Antibiotic CSPs

The macrocyclic antibiotic-based CSPs have not been used extensively in SFC. Two macrocyclic antibiotic CSPs, Chirobiotic T and Chirobiotic V, were included in a study of various CSPs in SFC. At least partial resolution of approximately half of the 44 test compounds could be obtained on these two CSPs in SFC [63]. A high concentration of modifier was necessary to elute some of the analytes. Enantioreso-lution of derivatized amino acids was also demonstrated in the same study. Flowever, a complex modifier comprised of methanol, water, and glycerol was required for separations performed on the Chirobiotic T CSP. The separation of coumachlor enantiomers on a vancomycin-based CSP (Chirobiotic V) in SFC is illustrated in Fig. 12-5 [32]. 

The PO mode is a specific elution condition in HPLC enantiomer separation, which has received remarkable popularity especially for macrocyclic antibiotics CSPs and cyclodextrin-based CSPs. It is also applicable and often preferred over RP and NP modes for the separation of chiral acids on the cinchonan carbamate-type CSPs. The beneficial characteristics of the PO mode may arise from (i) the offset of nonspecific hydrophobic interactions, (ii) the faster elution speed, (iii) sometimes enhanced enan-tioselectivities, (iv) favorable peak shapes due to improved diffusive mass transfer in the intraparticulate pores, and last but not least, (v) less stress to the column, which may extend the column lifetime. Hence, it is rational to start separation attempts with such elution conditions. Typical eluents are composed of methanol, acetonitrile (ACN), or methanol-acetonitrile mixtures and to account for the ion-exchange retention mechanism the addition of a competitor acid that acts also as counterion (e.g., 0.5-2% glacial acetic acid or 0.1% formic acid) is required. A good choice for initial tests turned out to be a mobile phase being composed of methanol-glacial acetic acid-ammonium acetate (98 2 0.5 v/v/w). 

Enantioseparation of nine amphetamine derivatives, methorphan, and propoxyphene was studied by comparing two different CSP typologies, a macrocyclic antibiotic CSP (vancomycin) and a native P-cyclodextrin CSP [123]. The suitability of the eluent systems to ESI interfacing was discussed, and a tandem mass spectrometric (MS/MS) detection method was developed. 

Three types of macrocyclic antibiotic CSPs have been commercialized by Astec (Whippany, New York) and show complementary enantioselec-tivity Chirobiotic V (selector vancomycin), Chirobiotic T (teicoplanin)... 

TABLE 1 The Selectivities of Antibiotic CSPs in the Three Mobile Phase Modes... 

Recently, Aboul-Enein and Ali reviewed the chiral resolution on antibiotic CSPs by HPLC [3,47]. It was observed that chiral resolution on antibiotic CSPs is governed by various HPLC parameters. The antibiotic CSPs may be used in normal, reversed, and new modified polar organic phase modes. The most important parameters which control the chiral resolution on antibiotic CSPs by HPLC are mobile phase composition, pH of the mobile phase, flow rate, temperature, structures of solutes, structures of antibiotics, and other parameters. These parameters are discussed herein. 

FIGURE 6 Effect of mobile phase composition on the resolution of enantiomers of different racemates in normal phase HPLC on antibiotic CSPs. (a) First ( ) and second ( ) eluted enantiomers of y-phenyl-y-butyrolactone and first ( ) and second (O) eluted enantiomers of 4-phenyl-2-methoxy-6-oxo-2,4,5,6-tetrahydropyridine-3-carbonitrile on Chirobiotic T column and (b) first ( ) and second (O) eluted enantiomers of mephenytoin on Chirobiotic V column. (From Refs. 1 and 21.)... 

Temperature is also an important parameter for controlling the resolution of enantiomers in HPLC. The enthalpy and entropy control of chiral resolution on antibiotic CSPs is similar to the case of polysaccharide-based CSPs (Chapter 2). Armstrong et al. [1] have studied the effect of temperature on the resolution behavior of proglumide, 5-methyl-5-phenylhydantoin and A-carbamyl-D-pheny-lalanine on the vancomycin column. The experiments were carried out from 0°C to 45°C. These results are given in Table 6 for three chiral compounds. It has been observed that the values of k, a, and Rs for the three studied molecules have decreased with the increase in temperature, indicating the enhancement of chiral resolution at low temperature. In another work, the same workers [22] have also studied the effect of temperature on the resolution of certain amino acid derivatives on the teicoplanin chiral stationary phase. They further observed poor resolution at ambient temperature, whereas the resolution increased at low... 

Figure 1 shows a variety of groups on antibiotic CSP that can take part in the bonding with the different groups of the racemates. Therefore, different structures of the racemates provide different types of bondings and, therefore, the different patterns of chiral resolution observed. Amino acids is the best class of com-... 

