PAG 9.3

Question 1

Equipment

Answer 1

• Benedict’s reagent
• 2 dropping pipettes
• 8 boiling tubes
• 8 x 5 cm3 syringes
• Samples in beakers labelled ‘A, B, C and D’ These are 1% glucose, 1% sucrose, 1% protein and distilled water but not necessarily in that order. By performing the qualitative tests in this activity you will identify which is glucose and which is sucrose.
• Water bath set to 100˚C
• 2 mol dm-3 hydrochloric acid
• Sodium hydrogen carbonate
• Small spatula
• pH paper
• Marker pen

Question 2

Method 1: Testing for reducing sugar

Answer 2

1. Label four boiling tubes A – D and place 4 cm3 of the corresponding samples into each using a fresh syringe each time.
2. Use a dropping pipette to add ̴̴ 2 cm3 Benedict’s reagent to each of the four samples.
3. Record your observations of each sample immediately after adding Benedict’s reagent.
4. Place all four boiling tubes into the water bath.
5. After 2 minutes record your observations of each sample, replacing the samples in the water bath afterwards.
6. After a further 2 minutes (4 minutes total incubation time) remove the tubes from the water bath and record your final observations of each sample and your conclusions about the presence or absence of reducing sugar in the samples (and therefore which letter corresponds to the glucose solution).

Question 3

Method 2: Testing for non-reducing sugar

Answer 3

Note: to carry out a test for non-reducing sugars you must first test the sample for reducing sugars. Only by obtaining a negative result for reducing sugar followed by a positive result for non-reducing sugar can you be sure you have a non-reducing sugar in your sample.

1. Put 4 cm3 of each of the samples, A – D, into its own labelled boiling tube using a fresh syringe each time.
2. Use a dropping pipette to add ̴̴ 2 cm3 of 2 mol dm-3 hydrochloric acid to each of the four samples.
3. Place all four boiling tubes into the water bath and incubate for 2 minutes.
4. Remove the tubes from the water bath and allow them to cool.
5. Next neutralise the acid by gradually adding sodium hydrogen carbonate to each sample until no more effervescence is observed.
6. Check the pH with indicator paper. If the pH is still lower than pH 7, repeat step 5. It does not matter if the pH has risen slightly above pH 7.
7. Use a dropping pipette to add ̴̴ 2 cm3 Benedict’s reagent to each of the four samples.
8. Record your observations of each sample immediately after adding Benedict’s reagent.
9. Place all four boiling tubes into the water bath.
10. After 2 minutes record your observations of each sample, replacing the samples in the water bath afterwards.
11. After a further 2 minutes (4 minutes total incubation time) remove the tubes from the water bath and record your final observations of each sample and your conclusions about the presence or absence of non-reducing sugar in the samples (and therefore which letter corresponds to the sucrose solution).

Question 4

1. What improvements can you suggest to the method you were given for this qualitative test?

Answer 4

1. More accurate measuring of the Benedict’s reagent would be an obvious improvement. Additional repeats might be suggested. Depending on the way in which the boiling tubes were labelled, students might comment on difficulties with labels becoming smudged or illegible in the water bath.

Question 5

2. What changes would you make to obtain semi-quantitative results?

Answer 5

2. This test is already effectively semi-quantitative. However, greater accuracy with administering the Benedict’s would be desirable. The key point is simply that to interpret the test semi-quantitatively the observer needs to know the sequence of colours that will be seen in the presence of increasing concentrations of reducing sugar: green, yellow, orange, red. Students might suggest using the rapidity with which a particular colour is achieved as a discriminator, and realise that it will allow discrimination even when the end point colours are indistinguishable.

Question 6

3. Outline a method for a fully quantitative test for reducing sugars.

Answer 6

3. Methods relying on colorimetry are good. If students have not previously been taught the theory of the quantitative test many will quite sensibly focus on the idea of a more intense red colour as the indicator of increased reduction (and hence greater reducing sugar concentration). They might suggest measuring absorbance of blue light. Details relating to the idea of filtering or centrifuging to remove the precipitate (and then realising that the paler the remaining solution the more reduction must have taken place) make this idea even better. Calibration curves made using known concentrations of a reducing sugar (e.g. glucose) will be needed to convert colorimeter readings to sugar concentration. Students could also suggest methods relying on weighing the mass of precipitate.

Question 7

4. Suggest how you could distinguish a sample of glucose from a sample of maltose using Benedict’s reagent. [Hint: maltose is a disaccharide reducing sugar].

Answer 7

4. A semi-quantitative test for reducing sugar followed by a semi-quantitative test for non-reducing sugar should allow glucose and maltose to be distinguished. Hydrolysis of maltose will yield two moles of glucose for every mole of maltose so there will be more reducing sugar following hydrolysis. Therefore there will be an observable difference between the two maltose tests.