Biological molecules = Biomolecules = Compounds of life
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2 Biological molecules = Biomolecules = Compounds of life Carbohydrates Proteins & Amino Acids Mono-saccharides Olego-saccharides Di-saccharides Poly-saccharides Lipids Oils & Fats Amino acids Proteins Enzymes Vitamins Ascorbic acid (Vit-C) Fatty acids Cholesterols
3 Before starting ANY test, you need a control test. The control tube contains everything that the assays contain except the substance to be tested. In the assays, water is the solvent for all of the other substance and will be in the control, along with the test reagents. The control test is the negative standard against which the assays are compared.
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5 Mono-saccharides Di-saccharides Sucrose = Glucose + Fructose Maltose = Glucose + Glucose Lactose = Glucose + Galactose Poly-saccharides Starch
6 Polysaccharidesaccharidesaccharides Di- Mono- Starch Maltose Glucose Cellulose Lactose Fructose Glycogene Sucrose Galactose Mannose
7 Tests to be done: Molisch s test (specific to all carbohydrates) Iodine s test (specific to starch) Barfoed s test (specific to mono-saccharides) Seliwanoff s test (specific to keto-sugars) Reducing sugars tests (specific to sugars with free carbonyl group) Benedict s test Fehling s test
8 Molisch s test Principle: Molisch test is Specific for all carbohydrates To differentiate between Proteins & Amino Acids (-ve), and Carbohydrates (+ve). The Molisch reagent reacts with all carbohydrates larger than tetroses. This test is based on the fact that pentoses and hexoses are dehydrated by conc. H 2 SO 4 acid to form furfural or O H hydroxymethylfurfural, respectively. O C H O Furfural α-naphthol Oligo-and polysaccharides first undergo hydrolysis with H 2 SO 4 acid to give pentoses /or hexoses, which on subsequent dehydration yield furfurals. These furfurals condense with α-naphthol to give colored complexes. Procedure & observation: To 2 ml of carbohydrate solution in a test tube, add 1 ml of Molisch reagent [95% Ethanole + 5% α-naphthol]. Mix well; add carefully at the side wall of the test tube 2 ml conc H 2 SO 4. - Blue to violet ring will be formed at the interface of the two solutions (carbohydrate + Molisch reagent and H 2 SO 4 )
9 Few drops of B Few ml of H 2 SO 4 On the side wall Carbohydrate Mix Mix Few ml of H 2 O g of A C H 2 SO 4 Unknown A Any Carbohydrate Reagent B Molisch reagent [Ethanolic α- Naphthol] then add conc H 2 SO 4 at the side wall of the test tube Result Observation C Blue-Violet ring at the interface Any Carbohydrate C No change Any Proteins or Amino Acids
10 Iodine s test Principle: Iodine test is Specific to Starch To differentiate between Mono- & Disaccharides (-ve), and Starch (+ve). The structure of starch is a such that the molecule is coiled. Iodine reagent (I 2 + KI) forms I 2 KI, which interact specifically with these coiled molecules to form a blue to black colored complex. A positive iodine assay indicates the presence of starch, so if the color turns dark blue, or black, this is a positive result. Procedure & observation: - To 2 ml of carbohydrate solution in a test tube, add 1 ml of Iodine solution -Mix well, a deep blue colour appears. -Warm up, the colour dissapears. - Cool down, the colour re-appears
11 Few drops of B Mix Mix Few ml of H 2 O g of A C Result Unknown A Starch & (Mono Di-Saccharides) Reagent B Iodine solution Observation C Deep blue colour Starch C No change Mono- or Di- Saccharides
12 Barfoed s test Principle: Barfoed test is Specific to Mono-saccharides To differentiate between Monosaccharides (+ve) and Di-saccharides (-ve). Barfoed reagent is formed from [Cu(CH 3 COO) 2 + CH 3 COOH]. Reducing monosaccharides are oxidized by the copper ion in solution to form a carboxylic acid and a reddish precipitate of copper (I) oxide within three minutes. In an acidic environment the reducing di-saccharides cannot reduce the cupric ion to red-brick ppt quickly, it does at a slower rate. If you see particles in the bottom of the test tube, it s a positive result. Procedure & observation: -To 2 ml of carbohydrate solution in a test tube, add 1 ml of Barfoed reagent - Mix well, and warm it up for 5 min - Observe red-brick ppt or a slight change in colour from blue to red/violet or green.
![[Cu(CH 3 COO) 2 + CH 3 COOH] Result Observation C Red brick ppt or red](/docs-images/93/112277979/images/13-2.jpg "colour Mono- (eg. Gluc, Fruc, Galac) C V. slow reaction Di- (e.g. Lact, Malt, Sucr)")
13 Few drops of B Mix Mix Heat 5-7 min Few ml of H 2 O g of A C Unknown A Mono- & Di- Saccharides Reagent B Barfoed reagent [Cu(CH 3 COO) 2 + CH 3 COOH] Result Observation C Red brick ppt or red colour Mono- (eg. Gluc, Fruc, Galac) C V. slow reaction Di- (e.g. Lact, Malt, Sucr)
14 Seliwanoff s test Principle: Seliwanoff test is Specific to Ketonic saccharides (Fructose or Sucrose). To differentiate between aldehydic and ketonic saccharides. When a ketose is heated with a strong mineral acid, hydroxymethylfurfural is formed. This compound forms a red complex with the organic compound, resorcinol. The Ketonic saccharides are more reactive than Aldehyde group of Aldose and it reacts rapidly with Seliwanoff's reagent [Contains resorcinol + conc. HCl] to form furfural derivative. Procedure & observation: - To 2 ml of carbohydrate solution in a test tube, add 1 ml of Seliwanoff reagent. - Mix well, and warm it up for 3-5 min - Observe red colour.
