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Study Notes: More on Sample Preparation for Chromatographic and Electrophoretic Techniques

Some sample preparation procedures commonly used with chromatographic and electrophoretic techniques are briefly introduced.

Dissolution
May be a simple dissolution of the entire sample into a simple solvent (water, detergent solution, ethanol) or may involve extraction by direct separation of the main components by dissolution into a suitable solvent leaving behind insoluble components.

Solvents may be complex such as the sample buffer used for protein SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), containing the following:

0.01M Tris HCl, pH 8.0, 0.001 M EDTA, 0.1% SDS

In some cases the solvent may need to be removed prior to analysis.

Concentration
A variation on dissolution is the precipitation of the sample in a solvent to concentrate the sample. For example, protein samples dissolved in an aqueous salts/detergent buffer may be precipitated in ice-cold acetone. The proteins precipitate out leaving the water and some of the other components in the acetone. The protein pellet is redissolved in a smaller volume of buffer.

Dialysis may also be used to concentrate a sample and to change the buffer in which the sample is dissolved. Choice of an appropriate dialysis buffer containing appropriate buffer components and high osmotic strength will cause the water and salts in the solution inside the dialysis bag to migrate out of the bag leading to a concentration of the sample.

Liquid-Liquid Extraction
A liquid sample (remember that this is also a solvent) is intimately mixed with an immiscible solvent to selectively transfer the analyte from the sample solvent into the immiscible solvent. The transfer occurs as a result of the greater solubility of the analyte in the immiscible solvent. The sample matrix is concentrated in the original sample solvent and the analyte in the immiscible solvent. After extraction, the immiscible solvent can be separated from the analyte (eg by evaporation) in readiness for analysis or for further processing.

This method requires a good knowledge of chemistry and the sample components need to be known in advance.

This procedure facilitates purification and/or concentration of the sample.

The first essential step in DNA analysis is to separate the DNA from the cellular components (matrix) in which it is contained. One of the most commonly used methods is to extract the DNA in a chloroform/phenol liquid-liquid mix.

Solid Phase Extraction and Affinity Chromatography
Adsorption of molecules of interest onto a solid, that is a stationary phase, of appropriate material in a column (column chromatography) or a planar surface (thin layer chromatography) are often used to clean up samples prior to further chromatographic or electrophoretic analysis. The molecules of interest are eluted (ie flushed off) from the solid phase using an appropriate solvent.

In affinity chromatography, a specific ligand is attached to the chromatographic bed material. The sample is passed through the bed and the molecules of interest bind to the bed via the specific ligand.

These molecules of interest can then be eluted from the column by changing the buffer conditions. Affinity chromatography and thin layer chromatography will be discussed further in Section 2 of this unit.

Filtration and Centrifugation

Chromatographic techniques such as HPLC and GC rely on fine tubing and columns with fine orifices. The presence of a precipitate or undissolved matter from some other source in the test sample may block or retard migration of the sample along the column. Removal of the undissolved material by filtering or centrifuging the sample can overcome this problem. Simple illustrations of a spinning centrifuge containing two tubes and a filtration system using a filter funnel fitted with filter paper through which clear solution is exiting.
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