Elution order and relative retention

Some optimization system models rely on predetermined libraries of compounds already calibrated on several common stationary phases. If a peak of interest is in such a library and the analyst is using one of the characterized phases, then no additional calibration may be necessary as long as (1) the calibration column and the experimental column dimensions are known with good accuracy, (2) the calibration gas chromatograph s oven temperature and experimental oven temperatures are standardized, and (3) the pressure drops and ambient pressures for the calibration and experimental systems are known accurately. If not, then the simulations will be less accurate. However, small errors in these areas will not distort simulated results so much that peak elution order and relative retention will be meaningless. Even when not exact down to the second, simulations provide a wealth of useful information about peak retention behavior under a range of test conditions. 

Up to this point we have discussed the optimization of gas chromatographic separations by manipulation of the column variables that do not affect peaks relative retentions. Changing the column dimensions, the stationary-phase film thickness or the carrier-gas velocity will affect retention times, but the peaks thermodynamic partition coefficients (K) remain constant a long as the colunm temperature and the stationary-phase chemistry remain unchanged. As a result, the peaks relative retentions—the ratios of their adjusted retention times (t )—also will not be affected by such manipulations, and so the peaks elution order and relative separations remain unchanged. This makes prediction of the effects of modifying these variables fairly simple to compute using relationships such as those presented thus far in this chapter. 

It should be noted that in this relative retention volume range elution does not necessarily take place in order of decreasing molecular size, because, as seen from Figure 12, functional groups may have a greater effect on elution behavior than molecular size (Forss, K. Talka, E., The Finnish Pulp and Paper Research Institute, unpublished results). 

Identification. The order of elution of isoflavones is largely independent of minor variations in the solvent system, and thus it is possible to make tentative identifications by comparing relative retention times and co-elution with pure isoflavone standards. The actual retention times will vary between different runs, usually within 1 min. Such variation could be cau sed by differences in mobile phase prepa-... 

Compound retention during RP-HPLC depends on the relative hydrophobicity of the sample compounds. As expected, the elution of phenolics for reversed-phase HPLC is in the order of decreasing polarity. Polarity is increased most by hydroxyls at the 4-position, followed by those at the 2- and 3-positions. Availability of the methoxy group and the acrylic substitution reduces polarity and increases retention times (4). Loss of polar hydroxy groups and/or addition of methoxy groups can decrease the polarity within each class of benzoic and cinnamic acid. Also, the presence of the ethylenic side chain in the cinnamic acids can reduce their polarity compared with similarly substituted benzoic acids (6). The elution order for benzoic acids is as follows (Table 1) gallic > a-resorcylic > protocatechuic > y-resorcylic > gentisic > p-hydroxyben-... 

The next step is to identify each peak. You dilute and inject each compound separately and compare retention times to identify the standard mixture run. The elution order is B, A, C, and D. The last thing you need to find is an internal standard with a retention time just longer than compound D. You find compound IS, make a 1-g/L solution. You will add 1 mL to each standard mixture before dilution. You run a standard run with IS and calibrate each peak relative to the peak height of IS (Fig. 12.3e). 

Since pH conditions can be readily chosen to ensure that peptides are significantly ionized, peptide elution orders from nonpolar phases with ion-pairing elution systems will be determined by their relative hydro-phobicities and the polarity or charge density of the counterion. Major changes in selectivity and retention times of peptides on reversed-phase... 

A database of relative retention times and co-elu-tions for all 209 congeners on 20 different stationary phases has been published [8]. For 12 of the most useful of these phases, the elution orders of 9 solutions of... 

The application of thermal FFF to a variety of colloids and particles has been demonstrated in both aqueous and organic carrier liquids. Figure 2 illustrates the dependence of retention on the surface composition of polystyrene particles. The three particles are similar in size, but the surface of one of the samples has been carboxylated, whereas another has been am-inated. The relative elution order of the three particles can be changed by modifying the carrier liquid [13]. 

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