Sugar tests: Molisch's, iodine, Benedict's, Fehling's, Barfoed's, Seliwanoff's
One ladder of tests for five sugars: Molisch's says carbohydrate, Benedict's and Fehling's say reducing, Barfoed's says monosaccharide, Seliwanoff's says ketose.
Aim and principle
Aim: To perform the qualitative tests on glucose, fructose, lactose, maltose and sucrose and record what each sugar shows. Principle: Each test picks out one feature of a sugar. Molisch's: concentrated sulphuric acid splits and dehydrates any carbohydrate to furfural or hydroxymethylfurfural, which condenses with α-naphthol to give a violet ring. Iodine: only starch-type polysaccharides trap iodine in their coils (blue-black), so simple sugars give no colour; the test rules starch out. Benedict's and Fehling's: in hot alkali, a sugar with a free aldehyde or keto group reduces blue Cu2+ to Cu+, which separates as red cuprous oxide (Cu2O). Barfoed's: the same copper reduction but in weak acid, where only the stronger-reducing monosaccharides react quickly (red precipitate within about 2-3 minutes). Seliwanoff's: hot HCl turns ketoses into hydroxymethylfurfural much faster than aldoses, and this gives a cherry-red colour with resorcinol within about a minute.
Requirements
| Reagent or item | Made of | What it does |
|---|---|---|
| Sugar solutions | 1-2% glucose, fructose, lactose, maltose and sucrose in water | The samples to be tested |
| Molisch reagent | α-naphthol (about 5%) in ethanol | Condenses with furfural compounds to give the violet colour |
| Conc. sulphuric acid | Concentrated H2SO4 | Hydrolyses and dehydrates the sugar; forms the heavy lower layer |
| Iodine solution | Iodine dissolved in potassium iodide | Blue-black with starch; rules starch out |
| Benedict's qualitative reagent | Copper sulphate, sodium citrate, sodium carbonate | Cu2+ is the oxidant; carbonate gives mild alkali; citrate keeps copper dissolved so Cu(OH)2 does not settle |
| Fehling's A and B | A: copper sulphate. B: sodium potassium tartrate (Rochelle salt) with sodium hydroxide. Mix equal volumes fresh | Tartrate holds Cu2+ in strong alkali; copper is reduced to red Cu2O |
| Barfoed's reagent | Copper acetate in dilute acetic acid | A weakly acidic copper reagent, reduced quickly only by monosaccharides |
| Seliwanoff's reagent | Resorcinol (about 0.05%) in dilute HCl | Acid dehydrates ketoses; resorcinol gives the red colour |
| Apparatus | Test tubes, stand, holder, boiling water bath, droppers, graduated pipettes with a pipette bulb, stopwatch | The stopwatch is needed for Barfoed's and Seliwanoff's |
| Safety | Gloves, goggles, fume hood | Concentrated acids burn; never pipette by mouth |
Procedure
Run each test on all five sugars side by side. Typical volumes are given. Follow the exact volumes in your lab manual.