15 Few drops of B Mix Mix Heat 3-5 min Few ml of H 2 O g of A C Result Unknown A Mono- & Di- Saccharides Reagent B Seliwanoff reagent [Resorcinol + HCl] Observation C Red colour in 2-3 min Mono- (Only Fructose due to keto gp) C Red colour after few min Di- (Only Sucrose due to keto gp) C No reaction Mono- and Di- (No keto gps )
16 Benedict s test Principle: Benedict test is Specific to reducing sugars, sugars with a free aldehyde or ketone group) such as Fructose, Glucose, Galactose, Lactose & Maltose. To differentiate between Sucrose & reducing sugars. Benedict is formed from sodium carbonate + sodium citrate dihydrate + copper sulfate pentahydrate. The copper sulfate (CuSO 4 ) present in Benedict's solution reacts with electrons from the aldehyde or ketone group of the reducing sugar to form cuprous oxide (Cu 2 O), Green, yellowish orange or red ppt. CuSO 4 Cu ++ + SO Cu ++ + Reducing Sugar (electron donor) Cu + Cu + Cu 2 O (precipitate) Procedure & observation: - To 2 ml of carbohydrate solution in a test tube, add 1 ml of Benedict reagent. - Mix well, and warm it up for 1 min - Observe Green, yellowish orange or red ppt.
17 Few drops of B Mix Mix Heat 2-3 min Few ml of H 2 O g of A C Unknown A Mono- & Di- Saccharides Reagent B Benedict reagent [Na 2 CO 3 + Na.citrate + CuSO 4.5H 2 0] Result Observation C Green, yellowish orange or red ppt Mono- (Fruc, Gluc, Galac) C Green, yellowish orange or red ppt Di- (Lact, Malt) C No reaction Only with Sucrose (not reducing sugar)
18 Mild Oxidation: Fehling s reduction test: Sugar solutions + Fehling s A (CuSO 4 ) + Fehling s B (NaOH, NaK tartarate rochell salt), heat on water bath Red Precipitate RCHO + Cu ++ RCOOH + Cu 2 O Procedure & observation: - To 2 ml of carbohydrate solution in a test tube, add 1 ml of Fehling reagent. - Mix well, and warm it up for 2-3 min - Observe red colour precipitate.
19 Few drops of B Mix Mix Heat 2-3 min Few ml of H 2 O g of A C Result Unknown A Mono- & Di- Saccharides Reagent B Fehling reagent [CuSO 4 + NaOH] Observation C Red ppt Mono- (Fruc, Gluc, Galac) C Red ppt Di- (Lact, Malt) C No reaction Only with Sucrose (not reducing sugar)
20 Polysaccharidesaccharidesaccharides Di- Mono- Starch Maltose Glucose Cellulose Lactose Fructose Glycogene Sucrose Galactose Mannose
21 Scheme for qualitative determination of sugars +ve -Starch -ve -Sucrose Iodine s test Confirmed by Benedict s test &Fehling s after hydrolysis Unknown sugar solution Benedict s test +ve Reducing sugars -Monosaccharides (glu. & fru.) -Disaccharides(lact.) Barfoed s test +ve -Reducing Monosaccharides -ve - Reducing disaccharides - (glu. & fru.) (lactose) Seliwanoff s test +ve fructose -ve glucose
22 Carbohydrates Procedure 1- Molisch's test Sample solution (1 ml) + 1 ml Molisch reagent (Ethanolic α- Naphthol) + Conc. H 2 S0 4 on the side of test tube (1-2) ml 2- Iodine test -Sample solution (1 ml) + Iodine reagent (1drop) and shake -Take small amount and Boil the solution (2 min) -Cool the solution 3- Barfoed's test Sample solution (1 ml) + Barfoed's reagent (1 ml) Boil for (5-7) min and cool it 4- Seliwanoff's test Sample solution (1 ml) + Seliwanoff''s reagent (1 ml), Boil in a water bath for (3-5) min 5- Benedict test Sample solution (1 ml) + Benedict's reagent (1 ml) Boil in a water bath for (1-2) min 6- Tollen s test Sample solution (0.5 ml) + Ammoniacal AgNO 3 (1 ml) Boil in a water-bath for (1-2) min. 7- Fehling test Sample solution (1 ml), Fehling reagent [Fehling's solution A (5 drops) + Fehling's solution B (5 drops)] Mix then Boil for (1-2) min then Cool Observation Formation of violet colour ring at the junction of two layers -Intense blue colouration -Colour disappears -Colour reappears Red-brick precipitate along the side and at the bottom of test tube Red color is formed immediately (presence of Ketone group) Green, yellowish orange or red precipitate A silver mirror is formed on the inner wall of the tube or red ppt Yellowish-red ppt Unknown Any Carbohydrate Starch Mono-Saccharides (fast) Disaccharides (slow) Mono- (Fructose) Di- (Sucrose) Reducing sugars Glucose, Frudtose, Galactose, Lactose, Maltose Reducing sugars Glucose, Frudtose, Galactose, Lactose, Maltose Reducing sugars Glucose, Frudtose, Galactose, Lactose, Maltose
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