Column chromatography, adsorbents analytical procedures

Analytical Procedures. The chemical compositions of both liquid and gas products were determined by gas chromatography (GC). The normal paraffinic concentration in the liquid products and their carbon number distribution were determined by a GC method (5). The sample was analyzed first with a 2-ft silicone gum rubber (SE-30) column and then analyzed again with the same column attached behind a 3-inch Ca/A zeolite column which adsorbed all n-paraffins. 

The four common causes for high-performance liquid chromatography (HPLC) column failure include column clogging at the inlet frit (from samples/mobile phase), voids generated in the column, strongly adsorbed impurities from solvent/sample, and chemical attack of the stationary phase from the mobile phase or analytes. Procedure for removal of strongly adsorbed impurities from sample/mobile phase was discussed in Chapter 3, Section 3.9.2. 

The second analytical procedure is somewhat similar, but a continuous stream of gas is passed over the sample and through the trap. This produces a much larger sample of the volatile substances of interest and, thus, can often detect trace materials. The adsorbed components are desorbed by heat in the same manner and passed directly onto a gas chromatography (GC) column. The results are stiU determined by the distribution coefficient of each solute between the sample matrix and the air and, thus, the quantitative results remain comparative or relative, but not absolute. 

Surface enhanced laser desorption/ionization (SE-LDI) is a variant of MALDI in which the MALDI probe is derivatized with various substances that have affinity for the analyte. The probes are then used to extract the analyte directly from mixtures thus avoiding sample loss through more complicated procedures such as column chromatography. Contaminants can be washed from the probe with appropriate buffers or solvents leaving the purified analyte ready for analysis. Many adsorbents have been used typical examples are hydrophobic or ionic compounds, enzymes, various receptors, antibodies, and nucleic acids. Although most applications have been reported with proteins, the technique is potentially applicable to any type of compound for which a specific adsorbent can be attached to the probe. 

Complex carbohydrates frequently contain adsorbed lipids, and this is especially the case where tissue, cell and membrane preparations are desialylated. For this reason, two extractions of the supernatant or dialysate containing free sialic acids with one volume each of diethyl ether or n-hexane is advisable, prior to ion-exchange chromatography (Schauer et al. 1975, Schauer 1978). If this extraction is not performed, erroneous results due to non-sialic acid compounds may be obtained in the analytical procedures, especially in the periodic acid/thiobarbituric acid assay of sialic acids (Schauer et al. 1975). Furthermore, lipid extraction improves sialic acid resolution on the following column chromatographic steps. 

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