Molisch's Test for Carbohydrates

Molisch's Test

• A group test for all carbohydrates that can be used to detect their presence
• Concentrated H2SO4 catalyses the dehydration of sugars
• Pentoses are dehydrated to form furfural
• Hexoses are dehydrated to form hydroxymethyl furfural
• Furfurals condense with sulfonated alpha-naphthol
• This forms a purple or violet colored product (furfuryl-diphenyl-methane-dyes)
• Positive reactions are shown for polysaccharides and glycoproteins
• Acid first hydrolyzes poly- or disaccharides into component monosaccharides
• Monosaccharides are then dehydrated to form furfural or derivatives

Importance of Molisch's Test

• Used as a general test for the presence of carbohydrates, superior to other tests
• General tests are important before specific carbohydrate tests
• Test is positive for all types of carbohydrates, including those in combined form like glycoproteins and glycolipids

Reagents

• Concentrated H2SO4 is required
• Molisch’s reagent consists of Alpha-naphthol 5%(w/v) in 95% ethanol

Procedure

• Take 1-2 mL of sample solution and add 2-3 drops of Molisch’s reagent (5% α-naphthol in 95% ethanol), then mix
• Incline the tube and carefully pour 1-2 mL of concentrated H2SO4 down the side
• This allows the acid to form a layer beneath the aqueous solution
• Observe the color at the interface between two layers and compare with a control test
• A purple or violet ring/zone at the junction indicates carbohydrates

Note

• Apply the test to monosaccharide and polysaccharide carbohydrate solutions
• Ignore brown color from charring and repeat the test with a more dilute sugar solution

Precautions

• Alpha-naphthol solution should be freshly prepared due to instability
• Add concentrated H2SO4 carefully along the test tube sides to minimize disturbance
• Few drops of Molisch’s reagent are sufficient
• Add acid carefully due to its corrosive nature
• Avoid shaking the test tube when the ring forms to prevent its destruction

Limitations

• Furfurals, organic acids, aldehydes, and ketones can also give this test
• Concentrated sugar solutions may yield a red color due to acid charring instead of purple

Tests for Reducing Sugars

• Reducing sugars possess a hemiacetal/hemiketal functional group in their molecular structure
• These groups are in equilibrium with a free aldehyde group, easily oxidized to a carboxylic acid
• Oxidation is accompanied by the reduction of an oxidizing agent, like copper(II) or silver(I) ions
• Copper(II) ions are reduced to copper(I) ions, and silver ions to silver metal
• This was a major method for silvering glass to make mirrors
• The oxidation number of the oxidizing agent decreases (is reduced)

Benedict’s Test

• Used to test for simple carbohydrates
• Identifies reducing sugars (monosaccharides/disaccharides) with free ketone or aldehyde groups
• Can be used to detect glucose in urine
• Reducing sugars transfer hydrogens (electrons) to other compounds

Reaction with Benedict's Reagent

• Benedict's reagent changes color when mixed with reducing sugars and heated because of a reduction reaction
• Color ranges from green to dark red/rusty-brown, depending on the sugar amount and type

Benedict's Quantitative Reagent

• Contains potassium thiocyanate
• Determines the amount of reducing sugar present
• Yields a copper thiocyanate precipitate (white), suitable for titration
• Titration repeated with 1% glucose for calibration

Principle of Benedict’s Test

• Benedict’s solution, when heated with carbohydrates, changes to orange/brick red
• Change caused by reducing properties of simple carbohydrates
• Copper (II) ions in Benedict’s solution reduce to Copper (I) ions, changing the color
• Red copper(I) oxide forms, is water-insoluble, and precipitates out
• Higher reducing sugar concentration leads to brick-red color and more precipitate
• Sometimes, brick-red copper oxide precipitates out and collects at the bottom

Reagents

• Sodium carbonate provides alkaline conditions for redox reaction
• Sodium citrate forms complexes with copper (II) ions, preventing deterioration during storage

Limitations

• Complex carbohydrates (starches) do not react positively unless broken down by heating or digestion
• Table sugar (disaccharide) is a non-reducing sugar
• Table sugar does not react with iodine or Benedict Reagent
• Glucose/fructose components must be decomposed for a positive glucose test; starch test will still be negative

Composition and Preparation of Benedict’s Solution

• Benedict’s solution is a deep-blue alkaline solution that is used to test for aldehyde functional group – CHO.
• 1 Litre requires:
  • 100 g anhydrous sodium carbonate
  • 173 g sodium citrate
  • 17.3 g copper(II) sulfate pentahydrate

Procedure of Benedict's Test

• Approximately 1 ml of sample is placed into a clean test tube.
• 2 ml (10 drops) of Benedict’s reagent (CuSO4) is placed in the test tube.
• The solution is then heated in a boiling water bath for 3-5 minutes.
• Observe for color change in the solution of test tubes or precipitate formation.

