Biochemistry Lab Report:

Thin Layer Chromatography of Amino Acids (TLC)

CHEM 451: Biochemistry Laboratory

November 24, 2015

Aim/Objective

 To understand the principles of Thin Layer Chromatography (TLC) and how to apply it.

 To identify and separate unknown amino acids in a mixture into their components using

amino acid standards.

Introduction

Chromatography is essentially the most useful available technique for the separation of closely

related compounds in a mixture. TLC technique provides quality information and with careful

attention to details, it is possible to obtain quantitative data. TLC consists of 3 steps; spotting,

development and visualization.

A TLC plate, made up of a thin layer of silica, is supported by glass or aluminum which it

adheres to. The components of the solvent mixture are soluble to different degrees. Separation

occurs as a mobile phase (usually the solvent mixture) flows through a stationary phase (solid or

liquid supported by solid) carrying components of the mixture with it. The stationary phase,

usually the silica gel, often contains a substance which fluoresces in UV light. The following are

some of the uses/value of TLC;

 To determine the purity of a substance.

 To identify and separate mixture components.

 To attain a quantitative analysis of the mixture components.

 To monitor the progress of a reaction.

 To examine fatty acids and ceramides.

Separation is dependent on several factors, some of which are;

 Size of the compound: The smaller a compound as a result of its side chain, the faster it

will move up the plate.

  Solubility: The less soluble a compound, the slower it moves up the plate.

 Polarity: Compounds with high polarity have high affinity for the silica plate, and will

move up the plate more slowly.

Other factors are temperature, solvent system, absorbent medium, etc.

Equipment/Reagent

Equipment includes TLC plate, TLC chamber, pencil, ruler, capillary tubes, fume cupboard,

reagent spray bottle, conical flask, beaker and developing tank with a lid.

Reagents include ninhydrin reagent, 2% solution of amino acids standards, solvent mixture of

butan-1-ol, water and 3% of glacial acetic acid (Solvent system consists butan-1-ol, acetic acid,

and water in the ratio 4:1:1 by volume).

Method/Procedure

1. A pencil and ruler was used to draw a line 2cm from one edge to another of the TLC

plate.

2. 5µl of the amino acid standards and unknown sample were applied (equally spaced) on

the drawn line using a micropipette, with the unknown sample at the middle spot.

3. A blow drier was used to speed up the drying of the spots.

4. Steps 2 and 3 were repeated.

5. The TLC plate was carried carefully by the edges to the fume hood and placed in one of

the provided racks in the tank.

6. The chromatogram was then left to develop for 8/9 hours.

7. The plate was then removed, sprayed with ninhydrin reagent and allowed to dry in the

fume cupboard.

8. A pencil was carefully used to mark the solvent front and the Rf values determined.
9. The unknown samples were identified by comparing the Rf values.

10. The identity of the unknown samples was then indicated on the data sheet.

Results/Observations

Attached below, are pictures my data sheet with the results of the experiment, and a photocopy of

my group’s TLC plate.

Conclusion

With the results on the result sheet, the Rf of the unknowns (ratio of the distance travelled to the

solvent front) was determined.

Further discussion

The TLC experiment has several clinical applications. Amongst them is its application in

“Lipids”. Lipids naturally occur and their biological functions include; energy storage, signaling
molecule and cell membranes structural component. Lipids are the most extensively examined

compounds by TLC because of some advantages. Lipid detection reagents are obtainable when

using TLC, and most of these reagents can be used to saturate layers to increase selectivity such

as argentation TLC. Several studies have been carried out to examine the clinical application of

TLC in lipids. For example, Taki et al, (1994), examined the purification of phospholipids and

GSLs suing TLC blotting technique.

Some other clinical applications include drug screening, doping control, presence of drug of

abuse, metabolism studies, etc. (Mohammad & Moheman, 2010).

 References

Buluma, E. M. (2015). Thin layer chromatography. Retrieved November 23, 2015 from

http://www.academia.edu/2112360/THIN_LAYER_CHROMATOGRAPHY.

Ghalayini, A., Wildman, T., James, K., Ikolo, T., Ikolo, F. (2015). Biochemistry laboratory manual,

Spring: Separation of amino acids by thin layer chromatography. Page 9. St. George’s

University.

Mohammad, A., & Moheman, A. (2010). TLC/HPTLC in Biomedical Applications. Chp. 10, High-

Performance Thin-Layer Chromatography (HPTLC). 151–178. doi:10.1007/978-3-642-

14025-9

vlab.amrita.edu,. (2011). Separation of Amino Acids by Thin Layer Chromatography. Retrieved

November 21, 2015, from vlab.amrita.edu/?sub=3&brch=63&sim=154&cnt=1