Reversed phase enantiomer separation

Fig. 7.7 Effect of various types of additives on the reversed-phase enantiomer separation of neutral, acidic and basic analytes on a polysaccharide-type CSP. Column CH I RALCEL AD-RH (150 x 4.6 mm i.d.), mobile phase aqueous mobile phase containing modifier indicated in the figure/acetonitrile (60/40 v/v), flow rate 0.5 mb min" , temp. 25 °C, detection UV 254 nm. (Reprinted with permission from [116]).

N. Nishi, N. Fujimora, Y. Yamaguchi, W. Jyomori, and T. Fukuyama, Reversed-phase HPLC separation of enantiomers of dena-pamine after derivatization with GITC chiral reagent, Chromato-grahic, 50 186 (1990). 

The efficiency of chiral stationary phase (CSP) is crucial in chromatographic technique. Recently, a new p-cyclodextrin phenyl isocyanate bonded chiral stationary phase (CSP) was developed. This CSP is quite stable and can be used in most of HPLC solvents. Many drug enantiomers that do not have enantioseparation effect on native P-cyclodextrin column in reversed phase were separated very well on this new CSP. 

The mixture of free amino acids is reacted with OPA (Fig. 7-8) and a thiol compound. When an achiral thiol compound is used, a racemic isoindole derivative results. These derivatives from different amino acids can be used to enhance the sensitivity of fluorescence detection. Figure 7-9 shows the separation of 15 amino acids after derivatization with OPA and mercaptothiol the racemic amino acids may be separated on a reversed-phase column. If the thiol compound is unichiral, the amino acid enantiomers may be separated as the resultant diastereomeric isoindole compound in the same system. Figure 7-10 shows the separation of the same set of amino acids after derivatization with the unichiral thiol compound Wisobutyryl-L-cysteine (IBLC). 

Comparisons of LC and SFC have also been performed on naphthylethylcar-bamoylated-(3-cyclodextrin CSPs. These multimodal CSPs can be used in conjunction with normal phase, reversed phase, and polar organic eluents. Discrete sets of chiral compounds tend to be resolved in each of the three mobile phase modes in LC. As demonstrated by Williams et al., separations obtained in each of the different mobile phase modes in LC could be replicated with a simple CO,-methanol eluent in SFC [54]. Separation of tropicamide enantiomers on a Cyclobond I SN CSP with a modified CO, eluent is illustrated in Fig. 12-4. An aqueous-organic mobile phase was required for enantioresolution of the same compound on the Cyclobond I SN CSP in LC. In this case, SFC offered a means of simplifying method development for the derivatized cyclodextrin CSPs. Higher resolution was also achieved in SFC. 

A simple and rapid method of separating optical isomers of amino acids on a reversed-phase plate, without using impregnated plates or a chiral mobile phase, was described by Nagata et al. [27]. Amino acids were derivatized with /-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA or Marfey s reagent). Each FDAA amino acid can be separated from the others by two-dimensional elution. Separation of L- and D-serine was achieved with 30% of acetonitrile solvent. The enantiomers of threonine, proline, and alanine were separated with 35% of acetonitrile solvent and those of methionine, valine, phenylalanine, and leucine with 40% of acetonitrile solvent. The spots were scraped off the plate after the... 

The most widely used approach for the separation of enantiomers by TLC is based on a ligand exchange mechanism using commercially available reversed-phase plates impregnated with a solution of copper acetate and (2S,4R,2 RS)-4-hydroxy-l-(2-hydroxydodecyl)proline in optimized amounts. Figure 7.9 (10,97,98,107-109). Enantiomers are separated based on the differences in the stability of the diastereomeric complexes formed between the sample, copper, and the proline selector. As a consequence, a prime requirement for separation is that the seumple must be able to form complexes with copper. Such compounds include... 

Figure 8.43 Separation of enantiomers using complexation chromatography. A, Separation of alkyloxiranes on a 42 m x 0.2S mm I.O. open tubular column coated with 0.06 M Mn(II) bis-3-(pentafluoro-propionyl)-IR-camphorate in OV-ioi at 40 C. B, Separation of D,L-amino acids by reversed-phase liquid chromatography using a mobile phase containing 0.005 M L-histidine methyl ester and 0.0025 M copper sulfate in an ammonium acetate buffer at pH 5.5. A stepwise gradient using increasing amounts of acetonitrile was used for this separation.

Ferretti et al. (1988) used an amino column coupled to a derivatized amylose column (Chiralpak AS) operated in the reverse-phase mode to separate the enantiomers of the antifungal agent voriconazole from several chiral impurities and one achiral impurity. Three of the chiral impurities are the other enantiomer and corresponding diastereomers of voriconazole. More chiral impurities result from a chlorinated voriconazole. Additionally, this multidimensional method could baseline separate all but two of the chiral impurities into their respective enantiomers. These separations are shown in Figure 14.5. 

