Botany lab fourteen chromatography

The day before laboratory Ace Hardware was searched for iso-octane, 2,2,4-Trimethylpentane, to no avail. Ninety minutes of perusing solvent and automotive additive ingredients surfaced nothing equivalent. The lab directions in the thin layer chromatography kit referenced acetone and isopropyl alcohol. 

Acetone was available at Ace Hardware, ethyl alcohol and isopropyl alcohol were available at Ace Office Supply. Cheesecloth was not located. White cloth from the house sufficed. The hardware store lacked lime - they had bags of lime for track and field at one time. The possibility of lime in the gardening section was overlooked. Black nitrile gloves were also purchased and provided. Nitrile has only moderate to good protection with respect to acetone, but no one was coming into direct intentional contact with the acetone in the class except the instructor. The leaf pulp was squeezed through a white cloth causing contact with the acetone. The gloves held up due to the brief nature of the contact. 

There is a possibility that the leaves could be mashed and squeezed without the acetone being added. The advantage to the acetone is that the acetone dries very quickly. Perhaps rubbing alcohol could be used, or perhaps nothing could be added. 

The right board was set up first prior to the start of the procedure.

The left board was set up after the chromatography runs were done.

Just ahead of lab Alternanthera sessilis, Volkameria inermis, and the flowers of Spathodea plicata were collected. The Volkameria went untested, the Spathoglottis yielded only a single rust red pigment.

A quarter teaspoon of calcium carbonate was used to conserve lime. This is said to stabilize the magnesium atom and keep the chlorophyll from disintegrating. This might have been too little as no group obtained clear chlorophyll a and b separation.

Acetone performed particularly poorly today for achieving pigment separation. The consistent failure of acetone today was unexpected. 

Ashley and Litana used acetone. The open air ventilation and closed TLC chamber reduced vapor exposure to a minimum. The chamber was prepared by the instructor and given to them already sealed. They were also on the upwind side of the classroom.

The new glass TLC plates are of variable width. The widest do not fit into the TLC chamber. The glass plates should apparently be left in until the solvent front hits the top of the plate. Then pull the plate out and dry as quickly as possible. 

Marstella and Jasery also worked with acetone 

Elavia and Elaine 

Reenomilyn Merma worked solo

Ethyl alcohol as the driving solvent with chlorophyll, carotenoids, and two xanthophylls

Isopropyl alcohol showing something at a low Rf along with chlorophyll, a probable carotenoid, and a likely xanthophyll.

The Spathoglottis was a wash out. There was only single extended rust red brown smudge in an ethyl alcohol solvent.

The performance of the ethyl and isopropanol alcohol, coupled with the relative safety of the compound, suggests that this be used in the future. If available, 90% isopropanol should also be tested. Acetone is the standard substance for thin layer chromatography. The failure of acetone to separate the pigments is puzzling. 

One future concern is that the new glass plates did not cleanly separate the pigments. There are hints of chlorophyll a in a streak on the right side of the chlorophyll, which might be chlorophyll b. There is a yellow patch, but whether this was a carotenoid or anthophyll is unclear. Perhaps extending the finish line closer to the end of the plate would help. The above plate was driven by ethyl alcohol. 

The key to using these plates will be to let the solvent front reach the top of the plate. Use as much plate as possible.