QUALITATIVE TESTS FOR CARBOHYDRATES Flashcards
BENEDICT’S TEST, BARFOED’S TEST, SELIWANOFF’S TEST, BIAL’S ORCINOL TEST
QUALITATIVE TESTS FOR
CARBOHYDRATES
are polyhydroxyaldehydes (aldoses) & polyhyroxyketones(ketoses)
General Formula: (CH20)n
Carbohydrates
based on the number of monosaccharide units they contain
Classification
one sugar molecule. Ex. Glucose, Fructose, Galactose
Monosaccharides
two sugar molecule. Ex. Sucrose, Lactose, Maltose
Disaccharides
two to ten sugar mol ex. Raffinose, Stachynose
Oligosaccharides
ten or more sugar molecules ex. Starch, glycogen, cellulose
Polysaccharides
simplest sugar and cannot be hydrolyzed further.
Classification is by the number of carbon atoms they contain
Monosaccharide
- Pentoses: five carbon atoms
- Aldopentoses: ribose & Xylose
- Hexoses: six carbon atoms
- Aldohexoses: glucose, galactose
- Ketohexose: fructose
On hydrolysis, disaccharides yield two monosaccharide units.
Classification is by the number of carbon atoms they contain
Dehydrating acids (2-step analysis)
Copper (Il) ions containing solutions
Two general classes of carbohydrate test reagents based on the type of reaction involved
- Converts pentoses into furfural and hexoses into 5-hydroxymethylfurfural which then reacts with phenolic compounds
- Production of highly colored products
- Molisch’s, Anthrone, Bial’s & Seliwanoff’s tests
Dehydrating acids (2-step analysis)
- CHO reduces copper (Il) ions into copper (1) oxide
- Reducing sugars include aldoses containing either a free aldehyde group or a cyclic hemiacetal
Copper (Il) ions containing solutions
- To determine the presence or absence of reducing sugar in the solution.
- All monosaccharides are reducing sugars
- A reducing sugar has a free aldehyde group or a free ketone group
Benedict’s Test
Principle of the Test
Benedict’s reagent contains copper (Il) ions in an alkaline solution with sodium citrate to keep the cupric ions in solution. Test is performed by heating the reducing sugar solution with Benedict’s reagent.
Alkaline condition of this test causes isomeric transformation of ketoses to aldoses resulting in the reduction of blue cupric ion to cuprous oxide
Benedict’s Test
- presence of reducing sugar with either ketone or aldehyde, positive color brick-red or brick red precipitate caused by reducing property of simple sugar
Alkaline reagent
Copper sulfate in benedict solutions are reduced to copper ions
Cupric to cuprous ions
Composition: Sodium carbonate 10g, sodium citrate 17.3g, distilled water 100ml, copper sulfate pentahydrate 1.73 g
Copper sulfate act as copper 2+ (Cu ^2+) ions
Sodium carbonate as an alkaline medium
Sodium citrate: forms complexes to Cu 2+ ions
Test tube
Test tube holder
Pippete
Urine sample
Burner
Beaker
2 ml of sample urine + 3mL of benedicts reagent mix heat
Brick red large amount of reducing sugar, >2 percent
Red - brick red
Blue indicate negative, 0 percent
Green has trace of reducing sugar, variable color, 0.5-1 percent
Yellow, is low 1.1-5 percent or orange, 1.5 - 2 percent moderate amount of reducing sugar
Benedict’s test
- check if its monosaccharide or disaccharide
Copper ions to copper oxide
Reducing sugar: aldehyde or ketones
Monosaccharides- reacts fast, all are reducing sugars ex. Lactose and maltose non reducing sucrose
Disaccharide - reacts slowly
Reagent: copper sulfate, acetic acid: provide acidic condition that would decrease pH
Monosaccharide + barfoed results to brick red or red or brown precipitate within 5 mins
Disaccharide + barfoed appear to precipitate within 10 mins brick red
What’s positive reaction for barfoed brick red or brown precipitate
4 test tube 1 percent per tube of glucose, lactose, fructose, negative control or distilled water lagay barfoed 2ml
Drum test tube in water butt or beaker
Barfoed’s test
distinguish if ketose or aldose
Ketose - contain ketone group
Ketose from aldose sugars
Aldose example glyceraldehyde, erythrose, ribose, arabinose, xylose, glucose, mannose, galactose
Ketose ex. dihydroxyacetone, erythrulose, ribulose, fructose, and sedoheptulose
Rapid state of hydration than aldose
Seliwanoffs reagent - orcinal and hydrochloric acid
Aldose - light pink - negative
Ketose - cherry red - positive
Seliwanoff’s test
check if pentose or hexose
Bial’s orcinal
Ribose and xylose - pentose
Blue - Green Positive color
Any color besides green negative
5 percent fructose, ribose, sucrose, glucose
2 ml bials reagent
Bial’s Orcinol test
- Benedict’s reagent
- Barfoed’s reagent
- Seliwanoff’s reagent
- Orcinol reagent
- Carbohydrate solutions (0.1 M glucose, fructose, xylose, galactose, lactose, sucrose and 1% starch)
- Beaker
- Alcohol lamp
- Hot plate
- Distilled Water
Benedict’s Test, Barfoed’s,
Seliwanoff’s and Bial’s Orcinol tests
REAGENTS & MATERIALS
Test Objective
* Barfoed’s test is a chemical test used to detect the presence of reducing monosaccharides.
