Thin Layer Chromatography with Plant Pigments

Thin layer chromatography is an important analytical test for identifying unknown compounds, monitoring reactions, and testing chemical purity. The purpose of this experiment was to acquire the TLC technique.

Chlorophyll: a common plant pigment

Analysis
Thin-layer chromatography (TLC) is an essential analytical technique for organic experiments. It is a quick and simple method for determining the number of compounds in a mixture, identifying an unknown, testing purity, and monitoring reactions. These tests occur on chromatographic sheets, commonly constructed of alumina or silica pasted on a metal backing. Alumina was selected for this experiment.

Basically, the compounds being tested are spotted near the bottom of 1” x 3” chromatographic sheets to be placed vertically in a small pool of solvent that initially does not submerse the solutes. The solvent will be carried through the sheet via capillary action, contacting the test compounds, and then continuing on. Several factors determine whether or not, and how far, the solutes will traverse the chromatographic plate; this is the non-trivial part. Differential partitioning occurs when the solvent reaches the test compounds, in which the solutes are competitively bound between the highly polar stationary phase (alumina) and the rising solvent. The compounds should rise some distance, D, and the solvent some terminal distance F. Comparison of polarity and solubility can be made by calculating the ratio of D/F, termed the R_f value. Good solvents will spatially separate a mixture of chemicals and yield R_f values within 0.3 and 0.7.

This experiment was designed to separate the pigments in plant leaves, like chlorophyll and carotenes, via TLC. Parsley was used for the pigment extraction, while most peers used common tree leaves. The chromatography chamber was prepared with a plate as discussed, spotted repeatedly (~1 mm in diameter) with the parsley extract and placed in a beaker containing a filter paper soaked with the solvent and isolated by a watch-glass lid. These precautions were taken to saturate the atmosphere with the solvent. When the solvents rose about 6 cm they were removed. The solvent front and solute spots were promptly marked with pencil.

An ethanol/acetone solvent pair was used for the chromatography and two plates were developed with differing concentrations. The first chromatography  was prepared with equal shares of the solvents and yielded an R_f of 0.21 with no separation. In the other development, the solvent consisted of 30% acetone and 70% ethanol, and without separation the R_f  was measured at 0.285 (see Table A). Initially, the spots presented as a darker green and afterward were observed as light green streaks (~2 cm in length). Other solvent pair systems employed by peers reveal some interesting results.

The chart shows several solvent systems in this experiment that yielded desirable R_f values (0.3-0.7), especially with ethanol. Remarkably, only two solvents were observed to separate the pigments, pure hexane; and 30% acetone, 70% hexane. At first glance, it appears that hexane was crucial for successful TLC separation. However, careful examination of the data and solvent pairs makes this assumption questionable (see Table A for complete experimental data). These tests with pigment separation were both conducted by Mr. Rodgers, who used an independent selection of leaves for extraction. Those leaves were notable because they had some yellow characteristics, unseen in the other leaf samples. Consider too, that hexane was used in 1:1 and 3:7 combinations with acetone by other experimenters, resulting in no visible separation.

Table A: Experimental TLC Data

In conclusion, it seems the acetone/ethanol systems failed to separate the pigments. It is unfortunate that time did not allow for further experimentation with Rodger’s hexane solvent pair.  However, the experiment was successful for the acquisition of the TLC technique.