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Chemical Tests for Biomolecules
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Laboratory Techniques
INTRODUCTION
Biomolecules are complex organic molecules. Carbon, hydrogen, oxygen, nitrogen and phosphorus are the atoms that make up most of the biomolecules. These molecules form the basic structure of a living cell. The compounds such as amino acids, nucleotides and monosaccharide’s serve as the building blocks of complex biomolecules. The important biomolecules are proteins, carbohydrates, fats, hormones and nucleic acids (Kimball, 2012).
Carbohydrates
Carbohydrates are substances which containing the elements carbon hydrogen and oxygen and they have the general formula of Cx (H2O) y. Simple carbohydrates or the entire carbohydrate family may also be called saccharides .They are the most abundant biomolecules belonging to class of organic compounds found in living organisms. The major source of metabolic energy for both animals and plants are carbohydrates (Churms, 1982). Carbohydrates link to with proteins forming glycoproteins and with lipids forming glycolipids. Moreover they are present in DNA and RNA, which are essentially polymers. More than 75% of the dry weight of the plant world is carbohydrate in nature mainly cellulose, hemicelluloses and lignin (Reed, 2005).
Carbohydrates are classified on the basis of their behavior on hydrolysis. They have been broadly divided into following three groups: Monosaccharide’s, Disaccharides, Oligosaccharides, and Polysaccharides.
Monosaccharide
A carbohydrate that cannot be hydrolyzed further to give simpler unit of polyhydroxy aldehyde or ketone is called a monosaccharide. Monosaccharides are single sugars units and there general formula is (CH20) n. Moreover they are colorless, crystalline solids that are freely soluble in water but insoluble in nonpolar solvents. The backbone of monosaccharide is an unbranched carbon chain in which all the carbon atoms are linked by single bonds (GyörgydeaÌk and PelyvaÌs, 1998). One of the carbon atoms is double-bonded to an oxygen atom to form a carbonyl group each of the other carbon atoms has a hydroxyl group. If the carbonyl group is at an end of the carbon chain, the monosaccharide is an aldehyde and is called an aldose, furthermore if the carbonyl group is at any other position the monosaccharide is a ketone and is called ketoses. Glucose, fructose, galactose, and ribose are some examples of monosaccharide. The building blocks of disaccharides like sucrose and polysaccharides such as cellulose and starch and hemicelluloses are monosaccharide (Ferrier, 1999).
Figure 1.1.1 ring structure of monosaccharide molecules.
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Figure 1.1.2 monosaccharide molecule showing the aldehyde and ketone group
http://academic.brooklyn.cuny.edu/biology/bio4fv/page/monosacchrides.html
Disaccharides
A Disaccharide is two monosaccharide units linked by an oxide linkage formed by the loss of a water molecule. Such a linkage between two monosaccharide units through oxygen atom is called glycoside linkage. Three most abundant disaccharides are sucrose, lactose, and maltose. In Maltose α (1→4) glycosidic linkage joins two glucose units, this occurs mainly as a breakdown product during digestion of starch by enzymes called amylases (Owusu-Apenten, 2005). Sucrose is the most abundant disaccharide in nature and it’s mostly found in plants which acts a good transport sugar since it is very soluble and can move in very high concentration. In Sucrose the anomeric carbon atoms of a glucose unit and fructose unit are joined. Moreover lactose the disaccharide of milk consists of galactose joined to glucose by β (1→4) glycosidic linkage (Denniston, Topping and Caret, 2004). In additionally Sucrose and lactose are heterosaccharides and maltose is homosaccharides as well as maltose and lactose are reducing sugars. Sucrose is the only common non reducing sugar.
Figure 1.3.1 disaccharides are formed by condensation of two monosaccharide.
https://www.google.lk/search?q=disaccharides&es_sm=122&source
Polysaccharides
Polysaccharides are complex carbohydrates made up of many monosaccharide joined together by glycosidic bond. They are large, often branched, macromolecules. Their large sizes make them more or less insoluble in water and have no sweet taste (Aspinall, 1982). When all the monosaccharide in a polysaccharide is of the same type, the polysaccharide is called a homopolysaccharide and when more than one type of monosaccharide is present, they are called heteropolysaccharides. Polysaccharides have a general formula Cn (H2O) n-1 where n can be any number between 200 and 2500. Starch glycogen and cellulose are the examples of polysaccharides (Tombs and Harding, 1998).
