UNIVERSITY OF NAIROBI

DEPARTMENT OF MECHANICAL ENGINEERING

BULUMA MARK EUGINE F18/1494/2011 GROUP 4

EXPERIMENT 6: THIN LAYER CHROMATOGRAPHY.

THE OBJECTIVE OF THE EXPERIMENT.

1. To separate the unknown amino acids mixture into its various components.
2. To identify the amino acids present in the unknown amino acid mixture.

THE THEORY BEHIND THE EXPERIMENT.

Chromatography is a method of separating a mixture into its components, by use of

heterogeneous equilibrium established during the flow of the solvent called a mobile phase
through a fixed (stationary) phase. The stationary phase can be either solid or liquid, while the
mobile phase can either be a liquid or a gas. Therefore, chromatography can be classified as;
solid- liquid, liquid- liquid, or gas- liquid.

Experimentally, chromatography can be carried out in columns or in layers. The column

chromatography uses a vertical tube packed with a medium/ adsorbent. The layer
chromatography uses a thin layer embedded unto a plate unto which the samples are introduced.

The thin film stationary phase may be:

1. A liquid (partition chromatography). Example is paper chromatography.

2. A finely divided adsorbent solid. (Adsorption chromatography). Example is Thin Layer
Chromatography.

INTRODUCTION TO THE EXPERIMENT.

Thin layer chromatography (TLC) is a chromatography technique used to separate

mixtures.[1] Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum
foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide,
or cellulose (blotter paper). This layer of adsorbent is known as the stationary phase.
After the sample has been applied on the plate, a solvent or solvent mixture (known as
the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend
the TLC plate at different rates, separation is achieved.[2]

Thin Layer Chromatography (TLC) is an example of adsorption chromatography. Adsorption

chromatography is the most common form of chromatography.

The following are the uses of Thin Layer Chromatography:

1. To check the purity of a substance by separating all the components of the substance.
2. To separate and identify components in a mixture.
3. To obtain a quantitative analysis of one or more of the components present in a mixture.
4. To monitor the progress of a reaction.
5. Analyzing Ceramides and Fatty acids.
6. Detection of insecticides and pesticides in food and water.
7. Analyzing the dye composition of fibers in forensics.
8. Assaying the radiochemical purity of radiopharmaceuticals.
9. Identification of medical plants and their constitutents.

The following are some of areas where Thin Layer Chromatography is applied:

1. Quality analysis and standardization bureaus, i.e. Kenya Bureau of Statistics.

2. Pharmaceutical industries and companies.
3. School laboratories during chemistry practical.
4. Detergent manufacturing industries and factories.
5. Water purification plants.

Here are a few of the numerous advantages of Thin Layer chromatography:

1. Simple and unsophisticated procedure of conducting thin layer chromatography makes it

easy to perform.
2. Its faster runs make it time saving.
3. Its cost effectiveness makes it affordable.
 4. It provides better separations than paper chromatography.
5. It allows a choice between different stationary phases

However, Thin Layer Chromatography has its disadvantages. Below is the main disadvantage of
Thin Film Chromatography:

1. It is dependent on other analytical methods to determine the quantities of components in a

mixture.
2. TLCs from low-temperature reactions may give misleading results, when used to
qualitatively monitor reactions i.e. DIBALH reduction of ester to aldehyde., because the
sample is warmed to room temperature in the capillary

PLATE PREPARATION

TLC plates are usually commercially available, with standard particle size ranges to improve
reproducibility. They are prepared by mixing the adsorbent, such as silica gel, with a small
amount of inert binder like calcium sulfate (gypsum) and water. This mixture is spread as a thick
slurry on a non-reactive carrier sheet, usually glass, thick aluminum foil, or plastic. The resultant
plate is dried and activated by heating in an oven for thirty minutes at 110 °C. The thickness of
the adsorbent layer is typically around 0.1 – 0.25 mm for analytical purposes and around 0.5 –
2.0 mm for preparative TLC.[4]

APPARATUS AND REAGENTS USED.

1. Pencil, Ruler, Glass capillary, Thin Layer Chromatography plate, developing tank with a
lid, an oven.
2. Developing solvent (n-butanol),4 Standard amino acids, Ninhydrin 0.3% in n-butanol
containing 3% of glacial acetic acid, Unknown amino acid mixture.

THE PROCEDURE OF THE EXPERIMENT.