* To distinguish reducing monosaccharides from disaccharides
Barfoed’s test
Principle of the Test
The Barfoed reagent is made up of copper acetate (cupric ions) in a dilute solution of acetic acid. (acidic medium)
Reducing Monosaccharides are strong reducing agent: React within 3 minutes
Reducing Disaccharides have to first to get hydrolyzed in the acidic solution and then react with the reagent: React in about >3 minutes
Barfoed’s test
Interpretation of Result
Positive Result: Brick red precipitate at the bottom of the tube.
Monosaccharide: ≤ 3 minutes
Disaccharides: >3 minutes
The difference in the time of appearance of precipitate thus helps distinguish reducing monosaccharides from reducing disaccharides.
Barfoed’s test
Test Objective
* To detect the presence of ketohexoses in a given sample.
* To distinguish ketoses from aldoses.
Sellwanoff’s Test
An aldose contains one aldehyde group per molecule.
A ketose contains one ketone group per molecule
In Seliwanoff’s test, Aldoses react slowly and produce a light pink colour.
In Seliwanoff’s test, Ketoses react with resorcinol to give a deep cherry-red colour.
ALDOSE VS KETOSE
Principle of the Test
* The reagent of this test consists of resorcinol in 6M HCI.
* The acid hydrolysis of polysaccharides and oligosaccharides yields simpler sugars.
* Ketoses are more rapidly dehydrated than aldoses.
* Ketoses undergo dehydration in the presence of concentrated acid to yield hydroxymethyl furfural that condense with resorcinol
Seliwanoff’s Test
Interpretation of Result
* Ketose reacts to produce a deep cherry red color
* Aldoses may react slightly to produce a faint pink to cherry red color if the test is prolonged.
* The product and reaction time of the oxidation reaction helps to distinguish between carbohydrates.
* Sucrose and inulin also give a positive result for this test as these are hydrolyzed by acid to give tructose.
Seliwanoff’s test
Test Objective
In chemistry, a pentose is a monosaccharide (simple sugar with five carbon atoms. The chemical formula of all pentoses is C5H100.
* To detect the presence of carbohydrates.
* To distinguish the pentoses and pentosans from other derivatives of carbohydrates like the hexoses.
Bial’s Orcinol Test
- This test is based on the principle that under hydrolysis pentosans are hydrolyzed into pentoses.
- Further, pentoses are dehydrated to yield furfural, which in turn condense with orcinol to form a blue-green precipitate. In the presence of hexoses, hydroxyfurfural is formed instead of furfural which upon condensation with orcinol forms a muddy brown colored precipitate
- The intensity of the precipitation is directly proportional to the concentration of the pentoses in the sample.
- The intensity of the color developed depends on the concentration of HCl, ferric chloride, orcinol, and the duration of boiling.
Bial’s Orcinol Test
Interpretation of Result
• The presence of a blue-green complex indicates the presence of pentoses in the sample.
Bial’s Orcinol Test
