ÇUKUROVA UNIVERSIY

SCIENCE AND LITERATURE FACULTY

CHEMISTRY DEPARTMENT.
BIOCHEMISTRY LABORATORY I

Chromatography of Amino Acids.

Daniel Alejandro MALAVER RANGEL

2019144315

Porpoise of the experiment

Performing a chromatography to several amino acids and a problem sample of an
unknown amino acid to develop a technique in such a technique.

Chromatography has been one of the most important methods that has been developed due to
its efficacy in separating different compounds with different polarities. The determination of
amino acids is extremely important for knowing basic things about the amino acid and protein
structure in several natural compounds that are found in nature.
During this experiment a chromatography will be applied to five amino acids. These amino
acids will be: (JDJASDFA). As a stationary phase the chromatography will be made in a
paper known as Whatman paper which is stronger and more resistant than normal filter paper.
Chromatography relies on the principle of differential partitioning between the stationary and
mobile phases. Components with different affinities for the two phases will move at different
rates, leading to separation. The stationary phase is the immobile part of the chromatographic
system. It can be a solid (as in column chromatography) or a liquid (as in thin-layer
chromatography or paper chromatography). The choice of stationary phase depends on the
type of chromatography and the properties of the analytes. The mobile phase is the moving
part of the chromatographic system. It carries the sample through the stationary phase. In
liquid chromatography, the mobile phase is typically a liquid solvent, while in gas
chromatography, it is a carrier gas.
During this experiment the mobile phase will consist on an polar solution of N-
butanol(100):NH3(3):H2O(50) and a stationary phase will consist on a typical Whatman filter
paper, the velocity of the amino acidic will depend basically of the type of amino acid that is.
 Aminoacids that are more polar will have a major displacement as the solution moves thanks
to the capillary effect of the paper.

for each spot, the Rf value (Rf means relative to front) will be calculated:

distance moved by spot

distance moved by solvent front
The Rf value is a constant that is recorded when
Amino acid Rf the chromatrogafy is made in optiam
value circumstances and thus it converts itself in a
alanine 0.38 constat. The values obtained in the
arginine 0.20 chromnatrography will be compared reference
asparagine 0.5 Rf values. Different solvents and different types
aspartic acid 0.24 or makes of chromatogaphy papers will give
slightly different results.
cysteine 0.4
glutamine 0.13 One or both of the spots from solution X may be
glutamic acid 0.30 at the same level as another (known) amino acid
glycine 0.26 alongside it. This should assist in identification.
histidine 0.11
isoleucine 0.72
leucine 0.73
lysine 0.14
methionine 0.55
phenylalanine 0.68
proline 0.43
not a true amino acid - shows up as
yellow
serine 0.27
threonine 0.35
tryptophan 0.66
tyrosine 0.45
valine 0.61
 Results and calculations

Amino acids have no colour. Therefore all of these procedures need to be carried out "blind",
and the results will be seen when a revealing agent (ninhydrin) is sprayed on the resulting
chromatogram.

Şekil 1 chromatrography papper with Şekil 2: meansurements taken in

contrast increased for having a the chromatrography
better quality of image

no. Of the aminoacid

Rf value
Distance (from
Rf value moved by the public Error
(Experimental) solvent source) porcentage
1 alanin 2.7 0.3913043478 6.9 0.38 2.63%
2 sistein 2.9 0.4202898551 6.9 0.4 5%
3 proline 3.2 0.4637681159 6.9 0.43 6.50%
4 glutanik asit 2.3 0.3333333333 6.9 0.3 11%
5 trionin 2.3 0.3333333333 6.9 0.35 5%
6 proline 3.2 0.4637681159 6.9

 According to the chart, the sample might be proline

  There is a contamination of another aminoacid having another leak in the glutaminic acid
identification lane. 5.00/6.9 = 0.72 prabli leucine or isoleucine.

Discussion of the results

At the moment of applying or conducting a paper chromatography, proper conditions should

be followed to achieve a successful outcome. These conditions are highly important for
obtaining an Rf value that aligns with the accurate standards documented in the literature. As
observed, several stains remain at the same level. To avoid choosing the incorrect center of
the stain, the highest and lowest peaks were subtracted, and the midpoint of the stain was
determined.
Two conditions were found to be favorable for the analysis: the first involves double staining
in the trionin stain, and the second pertains to the similarities between glutonic acid and
trionin. Both compounds seem to exhibit the same displacement, although such behavior was
unexpected.
Changes in temperature and humidity can affect the rate of solvent migration, leading to
variations in the separation. This may explain the 5 cm stain, and contamination from another
amino acid is also a possibility. Similarly, the drying process of the paper is crucial for
avoiding lane contamination. The quality of the paper plays an important role in achieving
stains with better resolution.

Overall, the objectives of the experiment were concluded successfully

 Javeed Ahmad Tantray, Sheikh
Mansoor, Rasy Fayaz Choh
Wani, Nighat Un Nissa,
Chapter 33 - Paper
chromatography of different
amino acid,
Editor(s): Javeed Ahmad Tantray,
Sheikh Mansoor, Rasy Fayaz
Choh Wani, Nighat Un Nissa,
Basic Life Science Methods,

Academic Press,Javeed
Ahmad Tantray, Sheikh
Mansoor, Rasy Fayaz Choh
Wani, Nighat Un Nissa,
Chapter 34 - Separation of
plant pigments using column
chromatography,
Editor(s): Javeed Ahmad
Tantray, Sheikh Mansoor,
Rasy Fayaz Choh Wani, Nighat Un Nissa,
Basic Life Science Methods,
Academic Press,

Javeed Ahmad Tantray,

Sheikh Mansoor, Rasy
Fayaz Choh Wani, Nighat
Un Nissa,
Chapter 35 - Paper
chromatography of
different amino acid,
Editor(s): Javeed Ahmad
Tantray, Sheikh Mansoor,
Rasy Fayaz Choh Wani,
Nighat Un Nissa,
Basic Life Science Methods,
Academic Press,
 Blibiography and sources

Amino acids commonly found in proteins. Amino acids. (n.d.).

https://www.biotopics.co.uk/jsmol/jsAmino_acids.html

Elgubbi, H. S., & Mlitan, A. M. (2015). Colorimetric Method for Determination of Amino
Acids on Thin Layer and Filter Paper Chromatography Using a Modified Ninhydrin
Reagent . Journal of Agricultural Science and Technology A 5 (2015) 190-193.
https://doi.org/10.17265/2161-6256/2015.03.005

Steane, R. (n.d.-a). Chromatography of amino acids.

https://www.biotopics.co.uk/as/amino_acid_chromatography.html

Steane, R. (n.d.-b). Water - in a class of its own. Water - its biological significance.
https://www.biotopics.co.uk/A15/water.html