Detoxicol SDLS 2008 Medicine for the intoxicated File Created on 10/04/2004 08:08 PM Transcriber(s): JC Tayco Editors: JC Tayco, Mark Lomboy, Pre Ausan
Topic: Biochemistry Laboratory Carbohydrates Lecturer: Prof. Patricia Generoso, Dr. Esperanza Uy No. of pages: 4
→ starch and glycogen will give a positive result becau bec ause se the su sulfu lfuric ric ac acid id is sti still ll ca capab pable le of breaking down the glycosidic bonds → Procedure: 1 mL test so sollut utio ion n + 2 dr drop ops s mo mollis isch ch’s ’s reagent + 1 mL concentrated H 2SO4 → nice to know: Sulfuric acid removes 3 molecules of water Sulfuric uric acid breaks glyc glycosid osidic ic bonds Sulf and acts as dehydrating agent
Carbohydrates → ar are e plo ployhy yhydr dric ic alc alcoho ohols ls hav having ing an al aldeh dehyde yde (aldoses) or a ketone ketone (ketoses) (ketoses) group → glucose is the most common monosaccharide in mammalian metabolism → gl gluc ucos ose e is th the e ma majo jorr so sour urce ce of en ener ergy gy fo forr various cells → ox oxid idat atio ion n of ca carb rboh ohyd ydra rate tes s is th the e ce cent ntra rall ener en erg gy yiel eld din ing g path thwa way y in mo mos st non on-photosynthetic cells → in th the e di diet et,, ca carb rboh ohyd ydra rate tes s ar are e ta take ken n up as starch Melliturias → co cond ndit itio ion n in wh whic ich h ab abno norm rmal al am amou ount nts s of specific sugars are excreted in the urine → the most common type is glucosuria in which D-glucose is excreted in large amounts in the urine * in Diabetes mellitus → glucosuria is evident → other sugars excreted include: Fructose Lactose Galactose Arabinose (a pentose) Glycosidic bonds → links a monosaccharide monosaccharide
to
another
GENERAL TESTS FOR CARBOHYDRATES MOLISCH’S T E ST (Alpha – Naphthol Reaction) → a ge gene nera rall te test st fo forr ca carb rboh ohyd ydra rate tes s in fr free ee or combined forms → conce concentra ntrated ted sulf sulfuric uric acid hydrolyzes hydrolyzes bound sugars (breaking of glycosidic bonds) → de dehy hydr drat ates es th the e mo mono nosa sacc ccha hari ride des s to fo form rm furf fu rfur ural al (i (iff a pe pent ntos ose) e) or hy hydr drox oxym ymet ethy hyll furfural (if a hexose) → the furfural derivatives reacts reacts with α- naphthol → reactions:
→ used in quantitative colorimetric determination of glycogen, insulin and blood sugar → 1 mole of anthrone is needed to react with 1 mole of sugar in the furfural form to give the color compound → remember that to form a furfural ring, at least 5 carbons are needed → Procedure: 1 mL anthrone solution + 3 drops test solution
→ 1 mole of sugar : 1 mole of α- naphthol
ANTHRONE TEST → another general test for carbohydrate carbohydrates s → very sensitive test → conce concentra ntrated ted sulf sulfuric uric acid hydrolyzes hydrolyzes bound sugars (breaking of glycosidic bonds) → de dehy hydr drat ates es th the e mo mono nosa sacc ccha hari ride des s to fo form rm furf fu rfur ural al (i (iff a pe pent ntos ose) e) or hy hydr drox oxym ymet ethy hyll furfural (if a hexose) → the furfural derivatives reacts reacts with anthrone → reactions:
TEST FOR REDUCING SUGARS → dep depend endent ent on the presence presence of ald aldehy ehyde de or ketone groups → sug sugar ars s hav have e red reduci ucing ng pro proper pertie ties s due to the presence of an aldehyde group or a ketone group → on only ly fr free ee al alde dehy hyde de or ke keto tone ne gr grou oup p is capable of reducing cupric ions into cuprous oxide BENEDICT’S TEST
→ under alkaline medium where sugars are more reactive → Benedict’s reagent is a solution of Cu(OH) 2, is stabilized by forming a soluble complex with the alcohol group of sodium citrate in an alkaline medium of sodium carbonate → this test is not specific for sugar → active component is cupric ion (Cu ++) → for qualitative and quantitative analysis of sugar in blood and urine → all monosaccharides are reducing sugars → disaccharides are reducing sugars except for sucrose
→ remember: Reactive part of aldoses like glucose is carbon number 1 Reactive part of ketoses like fructose is carbon number 2 → Procedure: 1 mL Benedict’s solution + 8 drops of test solution then boil in water bath → cupric ion is reduced and the resulting cuprous ion is less soluble and cuprous oxide precipitates as a brick red solid → reaction:
