Biochem Postlab - Carbohydrates PDF

BIOCHEMISTRY] POST-LABORATORY: CARBOHYDRATES CARBOHYDRATES All carbohydrates and glycoproteins will...

BIOCHEMISTRY] POST-LABORATORY: CARBOHYDRATES CARBOHYDRATES All carbohydrates and glycoproteins will Organic compounds containing hydrogen and Molisch's Test give a positive test with Molisch and oxygen with the ratio the same as in water, thus Anthrone, except triose and tetrose sugars called hydrates of carbon or carbohydrates Alcoholic alpha-naphthol Reagent/s Concentrated sulfuric acid o The definition is not perfect because there TEST FOR KETOSES are compound that comply with the Violet or purple colored ring at the point definitions but are not carbohydrate of junction between the carbohydrate Seliwanoff's Test solution and the acid ▪ Example: Acetic acid (CH3COOH) Resorcinol Carbohydrates are aldehyde or ketone Result Reagent/s Concentrated hydrochloric acid derivatives of polyhydric alcohol. A cherry red colored complex GENERAL TEST FOR CARBOHYDRATES Principle: Carbohydrates when treated with Result concentrated mineral acids undergo Reagents: Alcoholic alpha naphthol and dehydration to give furfural derivatives. concentrated sulfuric acid is allowed to flow along the side of the test tubes containing the The presence of carbohydrates in a carbohydrate. preparation can be detected by either Molisch Principle: Used to distinguish aldose from or Anthrone Test. Anthrone's Test ketose sugar. Ketose is more reactive than o Both tests are based on the hydrolyzing aldose and dehydrating action of concentrated Anthrone Reagent/s o Heating should not exceed 3 minutes. mineral acids Concentrated sulfuric acid Avoid overheating. On prolonged heating Polysaccharides: A blue green colored complex aldose sugar will give a positive result o first undergo hydrolysis of any glycosidic bonds present in the sample TEST FOR REDUCING PROPERTY OF o then the dehydration of furfural (from SUGARS pentoses) or hydromethyl furfurals Result (from hexoses) occurs. Reagents: Solution of metallic salts, usually in an alkaline medium mixed with an organic ▪ Both processes are catalyzed by the compound containing alcoholic groups concentrated mineral acid. These furfurals then condense with o The salt of copper, mercury, bismuth, and organic phenols to form colored A very sensitive test; gives a positive reaction silver has been used products with filter paper (cellulose) For quantitative determination of glycogen, Additional Reagents: Sodium potassium inulin, and blood sugar in the laboratory tartrate (Rochelle salt), sodium citrate or glycerol containing one or more hydroxyl V. ANGELS NMD - 1 1  BIOCHEM POSTLAB - CARBOHYDRATES Sugars with free aldehyde and ketone group B. Reducing Ketose Sugars are reducing sugars COLOR OF RESULT No. of Reducing Sugars When solution of such sugars is heated in the Apple green Traces presence of certain metallic ions, the active Yellow Plus 1 group is oxidized and the metallic ions are reduced to a lower valence state Orange Plus 2 Sugars containing free aldehyde or ketone Red Plus 3 group or potentially free reducing group (that is: hemiacetal or hemiketal carbons) are Cuprous hydroxide Cu(OH)2 forms a soluble subject to oxidation by a variety of general complex with the alcoholic group of sodium oxidizing agents C. Reducing Disaccharides citrate Principle: Reducing sugars under alkaline o Sugars lacking such groups (Ex: those condition undergo tautomerization and form containing only glycosidic linkage like enediol. Enediols are powerful reducing sucrose) are resistant to these changes agents. They reduce cupric ions to cuprous Initial attack of general oxidizing agents form. Cuprous ions combine with –OH forming (Cupric, Bismuth, Silver, Ferricyanide, and yellow cuprous hydroxide, which upon aromatic nitro compounds) occurs at the D. Non-Reducing Disaccharide heating is converted to red cuprous oxide. reducing group of the sugar (hence, the