AS Biology Practical Topic 1: Lifestyle Health and Risk
Background
Benedict’s solution reacts with reducing sugars on heating and reduces the Cu(II) ion to Cu(I) producing a precipitate of red copper oxide. The resulting colour change depends on the type and concentration of reducing sugar, so this test can be used semi-quantitatively to indicate approximate concentrations .
This investigation allows you to develop your skills in:

Following instructions

Constructing a calibration curve

Estimating unknown amounts.
AIM
You are required to estimate the reducing sugar content of samples of three different fruit juices, A,
B and C using two different methods
Method 1
This will be done in two stages.
In stage one, you will use a series of five glucose solutions of known concentrations to prepare five colour standards.
In stage two, you will use these colour standards to determine the reducing sugar content of the three fruit juices.
Stage 1
Carry out Benedict’s tests on the five different solutions of glucose with which you have been provided as follows:
1.
Label six test tubes 0%, 0.1%, 0.5%, 1%; 2% and 4% respectively
2.
Add 5cm 3 of Benedict’s solution to each tube
3.
Add 0.5 cm 3 of each of the glucose solutions to the appropriate test tubes, using a clean syringe each time.
4.
Stir the contents of each tube with a clean, dry, glass rod.
5.
Place all five test tubes in the boiling water for TWO minutes
6.
After two minutes, carefully remove the tubes from the boiling water and place them in a rack.
7.
Using a clean dry glass rod stir the contents of each tube.
8.
Observe the contents of the tubes carefully immediately after stirring.
9.
Record your observations in the table below.
Note particularly any differences you observe between the contents of the tubes, both in terms of colour and cloudiness.

Results glucose concentration(%) mass of glucose/mg
Colour
Cloudiness
0
0
0.1
0.5
0.5
2.5
1.0
5.0
2.0
10.0
4.0
20.0
Stage 2
Carry out Benedict’s tests on the samples of fruit juices A, B and C
Use 0.5cm 3 of each fruit juice and 5 cm 3 of Benedict’s solution
Refer to the colour standards that you prepared in Stage 1
Use the colour standards and the results from the fruit juices to estimate the mass of reducing sugar,
in mg, in each fruit juice.

METHOD 2
To measure the concentration of reducing sugar in the solutions we can use a colorimeter. This moves this practical from qualitative to quantitative.
Using Benedict’s test will reveal the presence of reducing sugars
 It results in an orange-red precipitate
 The more reducing sugar there is present, the more precipitate will be formed, and the more Benedict’s solution (copper sulphate) will be used up
 The precipitate is filtered out then the concentration of the remaining solution can be measured
 This will tell you how much Benedict’s solution has been used up allowing you to estimate the concentration of reducing sugar in the original sample
Using a colorimeter
A device called a colorimeter can be used to make more accurate measurements. This device shines a beam of light through a prepared sample, and a reading is measured of percentage light transmission or absorbance
The solution is placed in a clear plastic cuboid called a cuvette which then goes into a small chamber in the colorimeter
The colorimeter shines a beam of light through the sample.
A photoelectric cell picks up the light that is passed through the sample (on the other side) and will provide you with a reading of the amount of light that was passed through – transmitted or absorbed
The more copper sulphate that has been used in the Benedict’s test, the less light will be blocked out in the sample, and more transmitted/less absorbed. Therefore the reading gives a measure of the amount reducing sugar based on the Benedict’s reaction.
Method
For each of the known glucose concentrations – leave the precipitate to settle and pipette off the supernatant or spin in a centrifuge.
Zero the colorimeter with a blank – in this case Benedict’s solution with a red filter
Place the supernatant in a cuvette and read the absorbance with a red filter.
Plot the readings in a graph to show the absorbance versus reducing sugar concentration

Concentration of Glucose
Solution (%)
0
0.1
0.5
1.0
2.0
4.0
Replicate 1
________________
Replicate 2
Fruit Juice results – construct a table of results
Replicate 3
Mean value

Discussion
Method 1
1.
State how you made a comparison between your results from the fruit juices and the colour standards.
2.
Explain the difficulties you found above and describe how you tried to overcome them.
Method 2
3.
What are the mean concentrations of reducing sugar in the unknown fruit juice samples?
4.
What do the range bars tell you about the reliability of the results for both the calibration curve and for fruit juices A, B and C?
5.
Comment on the validity of method 2
6.
Suggest one reason why the reducing sugar content of fruit juices is likely to be different from their glucose content.