Figure 1.4.1 ring structure of polysaccharides molecules.
https://www.google.lk/search?q=polysaccahrides&es_sm=122&source=lnms&tbm=isch&sa
Proteins
Cells are made of protein. Proteins are the most versatile class of molecules in living organisms. All proteins contain C, H, N, O some S, P, Fe, Zn, Cu. Proteins contains 20 different amino acids which are encoded by the genetic code and which constitute the building blocks of the proteins in all living organisms (Walsh, 2002). Each protein species contains one or several polypeptide chains of defined amino acid sequence. Their functions are catalysis, transport, hormones and structure. Amino acids are molecules containing an amine group carboxylic acid group and a side chain. Simple proteins contain only polypeptide chains Proteins can be soluble (globular proteins) and insoluble (myosin, fibrinogen) (Whitford, 2005).
Figure 1.5.1 classification of proteins and there structures.
https://www.google.lk/search?q=protein structure&revid=120848340&tbm
OBJECTIVES
To distinguish between monosaccharide’s and disaccharides.
To differentiate between different types of amino acids.
To identify an unknown sample of carbohydrate and amino acid.
MATERIALS
Albumin solution
Arginine solution
Barfoed reagent
Beakers
Benedict’s solution
Bunsen burner
Burner stand
Concentrated sulphuric acid
Concentrated nitric acid
Copper sulphate
Fructose solution
Glucose solution
Glysin solution
Iodine solution
Lactose solution
Molisch’s reagent
Ninhydrin solution
Pipettes
Seliwanoff’s reagent
Sodium hydroxide
Starch
Sucrose solution
Test tubes
Tyrosine solution
Unknown solutions
Water bath
TEST FOR CARBOHYDRATES METHODOLOGY
Molisch’s Test
Five test tubes were taken with 1ml of carbohydrate solutions. Few drops of Molisch’s reagent were added to the testubes following with concen.sulphuric acid down the slide of the test tube. The colour change was observed.
Iodine test
Three drops of Iodine solution was added to each test tube with 1ml of each of the carbohydrate solutions. The colour change was observed.
Benedict’s test
1ml of each carbohydrate solutions was taken in five test tubes.5ml of Benedict’s reagent was added to all three test tubes. All five test tubes were placed in a water bath and heated for two minutes. The colour change was observed.
Barfoed test
5ml of Barfoed reagent was added with 1 ml of carbohydrate solutions. Test tubes were placed in water bath and heated for five minutes. The colour change was observed.
Seliwanoff test
1ml of each carbohydrate solution was added to the test tubes following with 4ml of Seliwanoff reagent. The test tubes were placed in the water bath and heated to two to three minutes. The colour change was observed.
Two unknown samples were taken in a test tubes and labeled A and B. Sample A was added to two test tubes. To the sample A the Iodine reagent was added and the colour change was observed. The Benedict’s reagent was added to the sample A of another test tube and was heated in general flame for two minutes and the colour change was observed.
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The sample B was added to four test tubes. One drop of Iodine reagent was added to the sample B test tube and colour change was observed following with Benedict’s reagent, Barfoed reagent and the Seliwanoff reagent were added to the remaining test tubes with sample B and was heated in the water bath for three minutes and the colour change was observed.
TEST FOR AMINO ACID METHODOLOGY
Ninhydrin test
1ml of Ninhydrin solution was added into 0.5 ml of 0.02 % amino acid solution in four test tubes. The test tubes were placed in water bath and heated for three to four minutes. The colour change was observed.
Xanthoproteic Test
2ml of conc. Nitric acid was added to 2ml of 0.02% amino acid solution in four test tubes. The test tubes were placed in water bath for two minutes and the colour change was observed.
Millon’s Test
Four drops of Millon’s reagent was added into 2ml of 0.02% of amino acid solution in four test tubes. The test tubes were placed in water bath for four minutes and the colour change was observed.
Biurete Test
3ml of 10% of sodium hydroxide was added drop wise to 1% of copper sulphate. The colour change was observed.