Result Interpretation

• Green upon boiling: 0.1-0.5% sugar
• Yellow upon boiling: 0.5-1% sugar
• Orange upon boiling: 1-1.5% sugar
• Red upon boiling: 1.5-2% sugar
• Brick red upon boiling: More than 2% sugar

Positive/Negative Results

• Positive: Formation of reddish precipitate within three minutes; reducing sugars are present, example: Glucose
• Negative: No color change (remains blue); reducing sugars absent, example: Sucrose

Barfoed's Carbohydrate Test

• Detects monosaccharide (reducing) sugars
• Barfoed's reagent (ethanoic/acetic acid and copper (II) acetate) is combined with the test solution and boiled
• Red copper (II) oxide precipitate indicates reducing sugar
• Disaccharides give negative results because they are weaker reducing agents
• Specific for monosaccharides due to the weakly acidic nature of Barfoed's reagent,
• It's reduced only by monosaccharides in a fast manner
• Monosaccharides react faster versus disaccharides to produce cuprous oxide
• Much brick-red precipitate indicates reducing monosaccharide
• Trace precipitates may result from hydrolysis of disaccharides
• The precipitate adheres to the walls of the test tube
• The precipitate is less voluminous than Benedict's test

Seliwanoff’s Test

• Color reaction specific for ketoses
• Concentrated HCl causes ketoses to undergo dehydration to yield furfural derivatives more rapidly than aldoses
• These derivatives create complexes with resorcinol to yield deep red color
• Test reagent causes ketohexoses to dehydrate into 5-hydroxymethylfurfural
• 5-hydroxymethylfurfural reacts with resorcinol; produces a red product within two minutes
• Aldohexoses react slower to form the same product

Interferences

• Sucrose may give a positive ketohexose test due to partial hydrolysis into glucose and fructose
• Other sugars give a red color upon prolonged heating
• Some sources say that an apricot color is negative
• The reaction depends on concentration, and sugars like glucose give little to no color even after ten minutes

Bial’s Test

• Distinguishes between pentoses and hexoses
• Pentose is one of the most important chemical substance
• Manfred Bial's test brought a revolution:
  • Orcinol 0.4g
  • 200 ml of hydrochloric acid
  • 0.5 ml of ferric chloride solution
• Pentoses react with Bial’s reagent and are converted to furfural
• Orcinol and furfural condense in the presence of ferric ion to form a colored product
• This procedure includes
  • 2 ml of sample solution is placed in a test tube
  • Add 2 ml of Bial’s reagent
  • Heat the solution by Bunsen burner or hot water bath
  • Changes in color are observed
• Appearance of green colour or precipitate indicates the presence of pentoses and
  • Muddy brown precipitate indicates the presence of hexoses

Iodine Test:

• Detects starch in the solution
• Blue-black color forms due to the starch-iodine complex:
  • α-amylose and amylopectin polymers form a complex with iodine
  • Yielding the blue-black color
• Polysaccharides like glycogen and cellulose do not show this blue-black color in the presence of iodine
• Iodines solution referred to as Lugol’s solution: -Contains iodine and potassium iodide (KI) -Distinguishes starch and glycogen from other polysaccharides -Yielding a blue-black color
• Glycogen reacts with Lugol's reagent to give a brown-blue color
• Other polysaccharides and monosaccharides yield no color change; the test solution remains the characteristic brown-yellow of the reagent

Reaction

• Starch and glycogen form helical coils that fit iodine atoms
• Starch-iodine or glycogen-iodine complex is generated
• Amylose and amylopectin (starch forms) have fewer branches than glycogen
• Starch helices are longer than glycogen:
  • Binding more iodine atoms
  • Resulting in more intense color
• Iodine Test (Starch/Amylose)
  • A few drops of 0.01 M iodine in 0.12 M KI are added to a 1% solution of the carbohydrate in question
• Immediate vivid blue indicates amylose
  • Starch forms a blue black due to the polyiodide complex formed

Osazone Test:

• Ketoses and aldoses react with phenylhydrazine
  • Yielding phenylhydrazone
• Phenylhydrazone reacts with two molecules of phenylhydrazine
  • Yielding osazone
• Needle-shaped yellow osazone crystals are produced by glucose, fructose and mannose
• Lactosazone produces mushroom shaped crystals
• Crystal shapes vary with different osazones -Flower-shaped produced by maltose
• Three molecules of phenyhyrazine are required
• Reacts with the first two carbons on the sugar -Used rarely for sugar ID today