Mobile glycopeptides

The macrocyclic glycopeptides CSPs arc capable of operating in three different mobile phase systems reversed phase, normal phase, and the new polar organic mode. The new polar organic mode refers to the approach when methanol is used as the mobile phase with small amounts of acid and/or base as the modifier to control... 

The enantioselectivity of the macrocyclic CSPs are different in each of the operating modes, probably because of different separation mechanisms functioning in the different solvent modes. The possible chiral recognition mechanisms for three mobile phase compositions on glycopeptide phases are listed in Table 2-3 in descending order of strength. 

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. 

Typical normal-phase operations involved combinations of alcohols and hexane or heptane. In many cases, the addition of small amounts (< 0.1 %) of acid and/or base is necessary to improve peak efficiency and selectivity. Usually, the concentration of polar solvents such as alcohol determines the retention and selectivity (Fig. 2-18). Since flow rate has no impact on selectivity (see Fig. 2-11), the most productive flow rate was determined to be 2 mL miiT. Ethanol normally gives the best efficiency and resolution with reasonable back-pressures. It has been reported that halogenated solvents have also been used successfully on these stationary phases as well as acetonitrile, dioxane and methyl tert-butyl ether, or combinations of the these. The optimization parameters under three different mobile phase modes on glycopeptide CSPs are summarized in Table 2-7. 

Table 2-3. Possible separation mechanisms for three mobile phase compositions on glycopeptide CSPs.

Table 2-4. Typical categories of racemic compounds resolved on glycopeptide CSPs in three mobile phase modes.

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).

Because plasma and urine are both aqueous matrixes, reverse-phase or polar organic mode enantiomeric separations are usually preferred as these approaches usually requires less elaborate sample preparation. Protein-, cyclodextrin-, and macrocyclic glycopeptide-based chiral stationary phases are the most commonly employed CSPs in the reverse phase mode. Also reverse phase and polar organic mode are more compatible mobile phases for mass spectrometers using electrospray ionization. Normal phase enantiomeric separations require more sample preparation (usually with at least one evaporation-to-dryness step). Therefore, normal phase CSPs are only used when a satisfactory enantiomeric separation cannot be obtained in reverse phase or polar organic mode. 

A simple and efficient alternative to the traditional UV detection of amino acids and related compounds is nowadays represented by the evaporative light scattering (ELS) detector, which allows the direct chromatographic separation, with no need for preliminary derivatization. In the field of glycopeptides-based CSPs, it was applied for the first time in the chromatographic resolution of carnitine and 0-acylcarnitine enantiomers on a TE CSP [61]. The considered compounds are nonvolatile solids and gave optimal ELS response under a variety of experimental conditions (buffered and unbuffered mobile phases, flow-rates from 0.5 to 1.5 mL/min, different kind and... 

A set of sulfoxides, tosylated sulfilimines, and sulfinate esters were separated using five different commercially available glycopeptides CSPs, namely ristocetin A, tei-coplanin, TAG, vancomycin, and VAG, and seven mobile phases (three NP, two RP,... 

Figure 4.10 shows the effect of additive concentration on the separation of clen-buterol enantiomers on a polysaccharide-based chiral stationary phase [79]. The peak shapes were dramatically improved by adding an amine additive and the separation time was also reduced from 14 to 7 min when 1.0% amine was added to the mobile phase. Phinney and Sander [100] investigated the effect of amine additives using chiral stationary phases having either a macrocyclic glycopeptide or a... 

Macrocyclic Glycopeptide Antibiotic Mobile Phase Additives... 

MACROCYCLIC GLYCOPEPTIDE ANTIBIOTIC MOBILE PHASE ADDITIVES... 

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. 

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