Chirobiotic columns

TABLE 8 Examples of Separations Obtained on Chirobiotic Columns... 

The two enantiomers of Clenbuterol have been separated using a chirobiotic column, which consists of a macrolide-type antibiotic stationary phase, using a mobile phase with composition of 70%MeOH, 30% acetonitrile, 0.3% acetic acid, and 0.2%triethylamine... 

Chirobiotic columns are used with polar-organic mobile phase. Chirobiotic T was for instance used for enantiomers of salbutamol and its sulfate metabolites [135], for the high-throughput analysis of albuterol in dog plasma [136], while Chirobiotic V was used for MPH (Ch. 11.4.4) [57]. 

Figure 6.9. An example of a chiral separation using a macrocyclic chirobiotic column of unnatural amino acids as chiral synthesis intermediates. Courtesy of Chirotech Technologies, UK.

Thirteen different enantiomers were tentatively used to obtain a significant number of A-V chiral solute descriptors. They were selected because they were well resolved on the four Chirobiotic columns. The set contained eight ionic or zwit-terionic amino acids or amino acid derivatives and five molecular solutes. The 13 enantiomers were separated on 5 well-characterized achiral columns, namely a classical polymeric C18, a diphenyl bonded column, a weak anion exchanger (Primesep D, SiELC), a divinyl benzene bonded column, and a strong anion exchanger (SAX, Alltech). Five different ethanol-water mobile phases were used making more than 300 chromatograms to develop and to analyze. 

Now that the e, s, a, b, and v system parameters are known for the four Chirobiotic columns and the A, B, E, S, and V solute descriptors are obtained for five enantiomeric compounds, it is possible to calculate the retention factor k of the five enantiomeric compounds using Eq. (5) and compare it with the experimental two retention factors obtained for the two enantiomers on the CSP. Figure 7 illustrates the results three situations were observed, (i) The retention factors predicted by LSER corresponded to the first eluting enantiomer (e.g., dihydrofurocoumarin. Fig. 7a) (ii) the LSER-predicted retention factors corresponded to the last eluting enantiomer (e.g., 5-methyl-5-phenyl hydantoin in the normal-phase mode. Fig. 7b) and (iii) the LSER-predicted retention factors did not correspond to a particular enantiomer for all mobile phase compositions (e.g., 5-methyl-5-phenyl hydantoin in the RPLC mode and bromacil. Fig. 7c and d). From a mechanistic point of view, it can be speculated that in case i, the chiral selector has overall attractive enan-tioselective interactions with the second enantiomer more retained than the LSER prediction in case ii, the chiral selector has overall repulsive enantioselective interactions with the first enantiomer less retained than the LSER prediction and in case iii, the chiral selector has enantioselective interactions with both enantiomers. 

The column was 25 cm long, 4.6 mm I.D. and packed with Partisil 10. It is seen that linear curves were obtained for three different solutes and two different moderators in n-heptane. Scott and Beesley [14] obtained retention data for the two enantiomers, (S) and (R) 4-benzyl-2-oxazolidinone. The column chosen was 25 cm long, 4.6 mm I.D. packed with 5 mm silica particles bonded with the stationary phase Vancomycin (Chirobiotic V provided by Advanced Separations Technology Inc., Whippany, New Jersey). This stationary phase is a macrocyclic glycopeptide Vancomycin that has a molecular weight of 1449.22, and an elemental composition of 54.69% carbon. 

FIGURE 14.4 Achiral separation (a) of rye grass extract containing 2-(2,4-diclorophenoxy) propionoic acid (2,4-DP) on a C18 column and subsequent chiral separation (b) of the heart-cut portion on a Chirobiotic T CSP. Reprinted from Schneiderheinze et al. (1999) with permission John Wiley Sons. 

D acquarica, I., Gasparrini, F., Giannoli, B., Badaloni, E., Galletti, B., Giorgi, F., Tinti, M.O., Vigevani, A. (2004). Enantio- and chemo-selective HPLC separations hy chiral-achiral tandem-columns approach the combination of CHIROBIOTIC TAG and SCX for the analysis of propionyl carnitine and related impurities. J. Chromatogr. A 1061, 167-173. 