FIGURE 10 Effect of temperature on enantiomeric resolution on antibiotic CSPs. (a) k, a, and Rs for proglumide (O), 5-methyl-5-phenylhydantoin ( ) and iV-corbyl-DL-pheny-lalanine (x) on Chirobiotic V column using acetonitrile-1 % triethylammonium acetate buffer (10 90, v/v) as the mobile phase and (b) separation of enantiomers of /1-methyl phenylalanine on the Chirobiotic T column using water-methanol (10 90, v/v) as the mobile phase at (A) 1°C, (B) 20°C, (C) 50°C. 1 = erythro-L 2 = erythro-D 3 = threo-L 4 = threo-D. (From Refs. 1 and 22.)... 

Apart from the above-discussed parameters for HPLC optimization of chiral resolution on antibiotic CSPs, some other HPLC conditions may be controlled to improve chiral resolution on these CSPs. The effect of the concentrations of antibiotics (on stationary phase) on enantioresolution varied depending on the type of racemates. The effect of the concentrations of teicoplanin has been studied on the retention (k), enantioselectivity (a), resolution (Rs), and theoretical plate number (N) for five racemates [21]. An increase in the concentration of teicoplanin resulted in an increase of a and Rs values. The most surprising fact is that the theoretical plate number (N) increases with the increase in the concentration of teicoplanin. It may be the result of the resistance of mass transfer resulting from analyte interaction with free silanol and/or the linkage chains (antibiotics linked with silica gel). This would tend to trap an analyte between the silica surface and the bulky chiral selector adhered to it. This is somewhat... 

Bhushan and Parsad [65] resolved dansyl amino acids on erythromycin impregnated thin-layer chromatographic (TLC) silica plates. The mobile phase used was different ratios of 0.5 M aqueous NaCl-acetonitrile-methanol. Further, Bhushan and Thiong o [66] achieved the chiral resolution of dansyl amino acids on silica TLC plates impregnated with vancomycin chiral selector. The mobile phase used for this study was acetonitrile-0.5 M aqueous NaCl (10 4 and 14 3, v/v). The chiral recognition mechanisms of antibiotic CSPs in sub-SFC, SFC, CEC, and TLC modes of chromatography were found to be similar to HPLC. 

Although the macrocyclic glycopeptide antibiotic CSPs are very effective for the chiral resolution of many racemic compounds, their use as chiral mobile phase additives is very limited. Only a few reports are available on this mode of chiral resolution. It is interesting to note that these antibiotics absorb UV radiation therefore, the use of these antibiotics as the CMPAs is restricted. However, Armstrong et al. used vancomycin as the CMPA for the chiral resolution of amino acids by thin-layer chromatography, which will be discussed in Section 10.7. 

In addition to the vancomycin and teicoplanin CSPs, ristocetin A (Chirobiotic R) [289] and recently avoparcin [280] have been evaluated as novel chiral SOs and CSPs. It turned out that within the large family of macrocyclic antibiotics complementarity of enantioselectivity exists for different glycopeptides. As a consequence, very often it is possible to obtain a complete resolution by switching to a congeneric antibiotic CSP, if after optimization no baseline, but partial. separation can be achieved on a certain macrocyclic antibiotic type CSP (see Fig. 9.22). It can be expected that the enantioselectivity potential of closely related antibiotics will be further exploited in the future leading to an increase in the number of macrocyclic antibiotic type CSPs. 

Modified-C02 mobile phases excel at stereochemical separations, more often than not outperforming traditional HPLC mobile phases. For the separation of diastereomers, silica, diol-bonded silica, graphitic carbon, and chiral stationary phases have all been successfully employed. For enantiomer separations, the derivatized polysaccharide, silica-based Chiralcel and Chiralpak chiral stationary phases (CSPs) have been most used, with many applications, particularly in pharmaceutical analysis, readily found in the recent literature (reviewed in Refs. 1 and 2). To a lesser extent, applications employing Pirkle brush-type, cyclodextrin and antibiotic CSPs have also been described. In addi-... 

Antibiotic-CSP Broad Mainly polar organic mode 0.1-1 Limited Yes... 

One significant commercial development which has been taking place is that it has been demonstrated that macrocyclic antibiotic CSP may be used successfully with normal phase solvents, mixtures of n-hexane and ethanol being the most frequently used. Most of the illustrative examples cited in the commercial literature feature non-polar analytes not all of which are of pharmaceutical interest (Fig. 3.6) but nonetheless it is a good selling point that these CSP may be used with the complete range of mobile phase polarities. 

Fig. 3.6 Illustrative analytes resolved on macrocyclic antibiotic CSP in the straight phase mode...

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