TLC of Organic Dyes h Undergraduate Labs T o acquaint elementary organic and biochemistry students with paper chromatography (PC) and thin-layer chromatography (TLC), I originally used a procedure that involved PC separation of food colors and TLC separation of an organic dye mixture.' The specified dye mixture was composed of rhodamine B, methylene blue, and butter yellow in a 3%wlv solution of 95%ethanol. The "developing" solvent system was an equal volume mixture of n-propanol, acetone, and benzene. Experience with this procedure suggested several changes: (a) substitution of toluene for benzene in the solvent svstem due to the recoaniwd carcinurenic putential of benzene! (b) rrduction of the concentration ot dyes used in the dye mixture, (c) replacement of butter yrlhw ta suspected rarvinog~niwith a nmhnzardous dye, and id, rclectiun uf a v~lventsystem that would give better resulution of the comDonenlu of the dve mixture. In using the original procedure, I noLd that the mmbnents of the dye mixture were not well resolved and that streaking or "tailing" of the spots occurred, leading to an overlap of the rhodamine B and methylene blue spots. Reducing the dye concentrations from 3%to as little as 0.17% wlv improved the appearance of the spots on the TLC plates and eliminated most of the streaking. Fluorescein was substituted for butter yellow in the dye mixture with acceptable results. Several solvent systems were examined and one of the simplest and best for this procedure is a 3 2 1 vlv mixture of acetone, n-propanol, and water. Developing time is reasonably short (about 25 min) and the three components of the dye mixture are well resolved. Table 1provides comparative data for some of the systems that were examined. TLC plates used were Anasil Type 0plates, 5 X 20 cm, precoated to a thickness of 260 p (available from Analahs, Inc.). Table 2 gives the compositions of the various solvent systems used. Table 2. Table 1. Resolving Ablllty of Dlflerent Solvent Systems
Comporltlon of Developing Solvent System Volume of Each Carnwnent
R#Values of Dvs Camwnents in
Fluorescein RhOdamineB Methylene hh.
yellow pink-red blue
0.97 0.51 0.01
0.93 0.53 0.02
0.97 0.73 0.00
0.95 0.79 0.00
0.97 0.17 0.03
0.96 0.12 0.02
A B
C D
3 3 1 1
2 2
1
1
1 3
1
2
0 2 0 0
I now usea0.1770 w ~ vpolucion of fl~~uresccin, rhodomine B,and methylme blue in 959 ethanola~rhcdyemixturef~,r the TLC procedure. Far separation ofthe componentsof the mixture, s d v m system A is used.'l.hii combination pnwldrs n mmple, convenient, and relatively nonhazardous system for the illustration of thc '!'LC separatim tcchniquc.
'
Linstromberg, Walter W.. and Baumgarten. Henry E.. "Organic Experiments for a Brief Course," 3rd ed., D. C. Heath and Company, Lexington, MA 1974, pp. 55, 258. This change was in fact made in the more recent fourth edition (1980) of the referenced laboratory manual.
Allen J. Scism Central Missouri State University Warrensburg. MO 64093
Volume 62
Number 4
April 1985 .
361
