Thin-Layer Chromatography and Quantitative Protein Analysis of Gluten

from Wheat Flour

Abigail Beatrice H. Lumbao, Reham B. Macadato, Ryan B. Manzano,

Lina Dae T. Maranga and Eunice Nicole A. Martin
Group 4 2D Pharmacy Biochemistry Laboratory

ABSTRACT
Gluten was the protein isolated found in the wheat flour and then was subjected for thin-layer paper
chromatography and quantitative protein analysis. TLC separates small molecules of amino acids.
Quantitative protein analysis on the other hand was used to determine the total protein concentration
from biological samples. Under TLC, standards were applied 5 times and the sample protein, 10 times and
were used to compare and determine which standard is present in the protein. R f values were computed.
In quantitative protein analysis, 6 test tube were prepared and diluted with with distilled water and biuret
reagent respectively. After subjecting to a spectrophotometer, the absorbance of each sample was
determined and plotted into a linear graph. Protein concentration shows that in standard 1, having the
smallest concentration gives the least absorbance of all standards and In the given absorbance, the
unknowns value is -0.038,z.

INTRODUCTION 600 nm to determine the quantity (Leah

Chromatography is used to separate mixtures
of coloured compounds. It is very suitable in
observing the different components of a mixture
or a solution. It is capable of separating all the
components of a multicomponent chemical
mixture without requiring an extensive
foreknowledge of the identity, number, or relative
amounts of the substances present. Mixtures that
are suitable for separation by chromatography
include inks, dyes and colouring agents in food.
Chromatography has two different phases,
namely the mobile phase and stationary phase.
The mobile phase is a liquid solvent and/or a
mixture of solvents. The mobile phase flows
through the packed bed or column. On the other
hand, the stationary phase is a porous solid that
is packed into a glass or metal tube or that
constitutes the walls of an open-tube capillary
Thin layer chromatography (TLC) is a
chromatographic technique used to separate the
components of a mixture using a thin stationary
phase supported by an inert backing. TLC is an
analytical tool widely used because of its
simplicity, relative low cost, high sensitivity, and
speed of separation.
In Biuret Total Protein Assay, protein solutions
turn purple after the polypeptide chain chelates
with a copper ion. An alkaline solution of Biuret
reagent including copper sulfate and Rochelle salt
is added to a protein solution. Uses the
absorption maximum at 540 nm to determine the
quantity .
In Bradford Total Protein Assay, the absorption
maximum of a protein shifts from 465 nm to 600
nm when the protein binds to the Coomassie
Brilliant Blue G250, one of triphenylmethanse
blue pigment. Uses the absorption maximum at
Pandiscia, 1998).
The objectives of this experiment are (1)
determine the amino acid components of proteins
using thin-layer chromatography; and (2)
quantitatively determine the protein
concentration of a given sample using commonly
used and microscale methods of total protein
analysis.
EXPERIMENTAL
A. Test Compound/s (or Sample/s)
used
The following samples were used for the
experiment proper: Hydrolized Protein
(Gluten), 2% w/v of tryptophan, arginine,
proline, cysteine, serine, aspartic acid,
tyrosine, histidine, glycine, and alanine,
Protein Sample (filtrate from casein
isolation).
B. Procedure
1. Separation and Identification of
Amino Acids by Thin-Layer
Chromatography
A 12 x 15 cm of paper
chromatography was prepared. A
margin of 1.5 cm-which will serve as
the point of origin was drawn
horizontally on the longer side of the
paper. 11 equidistant points were
made-these points will serve as the
point of origin of the sample and the
standards. The sample was applied 4
times while the standards were applied
8 times on the paper. The paper was
placed inside the pre-equilibrated
chamber. The solvent, 1-Butanol:
acetic acid: water (4:1:5), used inside
 the chamber should be below the point
of origin of the paper. The chamber
was tightly covered to make sure that
no air should come in and was also left
undisturbed until the solvent is 0.5 cm
away from the top edge of the paper.
The solvent line was marked then
after. After the paper was air-dry, it
was sprayed with 1% ninhydrin
reagent, which is a flammable and
corrosive reagent. The paper was then
placed in an oven for 1-3 minutes. The
colors blue, purple, or yellow should