term Benedict's solution is not readily reducible by reducing groups) known -carbohydrate reducing substances such as uric acid, creatine, conjugated o After, subsequent oxidation and glucuronate, and other products occurring isomerization may continue down the normally in the urine carbon chain yielding a large number of products with low molecular weight Barfoed’s Test All monosaccharides and disaccharides are reducing sugars except sucrose Benedict's Test Polysaccharides with four or more Copper acetate Reagent/s monosaccharide units are non-reducing Copper sulfate Acetic acid Sodium hydroxide Scanty red precipitate (cuprous Reagent/s Sodium nitrate oxide) is observed with Reducing Sugars (**except for sucrose) Anhydrous sodium carbonate (from monosaccharides Postlab notes) A. Reducing Aldose Sugars Depends on concentration of reducing sugars Result Result Reagents: Copper acetate and acetic acid (**From Postlab notes: Copper sulfate and acetic acid) V. ANGELS NMD - 1 2  BIOCHEM POSTLAB - CARBOHYDRATES Principle: Sugars are less reaction in acidic Principle: Reaction is accompanied by the OXIDATION OF CARBOHYDRATES than in alkaline medium. In acidic medium, reduction of silver ions into metallic ions monosaccharides are more reactive than deposited as a silver mirror on the walls of Aldonic Acids the test tube Sodium tartrate Aldonic Acids – Aldehyde or ketone group Reagent/s Sodium hydroxide Picric Acid Test oxidized to carboxylic acid Bismuth subnitrate Result Black metallic bismuth Saturated picric acid Reagent/s 10% sodium carbonate disaccharides. This test is used in distinguishing monosaccharides from disaccharides by Result Orange/red solution controlling pH and time of heating Prolong heating may give positive reaction with Principle: Reducing sugar when boiled with disaccharides sodium carbonate will reduce the nitrophenol Copper reducing test but different from picric acid to aminophenol, resulting to Benedict's because reduction of metallic picramic acid, converting the yellow solution occurs in an acidic medium to orange (**From Postlab notes: Result is colored red) Uronic Acids Nylander's Test Piccric acid is also called trinitrophenol Uronic Acids – Primary alcohol oxidized to Creatine will also give positive reaction to the carboxylic acid Reagents: Sodium tartrate, Sodium hydroxide test and Bismuth subnitrate (**From Postlab notes: Bismuth nitrate, potassium sodium tartrate and Moore's Test ammonium) Principle: When bismuth subnitrate comes in contact with reducing sugar in a boiling Reagent/s Concentrated sodium hydroxide alkaline solution it reduces to black metallic bismuth Formation of a substance called Result caramel; varies in yellow, orange Tollen’s Test and brown color Principle: Reducing sugars undergo oxidation in alkaline solution. When solution of reducing Aldaric Acid Aqueous silver nitrate Reagent/s sugar is boiled with an alkali, it undergoes Sodium hydroxide ammonia Aldaric Acid – both aldehyde or ketone group tautomerization and produces a resinous Formation of silver mirror substance called caramel (brown color) and the primary alcohol are oxidized to carboxylic acid Result Reagents: Aqueous silver nitrate, sodium Oxidation of most monosaccharides by nitric hydroxide ammonia (**From Postlab notes: acid yield soluble dicarboxylic acid Ammoniacal solution of silver nitrate) However, oxidation of galactose yield an V. ANGELS NMD - 1 3  BIOCHEM POSTLAB - CARBOHYDRATES insoluble mucic acid phenylhydrazone, which however, are soluble Lactose upon hydrolysis will also yield a in water, and therefore has little use in the mucic acid due to cleavage of the glycosidic identification of sugars linkage between the glucose and galactose o Only the first two carbons of the subunits sugars with free aldehyde or ketone group will react with phenylhydrazine Mucic Acid Test o In the presence of excess phenylhydrazine, the first molecule of Concentrated HNO3 has