobile phase is 1:1 hexane:dichloromethane containing 2% methanol. Bottom is sulpiride,
mobile phase = 10% methanol in dichloromethane. Other conditions are the same as those for Figure 3.
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Figure 4 shows an overlay of three gradient runs with three compounds, and Figure 5 shows the final isocratic separations.
The gradient was chosen to run from the least-polar solvent mixture (hexane containing dichloro-methane) to the most polar
(dichloro-methane containing methanol). A clear correlation exists between the elution time in the gradient and the solvent
composition that allows elution in a reasonable time. Of course, this is not only applicable to methylene chloride as a mobile
phase but also can be used with the other solvent systems. Such gradients are made possible only by the immobilization of
the CSP.
Supercritical fluid chromatography
Supercritical fluid chromatography (SFC) is important in enantioselective separations, especially for the preparative purification
and isolation of individual enantiomers. Mobile phases based on alcohols (methanol, 2-propanol) or acetonitrile have been
used. Of these, the alcohol-based solvents have been the most useful. Acetonitrile is a much weaker solvent in SFC than it
is in HPLC and is often used with added alcohol to improve its elution power. The major problem with existing solvents in
preparative separations is that many compounds have limited solubility in alcohols and are better soluble in solvents of intermediate
polarity such as chloroform or THF. The sample in this case may precipitate when it is mixed with the mobile phase, thus blocking
the inlet frit of the column. Under certain conditions, some coated polysaccharide stationary phases may be stable to small
amounts of, for example, chlorinated solvents in CO2. But problems arise if the column depressurizes during operation. The immobilized chiral selectors are not affected by these
considerations and allow any organic solvent as SFC cosolvent, thus eliminating solubility problems.
Figure 6: Influence of methanol additive in SFC separation of 1,2,3,4-tetrahydro-1-naphthol enantiomers. Column is Chiralpak
IA, 250 × 4.6 mm. Mobile phase for (a) is 25% THF in CO2 and for (b) is 25% of 5% methanol in THF in CO2. Flow rate = 4 mL/min, back pressure = 150 bar, and temperature = 35°C.
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Selectivity in SFC follows much the same trends as in HPLC, although as noted, the lower solvent strength of aprotic solvents
requires the addition of alcohols for the more-polar solutes, not only to ensure rapid elution but also obtain good peak shapes.
Figure 6 illustrates the effect on peak shape and retention of 1,2,3,4-tetrahydro-1-naphthol by the addition of 5% methanol
to the THF cosolvent. Compounds containing polar groups appear to tail in aprotic solvents. Addition of methanol (or other
alcohol) removes this effect.
Method development in SFC
Figure 7: SFC separation of sulfinpyrazone enantiomers. Column is Chiralpak IC, 250 × 4.6 mm. Mobile phase is CO2 containing 20% of 0.5% ethanesulfonic acid in 2-propanol. Flow rate = 5 mL/min. Back pressure = 100 bar, Temperature = 35°C.
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Separation method development in SFC usually is carried out by investigating the selectivity derived from alcohol–CO2 mixtures. This task may be modified by using suitable additives such as ethanesulphonic acid (9), trifluoroacetic acid, or
diethylamine (see Figure 7). Sulfinpyrazone is not eluted from the Chiralpak IC column with CO2–2-propanol as a mobile phase. Addition of 0.5% ethanesulfonic acid to the cosolvent allows elution.
Once the alcohols have been investigated, other solvents may be tried. The most useful to date are THF and methyl acetate,
both with an admixture of methanol to adjust the solvent strength. This approach gave at least partial separations for 92%
of a 38 sample set. This work continues with the study of additional cosolvents, particularly MTBE and the chlorinated solvents.
Preparative chromatography
An important goal in the industry is to bring products quickly to clinical trial. Enantioselective chromatography is used
extensively in this activity because it is usually much faster to develop a chromatographic separation that can be applicable
to the quantities required for discovery, toxicology, and early Phase 1 testing than it is to develop a crystallization, an
enzymatic resolution or, especially, an enantiospecific chemical route. The most accepted stationary phases for this activity
are the polysaccharide-based CSPs because they combine the widest range of selectivity with good loading characteristics (10).
The biggest challenge in the development of a preparative separation is to find suitable solubility. Many compounds show very
little solubility in hexane-containing mobile phases and even where polar solvents such as methanol or acetonitrile can be
used, the solubility of many products remains low. Thus, a major need for preparative chromatography is access to the solvents
in which most samples are well soluble. Several separations involved adding chloroform or methylene chloride to the sample,
and sometimes the mobile phase, to enhance sample solubility. Such operations are fraught with peril because the coated polysaccharide
CSPs can be damaged very easily by such mobile phases. With immobilized CSPs, this problem disappears, and in a number of
separations with HPLC and SFC, chlorocarbon-based mobile phases were used with great success.
Table III: Solubity, retention, selectivity, and productivity
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Studying the effects of solubility, however, we found other influences of the mobile phase on preparative separations. The
effects of the nature of the mobile phase on the separation of α-methyl-α-phenylsuccinimide enantiomers were studied with
a view to establishing the highest production rate for HPLC and SMB separations. Screening against a number of possible mobile
phases gave the solubility, retention, and selectivity shown in Table III, which shows considerable variations in the values
among the solvents.
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