BIOMOLECULE TEST

Dwi Purwanti Student of Bilingual Biology Department Faculty of Mathematics and Natural Science State University of Medan Jl.Willem Iskandar, Pasar V Medan Estate Kode Pos 20222 Telp. 061-6625970 email: dwiipurwantyy@gmail.com ABSTRACT Biomolecule test is the test that do to detect the presence of major organic compound that found in our living things. The research was conduct at the laboratory of Chemistry at University of Medan, on April, 19th 2013 at one past fifty p.m. the result of this experiment conclude that the different reagent give the different result in detected organic compound in the materials. Key Words: carbohydrate, protein, Molicsh test, Fehling test Biuret test, millon test INTRODUCTION The main objective for these tests is to determine whether specific organic compounds are present in different substances. I choose it because, I think this experiment is very attractive. The chemical compounds of living things are known as organic compounds because of their association with organisms. Organic compounds, which are the compounds associated with life processes, are the subject matter of organic chemistry. Among the numerous types of organic compounds, four major categories are found in all living things: carbohydrates, lipids, protein, and nucleic acids. A carbohydrate is an organic compound with the empirical formula Cn(H2O)n, that is, consists only of carbon, hydrogen and oxygen with the last two in the 2:1 atom ratio. Carbohydrates can be viewed as hydrates of carbon, hence their name. Structurally however, it is more accurate to view them as polyhydroxy aldehydes and ketones. The carbohydrates (saccharides) are divided into four chemical groupings: monosaccharides, disaccharides, oligosaccharides and polysaccharides. In general monosaccharides and disaccharides which are smaller (low molecular weight) are commonly referred to as sugars. The word saccharide comes from the Greek word (skkharon), meaning sugar. Carbohydrates perform numerous roles in living things. Polysaccharides serve for the storage of energy (e.g. starch and glycogen) and us structural components (e.g. cellulose in plants and chitin in anthropods). The 5-carbon monosaccharide ribose is an important component of coenzymes (e.g. ATP, FAD and NAD) and the genetic molecule known as RNA. ( Nigam,2007) Molisch test The Molisch test is a general test for the presence of carbohydrates. Molisch reagent is a solution of alpha-naphthol in 95% ethanol. This test is useful for identifying any compound which can be dehydrated to furfural or hydroxymethylfurfural in the presence of H2SO4. Furfural is derived from the dehydration of pentoses and pentosans, while hydroxymethylfurfural

is produced from hexoses and hexosans. Oligosaccharides and polysaccharides are hydrolyzed to yield their repeating monomers by the acid. The alpha-naphthol reacts with the cyclic aldehydes to form purple colored condensation products. Although this test will detect compounds other than carbohydrates (i.e. glycoproteins), a negative result indicates the ABSENCE of carbohydrates. Shows positive test for: All carbohydrate . Monosaccharides give a rapid positive test. Disaccharides and polysaccharides react slower. The test reagent dehydratespentoses to form furfural and dehydrates hexoses to form 5-hydroxymethyl furfural The furfurals further react with -naphthol present in the test reagent to produce a purple product (reaction not shown). To perform the test: Two ml of a sample solution is placed in a test tube. Two drops of the Molisch reagent (a solution of -napthol in 95% ethanol) is added. The solution is then poured slowly into a tube containing two ml of concentrated sulfuric acid so that two layers form. A positive test is indicated by: The formation of a purple product at the interface of the two layers.( Mittal, Arun.2000 ) Fehlings Test : This forms the reduction test of carbohydrates. Fehlings solution contains blue alkaline cupric hydroxide solution, heated with reducing sugars gets reduced to yellow or red cuprous oxide and is precipitated. Hence, formation of the yellow or brownish-red colored precipitate helps in the detection of reducing sugars in the test solution. Millonstest (a) Millons reagent consists of mercury dissolved in nitric acid (forming a mixture of mercuric & mercurous nitrates). (b) When millons reagent is added to a protein, a white ppt is formed, which turn brick red on heating. (c) This test is given by protein which yield tyrosine on hydrolysis (due to the presence of phenolic group) (Murray, Peter R. S. 1999) RESEARCH METODOLOGY Overview of the Experiment In this experiment, we using some apparatus and materials. The apparatus that we use in this experiment are Pyrex reaction tubes, 11 pieces; the water heater, 1 piece; and volumetric pipette size 10 ml, 1 piece.The materials that we use are Glucose, Fructose, Sucrose, starch solution, solution of Eggs yolk (albumin), solution of Milk and solution of honey. And as the reagent, we use :Molisch, Fehling A and B, Biuret, and Ninhydirin. The experiment is conduct in the laboratory of Chemistry at University of Medan, on April, 19th 2013 at one past fifty p.m. The procedure of this experiment, is doing in 4 ways that will be given as follows: First, Carbohydrate Test, by using Molisch Reagent. Firstly, we prepare the 5 clean of reaction tube, sequentially the 1- 5 test tube we fill by 2 ml of glucose solution, 2 ml of fructose solution, 2 ml of sucrose solution, 2 ml of starch solution and 2 ml of honey solution. And each part of the test tubes we drop by 2 drops of Molisch reagent and 2 ml of H2SO4 concentrated. Second, Carbohydrate Test, by using Fehling Reagent. In this reagent, firstly we must mix 1 ml of Fehling A reagent with 1 ml of Fehling B and add by 4 ml of distillated water. And then we share that mixture into three test tube. Sequentially, at first test tube we add by 2 ml of glucose

