ACID HYDROLYSIS OF INTACT PROTEIN AND SEPRATION AND IDENTIFICATION OF AMINO ACIDS BY THIN-LAYER CHROMATOGRAPHY Thryssa Johanna B.

Cardenas, Mae Genevieve G. Cheung, Michael G. Cuevas, Lovely Fe L. Cuison, Elijin Dai, Francesca Pauline Dela Cruz Group 2 2F Pharmacy Organic Chemistry Laboratory ABSTRACT
In this experiment, the protein gluten was isolated from whole wheat flour and some of it was hydrolyzed in acidic medium and some of it was kept intact. After the process of acid hydrolysis the gluten was then neutralized and was used in the thin-layer chromatography. The thin-layer chromatography is a separation technique used in amino acids which separates them according to their polarity or affinity with the stationary phase. In the experiment different amino acids standards was compared to the different amino acids that was eluted by the hydrolyzed gluten sample. These data then were analyzed to determine which amino acids were present in the hydrolyzed gluten sample.
the solution was adding 1 M NaOH. then neutralized by

INTRODUCTION
Proteins are organic macromolecules that are composed of one or more amino acids that are bonded through their peptide chain.[1] When a protein is hydrolyzed the polypeptide chains break and break down the proteins to its more basic structure which are the amino acids. Specific tests were used in the classification of the amino acids present in the gluten sample and one of these tests was the thin-layer chromatography.[2] The thin-layer chromatography is a separation technique which uses the polarity of the substance to separate them. The protein sample that was assigned to the group was gluten that was extracted from whole wheat flour and is a water insoluble protein made up of different types of amino acids.[3][4]

EXPERIMENTAL
A. Compounds Tested: Intact gluten sample Hydrolyzed gluten sample B. Procedures:
1. Acid Hydrolysis of Intact Protein The 0.5g of isolated gluten protein was placed in a hard glass test tube with a 5mL of 6 M HCl. This test tube was then autoclaved at 15 psi for 5 hours. Then after it was autoclaved 10 mL of distilled water was added and the mixture was transferred into a 250-mL beaker. Finally,

2. Separation and Identification of Amino Acids by Thin-Layer Chromatography The 12 by 15 cm TLC paper was first prepared by drawing the origin which is a straight line with a 1.5 cm margin from the bottom of the longer edge of the plate and then marking 13 equidistant points on the same line for the spotting of the amino acid standards (10) and 3 for the hydrolysate sample. Then the standards were applied 5 times and the samples were applied 10 times and allow each sample to dry in between the applications. The TLC paper was then placed in a pre-equilibrated chamber wherein the level of the solvent should be just below the origin. The chamber was then covered and the solvent was allowed to ascend undisturbed only to be removed when the solvent front was 0.5 cm from the top of the edge of the plate and the solvent front was then immediately marked with a pencil. The chromatogram was then air dried and was sprayed lightly with 1% ninhydrin reagent. After which, the chromatogram was placed inside an oven for 1-3 minutes and the amino acids appeared as blue, purple and yellow spots on the plate after which the Rf values were computed.

RESULTS AND DISCUSSION

The experiment involved series of tests to isolate and determine which amino acids are present in the hydrolyzed gluten sample.

Thin-Layer chromatography was a test that was used to determine which amino acids are present in the hydrolyzed gluten sample.[4] The process of chromatography is based on the affinity of the substance to the solvent or based on the substances polarity.[5][6] In the experiment 10 amino acid standards were used to be the basis of finding the amino acids present in the hydrolyzed gluten sample. The basis on determining which amino acid was present in the gluten sample is through comparing the solved Rf value of the amino acid standards to the Rf values that were computed from the gluten sample.[3][5] The results are as follow: Table 1.Obtained Rf Values in the Thin-Layer Chromatography
Amino Acid Standards Rf value of Amino Acid Standards Rf value of the hydrolyzed gluten

[4] Crisostomo, A.et al. (2013), Laboratory Manual in General Biochemistry. Quezon City: C&E Publishing, Inc. [5] Characterization and Isolation of proteins, Retrieved December 18, 2013, from http://www.scribd.com/doc/49052097/Isolationand-Characterization-of-Proteins [6] Pastro, D. J., John, C. R., & Miller,M. S. (1998). Experiment and Techniques in Organic Chemistry. New Jersey: Prentice Hall [7] Protein Purification, Retrieved December 18, 2013, from http://biotech.about.com/od/protocols/a/ProteinPurify.ht m

Alanine Arginine Glycine Histidine Proline

0.69 0.42 0.53 0.62 0.81

0. 491 0.317 0.384 0.41 0.579

Based from the results above 5 amino acids were found present in the hydrolyzed gluten sample which are Alanine, Arginine, Glycine, Histidine and Proline. Also based from the computed Rf values Proline has the highest Rf value and was the amino acid that travelled more from the origin hence it has a great affinity to the solvent so it was eluted the last so it is the most nonpolar among the five amino acids that were found in the hydrolyzed gluten sample while Arginine is the most polar among the five amino acids present.[5][7]

REFERENCES: [1] Protein, Retrieved December 18, 2013 , from http://en.wikipedia.org/wiki/Protein [2] Gluten, Retrieved December 18, 2013, from http://www.greatplainslaboratory.com/home/eng /peptide.asp [3] Isolation of Proteins , Retrieved December 18, 2013, from http://www.scribd.com/doc/46097718/Isolationand-Characterization-of-Proteins