Scheme to identify an unknown sugar
Run the tests in a fixed order so each result removes some sugars: Molisch's, Benedict's, Barfoed's, Seliwanoff's, then osazone for the disaccharides.
Aim and principle
Aim: To identify the given unknown carbohydrate (glucose, fructose, lactose, maltose or sucrose) by systematic qualitative analysis. Principle: The five sugars differ in four features: whether they reduce copper in alkali (free aldehyde or keto group), whether they reduce it in weak acid within 2-3 minutes (monosaccharide), whether they are ketoses (quick red colour with resorcinol in HCl) and the shape of their osazone. Doing the tests in this order means each result rules out some sugars until only one fits. A confirmatory test is then done for the sugar you suspect.
Requirements
- Unknown sugar solution (about 1-2%) and, if given, known sugars for comparison.
- Reagents: Molisch reagent and conc. H2SO4, iodine solution, Benedict's, Fehling's A and B, Barfoed's, Seliwanoff's, phenylhydrazine mixture with glacial acetic acid, conc. HCl and 10% NaOH (hydrolysis test), conc. HNO3 (mucic acid test).
- Apparatus: test tubes, stand, holder, boiling water bath, pipettes with a bulb, stopwatch, slides, coverslips, microscope.
- Safety: gloves, goggles and a fume hood for concentrated acids; never taste a sample or pipette by mouth.
Procedure
Typical volumes are given. Follow the exact volumes in your lab manual.
- 1Note the appearance: a colourless, odourless solution (or a white crystalline solid soluble in water). Never taste a lab sample.
- 2Molisch's test (2 mL unknown + 2 drops reagent, 2 mL conc. H2SO4 down the side) and iodine test (1 mL unknown + 2 drops iodine).Violet ring and no blue-black: a simple sugar
- 3Benedict's test: 5 mL reagent + 8 drops unknown, boiling water bath 5 minutes.Negative: go to step 7
- 4Benedict's positive: Barfoed's test (2 mL reagent + 1 mL unknown, 2-3 minutes only).Red precipitate: step 5. None: step 6
- 5Monosaccharide: Seliwanoff's test (3 mL reagent + 1 mL unknown, watch 1 minute); confirm with osazone (needles).Cherry red: fructose. No red: glucose
- 6Reducing disaccharide: osazone test, cool slowly, view under the microscope; confirm lactose with the mucic acid test.Sunflower: maltose. Powder-puff: lactose
- 7Benedict's negative: Seliwanoff's test and the hydrolysis test (boil with HCl, neutralise, repeat Benedict's).Red with Seliwanoff's and reducing after hydrolysis: sucrose
- 8Write every test as Test | Observation | Inference and report the sugar.
Flow scheme for an unknown sugar
Each arrow gives the result that sends you on; the grey note gives the other branch. In your record, draw it as a branching chart.
Test | Observation | Inference
| Test | Observation | Inference |
|---|---|---|
| Appearance | Colourless, odourless solution | May be a sugar |
| Molisch's test | Violet ring at the junction | Carbohydrate present |
| Iodine test | No blue-black colour | Starch absent |
| Benedict's test | (a) Green to brick-red precipitate (b) Stays blue | (a) Reducing sugar: glucose, fructose, lactose or maltose (b) Non-reducing: sucrose |
| Barfoed's test | (a) Red precipitate within 2-3 minutes (b) No precipitate | (a) Monosaccharide: glucose or fructose (b) Disaccharide: lactose or maltose |
| Seliwanoff's test | (a) Cherry red within 1 minute (b) No red colour | (a) Ketose: fructose (sucrose if Benedict's was negative) (b) Aldose: glucose |
| Osazone test | (a) Needles while hot (b) Sunflower crystals on cooling (c) Powder-puff crystals on cooling | (a) Glucose or fructose (b) Maltose (c) Lactose |
| Mucic acid test | Colourless rod- or prism-shaped crystals | Lactose confirmed |
| Hydrolysis test (only if Benedict's was negative) | Brick red with Benedict's after boiling with HCl and neutralising | Sucrose confirmed |
Telling look-alikes apart
| Pair | Deciding test | What you see |
|---|---|---|
| Glucose vs fructose | Seliwanoff's | Fructose cherry red within 1 minute; glucose no red |
| Glucose vs maltose | Barfoed's | Glucose red precipitate in 2-3 minutes; maltose none |
| Lactose vs maltose | Osazone; mucic acid | Lactose powder-puff and mucic acid crystals; maltose sunflower, no mucic acid |
| Fructose vs sucrose | Benedict's | Fructose brick red; sucrose stays blue (both red in Seliwanoff's) |
| Sucrose vs starch | Iodine | Starch blue-black; sucrose no colour |
Precautions and result
- Do the tests in order and write each inference before moving on; it saves reagent and time.
- Time Barfoed's (2-3 minutes) and Seliwanoff's (1 minute) strictly; over-heating gives false positives.
- Run a known sugar alongside whenever a result is doubtful.
- Gloves and goggles; acid down the side of the tube; fume hood for HCl and HNO3; never taste a sample or pipette by mouth.
- Result: The given unknown carbohydrate is identified as ______ (for example, maltose: Molisch's positive, reducing, Barfoed's negative, sunflower-shaped osazone).
The same reasoning is used when reading urine sugar results: glucose suggests diabetes, while galactose or lactose in a newborn's urine can point to galactosaemia or lactose overload. Knowing that milk carries lactose, malted foods maltose, fruit and honey fructose and table sugar sucrose helps you link each sugar to its food source in diet counselling.
Short answer: “Name the test that distinguishes glucose from fructose.” Seliwanoff's test: fructose, a ketose, gives cherry red within about a minute; glucose, an aldose, does not.
Short answer: “How will you distinguish lactose from maltose?” By the osazone test: lactose gives powder-puff crystals and maltose sunflower crystals. Lactose also gives mucic acid crystals; maltose does not.
Structured question: “Give a scheme for the identification of an unknown sugar.” Flow chart: Molisch's (carbohydrate) → iodine (not starch) → Benedict's (negative = sucrose, confirm by hydrolysis and Seliwanoff's) → Barfoed's (monosaccharide or disaccharide) → Seliwanoff's (fructose vs glucose) → osazone (maltose sunflower, lactose powder-puff) → mucic acid for lactose.
My Big Brown Sack Opens: Molisch's, Benedict's, Barfoed's, Seliwanoff's, Osazone, in that order.