Column chromatography sample application

A third parameter to consider is the column construction. Thus the sample applicator should provide optimal sample application to give the most performance possible out of the packed bed. Constructions should also allow simple, fast, and reproducible packing of the column. Because costs for repacking of columns are a substantial operating cost item in industrial chromatography, the selection of column construction from this point of view is also important. Some novel column constructions allow very simple procedures both for laboratory and for industrial scale (e.g., INdEX columns, see Section V). 

Dry column chromatography [528] provides several improvements over traditional column chromatography, such as better resolution and high speed. Another important characteristic is the near-quantitative applicability of TLC results in dry column analysis. Knowledge of the TLC characteristics of a sample is useful before column chromatography is employed. Careful control of the moisture content of the adsorbent is crucial to the dry column as well as other types of chromatography. 

Figure 13.2. Separation of a complex mixture by chromatography. The top shows the column just after application of mixture. As the sample progresses through the column, separation of mixture occurs as a result of the chromatography. The first component exits the column and is detected, followed by the other two components. 

Three gradients of 0.0-0.5 M sodium chloride were run consecutively at 4°C in 0.05 M sodium acetate-acetic acid, 1 mM sodium azide, pH 5.25, followed by 0.05 M sodium acetate-acetic acid, 1 mM sodium azide, pH 3.5, and finally by 0.05 M sodium dihydrogen phosphate-disodium hydrogen phosphate (approx. 1 3), 1 mM sodium azide, pH 7.0. After sample application, the column was washed with the starting buffer to remove any non-bound compounds. Elution was continued with the high salt buffer. Fractions of 4 ml were collected and assayed for reactivity towards ninhydrin and for electric conductivity (salt concentration) after 75-fold dilution of a 100-pl aliquot. Ninhydrin-positive fractions were pooled for each peak, concentrated, and desalted by size exclusion chromatography (see above). 

Elution is one of the critical step for successful separation. Sample application in affinity chromatography is performed usually by injection or application in the presence of mobile phase which is prepared in appropriate pH, ionic strength and solvent composition for solute-ligand binding. This solvent is referred as application buffer [8]. In the presence of application buffer, compounds which are complementary to the affinity ligand will bind while the other solutes in the sample will tend to pass through the column as nonretained compounds. After... 

Multidimensional (or coupled) column chromatography is a technique in which fractions from a separation system are selectively transferred to one or more secondary separating systems to increase resolution and sensitivity, and/or to reduce analysis time. The application of secondary columns is illustrated schematically in Figure 8.1. The smaller the AtT value applied, then the greater is the resolution and number of runs needed to check a certain portion of the sample (5). 

The solvent extracts can be cleaned up by traditional column chromatography or by solid-phase extraction cartridges. This is a common cleanup method that is widely used in biological, clinical, and environmental sample preparation. More details are presented in Chapter 2. Some examples include the cleanup of pesticide residues and chlorinated hydrocarbons, the separation of nitrogen compounds from hydrocarbons, the separation of aromatic compounds from an aliphatic-aromatic mixture, and similar applications for use with fats, oils, and waxes. This approach provides efficient cleanup of steroids, esters, ketones, glycerides, alkaloids, and carbohydrates as well. Cations, anions, metals, and inorganic compounds are also candidates for this method [7],... 

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