Column selectivity effects

Equations 12.21 and 12.22 contain terms corresponding to column efficiency, column selectivity, and capacity factor. These terms can be varied, more or less independently, to obtain the desired resolution and analysis time for a pair of solutes. The first term, which is a function of the number of theoretical plates or the height of a theoretical plate, accounts for the effect of column efficiency. The second term is a function of a and accounts for the influence of column selectivity. Finally, the third term in both equations is a function of b, and accounts for the effect of solute B s capacity factor. Manipulating these parameters to improve resolution is the subject of the remainder of this section. 

An example may show how the different concepts come into effect in a real-life laboratory environment. This example is based on column selections that many laboratories use for ordinary, general-purpose work. 

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.

Column selection remains the most important factor in successful enantiomeric separations. The CSPs most likely to be effective in SFC are those that have been employed under normal phase conditions in LC. In fact, the tremendous body of knowledge that has been accumulated for LC can also guide column selection in SFC [66]. The likelihood of success with a particular CSP can generally be gauged after one or two injections [67]. If no evidence of separation is observed, another CSP should be investigated. 

Trying to determine which column is ideal for a specific analysis can be difficult with over 1000 different columns on the market [74]. A proper choice implies a definition of parameters such as column material, stationary phase (polarity), i.d., film thickness and column length. Guides to column selection are available [74,75]. The most important consideration is the stationary phase. When selecting an i.d., sample concentration and instrumentation must be considered. If the concentration of the sample exceeds the column s capacity, then loss of resolution, poor reproducibility and peak distortion will result. Film thickness has a direct effect on retention and the elution temperature for each sample compound. Longer columns provide more resolving probe, increase analysis times and cost. 

Obviously, the magnitude of the temperature effect on retention depends on the difference in the enthalpy of the solute in either phase, and is specific for each solute. Therefore, it also changes the column selectivity. There is no retention and no temperature effect for AH=0. 

Sander, L.C. and Wise, S.A., Effect ofphase length on column selectivity for the separation of polycychc aromatic hydrocarbons by reversed-phase hquid chromatography, Anal. Chem., 59, 2309, 1987. 

An advanced type of column selectivity is chiral discrimination. Since enantiomers have identical physical properties they are not separable on conventional GC columns. However, if chiral analytes are allowed to interact with a chiral environment they will form transitory diastereomeric complexes which result in their being retained by the column to a different extent. As increasing numbers of enantiomerically pure drugs are synthesised in order to reduce side-effects, this type of separation will become increasingly important. 

Tests of the reproducibility of retention times, retention factors, separation selec-tivities, and column efficiencies for our methacrylate monolithic capillary columns are summarized in Table 6.2. This table shows averaged data obtained for 9 different analytes injected 14 times repeatedly every other day over a period of 6 days, as well as for 7 different capillary columns prepared from the same polymerization mixture. As expected, both injection-to-injection and day-to-day reproducibilities measured for the same column are very good. Slightly larger RSD values were observed for col-umn-to-column reproducibility. While the selectivity effectively did not change, larger differences were found for the efficiencies of the columns. 

Figure 3. Selectivity of modifier solvent (column 250 X 4.6 mm l.D. 10-pm Lichrosorb RP-8 other columns provide differing degrees of selectivity effects with...

Step 8. Pass each eluting agent through the resin column at flow of 5 mL per minute. Collect sequential 5-ml fractions of each eluent and measure the radio-iodine net count rate in each eluent fraction. Draw curve of net radioiodine count rate vs. eluted volume. Select the eluting solution that removes radio-iodine from the resin column most effectively, i.e., in the smallest volume. 

Column Selectivity. Separations can be effected by differences in partition coefficients as well as by the efficiencies of the columns in which they... 

Such approaches underpin the current popularity of RP-HPLC procedures for the purification of synthetic or recombinant polypeptides at the production scale, or analogous approaches employed in the HP-IEX of commercially valuable proteins. However, in some cases when linear scale-up methods are applied to higher molecular weight polypeptides or proteins, their biological activity may be lost due to unfavorable column residency effects and sorbent surface area dependencies. It is thus mandatory that the design and selection of preparative separation system specifically address the issues of recovery of bioactivity. Often some key parameters can be easily controlled, i.e., by operating the preparative separation at lower temperatures such a 4°C, or by minimizing column residency times. 

The final part of the chapter provides a refresher on pA from an analytical chemist s perspective, the drivers for choosing normal phase versus reversed phase as a separation mode for a particular analysis, and instru-ment/system consideration, and it concludes with a very interesting section on column testing within the framework of bonded phase stability (effect of pH and type of buffer) and probing column selectivity. 

Figure 6-8 Effect of selectivity and efficiency on chromatographic resolution. A, Poor resolution. B, Good resolution because of column efficiency. C, Good resolution because of column selectivity. (From Johnson EL, Stevenson R Basic liquid chromatography, Palo Alto, Calif, Varian Associates, 1978.)...

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