Quantitative evaluation of models for solvent-based, on-column focusing in liquid chromatography

Abstract

On-column focusing or preconcentration is a well-known approach to increase concentration sensitivity by generating transient conditions during the injection that result in high solute retention. Preconcentration results from two phenomena: 1) solutes are retained as they enter the column. Their velocities are k′-dependent and lower than the mobile phase velocity and 2) zones are compressed due to the step-gradient resulting from the higher elution strength mobile phase passing through the solute zones. Several workers have derived the result that the ratio of the eluted zone width (in time) to the injected time width is the ratio k2/k1 where k1 is the retention factor of a solute in the sample solvent and k2 is the retention factor in the mobile phase (isocratic). Mills et al. proposed a different factor. To date, neither of the models has been adequately tested. The goal of this work was to evaluate quantitatively these two models. We used n-alkyl esters of p-hydroxybenzoic acid (parabens) as solutes. By making large injections to create obvious volume overload, we could measure accurately the ratio of widths (eluted/injected) over a range of values of k1 and k2. The Mills et al. model does not fit the data. The data are in general agreement with the factor k2/k1, but focusing is about 10% better than the prediction. We attribute the extra focusing to the fact that the second, compression, phenomenon provides a narrower zone than that expected for the passage of a step gradient through the zone.

Graphical Method for Choosing Optimized Conditions Given a Pump Pressure and a Particle Diameter in Liquid Chromatography

“…3" rend="display" xml:id="FD3">$N_a=\frac{L}{H_a}=\frac{L}{H+\left(\raisebox{1ex}{${\sigma }_{\mathit{inj}}^2$}\!\left/ \!\raisebox{-1ex}{$L$}\right.\right)}$ where L is column length, H a is the apparent plate height, H is the non-overload plate height (calculated as above), and ${\sigma }_{\mathit{inj}}^2$ is the injection variance. The injection variance is a function of the injection volume, column volume and solute retention factor in the sample solvent 40 . Figure 3 shows the plot with contours of N a , rather than the column’s intrinsic plate count, N. The apparent plate count has been calculated for fixed 500 nL injections into 100 (A), 150 (B) and 250 (C) μm ID columns while the remaining parameters are as in Figure 2B.…”

“…4" rend="display" xml:id="FD4">$$n_c=1+\frac{1}{4}N{\left(t_o,L,u_e\right)}^{\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$2$}\right.}\frac{1}{G+1}\text{ln}\left[\frac{G+1}{G}{e}^{S\text{Δ}\varphi }-\frac{1}{G}\right]$$ were N is the column efficiency, a function of t 0 , L, and u e , S is the slope of the log of solute retention factor vs eluent composition from Snyder’s linear solvent strength theory 40 , Δϕ is the range in eluent composition and G = SΔϕt 0 /t G , t G is the gradient time.…”

Temperature-assisted solute focusing with sequential trap/release zones in isocratic and gradient capillary liquid chromatography: Simulation and experiment

“…Fused silica capillary columns were packed in-house using a previously described procedure [28, 53, 55]. Briefly, Waters Acquity BEH C18, 1.7 µm d p , particles were packed into 150 µm diameter fused silica capillaries from Polymicro Technologies (Phoenix, AZ).…”

“…Pre-column strategies can be particularly effective because they may involve solute-specific, high-affinity focusing [21–25] or fractionation, e.g., in proteomics (MudPIT [26, 27]). On-column focusing or preconcentration is a consequence of generating transient conditions during the injection that result in high solute retention at the head of the column [28]. For example, focusing can be induced by injecting aqueous samples onto a reversed phase column or application of a solvent gradient [29].…”

A twin‐column recycling chromatography with solvent gradient for reinforcing the isolation of minor impurities

“…For example, the introduction of water as the modifier in the reversed phase chromatography mode can reduce the eluotropic strength of the downstream liquid. As a result, the movement of the band front is slowed down and the tail will move faster than the front, generating some band compression effect to offset the band broadening caused by both nonlinear adsorption and nonideal factors. Accordingly, the separation will be improved, leading to larger amount of feeding and higher concentration of product.…”