Separation, Identification and Analysis of the Amino Acid Components of Gluten

by Paper Chromatography

C.A.M. Dimatatac, M.J.T. Dy, *R.D.V. Figuerroa, J.M.T. Geotina, V.D.V Lazatin

Abstract

Paper chromatography is an analytical chemistry technique for separating and identifying

mixtures that are or can be colored, especially pigments. In this experiment, paper chromatography was
utilized for the qualitative analysis of amino acids namely: Aspartic acid, Arginine, Cysteine, Histidine and
Proline. The acidic, basic and enzymatic hydrolysates of gluten where also tested for the presence of
these five amino acids. From the chromatogram, amino acids and hydrolysates were also characterized
based on their affinity toward the stationary and mobile phases. From the results of the experiment,
Proline had greater affinity toward the mobile phase while Histidine had greater affinity toward the
stationary phase. Most of the component that belonged to the first group of amino acids, the hydrophobic
non-polar amino acids, moved farthest from the point of origin while the components that had the lowest
Rf values belonged to the 2 nd, 3rd and 4th groups of amino acids: the polar uncharged, acidic and the basic
amino acids, respectively. For the basic and acidic hydrolysates o Gluten, it was found out that the acidic
hydrolysate, which contained noticeable amount of Aspartic acid, is polar while the basic hydrolysate
which comprised mostly of Proline, is non-polar.

Introduction

Amino acids are the building

blocks of peptides and proteins. They
possess two functional groups—the
carboxylic acid group gives the acidic
character, and the amino group provides
the basic character (1).
Figure 1. General structure of Amino Acids
The R represents the side chain
that is different for each of the amino
acids that are commonly found in
proteins. Refer to Figure 1. However, all
20 amino acids have a free carboxylic
acid group and a free amino (primary
amine) group, except Proline which has
a cyclic side chain and a secondary
amino group (2). Refer to Figure 2.
Figure 2. Structure of Proline
The common carboxylic acid and
amino groups provide the acid–base
nature of the amino acids. The different
side chains, and their solubilities
provided by these side chains, can be
utilized to identify the different amino
acids by their rate of migration in paper
chromatography (3).
In this experiment, paper
chromatography was employed to
determine the amino acid components
of the protein, Gluten. Paper
chromatography is a useful technique
because it is relatively quick and
requires small quantities of material.
Separations in paper chromatography
 involve the same principles as those in
thin layer chromatography (4). In this
kind of chromatography, substances are
distributed between a stationary phase
and a mobile phase. The stationary
phase is usually a piece of high quality
filter paper. The mobile phase is a
developing solution that travels up the
stationary phase, carrying the samples
with it. In this experiment, a solution of
1-Butanol, acetic acid and water in 4:1:5
ratio served as the mobile phase.
Components of the sample will separate
readily according to how strongly they
adsorb on the stationary phase versus
how readily they dissolve in the mobile
phase. Thus, components in this
separation technique are characterized
based on their polarities toward the two
phases.
Stationary
phase
Components
separated
Mobile
phase
Figure 3. Paper Chromatography diagram
Gluten is a protein composite
found in foods processed from wheat
and related grain species, including
barley and rye (5). It is extracted from
flour by kneading the flour,
agglomerating the gluten into an elastic
network, a dough, and then washing out
the starch. It is commonly used in bread
products, imitation meats and as an
ingredient in ketchup and ice cream.
This experiment aims (1) to
determine the amino acid components
of the hydrolysates of the protein Gluten
by paper chromatography and to (2)
characterize amino acids through their
polarities toward the stationary and
mobile phase.
Methodology
To facilitate paper
chromatography, a 12 x 15 cm filter
paper was used. A 1.0 cm margin from
the top of the longer side of the filter
paper was drawn using a pencil to mark
the point of origin. Eight equidistant
points were marked to indicate where
the five chosen amino acids and the
other three hydrolyzed proteins were to
be spotted. After this, the five amino
acid standards were alternately spotted
three times using a capillary tube. The
acidic, basic and enzymatic protein
hydrolysate were spotted five times
using the same technique. The dry filter
paper was then stapled on one edge to
create a cylinder. A 1000-ml beaker and
a watch glass were prepared to serve as
the developing chamber. The prepared
solvent system (a mixture of 1-Butanol,
acetic acid and water in 4:1:5 ratio) was
poured and left undisturbed for five
minutes inside the chamber. Afterwards,
the cylindered filter paper was placed
inside the beaker, covered with a watch
glass and allowed to ascend
uninterrupted for an hour. Refer to
Figure 4.

