Retention factor resolution

Table 17-4 summarizes the qualitative influence of changing the gradient parameters on resolution, retention factor, and run time. The bold rows of Table 17-4 offer the best approach to reducing analysis time and will be discussed in more detail. The nonbold rows represent parameters that should be optimized during the initial stages of method development to optimize resolution. 

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... 

According to Equation 3, the resolution of two peaks in column separation is controlled by three major variables retention defined in terms of the retention factor k column efficiency expressed as the number of theoretical plates N and selectivity characterized by the selectivity factor a [48] ... 

The modern analytical laboratory employing instrumental chromatography uses a computer data collection system and associated software to acquire the data and display the chromatogram on the monitor. Parameters important for qualitative and quantitative analysis, including retention times and peak areas, are also measured and displayed. The software can also analyze the data to determine resolution, capacity factor, theoretical plates, and selectivity. 

The resolution can be improved by increasing the column plate number, N, and/ or the separation factor, a (a = the ratio of the retention factors of the two compounds). N is the physical parameter and a is the chemical parameter for the separation. Higher N and a values give a better separation. 

On the other hand, optionally added co-ions of the eluent may also interfere with the ion-exchange process through competitive ion-pairing equilibria in the mobile phase. The effect of various amines added as co-ions to the polar-organic mobile phase was systematically studied by Xiong et al. [47]. While retention factors of 9-fluorenylmethoxycarbonyl (FMOC)-amino acids were indeed affected by the type of co-ion, enantioselectivities a and resolution values Rs remained nearly constant. For example, retention factors k for FMOC-Met decreased from 17.4 to 9.8 in the order... 

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]. 

FIGURE 4.1 Effect of the plate number (N), the separation factor (a ), and the retention factor (k) on resolution (Rs). (Adapted from Sandra, P.J. 1989. High Resolut. Chromatogr. 12 82-86. With permission.)... 

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

Efficiency or plate count (N)—an assessment of column performance. N should be fairly constant for a particular column and can be calculated from the retention time and the peak widths. Selectivity (a)—the ratio of retention k ) of two adjacent peaks. Sample capacity— the maximum mass of sample that can be loaded on the column without destroying peak resolution. Capacity factor k )—a measure of solute retention obtained by dividing the net retention time by the void time. 

In the System Suitability section, different parameters are described which can be applied in order to check the behavior of the CE system. The choice of the appropriate parameters depends on the mode of CE used. The system suitability parameters include retention factor (k) (only for MEKC), apparent number of theoretical plates (N), symmetry factor (Af), resolution (Rs)> Rtea repeatability, migration time repeatability, and signal-to-noise ratio. Practical equations to calculate different system suitability parameters from the electropherograms are presented, which are also included in Table 3. 

DL-Leucine, resolution of, 222, 262 Leucine encephalin, 290 LFER, see Linear free energy relationships LH-releasing hormone, 263, 290 Ligate, carbon number of, 153 Ligates, solute binding to, 213 Limiting retention factors, 239 Linear elution adsorption chromatography, 58... 

Gant et al. (175) examined the effect of temperature on resolution and on selectivity, retention factors, and plate number, which determine the magnitude of resolution. They found that these data can be used together with the lempeniliire dependence of solvent viscosity to optimize iinaivsis rate with required resolution. This is of particular interest when RFC is used for automated repetitive analyses of lar e numbers of samples. 

Hong et al. applied capillary EKC with dodecyltrimethylammonium-bromide/sodium dodecylsulfate (12.7/21.1 mM) vesicles to the separation of alkylphenones (Fig. 8A) and obtained better resolution than with sodium dodecylsulfate micelles (59). The logarithms of the retention factors for 20 neutral compounds of similar structures showed an excellent linear correlation with log Poct (R2 = 0.98). Similarly, Razak et al. (60) showed that the log capacity factors for interaction between neutral and positively charged analytes and cetyltrimethylammoniumbromide/sodium octylsulfate vesicles correlated linearly with the log Poa values. 

EKC in the reversed direction mode is performed when analytes and pseudostationary phase move at different velocities in the same direction, which is opposite to that of EOF. In this case, retention factor and resolution are expressed by the following equations [211] ... 

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. 

Shown in Figure 10 is the chromatogram acquired at the optimum predicted by CRF-4. Baseline resolution of all 8 components was achieved in about 27 minutes, except for components 2-4 which were almost baseline resolved. Additional evidence for the accuracy of the retention model (equation 9 and Table VI) employed for this window diagram optimization is evident in Table VIII, where predicted and measured retention factors differed by less than 15%. The slight positive bias observed for all solutes at the optimum conditions in Table VIII was coincidental averaged over the entire parameter space the bias was almost completely random. 

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