Isobutyric acid-ammonium hydroxide

Suspend lyophilized crude or freshly washed bacterial cells (10 mg) in 400 pL of isobutyric acid-ammonium hydroxide 1 M (5 3 v/v) and keep for 2 h at 100°C in a screw-cap test tube under magnetic stirring. 

The nucleosides obtained by enzymatic digestion of t-RNA were separated by bidimensional chromatography using Merck cellulose layered on 20 x 50 cm aluminum sheets. The elution in the first direction was performed with n-butanol-isobutyric acid-ammonium hydroxide, conc.-water (75 37.5 2.5 25, v/v) mixture for 24 hr at 20-21°C, in an unsaturated chamber. The chromatographic plate was dried with a fan for 3 hr at 20°C, after the first elution. Elution in the second direction was performed for 4 hr with the mixture ammonium sulfate (saturated solution)-0.1 M sodium acetate (pH 6)-isopropanol (79 19 2, v/v). The separated compounds were visualized in UV light at 254 mn. 

Paper chromatography has also been successfully used for sequence isomer separation. For example, Fiers et al. (1965b) separated (Ap, Gp)Cp by paper chromatography on Whatman 40 paper eluted with isobutyric acid 0.5 N ammonium hydroxide (5 3 v/v), 5 -terminal Ap running faster in this system. 

Water has been used as solvent on DEAE- and ECTEOLA-cellulose layers [11, 59] and also 0.005N hydrochloric acid [11] and isobutyric acid-conc. ammonium hydroxide-water (66 + 2 + 32) on the former [11]. Saturated ammonium sulphate solution-N sodium acetate-isopropanol (80 -f 18 + 2), a solvent which was developed for the PC [45] and TLC (cellulose) [60] of nucleotides, is particularly good for separating purine and pyrimidine bases on layers of DEAE-cellulose [11]. 

Table 3 demonstrates the utility of cellulose in the relative retention of amino groups, regardless of purine/pyrimindine structure in either acid (HCI, isobutyric acid) or ammonium hydroxide mixtures. Ammonium carbonate (less so formate) does effect purine/pyrimidine separations, with Rfi greater for pyrimidines. 

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