DEPARTMENT OF BIOMEDICAL ENGINEERING FACULTY OF ENGINEERING UNIVERSITY MALAYA

Lab 12 : Detections of Fats, Proteins, and Carbohydrates Objective

The main purpose of this experiment is to understand some general tests that can detect fats, proteins and carbohydrates in foods

Introduction
Carbohydrates are also known as sacchrides. There are 4 main groups of carbohydrates, which are monosaccharides, disaccharides, oligosaccharides and polysaccharides. Carbohydrates play an important role in living organism as it is the energy storage, and it also plays an important role in structural of living organisms. There are various of test can be done to identified the present of carbohydrates in foods but the few most well-know are the Molisch test, Bennedict test and the iodine test. Proteins are made up of amino acids, which are the simplest unit of proteins. The few main tests to identify the present of the proteins are Xanthoprotic test, Biuret test and also Ninhydrin

test. The test used to identify the present of fats are the Grease spot test. This can be carried out by using a piece of filter paper. Although it cannot identify the types of fats present but it can show the present of fats.

Materials
Distilled water 1% of glucose solution 1% of sucrose solution 1% of lactose solution 1% of starch solution Milk Molisch reagent Concentrated sulfuric acid, H2SO4 solution Benedict reagent Iodine solution Oil 1% of biuret solution 2% of biuret solution 2% of tyrosine solution 2% of egg-albumine solution 2% of glycine solution Concentrated nitric acid, HNO3 solution 6M sodium hydroxide, NaOH solution 2% of copper(ll) sulfate, CuSO4 solution 0.1% of ninhydrin solution Acetone solution

Apparatus
pH papers Parafilm sheets Filter paper Test tubes and holders Stir rod Droppers 25ml of beaker 10ml measuring cylinder

Procedure
Part A : Tests for Detecting Carbohydrates
1. Molisch Test

(a) Six test tubes are needed to perform this test. By using small labels paper, the test tubes are named as below 1. 20 drops of deionized water (control) 2. 20 drops of 1% glucose solution 3. 20 drops of 1% sucrose solution 4. 20 drops of 1% lactose solution 5. 20 drops of starch solution 6. 20 drops of milk (b) For each of the test tubes, 3 drops of the Molisch reagent (-naphthol dissolved in ethanol) are added, and all the six tube are sealed with parafilm. The test tubes are then shaked to ensure mixing of the solutions. By inclining the test tubes at 45, 10 drops of concentrated sulfuric acid solution, H2SO4 are added to the test tube slowly and carefully to ensure the forming of two layer. Be sure to keep a distance with the mouth of the test tube as the concentrated sulfuric acid solution, H2SO4 is very dangerous and harmful. The color produced where the two layer of the solution meet are noted and the observations are recorded into the lab sheet. (c) The reactions will be discussed in the discussion part. 2. Molisch Test (a) Boiling water bath are set up in a beaker half-filled with water. Boiling chips can be added to ensure smooth boiling. (b) 6 clean and labeled test tubes are prepared and are filled with the fresh solutions as stated in the Molisch Test. 5 drops of Benedicts reagent are added to each of the test tube and shaking are needed to mix the solution. At the mean time, all the 6 test tubes are put inside the hot water bath for around 5 minutes. After 5 minutes, all the 6 test tubes are removed and the observation are done and recorded into the lab sheet. (c) The reactions happened will be discussed in the discussion part.

3. Iodine (a) 6 clean and labeled test tubes are prepared and are filled with the fresh solutions as stated in the Molisch Test. Each test tube is added with a drop of iodine solution and shakes in order to mix the solution. The color changed in the samples were noted and recorded in the lab sheet. If none of the samples has a color differ from the control, another 10 drops of distilled water are added to the test tube and 1 more iodine solution is added. The changes in the solution are observed and recorded into the lab sheet. (b) The reactions happen will be discussed in the discussion part.

Part B : Test for Detecting Fats (The Grease Spot Test)

1. A small drop of oil is placed on a piece of filter paper. By holding the filter paper under the light, observation is done and the result is record into the lab sheet. 2. The oil is the replace with a drop of milk with acetone and is placed on a new piece of filter paper. Observation is done and the result is recorded into the lab sheet.

