Retention variation with stationary phase composition

Figure 5.16a can be constructed once the retention data of all solutes have been measured on the two pure phases. It is assumed that retention (K) varies linearly with composition (

straight lines, which represent the (expected) variation of retention with stationary phase composition for the four solutes W,X, Y and Z. 

Figure 5.16 Example of a window diagram for optimizing the stationary phase composition in GLC. (a) (top) variation of the retention (distribution coefficient K) with composition for the individual solutes W,X, Yand Z. (b) (bottom) window diagram showing grey areas ( windows ) at compositions where all components may be separated. Figure taken from ref. [545]. Reprinted with permission.

Figure 6.8 Experimental variation of the retention of 23 phenylthiohydantoin (PTH) derivatives of amino acids with mobile phase composition in RPLC. Mobile phase mixtures of acetonitrile and 0.05M aqueous sodium nitrate buffer (pH — 5.81). All mobile phases contain 3% THF. Stationary phase ODS silica. Solutes D = aspartic acid C-OH = cysteic acid E = glutamic acid N = asparagine S = serine T = threonine G = glycine H = histidine Q = glutamine R = arginine A = alanine METS = methionine sulphone ABA = a-aminobutyric acid Y = tyrosine P = proline V = valine M = methionine NV = norvaline I = isoleucine F = phenylalanine L = leucine W = tryptophan K = lysine. Figure taken from ref. [610]. Reprinted with permission.

Figure 3.3 Variation of retention (distribution coefficient) with the composition of the stationary phase in GLC at three different temperatures (indicated in the figure in °C). Stationary phase mixtures of squalane and dinonylphthalate (DNP). Solutes (a) n-octane, (b) cyclohexane, (c) methylcyclohexane and (d) tetrahydrofuran. Straight lines observe eqn.(3.14). Figure taken from ref. [304]. Reprinted with permission. 

Figure 3.6 Variation of retention with the composition of the stationary phase in GLC. Stationary phase styrene-butadiene polymer blends and copolymers, the butadiene fraction is plotted on the horizontal axis, (a) Specific retention volumes for three n-alkanes and benzene. V is proportional to the capacity factor, (b) the retention index for benzene. The solid line is calculated from the straight lines in figure 3.6a. The circles (polymer blends) and triangles (copolymers) represent experimental data. Figure taken from ref. [310], Reprinted with permission.

Figure 3.6 illustrates the variation of (a) the specific retention volume (Vff which is proportional to the capacity factor) with the composition of a mixed stationary phase, and (b) the variation of the retention index for benzene with the composition. It is clear from these figures that, whereas straight lines are observed for the variation of the capacity factor with the composition, the retention index varies in a highly non-linear manner. 

Figure 3.19 Variation of retention with composition in LSC according to the simplified linear relationship of eqn.(3.74). Stationary phase Lichrosorb ALOX T (Alumina). Mobile phase n-propanol (

volume fraction) in n-heptane. Solutes lumisterol (1), tachysterol (2), calciferol (3) and ergosterol (4). Figure taken from ref. [357]. Reprinted with permission. 

It will be clear from figure 6.7 that the nature of the mobile phase (compare figures 6.7a and 6.7b) and the stationary phase (compare figure 6.7c with figures 6.7a and 6.7b) have a great effect on the character of the retention vs. composition plots and hence on the shape of the required (optimum) gradient. It will also be clear that, unlike the situation in GC, the selectivity may be greatly influenced by variations in the mobile phase. 

This is largely due to the fact that retention data depend on certain factors the effects of which are difficult to eliminate completely or control and which are normally neglected. These factors are the imperfections in the gas phase and the compressibility of the stationary phase (cf., the quantities vh v , zq and 0 in eqn. 1), the finite rate of equilibration of the solute, variations in the composition of the sorbent, spurious sorption of the solute, solubility of the carrier gas in the stationary phase, etc. Hence, even relative retention volumes and/or retention indices must depend to some extent on the kind, flow-rate and absolute pressure of the carrier gas, the load of the liquid stationary phase on the support, which production batch of the stationary phase has been used and the kind of support. The absolute column pressure will obviously vary with the column length and particle size of the support. Moreover, adjusted retention data are required in all instances, which renders it necessary to measure the dead retention time. This is a crucial step in obtaining accurate retention data and presents a problem per se. 

Equation (10-1) is based on the assumption of simple additivity of all interactions and a competitive nature of analyte/eluent interactions with the stationary phase. The paradox is that these assumptions are usually acceptable only as a first approximation, and their application in HPLC sometimes allows the description and prediction of the analyte retention versus the variation in elution composition or temperature. For most demanding separations where discrimination of related components is necessary, the accuracy of such prediction is not acceptable. It is obvious from the exponential nature of equation (10-1) that any minor errors in the estimation of interaction energy, or simple underestimation of mutual influence of molecular fragments (neglected in this model), will generate significant deviation from predicted retention factors. 

The kinetic characteristics are determined in GC methods both directly and indirectly. In direct pulse chromatographic methods the reaction rate can be established by the direct determination of the amount (concentration) of the reacting component, whereas in indirect methods this is done on the basis of the variations with time of the chromatographic properties of the reacting system, which are usually determined from the relationship between and the retention times of the non-reacting components and the composition of the reaction mixture used as the stationary phase [58]. Pulse chromatography... 

Figure IB displays the similar effect with temperature variation at the fixed eluent composition (CH2Cl2/CH3CN=57/43, v/v). The overall temperature dependence, i.e., decrease of retention with temperature, indicates that the interaction of polymer chains with CIS silica stationary phase is exothermic. Provided that the...

The pattern of the variation of retention with composition in LC is affected by the choice of both the stationary and the mobile phase. The optimum shape of the gradient for unknown wide range samples is dictated by the phase system. Linear or slightly convex gradients are optimal for RPLC. Concave gradients are optimal for LSC. 

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