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Lab Activity 7Determination of Reducing & Nonreducing Sugars in Honey

IUG, Fall 2013Dr. Tarek Zaida

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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:1. Fructose (38%) & glucose (31%)2. Nonreducing sugars (1.3%)3. Other content (18%)

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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.

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• 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 + 2S2O3

2- 2I- + S4O6 2-

Tetrathionate ionThiosulfate ion

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• 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).

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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

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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 H2O & stir with stirring

rod until honey is completely dissolved.

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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)

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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

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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.

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• 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).

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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).