Double development chromatography

As a fractionation method, I developed chromatography of nucleic acids on hydroxyapatite, a calcium phosphate which had been used by Tiselius et al. (1956) for the fractionation of proteins. Previous observations (Bernardi and Cook, 1960a,b,c) that hydroxyapatite was particularly good as a chromatographic substrate for fractionating of phospholipoproteins characterized by different phosphorylation levels convinced me to try it on DNA. The main discovery was that hydroxyapatite could fractionate single- from double-stranded DNA (Fig. 1.4 left panel), the former being eluted by a lower phosphate... 

Chamber saturation (CS) (the solvent was introduced into the chamber 30 min before chromatography and shaken energetically in order to improve the reproducibility of the hRf-values) solvent I = benzene II chloroform III n-hexane-diethyl ether (80 + 20), double development, 10 cm. 

Soczewinski, E., Wojciak-Kosior, M., and Matysik, G., Analysis of glycosides and aglycones of flavonoid compounds by double-development thin-layer chromatography, J. Planar Chromatogr., 17, 261, 2004. 

Argentation thin-layer chromatography is an extemely useful procedure for the separation of methyl esters of fatty acids. Saturated fatty acids have the highest Rf values, which decrease with the increasing degree of unsaturation, and for a particular acid, the trans isomer usually travels ahead of its corresponding cis isomer. The solvents most commonly used contain hexane and diethyl ether (9 1) although a mixture of 4 6 is used to separate compounds with more than two double bonds. In order to separate positional isomers of the same acid, conditions must be carefully controlled and multiple development in toluene at low temperatures is often necessary. 

While you are waiting for the paper chromatogram to develop (step no. 7), you can perform the column chromatography experiment. Take a 25-mL buret. (You may use a chromatographic column, if available, of 1.6 cm diameter and about 13 cm long see Fig. 29.8. If you use the column instead of the buret, all subsequent quantities below should be doubled.)... 

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