Exercise

Practical 9 - Estimation of sugars (non-reducing and total sugars)

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

Theory: Glucose and fructose in the fruits represent reducing sugars while sucrose or cane sugar added to the fruit products 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. Non-reducing sugar is determined by subtracting the total reducing sugar from reducing sugar and multiplying the remainder with 0.95 factor.

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.

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
  • Burette - 50ml
  • Hot plate
  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)
  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)
  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 250ml increase in 1 litre flask, add 100ml water and 5ml concentrated HCl for hydrolysis. Allow to stand for 3 days at 20-25OC or 7 days at 15oC for 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)= ------------------× 100
1000


Procedure for estimation of Total Sugar
  1. Follow the method for preparation of deleaded samples from fruits or fruit products as explained under reducing sugars (Practical-8).
  2. Take 50 ml of the clarified deleaded solution from step 1 into a 250 ml conical flask
  3. Add 5 g of citric acid and 50 ml of water boil gently for 10 minutes to complete the inversion of sucrose and allow to cool.
  4. Transfer the inverted solution to a 250 ml volumetric flask and neutralize with 1 N NaOH using phenolphthalein as indicator and make up the volume to 250 ml with water. Place this solution into the burette and use for filtration.
  5. Pipette 10ml of mixed Fehling’s solution (5 ml of Fehling’s A and B) and few ml of water into 250 ml conical flask.
  6. Heat the flask containing Fehling’s solution on hot plate and add sample (clarified sugar) solution drop wise from the burette till faintest blue color remains.
  7. Add 2-3 drops of methylene blue indicator and complete the titration till the colour changes to brick red precipitates.
  8. Note the titre value and calculate the total as well as non –reducing sugars from the following equations.

Observations

Sample No

1

2

3

Mean

1.Weight of sample taken(ml/g)





2. Volume made





3.Volume of sample taken for deleading





4.Volume made up





5. Volume of deleaded solution taken for inversion





6. Volume made up





7. Titre (ml of sugar solution used in titration)





8. % Total sugars as invert sugar





9.% Reducing sugars (From chapter 8)





10.% Non reducing sugars (sucrose)





11. Total sugars.








Calculations
mg of invert sugar x dilution x dilution x 100
Total sugar as invert sugars (%) = --------------------------------------------------------------------------------------- x 100
titre x weight of sample taken x Volume of aliquot taken for hydrolysis ×1000


Sucrose or non-reducing sugar (%) = (% total sugar as invert sugar ¬¬­ % reducing sugar) x 0.95

Total sugar (%) = % Reducing sugars + % sucrose

Non-reducing sugars (%) = Total sugars - reducing sugar

Results: Fruit contain more reducing sugar while fruit product prepared by using cane sugar contains more non-reducing sugars.

Problem: 25gm of mango juice was neutralized with 1N NaOH and made to 250 ml and filtered. 50ml of filtrate was deleaded and made to 100ml followed by filtration. The solution was utilized for estimation of reducing sugar whose titre was 15ml. Out of this deleaded filtrate 50ml was acidified for inversion and neutralized with NaOH and made to 100ml. The sample was utilized for estimation of total sugar, the titre value of which was found to be 18ml. Calculate total and non-reducing sugars of the given mango squash. Factor for Fehling’s solution is 0.05.

Solution:
Given
  • Weight of sample taken 25g
  • Volume made 250ml
  • Volume of sample taken 50ml for deleading
  • Volume made up 100ml
  • Volume of deleaded solution taken for inversion =50ml
  • Volume made up =100ml
  • Titre for Total sugar= 20ml
  • Titre of reducing sugar = 15 ml

For total sugar
Volume of deleaded sample taken for total sugar 50 ml

Factor for Fehling=0.05

0.05 × 250 × 100 × 100
% reducing sugar = ----------------------------------------× 100
15 × 25 × 50

Total sugar as reducing sugars (%) = 6.67%

% 0.05 × 250 × 100 × 100 × 100
% Total sugar as invert sugar = ------------------------------------------× 100
18 × 25 ×50 × 50

Total sugar as invert sugar = 11.11%

% Sucrose (Non-reducing sugar)= (Total sugar as invert sugar - reducing sugar) × 0.95
= (11.11 – 6.67) × 0.95
= 4.44 × 0.95
= 4.22%
Total sugar= % reducing sugar + % non-reducing sugars

= 6. 67 + 4.22
= 10.89%

Inference: Thus, the sample of mango juice contained 6.67% reducing sugars, 4.22% non-reducing sugars and 10.89% total sugars.
Last modified: Wednesday, 7 March 2012, 10:33 AM