Retention factor analyte

Other cases, involving an arbitrary relationship between the solute retention factor and the modulator concentration can be handled analytically using the approaches of Frey [Biotechnol. Bioeng., 35, 1055 (1990)] and Carta and Striugfield []. Chromatogr, 605, 151 (1992)]. 

Lochmiiller, C. H., Hsu, S.-H., and Reese, C., Prediction of the retention behavior of ionizable compounds in reversed-phase LC using factor-analytical modeling, /. Chromatogr. Sci., 34, 77, 1996. 

Where a, b, and c = van Deemter coefficients, dp = particle size of column, L = column length, Dm = diffusion coefficients of analytes, t = column dead time (depends on flow rate F), tg= gradient time (determines analysis time via tA = tg + t0), Ac = difference in concentrations of the organic modifier at the end and the beginning of the gradient (a continuous linear gradient is assumed), and B = slope of the linear relationship between the logarithm of the retention factor and the solvent composition. 

The adjusted retention time provides a measure of the strength of intermolecular interaction between the analyte and the stationary phase, with stronger interactions giving a longer time. The gas hold-up time is derived from the flow rate and the column dimensions and is often measured by injecting a non-retained compound. The retention factor, which represents a ratio of the mass of analyte dissolved in the stationary phase to the mass in the mobile phase, can be calculated from the adjusted retention time and the gas hold-up time. 

Common standard compounds for reversed phase columns are toluene and naphthalene, which have retention factors, k, of about 3. The eluent modifier is methanol or acetonitrile at a concentration of 50-80%, depending on the hydrophobicity of the stationary phase material. For other stationary phase materials, corresponding analytes, with k = 3-5, can be used. 

The carbon content of a stationary phase is measured by an elemental analyser, as a weight balance before and after heating at 800 °C. Particle size, pore size, and surface area are measured by specific instruments, such as a particle size analyser, nitrogen adsorption porosimeter, and mercury depression analyser, respectively. The precision of the measurement of carbon content is high however, that of the other measurements is relatively poor. Therefore, it is difficult to relate the surface area of different silica gels to analyte retention factors. 

The retention factor, k, is the basic value in chromatography, and is related to the void volume (dead volume). The void volume is the space inside the column, where no retention of solutes has occurred and can be measured on a chromatogram, as shown in Figure 1.3. The void volume is about half the total volume of the column when it is packed with porous stationary phase materials. In practice, the effective void experienced by the analyte is smaller because the molecular mass of the analyte is usually much greater than that of the eluent molecule. In a model of porous stationary phase material, the pores can be represented as V-shape valleys (Figure 3.8), where region a is a support, such as... 

The relationship of the selectivity to the polarity of the analytes can be understood from the differences in the retention factors of homologous alkanols (Figure 3.12). The polar alkanols are relatively more retained on the non-endcapped bonded phases (LOC-ODS-NE and HIC-ODS-NE) because smaller-size alkanols can reach the unreacted silanol groups on the surface of silica gels. 

The above results only demonstrate the chromatographic behaviour of compounds with logP = 3. Their relative retention factors thus are influenced by the properties of solvents in the eluent. The relative values cannot be controlled by only one property of the solvents and chromatographic behaviour therefore also depends on the properties of the analytes. 

The retention factor of the molecular form of the analyte, km, and the retention factor of the paired-ion, kip, can be easily measured experimentally. When the retention factor is measured at low and high pH, and the pKa value is known, A at a given pH is roughly estimated from the following equation ... 

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]. 

H is the plate height (cm) u is linear velocity (cm/s) dp is particle diameter, and >ni is the diffusion coefficient of analyte (cm /s). By combining the relationships between retention time, U, and retention factor, k tt = to(l + k), the definition of dead time, to, to = L u where L is the length of the column, and H = LIN where N is chromatographic efficiency with Equations 9.2 and 9.3, a relationship (Equation 9.4) for retention time, tt, in terms of diffusion coefficient, efficiency, particle size, and reduced variables (h and v) and retention factor results. Equation 9.4 illustrates that mobile phases with large diffusion coefficients are preferred if short retention times are desired. 

While retention time is used for peak identification, it is dependent on the flow rate, the column dimension, and other parameters. A more fundamental term that measures the degree of retention of the analyte is the capacity factor or retention factor (k ), calculated by normalizing the net retention time (% > retention time minus the void time) by the void time. The capacity factor measures how many times the analyte is retained relative to an unretained component. ... 

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. 

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. 

Solvent gradients are generally mnch more efficient to decrease the retention than programmed temperature. For example, the retention factors k of low-molecnlar-weight analytes in reversed-phase... 

The effect of the dwell volume on the retention times of analytes increases with decreasing retention factor at the start of gradient elution and with increasing ratio VpIV, and becomes very significant in the instrumental setup with the dwell volume comparable to or larger than the column hold-up volume, which is more likely to occur in micro- or in capillary LC than in conventional analytical LC (see Figure 5.4) [12]. 

The migration behavior of neutral analytes in MEKC can be described by parameters similar to those employed in liquid chromatography [209], Hence, the ratio of the number of moles of the analyte in the micellar phase, the pseudostationary phase, (ttp p) to those in the surrounding solution (nip) defines the retention factor t... 

The retention factor is related to the distribution coefficient K of the analyte between the micellar phase and the aqueous phase, according to the following equation ... 

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] ... 

EKC is not restricted to the separation of neutral analytes, as it is widely employed for the simultaneous separations of charged and neutral analytes as well as of ionizable compounds having similar electrophoretic mobility. The separation of ionizable analytes by EKC is governed by differences in the partitioning between the pseudostationary phase and the surrounding electrolyte solution as well as electrophoretic mobility. For these analytes, the retention factor can be described by the following mathematical model ... 

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