Exercise 3

CHROMATOGRAPHIC ANALYSIS OF PEPTIDE HYDROLYSATE

Bate AC, Enriquez IJ, Loberiano AK, Ochea C

OBJECTIVES
The objective(s) of the experiment is/are as follows:
● To perform paper chromatography with the sample amino acid mixtures, and
● To identify the amino acids used in the experiment

RESULTS AND DISCUSSION

Chromatography comes from the Greek word chroma which means “color,” and graphein which
means “to write”. This means that in the process of chromatography, chemists ought to write
colors. The technique, chromatography, was first used in order to easily separate plant
pigments with visible colors around the beginning of the 20th century. Chromatography is used
because of the assumption that different compounds distribute themselves to various extents
between different phases of matter. Chromatography is divided into two phases. The first one
phase is the stationary phase and the second one is the mobile phase. The mobile phase
carries the sample to be separated and flow over the stationary phase, where interactions
between components happen. In this process, the movement of the flow of interaction depends
on how the components interact with the stationary phase. If it strongly interacts with the
stationary phase then components that were carried along by the mobile phase will move slowly
than are those that interact less strongly. This differing mobilities of the components are the
basis of the separation in the process of chromatography.​8 ​This separation process can be
performed either in ascending or descending manner. In ascending chromatography, the
solvent is at the bottom and moves up by capillarity until it reaches the final point while in
descending chromatography the solvent is at the top and flows down by capillarity and with the
help of gravity.​9 ​The frequently used technique of chromatography is paper chromatography.

Paper chromatography follows the same principle as the other chromatography techniques. And
from the title itself, paper chromatography uses paper as its stationary phase. In the experiment,
the mobile phase was the eluting solution made up of the mixture of ethanol, water and
ammonia. The paper and solution were used to determine the Rf values of the amino acid
samples by measuring the distance travelled by the sample over the distance travelled by the
solvent.

Amino Acid Distance Travelled (cm) Theoretical Rf Value Experimental Rf Value

1 ALB 0 cm 0 0

2 ASP 15 cm 0.24 0.13

3 THR 36 cm 0.35 0.31

4 LYS 25 cm 0.14 0.21

5 MET 56 cm 0.55 0.47

6 TYR 40 cm 0.45 0.34

Distance travelled by solvent (cm): ​116-118 cm

Based on the recorded results from the experiment, Methionine was observed to have the most
distance travelled while the Albumin exhibited the least. The farther the distance of the amino
acid is expected to travel, the higher its theoretical Rf value. Threonine exhibited the most
satisfactory result with the nearest obtained Experimental Rf Value of 0.31 to its Theoretical Rf
value, 0.35. On the other hand, Lysine exhibited the least ideal result, obtaining 50% error from
its theoretical value. While the results are not of high accuracy, most of the amino acid samples
conformed to their expected results, except amino acids 2 and 4. Aspartic acid should be
expected to have longer travel distance than Lysine because it has higher Rf value, however,
results exhibited oppositely. This has caused high percent errors to the experimental values of
two amino acids.
The solvent front was allowed to ascend in the paper and was dried before putting the protein
samples in it. It was observed that the solvent did not move up in a perfectly straight line, this
could pose an error in determining the actual distance travelled by the solvent which is used to
calculate the Rf.​11 ​Another possible error could also be caused by inaccurate measurements of
the distances traveled by the protein. The unequal amount of protein used may also cause
errors as well. 10
​ ​Spraying the ninhydrin reagent may not be equally distributed within the paper
thus causing not compact or significant spots. The solubility of the amino acids in water
absorbed by the paper leads to the formation of diffuse spots.

