QUALITATIVE COLOR REACTIONS FOR CASEIN Anneliese Y. Abalos, Princess Leigh A. Acosta, Acosta, Lia Carmela S. Aranton, Grace Marie Nicole Biso, and Zillah Glarilyn Calaunan Group 1 2G Medical Technology Biochemistry Laboratory
ABSTRACT The experiment revolved mainly on the reactions of intact casein and acid hydrolyzed casein to the different tests. Positive results were manifested by both of the samples upon subjecting them to Biuret Test, Xanthoproteic Test, Millon¶s Test, Fohl¶s Test, and Test for Amides. The intact protein obtained a positive result in Ninhydrin Test, and a negative result in Hopkins-Cole Test, while the hydrolyzed casein obtained a positive result in Hopkins-Cole Test, and a negative result in Ninhydrin Test.
INTRODUCTION Casein is a protein that is found in milk and used independently in many foods as a binding agent. The experiment shows the qualitative tests done in intact casein and acidic hydrolyzed casein using different reagents. The objective of the experiment is to characterize the properties of the protein casein by subjecting it to different color reactions as well as to compare the reactions of the intact casein and the acidic hydrolyzed casein with the same qualitative tests.
EXPERIMENTAL A. Compounds Tested The compounds tested were the intact casein and hydrolyzed casein. B. Procedure 1. Biuret Test In this test, 20 drops of 2.5 M NaOH was introduced to the samples and mixed well. Then, 3 drops of 0.1 M CuSO4 solution was added to the solutions. The test tubes were then shaken to mix the solution well and the colors were noted down. 2. Ninhydrin Test In two separate test tubes containing the samples, 10 drops of 0.1% Ninhydrin solution was added. The test tubes were then heated in a boiling water bath and the appearance of a blueviolet coloration was noted. 3. Xanthoproteic Test In two separate test tubes containing the samples, 10 drops of concentrated HNO3 was slowly added and well mixed. The colors of the each solution were noted. Then, 10 drops of concentrated NaOH was slowly added into the solutions. Lastly, the test tubes were mixed well and the changes in color were noted. 4. Millon¶s Test In this test, 5 drops of the Millon¶s reagent was added to each of the samples in separate test tubes. Changes in color were noted.
5. Hopkins-Cole Test In two separate test tubes containing the samples, 20 drops of Hopkins-Cole reagent was slowly added. Then, the test tubes were inclined and 20 drops of concentrated H 2SO4 was added. The test tubes were not shaken. Only the colors of the interface were noted. 6. Fohl¶s Test In this test, 5 drops of 30% NaOH and 2 drops of 5% (CH3COO)2Pb to each of the samples in separate test tubes 7. Test for Amides In 2 dry test tubes, 10 drops of each of the samples were placed separately. To each test tube, 1 mL of 20% NaOH was placed and the solutions were subjected to a boiling water bath. A moistened red litmus paper was placed over the mouth of each test tube and the changes in color of the litmus papers were noted down.
RESULTS AND DISCUSSION 1. Biuret Test Biuret test is used for detecting the presence of peptide bonds. The biuret test relies on the reaction between copper (II) ions and peptide bonds in an alkaline solution. A violet coloration indicates the presence of proteins. Both the intact and the hydrolyzed casein indicated a positive result in this test. Table 1. Results obtained from Biuret Test Color Reaction Intact Protein Protein Hydrosylate Biuret Test Pale purple Blue suspension solution in purple solution
2. Ninhydrin Test Amino acids contain a free amino group and free carboxylic acid group that react together with ninhydrin to produce a colored product. When an amino group is attached to the first, or alpha, carbon on the amino acids¶ carbon chain, the amino acids¶ nitrogen atom is part of the blue-purple product. Proteins also contain free
amino groups on the alpha-carbon and can react with ninhydrin to produce a blue-purple product. A blue-violet coloration was observed in the intact protein upon adding ninhydrin reagent. No changes were obtained from the protein hydrosylate when in fact there should be a blueviolet coloration because it still contains free amino groups. In this part, there was an error committed by the group which can be an error from the preparation of samples or reagents, or it can be an error in the execution of this part of the experiment. Table 2. Results obtained from Ninhydrin Test Color Reaction Intact Protein Protein Hydrosylate Ninhydrin Test Blue-violet Cloudy solution coloration
3. Xanthoproteic Test This test is used to determine the presence of aromatic rings in a given sample or the presence of residues of tyrosine or tryptophan. The solution to be tested is treated with concentrated nitric acid, which will nitrate the benzene rings of those residues. The nitrated aromatic rings are yellow in color and are called Xanthoproteic acids (Xantho, = yellow, Greek ). Both the intact protein and the protein hydrolysate turned to a yellow solution which indicates a positive result for the presence of aromatic rings. Table 3. Results obtained from Xanthoproteic Test Color Reaction Intact Protein Protein Hydrosylate Xanthoproteic Clear yellow Yellow solution Test solution
4. Millon¶s Test Millon¶s test is specific for tyrosine, the amino acid containing a phenol group, a hydroxyl group attached to a benzene ring. In this test, the phenol group of tyrosine is first nitrated by nitric acid in the test solution. Then the nitrated tyrosine complexes mercury (I) and mercury (II) ions in the solution to form either a red precipitate or a red solution. Proteins that contain tyrosine will therefore yield a positive result. However, some proteins containing tyrosine initially form a white precipitate that turns red when heated. In the case of casein, both the intact and the hydrolyzed casein resulted to a white cloudy solution which means that both samples contain tyrosine. Table 4. Results obtain from Millon¶s Test Color Reaction Intact Protein Protein Hydrosylate
Millon¶s Test
White cloudy solution
White cloudy solution
5. Hopkins-Cole Test The Hopkins-Cole reagent only reacts with proteins containing tryptophan. The protein solution is hydrolyzed by the concentrated sulfuric acid at the solution interface. Once the tryptophan is free, it reacts with glyoxylic acid to form the violet product. A negative result was given by the intact protein because the tryptophan was not free or was not hydrolyzed enough for it to react with the glyoxylic acid, while the hydrolyzed protein gave a positive result which is a violet coloration at the interface. Table 5. Results obtained from Hopkins-Cole Test Color Reaction Intact Protein Protein Hydrosylate Hopkins-Cole Yellow coloration Violet coloration Test at the interface at the interface
6. Fohl¶s Test Fohl's test is used to know if sulfur-containing amino acids are present. The positive result for this test is the appearance of dark (black or brown) sediment. Upon subjecting the samples to this test, both manifested a positive result. Table 6. Results obtained from Fohl¶s Test Color Reaction Intact Protein Protein Hydrosylate Fohl¶s Test Brown cloudy Brown Solution solution
7. Test for Amides As the title suggests, this test is used to determine the presence of amides. Both samples showed positive results. However, the litmus paper used for the hydrolyzed protein took longer to change in color than that of the litmus paper used for the intact protein. Table 7. Results obtained from Fohl¶s Test Color Reaction Intact Protein Protein Hydrosylate Test for Amides Red litmus Red litmus (Litmus paper paper to blue paper to blue test) litmus paper litmus paper
REFERENCES Milio, Frank R., Towson State University and Loffredo, William M., East Stroudsburg University http://www.cerlabs.com/experiments/108754044 80.pdf 1/08/2011 McGuigan, Brendan http://www.wisegeek.com/what-is-casein.htm 1/08/2011
Palleros, Daniel R., Experimental Organic Chemistry http://www.uwlax.edu/faculty/koster/newlactose. htm 1/08/2011 http://www.mondofacto.com/facts/dictionary?biu ret+test 1/08/2011
