Vancomycin phase

A comprehensive study on the temperature effect was done in 2004 for 71 chiral compounds on four glycopeptide CSPs TE, TAG, ristocetin A, and vancomycin phases, using the three RP, POM, and NP elution systems [95]. The separations were studied in the 5 5°C temperature range. Peak efficiencies always increased with temperature, but in only 17% of the separations studied, a small increase of the resolution was observed. In the rest of the cases, the resolution decreased or even vanished when temperature increased. All van t Hoff plots were linear, showing that... 

Figure 3 Separation of citalopram and its demethylated metabolites in a human plasma extract on a vancomycin phase. Column 4.6 mm x 15 cm stationary phase Chirobiotic V, 5 pm mobile phase methanol-acetic acid-triethylamine (99.9 0.055 0.06, v/v/v), 1 ml min detector fluorescence 240/296 nm. The structure of the drug is shown it is used as the racemate. Peaks 1, alprenolol (internal standard) 2, R- - )-didesmethylcitalopram 3, R-( - )-desmethylcitalopram 4, R- - )-citalopram 5, S-( + )-citalopram 6, S-(+ )-desmethylcitalopram. S- + )-didesmethylcitalopram is not present. (Reproduced with permission from Kosel M, Eap CB, Amey M, and Baumann P (1998) Analysis of the enantiomers of citalopram. Journal of Chromatography B 719 234.)...

The chirobiotic chiral stationary phases (103,104) are based on macrocycHc antibiotics such as vancomycin (4) and teicoplanin (5). 

Only three dalbaheptides are commercialized vancomycin (39) and teicoplanin (18—22) for human health, and avoparcin (63—65) for animal usage. Vancomycin, the main trademark of which is EH Lilly s Vancocin had 1990 sales around 160 million. Total annual production is in the vicinity of 8 t. Teicoplanin, trademarked Targocid, had 1990 sales of 35 million corresponding to 200 kg. Teicoplanin is commercialized in Europe, Hong Kong, Korea, and the Middle East. It is at the late developmental clinical phase in North America and Japan. Avoparcin is used as a growth promoting feed additive (see... 

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. 

Fig. 2-3. The enantiomeric sepai ation of profens on vancomycin CSP (250 x 4.6 mm) with the same mobile phase composition tetrahydrofu-ran 20 mM sodium citrate (10/90 v/v) pH 6.3. The flow rate was 1.0 mL min at ambient temperature (23 C).

When analytes lack the selectivity in the new polar organic mode or reversed-phase mode, typical normal phase (hexane with ethanol or isopropanol) can also be tested. Normally, 20 % ethanol will give a reasonable retention time for most analytes on vancomycin and teicoplanin, while 40 % ethanol is more appropriate for ristocetin A CSP. The hexane/alcohol composition is favored on many occasions (preparative scale, for example) and offers better selectivity for some less polar compounds. Those compounds with a carbonyl group in the a or (3 position to the chiral center have an excellent chance to be resolved in this mode. The simplified method development protocols are illustrated in Fig. 2-6. The optimization will be discussed in detail later in this chapter. 

Similar to the new polar organic mode, the retention of analytes in normal phase is not difficult to predict. For all the compounds, the average of the retention on individual columns is fairly close to the retention on the coupled columns. The selectivity of most compounds on coupled columns is an average of the selectivities of individual columns (Fig. 2-9). However, it was found that the elution order for some compounds was reversed on ristocetin A and teieoplanin or vancomycin. As a result. 

Fig. 2-9. Chromatograms of phensuximide in normal phase on vancomycin (A), teicoplanin (B), ristocetin A (C), vancomycin + teicoplanin (D), ristocetin A + vancomycin (E), ristocetin A + teicoplanin (F), and ristocetin A + vancomycin + teicoplanin (G). All columns were 100 x 4.6 mm. The numbers by the peaks refer to the retention time in minutes. The mobile phase was ethanol hexane (60/40 v/v) and the flow rate was 1.5 mL min at ambient temperature (23 °C).

Fig. 2-10. The effect of flow rate on the resolution of methylphenidate enantiomers on vancomycin CSP (250 x 4.6 mm). The mobile phase was methanol 1.0 % triethyl-ammonium acetate (95/5 v/v) pH 4.1 at ambient temperature (23 °C).

For most free amino acids and small peptides, a mixture of alcohol with water is a typical mobile phase composition in the reversed-phase mode for glycopeptide CSPs. For some bifunctional amino acids and most other compounds, however, aqueous buffer is usually necessary to enhance resolution. The types of buffers dictate the retention, efficiency and - to a lesser effect - selectivity of analytes. Tri-ethylammonium acetate and ammonium nitrate are the most effective buffer systems, while sodium citrate is also effective for the separation of profens on vancomycin CSP, and ammonium acetate is the most appropriate for LC/MS applications. 

Macrocyclic glycopeptides. The first of these CSPs - based on the cavity of the antibiotic vancomycin bound to silica - was introduced by Armstrong [25]. Two more polycyclic antibiotics teicoplanin and ristocetin A, were also demonstrated later. These selectors are quite rugged and operate adequately in both normal-phase and reversed-phase chromatographic modes. However, only a limited number of such selectors is available, and their cost is rather high. 

The reaction can be carried out efficiently using aryl diazonium tetrafluoroborates with crown ethers, polyethers, or phase transfer catalysts.103 In solvents that can act as halogen atom donors, the radicals react to give aryl halides. Bromotrichloromethane gives aryl bromides, whereas methyl iodide and diiodomethane give iodides.104 The diazonium ions can also be generated by in situ methods. Under these conditions bromoform and bromotrichloromethane have been used as bromine donors and carbon tetrachloride is the best chlorine donor.105 This method was used successfully for a challenging chlorodeamination in the vancomycin system. 

Fig. 2-5. Examples showing the complementary separations on glycopeptide CSPs. (A) Separation of N-CBZ-norvaline on vancomycin (left) and teicoplanin (right). The mobile phase was methanol 1 % triethylammonium acetate (20/80 v/v) pH 4.1. (B) Separation of warfarin on teicoplanin (left) and vancomycin (right) CSPs. The mobile phase was acetonitrile 1 % triethylammonium acetate (10/90 v/v) pH 4.1. (C) Separation of naproxen on teicoplanin (left) and ristocetin A (right). The mobile phase was methanol 0.1 % triethylammonium acetate (30/70 v/v) pH 4.1. All columns were 250 x 4.6 mm i.d. The flow rate for all the separations was 1 mL min1 at ambient temperature (23 °C).

Fig. 2-15. Reversed-phase retention of the first eluted and the second eluted enantiomers of 5-methyl-5-phenylhydantoin as a function of mobile phase composition. The column was a 250 x 4.6 mm vancomycin CSR The buffer was triethylammonium acetate at pH 7.0. The flow rate was 1.0 mL min-1 at ambient temperature (23 °C).

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