Introduction

Thin layer chromatography (tlc) is a chromatography technique used to separate mixtures ( Archana
Bele et’al,2011). Chromatography was discovered in 1906 by M Tswett. Normally a suitable closed
vessel containing a solvent and a coated plate are all that are required to carry out the sepation and
qualitative and semi quantitative analysis (J. Sherma 2003). The stationary phase (coated plate) is
usually a sheet of glass, plastic or aluminium foil, which is covered or coated with a thin layer of
adsorbent material which in most cases can be found to be silica gel, aluminium oxide or cellulose
(blotte paper). The mobile phase is usually a solvent or solvent mixture (A.Bele et’al 2011). A basic
thin layer chromatography is carried out as follows, a small aliquot sample is placed near one end of
the stationary phase,a thin layer of sorbent to form the initial zone. The sample is the dried. The end
of the stationary phase the initial zone is then placed carefully into the mobile phase (usually a
mixture of 2-4 pure solvents) inside a closed chamber (J.Sherma 2003). If the thin layer and solvent
were chosen correctly, the solvent is drawn up the plate via capillary action, this process is termed
the development of the chromatogram. Because different analytes ascend the TLC plate at different
rates separation is archived. Thin layer chromatography can be used to monitor the progress of a
reaction, identify components or compouds present in a given substance hence the aim of this
experiment is to use thin layer chromatography to separate and identify the componets that make up
the amino acids provide using 2 different developing solvents and ninhydrin as a locating agent.
EXPERIMENT SECTION

APPARATUS

2 TLC plates (20cm x 20cm)

Sprayer (for application of locating agent)

Developing apparatus

Rule (30cm)

Pencil

REAGANTS

Amino acids

Developing solvent 1; butyl alcohol/acertic acid /water (80,20,20 vol./vol)

Solvent 2; propyl alcohol/water(7:3 vol./vol)

Locating agent/ ninhydrinsolution-0.3% ninhydrin (1,2,3triketohydrine, in butyl alcohol containing

3% glacial acetic acid.)

PROCEDURE

Unknown and known standards of amino acid were provided in which the mixtures to be separated
contained approximately 1 mg of each amino acid per mL of 0.5 M alcoholic hydrochloric acid
solution. Two TLC plates were collected and carefully held by the sides to avoid disturbing the
silica gel, a line was drawn 2cm from the bottom edge using an hb pencil. On the line that was
created a point was created on the line for each of the known and unknown amino acids to be
placed at, this was done leaving a 1 cm gap both sides of the TLC plate. Each point was labelled
accordingly for the amino acids to be placed at. The known amino acids were then dropped on their
points using a capillary tube while ensuring a diameter of 2-3mm. The amino acids were then
dropped again after they evaporated on the silica gel. The process was repeated for the unknown
amino acid mixtures. After applying the amino acids to the TLC plate, the plates were then allowed
to dry of for about 10-15 minutes. After drying the TLC plates were placed in a the developing
solvents to develop the chromatogram plates. The plates were left for approximately 2 hrs so that the
solvent can elude to a distance of ¾ of the total height of the TLC plate. The plates were then
removed from the developing solvents and the hieghest point reached by the solvent (solvent front)
was the marked with a pencil. The chromatogram plates were then allowed to dry. Once dried they
were the taken to the fume hood to spray with the locating agent ninhydrin solution. The plates
were then air dried for a few minutes and then they were gently heated in theoven for several
minutes until the separated zones appeared clearly. The plates were then removed quickly from the
oven and the coloured spots were circled with a pencil.

RESULTS

AMINO ACIDS Rf in solvent 1 Rf in solvent 2

LA 0.33 0.31
0.65 0.52
0.77
LC 0.14 0.14
0.35 0.23
0.56
LV 0.35 0.38
0.50 0.63
0.62 0.81
0.71
LM 0.20 0.5
0.55 0.71
0.73 0.85

It was found that the unknown amino acids in mixture A were (LA,LC) and the mixture B was made
of (LC,LV) whereas mixture C was( LA,LC & LA).

When comparing the Rf values with the obtained with approximate one it was found that the Rf
values were not all accurate and most of them did not match any amino acid Rf values.
DISCUSSION

Many errors might have affected the results that were obtained. One of the errors might be because
of the diameter of the amino acids when dropping them on the starting points. This error might have
made the separated amino acids on the chromatogram more difficult to differentiate since it might
have been difficult to separate bigger dots thus affecting accuracy of separation. The other error that
might have affected the chromatogram and separation would have been touching the chromatogram
with fingers while marking the starting point and the solvent front of the chromatogram. The other
thing that could have affected the results and accuracy of the chromatogram would be holding the
chromatogram plates vertically when applying the ninhydrin locating agent since it would drip down
the chromatogram plate. The results were also inaccurate because of lack of knowledge when
identifying the and marking the separated amino acids. Since developing solvent 1 and 2 differ they
did not separate the chromatogram plate in the same manner, solvent 1 left a more distinguishable
dots on the chromatogram whereas solvent 2 left some sort of a trailing mark on the chromatogram.
This trailing mark left was then not circled properly which might have affected the results. This
tailing is caused by the partial retention of the amino acids since some molecules might react
accordingly with the solvent 2.

CONCLUSION

It was concluded that the unknown amino acids in mixture A were (LA,LC) and the mixture B was
made of (LC,LV) whereas mixture C was( LA,LC & LA).

When comparing the Rf values with the obtained with approximate one it was found that the Rf
values were not all accurate and most of them did not match any amino acid Rf values.
REFERENCES
 DEPARTMENT OF CHEMISTRY

CHE 314

EXPERIMENT 4
SURNAME: SAMAKWATI

NAME : BOTLHE KGOTLA

ID : 202003096

LAB DAY: WEDNESDAY GROUP B

DATE: 22/02/23

GROUPTIME: 1500-1800 HRS

THIN LAYER CHROMATOGRAPHY SEPARATION OF AMINO

ACIDS