Sodium Deoxycholate Micelles Activated Separation of Coexisting Five- nucleobases by High-performance Thin-layer Chromatography

High-performance thin-layer chromatography (HPTLC) is well suited to the separation of nucleobases. Most of reports on this topic are limited to the use of PEI-cellulose, ODS (octadecyl silica), and silica gel as layer materials (Randerath and Struck, 1961; Bij and Lederer, 1983; Steinberg et al., 1996). Chiral plates have also been used for separation of nucleobases, and enantiomers (Hatzack and Rasmuseen, 1999). The first use of micellar mobile phase by Armstrong in 1979 for the analysis of nucleotides was 1.3 AOT in cyclohexane-water mixture. The suggested mobile phase was capable to resolve a mixture of nucleotides on silanized silica gel. Compared to normal micelle forming surfactants, bile salts are unique in forming helical aggregates in solution (Gillio et al., 1988; Campanelli et al., 1989; Williams et al., 1990). In nature, sodium deoxycholate (Figure 1) referred as “secondary bile acid” is produced in the intestine from the salts of glycocholic, and taurocholic acid by the action of bacterial enzymes. Applications of sodium deoxycholate range from cell lyses, liposome preparation, isolation of membrane proteins and lipids, a cell culture media as supplement, preventing non specific binding in affinity chromatography, micellar electrokinetic chromatography and other chromatographic techniques (Hofmann and Mysels, 1987; Williams et al., 1990; Thompson et al., 1995 Morgan et al., 2008). Versatility of bile salts led us to utilize its analytical potential as mobile phase for the separation of coexisting nucleobases from their mixtures. This is probably the first report to separate five nucleobases (two purines, and three pyrimidines) on cellulose 60 F254 HPTLC plates by utilizing sodium deoxycholate (aqueous 5.0%) plus acetonitrile (1:3, v/v) as mobile phase. Furthermore, we have successfully achieved an interesting separation of thymine from uracil. This separation is important because both pyrimidines are used to differentiate between the structures of DNA and RNA (Figure 2).