1 mL Barfoed’s reagent + 6 drops of test solution + boil in water for 3 minutes then cool → reaction:
TEST FOR PENTOSES → positive result only for pentose thus eliminates the possibility that the sugar is hexose → pentoses are monosaccharides containing 5 carbon atoms → prolonged heating with mineral acids will yield furfural → complex carbohydrates in the form of pentosamine, gums, xylans and arabans, wchich on hydrolysis yield pantoses
TAUBER’S BENZIDINE TEST → more specific for pentoses → can detect up to 0.01 mg of the pentose either in its free of combined form → Procedure: 0.5 mL Tauber’s reagent + 5 drops test solution, boil over open flame for 1-2 minutes
→ → → →
→ →
→ sucrose is a non-reducing disaccharide because the reducing groups in both glucose and fructose are involved in the linkage between the two sugar units
→
→
→
BIAL’S ORCINOL TEST based on the reaction with strong acid and phenol hexuronic acid is positive because they are decarboxylated and forms pentose pentose should not be bound in order to react with Bial’s reagent pentose should form a furfural ring to bind with orcinol to produce a blue colored product at C1 Bial’s reagent dehydrates the sugar complex carbohydrates in the form of pentosans, gums, xylans and arabans, wchich on hydrolysis yield pantoses involves the decomposition o the sugars when heated with concentrated HCl to a furfural which condenses with orcinol (3,5 dihydroxy toluene) Procedure: 1 mL Bial’s reagent + 1 mL test solution, boil until bubles come then dilute to 10 mL with water reaction:
BARFOED’S TEST → also a copper reduction test but it is carried out in acidic medium where sugar is less reactive → Barfoed’s solution consists of: copper acetate and glacial acetic acid → this test differentiates monosaccharides from disaccharides → in acidic medium, monosaccharides are much reactive than disaccharides → prolonged heating of disaccharides gives a positive result because they are already broken down into their monosaccharide components → Procedure:
→ →
→
→
TOLLEN’S PHLOROGLUCINOL TEST based on the formation of similar intermediate furfurals which condenses with phloroglucinol in the presence of resorcinol and an acid, ketohexoses form a red color in a much faster rate than aldohexoses sucrose, when heated longer also shows a red color since the prolonged heating hydrolyzes sucrose into glucose and fructose fructose is a ketohexose
2
→ galactose and glucoronic acid give positive Tollen’s test → Procedure: 1 mL test solution + 1 mL concentrated HCl + 2-3 drops Tollen’s reagent → reaction:
TEST FOR KETOSES SELIWANOFF’S TEST → no acid was used instead heat was applied → the formation of the furfural ring is due to heat → 2 moles of resorcinol : 1 mole of 5hydroxymethyl furfural → sucrose will give a positive result only if heated longer → the ketone group of the ketohexoses is the active part in the reaction (dehydration) → ketoses are dehydrated much faster than aldohexoses → Procedure: 1 mL of Seliwanoff’s reagent + 7 drops of test solution, then heat in water bath for 3 minutes → reaction:
→ Interferences: → False positive: Contamination by oxidizing agents detergents → False negative High levels of ascorbic acid High levels of ketones High specific gravity Low temperature Improperly preserved specimens
ENZYME TEST FOR GLUCOSE → quantitative and qualitative test → most specific test because of the use of an enzyme reagent (glucose oxidase) → glucose oxidase oxidizes glucose to gluconic acid and hydrogen peroxide → Determines the amount of glucose in urine → DOUBLE SEQUENTIAL ENZYME REACTION → Glucose oxidase catalyzes the reaction between glucose and room air to produce gluconic acid and peroxide → Peroxide catalyzes the reaction between peroxide and chromogen to form an oxidized colored compound representing the presence of glucose → Positive result (if the reagent strip is): → Potassium iodide: Green → brown → Tetramethylbenzidine: yellow → green → Reactions involved:
and
