Elution problems

The general elution problem in chromatography. Improving the resolution of the overlapping bands in chromatogram (a) results in a longer analysis time for chromatogram (b). 

Separation scientists speak of a general elution problem when asked to develop a universal separation method using chromatography. What is the problem (Hint you may need to consult works by Snyder or Heftmann in the library.)... 

General elution problem For a complex mixture, isocratic conditions can often be found to produce adequate separation of early-eluting peaks or late-eluting peaks, but not both. This problem drives us to use gradient elution. 

The general elution problem. A Normal elution. B Gradient elution. From L. Ft. Snyder, J. Chromatogr. Sci. 8, 692 (1970) Reproduced by permission of Preston Publications. 

Figure 6.1 Illustration of the general elution problem in chromatography. Chromatograms a and b constant elution conditions. Chromatogram c (opposite page) programmed analysis.

The vertical lines a and b correspond to temperatures at which chromatograms would be obtained which are similar to the chromatograms a and b in Figure 6.1. Hence, we are confronted with the general elution problem . 

Organic analytes must be separated from nonvolatile materials that may affect the performance of GC columns, such as pigments, inorganic sulfur, and triglycerides. Also, there is a need to separate, as much as possible, the analytes from each other before GC analysis in order to limit co-elution problems. Typical techniques used for cleanup are column chromatography or SPE and/or acid/base washes. 

While such large changes in a are limited to compounds of quite different structure, it is likely that some preparative separations can be greatly facilitated by maximizing a in this way without limit. In analytical separations, however, values of a larger than 5-10 can actually be detrimental, because they result in too large a difference in retention times for the two solutes (the general elution problem, Ref. 1). 

For some very non-polar compounds, even small quantities of dichloromethane may cause elution problems and an alternative method of loading can be used. Thus, a solution of the sample is added to a small amount of silica in a round bottom flask and the mixture is evaporated to dryness. The dry impregnated silica is then added to the top of the pre-packed column. 

Fig. 1.25. An example of ihe general elution problem with ihc separation of a homologous series of l,2-naph(hoylenebenzimida/,olesulphonamide derivatives of ten homologous alkylamines — methyl (/) to n-decyl (/O) — by reversed-phase HPLC on a Liehrosorb RP-18. 10 pm, column (300 x 4 mm i.d.). (A) Isocratic separation with methanol-water 95 5 as the mobile phase. (B) Isocraiic separation with methanol-water 80 20 as the mobile phase. (C) Gradient-elution separation with linear concentration gradient from methanol-water 70 30 to l(X) methanol in 20 min. Flow rale I ml/min. fluorimctric detection, ycx = 365 nm, ytm >410 nm. The numbers of peaks agree with the number of carbon atoms in n-alkylamines.

To illustrate the general elution problem and its solution, let us consider the following situation. Consider a 20-component mixture with capacity factors k of the components forming a geometrical progression and exponentially dependent on the modifier concentration (molar or volume fraction c), in accordance with the Snyder-Soczewinski model of adsorption [2]. The log k versus log c plots of the 20 solutes are given in Fig. 1, which has a parallel Rf axis subordinated to the right-hand-side log k axis. It can be seen that no isocratic eluent can separate all the components. A pure modifier [c = 1.0 (100%)] separates well solutes 1-7, and the less polar solutes are accumulated near the solvent front for c = 0.1 (10%), solutes 8-14 are... 

General elution problem The compromise between elution time and resolution addressed through gradient elution (for liquid chromatography) or temperature programming (for gas chromatography). 

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