Polar organic mode

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... 

Statistically, of the compounds enantioresolved by macrocyclic glycopeptide CSPs, new polar organic mode accounts for more than 40 %, balanced by reversed-phase mode, while typical normal-phase operation resulted in approximately 5 % of separations. Some categories of racemic compounds that are resolved on the glycopeptide CSPs at different operating modes are listed in Table 2-4. 

In the new polar organic mode, the ratio of acid/base in the mobile phase affects the selectivity and the concentration of acid and base controls the retention. It is suggested to start the method development with a medium concentration (0.1 %) for both acid and base. If retention is too long or too short, the concentration can be increased to 1 % or reduced to 0.01 %. If no selectivity is observed in this mode, reversed phase is recommended as the next step in the protocols. 

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. 

One potential problem associated with column coupling in reversed phase is relatively high back-pressure ( 2600 psi at 1 mL miir ). This will place a limit on the flow rate, which in turn limits the further reduction of analysis time. Also, compared to the new polar organic mode, the retention in reversed phase on coupled columns is deviated more from the average retention on the individual stationary phases. 

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. 

This is because the increased turbulence from higher flow rates decreases the possibility for inclusion complexation, a necessary event for chiral recognition in reversed phase. Some effect has also been observed in the new polar organic mode when (capacity factor) is small (< 1). Flow rate has no effect on selectivity in the typic normal-phase system, even at flow rates up to 3 inL miir (see Fig. 2-11). 

Optimization of Enantiomeric Separations in the New Polar Organic Mode... 

Fig. 2-12. The effect of the acid/ base ratio on the selectivity of sotalol on teicoplanin CSP (250 x 4.6 mm) in the new polar organic mode. The flow rate was 1.0 mL min at ambient temperature (23 °C).

This relatively new class of CSPs incorporates glycopeptides attached covalently to silica gel. These CSPs can be used in the normal phase, reversed phase, and polar organic modes in LC [62]. Various functional groups on the macrocyclic antibiotic molecule provide opportunities for tt-tt complexation, hydrogen bonding, and steric interactions between the analyte and the chiral selector. Association of the analyte... 

New polar organic mode a. Type of acid and base... 

In achiral-chiral LC-LC, the mobile phases used with the achiral and chiral columns must be miscible with one another. Since the enantiomeric separation is usually the most difficult to optimize, it is usually the separation that dictates the mode of operation of the total analysis. Thus, it makes sense that a chiral column that operates in the normal phase mode would require an achiral column that also works in the normal phase mode. Polar organic mode chiral separations are universal in that they can be paired with an achiral column that operates in either the reverse phase or normal phase mode. The choice of the achiral column is always determined after selecting the chiral column and the mode of operation. As with traditional liquid chromatography, different achiral columns will give different selectivity. 

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. 

Ekgorg-Ott et al. (1997). An interesting trend was discovered when considering the relative amount of D-theanine present in the samples. The teas of the highest grades consistently contained the lowest amounts of D-theanine. The theanine achiral-chiral system configuration included a C18 column operated in the reverse-phase mode and a y-cyclodextrin CSP in the polar organic mode. 

FIGURE 1.32 Stereoisomer separations of chrysanthemic acid (a) and fenvaleric acid (b) employing an 0-9-(tert-bntylcarbamoyl)qninine-based CSP in the polar-organic mode (a) and an 0-9-(2,6-diisopropylphenylcarbamoyl)quinine based CSP (b). Experimental conditions Column dimensions, 150 mm x 4mmID eluents(a)0.06%aceticacidin acetonitrile-methanol (95 5 v/v) (b) acetonitrile-0.3 M ammoninm acetate buffer (90 10 v/v) (pHa 6.0) flow rate, 0.65 mLmin temperature, 25°C detection, UV at 230 nm. (Reproduced from W. Bicker et al., J. Chromatogr. A, 1035 37 (2004). With permission.)... 

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