and Identification by Thin-Layer Chromatography LAB. C1 Separation of Azo Dyes Using Column Chromatography and Identification by Thin-Layer Chromatography

Objectives Familiarize with the column chromatography (CC) technique and its application. Use the CC technique to separate the components of a mixture of azo dyes. Recognize, improve and optimize basic parameters of CC. Use the thin-layer chromatography (TLC) technique to identify fractions collected from CC. Assess the quality of column-chromatographic separation by TLC.

Principle Column chromatography makes use of a stationary phase, consisting of small particles that are packed in a glass tube to build a column and a mobile phase or eluent which is carrying the sample and moves through the stationary phase by gravity action.   Different molecules are retained differently by the stationary phase according to their different physico-chemical properties. This retention is a result of mainly (1) adsorption of a substance on a surface and (2) partition (or distribution) of a substance between physical phases. Contrary to thin-layer chromatography, at CC the process of chromatography is performed until the analytes have been eluted, i.e. they have been washed from the stationary phase by the eluent. The mobile phase is called eluate when it has passed the column. (At TLC, the analytes are still in the stationary phase after complete development)

Procedure A Column Chromatography:   stationary phase: silica gel 60, 70-230 mesh mobile phase: petroleum ether / diethyl ether (90 + 10) mixture solution of azo dyes The eluting dyes will be spotted on normal and reversed sheets TLC sheets.

B Normal Phase TLC: Stationary phase: TLC plates 20x20 cm, Merck™ KG 60 F254 on aluminum. DO NOT SCRATCH THEM OR TOUCH THEM ON THE SURFACE OR CONTAMINATE THEM WITH ANYTHING. mobile phase: petroleum ether / diethyl ether (70 + 30) samples: Azobenzene soln. 4-methoxyazobenzene soln. Orange G soln. unknown mixture fractions collected from column chromatography   C Reversed Phase TLC: stationary phase: TLC plates 20x20 cm, Merck™ KG 60 F254 on aluminum. impregnated by petroleum ether / liquid paraffin (95 + 5) mobile phase: methanol / acetone / water (70 + 8 + 22) azobenzene soln. unknown mixture (the same as used for normal phase TLC!)

Results 1- From the differently developed plates, calculate the Rf values for each dye, for each spot in the unknown mixture and if present for each spot of the collected fractions from the column chromatography and table them.   2- Comparing Rf values, find out which substances your unknown mixture most probably consisted of. 3- Judge the quality of your column-chromatographic separation.