Part C : Test for Detecting Protein

1. Xanthoprotic Test (d) 7 test tubes are needed to perform this test. By using small labels paper, the test tubes are named as below i. 20 drops of deionized water (control) ii. 20 drops of 1% biuret solution iii. 20 drops of 2% biuret solution iv. 20 drops of 1% tyrosine solution v. 20 drops of egg-albumine solution vi. 20 drops of glycine solution vii. 20 drops of milk (a) For each of the test tubes, 5 drops of concentrated nitric acid, HNO3 are added. The concentrated nitric acid, HNO3 solution must be handled with extra careful. The solutions are heated in a hot water bath for around 3 minutes. The color produces are recorded into the lab sheet. (b) The test tubes are then removed and allow them to cool for 5 minutes. 15 drops of sodium hydroxide, NaOH solution are added to the test tubes carefully and cautiously. By using a stir rod, the solutions are mixed and pH papers are used to test each of the solutions. If the result from pH papers shows that the solutions are not strongly basic, then recheck the pH and more NaOH solutions are added if necessary until the solution shows strongly basic when test with pH paper. The color changes are recorded into the lab sheet. (c) The reactions happen will be discussed in the discussion part. 2. Biuret Test (a) 7 clean and labeled test tubes are prepared and are filled with the fresh solutions as stated in the Xanthoprotic Test. 5 drops of 6M NaOH solution and 3 drops of 2% copper(ll) sulfate, CuSO4 solution are added. The test tubes are shakes to ensure mixing of the solutions. After 3 minutes, the color produced are noted and recorded into the lab sheet. If any of the solution, except control, if did not undergoes any changes in color, then it is checked with pH paper to identify whether the solution is basic or not. If the solution are not basic then 2 additional drops of copper(ll) sulfate, CuSO4 solution are added and the observation are done and recorded into the lab sheet. (b) The reactions happened will be discussed in the discussion part. 3. Ninhydrin Test (a) 5 clean and labeled test tubes are prepared and are filled with the fresh solutions as stated in the Xanthoprotic Test except for the 1% and 2% of the biuret solution. 2 drops of 0.1% ninhydrin solution are added to each of the test tubes. The test tubes are heated by placing it in a hot water bath for several minutes. After allow the solution turns cold, the observations to the test tubes are done and the observations are recorded into the lab sheet. (b) The reactions happened will be discussed in the discussion part.

Result
Part A : Tests for Detecting Carbohydrates
Molisch Test Distilled water(control) 1% glucose solution 1% sucrose solution 1% lactose solution 1% starch solution milk Positive test Dark brown Dark brown More darker Light brown Light brown Reddish orange all Benedict Test Clear blue Orange greenish Orange Pale grey Clear blue White with blue layer glucose Iodine Test Clear yellow Clear yellow Clear yellow Clear yellow Blue black No change Starch

Part B : Test for Detecting Fats (The Grease Spot Test)

Oil Filter paper Transparent Milk with acetone Not transparent

Part C : Test for Detecting Protein

Xanthoprotic Test Distilled water(control) 1% biuret solution 2% biuret solution 2% tyrosine solution 2% egg-albumine solution 2% glycine solution Milk Colourless Colourless Colourless Reddish yellow Yellownish Colourless Sleihgtly reddish orange Biuret Test Light Blue Colourless with slightly blue Cloudish blue Colourless with a bit blue Purple Blue Purple Ninhydrin Test Colourless Slightly pink Slightly pink Colourless No change

(Molisch Test)

From left to right : Distilled water (control) , 1% of glucose solution, 1% of sucrose solution, 1% of lactose solution, 1% of starch solution, Milk (Benedict Test & Iodine Test)

From left to right : Distilled water (control) , 1% of glucose solution, 1% of sucrose solution, 1% of lactose solution, 1% of starch solution, Milk Picture at left is Benedict Test while at right is Iodine Test (Xanthoprotic Test)

From left to right : milk, glycine solution, egg-albumine solution, 1% tyrosine solution, 2% biuret solution, 1% biuret solution, deionized water (control) Picture at left is Xanthoprotic Test while picture at right is after added NaOH solution

Picture at right is the pH value of each solution while picture at right is the pH scale of the pH paper. (Biuret Test & ninhydrin Test)

Left picture, from left to right : milk, glycine solution, egg-albumine solution, 1% tyrosine solution, 2% biuret solution, 1% biuret solution, deionized water (control) Right picture, from left to right : milk, glycine solution, egg-albumine solution, 1% tyrosine solution, deionized water (control) (Fats Test)

Filter paper at left is milk with acetone and at right is oil.

Discussion
Part A : Tests for Detecting Carbohydrates
1. Molisch Test Molisch test is a sensitive chemical test used to identify the present of carbohydrates. Based on the theory, all types of carbohydrates such as monosaccharide, disaccharide, oligosaccharide and polysaccharide will give a positive test. The monosaccharide will give the most rapid positive test, followed by disaccharide, oligosaccharide and polysaccharide. Besides that, glycoproteins and nucleic acid also will give a positive result. The mechanism is the test reagent will first dehydrated pentoses to form furfural and it also will dehydrates the hexoses to form 5-hydroxyl furfural. The furfural produces then will react with -napthanol(present in the Molisch reagent) to produce a purple product. Molisch reagent is a solution of -napthol in 95% of ethanol.

