Elution zonal

Zonal chromatography is used predominantly in both gas and liquid chromatography. The separated zones are swept (eluted) from the column into a detector where they are sensed and recorded as the sequential peaks of a chromatogram. Typical chromatograms are shown in Figure 10.3. 

Several theoretical models were constructed to describe the chromatographic process in the frontal 116.191 and the zonal elution mode 20. The conventional method of obtaining the kinetic parameters consists in fitting the model to the experimental breakthrough curves. Another method based on the split-peak effect is a direct measurement of the apparent association rate constant (7,211. Because of the slow adsorption process, a fraction of the solute injected as a pulse into the immunochromatographic column is eluted as a nonretained peak. This behavior is observed at high flow rates, with very short or low-capacity columns 121—251. 

Although the use of zonal rotors is somewhat more involved than conventional swinging bucket rotors it is important to emphasize that the end result is the same. An example of this is shown in Figure 9-32, which depicts the elution profile of a stepped sucrose gradient that was used to separate mitochondria, proplastids, and glyoxysomes. This profile is very similar to that shown in Figure 9-16A, but in that experiment the gradient volume was 54 ml. Here it is 1760 ml and accommodates the crude particulate fraction yielded from 1 lb of fresh tissue. 

The method of zonal elution is one of the most common techniques used in affinity chromatography to examine biological interactions. An example of this type of experiment is shown in Fig. la. In its usual form, zonal elution involves the application of a small amount of analyte (in the absence or presence of a competing agent) to a column that contains an immobilized ligand. The retention of the analyte in this case wiU depend on how strongly the... 

Equation (1) represents one specific type of zonal elution study, in which the injected analyte and competing agent bind at a single common site on the immobilized ligand ... 

Another group of methods in analytical affinity chromatography are those that examine the kinetics of biological interactions. Band-broadening measurements (also known as the isocratic method) represent one such approach. This is really a modification of the zonal elution method in which the widths of the eluting peaks are measured along with their retention times. 

Figure 5 An example of a zonal elution experiment, in which small injections of L-tryptophan are made on to an immobilized human serum albumin column in the presence of increasing amounts of phenytoin in the mobile phase.

Temperature is another factor that can be varied during zonal elution studies. For instance, Eq. 3 can be used for a system with 1 1 binding... 

Some methods for obtaining kinetic information by affinity chromatography fit experimental elution data to empirical equations that can be used to describe profiles for a given set of application conditions. For instance, this approach has been used with zonal elution data obtained imder nonlinear elution conditions by using the following function. ... 

Fig. 5 (A) Densitogram obtained from micropreparative zonal chromatography of a mixture of quaternary alkaloids on silica plate with toluene/EtOAc/MeOH (83 15 2) as mobile phase. Detection by UV at A = 254 nm. (B) Densitogram obtained from micropreparative zonal chromatography of a quaternary alkaloid mixture on silica plate. Gradient elution with T/EtOAc/MeOH, ra=l 75 25 5, n = 2 70 20 10, n — 3 70 15 15, n = 4 EtOH/CHCla/AcOH (67 30 3) as mobile phases. Detection by UV at A = 254 nm.

Soczewinski, E. Matysik, G. Polak, B. Improvement of separation in zonal preparative thin-layer chromtography by gradient elution. Chromatographia 1994, 39 (7-8), 497. 

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