Biochemistry Laboratory Experiment #4 PDF

Preview

Summary

Download Biochemistry Laboratory Experiment #4 PDF

Description

1

Qualitative Analysis of Amino acids Objective The aim of this laboratory report is to detect the presence of amino acid from a given unknown sample. Amino acids are building blocks of all proteins and are linked in series by peptide bonds (CONH-) to form the primary structure of a protein. Amino acids possess an amine group, a carboxylic acid group and a varying side chain that differs between different amino acids. There are 20 naturally occurring amino acids, which vary from one another with respect to their side chains. Their melting points are extremely high (usually exceeding 200°C), and at their pI, they exist as zwitterions, rather than as unionized molecules. Amino acids respond to all typical chemical reactions associated with compounds that contain carboxylic acid and amino groups, usually under conditions where the zwitterions form is present in only small quantities. All amino acids (except glycine) exhibit optical activity due to the presence of an asymmetric α – Carbon atom. Amino acids with an L – configuration is present in all naturally occurring proteins, whereas those with D – forms are found in antibiotics and in bacterial cell walls.

Procedure The following are the different preparations and procedures of the amino acids needed to be analyzed in this laboratory report. 1. Ninhydrin Test To 1ml of amino acid solution taken in a test tube, add a few drops of ninhydrin reagent and vortex the contents. Place the test tube in a boiling water bath for 5 minutes and cool to room temperature. 2. Xanthoproteic acid Test To 1ml of the amino acid solution taken in a test tube, add a few drops of nitric acid and vortex the contents. Boil the contents over a Bunsen flame, using a test tube holder, for a few minutes. Cool the test tube under running tap water and add a few drops of alkali. 3. Pauly’s diazo Test Take 1ml of sulphanilic acid reagent in a test tube and chill the contents in a small ice bucket. Add a few drops of prechilled sodium nitrite solution and vortex. Add immediately a few drops of pre chilled amino acid solution and vortex. This is followed by dropwise addition of sodium carbonate solution until the color appears. 4. Millon’s Test

2

To 1ml of the amino acid solution in a test tube, add a few drops of Millon’s reagent and vortex. Boil the contents over a Bunsen flame for 3-5 minutes. Cool the contents under running tap water and add a few drops of sodium nitrite solution.

5. Histidine Test To 1ml of amino acid solution, add 5% bromine in 33% acetic acid until a yellow color is formed. After 10 minutes, add 2ml of 5% ammonium carbonate solution and placed in a boiling water bath for 10 minutes. 6. Hopkins-Cole Test Mix 1 ml of the amino acid solution with 1 ml acetic acid – glyoxylic acid reagent, in a test tube, vortex. Then carefully, add conc. Sulphuric acid along the side of the test tube, keeping the tube in an inclined position (do not shake the test tube, while adding acid). 7. Sakaguchi Test To 1 ml of prechilled amino acid solution and a few drops of NaOH is mixed and 2 drops of alpha naphthol is added. Mix thoroughly and add 4-5 drops of hypobromite reagent and observe. 8. Lead sulphide Test To 1ml of the amino acid solution taken in a test tube, add a few drops of sodium hydroxide (40%) and boil the contents for 5 – 10min over a bunsen burner. Cool the contents and add a few drops of 10% Lead acetate solution and observe. 9. Folin’s McCarthy Sullivan Test To 1ml of the amino acid solution taken in a test tube, add a few drops of sodium hydroxide (5N), followed by addition of a few drops of glycine (1%) and 10% sodium nitroprusside solution and vortex. Place the test tube in a hot water bath, maintained at 40°C, for 15 minutes. Cool the test tube in ice cold water for 5 minutes and add 0.5ml of 6N HCl. Vortex the contents and allow to stand for 15 minutes at room temperature. 10. Isatin Test Apply a drop of amino acid solution on a filter paper strip and dry the spot using a hot air gun / Hair dryer or hot air oven. Apply a drop of isatin reagent on to the dried spot. Repeat the drying procedure with a hot air gun for a few minutes and observe.

3

Results

Figure 1. Ninhydrin test. The deep blue color indicates the presence of alpha amino acid.

Figure 2. Xanthoproteic acid test. The yellow-orange color indicates the presence of aromatic amino acid.

4

Figure 3. Pauly’s diazo test. The red color indicates the presence of tyrosine or histidine.

Figure 4. Millon’s test. The red solution or precipitate that is formed in heating indicates the presence of tyrosine.

5

Figure 5. Hopkins cole test. The purple violet ring appears between the two layers which indicates the presence of tryptophan.

Figure 6. Lead sulphide test. The black precipitate formation at the bottom of the test tube indicates the presence of cysteine.

