Lab Activity 2 Determination of Reducing & Nonreducing Sugars in Honey IUG, 2016-2017 Dr. Tarek Zaida

Background Carbohydrates with free aldehyde or ketone group have the ability to reduce Cu2+ to Cu+. Thus all monosaccharides and certain disaccharides are reducing sugars. Honey contains reducing sugars: Fructose (38%) & glucose (31%) Nonreducing sugars (1.3%) Other content (18%)

Principle of Determination 1. CuSO4 + 2NaOH Cu(OH)2 + Na2SO4 2. + Cu (OH)2 Cu(O)2(CHCOO)2NaK+ H2O 3. Cu(O)2(CHCOO)2NaK + R-CHO + H2O Cu2O + R-COOH + Rochelle salt Basically what happens is: alkaline copper solution reacts with aldehyde (reducing sugar), so that copper will be reduced.

The following would be the oxidation of Cu+ by iodine liberated from a standard solution of KI/KIO3. KIO3 + 5 KI + 3H2SO4 3 K2SO4 + 3 I2 + 3 H2O 2 Cu+ + I2 2 Cu2+ + 2I- The excess iodine is then measured by titration with standard sodium thiosulfate solution: I2 + 2S2O32- 2I- + S4O6 2- Thiosulfate ion Tetrathionate ion

Based on the above reactions, a blank titration is done. The difference between the 2 titrations gives the amount of I2 used to reoxidize Cu+ (which is proportional to the amount of reducing sugars in the sample).

Experiment Materials: Honey, Copper reagent (ready to use) 125ml erlenmeyer flask, 500ml volumetric flask, pipets 5, 50ml, graduated cylinder,100ml, 250ml beaker, funnel, 2.5x20 cm test tubes, water bath

Procedure For Total sugars: In a suitable cup weigh exact 2.0 g of honey to be tested. Transfer the 2 g of honey into a 125ml Erlenmeyer flask. Add exactly 50 ml of H2O & stir with stirring rod until honey completely dissolved.

4. From solution of number 3 transfer 5ml into a 500ml volumetric flask. 5. Add exact 5ml of 1 N H2SO4 and heat the flask for 10 min in a boiling water-bath. 6. Cool the flask under water tap & neutralize the acidity by using 1 N NaOH (using phenol-phthalein as indicator to the pink color) and dilute to the mark with water. (total solution is 500ml)

7. Take 5ml of the solution (the one in the volumetric flask) and transfer into a 2.5x20 cm test tube, then exactly add 5ml of copper reagent. 8. Mix well & loosely cover the tube, then immerse in a boiling water-bath for exact 15 min. 9. Remove the tube from water-bath, cool under water tap. Then add 5ml 1 N H2SO4 .

10. After 1-2 min and with occasional swirling, the liberated I2 is then titrated with 0.005 N Na2S2O3 solution until the yellow-brown iodine color is almost gone. 11. A few drops of starch indicator are then added and the titration is continued until the blue starch iodine color is just discharged.

For the blank titration: Using 5ml of water instead of 5 ml of sample of step 7, then continue steps to the end. The difference between blank titration & the titration of sample (represents the I2 utilized to reoxidize Cu+ ). This amount of titrant is then converted into the corresponding weight of reducing sugars. (use Shaffer-Somogyi Table for this purpose).

Determination of reducing sugars in honey: Steps 1, 2, 3, 4 are done same with total sugars, but then (5ml of sample are transferred in 500ml volumetric flask and diluted to the mark with distilled water and continue steps 7 11. The difference of the amount of sugars in hydrolyzed sample with H2SO4 and non hydrolyzed sample gives the amount of non-reducing sugars (namely sucrose).