Chromatographic separation, elution curve

Primarily the Plate Theory provides the equation for the elution curve of a solute. Such an equation describes the concentration of a solute leaving a column, in terms of the volume of mobile phase that has passed through it. It is from this equation, that the various characteristics of a chromatographic system can be determined using the data that is provided by the chromatogram. The Plate Theory, for example, will provide an equation for the retention volume of a solute, show how the column efficiency can be calculated, determine the maximum volume of charge that can be placed on the column and permit the calculation of the number of theoretical plates required to effect a given separation. 

Solute equilibrium between the mobile and stationary phases is never achieved in the chromatographic column except possibly (as Giddings points out) at the maximum of a peak (1). As stated before, to circumvent this non equilibrium condition and allow a simple mathematical treatment of the chromatographic process, Martin and Synge (2) borrowed the plate concept from distillation theory and considered the column consisted of a series of theoretical plates in which equilibrium could be assumed to occur. In fact each plate represented a dwell time for the solute to achieve equilibrium at that point in the column and the process of distribution could be considered as incremental. It has been shown that employing this concept an equation for the elution curve can be easily obtained and, from that basic equation, others can be developed that describe the various properties of a chromatogram. Such equations will permit the calculation of efficiency, the calculation of the number of theoretical plates required to achieve a specific separation and among many applications, elucidate the function of the heat of absorption detector. 

On examination of the curve in figure 4 the problem associated with high efficiencies becomes apparent. The elution time for the short column having about 3500 theoretical plates is only just about one half minute. The elution time from the 1.5 million plate column, however, is about 54 days, a rather long time to wait for a chromatographic separation. It is also seen that the higher the efficiencies that are required (the more difficult the separation problem) the longer the separation time and this is inevitable as a result of practical limits to the column inlet pressure. 

Qualitatively, the influence of the eluent gradient on chromatographic separation according to functionality is shown in Fig. 24. The initial point on the sab axis roughly determines the absolute values of retention volumes at which the macromolecules are eluted, and the slope of eab vs. V curve the distance between the zones of different functionality. Unpublished experimental data obtained in gradient chromatography of PBTP in a binary heptane — tetrahydrofuran eluent fully support this conclusion. 

Procedure Transfer 2.00 mL of each sample to separate test tubes, and equilibrate in the 37° water bath for at least 10 min. At the same time, equilibrate the Substrate Solution in the same water bath. At zero time, transfer 2.0 mL of the equilibrated Substrate Solution to the first sample tube, mix thoroughly, and return the tube to the 37° bath. Repeat the process for each sample. After exactly 30.0 min, transfer the test tube to a boiling water bath for 15 min, then remove and cool to room temperature. Add approximately 100 mg of Amberlite MB-1 Ion Exchange Resin to each tube, place the tubes on the shaker, and mix for at least 15 min. Filter the treated solution through a 0.45-p.m filter. Use a separate filter for each sample. Inject a 5-p.L portion of each filtered sample into a previously equilibrated high-performance liquid chromatograph equipped with an HPX 87C column (Biorad, or equivalent) and a differential refractometer. Filtered, degassed water is the mobile phase. Record the elution curve. 

Chromatographic Separation. The LC elution curve is shown in Figure 2, and the elution range of individual fractions designated Fr-P, M, D, T, and PP are indicated. Elution curves from GPC and the range of elution volume for GPC subfractions are shown in Figure 3. 

Figure 2 illustrates serum protein separation by centrifugal precipitation chromatography the chromatographic tracing of the elution curve in Fig. 2a and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of separated fractions in Fig. 2b. In this example, lOOmL of normal human serum (pooled) was diluted to 1 mL and introduced into the separation channel. The experiment was initiated by filling both upper and lower channel with 75% AS solution followed by sample...

Isothermal chromatographic experiments with the detection of elution curves at several column temperatures are feasible in the separation practice of numerous fields of chemistry, and Eq. 5.15 has been widely exploited. In the radiochemical studies of our concern, such an approach could be realized only if long-lived nuclides with high enough activity were available this, unfortunately, is not the case. 

In the top segment of Table 11.2 we display data for peaks 2, 3, and IS 6 from the standard curve mns, and for three unknown peaks (A, B, C) in a separate chromatographic run eluting with similar RT, plus D, which is the IS 6 peak in that mn. In both runs we use IS 6 as a retention internal standard to calculate RRT values displayed in the third column of the table. If we only compared RTs (i.e., t ), we might say A is 2 and B is 3. We note, however, that IS D elutes 0.2 min later than it did in the standard mns. Something has caused a shift in retention between the mns, perhaps the flow has slowed a little, or the GC oven temperature dropped a bit. If we compare RRTs we see that B and C are better matches to 2 and 3, respectively. 

Liquid chromatographic separations of the isotopes of elements other than hydrogen have been rather rare. A high-efficiency liquid-liquid chromatographic system consisting of porous silica microspheres covered with 25% (w/w) bis(2-ethylhexyl)phosphor-ic acid in dodecane as the stationary phase and nitric acid as the mobile phase provided a certain enrichment of heavier isotopes of calcium in front of the elution curve. Separation factors calculated by Glue-ckauf for " Ca, " Ca, " Ca versus were 1.0012-1.0029. 

The distribution ratio, extraction rate and extraction volume, optimum extraction pH were determined, cobalt and nickel elution curves were obtained and the process conditions for chromatographic separation were defined. 

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