Isocratic system retention factor

The variety of stationary phases available commercially and the lack of standardisation between different laboratories makes it difficult to compare the retention factors k directly as comparison of lipophilicity. Therefore different lipophilicity scales obtained from chromatographic data (isocratic or gradient elution) have been introduced. A calibration of the individual chromatographic system with compounds of known lipophilicity is used in all cases. 

Once the probe gradient is run, check the diode array purity and if LC-MS is available, run as well to check for peak homogeneity. If you have any known precursors or impurities, run them as well to ensure resolution from the main component and to make sure they are adequately retained. The main analyte should elute between k 2-5. If the main component elutes at A = 2-5 and is spectrally pure and the impurities all elute k > 1, the method is complete. If the retention factor of the impurities is below 1, then an isocratic hold at the initial organic composition should be implemented until the minor component (impurity) elutes k > 1 and then a linear gradient can be implemented. The method could be further optimized by increasing the flow rate as long as the backpressure limitation of the system has not been reached. A general rule of thumb is that the backpressure should not exceed 85% of the maximum backpressure for a particular HPLC system. 

Similar to the log D-pH profile, the distribution of the compounds in a chromatographic partition system is also influenced by the pH. Charged species have much shorter retention times than their uncharged parent compounds. Horvath et al. [105 described first the effect of solute ionisation on the retention of weak acids, bases and ampholytes on octadecyl silica, both theoretically and experimentally. They have found a similar equation to Eqs. (12.11) and (12.12) that describes the pH dependence of the isocratic retention factor (A) for weak acids as shown in Eq. (12.1.3) ... 

Seshadri and Deming [44] have used the Craig model to calculate band profiles in chromatographic systems. However, they selected an unrealistic isotherm, (Ji = ( j + biCj)Ci, i.e., an isotherm which, for each component i, is linear in respect to its concentration, but with a retention factor that is a linear function of the other component concentration. There is little physical basis for this model, and this prevented them from deriving any useful conclusions. More recently, Eble et al. [45] have used the Craig model to calculate band profiles in isocratic elution and to develop general correlations between the sample size on the one hand and the apparent retention factor and the column efficiency on the other. Experimental data confirm the approximate validity of the relationships obtained [24,25] (Eig-ure 10.3). The use of such empirical relationships allowed an estimation of the band shape on a personal computer for column efficiencies not exceeding a few hxmdred theoretical plates. 

Figure 2.31 Peak capacity as a function of analytical run time. The graph is valid for isocratic reversed-phase systems which are run at their van Deemter optimum. The maximum retention factor /ris 20, i.e. the maximum retention time is to 21, then the separation ends. The figure is only valid for small analytes with a diffusion coefficient ofapprox. 1-10 m s and not for macromolecules. Dotted lines represent the particle diameter, dashed lines the column length, and solid lines the pressure, respectively.

Here, a, b, and m are experimental constants depending on the solute and on the chromatographic system [a = l/(fea) , fea is the retention factor in pure non-polar solvent]. Usually, Eq. 7 slightly improves the description of the experimental data with respect to Eq. 5. In NP systems where Eq. 7 applies under isocratic conditions, the elution volume Ur of a sample solute in NPLC with... 

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