Tes Tape → paper that contains glucose oxidase COMBISTIX → with additional enzyme peroxidase to catalyze reaction between peroxide and potassium iodide chromogen → Oxidizing the chromogen to give a green to brown color → ascorbic acid and bilirubin gives false positive result because they will compete with chromogen Answers to Questions: Give the reason/rationale for each of the following: a. sucrose gives a negative result to Benedict’s test → Benedict’s test is used to detect reducing sugars, sucrose is a non-reducing disaccharide because the reducing groups in both glucose and fructose are involved in the linkage between the two sugar units. b. arabinose yields positive result to Orcinol Test → Orcinol Test is used to detect the presence of pentoses, arabinose is a pentose thus yields a positive Orcinol Test c. no brick red color is formed by lactose when heated with Barfoed’s Test → Barfoed’s test is used to detect the presence of reducing monosaccharides, lactose is a disaccharide thus will give a negative Barfoed’d test d. Seliwanoff’s Test is positive with the sugar fructose → Seliwanoff’s test is used to detect the presence of ketoses, fructose is a ketose thus will give a positive Seliwanoff’s test
Differentiate Benedict’s Test from Barfoed’s Test in terms of: reaction medium, and reactivity of the monosaccharides and disaccharides. → Benedict’s test is done in an alkaline medium thus both monosaccharides and disaccharides
3
are reactive. Barfoed’s test on the other hand, is done in an acidic medium. In acidic medium, sugars are less reactive but between monosaccharides and disaccharides, monosaccharides are more reactive.
Differentiate fructose and sucrose using Seliwanoff’s test. → Seliwanoff’s test is used to detect the presence of ketoses, fructose is a ketose thus will give a positive Seliwanoff’s test. Sucrose on the other hand would not give a positive Seliwanoff’s test but if sucrose is heated longer, HCl in Seliwanoff’s reagent will break down sucrose to glucose and fructose only then will it give a positive Seliwanoff’s test.
Why is Glucose Oxidase Test (Testape) a more specific test for urinary glucose than Benedict’s Test? Explain. → Benedict’s test is used to detect the presence of reducing sugars, glucose will give a positive Benedict’s test but the test is not specific for glucose since glucose is not the only reducing sugar. Glucose Oxidase Test is more specific for glucose because the enzyme oxidizes selectively glucose.
Summary Schematic diagram for the determination of unknown sugars:
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Tests
Reagents Used
Principle
Visible Results
Detected
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Molisch’s Test (α – naphthol reaction)
Molisch’s reagent (5% α – naphthol in 95 % Alcohol)
Anthrone Test
Anthrone solution (0.2% Anthrone in concentrated H2SO4) Benedict’s solution (cupric carbonate, sodium carbonate, citrate)
Benedict’s Test
Barfoed’s Test
Barfoed’s reagent (copper acetate and glacial acetic acid)
Involved Dehydration with subsequent condensation
Green or blue green color
All Carbohydrates
Reduction of sugars (Oxidation Reduction Reactions)
Greenish precipitate (+) Yellowish brown precipitate (++) Brownish red precipitate (++ +) Brick red precipitate (+++ +) Brick red precipitate
Reducing sugars (both monosaccharides and disaccharides)
Cherry red color
Pentoses
Blue green
Pentoses
Reduction of sugars (Oxidation Reduction Reactions)
Tauber’s reagent (4% benzidine dihydrochloride in glacial acetic acid)
Bial’s Orcinol Test
Bial’s reagent (0.3% orcinol and 0.02%FeCl3 in 10N HCl)
Dehydration with subsequent condensation
Concentrated HCl Tollen’s reagent (2% Phloroglucinol in 95% Alcohol)
Dehydration with subsequent condensation
Seliwanoff’s reagent (0.05% resorcinol in 12% HCl) Tes tape
Dehydration with subsequent condensation
Seliwanoff’s Test
Tes Tape
All Carbohydrates
Dehydration with subsequent condensation
Tauber’s Benzidine Test
Tollen’s Phloroglucinol Test
Purple ring at the junction
Oxidation
* brown to greenbrown for hexoses * hexuronic acid gives positive result due to conditions of the test Red color
*sucrose is not a reducing sugar
Reducing sugars (monosaccharides ONLY!)
Pentoses (ketohexoses has faster reaction than aldohexoses) Galactose Glucoronic acid
Red color Ketoses (sometimes there may be brown red precipitate) Changes into green Glucose after 30 seconds
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