- 1Molisch's: 2 mL sugar solution + 2 drops Molisch reagent; mix. Tilt the tube and run 2 mL conc. H2SO4 slowly down the side so it forms a layer below.Look at the junction of the two layers
- 2Iodine: 1 mL sugar solution + 2 drops iodine solution.Compare with a starch tube if one is given
- 3Benedict's: 5 mL Benedict's reagent + 8 drops (about 0.5 mL) sugar solution. Keep in a boiling water bath for 5 minutes; cool.Green, yellow, orange or brick red = positive
- 4Fehling's: mix 1 mL Fehling's A + 1 mL Fehling's B and boil (it must stay blue). Add 1 mL sugar solution and heat in a boiling water bath for a few minutes.Red precipitate = positive
- 5Barfoed's: 2 mL Barfoed's reagent + 1 mL sugar solution. Heat in a boiling water bath for 2-3 minutes only; cool under the tap.Look for a thin red layer at the bottom
- 6Seliwanoff's: 3 mL Seliwanoff's reagent + 1 mL sugar solution. Heat in a boiling water bath and watch for 1 minute.Note the time the red colour appears
- 7Write every result in the Test | Observation | Inference format.Write exact colours, not just 'positive'
Test | Observation | Inference
| Test | Observation | Inference |
|---|---|---|
| Molisch's test | Violet (purple) ring at the junction of the two layers, with all five sugars | Carbohydrate present |
| Iodine test | No blue-black colour; tube stays pale yellow-brown | Starch absent; a simple sugar |
| Benedict's test | Glucose, fructose, lactose, maltose: green to brick-red precipitate. Sucrose: stays clear blue | First four are reducing sugars; sucrose is non-reducing |
| Fehling's test | Glucose, fructose, lactose, maltose: red precipitate. Sucrose: stays blue | Confirms the reducing sugars |
| Barfoed's test | Glucose and fructose: thin brick-red precipitate at the bottom within 2-3 minutes. Lactose, maltose, sucrose: none | Glucose and fructose are monosaccharides; lactose and maltose are reducing disaccharides |
| Seliwanoff's test | Fructose: cherry red within about 1 minute. Sucrose: red too. Glucose, lactose, maltose: no red (faint pink only after long heating) | Fructose is a ketose; sucrose contains fructose; the others are aldoses |
Results at a glance
| Sugar | Molisch's | Iodine | Benedict's / Fehling's | Barfoed's | Seliwanoff's |
|---|---|---|---|---|---|
| Glucose | + | − | + | + | − |
| Fructose | + | − | + | + | + (cherry red) |
| Lactose | + | − | + | − | − |
| Maltose | + | − | + | − | − |
| Sucrose | + | − | − | − | + (frees fructose) |
Precautions and result
- Wear gloves and goggles. Run conc. H2SO4 slowly down the side of a tilted tube pointed away from you; use the HCl reagent in a fume hood.
- Never pipette acids or reagents by mouth; use a pipette bulb or a dispenser.
- Time Barfoed's (2-3 minutes; some manuals allow up to 5) and Seliwanoff's (about 1 minute) with a watch. Longer heating splits disaccharides and gives false positives.
- Mix Fehling's A and B fresh and boil them alone first; if the mixture turns red by itself, it is spoilt.
- Use clean tubes and the same volumes for every sugar so results can be compared.
- Result: Glucose and fructose are reducing monosaccharides (fructose is a ketose); lactose and maltose are reducing disaccharides; sucrose is a non-reducing disaccharide.
Benedict's test was the old bedside test for sugar in urine (glycosuria) in diabetes; the colour grade from green to brick red gives a rough amount. The copper tests also explain why lactose in milk and fructose in fruit and honey react as reducing sugars, while table sugar (sucrose) does not react until it is split.
Short answer: “Why is sucrose a non-reducing sugar?” Its glucose and fructose are joined through both of their reducing groups (C1 of glucose to C2 of fructose), so no free aldehyde or keto group is left to reduce Cu2+.
Short answer: “How does Barfoed's test differ from Benedict's test?” Both rely on copper reduction. Benedict's is alkaline and is given by all reducing sugars; Barfoed's is weakly acidic, so only monosaccharides reduce it within 2-3 minutes.
Structured question: “Explain the principle and procedure of Molisch's test and Seliwanoff's test.” Molisch's: conc. H2SO4 forms furfural derivatives that condense with α-naphthol; 2 mL sugar + 2 drops reagent, acid layered down the side; violet ring = carbohydrate. Seliwanoff's: ketoses dehydrate fast in HCl to hydroxymethylfurfural, which gives cherry red with resorcinol; 3 mL reagent + 1 mL sugar in a boiling water bath; red within 1 minute = fructose.
Copper in alkali (Benedict's, Fehling's) = any reducing sugar; copper in acid (Barfoed's) = only the quick monosaccharides; resorcinol in acid (Seliwanoff's) = ketoses.