solution, second by sucrose solution, and the last by starch solution. And then, the three of that test tube we put into the water heater 600 C during 10 minutes. Third, Protein and Amino acid Test, by using Biuret Reagent. We take 4 test tubes and fill each part sequentially by solution of Eggs yolk (albumin), milk solution, broth solution, and honey solution. And then, into them add 5 drops of CuSO4 0.05 M and 2 ml of NaOH 10 M and shake them. Fourth experiment, Protein and amino Acid Test, by using Millon Reagent. The same way like Protein and Amino acid Test, by using Biuret Reagent, we must take test tubes and fill each part sequentially by solution of Eggs yolk (albumin), milk solution, broth solution, and honey solution. And then, we put into them 5 drops of Millon reagent. We heat the fourth of test tube into water heater during 10 minutes and let cool. After that we add 5 drops of NaNO2 0.15 M. RESULTS AND DISSCUSSION Result that we get from first experiment, Carbohydrate Test, by using Molisch Reagent will be given as follows : At first tube that filled by 2 ml of Glucose, there is the formation of a reddish pink product at the interface of the two layers. At second tube that filled by 2 ml of Sucrose, we get the formation of brownish product at the interface of the two layers. At third tube that filled by fructose, we get the formation of pinkish product at the interface of the two layer. At fourth tube that filled by starch solution, we get the formation of pink light product at the interface of the two layer. At fifth tube that filled by solution of honey, we get the changing color of honey become black. Discussion The presence of the reddish pink, pinkish, pink light color at the interface of the two layer, indicated the presence of condensation between furfural or hydroxymethyl furfural with alpha-naphthol in molisch reagent were dehydrated by concentrated sulfuric acid. Actually, the color of this furfural condensation is purple red, but in our experiment we get the variety of color. I think the color that we get is enough to represent this test that the material is consist of carbohydrate. For sucrose that give brownish color, it may be happen because sucrose is different carbohydrate than another carbohydrate that we use as materials (glucose, and fructose belong to monosaccharide, starch solution belong to polysaccharides that consist of glucose) while sucrose is belong to disaccharides, same with honey that consist of fructose + glucose = sucrose. So, in this test we get conclusion that all of material that we test by molish reagent positive belong to carbohydrate. For second experiment, Carbohydrate Test, by using Fehling Reagent We get the color of glucose, sucrose and starch solution are colorless( no changing color happen). Actually Fehling reagent is use to test the presence of aldehydes but not ketones that detected by reduction of the deep blue solution of copper(II) to a red precipitate of insoluble copper oxide. The test is commonly used for reducing sugars but is known to be NOT specific for aldehydes, so sucrose give negative result for this test. But in this experiment, we also get

