Preparative chromatography sample volume

Preparative chromatography involves the collection of individual solutes as they are eluted from the column for further use, but does not necessarily entail the separation of large samples. Special columns can be designed and fabricated for preparative use, but for small samples the analytical column can often be overloaded for preparative purposes. Columns can be either volume overloaded or mass overloaded. Volume overload causes the peak to broaden, but the retention time of the front of the peak... 

For the analysis of americium in water, there is a broad array of sample preparation and detection methodologies that are available (see Table 7-2). Many of the common and standardized analytical methodologies typically include the minimization of sample volume, purification through co-precipitation, anion exchange column chromatography, and solvent extraction techniques followed by radiochemical detection of americium in the purified sample. Gross alpha analysis or liquid scintillation are common... 

Headspace-GC-MS analysis is useful for the determination of volatile compounds in samples that are difficult to analyze by conventional chromatographic means, e.g., when the matrix is too complex or contains substances that seriously interfere with the analysis or even damage the column. Peak area for equilibrium headspace gas chromatography depends on, e.g., sample volume and the partition coefficient of the compound of interest between the gas phase and matrix. The need to include the partition coefficient and thus the sample matrix into the calibration procedure causes serious problems with certain sample types, for which no calibration sample can be prepared. These problems can, however, be handled with multiple headspace extraction (MHE) [118]. Headspace-GC-MS has been used for studying the volatile organic compounds in polymers [119]. The degradation products of starch/polyethylene blends [120] and PHB [121] have also been identified. 

There is frequently a need to change buffer conditions during protein preparations a typical example is when an ammonium sulphate precipitation step is followed by ion-exchange chromatography. The ammonium sulphate can be removed from the re-dissolved pellet by prolonged dialysis, but it is preferable, for reasons of speed and stability, to pass the solution over a Sephadex G-25 coarse column, preequilibrated with the start buffer for the next column for such coarse operations the sample volume can amount to ca. 15-20 % of the column volume. 

The method used for application of sample solutions is determined by whether HPTLC, TLC, or preparative layer chromatography (PLC) and qualitative or quantitative analysis are being performed. Sample volumes of 0.5-5 pi for TLC and 0.1-1 pi for HPTLC are applied manually to the layer origin as spots using fixed volume glass micropipets, such as Drummond Microcaps or selectable volume 10 or 25 pi digital microdispensers. In addition, many manual and automated instruments are available for sample application, especially for quantitative HPTLC. 

As the analytes to be assayed are nearly always present in minute amounts, preconcentration steps are almost always necessary. The techniques used for this purpose have been mostly adopted from analytical procedures using gas or liquid chromatography as the separation step [5]. This fact is emphasized here because the appropriate adjustment of the sample preparation is necessary because the original procedures do not respect the fact that the sample volume injected into a CE system represents a few nanoliters only and requires a relatively high concentration of analytes assayed. However, in selected types of analysis, direct sample application is also possible (for a review, see Ref. 3). 

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