Osazone, sucrose hydrolysis and mucic acid tests
Reducing sugars boiled with phenylhydrazine form yellow osazone crystals whose shape and timing name the sugar; acid splits sucrose; mucic acid crystals point to lactose.
Aim and principle
Aim: To prepare and identify the osazones of glucose, fructose, lactose and maltose, to show that sucrose becomes reducing after acid hydrolysis, and to perform the mucic acid test for lactose. Principle: Osazone: on heating, one molecule of a reducing sugar reacts with three molecules of phenylhydrazine. Carbons 1 and 2 both end up joined to phenylhydrazine, giving a yellow, poorly soluble osazone that crystallises in a shape typical of the sugar. Because carbons 1 and 2 are used up, glucose, fructose and mannose give the same osazone (needles). Disaccharide osazones dissolve in hot water and appear only on cooling. Sucrose hydrolysis (inversion): boiling with dilute HCl splits non-reducing sucrose into glucose and fructose; after neutralising, the mixture reduces Benedict's reagent. The optical rotation changes from right (+66.5°) to left (about −20°), so the product is called invert sugar. Mucic acid: hot concentrated nitric acid oxidises both ends of galactose to mucic acid (galactaric acid), which is insoluble in cold water and crystallises. Lactose answers because it contains galactose; glucose gives soluble saccharic acid, so no crystals form.
Requirements
| Reagent or item | Made of | What it does |
|---|---|---|
| Sugar solutions | Glucose, fructose, lactose, maltose, sucrose (about 2-5%) | The samples |
| Phenylhydrazine mixture | Phenylhydrazine hydrochloride mixed with sodium acetate | Sodium acetate frees phenylhydrazine from its salt and buffers the mixture; phenylhydrazine forms the osazone |
| Glacial acetic acid | A few drops | Keeps the mixture slightly acidic, which favours osazone formation |
| Hydrochloric acid | Conc. HCl (a few drops) or dilute HCl | Hydrolyses sucrose into glucose and fructose |
| 10% NaOH or sodium carbonate | In water | Neutralises the acid before Benedict's test, which needs alkali |
| Benedict's reagent | Copper sulphate, sodium citrate, sodium carbonate | Detects the reducing sugars formed |
| Conc. nitric acid | Concentrated HNO3 | Oxidises galactose to mucic acid |
| Apparatus | Boiling water bath, test tubes, funnel and filter paper, glass slides, coverslips, microscope (low power), litmus paper, fume hood | Crystals are seen and drawn under the microscope |
| Safety | Gloves, goggles, fume hood | Phenylhydrazine is toxic and passes through skin; nitric acid gives brown toxic fumes |
Procedure
Typical volumes are given. Follow the exact volumes in your lab manual.
- 1Osazone: to 5 mL of each sugar solution add about 0.5 g phenylhydrazine mixture and 5 drops glacial acetic acid. Warm and shake to dissolve; filter if cloudy.Label the tubes; wear gloves
- 2Put all tubes together in a boiling water bath and note the time at which yellow crystals appear in each.Fructose about 2 min, glucose about 4-5 min
- 3After 30-45 minutes, take the tubes out and let them cool slowly in air, not under the tap.Lactose and maltose osazones appear only on cooling
- 4Place a drop of crystals on a slide, cover with a coverslip and view under low power. Draw the shapes.Needles, sunflowers or powder-puffs
- 5Sucrose hydrolysis: 5 mL sucrose solution + 5 drops conc. HCl. Heat in a boiling water bath for 10 minutes; cool.Keep plain sucrose as a control
- 6Neutralise with 10% NaOH until just alkaline to litmus. Do Benedict's test on 0.5 mL of it (5 mL reagent) and on the control.Compare the two tubes
- 7Mucic acid: 5 mL lactose solution + 1 mL conc. HNO3 in a fume hood. Heat in a boiling water bath for 1-1½ hours, until only about 1-2 mL is left. Cool, leave overnight and view under the microscope.Run glucose alongside for comparison
Test | Observation | Inference
| Test | Observation | Inference |
|---|---|---|
| Osazone: fructose | Yellow needle-shaped crystals in about 2 minutes, while hot | Fructose (fastest; same osazone as glucose) |
| Osazone: glucose | Yellow needles in sheaves, like a broom, in about 4-5 minutes, while hot | Glucose |
| Osazone: maltose | None while hot; yellow sunflower- (petal-) shaped crystals on cooling | Maltose |
| Osazone: lactose | None while hot; yellow powder-puff (hedgehog) crystals on cooling | Lactose |
| Osazone: sucrose | No osazone (needles may appear after long heating, from partial hydrolysis) | Non-reducing sugar |
| Sucrose hydrolysis | Hydrolysed and neutralised sample: brick red with Benedict's. Unhydrolysed control: stays blue | Sucrose was split into reducing glucose and fructose (invert sugar) |
| Mucic acid test | Lactose: colourless rod- or prism-shaped crystals after standing. Glucose: no crystals | Galactose present: lactose (or galactose) |
Precautions and result
- Wear gloves and goggles: phenylhydrazine is toxic and passes through skin. Do the mucic acid heating in a fume hood (brown nitrogen dioxide fumes).
- Cool the osazone tubes slowly in air; quick cooling under the tap gives broken, shapeless crystals.
- Heat all osazone tubes together and note the time; sucrose heated too long may give needles because the acid splits it.
- Neutralise the hydrolysed sucrose before Benedict's test, because the reagent needs alkali.
- Never pipette acids by mouth; use a bulb.
- Result: Osazones: glucose and fructose, needles; maltose, sunflower; lactose, powder-puff. Sucrose gave a positive Benedict's test only after hydrolysis. Lactose gave mucic acid crystals.
Sucrose hydrolysis is what sucrase (invertase) does in the gut and what happens when sugar syrup is boiled with lemon juice. Invert sugar (glucose + fructose) is sweeter than sucrose and does not crystallise easily, so it keeps sweets and syrups smooth; it is also the main sugar in honey. The mucic acid test points to galactose, which matters in galactosaemia, where milk sugar must be removed from the diet.
Short answer: “Why do glucose and fructose form the same osazone?” Osazone formation uses carbons 1 and 2, the only carbons where glucose and fructose differ. The rest of the molecule (C3-C6) is identical, so both give needle-shaped glucosazone.
Short answer: “Why is hydrolysed sucrose called invert sugar?” Sucrose turns plane-polarised light to the right (+66.5°). After hydrolysis, the strong left rotation of fructose outweighs glucose, so the mixture turns light to the left (about −20°): the sign is inverted.
Structured question: “Describe the osazone test with the crystal shapes of common sugars.” Principle: a reducing sugar reacts with three molecules of phenylhydrazine at C1 and C2 to give a yellow osazone. Procedure: 5 mL sugar + phenylhydrazine mixture + acetic acid, boiling water bath, note time, cool slowly, view under microscope. Shapes: fructose and glucose needles (about 2 and 4-5 min); maltose sunflower and lactose powder-puff on cooling; sucrose none.
Nice Sunny Puffs: Needles (glucose, fructose) come hot and fast; Sunflowers (maltose) and Powder-puffs (lactose) wait for cooling.