Two unknown samples were taken in test tubes and labeled C and D. Sample C was added into two test tubes. To the sample C the Biurete reagent was added and the colour change was observed. The Millon’s reagent was added to the sample C of another test tube and was heated in general flame for two minutes and the colour change was observed.
The sample D was also added into two test tubes. Biurete reagent was added to the sample B test tube and colour change was observed. Besides Millon’s reagent were added to the remaining test tube with sample B and was heated in the water bath for three minutes and the colour change was observed.
RESULTS
Test for carbohydrates
Sugar type
Test performed
Observation
Inferences
Starch
Molisch’s test
Green precipitate at the bottom and thick purple ring formed
All are positive hence it’s a carbohydrate solution
Lactose
Green precipitate at the bottom and purple ring formed
Fructose
Green precipitate at the bottom and purple ring formed
Glucose
Green precipitate at the bottom and light purple ring formed
Sucrose
Green precipitate at the bottom and purple ring formed
Starch
Iodine test
Brown to dark blue solution
Positive –unbranched polysaccharide
Lactose
Remains brown colour, no colour change
These three results in negative as there is a possibility of becoming either monosaccharide or disaccharide.
Fructose
Remains brown colour, no colour change
Glucose
Remains brown colour, no colour change
Sucrose
Remains brown colour, no colour change
Starch
Benedict’s test
Remained blue, no colour change.
Negative-non reducing sugar
Lactose
Green precipitate turned into reddish orange colour
Positive-reducing sugar
Fructose
Green precipitate turned into reddish orange colour
Positive-reducing sugar
Glucose
Green precipitate turned into reddish orange colour
Positive-reducing sugar
Sucrose
Remained blue, no colour change.
Negative- reducing sugar
Starch
Barfoed test
Remained blue, no colour change
Negative-non reducing disaccharide
Lactose
Remained blue, no colour change
Negative- reducing disaccharide
Fructose
Blue colour turned to reddish colour precipitate
Positive- reducing monosaccharide
Glucose
Blue colour turned to reddish colour precipitate
Positive- reducing monosaccharide
Sucrose
Remained blue, no colour change
Negative-non reducing disaccharide
Starch
Seliwanoff test
No colour change, remains colourless
Negative- not reducing sugar
Lactose
No colour change, remains colourless
Negative- not reducing sugar
Fructose
Reddish colour
Positive-ketone
Glucose
No colour change, remains colourless
Negative -aldose
Sucrose
No colour change, remains colourless
Negative- not reducing sugar
Unknown solution
Iodine test
Benedict’s test
Barfoed test
Seliwanoff test
A
No colour change. (monosaccharide or disaccharide)
No colour change
(negative-sucrose)
——–
——–
B
No colour change. (monosaccharide or disaccharide)
Colour change. (Green precipitate). Positive- either glucose, lactose or fructose)
Red precipitate
Positive -(glucose or fructose)
Remains colourless. Negative
Test for amino acids
Amino type
Test performed
Observation
Inferences
Arginine
Ninhydrin test
Violet colour solution
Positive-protein, peptide or amino acid.
Tyrosine
Albumin
Glysin
Arginine
Biurete test
Blue colour precipitate formed
Negative- since copper hydroxide formed.
Tyrosine
No colour changed
Negative – amino acid present
Albumin
Violet colour solution
Positive- protein or peptide
Glysin
No colour changed
Negative – amino acid present
Arginine
Xanthoproteic test
initial
colourless
Final
colourless
Negative –not aromatic amino acid
Tyrosine
Turned yellow
Remained yellow
Positive-aromatic amino acid
Albumin
Milky precipitate
colourless
Negative-not aromatic amino acid
Glysin
Colourless
Colourless
Negative-not aromatic amino acid
Arginine
Millon’s test
Milky solution
Colourless
Negative-Absent of phenolic OH
Tyrosine
Pink to red precipitate
Positive-protein containing phenolic OH
Albumin
Light pink precipitate
Positive-protein containing tyrosine
Glysin
Colourless
Negative-absent of phenolic OH
Unknown solution
Biurte test
Millon’s test
C
Purple (violet colour formed)
Positive-protein or peptide
Cloudy solution turns pinkish
Positive-protein contain tyrosine
D
Light blue
Negative-amino acid
Colourless
Negative-amino acid without phenolic OH
DISCUSSION
In Molisch’s test all the carbohydrate solution gave a positive result, so as it’s a general test to confirm the molecule is carbohydrate. Iodine test is performed to separate the polysaccharide from monosaccharide and disaccharide as a result in this test only starch gave a positive result since its unbranched molecule. Glucose has a free aldehyde group and fructose has a free ketone group. Thus they react with Benedict’s reagent and reduce it to form a reddish orange colour, which is a positive indication of Benedict’s reaction .The copper (II) ions in the Benedict’s solution are reduced to Copper (I) ions, which causes the colour change. Complex carbohydrates such as starches do not react positive with the Benedict’s test.