Watch glass

Filter paper

Beaker

Stationary
phase

Point of Figure 6. The chromatogram

origin

Solvent The distance travelled by each

(mobile component and the solvent front were
phase)
measured using a ruler. Rf values were
Figure 4. The developing chamber computed using this formula:

The solvent front was

immediately marked using a pencil and
Figure 6. Rf value formula
allowed to dry. Subsequently, the
chromatogram was lightly sprayed with Results
1% Ninhydrin reagent and heated using
a hot plate for 5 minutes. Refer to Figure Table 1. Rf values of each component

5.
Distance Solvent
Rf
Component traveled front
value
(cm) (cm)
Aspartic
3.2 8.7 0.37
Acid
Arginine 3.1 8.7 0.36

Cysteine 3.0 8.7 0.34

Histidine 2.9 8.7 0.33

Proline 3.5 8.7 0.40

Figure 5. Chromatogram being heated in the hot
plate
Acidic
3.2 8.7 0.37
gluten
Blue, purple and yellow spots Basic gluten 3.5 8.7 0.40
were encircled. Refer to Figure 6. Enzymatic
n/a 8.7 n/a
gluten
Discussion
Paper chromatography is a
separation technique facilitated by the
principle of polarity toward the two
phases, the stationary and mobile
phase. As seen from Figure 5, Histidine,
a basic amino acid, moved slowest from
the point of origin. Thus, it has the
lowest Rf value. This means that it is the
amino acid among the five used which
has the greatest affinity toward the
stationary phase. On the other hand,
Proline, a hydrophobic non-polar amino
acid moved farthest from the point of
origin. Consequently, it has the greatest
Rf value. This means that Proline has
the greatest affinity toward the mobile
phase. Histidine has the highest affinity
toward water while Proline has the
highest affinity to the n- butanol. Looking
closely at the chromatogram, most of
the amino acids that moved least from
the point of origin are from the 2 nd, 3rd
and 4th group of amino acids which are
the polar uncharged, acidic and the
basic amino acids. Meanwhile, most of
the components that moved farthest
from the point of origin are from the 1 st
group of amino acids, the hydrophobic
non-polar amino acids. The Rf values of
each component indicated the polarities
of these amino acids. A higher Rf value
denote lower polarity while a lower Rf
value imply that the component is polar.
N-butanol (C4H9OH), a non-polar
solvent, carries the non-polar amino
acids up the chromatogram while acetic
acid (CH3COOH), a polar amino acid
keeps the polar amino acids near the
baseline. Due to their quantitative
difference in the solvent system mixture
which is 4:1 or four measures of Butanol
for every one measure of acetic acid,
non-polar amino acids are favored than
polar amino acids. The chromatogram
was sprayed evenly with 1% Ninhydrin
solution to give them the distinct yellow
and violet colors. Only Proline produced
a distinct yellow spot. For the acidic
hydrolysate of Gluten, Aspartic acid was
found to be a major component. The
basic hydrolysate contained prominent
amount of Proline because of the
distinct yellow spot that can be seen.
The basic hydrolysate has greater
affinity toward the mobile phase while
the acidic hydrolysate has greater
affinity toward the stationary phase. This
means that the acidic hydrolysate of
gluten is polar while the basic
hydrolysate is non-polar. No spot or
coloration for the enzymatic hydrolysate
can be observed from the
chromatogram.
References
(1) http://www.macalester.edu/.../
Revised%20Amino%20Acids
%20(9%201%2001).pdf
(2) http://www.docstoc.com/docs/54003
970/Identification-of-Amino-Acids-Using-
Paper-Chromatography
(3) http://en.wikipedia.org/wiki/Paper_
chromatography
(4) Bettelheim, F. & Landesberg, J.
(2004). Laboratory Experiments for
General, Organic and Biochemistry. 5th
Ed. Brooks/Cole, pp. 426-447.
(5) http://www.friedli.com/research/PhD
/gluten/chap2.html