Name: Tinashe Katsuro

Student ID: 210304

Email: katsurot@africau.edu
Course title: Agricultural Biochemistry

Assignment 3

TITLE
To separate and identify components from a mixture

METHOD
Separate a mixture using paper chromatography

OBJECTIVES
● To separate the various substances that make up the mixture
● To identify the substances (amino acids) in a mixture
● To purify the components of a mixture for qualitative and quantitative
analysis

INTRODUCTION

Chromatography is a technique that is used to separate and to identify

components of a mixture. This analytical technique has a wide range of
applications in the real world since many substances are mixtures of chemical
compounds. In this lab, you will explore two applications of chromatography -
identification of an unknown ink sample and the separation of food colorings. In
paper chromatography, the sample mixture is applied to a piece of filter paper,
the edge of the paper is immersed in a solvent, and the solvent moves up the
paper by capillary action. Components of the mixture are carried along with the
solvent up the paper to varying degrees, depending on the compound's
preference to be adsorbed onto the paper versus being carried along with the
solvent. The paper is composed of cellulose to which polar water molecules are
adsorbed, while the solvent is less polar, usually consisting of a mixture of water
and an organic liquid. The paper is called the stationary phase while the solvent is
referred to as the mobile phase. Performing a chromatographic experiment is
basically a three-step process: 1) application of the sample, 2) "developing" the
chromatogram by allowing the mobile phase to move up the paper, and 3)
calculating Rf values and making conclusions. In order to obtain a measure of the
extent of movement of a component in a paper chromatography experiment, we
can calculate an "Rf value" for each separated component in the developed
chromatogram. An Rf value is a number that is defined as:

distance traveled by component from application point

Rf = ------------------------------------------------------------------------
distance traveled by solvent from application point

The distance traveled by the spot is measured to the MIDDLE of the spot.

MATERIALS
1. Chromatography Paper (Stationary phase)
2. Solvents (Mobile phase)
3. Ruler
4. Pencil
5. 4 unknown sample solutions
6. Oven Dryer
7. 4 Inverted cups
8. Beaker
9. Aluminium foil paper
10. Ninhydrin
METHODS
1. A pencil line was drawn along the bottom of the chromatography paper,
this line was 2cm from the bottom edge
2. 1 pencil dot was placed along this line about 2 cm apart and was numbered
on the bottom of the sheet
3. Spots of amino acids was placed on the paper at the pencil dots busing
inverted cups, was let to dry the spot and repeated 3 times(at least)
4. The paper was transferred into a beaker with the solvent
5. The top of the beaker was covered with the aluminum foil to allow solvent
to move
6. The paper was removed after the solvent have travelled for about 8 cm and
the point moved by solvent was marked
7. The paper was dried in an oven
8. The paper was sprayed with ninhydrin
9. Th paper was dried
10.The distance travelled by each spot was recorded
11.The distance travelled by the solvent was measured and the Rf value was
calculated and the amino acids were identified in each sample

RESULTS
Solution/Sample Distance travelled by Distance travelled by Rf value
Solvent Spot

A 5.5cm 4.4cm 0.8

B 5.5cm 3.5cm 0.64

C 5.5cm 3.8cm 0.69

D 5.5cm 2.8cm 0.51

A is isoleucine
B is Norleucine
C is phenylalanine
D is Methionine/ asparagine
DISCUSSION
Paper chromatography is a chromatography technique that is used in separating
organic compounds and identifying the compounds present in them.

Sample A travelled 4.4cm meaning the compounds are very mobile and the Rf
value is 0.8. The amino acid found in this compound is isoleucine. This amino acid
may help how hemoglobin is made.

Sample B travelled 3.5cm, its Rf value was 0.64 and the amino acids in that
compound is Norleucine. It is used to probe the role of methionine in Amyloid-β
peptide (AβP) , the central constituent of senile plaques in Alzheimer's disease.

Sample C travelled 3.8cm, its Rf value is 0.69 and the amino acids in that
compound is phenylalanine. There are three forms: L-, D-, and DL- phenylalanine.
L-phenylalanine is found naturally in foods such as meat and eggs. Amino acids
are the building blocks of protein. L-phenylalanine is an essential amino acid.

Sample D travelled 2.8cm, its Rf value is 0.51 and the amino acids found in that
sample are Methionine/ asparagine. Asparagine is an alpha-amino acid among
twenty amino acids that are found in animal proteins. It is used in the
biosynthesis of proteins. Methionine is an essential amino acid found in meat,
fish, and dairy products. Amino acids are the building blocks used to make
proteins. Methionine cannot be made by the body, so it must be consumed in the
diet. It plays an important role in the many functions within the body.

CONCLUSION
In conclusion, these samples separated by paper chromatography and identified
are amino acids essential for humans and animals and are found in food also. The
paper chromatography may not give exact results as compared to thin layer
chromatography.
REFERENCES
https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-
biology/paper-chromatography#:~:text=Paper%20chromatography%20is%20a
%20technique%20used%20for%20the%20separation%20of,the%20amino
%20acids%20with%20it. Friday 17 March 2023, 12:33pm

https://chemlab.truman.edu/files/2015/07/PAPER-CHROMATOGRAPHY.pdf
Monday 20 March 2023, 19:07pm

http://geo1.tcu.edu/richards/Lab%206.pdf Tuesday 21 March 2023, 6:14am