Estimation of reducing sugar by Benedict's titration
Boiling Benedict's quantitative reagent is titrated with the sugar solution until the blue just disappears; a standard glucose titre turns the burette reading into mg of sugar.
Aim and principle
Aim: To estimate the amount of reducing sugar in the given solution (or food sample) by Benedict's quantitative method, using a standard glucose solution. Principle: In hot alkaline solution a reducing sugar reduces blue Cu2+ to Cu+. Benedict's quantitative reagent also contains potassium thiocyanate, which traps Cu+ as a white precipitate of cuprous thiocyanate instead of red Cu2O, so the end point is simply the disappearance of the blue colour. The reagent is made so that 25 mL is reduced by 50 mg of glucose (10 mL by 20 mg). The exact value depends on the batch and on how you boil, so the reagent is first titrated against a standard glucose solution, and the same volume is then titrated with the unknown. A smaller titre means more sugar. Other methods: Lane-Eynon titration with Fehling's solution and methylene blue indicator; colorimetric DNS (3,5-dinitrosalicylic acid, orange-red, read at 540 nm) and Nelson-Somogyi (arsenomolybdate blue).
Requirements
| Reagent or item | Made of | What it does |
|---|---|---|
| Benedict's quantitative reagent | Copper sulphate, sodium carbonate, sodium citrate, potassium thiocyanate and a little potassium ferrocyanide | Cu2+ is reduced by the sugar; thiocyanate gives white CuSCN; ferrocyanide keeps cuprous oxide from separating; citrate keeps copper dissolved |
| Anhydrous sodium carbonate | About 1-2 g per titration | Extra alkali for a sharp end point |
| Porcelain bits or pumice | A few small pieces | Prevent bumping while boiling |
| Standard glucose | 1 mg/mL (100 mg glucose in 100 mL water) | Finds how many mg of glucose reduce 10 mL of this reagent |
| Sample | Unknown made up to 100 mL, or a food extract clarified and made up to volume | Filled in the burette and titrated |
| Apparatus | 50 mL burette, 10 mL pipette with a bulb, 250 mL conical flasks, standard flasks, wire gauze, tripod, burner or hot plate | Titration at boiling point |
| Safety | Goggles, gloves, tongs or a flask holder | The flask is kept boiling and hot liquid can spurt |
Procedure
Typical volumes are given. Follow the exact volumes in your lab manual.
- 1Rinse and fill the burette with standard glucose (1 mg/mL).
- 2Pipette 10 mL Benedict's quantitative reagent (using a bulb) into a conical flask. Add about 1-2 g anhydrous sodium carbonate and a few porcelain bits.
- 3Heat to boiling on a wire gauze and keep it boiling throughout the titration.
- 4Run in glucose quickly at first, then drop by drop near the end, still boiling.Blue fades as a white precipitate forms
- 5Stop when the last trace of blue disappears.End point: chalky white precipitate, no blue tint
- 6Do one rough titration, then repeat until two titres agree within 0.1 mL.This titre gives mg glucose equal to 10 mL reagent
- 7Make the unknown up to 100 mL (or prepare the food extract), fill the burette with it and titrate fresh 10 mL reagent the same way.Take concordant titres
Observation table
| Titration | Trial | Initial reading (mL) | Final reading (mL) | Titre (mL) |
|---|---|---|---|---|
| Standard glucose (1 mg/mL) | Rough | 0.0 | 20.0 | 20.0 |
| Standard glucose (1 mg/mL) | 1 | 0.0 | 19.6 | 19.6 |
| Standard glucose (1 mg/mL) | 2 | 0.0 | 19.6 | 19.6 |
| Unknown sugar solution | Rough | 0.0 | 13.0 | 13.0 |
| Unknown sugar solution | 1 | 0.0 | 12.4 | 12.4 |
| Unknown sugar solution | 2 | 0.0 | 12.4 | 12.4 |
Worked example 1: the given sugar solution
10 mL of Benedict's quantitative reagent needed 19.6 mL of standard glucose (1 mg/mL). The given unknown was made up to 100 mL, and 10 mL of the same reagent needed 12.4 mL of it. Find the reducing sugar (as glucose) in the 100 mL.