appear in the paper. The spots were
encircled and the Rf values of each
sample or standard was computed.
To compute for the Rf value of the
sample, the distance traveled by the
solute was divided by the distance
traveled by the solvent. The solute in
this experiment is the sample or the
standard while the solvent used is the
solvent front.
2. Quantitative Protein Analysis
a. Preparation of Sample and
Protein Standards
The standard protein
concentrations were prepared by
mixing the following solutions in
the labeled test tubes:
Test Tube BSA Std. Distilled
Stock Soln Water
(mL)
Blank 0 2.50
Standard 1 0.10 2.40
Standard 2 0.50 2.00
Standard 3 1.00 1.50
Standard 4 1.50 1.0
Standard 5 2.50 0
The unknown protein solution
were diluted with distilled water to
produce 1:100 and 1:1000
samples.
b. Biuret Total Protein Assay
The blank and standard protein
solutions were prepared as
described in procedure 2.a. 2.50
mL of the 1:100 diluted unknown
protein solution was transferred
into another test tube. 2.5 mL of
the Biuret reagent was added into
each test tube. The test tubes were
heated in a 40-50 degree Celsius
water bath for five minutes. The
solutions were cooled and the
contents of the test tube were
transferred into a cuvette. The
cuvette was then placed in the
spectrophotometer.The wavelength
was set to 540 nm. The reading
was set to zero using the blank.
The absorbance of each samples
were read. The concentration of
the unknown protein solution was
determined using the graphical
linear regression analysis.
RESULTS AND DISCUSSION
Table 1: Shows the Rf value of the
different amino acid mixtures including the
solvent front.
Rf Values
Amino Acid Standard Acid
Hydrolysate
Trypthophan 0.9135
Arginine 0.9375
Proline 0.96
Cysteine 0.9375
Serine 0.9375
Aspartic Acid 0.925
Tyrosine 0.925
Histidine 0.9125
Glycine 0.9375
1.00
Alanine 0.975
Analysis on the Chromatography Values
The chromatography is a kind of filtration wherein
specific mixtures are separated by means of
passing it to a solution into a medium that can
make the different substances move at different
rates. Different amino acid mixtures were
dropped at the chromatography paper. The
unknown protein mixture was also dropped and
let stand. The Ninhydrin solution serves as the
key factor to separate and differentiate the
mixtures. The depiction of results serves as a
determining factor on what is/are the major
amino acids contributing on the unknown
substance, together with the help of the chemical
tests where the unknown substance underwent,
results were finally concluded. The unknown
substance giving the RF value of 1.00,
determined the amino acid that has the RF value
closest to the RF value of the unknown
substance. The Retardation factor (RF) value of a
chromatogram analysis is a value that depicts on
the distance traveledby the center of the spot to
the distance traveled by the solvent font. The RF
value of the different amino mixtures is defined
as the ratio of the distance moved by the solute
and the distance moved by the solvent, where
both distance were measured from the common
origin. The distance traveled by the solvent front
was 8. The table shows the different Rf values
naming Tryptophan (0.9135), Arginine (0.9375), of -0.029at 0.02 concentration. Standard 4 has
Proline (0.96), Cysteine (0.9375), Serine an absorbance of 0.032 at 0.03 concentration,
(0.9375), Aspartic acid (0.925), Tyrosine (0.925), and lastly, standard 5 has an absorbance of
Histidine (0.9125), Glycine (0.9375), and Alanine 0.032 in protein concentration. In the given
(0.975). The unknown sample, was able to reach absorbance, the unknowns value is -0.038,z
the solvent front,measuring at 8 cm, and so, the
Rf value as 1. REFERENCES
Crisostomo, A.C., et. al. (2010). Laboratory
Manual in General Biochemistry. Quezon City: C
Protein & E Publishing, Inc. Pages 26-29.
Test Concentratio Absorbance
tube/standar n (mg/mL) N.A. Thin Layer Chromatography.
d http://chem.libretexts.org/Core/Analytical_Chemi
Blank 0 0.000 stry/Lab_Techniques/Thin_Layer_Chromatograph
Standard 1 0.002 0.017 y 03/22/2017
Standard 2 0.01 - 0.036
Standard 3 0.02 - 0.029 N.A. Quantitative Determination of Proteins.
Standard 4 0.03 0.032 http://www.jascoinc.com/uv-vis/quantitative-
Standard 5 0.05 0.032 determination-of-proteins 03/22/2017
Unknown - - 0.038
Protein concentration shows that in standard 1,
having the smallest concentration gives the least
absorbance of all standards. While standard 2
obtain a absorbance of -0.036 from a 0.01
concentration. The standard 3 has an absorbance