Separation and Identification of Amino Acids by Paper Chromatography

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.

Stationar y phase

Component s 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 Beake r Stationa ry phase Point of origin Solvent (mobile phase)

Figure 4. The developing chamber

The solvent front was immediately marked using a pencil and allowed to dry. Subsequently, the chromatogram was lightly sprayed with 1% Ninhydrin reagent and heated using a hot plate for 5 minutes. Refer to Figure 5.

Figure 5. Chromatogram being heated in the hot plate

Blue, purple and yellow spots were encircled. Refer to Figure 6.

Figure 6. The chromatogram

The distance travelled by each component and the solvent front were measured using a ruler. Rf values were computed using this formula:

Figure 6. Rf value formula

Results Table 1. Rf values of each component

Component

Distance traveled (cm)

Solvent front (cm)

Rf value

Aspartic Acid

3.2

8.7

0.37

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

Acidic gluten

3.2

8.7

0.37

Basic gluten

3.5

8.7

0.40

Enzymatic gluten

n/a

8.7

n/a

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 2nd, 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 1st 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-UsingPaper-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