Biological molecules = Biomolecules = Compounds of life
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2 Biological molecules = Biomolecules = Compounds of life Carbohydrates Proteins & Amino Acids Monosaccharides Oligosaccharides Disaccharides Polysaccharides Lipids Oils & Fats Amino acids Proteins Enzymes Vitamins Ascorbic acid (Vit-C) Fatty acids Cholesterol
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 Monosaccharides Di-saccharides Sucrose = Glucose + Fructose Maltose = Glucose + Glucose Lactose = Glucose + Galactose Polysaccharides Starch
6 Polysaccharidesaccharidesaccharides Di- Mono- Starch Maltose Glucose Cellulose Lactose Fructose Glycogen 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 2 drops of Molisch s reagent [1% α-naphthol in absolute alcohol]. well; add carefully at the side wall of the test tube 2 ml conc. H 2 SO 4 without mixing. - Reddish violet ring will be formed at the interface of the two solutions
9 2-3 drops of B 2 ml of H 2 SO 4 On the side wall Carbohydrate Sugar solution C H 2 SO 4 Unknown A Any Carbohydrate Reagent B Molisch s reagent [Ethanolic α- Naphthol] then add conc H 2 SO 4 at the side wall of the test tube Result Observation C ReddishViolet ring at the interface Any Carbohydrate C No change Non carbohydrate
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 colored complex. A positive iodine assay indicates the presence of starch, so if the color turns dark blue, this is a positive result. Procedure & observation: - To 2 ml of carbohydrate solution in a test tube, add 1-2 drops of Iodine solution - well, a deep blue colour appears. -Warm up, the colour disappears. (why?) - Cool down, the colour re-appears
11 Few drops of B 2ml sugar solution 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 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. It is used to differentiate between non-reducing (e.g. 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), of yellowish orange or red ppt. CuSO 4 Cu ++ + SO Cu ++ + Reducing Sugar (electron donor) Cu + (CuOH) Cu + Cu 2 O (precipitate) Procedure & observation: - To 2 ml of carbohydrate solution in a test tube, add 1 ml of Benedict reagent. - well, and boil it up for 2 min - Observe yellowish orange or red ppt.
13 Few drops of B boil 2 min 2ml sugar solution C Unknown A Mono- & Di- Saccharides Reagent B Benedict reagent [Na 2 CO 3 + Na.citrate + CuSO 4.5H 2 0] Result Observation C yellowish orange or red ppt Mono- (Fruc, Gluc, Galac) C yellowish orange or red ppt Di- (Lact, Malt) C No reaction Sucrose (not reducing sugar)
14 1ml of sugar solution Boil 2 min 1ml Fehling A + 1ml Fehling B C Result Unknown A Mono- & Di- Saccharides Reagent B Fehling reagent [CuSO 4 + NaOH] Observation C Orange or Red ppt Mono- (Fruc, Gluc, Galac) C Orange or Red ppt Di- (Lact, Malt) C No reaction with Sucrose (not reducing sugar)
15 Barfoed s test Principle: Barfoed test is Specific to Mono-saccharides To differentiate between Monosaccharides (+ve) and Disaccharides (-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 disaccharides 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 s reagent - well, and boil it up for 5 min - Observe red-brick ppt or a slight change in colour from blue to red/violet
16 1ml of B boil 5 min only 2 ml of sugar solution (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)
17 Seliwanoff s test Principle: Seliwanoff test is Specific to Keto sugars (Fructose or fructose-containing carbohydrates such as Sucrose). To differentiate between aldehydic and ketonic saccharides. When a ketose is heated with a strong mineral acid, hydroxymethylfurfural is formed. This compound (Sucrose or Fructose) forms a red complex with the organic compound, resorcinol. The Ketonic saccharides are more reactive than Aldehyde group of Aldoses 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 s reagent. - well, and boil it up for 1 min only. - Observe red colour.
18 1 ml of B boil < 1 min 2 ml of sugar solution (A) C Result Unknown A Mono- & Di- Saccharides Reagent B Seliwanoff reagent [Resorcinol + HCl] Observation C Red colour in 1min Mono- (Only Fructose due to keto gp) C Red colour in 1min Di- (Only Sucrose due to keto gp) C No reaction Mono- and Di- (No keto gps )
19 Polysaccharidesaccharidesaccharides Di- Mono- Starch Maltose Glucose Cellulose Lactose Fructose Glycogen Sucrose Galactose Mannose
20 Scheme for qualitative determination of sugars +ve -Starch -ve -Sucrose Iodine s test Unknown sugar solution Benedict s test +ve Reducing sugars -Monosaccharides (glu. & fru.) Confirmed by Benedict s test -Disaccharides(lact.) &Fehling s after hydrolysis.). Barfoed s test +ve -Reducing Monosaccharides - Reducing disaccharides - (glu. & fru.) (lact.) Seliwanoff s test -ve +ve fru. -ve lact.
21 Carbohydrates Procedure 1- Molisch's test Sample solution (2 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 (2 ml) + Iodine reagent (1-2 drops) and shake -Take small amount and Boil the solution (2 min) Cool the solution 3- Barfoed's test Sample solution (2 ml) + Barfoed's reagent (1 ml) Boil for (5) min and cool it 4- Seliwanoff's test Sample solution (2 ml) + Seliwanoff''s reagent (1 ml), Boil in a water bath for (1) min 5- Benedict test Sample solution (2 ml) + Benedict's reagent (1 ml) Boil in a water bath for (1-2) min 6- Tollen s test Sample solution (2 ml) + Ammoniacal AgNO 3 (1 ml) Boil in a water-bath for (1-2) min. 7- Fehling test Sample solution (2 ml) + Fehling reagent [Fehling's solution A (1 ml) + Fehling's solution B (1ml)] 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 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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