Retention and resolution

Enantioresolution in capillary electrophoresis (CE) is typically achieved with the help of chiral additives dissolved in the background electrolyte. A number of low as well as high molecular weight compounds such as proteins, antibiotics, crown ethers, and cyclodextrins have already been tested and optimized. Since the mechanism of retention and resolution remains ambiguous, the selection of an additive best suited for the specific separation relies on the one-at-a-time testing of each individual compound, a tedious process at best. Obviously, the use of a mixed library of chiral additives combined with an efficient deconvolution strategy has the potential to accelerate this selection. 

In 1987, Ken Setchell first described the method for the isolation of phytoestrogen in soy (Setchell et al., 1987). The phytoestrogens daidzein, genistein, coumestrol, formononetin, and biochanin-A were separated on a Cl8 reversed-phase column (Hypersil ODS) with methanol-0.1 M ammonium acetate buffer, pH 4.6 (60 40 v/v), as eluent. The retention and resolution were affected by buffer concentrations, pH type, and proportion of organic solvent in the mobile phase. Detection in the low picograms range was achieved with an electrochemical detector, and the compounds were positively identified by HPLC-thermospray mass spectrometry. 

TLC is a simple, cheap and fast analytical chemical technique which is often used for screening purposes. Due to the set-up of an TLC experiment, not only the mobile phase composition has influence on retention (and resolution) but also temperature and humidity. Of course, a... 

Case III chromatographic retention and resolution is sum total of nonstereoselective and stereoselective SO-SA interactions. 

WAX (weak anion exchange). Water only, retention and resolution are modified by pH and ionic strength Ionic compounds. 

Multiparameter Control of Retention and Resolution. Although the key SFC variables—density (pressure), temperature, composition, and their respective gradients—can be utilized individually to vary retention, selectivity, and hence resolution, they can be employed collectively to exert even greater control. The simultaneous use of two or three variables to vary retention and selectivity instead of one is, however, an obviously more complex situation. Under ideal circumstances (in the absence of interaction), the relationships expressed in equations 2-4 might be written as... 

Retention and resolution data corresponding to the chromatogram of figure 4.13. The relevant peaks and the relevant values for k and S are underlined, t was calculated from eqns.(4.55a) and (4.56a). r ( was calculated from eqn.(4.57). 

A strategy for the optimization of gradient programs based on the actual retention behaviour of some sample components has been described by Jandera and Chura5ek [623, 624]. This approach relies on the possibility to calculate retention and resolution under gradient conditions from known retention vs. composition relationships and plate numbers. Both typical RPLC (eqn.3.45) and LSC (eqn.3.74) relationships can be accommodated in the calculations and linear, convex and concave gradients are all possible because of the use of a flexible equation to describe the gradient function. This equation reads... 

Several modifications to the Fl-FFF method have been developed to enhance operation. Shortly after the introduction of Fl-FFF, the effect of relaxation on the retention and resolution was studied [245]. A substantial improvement in the... 

The effect of different chaotropic mobile-phase additives can also assist the chromatographer in achieving adequate retention and resolution of critical pairs in complex mixtures [156]. 

As shown in Figure 4-63 at an equimolar concentration of chaotropic mobile-phase additives, the greatest increases in retention and resolution between critical pairs of components was achieved employing a 30 mM PFe mobile-phase additive. An increase in retention and an increase in the peak efficiency were obtained, leading to an increase in the resolution of critical pairs of components (A/B and C/D). 

The surface of the silica may be dynamically coated with transition metals, and the selectivities observed can be attributed to the complexes between the metal ions and the analyte species [56], The use of silver-impregnated silica (adsorption of salts of transition metals on the silica surface) has been used for the analysis of saturated and unsaturated fatty acid methyl esters (FAME) and triacylglycerols (TAG) [57], The retention of the unsaturated FAME and TAG can be attributed to the stability of the complex that is formed between the K electrons of the carbon-carbon double bonds and the silver ions. The predominant interaction for saturated analytes is with the polar silanol groups. The secondary interactions are those of the silver ions with the unpaired electrons of the carbonyl oxygens of the analytes. The amount of silver adsorbed onto the silica and the pH (employment of acidic or basic modifiers) have been determined to have an effect on the retention and resolution of certain acidic and basic compounds and fatty acids [58]. 

The most evident design would appear to be a three-level factorial design. An example of a three-level factorial design is shown in Fig. 6.1.. A full three-level factorial design, 3, can be used to obtain quadratic models. However, unless / is small (/ = 2) the design requires a number of experiments (= V) that is not often feasible. An example of the use of a 3" design can be found in Ref. 51]. The two factors, the pH and the percentage acetonitrile were examined to evaluate their influence on the retention and resolution of three isoxazolyl penicillin antibiotics. The centre point experiment was triplicated to evaluate experimental error. 

The column is arguably the most important component in HPLC separations. The availability of a stable, high-performance column is essential for developing a rugged, reproducible analytical method. Performance of columns from different vendors can vary widely. Separation selectivity, resolution, and efficiency depend on the type and quality of the column. Proper column maintenance is the key to ensure optimum column performance as well as an extended column lifetime. It ensures stability of column plate number, band symmetry, retention, and resolution. The major issues related to column performance and maintenance are discussed here. 

How to Ensure Retention and Resolution Reproducibiiity of HPLC Coiumns... 

A reverse-phase HPLC procedure was proposed as a suitable method for the simultaneous determination of metformin and glibenclamide in a combined dosage form. The chromatographic conditions were adjusted to provide adequate retention and resolution of metformin and glibenclamide. A mixture of buffer and acetonitrile, in... 

Figure 2.10. Six RPLC chromatograms illustrating the effect of mobile phase solvent strength on solute retention and resolution. LC conditions were column Waters Symmetry C18, 3pm, 75x4.6 mm, 1 mL/min, 40°C, Detection at258nm. Mobile phase is mixture of acetonitrile (ACN) and water. Solutes were nitrobenzene (NB) and propylparaben (PP).

Figure 3.6. Examples illustrating the increase of retention and resolution with increasing surface area of the silica support. Diagram courtesy of Supelco Inc.

Temperature has only a small effect on retention and resolution in liquid chromatography, since the enthalpy of solution from the mobile phase to the stationary phase is quite small unlike it is the case for gas chromatography. Thus analysis by liquid chromatography is most often carried out at room or relatively low temperature. 

Table 24.4 summarizes the features of selected methods for the LC determination of carbamates in environmental samples. As can be seen, the most used reversed-phase chromatography is with Ci8 or Cg columns and aqueous mobile phases. Other reversed-phase chromatography such as with phenyl has also been used. Some methods use normal-phase silica or diol and nitrile." Sparcino and Hines studied the retention and resolution of 30 A-methylcarbamates in the normal (e.g., silica, cyanopropyl, and propylamine) and reversed-phase mode (Cig). The authors concluded that the reversed-phase columns generally provided better results and acetonitrile-water gave the best overall results on a Cig column. 

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