sdls 2008 biochemistry laboratory - carbohydrates

DetoxicolSDLS 2008

Medicine for the intoxicated

File Created on 10/04/2004 08:08 PM Topic: Biochemistry Laboratory CarbohydratesTranscriber(s): JC Tayco Lecturer: Prof. Patricia Generoso, Dr. Esperanza UyEditors: JC Tayco, Mark Lomboy, Pre Ausan No. of pages: 4

Carbohydrates→ are ployhydric alcohols having an aldehyde (aldoses) or a ketone

(ketoses) group→ glucose is the most common monosaccharide in mammalian

metabolism→ glucose is the major source of energy for various cells→ oxidation of carbohydrates is the central energy yielding pathway

in most non-photosynthetic cells→ in the diet, carbohydrates are taken up as starch

Melliturias→ condition in which abnormal amounts 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 to another monosaccharide

GENERAL TESTS FOR CARBOHYDRATES MOLISCH’S TEST (Alpha – Naphthol Reaction)

→ a general test for carbohydrates in free or combined forms→ concentrated sulfuric acid hydrolyzes bound sugars (breaking of

glycosidic bonds)→ dehydrates the monosaccharides to form furfural (if a pentose) or

hydroxymethyl furfural (if a hexose)→ the furfural derivatives reacts with α- naphthol→ reactions:

→ 1 mole of sugar : 1 mole of α- naphthol→ starch and glycogen will give a positive result because the sulfuric

acid is still capable of breaking down the glycosidic bonds→ Procedure:

1 mL test solution + 2 drops molisch’s reagent + 1 mL concentrated H2SO4

→ nice to know: Sulfuric acid removes 3 molecules of water Sulfuric acid breaks glycosidic bonds and acts as

dehydrating agent

ANTHRONE TEST→ another general test for carbohydrates→ very sensitive test

→ concentrated sulfuric acid hydrolyzes bound sugars (breaking of glycosidic bonds)

→ dehydrates the monosaccharides to form furfural (if a pentose) or hydroxymethyl furfural (if a hexose)

→ the furfural derivatives reacts with anthrone→ 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

TEST FOR REDUCING SUGARS→ dependent on the presence of aldehyde or ketone groups→ sugars have reducing properties due to the presence of an

aldehyde group or a ketone group→ only free aldehyde or ketone group 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:

→ 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

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: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

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:

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→ 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 5-hydroxymethyl 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:

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:

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→ Interferences:→ False positive:

Contamination by oxidizing agents and detergents

→ False negative High levels of ascorbic acid High levels of ketones High specific gravity Low temperature Improperly preserved specimens

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 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 (Tes-tape) 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.

SummarySchematic diagram for the determination of unknown sugars:

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Tests Reagents Used Principle Involved Visible Results DetectedMolisch’s Test Molisch’s reagent Dehydration with Purple ring at the junction All Carbohydrates

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(α – naphthol reaction) (5% α – naphthol in 95 % Alcohol)

subsequent condensation

Anthrone Test Anthrone solution(0.2% Anthrone in concentrated H2SO4)

Dehydration with subsequent condensation

Green or blue green color All Carbohydrates

Benedict’s Test Benedict’s solution(cupric carbonate, sodium carbonate, citrate)

Reduction of sugars(Oxidation Reduction Reactions)

Greenish precipitate (+) Yellowish brown

precipitate (++) Brownish red

precipitate (+++) Brick red precipitate (++

++)

Reducing sugars(both monosaccharides and disaccharides)

*sucrose is not a reducing sugar

Barfoed’s Test Barfoed’s reagent(copper acetate and glacial acetic acid)

Reduction of sugars(Oxidation Reduction Reactions)

Brick red precipitate Reducing sugars(monosaccharides ONLY!)

Tauber’s Benzidine Test Tauber’s reagent(4% benzidine dihydrochloride in glacial acetic acid)

Cherry red color Pentoses

Bial’s Orcinol Test Bial’s reagent(0.3% orcinol and 0.02%FeCl3 in 10N HCl)

Dehydration with subsequent condensation

Blue green

* brown to green-brown for hexoses* hexuronic acid gives positive result due to conditions of the test

Pentoses

Tollen’s Phloroglucinol Test

Concentrated HClTollen’s reagent(2% Phloroglucinol in 95% Alcohol)

Dehydration with subsequent condensation

Red color Pentoses(ketohexoses has faster reaction than aldohexoses)GalactoseGlucoronic acid

Seliwanoff’s Test Seliwanoff’s reagent(0.05% resorcinol in 12% HCl)

Dehydration with subsequent condensation

Red color(sometimes there may be brown red precipitate)

Ketoses

Tes Tape Tes tape Oxidation Changes into green after 30 seconds

Glucose

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