Column chromatography tailing

The method is based upon the ability of low concentrations of cyclic nucleotides to activate protein kinases which catalyse the phosphorylation of protein substrates, such as histone, by ATP [156,157]. The extent of phosphorylation is proportional to the amount of the cyclic nucleotides. The limits of sensitivity of the method are about 0.3 pmol for cyclic AMP and 0.5 pmol for cyclic GMP. Purification on a Dowex 50 column separates the two cyclic nucleotides from each other and removes any substances, such as ATP, which might interfere with the assay. Cyclic GMP is further purified by column chromatography on aluminum oxide and Dowex 1. Cyclic AMP-activated protein kinase is prepared from bovine heart and cyclic GMP-activated protein kinase from lobster tail. 

In reversed-phase chromatography, tailing may show up or increase as the column ages. Tliis is due to a partial and slow hydrolysis of the stationary phase. This process exposes additional silanols, which cause or increase peak tailing. This is a natural part of the column aging process. If the column lifetime is too short as a result of this effect, you may want to incorporate amine modifiers into the mobile phase, even if they are not needed with a brand-new column. 

Fig. 5-14 [63] shows van Deemter plots obtained for columns packed with 5, 10 and 30 p,m oc-tadecylsilica. The smaller the particle the less was the dependence of linear velocity on the theoretical plate height. Chromatograms of aliphatic and aromatic hydrocarbons are shown in Fig. 5-15 [65]. Use of the octadecylsilica in micropacked columns allows improvement of the efficiency of capillary packed columns. No tailing was observed. Application of these sorbents to gas chromatography, especially in packed capillary columns, is, in our opinion, a very promising direction [55, 63-69]. We also think that this type of modified adsorbents can be successfully used in gas adsorption on open capillary columns.

Sterically hindered 1,3-diphosphaallenes, such as 6, are air- and moisture-stable and can be purified by column chromatography. Less hindered 1,3-diphosphaallenes are isolated as the head-to-tail dimers. 

When large numbers of samples have to be evaluated, such as fractions in a column chromatography purification study, the lethality endpoint calculations require an excessive number of mice because a series of dilutions of each sample have to be injected into a number of mice. This problem has been alleviated to some extent by using intravenous injection (tail vein) of a single dilution of a preparation into several mice and determining the time to death (3,61). The LD5o/ml of the sample is calculated from the linear relationship between LD50 and time to death determined for a standard toxin preparation. 

The assay of ethyleneamines is usually done by gas chromatography. Compared to packed columns, in which severe tailing is often encountered due to the high polarity of the ethyleneamines, capillary columns provide better component separation and quantification. Typically, amines can be analyzed using fused siUca capillary columns with dimethyl silicones, substituted dimethyl silicones or PEG Compound 20 M as the stationary phase (150). 

The purity of the product was checked by vapor phase chromatography on a polyethylene glycol on Teflon column at 72°, 15 p.s.i., and a flow rate of 102 ml. of helium per minute. The sample appeared to be homogeneous, but, since the amine tails badly on the column, it is not possible to detect the presence of a small amount of water (less than 3%). 

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