Separation time retention factor

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. 

As is well known, a good selectivity is not the same as a good separation in all cases. In addition to a low efficiency (band broadening, tailing), which can result in an insufficient resolution, a large retention factor (run time) also proves a disadvantage. As a criterion for the effectivity of a separation, the quotient of retention and separation factor could be used see the example in Fig. 14. 

The stationary phase is selected to provide the maximum selectivity. Where possible, the retention factor is adjusted (by varying the mobile phase composition, temperature, or pressure) to an optimum value that generally falls between 2 and 10. Resolution is adversely affected when k 2, while product dilution and separation time... 

In addition to the above strategies, the use of higher column temperatures is another approach that may decrease analysis time and improve sample throughput. The relationship between the chromatographic retention factor, k, and separation temperature is shown in Equation 13.1 ... 

Figure 7.2 Diagram of a TLC plate. The plate is suspended vertically in the solvent containing the compounds to be separated (the solvent level or origin), and over time the compounds and solvent migrate up the plate to different heights and are separated. The retention factor (Rf) for compound 1 is calculated as AB/ AD, and for compound 2 AC/AD.

Retention distance (or time) is normally used to aid the identification of a component of a mixture, provided that a known sample of the component has been subjected to separation under identical conditions. Because of the variations that can occur in the retention time due to technical factors, e.g. fluctuations in flow rate, condition of the column, the relative retention or selectivity factor (a) is sometimes used. This expresses the test retention time as a ratio of the retention time of another component or reference compound when both are injected as a mixture ... 

Typical NP conditions involve mixtures of n-hexane or -heptane with alcohols (EtOH and 2-propanol). In many cases, the addition of small amounts (<0.1%) of acid and/or base is necessary to improve peak efficiency and selectivity. Usually, the concentration of alcohols tunes the retention and selectivity the highest values are reached when the mobile phase consists mainly of the nonpolar component (i.e., n-hexane). Consequently, optimization in NP mode simply consists of finding the ratio n-hexane/alcohol that gives an adequate separation with the shortest possible analysis time [30]. Normally, 20% EtOH gives a reasonable retention factor for most analytes on vancomycin and TE CSPs, while 40% is more appropriate for ristocetin A-based CSPs. Ethanol normally gives the best efficiency and resolution with reasonable backpressures. Other combinations of organic solvents (ACN, dioxane, methyl tert-butyl ether) have successfully been used in the separation of chiral sulfoxides on five differenf glycopepfide CSPs, namely, ristocetin A, teicoplanin, TAG, vancomycin, and VAG CSPs [46]. 

Similarly, ahigh retention factor also favors high resolution. However, a high retention factor results in increased retention time. The separation time is given as... 

First, we look at isocratic separations. Let us assume that the analysis can be accomplished within a retention factor of 10. We also suppose that the analysis is carried out with a typical reversed-phase solvent and a sample with a typical molecular weight of a pharmaceutical entity. In order to manipulate the analysis time, we will keep the mobile phase composition the same and vary the flow rate. The maximum backpressure that we will be able to apply is 25MPa (250 bar, 4000psi). In Figure 1, we have plotted the plate count as a function of the analysis time for a 5 J,m 15-cm column. We see that the column plate count is low at short analysis times and reaches a maximum at an analysis time of about 1 h. A further increase in analysis time is not useful, since the column plate count declines again. This is the point where longitudinal diffusion limits the column performance. The graph also stops at an analysis time of just under 5 min. This is the point when the maximum allowable pressure drop has been reached. 

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. 

The maximum value of CRF-4 will be obtained for a separation that provides the required resolution in the shortest amount of time, assuming that column parameters remain the same. Note therefore, that the retention factors (k s) of equation 9 must be converted to retention times (t s) via the simple relationship tR = (1 + k ). Direct use of retention factors instead of times in equation 10a is not generally recommended unless it is known that t0 is constant over the range of densities and temperatures employed. 

As mentioned above, the basic principle of NLC is the same as for conventional techniques. The separation is identified and characterized by measuring retention times, capacity, separation, and resolution factors. Therefore, it is necessary to explain the chromatographic terms and symbols by which the chromatographic speciation can be understood and explained. Some of the important terms and equations of the chromatographic separations are discussed below. The chromatographic separations are characterized by retention (k), separation (a), and resolution factors (Rs). The values of these parameters can be calculated by the following standard equations [92]. 

Preparative-scale chromatography relies on a compromise between three variables (cf. Figure 1) (i) component resolution (determined by selectivity, efficiency and retention factor), (ii) speed of analysis and (iii) column sample capacity (Pescar, 1971). Any two of the desired goals may be realized only at the expense of the third. If a large amount of sample is required in a short time, resolution must be high. If resolution is insufficient, either the column load is limited or the time required for separation is long. 

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