Reversed-phase selectivity

Sugar analysis by hplc has advanced greatly as a result of the development of columns specifically designed for carbohydrate separation. These columns fall into several categories. (/) Aminopropyl-bonded siHca used in reverse-phase mode with acetonitrile—water as the eluent. (2) Ion-moderated cation-exchange resins using water as the eluent. Efficiency of these columns is enhanced at elevated temperature, ca 80—90°C. Calcium is the usual counterion for carbohydrate analysis, but lead, silver, hydrogen, sodium, and potassium are used to confer specific selectivities for mono-, di-, and... 

An on-line concentration, isolation, and Hquid chromatographic separation method for the analysis of trace organics in natural waters has been described (63). Concentration and isolation are accompHshed with two precolumns connected in series the first acts as a filter for removal of interferences the second actually concentrates target solutes. The technique is appHcable even if no selective sorbent is available for the specific analyte of interest. Detection limits of less than 0.1 ppb were achieved for polar herbicides (qv) in the chlorotriazine and phenylurea classes. A novel method for deterrnination of tetracyclines in animal tissues and fluids was developed with sample extraction and cleanup based on tendency of tetracyclines to chelate with divalent metal ions (64). The metal chelate affinity precolumn was connected on-line to reversed-phase hplc column, and detection limits for several different tetracyclines in a variety of matrices were in the 10—50 ppb range. 

Hplc techniques are used to routinely separate and quantify less volatile compounds. The hplc columns used to affect this separation are selected based on the constituents of interest. They are typically reverse phase or anion exchange in nature. The constituents routinely assayed in this type of analysis are those high in molecular weight or low in volatility. Specific compounds of interest include wood sugars, vanillin, and tannin complexes. The most common types of hplc detectors employed in the analysis of distilled spirits are the refractive index detector and the ultraviolet detector. Additionally, the recent introduction of the photodiode array detector is making a significant impact in the analysis of distilled spirits. 

The understanding of retention and selectivity behaviour in reversed-phase HPLC in order to control and predict chromatographic properties ai e interesting for both academic scientists and manufacturers. A number of retention and selectivity models are the subject of ongoing debate. The theoretical understanding of retention and selectivity, however, still lags behind the practical application of RP HPLC. In fact, many users of RP HPLC techniques very often select stationary phases and other experimental conditions by experience and intuition rather than by objective criteria. 

A selective, sensitive and stability indicating reversed phase-HPLC method was developed for the determination of clarithromycin antibiotic in human plasma. 

Cytochalasin B (from dehydrated mould matter) [14930-96-2] M 479.6. Purified by MeOH extraction, reverse phase Cl8 silica gel batch extraction, selective elution with 1 1 v/v hexane/tetrahydrofuran, crystn, subjected to TLC and recrystallised [Lipski et al. Aruil Biochem 161 332 1987]. 

In most situations the eluent composition is chosen to minimize the effects of hydrophobic interaction, but these secondary effects can be used to advantage. By careful selection of a salt and its concentration, specific selectivities for analytes can be achieved without the use of organic solvents. Therefore, many separations usually run by solvent gradient reversed-phase methods can be completed with a purely aqueous isocratic eluent (13,14). 

D. E. Martire and R. E. Boehm, A unified theory of retention and selectivity in liquid chromatography. 2. Reversed-phase liquid clrromatography with chemically bonded phases , J. Phys. Chem. 87 1045-1062 (1983). 

H. M. M. Arafa, E. M. A. Hamada, M. M. A. Elzamai and H. Nau, Eully automated detemination of selective retinoic acid receptor ligands in mouse plasma and tissue by reversed-phase liquid chi omatography coupled on-line with solid-phase extraction , 7. Chromatogr. A 729 125-136 (1996). 

Figure 13.7 Selectivity effected by employing different step gradients in the coupled-column RPLC analysis of a surface water containing 0.40 p-g 1 bentazone, by using direct sample injection (2.00 ml). Clean-up volumes, (a), (c) and (d) 4.65 ml of M-1, and (b) 3.75 ml of M-1 transfer volumes, (a), (c) and (d), 0.50 ml of M-1, and (b), 0.40 ml of M-1. The displayed cliromatograms start after clean-up on the first column. Reprinted from Journal of Chromatography, A 644, E. A. Hogendoom et al, Coupled-column reversed-phase liquid chromatography-UV analyser for the determination of polar pesticides in water , pp. 307-314, copyright 1993, with permission from Elsevier Science.

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. 

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). 

In addition to temperature and flow rate, the retention and selectivity in reversed phase are controlled by (i) the concentration and type of organic modifier and (ii) the type, concentration and pH of the buffer. 

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