Exercise

Practical 8 - Estimation of Sugars (Reducing Sugar)

Aim: Estimation of reducing sugars in fresh fruits and processed products using Lane and Eynon method.

Theory: Sugars are inherently present in different fruits and vegetables or added during preparation of different products. They are added to the fruit products to improve the taste and also to act as a preservative. Glucose and fructose in the fruits represent reducing sugars while sucrose or cane sugar added represents the non-reducing sugar. They are estimated by using Lane and Eynon method which measures sugar as reducing sugar and total sugar as invert sugar.

Principle:
Invert sugar reduces the copper in Fehling’s solution to red, insoluble cuprous oxide. The sugar content in a food sample is estimated by determining the volume of the unknown sugar solution required to completely reduce a known volume of Fehling’s solution. Glucose and other sugars are capable of reducing oxidizing agents and are called reducing sugars and this property is used for the estimation of sugars. The cupric ion in Fehling’s solution is reduced to cuprous state which precipitates as red cuprous oxide (Cu2O). Only reducing sugars reduce the copper solution. The method is suitable for estimation of sugars in fruit and fruit products.


CuSO4 + 2NaOH → Cu (OH)2 + Na2SO4

Cu (OH)2 → CuO (Cupric oxide)
2CuO + CHO → Cu2O (Cuprous oxide) + COOH

Apparatus, reagents and glassware required

• Beakers - 250ml

• Volumetric flasks - 250ml
• Measuring cylinder- 250ml
• Pipette - 10ml
• Hot plate

Preparation of Reagents
  1. Fehling’s solution-A: Dissolve 69.28g copper sulphate (CuSO4.5 H2O) in water, dilute to 1 litre and filter.(Standard Fehling’s solution A or 1, ampules can be used also)
  2. Fehling’s solution-B: Dissolve 346g of Rochelle salt (Potassium Sodium Tartrate, KNa C4H4O6.4H2O) and 100g NaOH in water, make volume to 1 litre. (Standard Fehling’s solution B or 2, ampules can also be used)
  3. Methylene blue indicator (1%): Dissolve 1g in 100ml water.
  4. 45% neutral lead acetate solution: Dissolve 225g of neutral lead acetate in water and make up to 500ml. It is used as clarifying agent.
  5. 22% Potassium oxalate solution: Dissolve 110g Potassium oxalate (K2C2O4. H2O) in water and make volume to 500ml. This is used for neutralizing excess of lead acetate.
  6. Standard invert sugar Weigh 10g of sucrose into 1 litre flask, add 100ml water and 5ml concentrated HCl for hydrolysis. Allow to stand for 3 days at 20-250C or 7 days at 15oC inversion to take place and then make up to volume.

Standardization of standard invert sugar: Pipette 25ml of standard invert solution in to a 100ml volumetric flask; add 50ml water and few drops of Phenolphthalein indicator. Neutralize with 20% NaOH until solution turns pink. Acidify with 1N HCl adding it drop wise until pink color disappears. Make up to 100ml with water (1ml=2.5mg invert sugar).

Standardization of Fehling’s solution: Mix 5ml Fehling A + 5ml Fehling B solution in 250ml conical flask. Add 25-50ml water and heat the flask. Add standard invert sugar solution from the burette dropwise till the solution turns brick red. Add few drops of Methylene blue indicator and add drop-wise invert solution, when the blue color disappears, note the titre value of invert solution, repeat the titration and calculate factor for Fehling’s solution as under:-



Titre x 2.5
Factor for Fehling solution((g of invert sugar) ) = --------------------
1000



Procedure for estimation of reducing sugars

Take 10-20g juice/squash/drink in 250 ml volumetric flask, add 100 ml water neutralize with 1 N NaOH. Add 2ml 45% lead acetate. Shake well and keep for 10 minutes. Add few drops of potassium oxalate solution to remove excess of lead acetate. Make volume to 250ml with water and filter.

OR


Take 10-25 g of sample (fresh fruit or fruit product) and grind in a pestle and mortar, blend in blender, add 100 ml water. Neutralize solution with 1 N NaOH. Boil gently for 1 hour with stirring. Replace water lost during evaporation, cool and transfer to 500 ml volumetric flask. Make volume 500 ml and filter through whatman filter paper.
  1. Pipette 100 ml aliquot from filtrate in 250ml flask, add 2 ml 45% lead acetate, let it stand for 10 minutes then add few drops of 22% potassium oxalate solution and made volume to 250 ml with water and filter.
  2. Add 2 ml 45% neutral lead acetate solution.
  3. Pipette 10ml of mixed Fehling’s solution (5 ml each of Fehling A and B) and few ml of water into 250ml conical flask.
  4. Heat the flask containing mixed Fehling’s solution on hot plate and add the sample (clarified sugar) solution drop wise from the burette/pipette till faintest blue colour remains.
  5. Add 2-3 drops of methylene blue indicator and complete the titration till the color changes to brick red.
  6. At the end point, note the readings and calculate the reducing sugars.

Calculations
mg of invert sugar × dilution × 100
Reducing sugar (%) = --------- ---------------------------------------
titre × weight of the sample × 100

Or

Factor for Fehling solution × Volume made × 100
% Reducing Sugar = --------------------------------------------------------
Titre × wt of sample × ml of aliquot

Problem: 10 g of fruit jam was made into 500 ml after neutralization with 1 N NaOH and filtered. From the filtrate 100ml of aliquot was clarified using lead acetate and potassium oxalate and volume made to 250 ml and filtered. 10 ml Fehling’s solution (A+B) was titrate against clarified sample. The titre value was 20 ml. Calculate the reducing sugar when factor for Fehling’s solution is 0.05.

Solution:
  • Given 10g made to 500ml
  • 100 ml aliquot made to 250 ml
  • Titre 20ml
  • Factor for Fehling’s solution 0.05

Factor× vol. made for 10g to 500ml × vol made100ml to 250 ml × 100
% Reducing Sugar = --------------------------------------------------------
Titre × wt of sample (10) × ml of aliquot 100

0.05 × 500 × 250 × 100
= ---------------------------------
20 × 10 × 100

= 31.25%





Last modified: Wednesday, 7 March 2012, 9:59 AM