NAME: TSHEPHANG THUTHUKA

STUDENT ID: 202001314

GROUP /DAY: MONDAY
DATE:27 FEBRUARY 2023
TITLLE: SEPARATION OF AMINO ACIDS USING THIN LAYER
CHROMATOGRAPHY
AIM:

To identify the amino acids in the unknowns by comparing to the Rf of the standards and
compare the experimental Rf values obtained with the approximate Rf values of the standards
INTRODUCTION

Thin Layer Chromatography is a technique used to isolate non-volatile mixtures. The

experiment is conducted on a sheet of aluminium foil, plastic, or glass which is coated with a
thin layer of adsorbent material(Bhawani et., al 2012) . The material usually used is
aluminium oxide, cellulose, or silica gel. On completion of the separation, each component
appears as spots separated vertically. Each spot has a retention factor (Rf) expressed as:

Rf = distance travelled by sample / distance travelled by solvent

The factors affecting retardation factor are the solvent system, amount of material spotted,
adsorbent and temperature. Like other chromatographic techniques, thin-layer
chromatography (TLC) depends on the separation principle( Silver, 2020). The separation
relies on the relative affinity of compounds towards both the phases. The compounds in the
mobile phase move over the surface of the stationary phase. The movement occurs in such a
way that the compounds which have a higher affinity to the stationary phase move slowly
while the other compounds travel fast. Therefore, the separation of the mixture is attained. On
completion of the separation process, the individual components from the mixture appear as
spots at respective levels on the plates. Their character and nature are identified by suitable
detection techniques.

Diagram of Thin Layer Chromatography

MATERIALS AND METHOUDS

REAGENTS APPARATUS
Developing solvents: TLC plate 20x20 cm
-butyl alcohol / acetic acid/ water(NO 1)
-propyl alcohol /water(NO 2)
Locating reagent: ninhydrin solution Sprayer
- 0.3% ninhydrin
Developing apparatus

A line was drawn using a pencil about 2cm from the bottom edge of a TLC plate . One point
was marked for each one of the known and unknown acids, leaving at least 1cm between
them. Every point was labelled, and the amino acids were spotted at their respective points.
The TLC paper was allowed to dry for about 15 minutes to ensure complete evaporation of
the HCL. The TLC plates were then placed into two solvents were they develop a distance of
about ¾ of total height of the TLC plates for 1 hour. After an hour the plate was removed,
and the farthest advance of solvent front was marked using a pencil. The plate was allowed
to dry. Once dried the plate was sprayed using ninhydrin solution in the fume hood and later
placed in the oven for 2 minutes. Once dried each coloured spot was circled using a pencil.
The amino acids of the unknowns were compared to the Rf of the standards and the
experimental Rf values obtained were compared with the approximate Rf values of the
standards.
RESULTS AND ANALYSIS

Sample calculation

Cystine

Rf= distance of analyte / distance of solvent

=1.9 cm/6.0cm

= 0.32

TABLE 1: CALCULATED RF VALUES OF AMINO ACIDS IN TWO DIFFERENT

SOLVENT SYSTEMS

Amino acid Rf value developing solvent Rf value developing solvent

no.1 no. 2
Alanine 0,22, 0.29 & 0.58 0.69& 0.84
Cystine 0.32 0.23
Methionine 0.5& 0.7 0.81, &0.90
Valine 0.46 & 0.56 0.73
A 0.19, 0.32 & 0.56 0.16 , 0.66, 0.85
B 0.17,0.46& 0.58 0.16 &0.70
C 0.22,0.30,0.51,0.59 &0.69 0.72 &0.90
DISSCUSSION

According to Sherma, & Fried ( 2003)pure compounds produce a single spot. In the table
above cystine showed on one spot with an Rf value of 0.32 in solvent no. 1 and 0.23 on
solvent no. 2. This indicates that there are no other substances present, reinforcing that the
cystine is pure. However, the other amino acids beside cystine produced multiple spots hence
this amino acids were not pure. In contrast to these substances create a series of spots. When
chromatography is carried out on a compound, the substances separate out to create a series
of spots. Each spot will represent one specific substance. This applies mainly in the unknown
amino acids :A, B & C. the unknown acids have series of spots hence this will help in
identifying which amino acids were contained in them.

The thin layer chromatography plate itself can affect the retention factor value obtained for a
given chemical. Thin layer chromatography plates can be coated with a variety of absorbent
solids, most frequently silica or alumina (Truter et., al 1965) . This could have caused the
differences in the Rf obtained in the experiment and the standards. For example, the results
show that methionine had a Rf of 0.5 and 0.7 in developing solvent no. 1 whilst the standards
show that it has a Rf of 0.47. this is a very huge difference the Rf are not even close. This
could have been caused by a change in the TLC coat since the experiment was carried out
using silica as an adsorbent ,the standards could have used alumina. Since the retention factor
is based on the relative affinity of the chemical for the absorbent compared to the solvent,
changing the absorbent greatly change the retention factor.

When spotting the sample may have been applied too much, this leads to diffuse bands of
chemical moving up the plate, making it difficult to accurately measure the distance the
chemical has been transported(Bhawani et., al 2012). When analysing the results it was
noticed that the solvent did not have the same distance on all the amino acids , for unknown
acids it was 5.9cm whilst for cystine it was 6.0 cm. this resulted from the diffusion of the
bands of the chemical moving up the plate making it hard to measure its distance.

When several students share a TLC chamber, all plates should be placed inside quickly, and
removed at the same time, since constantly opening the tank to insert or remove plates will
affect the solvent vapours in the tank. The technician who was placing the TLC plates inside
the chamber must have been not quick enough hence the variations in the experimental and
the standard Rf.
When comparing the unknown amino acids Rf with the standards the unknown A could have
contained arginine monohydrochloride since its standard Rf was 0.15 in both the developing
solvent no.1 and no.2. this is quite close to the experimental Rf which in the developing
solvent no. 1 was 0.19 and in the developing solvent no. 2 it was 0.16. Unknown B also
could have contained arginine monohydrochloride since its standard Rf was 0.15 in both the
developing solvent no.1 and no.2, the experimental results show that its Rf was 0.17 and 0.16
respectively. It might have also contained tryptophan the Rf for standard in developing
solvent no 1 is 0.52 whilst the results is shown to be 0.58. Lastly unknown C might have
contained leucine which its standard Rf in developing solvent no. 1 is 0.57 and that of
developing solvent no . 2 is 0.69 , the results indicate an Rf of 0.59 and 0.72 respectively and
tryptophan the Rf for standard in developing solvent no 1 is 0.52 whilst the results is shown
to be 0.51

CONCLUSION

The unknown A could have contained arginine monohydrochloride, unknown B: arginine

monohydrochloride and tryptophan and unknown C might have contained leucine and
tryptophan .
REFERENCES

Bhawani, S. A., Mohamad Ibrahim, M. N., Sulaiman, O., Hashim, R., Mohammad, A., &
Hena, S. (2012). Thin-layer chromatography of amino acids: a review. Journal of liquid
chromatography & related technologies, 35(11), 1497-1516.

Jack Silver. Journal of Chemical Education 2020 97 (12), 4217-4219DOI:

10.1021/acs.jchemed.0c00437

Truter, E. V., Morris, L. J., Criddle, W. J., Halpaap, H., & Shellard, E. J. (1965). Thin-layer
chromatography. Proceedings of the Society for Analytical Chemistry, 2(7), 113-116.

Sherma, J., & Fried, B. (Eds.). (2003). Handbook of thin-layer chromatography. CRC press.