1. IUG, Fall 2013 Dr. Tarek Zaida 1

2. Background Carbohydrates with free aldehyde or ketone group have the ability to reduce Cu 2+ to Cu +. Thus all monosaccharides and certain disaccharides are reducing sugars. Honey contains reducing sugars: 1.Fructose (38%) & glucose (31%) 2.Nonreducing sugars (1.3%) 3.Other content (18%) Carbohydrates with free aldehyde or ketone group have the ability to reduce Cu 2+ to Cu +. Thus all monosaccharides and certain disaccharides are reducing sugars. Honey contains reducing sugars: 1.Fructose (38%) & glucose (31%) 2.Nonreducing sugars (1.3%) 3.Other content (18%) 2

3. Principle of Determination 1. CuSO 4 + 2NaOH Cu(OH) 2 + Na 2 SO 4 2. + Cu (OH) 2 Cu(O) 2 (CHCOO) 2 NaK + H 2 O 3. Cu(O) 2 (CHCOO) 2 NaK + R-CHO + H 2 O Cu 2 O + R-COOH + Rochelle salt Basically what happens is: alkaline copper solution reacts with aldehyde (reducing sugar), so that copper will be reduced. 1. CuSO 4 + 2NaOH Cu(OH) 2 + Na 2 SO 4 2. + Cu (OH) 2 Cu(O) 2 (CHCOO) 2 NaK + H 2 O 3. Cu(O) 2 (CHCOO) 2 NaK + R-CHO + H 2 O Cu 2 O + R-COOH + Rochelle salt Basically what happens is: alkaline copper solution reacts with aldehyde (reducing sugar), so that copper will be reduced. 3

4. The following would be the oxidation of Cu + by iodine liberated from a standard solution of KI/KIO 3. KIO 3 + 5 KI + 3H 2 SO 4 3 K 2 SO 4 + 3 I 2 + 3 H 2 O 2 Cu + + I 2 2 Cu 2+ + 2I - The excess iodine is then measured by titration with standard sodium thiosulfate solution: I 2 + 2S 2 O 3 2- 2I - + S 4 O 6 2- The following would be the oxidation of Cu + by iodine liberated from a standard solution of KI/KIO 3. KIO 3 + 5 KI + 3H 2 SO 4 3 K 2 SO 4 + 3 I 2 + 3 H 2 O 2 Cu + + I 2 2 Cu 2+ + 2I - The excess iodine is then measured by titration with standard sodium thiosulfate solution: I 2 + 2S 2 O 3 2- 2I - + S 4 O 6 2- Tetrathionate ionThiosulfate ion 4

5. Based on the above reactions, a blank titration is done. The difference between the 2 titrations gives the amount of I 2 used to reoxidize Cu + (which is proportional to the amount of reducing sugars in the sample). Based on the above reactions, a blank titration is done. The difference between the 2 titrations gives the amount of I 2 used to reoxidize Cu + (which is proportional to the amount of reducing sugars in the sample). 5

6. 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 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 6

7. Procedure  For Total sugars: 1.In a suitable cup weigh exact 2.0 g of honey to be tested. 2.Transfer the 2 g of honey into a 125ml Erlenmeyer flask. 3.Add exactly 50 ml of H 2 O & stir with stirring rod until honey is completely dissolved.  For Total sugars: 1.In a suitable cup weigh exact 2.0 g of honey to be tested. 2.Transfer the 2 g of honey into a 125ml Erlenmeyer flask. 3.Add exactly 50 ml of H 2 O & stir with stirring rod until honey is completely dissolved. 7

8. 4. From solution of number 3 transfer 5ml into a 500ml volumetric flask. 5. Add exact 5ml of 1 N H 2 SO 4 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) 4. From solution of number 3 transfer 5ml into a 500ml volumetric flask. 5. Add exact 5ml of 1 N H 2 SO 4 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) 8

9. 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 H 2 SO 4. 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 H 2 SO 4. 9

10. 10. After 1-2 min and with occasional swirling, the liberated I 2 is then titrated with 0.005 N Na 2 S 2 O 3 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. 10. After 1-2 min and with occasional swirling, the liberated I 2 is then titrated with 0.005 N Na 2 S 2 O 3 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. 10

11. 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 I 2 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). 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 I 2 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). 11

12.  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 H 2 SO 4 and non hydrolyzed sample gives the amount of non- reducing sugars (namely sucrose).  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 H 2 SO 4 and non hydrolyzed sample gives the amount of non- reducing sugars (namely sucrose). 12