6

Discussion Amino acids are molecules that contain amine group, carboxylic acid group, and a side chain that varies among different amino acids. This procedure is usually done for a qualitative test to detect the presence of amino acids in a given sample. In the Ninhydrin test (figure 1), this test is to detect the presence of amino acids. It involves a ninhydrin solution that is an oxidizing agent used for detection of α-amino acids and free amino acids on proteins. Thus, mixing a known amino acid solution and ninhydrin reagent will result in purple color. For Xanthoproteic Test (figure 2), it is a test in which amino acid that has an aromatic ring is used and it is seen with yellow-orange color. This test will provide information only on the presence and absences of the amino acids. Nitric acid and vortex content are utilized for this, getting a result that has a yellow-orange color solution indicates the presence of the aromatic group. Pauly's diazo Test (figure 3), is a test to detect amino acids in tyrosine and histidine. The tests will diazotize the acid with addition of sodium nitrite and sodium carbonate solution. The component will react and form red color which will pertain to the presence of histidine or tyrosine. Millon’s test (figure 4), is focused on tyrosine, the only amino acid that contains the phenol group. This test is useful as many proteins consist of tyrosine. Tyrosine will then react with the sodium nitrate solution and heating, having the presence of red color meaning there is a tyrosine in the solution. Hopkins Cole Test (figure 5), the test is focused on the reaction of the tryptophan. The test is based upon dehydration reaction, the amino acid reacts with the reagent glyoxylic acid in the presence of concentrated sulphuric acid. Mixing it in the amino acids will react and cause a formation of purple colored ring or the indole ring that indicates the presence of tryptophan. The Lead Sulphide Test (figure 6), is the test focused in the detection of cysteine and cystine in the amino acids. The test is based on the precipitation reaction, by adding sodium hydroxide and boiled it gets converted into sodium sulphide. Then, sodium sulphide reacts with the lead acetate and undergo precipitation reaction by forming black precipitates as lead sulphide. Histidine test, the test is used to detect the presence of histidine. After adding bromine in the acetic acid and forming a yellow color, add carbonate solution then put in boiling water bath, the component will react and form a color blue that indicates the presence of histidine in the amino acid. Sakaguchi Test, is a specific test to determine the presence of arginine. Arginine reacts with α-naphthol in presence of an oxidizing agent such as bromine water or sodium hypochlorite to give a red coloured product that indicates the presence of arginine.

7

Folin's McCarthy Sullivan Test is used to detect the presence of methionine. Methionine is a unique sulfur-containing amino acid that can be used to build proteins and produce many molecules in the body. Thus, mixing an unknown amino acid solution,40% of NaOH, glycine and sodium nitroprusside will result in a red color that indicates the presence of methionine. The last test is the Isatin test which also is specifically used to detect the presence of methionine. Mixing a few drops of 5N NaOh and 6N HCI will result in red color indicating the presence of methionine.

Conclusion In using the qualitative test, we are able to detect the presence of amino acid in an unknown sample. We therefore conclude that the different kinds of tests we conducted the amino acid shows significant reaction through variation of colors it produces. Using different principles, we've been able to identify the different kinds of amino acid that exist in a certain sample. In the Ninhydrin test, we've been able to detect the presence of amino acids. The Xanthoproteic Test detects the amino acid that has an aromatic ring. For the Pauly's diazo Test, it detects the amino acid in tyrosine and histidine. In the Millon Test, the only amino acid it detects is the one that contains the phenol group. The Hopkins Cole Test focused on the reaction of tryptophan which is the mix of amino acid, reagent glyoxylic acid in concentrated sulphuric acid. In Lead Sulphide Test, it detects the cysteine and cystine on the amino acid. And lastly, the Folin's McCarthy Sullivan Test, detects the presence of methionine.

Appendix A. Answers to Questions/Assignment 1. Write the reaction involved in the Ninhydrin reaction? Ninhydrin is a powerful oxidizing agent that gives off a purple product (diketo hydrin) termed as Rheumann's purple. When contact with primary amines and ammonia it reacts similarly but does not liberate with carbon dioxide. The amino acids proline and hydroxyproline, when intact with ninhydrin, produces the yellow color. 2. Do all the amino acids with aromatic side chains give positive test to Xanthoproteic acid test. Why? Since Xanthoproteic test uses a nitration reaction, when contact with nitric acid it reacts with aromatic amino acids and forms yellow colored products called as Xanthoprotein.

8

3. You have Cysteine, Tyrosine, Histidine in separate test tubes. By using which test would you find Cysteine containing test tubes? The Hopkins-Cole Test is the tryptophan indole group that reacts with glyoxylic acid (glacial acetic acid) When exposed to light, it often contains glyoxylic acid CHOCOOH as an impurity) in the presence of concentrated H2SO4 to give a purple tint. Apply the test to glycine, Tyrosine and tryptophan. 4. What is the role of H2SO4 in Hopkins-Cole test? H2SO4 or sulfuric acid is used to give it two layers and purple rings, then appear between the two layers if the test is positive for tryptophan. 5. Take an Unknown amino acid in your lab and Carry out the amino acid tests in a responsible sequence to determine your unknown solution? An unknown amino acid is to be subjected for the lab test. Using a dropper get 1ml of the unknown amino acid and transfer to an empty test tube, next is get the reagent to be use for the procedure - in this case we used the Ninhydrin, using another dropper, take a few drops of it and put it to the test tube where 1ml of the unknown amino acid is placed. After doing so place the test tube in the boiler for about 5 minutes, and after that let cool down to room temperature. By now, the amino acid will be determined by its color, in our experiment the unknown amino acid showed a yellow color after all the procedures, indicating the proline amino acid.

B. Self-Assessment 1. Triketohydrindene hydrate otherwise called? A. Millon’s reagent C. Ninhydrin reagent B. Hypobromite solution D. Isatin reagent 2. the

Which test is specific for detection of Tryptophan?

9

A. Pauly’s Diazo test B. Lead sulphide test

C. Xanthoproteic acid test D. Hopkins-Cole test

3. What is the precipitate obtained during the lead sulphide test? A. Lead acetate B. Lead sulphide

C. Sodium acetate D. Sodium sulphide

4. Which test is used as the confirmatory test for Methionine? A. Millon’s test C. Folin's Mccarthy Sullivan’s Test B. Sakaguchi test D. Hopkins-Cole test 5. Diazotization reaction is involved in which test? A. Pauly’s Diazo test B. Millon’s test

C. Lead sulphide test D. Sackaguchi test

6. Phenolic amino acids respond to which test? A. Isatin test B. Millon’s test

C. Lead sulphide test D. Pauly’s Diazo Test...