Buiret solution is a blue liquid that changes to purple when proteins are present and to pink in the presence of short chains of polypeptides. The cause of this colour change is because of the copper atom of the Biuret solution reacts with the peptide bonds.
Avoid spilling Ninhydrin solutions on your skin, as the resulting stains are difficult to remove. When handling with Concentrated Sulphuric acid wear safety garments to avoid Sulphuric acid getting on self. Do not over heat the amino solutions in water bath since all the proteins may denature moreover colour change cannot be observed.
CONCLUSION
The unknown solution A is sucrose and it’s a non reducing sugar since in Iodine and Benedict’s test it showed a negative result where there was no colour change in addition to unknown solution B is glucose which is a reducing sugar because in Iodine and Seliwanoff test it gave a negative result remaining colourless and in Benedict’s and Barfoed test it gave a positive result changing its colour from green precipitate to reddish colour solution concluding solution B is glucose.
The unknown solution C is protein since positive result was obtained and the solution turned pink in Biurete and Millon’s reagent along with the solution D is an amino acid because it remained colourless in Millon’s test and turned light blue in Biurete test resulting both in negative.
References
Aspinall, G. (1982). The Polysaccharides. 1st ed. New York: Academic Press. Google books [Online books] Available at: http://books.google.lk (Accessed: 3rd July 2014).
Churms, S. (1982). Carbohydrates. 1st ed. Boca Raton, Fla.: CRC Press. Google books [Online books] Available at: http://books.google.lk (Accessed: 3rd July 2014).
Denniston, K., Topping, J. and Caret, R. (2004). General, organic, and biochemistry. 1st ed. Boston: McGraw-Hill Higher Education. Google books [Online books] Available at: http://books.google.lk (Accessed: 3rd July 2014).
Ferrier, R. (1999). Carbohydrate chemistry. 1st ed. Google books [Online books] Available at: http://books.google.lk (Accessed: 3rd July 2014).
GyörgydeaÌk, Z. and PelyvaÌs, I. (1998). Monosaccharide sugars. 1st ed. San Diego: Academic Press. Google books [Online books] Available at: http://books.google.lk (Accessed: 3rd July 2014).
Kimball, L. (2012). Biomolecules. 1st ed. Delhi: Research World. Google books [Online books] Available at: http://books.google.lk (Accessed: 3rd July 2014).
Owusu-Apenten, R. (2005). Introduction to food chemistry. 1st ed. Boca Raton, Fla.: CRC Press. Google books [Online books] Available at: http://books.google.lk (Accessed: 4th July 2014).
Reed, D. (2005). Biomolecular archaeology. 1st ed. Carbondale: Center for Archaeological Investigations, Southern Illinois University, Carbondale. Google books [Online books] Available at: http://books.google.lk (Accessed: 3rd July 2014).
Tombs, M. and Harding, S. (1998). An introduction to polysaccharide biotechnology. 1st ed. London: Taylor & Francis. Google books [Online books] Available at: http://books.google.lk (Accessed: 4th july2014).
Walsh, G. (2002). Proteins. 1st ed. Chichester: J. Wiley. Google books [Online books] Available at: http://books.google.lk (Accessed: 6th July 2014).
Whitford, D. (2005). Proteins. 1st ed. Hoboken, NJ: J. Wiley & Sons. Google books [Online books] Available at: http://books.google.lk (Accessed: 6th July 2014).
