Elution, isocratic versus gradient

When performing HPLC (see Basic Protocol 2) the time of analysis will depend on the conditions used—i.e., isocratic versus gradient. Isocratic separation of the individual betacyanins, as shown in Figure F3.1.2, requires 20 min, compared to 9 min using a gradient elution system. 

Isocratic versus Gradient Elution. The principles of chromatography just described hold true for isocratic elution schemes, in which the nature of the mobile phase remains constant throughout the de-... 

Fig-1 Fractional migration X L) of solute bands along the column and resulting chromatograms for isocratic versus gradient elution. (Reprinted with permission from L. R. Snyder, M. A. Stadalius, and M. A. Quarry, Analytical Chemistry,... 

Considerations in the Choice of Isocratic versus Gradient Elution... 

Two analytical methods for priority pollutants specified by the USEPA (38) use HPLC separation and fluorescence or electrochemical detection. Method 605, 40 CFR Part 136, determines benzidine and 3,3-dichlorobenzidine by amperometric detection at +0.80 V, versus a silver/silver chloride reference electrode, at a glassy carbon electrode. Separation is achieved with a 1 1 (v/v) mixture of acetonitrile and a pH 4.7 acetate buffer (1 M) under isocratic conditions on an ethyl-bonded reversed-phase column. Lower limits of detection are reported to be 0.05 /xg/L for benzidine and 0.1 /xg/L for 3,3-dichlorobenzidine. Method 610, 40 CFR Part 136, determines 16 PAHs by either GC or HPLC. The HPLC method is required when all 16 PAHs need to be individually determined. The GC method, which uses a packed column, cannot adequately individually resolve all 16 PAHs. The method specifies gradient elution of the PAHs from a reversed-phase analytical column and fluorescence detection with an excitation wavelength of 280 nm and an emission wavelength of 389 nm for all but three PAHs naphthalene, acenaphthylene, and acenaphthene. As a result of weak fluorescence, these three PAHs are detected with greater sensitivity by UV-absorption detection at 254 nm. Thus, the method requires that fluores-... 

Gradient elution versus isocratic elution — effects of the gradient profile on separation... 

Abbott et al. [4] devised a method designed to predict the retention times in gradient elution under the assumption that the retention factor as determined under isocratic conditions is a log-linear function of solvent composition according to Eq. (5), where k is the retention factor obtained in water, ipo refers to the volume fraction of the organic component, and S refers to the solvent strength for which the values can be obtained as the negative slope of plots of log k versus volume fraction ... 

A mapping of the dependence of analyte retention (expressed as the natural logarithm of the retention factor, k) on the mobile phase composition (expressed as the volume fraction of solvent in the mobile phase, (p) in isocratic elution (or as k versus ip in gradient elution) with a minimum of two initial experiments can be used to define the useful range of mobile phase conditions, and can indicate the mobile phase composition at which the band spacing is optimal (see Figure 10). 

The dependence of the initial slope of the isotherm on the mobile phase composition can be derived in two different ways [29]. The retention time, Ir , of analytical pulses eluted imder isocratic conditions is a function of the mobile phase composition. Since Ir — to is proportional to Eq. 15.29 permits the determination of Sj as the slope of a plot of log(fR / — to)/to versus mobile phase composition at which analytical pulses are eluted rmder conditions of linear eluotropic-strength gradient. In this case, it has been shown [3] that the retention time of an analytical pulse is given by Eq. 15.6. Using this equation, it is simple to derive S, since all the other parameters are knovm. This method of estimation of S seems to give the best results [20,24,29]. 

If the Rf values obtained in several isocratic elution steps are known, the program for gradient elution can be constructed (11,12). Results of stepwise gradient elution of DABS-amino acids are presented in Fig. 13 (12). The solvent concentration for the first step was chosen from the plot of B/ versus percent of the more efficient solvent (it is still better in normal-phase TLC to use R versus log % of the more polar solvent), by assuming that for the first eluted compound the value should be equal to 0.25. In fact, half of the dead volume V of the eluent was used, so the B/value in the first step is equal to 0.25/2 = 0.125 (see Fig. 13). Knowing the concentration in which the B/for the first compound is equal to 0.25, the By values for the rest of the compounds were obtained in the same way from a plot of By versus %B. In the next steps, 0.5 V of 10% and 0.5 V of 15% concentrations were used (12). 

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