- Glucose equal to 10 mL reagent = standard titre × strength = 19.6 mL × 1 mg/mL = 19.6 mg.
- So 12.4 mL of the unknown also contains 19.6 mg reducing sugar.
- Sugar in 1 mL = 19.6 ÷ 12.4 = 1.581 mg.
- Sugar in 100 mL = 1.581 × 100 = 158.1 mg.
- Formula: reducing sugar (mg/100 mL) = (standard titre × standard strength ÷ sample titre) × 100 = (19.6 × 1 ÷ 12.4) × 100.
158.1 mg reducing sugar (as glucose) in 100 mL, i.e. 0.158 g/100 mL.
Worked example 2: reducing sugar in honey
0.5 g honey was dissolved and made up to 250 mL with water. 10 mL Benedict's quantitative reagent (equal to 19.6 mg glucose, from the standard) needed 13.8 mL of this solution. Find the reducing sugar in g per 100 g honey.
- 13.8 mL of honey solution contains 19.6 mg reducing sugar.
- Sugar in 250 mL = 19.6 × 250 ÷ 13.8 = 355.1 mg.
- This came from 0.5 g honey, so per gram = 355.1 ÷ 0.5 = 710.1 mg.
- Per 100 g = 710.1 × 100 = 71,014 mg = 71.0 g.
- Formula: g/100 g = (F × V ÷ T) × (100 ÷ W) ÷ 1000, where F = mg glucose equal to 10 mL reagent, V = volume made up (mL), T = sample titre (mL), W = sample weight (g).
About 71.0 g reducing sugar (as glucose) per 100 g honey, within the usual range for pure honey (Indian food standards ask for at least 65 g).
Precautions and result
- Keep the flask boiling throughout; if boiling stops, air can re-oxidise Cu+ and the end point drifts.
- Add sugar drop by drop near the end and stop at the last trace of blue; standardise with glucose on the same day, in the same way, with titres agreeing within 0.1 mL.
- Dilute the sample so the titre falls between about 10 and 25 mL; clarify coloured or protein-rich food extracts first (neutral lead acetate, with excess lead removed by potassium oxalate).
- Wear goggles and gloves; hold the hot flask with tongs; use a pipette bulb, never your mouth.
- Result: The amount of reducing sugar (as glucose) in the given solution is ____ mg per 100 mL (or ____ g per 100 g of the food).
Reducing sugar values go into food composition tables and sugar figures on nutrition labels. They help judge honey quality (pure honey is mostly glucose and fructose, so a low reducing sugar suggests added cane sugar or jaggery syrup), the ripeness of fruit and browning (Maillard reaction) in baked and fried foods. The same copper chemistry was once used for blood and urine glucose before enzyme strips.
Short answer: “Why is potassium thiocyanate added to Benedict's quantitative reagent?” It precipitates Cu+ as white cuprous thiocyanate, so red Cu2O does not form and the end point is seen as the disappearance of blue.
Short answer: “Why is the solution kept boiling throughout the titration?” The reduction needs heat, and steady boiling keeps air out so the Cu+ formed is not re-oxidised; otherwise the end point drifts.
Structured question: “Explain the principle and calculation of reducing sugar estimation by Benedict's method.” Principle: reducing sugar reduces Cu2+ in boiling alkali; thiocyanate gives white CuSCN, so blue disappears at the end point. 25 mL reagent ≡ 50 mg glucose, checked with a 1 mg/mL glucose standard. Calculation: mg/100 mL = (standard titre × strength ÷ sample titre) × 100, e.g. 19.6 × 1 ÷ 12.4 × 100 = 158.1 mg/100 mL.
Blue gone, sugar done. Standard first, sample second; the smaller titre has more sugar.