A chiral GC column is able to separate enantiomers of epoxy pheromones in the Type II class, but the applications are very limited as follows a custom-made column packed with a p-cyclodextrin derivative as a liquid phase for the stereochemical identification of natural 3,4- and 6,7-epoxydienes [73, 74] and a commercialized column of an a-cyclodextrin type (Chiraldex A-PH) for the 3,4-epoxydiene [71] (See Table 3). The resolution abilities of chiral HPLC columns have been examined in detail, as shown in Table 7 and Fig. 14 [75,76, 179]. The Chiralpak AD column operated under a normal-phase condition separates well two enantiomers of 9,10-epoxydienes, 6,7-epoxymonoenes and 9,10-epoxymonoenes. Another normal-phase column, the Chiralpak AS column, is suitable for the resolution of the 3,4-epoxydienes. The Chiralcel OJ-R column operated under a reversed-phase condition sufficiently accomplishes enantiomeric separation of the 6,7-epoxydienes and 6,7-epoxymonoenes. 

The stereochemistry of each enantiomer separated by the chiral HPLC has been studied after methanolysis of the epoxy ring. Examining the H NMR data of esters of the produced methoxyalcohols with (S)- and (R)-a-methoxy-a-(tri-fluoromethyl) phenylacetic acid by a modified Mosher s method [181], it has been indicated that the earlier eluting parent epoxides are (3S,4R)-, (6S,7R)-, and (9R,10S)-isomers (Table 7) [75, 76, 179]. The above three chiral HPLC columns show different resolution abilities but a different elution order is not observed. The resolution profile by the reversed-phase OJ-R column has been generalized with molecular shapes of the epoxy compounds considering the... 

It must be kept in mind that the ( )-diastereomer is a mixture of enantiomers that were not separable by reverse phase HPLC. Since the method for separation of (+)- and meso-diastereomers (reverse phase HPLC, C-18 column) is based on the difference in conformation between isomers (related ultimately to their differential attraction to the column), it is unlikely that the two enantiomers of the (+ )-diastereomer have different conformations at the air-water interface. It is not known, however, how the energetics of compression and expansion will differ for films cast from either R,R or S,S enantiomer from those cast from the racemic mixture. 

Chiral stationary phases for the separation of enantiomers (optically active isomers) are becoming increasingly important. Among the first types to be synthesized were chiral amino acids ionically or covalently bound to amino-propyl silica and named Pirkle phases after their originator. The ionic form is susceptable to hydrolysis and can be used only in normal phase HPLC whereas the more stable covalent type can be used in reverse phase separations but is less stereoselective. Polymeric phases based on chiral peptides such as bovine serum albumin or a -acid glycoproteins bonded to... 

S Einarsson, B Josefsson, P Moller, D Sanchez. Separation of amino acid enantiomers and chiral amines using precolumn derivatization with (+)-l-(9-fluorenyl)ethyl chlo-roformate and reversed phase liquid chromatography. Anal Chem 59, 1191, 1987. 

FIGURE 1.7 Enantiomer separation of 2-(4-hydroxyphenyl)-imidazo[l,5-b]-quinazoline-1,5-dione on CHIRALPAK QN-AX under reversed conditions. CSP Chiralpak QN-AX (150 X 4 mm ID) mobile phase, 0.1 M ammonium acetate-methanol (60 40 v/v) pH = 6.0 ... 

OPA in combination with chiral thiols is one method used to determine amino acid enantiomers. A highly fluorescent diastereomeric isoindole is formed and can be separated on a reverse-phase column. Some of these chiral thiols include N-acetyl-L-cysteine (NAC), N-tert-butyloxy-carbonyl- L-cysteine (Boc-L-Cys), N-isobutyryl- L-cysteine (IBLC), and N-isobutyryl- D -cysteine (IBDC). Replacing OPA-IBLC with OPA-IBDC causes a reversal in the elution order of the derivatives of D- and L-amino acids on an ODS column (Hamase et al., 2002). Nimura and colleagues (2003) developed a novel, optically active thiol compound, N-(tert-butylthiocarbamoyl)- L-cysteine ethyl ester (BTCC). This reagent was applied to the measurement of D-Asp with a detection limit of approximately 1 pmol, even in the presence of large quantities of L-ASP. 

CSPs and chiral mobile phase additives have also been used in the separation of amino acid enantiomers. Another technique that should be mentioned is an analysis system employing column-switching. D-and L- amino acids are first isolated as the racemic mixture by reverse-phase HPLC. The isolated fractions are introduced to a second column (a CSP or a mobile phase containing a chiral selector) for separation of enantiomers. Long et al. (2001) applied this technique to the determination of D- and L-Asp in cell culture medium, within cells and in rat blood. 

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