PAG 5.2

Question 1

Equipment

Answer 1

• Benedict’s reagent
• Distilled water
• 1% glucose solution
• Unknown glucose solution
• 5 x 50 cm3 beakers
• 7 boiling tubes
• 6 x 5 cm3 syringes
• Water bath set at 100˚C
• 14 dropping pipettes
• Centrifuge
• 8 centrifuge tubes
• 8 clean cuvettes
• Colorimeter

Question 2

Method

Answer 2

1. Collect 10 cm3 of 1% glucose solution in a 50 cm3 beaker.
2. Using a clean syringe for each transfer and mixing thoroughly at each stage, make a dilution series of glucose in distilled water to give the following concentrations: 1.00%, 0.50%, 0.25%, 0.13%, 0.06%. Label these as you create them.
3. Label the 7 boiling tubes: 1.00%, 0.50%, 0.25%, 0.13%, 0.06%, 0.00% and UNKNOWN.
4. Next add 5 cm3 of the corresponding glucose concentration to each tube. Put 5 cm3 distilled water in the 0.00% tube and 5 cm3 of the Unknown glucose solution in the UNKNOWN tube.
5. Then add 2 cm3 Benedict’s reagent to each tube.
6. Place all 7 boiling tubes into the water bath and incubate for 15 minutes.
7. Remove the boiling tubes from the water bath and stand them in a rack to cool.
8. Pipette 2 cm3 of each solution into a labelled 2 cm3 centrifuge tube. It does not matter if any precipitate in the boiling tube has started to settle out.
9. Create an eighth centrifuge tube with 2 cm3 water to act as a balance tube. Then centrifuge all 8 centrifuge tubes for 2 minutes ensuring that every tube has a balancing partner opposite it in the rotor head.
10. Set the colorimeter to ‘red light’ and use distilled water in a cuvette to set 100% transmission.
11. Carefully pipette the supernatant from each centrifuge tube in turn into a clean cuvette, measure the transmission in the colorimeter and record the results in a suitable table.
12. With the results from 1.00%, 0.50%, 0.25%, 0.13%, 0.06% and 0.00% create a calibration curve.
13. Use your calibration curve to find the glucose concentration of the Unknown glucose solution and record this.

Question 3

1. Explain why the transmission of red light increases as the glucose concentration in the sample rises.

Answer 3

1. More glucose means more precipitated Cu1+ which means less dissolved Cu2+. This means less intensity of blue colouration which in turn indicates that more red light is transmitted.

Question 4

2. Why is it necessary to centrifuge the sample before taking colorimeter readings?

Answer 4

2. The precipitate must be removed from the sample before colorimetry because any suspended particles will scatter light creating a false low transmission reading.

Question 5

3. If the reading for your unknown glucose solution did not fall within the range of your calibration curve (i.e. suggests a concentration higher than 1%) what could you do to obtain an accurate value for its concentration?

Answer 5

There are several possibilities here with different burdens in terms of how much extra work is required. The easiest but least accurate (unless the unknown concentration is only just greater than 1.00%) would be to extrapolate the calibration curve. Alternatively, creating a dilution series with the unknown sample could be suggested, guaranteeing that at least one of the diluted samples will fall within the range of the existing calibration curve and then doing the maths to work out the original concentration from that. Or creating an extended calibration curve using higher concentrations of known samples would also work.