Retention factor in HPLC

Nonlinear regression Nonlinear regression Nonlinear regression Nonlinear regression x-reciprocal 74 addition of urea, - -correction of velocities 75 76 influence of counterions, comparison with retention factors in HPLC 77 pH dependence 49 pH and T dependence of KB... 

In view of Eq. (1), the retention factor in HPLC can be expressed in terms of the two virtual length components as [1]... 

The k" measures the magnitude of retention in CEC due to reversible binding of the analyte to the stationary phase. Inspection of Eq. (11) shows that for all components (neutral or charged), k" is always positive, as a chromatographic retention factor should be. Further, while the retention factor in HPLC and the velocity factor in CZE are able to characterize the respective differential migration processes alone, both of them are required to characterize CEC. 

Similar to the retention factor in HPLC the retardation factor Rf describes retention behavior during TLC experiments. It represents the ratio of the distance migrated by the sample to the distance traveled by the solvent front. Boundaries are 0 < R < 1. For Rf = 0, the product does not migrate from the origin and for R = 1, the product is not retained. Rf values are calculated to two decimal places, while some authors prefer to tabulate values as whole numbers, as hR values (equivalent to 100Rf) (Poole, 2003). 

Martel, S., Carrupt, P. A. Retention factors in RP-HPLC as lipophilicity indices comparison between extrapolated and non-extrapolated logfc . Unpublished results. 

First, we will explore the three fundamental factors in HPLC retention, selectivity, and efficiency. These three factors ultimately control the separation (resolution) of the analyte(s). We will then discuss the van Deemter equation and demonstrate how the particle diameter of the packing material and flow rate affect column efficiencies. 

The parameters basic to this discussion are the resolution coefficients (a), defined as the ratio of the retention volumes, measured from the air or solvent peak in GC, and of the capacity factors in HPLC. From these data, readily obtained from the chromatograms (see e.g. Fig. 1), also the difference in free energy of solution or adsorption of the enantiomers can be calculated by the relationship AAG = -RTlna. 

Capacity factor The parameter used in HPLC to measure the retention of an analyte. 

Temperature has an influence on the retention and consequently on the capacity factors of carotenoids in HPLC columns. Usually, as the column temperature increases, the retention decreases however, in a polymeric C30 column, after an initial decrease of the t values of cis isomers of carotenoids, the retention of cis isomers actually increases at temperatures above 35°C. This different behavior can be explained by the increased order and rigidity of the C30 stationary phase at lower temperatures that in turn induce preferential retention of long, narrow solutes as the trans isomer and partial exclusion of bent and bulky cis isomers. The greater chain mobihty and less rigid conformation of the C30 at higher temperatures may increase the contact area available for interaction with the cis isomers and also may lower... 

In the simplest scheme of 2D HPLC, effluent of the first dimension (lst-D) was directly loaded into an injector loop (500 pL) of the 2nd-D HPLC for 28 s, and 2 s were allowed for injection. This operation was accompanied by the loss of lst-D effluent for 2 s out of 30 s in each cycle. The flow rate of 10 mL/min allowed the elution of solutes having retention factors (k values) up to 8 for the 2nd-D within the 30-s separation window, with f0 of 3.5 s. Figure 7.7 a and b shows the chromatograms for the 1 st-D and the 2nd-D, respectively, obtained for a mixture of hydrocarbons and benzene derivatives. The lst-D chromatogram showed many overlapping peaks. PAHs were eluted as mixtures from the FR column, and some are separated in the 2nd-D. 

Methods similar to those used in GC are applicable to HPLC. Thus, comparison of retention data is the most useful means of qualitative identification, the retention factor, , generally being used in preference to... 

Finally, a parameter known as the capacity factor may be determined. The capacity factor, symbolized k (k-prime) is the adjusted retention time divided by the retention time of an unretained substance, tM, such as air in GC or the sample solvent in HPLC. 

The retention of analyses in RP-HPLC markedly depends on the adsorption of the organic constituent of the mobile phase on the surface of the stationary phase. The excess adsorption isotherms of ACN, THF and methanol were measured on silica support modified with C, C6, C8, C10, C12 and C18 monomeric phase and a model was developed for the description of the retention of solutes from the binary mobile phase. The dependence of the retention factor on the partition coefficient can be described by... 

Pirkle and coworkers [59] compared retention and selectivity factors between HPLC and SFC using Poly Whelk-O chiral stationary phases and a-naphthyl-1-ethylamine carbamates. The results indicate that both retention and selectivity factors in SFC were higher than those in HPLC. This can be mainly attributed to the weaker solvating power of the carbon dioxide supercritical fluid as compared to a liquid such as methanol or hexane. 

The effectiveness of the separation (R ) in HPLC analysis is dependent on both thermodynamic factors (retention and selectivity) and kinetics factors (peak width and column efficiency)d° The relationship of resolution to other parameters can be expressed somewhat quantitatively in the resolution equation ... 

The silanol induced peak tailing is also a function of the pH of the mobile phase. It is much less pronounced at acidic pH than at neutral pH. Therefore many of the older HPLC methods use acidified mobile phases. However, pH is an important and very valuable tool in methods development. The selectivity of a separation of ionizable compounds is best adjusted by a manipulation of the pH value. The retention factor of the non-ionized form of an analyte is often by a factor of 30 larger than the one of the ionized form, and it can be adjusted to any value in between by careful control of the mobile phase pH. This control must include a good buffering capacity of the buffer to avoid random fluctuations of retention times. 

By simultaneous optimization of the percent organic modifier in the eluent and the column temperature to keep the retention factors fixed, very efficient, ultrafast separation can be achieved. The researchers conclude that for fast separations, the relationship between retention, temperature, and volume fraction of organic modifier needs to be taken into account. As the temperature increases, a lower volume of organic modifier is needed to speed up HPLC. Therefore, a highly retentive column... 

When a combination of CE and HPLC systems would be considered, the most dissimilar from the global set could be selected according to the above approach. For the CE methods, a response should be selected and applied with values in the same order of magnitude as the retention factors of the CSs, e.g., the migration times. Another possibility would be to use the so-called normalized migration indexes (see further Section III.C) for both the CE and the HPLC measurements. ... 

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