ANSWERS TO POST-LAB QUESTIONS

1. Discuss the principles involved in paper chromatography.

- Chromatography is a technique based on the fact that proteins separate from each other
to varying extents between different phases.​1 One phase is the stationary phase and the
other is the mobile phase. The mobile phase flows over the stationary material and
carries the sample to be separated along with it. If the sample reacts strongly with the
stationary phase, it is moved up the paper more slowly, by capillary action, than the
components reacting weakly. The varying mobilities are the basis of the separation.​2

2. Why do different compounds travel different distances on the piece of paper?

- The solutions used contain different types of amino acids, and these amino acids vary in
charge, size, binding affinity, and hydrophobocity.​3

3. What is the purpose of the ninhydrin solution? Discuss the principle behind developing
the chromatogram using this reagent.
- Ninhydrin reacts with alpha-amino groups, peptides, and proteins to form a purple color
called Ruhemann’s purple (RP).​4 Ninhydrin reacts with an amino acid, generating an
aldehyde, a reduced ninhydrin, CO​2​, and H​2​O. The resulting NH​3,​then reacts with
another ninhydrin, giving the purple-colored ion (Ruhemann’s complex).​5 If the sample
gives off a purple color, this means that the sample contains amino acids.

4. Give two other methods of protein analysis. Discuss the principles behind each.
- There are 3 main methods of protein analysis: through protein identification, protein
separation and purification, and western blotting. Chromatography is an example of
protein analysis through protein separation and purification.

A protein’s structure is determined by its amino acid composition. There are two main
methods used in identifying proteins: (1) The Edman Degradation Reaction is a series of
chemical reactions that remove amino acids from proteins without disrupting the peptide
bonds; and, (2) Mass Spectrometry is a detection technique that measures the
mass-to-charge ratio of molecules with high energy electrons to break a molecule into
 fragments. The fragments provide information about the molecular weights and chemical
structures of the proteins.​6

The western blotting is used to identify specific proteins from a mixture of proteins
extracted from cells. It separates the proteins based on molecular weight, and by type,
through gel electrophoresis. Then, they are transferred to a membrane producing a band
for each protein. This membrane is incubated with labels antibodies specific to the
protein of interest.​7

REFERENCES

1. Rodwell, V. W.; Bender, D. A.; Botham, K. M.; Kennelly, P. J.; Weil, P. A. In ​Harper's
Illustrated Biochemistry​; p 24.
2. Campbell, M. K.; Farrell, S. O. In ​Biochemistry;​ p 118.
3. Nelson, D. L.; Cox, M. M.; Lehninger, A. L. ​Lehninger principles of biochemistry​; W.H.
Freeman and Company: New York, NY, 2017.
4. Friedman, M. Applications of the Ninhydrin Reaction for Analysis of Amino Acids,
Peptides, and Proteins to Agricultural and Biomedical Sciences. ​Journal of Agricultural
and Food Chemistry​ 2004, ​52​ (3), 385–406.
5. Bhagavan, N. Amino Acids. Medical Biochemistry 2002, 17–33.
6. Buyukkoroglu, G.; Dora, D. D.; Ozdemir, F.; Hizel, C. In ​Omics Technology and
Bioengineering;​ 2018; pp 317–351.
7. Yang, P.-C.; Liu, Z.-Q.; Mahmood, T. Western Blot: Technique, Theory and Trouble
Shooting. North American Journal of Medical Sciences 2014, 6 (3), 160.
8. Campbell, M. K.; Farrell, S. O. In ​Biochemistry;​ Mary Finch, 2012; p 118.
9. How Does Chromatography Work?​ SAPS 2020.
https://www.saps.org.uk/saps-associates/browse-q-and-a/387-how-does-chromatograph
y-work​ (Accessed February 5, 2020).
10. Hackman, R.H. ;Lazarus M. Quantitative Analysis of Amino Acids Using Paper
Chromatography , 1955 , 289-291
11. Identifying Amino Acids By Using Paper Chromatography Biology Essay
https://www.ukessays.com/essays/biology/identifying-amino-acids-by-using-paper-chrom
atography-biology-essay.php​ (accessed Feb 6, 2020).