Detection of fats ,proteins and carbohydrates

T2:BIOCHEMISTRY AND ANALYSIS:DETECTION OF FATS,PROTEINS AND CARBOHYDRATES

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

INTRODUCTION
Carbohydrates are also known as sacharides. 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 maintests 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.

APPARATUS

1.pH papers
2.Parafilm sheets
3.Filter paper
4.Test tubes and holder
5.Stir rod
6.Droppers
7.25ml of beaker
8.10ml measuring cylinder

PROCEDURE

PART A:TEST FOR DETECTING CARBOHYDRATES

1. MOLISCH TEST
a)Small paper labels,number five clean test tubes are using as directed below: i. 20 drops of deionized water(control)
ii.20 drops of 1% glucose solution(1g of glucose is added in 99ml distilled water) iii.20 drops of 1% sucrose solution
iv.20 drops of 1% lactose solution
v.20 drops of 1% starch solution(1g of starch is added in 99ml distilled water and put in boil) b) Add 3 drops of Molisch reagent to each of the five test tubes,3 drops of Molisch reagent is added.Each test tube is seal with Parafilm® and each tube is shook to ensure mixing.Then,the test tube is inclined at 45% angle,10 drops of concentrated sulfuric acid (H2SO4) is slowly added,so two layers is formed. c) The reaction happens in this test is discussed.

2.BENEDICTS TEST
a)Boiling water bath is set up in a 250ml beaker half-filled with water.Two or three boiling chips are added to ensure smooth boiling. b) 5 clean and labeled test tubes are prepared with fresh samples of the solution used in the Molisch test.5 drops of benedicts reagent is added to each test tube,and it is shook to mix.At the same time,all five test tubes is placed in the boiling water bath.The five test tubes is removed after 5 minutes and our observation is recorded. c)The reaction happend in this test is recorded.

2. IODINE
a)5 clean and labeled test tubes is prepared with fresh samples of the carbohydrates solutions used in the Molisch test.1 drop of iodine solution is added to each and it is shook to mix.The control sample is observed.10 drops of distilled water is added to each test tube and 1 more drop of the iodine solution after none of the other samples has a color different that the control.Each solution is mixed and our observations is recorded on the Report Sheet. b)The reactions happend in this test is discussed.

PART B:TEST FOR DETECTING FATS:THE GREASE SPOT TEST
1)A small drop of oil is placed on a piece of filter paper and the paper is holded up to the light.Our observation is recorded.The reaction happens in this test is recorded.

PART C:TEST FOR DETECTING PROTEIN
1. XANTHOPROTIC TEST
a)6 test tube is prepared and labeled with the following solutions: 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 2% tyrosine solution
v)20 drops of 2% egg-albumine solution
vi)20 drops of 2% glycine solution
b)5 drops of concentrated nitric acid,HNO3 is added to each of the 6 solutions.The solutions is heated in a boiling water bath for 2 minutes.The color changed is recorded. c)The test tubes is removed and it is cool for 5 minutes and cautiously and 15 drops of 6M sodium hydroxide,NaOH is added to each.Each is stired with a glass stirring rod,then a droplet of each solution is removed and touched to a piece of pH paper.5 more drops of Naoh is added to any solution that does not test strongly basic.The pH is recheck and more NaOH is added until each solution test strongly basic with pH paper.Any color changes is recorded and the reaction happens in this test is discussed.

2. BIURET TEST
a)Fresh samples of the solutions listed in the xanthoprotic test in 6 clean and labeled test tubes is obtained.5 drops of 6M Naoh and 3 drops of 2% copper(II)sulphate(CuSo4) solution is added.Each test tube is seal and each mixture is shook.after 3 minutes,the colors produced.All observations is recorded.

3. NINHYDRIN TEST
Fresh sample of the control and solutions in the xanthoproteic test is obtained except for the 1% and 2% biuret solutions.Then,to each 4 samples,2 drops of 0.1% ninhydrin solution is added.Each test tube cautiously and
slowly to boiling over a burner for 1 minute.The solution is allowed to coole and the observation is recorded.

TESTING MILK
1)1ml of milk is placed in each of the three clean test tubes.The three carbohydrate test is performed. 2)1ml of milk is mixed with 2ml of acetone.A few drops of the solution is placed on a piece of filter paper and the results is compared with that of the grease spot test obtained in part B. 3)1ml samples of milk carry out the three proteins test is used. 4)All observations is recorded and discussed.

