The purpose of the document is to establish a general and uniform method for the determination of total reducing sugar and sucrose in honey.
2.0 SCOPE
This standard testing procedure is suitable for the determination of total reducing sugar and sucrose in honey.
3.0 RESPONSIBILITY
The expert and trained chemist are responsible for following this procedure. The technical manager is responsible for implementing this procedure.
4.0 REQUIREMENTS
4.1 Apparatus
4.1.1 Heating Mantle
4.2 Glassware
4.2.1 Burette-50 ml
4.2.2 Pipette-5 ml,10 ml
4.2.3 Conical flask-250 ml
4.2.4 Volumetric flask-250 ml,100ml
4.3 Reagent
4.3.1 Fehling’s solution: equal volumes of Solution A+ Solution B (immediately before use.)
4.3.2 Solution A: 34.639 g of copper sulfate crystals in water dilute to 500 ml and filter.
4.3.3 Solution B: Potassium Sodium tartrate (Rochelle salt) 173 g of Potassium sodium tartrate, 50 g of sodium hydroxide in water, dilute to 500 ml. Let the solution stand for a day and filter.
4.3.4 Hydrochloric acid: (Conc.)
4.3.5 Methylene blue indicator: 0.2 % in water
5.0 PROCEDURE FOR TOTAL REDUCING SUGAR
5.1 Take 1g of the sample into a 250 ml volumetric flask, and dilute with 150 ml of distilled water.
5.2 Mix & make up to 250 ml with distilled water (stock solution).
5.3 Take 5 ml each of solution A and solution B in a 250 ml conical flask, add 12 ml honey solution from a burette in it & heat it on asbestos gauze.
5.4 Add 1 ml of methylene blue indicator and complete the titration within 3 min in boiling condition.
5.5 The endpoint is brick red. Note the volume.
5.6 CALCULATION
Total reducing sugars (%by mass) = 250x100xS
H1 x M
Where,
S = Strength of copper sulfate solution
H1 = Volume of Honey solution required for titration (in ml) (Before inversion)
M = Weight of honey (in g)
· Preparation of Standard Invert Sugar solution: Dissolve 0.95 g Sucrose in 500 ml of water, add 2 ml conc. HCl, boil gently for 30 min and kept for 24 hrs. Neutralize with Na2CO3 and makeup to 1000 ml.50 ml of this solution contains 0.05g inverted sugar.
· Standardization of Copper sulfate solution: Take 5 ml solution A and 5 ml solution B into a 250 ml conical flask, add approx.50 ml of standard Invert sugar solution from a burette, and boil. Add 1 ml of Methylene blue indicator and titrate again till the endpoint is blue to red. From the volume of inverted sugar solution used calculate the strength of copper sulfate solution by multiplying the titer value with 0.001(mg/ml of the standard. Invert sugar solution). This gives the quantity of inverted sugar required to reduce the Copper in 5ml of copper sulfate solution.
6.0 PROCEDURE FOR SUCROSE
6.1 Take 100 ml of honey solution from the 250 ml stock solution.
6.2 Add 1 ml conc. HCl & heat to boiling.
6.3 Keep undisturbed overnight.
6.4 Neutralize with sodium carbonate and determine total reducing sugars(after inversion),as described in clause 5.0(5.3 to 5.5).
6.5 CALCULATION
Total reducing sugars (%by mass) = 250x100xS
H2 x M
Where,
S = Strength of copper sulfate solution
H2 = Volume of Honey solution required for titration (in ml) (After inversion)
M = Weight of honey (in g)
Sucrose % by mass = {(reducing Sugars after inversion, % by mass) – (Reducing sugar before inversion, % by mass)} x 0.95.
7.0 REVISION/CHANGE HISTORY
8.0 ATTACHMENTS
· Appendix-1: FLOW CHART FOR TOTAL REDUCING SUGAR
· Appendix-2: FLOW CHART FOR SUCROSE
9.0 REFERENCES
· Annex C of IS 4941:1994;RA 2008; Amendment -1,2011
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