negative result for glucose and fructose that actually we must get positive result that characterized by the presence of red bata precipitation. So in this test we get fault. For third experiment, Protein and Amino acid Test, by using Biuret Reagent At first tube that filled by albumin, we get the changing color of albumin from colorless to dark blue. At second tube that filled by milk solution, we get the changing color of milk from white to blue color. At third tube that filled by broth solution, we get the changing color of broth solution from colorless to bluish color. At fourth tube that filled by honey solution, we get the changing color of honey solution from colorless to blue + color. Discussion The Biuret Test is done to show the presence of peptide bonds, which are the basis for the formation of proteins. These bonds will make the blue Biuret reagent turn purple. The reagent used in the Biuret Test is a solution of copper sulfate (CuSO4) and sodiumhydroxide (NaOH).The NaOH is there to raise the pH of the solution to alkaline levels; the crucial component is the copper II ion (Cu2+) from the CuSO4. When peptide bonds are present in this alkaline solution,the Cu2+ions will form a coordination complex with 4 nitrogen atoms from peptide bonds. The complex of Cu2+ ions and nitrogen atoms make the color of CuSO4 solution changes from blue to violet. This color change is dependent on the number of peptide bonds in the solution, so the more protein, the more intense the change. So in this experiment, we get that, all of the material that tested are consist of peptide bond because all of the materials give the blue color.. For the fourth experiment, Protein and amino Acid Test, by using Millon Reagent At first tube that filled by albumin, we get the color of solution is colorless At second tube that filled by milk solution, we get the orange precipitation At third tube that filled by broth solution, we get also the orange precipitation At fourth tube that filled by honey solution, we get the color of honey solution is colorless Discussion. Millons test is chemical test that detected the presence of Tyrosine. Millons test is given by any compound containing a phenolic hydroxy group. Consequently, any protein containing tyrosine will give a positive test of a pink to dark-red colour. The Millon reagent is a solution of mercuric and mercurous ions in nitric and nitrous acids (CAUTION: MILLONS REAGENT IS HIGHLY TOXIC AND HIGHLY CORROSIVE). The red colour is probably due to a mercury salt of nitrated tyrosine. So, in this test we get the conclusion that milk and broth solution are solution that contain protein. CONCLUSION AND SUGGESTION From the result of the research, it can be conclude that at first experiment, Carbohydrate Test, by using Molisch Reagent we get that the using of Molisch reagent to detect Carbohydrate will produce purple red furfural condensation at the interface of the two layer, but in our experiment we get pink to purple color, and all of the materials are belong to carbohydrate.

At second experiment , Carbohydrate Test, by using Fehling Reagent, we get that the using of Fehling reagent to detect the Carbohydrate will produce red bata precipitation, sucrose give negative result for this test and we get fault doing this test, because our result is not coherence with the theory. At third experiment Protein and Amino acid Test, by using Biuret Reagent, we get that the using of Biuret Reagent to detect the peptide bond, will give the blue color. In this test we get it with the variety blue color of the materials. All of the materials are protein. At fourth experiment Protein and amino Acid Test, by using Millon Reagent, we get that When millons reagent is added to a protein, a white ppt is formed, which turn brick red on heating. In this test, we get orange precipitation at milk solution and broth solution, so milk and broth solution contain protein and, actually we must get same result to Albumin of eggs but we get fault doing this, the color of the solution not presence the brick red or orange when heating. For improving the quality of experiment, it is recommended to do not use many sample to doing this biomolecules test, because it can make the confusing of the practican to detecting many sample. ACKNOWLEDGMENTS I would like to say thanks to ALLAH SWT that always give me the blessing so I can doing this journal, although so many mistake in it, to my parents who always support me and especially for my assistance Sister Deby Suci Martalina who gives a chance to do this journal and all of my friends in Bilingual Biology Education 2012 that always remind me to finish this journal. REFERENCES Nigam. 2007.Lab Manual in Biochemistry: Immunology and Biotechnology.USA: Tata McGraw-Hill Education.p.23 Murray, Peter R. S. 1999.Principles of Organic Chemistry: A Modern and Comprehensive Text for Schools. Chicago: The university of Chicago Press.p.67 Mittal, Arun.2000.Chemistry. USA: APH Publishing. p.439 http://www.askiitians.com/iit-jee-carbohydrates-amino-acids-peptides/tests-of-proteins/