RESULTS
PART A:TEST FOR DETECTING CARBOHYDRATES
TABLE 1

MOLISCH TEST
BENEDICT TEST
IODINE
Distilled water(control)
colourless
blue colour
yellow colour
1% glucose solution
purple

brick red precipitate
yellow colour
1% sucrose solution
purple
Blue colour
yellow colour
1% lactose solution
purple
brick red precipitate
yellow colour
1% starch solution
purple
blue colour
dark blue precipitate
Milk
purple precipitate

yellow precipitate
yellow-orange precipitate
Positive test
All

1% glucose solution and 1% sucrose solution
1% sucrose solution

PART B:TEST FOR DETECTING FATS:GREASE SPOT TEST
TABLE 2

OIL
MILK WITH ACETONE
Filter paper
translucent spot
not translucent

PART C:TESTS FOR DETECTING PROTEINS
TABLE 3

XANTHOPTOTIC TEST
BIURET TEST
NIHYDRIN TEST
20 drops of deionized water (control)
No change ph 14
light blue
No change
Light brown
20 drops of 1% biuret solution
No change ph14
Light blue with precipitate
Second test:light blue

20 drops of 2% biuret solution
No change ph14
Green precipitate
Second test:light blue

20 drops of 2% tyrosine solution
Reddish brown ph14
Light blue
White precipitate
20 drops of 2% egg-albumine solution
Yellowish ph13
Purple
Cloudy
20 drops of 2% glycine solution
No change ph13
Light blue
Dark blue

DISSCUSSION

PART A:TEST FOR DETECTING CARBOHYDRATES

A.Molisch Test
Molisch test is 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,followed by disaccharides,oligosaccharide and polysaccharide.Besides that,glycoproteins and nucleic acid also will give a positive result.The mechanism is the test reagent will first dehydrates pentoses to form furfural and it also dehydrates the hexoses to form 5-hydroxyl furfural.The furfural produces then will react with α-napthanol(present in the Molisch reagent) to produce a purpl product,Molisch reagent is a solution of α-napthol is 95% of ethanol.

The results are follow:
Positive test: glucose,lactose,sucrose,starch and milk.

B.Benedict Test
Benedict solution(1 litre) is prepared from 100g of anhydrous sodium carbonate,173g of sodium citrate and 17.3g of copper(II) sulfate pentahydrate.Benedict test are used to test for the present of reducing sugars.For example,lactose and maltose and othe monosaccharide and disaccharide are the example of reducing sugar.Benedict’s test is also used to detect the presence of aldehydes.Copper sulfate serveas oxidant to reduce the sugars.Benedict quantitative testcan be used to calculate how much of the reducing sugar present.Besides that,Benedict solution also can used in place of Fehling’s solution.Carbohydrates that contain aldehydes or α-hydromethyl ketones groups can be oxidized by Cu(II) ion.Thus,these type of carbohydrates consider as reducing sugars as they reduced the Cu(II) ion into Cu(I).

The results as follows:
Positive:glucose and lactose

C.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 starc) will forms helices where iodine molecules assemble and gives dark blue with precipitate.The amylopectin (branched portion of starch) will give an yellow color). The results are as follows:

Positive: starch and mik

PART B:TEST FOR DETECTING FATS: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 does the solution will be lipids. The results are as follows:

Positive:oil drops
Negative:milk with acetone
PART C:TESTS FOR DETECTING PROTEINS
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 ring by using the nitric acid.Only activated benzene ring will gives out positive result and for deactived 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,egg albumin and milk
Negative:biuret and glysine
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(II) 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.The 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 is the purple complex ion
The results are as follws:
Positive:egg albumin,milk
Negative :tyrosine
Test with solution 20 drops of 1% biuret solution and 20% drops of 2% biuret
solution are repeated to get the real result.This is because the first test for this solution we got the wrong result.this might because of human error as contamination of the solution and samples during the experiments.

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 group will form different color products. Amino group at α-carbon:blue-purple product

Secondary amino acid:yellow
The results are as follows:
Positive:tyrosine,egg albumin and glycine,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 curtain test. 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.The objective had achieved. REFERENCES

1.Division of Chemical Education,Inc,American Chemical Society.Benedict’s test for reducing Sugar.2001. 2.http://www.chem.ucalgary.ca/course/351/Carey5th/Ch27/ch27-3-3.html 3. http://homepages.ius.edu/dspurloc/c122/casein.htm