Amino acid formation was monitored over 24 h by chiral high-performance liquid chromatography (CHIROBIOTIC T column) with samples diluted 10-fold (in H2O) to a suitable concentration. 

Aboul-Enein, H.Y. and Imran, A., Chiral resolution of clenbuterol, cimaterol, and mabuterol on CHIROBIOTIC V, T and TAG columns, J. Sep. Set, 25, 851,2002. Berthod, A. et al.. Facile RPLC enantioresolution of native amino-acids and peptides using a teicoplanin chiral stationary phase, J. Chromatogr. A, 731, 123, 1996. 

Arki, A. et al.. High-performance liquid chromatographic separation of stereoisomers of P-amino acids and a comparison of separation efficiencies on CHIROBIOTIC T and TAG columns, Chromatographia, 60, S43, 2004. 

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.)... 

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.)... 

Metoprolol enantiomers/urine HPLCMS Column Chirobiotic T (250 x 4.6 mm, 5 pm) Mobile phase MeOH CH3COOH NH3 (100 0.15 0.15 v/v/v) (isocratic elution) Detection MS, ionization ESI Extraction LLE/ethyl acetate LOQ 0.5 ng/mL [60]... 

These data were generated with a 250 x 4.6 mm Chirobiotic T (5-nm Teicoplanin bonded silica particles) column, methanol-water (60 40, v/v) mobile pha.se. 1 ml/min, 210 nm UV detection of underivalized solutes. 

The group of Bakhtiar [57-59] described the chiral bioanalysis of MPH in various matrices, utilizing a number of sample pretreatment strategies, separation on a Chirobiotic V columns, and the use of positive-ion APCI-MS in SRM mode. The chiral selectivity of the Chirobiotic V column is based on the use of the macrocyclic glycopeptide antibiotic vancomycin. The column can be used in both aqueous and organic mobile phase. 

Initially, MPH was extracted from plasma by LLE with cyclohexane. The reconstituted analytes were analysed using LC-MS [57]. A [ CDg]-analogue was used as ILIS. LC was performed using a 150x4.6-nun-ID Chirobiotic V column (5 pm) and methanol with 0.05% ammonium trifluoroacetate as mobile phase at a flow-rate of 1.0 ml/min. The first 3 min of the run were diverted to waste. The total ran time was only 7.5 min. The plasma-concentration-time profile for a child with ADHD after an oral administration of 17.5 mg of racemic MPH showed considerably higher plasma levels of d-threo-MPYi, as expected. The LOQ of this method was 87 pg/ml. This method was also applied in toxicokinetic studies in rat, rabbit, and dog [59]. In a further study [58], a semi-automated LLE in 96-well plate format was developed and validated. No chiral column was used in this case. 

Desai and Armstrong [139] discussed the optimization of mobile-phase conditions for enantioselective separations in combination with LC-MS. Their conclusions can be summarized as (a) polar organic mobile phases as used with Chirobiotic T and V columns are best compatible with ESl-MS, (b) normal-phase mobile phases require post-colunm addition, (c) high-aqueous mobile phases compromise ESl-MS sensitivity, and (d) ammonium tiifluoroacetate provides response enhancement for analytes with amine or amide groups. 

Figure 6.15 shows a chiral separation, where models of increasing complexity were fitted to the tailing peak of the more retained phenylglycine enantiomer measured on a Chirobiotic T column. In order to reach a good fit, the presence of at least three t5q es of sites (one nonselective and two enantioselective sites) had to be assumed [105]. 

Column CHIROBIOTIC Tcolumn (250 x4.6mm) Mobile ph. 70% methanol, 30% water,... 

The separations were carried out on a column 25 cm long, 4.6 mm ID., packed with Chirobiotic T. The mobile phase was methanol containing acetic acid and triethylamine in the concentrations shown in figure 8.12. 

It is usually monitored in blood or urine samples, using a chiral LC technique. It is necessary to resolve, and determine the enantiomers of the free Citalopram (CIT), its dimethylated metabolites (DCIT) and its didimethylmetabolites (DDCIT). A Chirobiotic V column (Vancomycin bonded to silica) was used for this purpose which was 25 cm long, 4.6 mm ID., and packed with 5 pm particles. 

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