Elution, Process

Elution development is by far the most common method of processing a chromatographic separation and is used in all types of chromatography. Elution development is best described as a series of absorption-extraction processes which are continuous from the time the sample is injected into the distribution system until the time the solutes exit from it. The elution process is depicted in Figure 1. 

Mass spectrometers that use electrospray ionization (ESI) do not function well if the eluent contains low volatility salts. This is a major concern when an ion-exchange column is used as a first-dimension column and the salt concentration is used to modulate the retention in this column. In this case, another valve can be connected between the second-dimension column and the detector so that any salt from the second-dimension elution process that is either unretained or weakly retained can be diverted prior to feeding zones to the mass spectrometer. 

To ensure operation under reproducible conditions, the column is enclosed in a thermostatically controlled oven whose temperature can be held constant to within 0.1°C. Operating temperatures range from ambient to over 400°C and may remain constant during a separation - isothermal operation - or automatically increased at a predetermined rate to speed the elution process - temperature programming (p. 106). The latter is a form of gradient elution. Rapid temperature equili-... 

The target dipeptide product was purified on a SephadexG-10 column (16 mm x 1000 mm) equilibrated and eluted with water at the elution rate of 1.0 mL min . The elution process was monitored at 220 nm. The fractions collected were lyophihzed to afford the desired product (29.6 mg). The HPLC purity and the yield of the product were 93.5 % and 82.9 % respectively. 

The equipment required to develop this type of sensor is very simple and resembles closely that used to implement ordinary liquid-solid separations in FI manifolds. The only difference lies in the replacement of the packed reactor located in the transport-reaction zone with a packed (usually photometric or fluorimetric) flow-cell accommodated in the detector. Whether the packing material is inert or active, it should meet the following requirements (a) its particle diameter should be large enough (< 80-100 fim) to avoid overpressure (b) it should be made of a material compatible with the nature of the integrated detection system e.g. almost transparent for absorbance measurements) and, (c) the retention/elution process should be fast enough to avoid kinetic problems. 

The second aliquot was passed onto another cation exchange resin and eluted as before, the effluent solution was passed onto a second cation exchange column, and the elution process was repeated. No dark band was left on the second column, and analysis of a portion of the new effluent solution showed that there had been no further loss of chromium on passage through the second column. Two additional portions of the last-mentioned solution were withdrawn for examination. The first portion was analyzed for oxalate, as before, and chromium-oxalate ratios of 1.00 to 1.02 were obtained. The spectrum of the second portion was measured through the visible range and found to agree well with the spectrum of the mono-oxalatotetraaquochromium (III) ion at the same chromium concentration. 

Model solutes recovered at levels in excess of 50 by two or more methods were 2,4-dichlorophenol, isophorone, biphenyl, 1-chlorododecane, 2,4 -dichlorobiphenyl, 2,2, 5,5 -tetrachlorobiphenyl, anthraquinone, bis(2-ethylhexyl) phthalate, and quinoline. Except for anthraquinone and quinoline, these solutes are hydrophobic. Porous PTFE effectively recovered stearic acid in addition to each of the hydrophobic solutes mentioned earlier. The higher recoveries by porous PTFE are not attributed to a greater adsorptive capacity but rather to the ease with which the hydrophobic solutes can be desorbed by conventional elution processes. 

The quantitation of substances separated by TLC may be carried out in several ways. The most common method is to remove the spot from the plate, elute the compound from the adsorbent and measure the concentration of the compound in solution by spectrophotometry, fluorimetry, etc. The elution process has been significantly improved and facilitated with the Eluchrom instrument developed by Sandoz and marketed by Camag (see Fig.3.6). This instrument permits direct elution from the plates via small PTFE cups in a continuous flow-through mode without the necessity of removal of the adsorbent and with the minimum requirement of solvent (usually less than 1 ml). The measuring instruments used are those available for classical solution analysis. A discussion of these instruments is beyond the scope of this book. 

A qualitative thin-layer chromatography method has been described by Kleef et al. [27] for the detection of rocuronium bromide and its metabolites in biological samples. This method was developed to confirm the identity of rocuronium bromide and its metabolites prior to their determination using HPLC coupled with fluorescence detection. In this method, the dried residue from the extraction process was dissolved in 0.05 ml of 0.01 M HC1. The stationary phase used was silicagel plates, that were developed in a mobile phase consisting of 2% solution of Nal in 2-propanol. The elution process was run for 4 h, and after the elution... 

For porewater extraction, whole wet sediment is centrifuged at 17,000 x g for 20 min. An elutriate (aqueous extract) is obtained by shaking the wet sediment sample in an aerobic milieu for 24 h. Soluble substances are extracted under such conditions. The elution process is performed with the original wet sediment and the... 

Displacement chromatography is commonly used for preparative-scale separations, but, because of its focusing or concentrating effect, it also shows potential on the analytical scale, for example, for the concentration of minor components in complex mixtures.24,25 Operationally, displacement chromatography is similar to the step elution process, except that in the displacement process the mobile phase has a greater affinity for the stationary phase than for the sample components, and therefore the components are eluted ahead of the displacer front. The focusing effect of displacement chromatography is due to the fact that the concentration of the displacer determines the concentration of the product bands.26... 

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