This experiment is entitled "Analysis of Amino Acids by Chromatography" which

aims to identify amino acids by thin layer chromatography based on their Rf values
​compared to the Rf values ​of pure amino acids.
Chromatography is one of the analytical methods used to determine the
components in a mixture by comparing the displacement distance of the investigated
substance in a solid matrix with the known displacement distance of the standard
substances. The solid matrix that is usually used is paper or thin layers and the
technique is called paper chromatography or thin layer chromatography (Mulyani, 2021).
Amino acids are organic compounds that have a carboxyl (-COOH) and an amine
(-NH2) functional group. The carboxyl group gives acidic properties and the amine group
gives basic properties. amino acid structure :

Ninhydrin is a chemical used to detect ammonia or primary and secondary

amines. When ninhydrin reacts with amino acids it produces a stain color. The general
formula for ninhydrin compounds is as follows:

Thin layer chromatography separates components from a mixture based on

differences in polarity. Components with the same polarity as the stationary phase will
tend to be retained, so they will separate from the final mixture with the smallest Rf
value. Thin layer chromatography is used to separate the components on the basis of
the difference in adsorption/partition by the stationary phase under the movement of the
developer solvent.
To identify the substances present can be calculated from the Rf value of each
substance in the chromatogram. From the Rf value obtained, if the Rf value is the same,
it can be said that the compounds analyzed are identical. (Day, 2002).
This experiment was carried out in two stages, namely:
a. Preparation with
Eluent Although when the experimental eluent used was provided, it can be seen that
this eluent was prepared by mixing 100 mL of n-butanol + 100 mL of distilled water + 24
mL of glacial acetic acid. Then shake it and wait for the separation to occur in the
 solution. Then take the top solution as the eluent. The bottom layer was collected in a
beaker and placed in a chromatography cabinet to saturate the chamber with its vapors.
The top layer is also collected and this is used as the eluent solution. That was the
method of preparing the eluent used in the analysis of amino acids using thin layer
chromatography.

b. Elution Process
This experiment aims to identify amino acids by thin layer chromatography based
on the comparison of the Rf value of the sample with the Rf of the amino acid.
The working procedure of this experiment is to place the sample to be analyzed
on a silica plate as follows:

After it is dripped with sample, then insert the plate into a chamber that has been filled
with eluent. Then wait for the eluent to rise. After it was felt to rise, take the plate and dry
the silica plate, then spray the silica plate with ninhydrin, then dry it in an oven at a
temperature of 100 degrees Celsius and then measure the distance of the stain formed.
The results of these steps are as follows:
 No. Sample solution Spot distance (cm) Eluent distance Rf
(cm)

1. Leucine 2.4 cm 5.3 cm 0.4528

2. Glycine 0.3 cm 5.3 cm 0.0566

3. Alanine 0.5 cm 5 ,3 cm 0.0943

4. Unknown solution Top part : 2.4 cm 5.3 cm Top part : 0.4528

Bottom part : 0.3 cm Bottom part : 0.0566

From the results of calculations that have been carried out, the Rf value of
leucine is obtained is 0.4528, the Rf value of glycine is 0.0566, the Rf value of alanine is
0.0943, and the Rf value for the Unknown sample is 0.4528 and the Rf is 0.0566 for the
bottom part. Determination of the type of amino acid can be determined by the Rf value,
namely by comparing the sample Rf value with the standard Rf value or standard amino
acids. Standard solutions usually have the same structure and properties as the
substances that can be separated or analyzed. Judging from the calculated Rf value, the
Rf value in the Unknown solution sample in the bottom part has the same Rf value as
the glycine solution, which is 0.0566. Then the Rf value in the unknown top part solution
has the same solution as the leucine solution, which is 0.4528. This means that the
unknown solution sample is a mixture of the amino acid compounds glycine and leucine.
Based on the Rf value, then compounds, compounds that have the same Rf value then
they also have the same level of polarity as well.
In thin layer chromatography, the stationary phase in the form of cellulose on the
plate must have a higher polarity than the mobile phase. Because the stationary phase
is more polar, the polar components will be stuck and difficult to move up, while the
non-polar components will separate faster, so the Rf value is larger. In other words, if the
substance has a low Rf value, then the polarity is high. And vice versa, if it has a high Rf
value, then the polarity is low.
Based on the calculation results, the Rf value in the glycine solution and the
unknown solution is the lowest Rf value compared to other solutions, which is 0.0566. So
this means that glycine and the unknown bottom part solution have the highest level of
polarity when compared to other solutions. So that the order of polarity in the samples
seen from the Rf value is Glycine and Unknown bottom part solution > alanine > Leucine
and unknown top part sample solution. This is in accordance with the theory where
according to theory, compounds that have a low Rf value have a high level of polarity,
and compounds that have a high Rf value have a low level of polarity.
The Rf value obtained in the experiment shows that the Rf value is in the 0 .
range ≤ Rf ≤ 1, so this experiment is in accordance with the theory where according to
the theory the value of Rf is in the range 0 ≤ Rf ≤ 1. If an experiment produces an Rf
value of ≥ 1 , it means that the polarity of the eluent itself must be reduced.
In this experiment, spraying was carried out with a solution of ninhydrin to form a
colored product (blue-purple).
 The reaction that occurs when ninhydrin + with amino acids (various types).

The functions of adding substances in this experiment include:

1. Glycine, Leucine, Alanine as standard amino acids.
2. Unknown solution as analyzed solution.
3. Ninhydrin functions as a colorant for amino acids when forming purple complex
compounds.
4. Drying aims to dry the eluent and ninhydrin so that it can make the stain more visible.