1. A light pencil line was drawn 1cm from the bottom of the TLC plate.
2. Spotting points were marked on the light line 2mm apart except for the point for spotting
of unknown mixture, which was marked 3mm from the fourth point.
 3. Using different glass capillaries, the four standard amino acids and the unknown amino
acids mixture were spotted unto the chromatography plate.
4. Some of the developing solvent was poured into the developing tank. The tank was then
covered for about 10 minutes to allow the atmosphere in the tank to be saturated with the
vapor of the developing solvent.
5. The spotted TLC plate was inserted into the developing tank with the spots towards the
bottom of the tank.
6. The plate was then tilted such that the uncoated face was uppermost.
7. More of the developing solvent was carefully poured into the developing tank to cover
the bottom of the adsorbent layer.
8. The developing tank was covered with a lid and the developing solvent allowed
ascending by capillary action for about 1.5 hours.
9. The developed chromatography sheet was removed after the 1.5 hrs, the solvent front was
marked then the sheet was dried in an oven.
10. The dry chromatogram was sprayed with ninhydrin solution. The chromatogram was then
heated gently for until separate zones were visible. Then they were circled using a pencil.
11. The lengths of the zones from the spotted points were measured and recorded.

The diagram on the results sheet indicates a drawing of the chromatogram.

ANALYSIS OF THE RESULTS.

Having the results as indicated on the result sheet, calculation of the movement of the amino
acids in relation to the solvent front, or retardation factor (Rf value) was possible.

Rf of
DISCUSSION.

Different compounds in the sample mixture travel at different rates due to the differences in their
attraction to the stationary phase, and because of differences in solubility in the solvent. By
changing the solvent, or perhaps using a mixture, the separation of components (measured by
the Rfvalue) can be adjusted. Also, the separation achieved with a TLC plate can be used to
estimate the separation of a flash chromatographycolumn.[6]
Separation of compounds is based on the competition of the solute and the mobile phase for
binding places on the stationary phase. For instance, if normal phase silica gel is used as the
stationary phase it can be considered polar. Given two compounds which differ in polarity, the
more polar compound has a stronger interaction with the silica and is therefore more capable to
dispel the mobile phase from the binding places. Consequently, the less polar compound moves
higher up the plate (resulting in a higher Rf value). If the mobile phase is changed to a more
polar solvent or mixture of solvents, it is more capable of dispelling solutes from the silica
binding places and all compounds on the TLC plate will move higher up the plate. It is
commonly said that "strong" solvents (elutants) push the analyzed compounds up the plate, while
"weak" elutants barely move them. The order of strength/weakness depends on the coating
(stationary phase) of the TLC plate. For silica gel coated TLC plates, the elutant strength
increases in the following order: Perfluoroalkane (weakest), Hexane, Pentane,Carbon
tetrachloride, Benzene/Toluene, Dichloromethane, Diethyl
ether, Ethylacetate, Acetonitrile, Acetone, 2-Propanol/n-
Butanol, Water, Methanol, Triethylamine, Acetic acid, Formic acid (strongest). For C18 coated
plates the order is reverse. Practically this means that if you use a mixture of ethyl acetate and
hexane as the mobile phase, adding more ethyl acetate results in higher Rf values for all
compounds on the TLC plate. Changing the polarity of the mobile phase will normally not result
in reversed order of running of the compounds on the TLC plate. An eluotropic series can be
used as a guide in selecting a mobile phase. If a reversed order of running of the compounds is
desired, an a polar stationary phase should be used, such as C18-functionalized silica.

References

^ Laurence M. Harwood, Christopher J. Moody. Experimental organic chemistry: Principles and

Practice (Illustrated edition ed.). pp. 159–173. ISBN 978-0632020171.
^ Vogel's Textbook of Practical Organic Chemistry (5th Edition) (Hardcover) by A.I. Vogel
(Author), A.R. Tatchell (Author), B.S. Furnis (Author), A.J. Hannaford (Author), P.W.G.
Smith ISBN 0582462363

^ Reich, E.; Schibli A. High-performance thin-layer chromatography for the analysis of

medicinal plants (illustrated edition). Thieme: New York, 2007. ISBN 3-13-141601-7

^ Tables showing the thickness value of commercial regular and preparative Thin Layer
Chromatography plates

^ Thin Layer Chromatography: How To http://www.reachdevices.com/TLC.html

^ Fair, J. D.; Kormos, C. M. J. Chromatogr. A 2008, 1211(1-2), 49-54.

(doi:10.1016/j.chroma.2008.09.085)

^ Thin Layer Chromatography stains http://www.reachdevices.com/TLC_stains.html

^ TLC plates as a convenient platform for solvent-free reactions Jonathan M. Stoddard, Lien
Nguyen, Hector Mata-Chavez and Kelly Nguyen Chem. Commun., 2007, 1240 -
1241,doi:10.1039/b616311d