The results are as follows: Positive : glusoce, lactose, sucrose and starch milk Negative : 2. Benedict Test Benedict solution(1 litre) is prepared from 100g of anhydrous sodium carbonate, 173g of sodium citrate and 17.3g of copper(ll) sulfate pentahydrate. Benedict test are used to test for the present of reducing sugars. For examples, lactose and maltose and other monosaccharide and disaccharide are the example of reducing sugar. Benedicts test is also used to detect the presence of aldehydes.Copper sulfate serveas oxidant to reduce the sugars. Benedict quantitative test can be used to calculate how much of the reducing sugar present. Besides that, Benedict solution also can used in place of Fehlings solution. Carbohydrates that contain aldehydes or -hydromethyl ketones groups can be oxidized by Cu(ll) ion. Thus, these type of carbohydrates consider as reducing sugars as they reduced the Cu(ll) ion into Cu(l).

The results are as follows: Positive : glucose and lactose Negative : sucrose, starch and milk 3. Iodine test The iodine test is used to test for the presence of the starch. The iodine solution is prepared by dissolving iodine in potassium iodide (aqueous). The intensity of the color decreases with the increasing of the presence of organic solvents. Amylase (straight chain portion of starch) will forms helices where iodine molecules assemble and gives dark purple or black color. The amylopectin (branched portion of starch) will give an orange or yellow hue.

The results are as follows: Positive : starch and milk Negative : glucose, sucrose and lactose

Part B : Test for Detecting Fats (The Grease Spot Test)

The grease spot test is being used to detect lipid. Lipids will leave a translucent or semi-transparent on the filter paper. The filter paper can be held under the light to checked whether the solution leave a semi-transparent mark. If it does then the solution will be lipids. The results are as follows: Positive : oil drops Negative : milk with acetone

Part C : Test for Detecting Protein

1. Xanthoprotic Test The Xanthoprotic tests are used to determine the presence of the proteins. This reaction is based on the nitration of aromatic benzene derivatives. This reaction can said to be the nitration of the benzene ring by using the nitric acid. Only activated benzene ring will gives out positive result and for deactivated benzene ring, there would not be any reaction of nitration occurs. The Sodium hydroxide solution, NaOH provides a basic medium for the reaction to start. The results are as follows: Positive : tyrosine and egg albumin Negative : biuret and glysine, milk? 2. Biuret Test The biuret test is an assay to determine the presence of the proteins in certain solution. The biuret test in other words is actually detecting the presence of peptide bonds. If a peptide bond is present, then the copper(ll) ion will forms a violet color complex in an alkaline solution. The biuret reagent is made up of sodium hydroxide solution added with copper sulfate. This blue reagent will turns violet if there is the presence of proteins it also would change to pink if combined with short-chain polypeptides.

The figure shown is the purple complex ion.

The results are as follows: Positive : biuret, egg albumin, glysine and milk Negative : tyrosine 3. Ninhydrin Test Ninhydrin test is used to determine the presence of proteins. Amines will react with ninhydrin to give a positive result. This test can be used for chromatographic visualization (qualitatively) and for peptide sequencing (quantitatively). The amino acids that contain a free carboxylic group and a free amino group will form different color products. Amino group at -carbon : blue-purple product Secondary amino acid : yellow The results are as follows: Positive : tyrosine, egg albumin and glycine Negative : milk?

There might be some incorrect results is compared to the theoretically results. This might because of human error as contamination of the solution and samples during the experiments. As to ensure getting the correct results, contamination of samples and solutions should be avoids by preparing each solutions and samples with a droppers. Besides that, proper heating also should take into considerations as over heat may be will damage the solutions and thus getting incorrect results. Proper volume of solution during preparation also should be taken into consideration and mixing the solutions properly can ensure getting the correct results for certain tests.

Conclusion
As conclusion, the several tests used to determine carbohydrates, fats and proteins are learned. There are some error during the experiment, thus more precaution should be taken.

References
1. James O. Schreck, William M. Loffredo, Qualitative Testing For Carbohydrates, University of Northern Colorado and East Stoudsburg university, 1995, United State of America. 2. Frank R. Milio, William M. Loffredo, Qualitative Testing For Amino Acid and Proteins, Towson State University and East Stoudsburg university, 1995, United State of America. 3. Division of Chemical Education, Inc., American Chemical Society. Benedict's Test for Reducing Sugars. 2001. http://jchemed.chem.wisc.edu/jcesoft/CCA/CCA5/MAIN/1ORGANIC/ORG18/TRAM18/B/ME NU.HTM 4. http://www.chem.ucalgary.ca/courses/351/Carey5th/Ch27/ch27-3-3.html 5. http://www.harpercollege.edu/tm-ps/chm/100/dgodambe/thedisk/carbo/molisch/molisch.h tm