a unique property phenylhydrazine will that it oxidizes two terminal carbons of react with the carbon containing the Osazone Structure Time formed aldohexoses, forming the corresponding aldehyde or ketone group to yield a dicarboxylic acids (saccharic acid) soluble hydrazone. The second Glucosazone Needle shaped 4 to 5 minutes molecule of phenylhydrazine will react or broomstick In general, saccharic acid are soluble in dilute nitric acid, but mucic acid, the particular with the adjacent carbon to yield a Galactosazone Rhombic 7 minutes crystalline osazone plates saccharic acid formed by galactose (either free or as released by hydrolysis) is insoluble in Maltosazone Sun flower dilute acids shaped Thus, the formation of an insoluble Lactose Powder puff or saccharic acid when concentrated nitric acid hedged hog is heated with a carbohydrate and Fructosazone 2 minutes subsequently diluted signifies the presence of Sucrose 30-35 minutes galactose and hydrolyzed lactose (after being hydrolyzed) Phenylhydrazine Test Phenylhydrazine hydrochloride Reagent/s Glacial acetic acid Osazones Sodium acetate Valuable compounds for identification of Result Formation of osazones sugars for the ff. reasons: Reagents: Phenylhydrazine hydrochloride, o The time formation of each osazone is glacial acetic acid, sodium acetate quite definite Principle: Solution of reducing sugars with o Some osazones are relatively insoluble phenylhydrazine hydrochloride, characteristic o Osazone crystallizes in specific forms o Each osazone, if carefully purified by o Solubility of Osazone yellow crystalline compounds called osazone are formed. These crystals have definite recrystallization and dried, has definite ▪ Both lactosazone and maltosazone crystalline structures, precipitation time melting time are soluble in hot water and melting point for different reducing Osazone Structure ▪ Osazone have characteristic sugars (**Postlab notes cuts it here) o Glucose, fructose and mannose will yield Phenylhydrazine reaction: similar osazone Most sugars have a free carbonyl group which can react with phenylhydrazine to form V. ANGELS NMD - 1 4  BIOCHEM POSTLAB - CARBOHYDRATES Interconversion of Sugars ▪ Principle: Iodine forms a coordinate o Results complex between helically coiled Aldoses in the presence of a weak alkali may polysaccharide chain and iodine ▪ Iodine test – Colorless be converted into ketoses, or other aldoses centrally located within the helix due ▪ Yields glucose or vice versa to its absorption ▪ Benedict's test – positive Hence, starting with either glucose, mannose ▪ Phenylhydrazine test – glucosazone or fructose, after interconversion at equilibrium, crystals Iodine Test Reaction there will be presence of equal amounts of the Amylose Deep blue complex three sugars Amylopectin Purple complex Hexose Sugars Glycogen Reddish brown complex Before After Erythrodextrin Red complex Inversion inversion Achrodextrin Colorless Glucose + Yellow Orange Ba(OH)2 B. Starch Hydrolysis Glucose + Yellow Yellow Water Fructose + Red Orange Ba(OH)2 Fructose + Red Red Tip: Use pattern of hydroxyl group to identify Water sugar by its structure. Inversion of Sucrose C. After Hydrolysis Invert sugar – is a solution of equimolar o Iodine test – Colorless mixture of glucose and fructose resulting from o Benedict's test – positive the hydrolysis of sucrose catalyzed by the o Phenylhydrazine test – glucosazone enzyme invertase or by an acid and heat Glycogen The storage form of carbohydrate in animals Stored mainly in the liver and the skeletal muscles A. Preparation of Glycogen from animal liver Polysaccharides B. Reactions of Glycogen o Iodine Test – Reddish brown iodine Starch complex o Benedict's Test – Negative Starch – storage form of carbohydrate in plants A. Reactions of Starch C. Hydrolysis of Glycogen o Benedict's Test – negative o Attained after boiling o Iodine test – blue color o Results similar to starch V. ANGELS NMD - 1 5