1. Testing for Reducing sugars Benedict’s Test
All monosaccharides and some disaccharides are reducing sugars
(This means that they provide electrons that can carry out a reduction)
Benedict’s Reagent detects the presence of a reducing sugar
Lactose and maltose are directly detected by Benedict's reagent, because each contains glucose with a free reducing group
Sucrose is a non-reducing sugar

2. Benedict’s test Benedict’s reagent contains copper(II)sulphate CuSO4
Heat a reducing sugar with Benedict’s reagent and an insoluble precipitate is formed
The copper is reduced by the sugar and insoluble copper(I)oxide Cu2O produced
(OILRIG: Reduction is gain: Cu2+ gains an electron and becomes Cu+)
The clear blue solution changes colour depending on the quantity of reducing sugar present
Textbook pp

3. Benedict test for reducing sugars – risk assessment
Hazard
Precautions
Remedial measures
Glassware breakage
Keep test tubes in rack/waterbath to prevent rolling of bench
Staff to clear up any broken glass
Cuts – seek first aid
Copper sulphate solution - toxic
Wear safety goggles
Carry dropper bottle by glass
Wash hands after practical
Wash off with copious amounts of water
Ingested – seek first aid
Waterbath – hot
Care with hot water
Scalds – run under cold water and seek first aid

4. Learning Objectives
You will have been successful if by the end of the lesson you can:
Explain how to test for reducing sugars
Demonstrate skilful and safe practical techniques using suitable qualitative/quantitative method
Follow instructions to practise the preparation of a serial dilution
Make and record valid observations.
Use the Benedict’s Test to carry out semi-quantitative method of determining concentration of a reducing sugar and explain the results.

5. Semi-quantitative assessment of reducing sugar content
No reducing sugar High
Produce a range of colour standards with known concentrations of glucose and these can be used to compare the result of a test carried out with an equal volume of an unknown solution e.g. orange juice
Textbook pp

6. Concentration of sugar

7. One method for creating a range of dilutions
Volume of 0.08 mol dm-3 glucose solution (cm-3)
Volume of deionised water (cm-3)
Final concentration of glucose solution (mol dm-3)
10.0
0.0
0.08
7.5
2.5
5.0

8. Serial dilution Making progressively more dilute solutions from the previous concentration
4% glucose solution = 4g glucose in 100cm3 de-ionised water
We can produce a 2% solution from a 4% solution :
5cm3 of 4% glucose solution
+ 5cm3 de-ionised water
= 10cm3 of 2% glucose solution
5 cm3 original concentration
In a total of 10cm3
5/10 = 1/2 original concentration
More concentrated solution
More dilute solution
+5cm3 de-ionised water
5cm3 8

9. Making Glucose Concentrations.
Notice that the table is arranged with the lowest glucose concentration at the top even though you will start with the greatest concentration
Volume of
de-ionised water
(cm3)
Volume and conc. of glucose solution
Total volume (cm3)
Concentration of glucose solution (%) 10
0.00 5
0.25
0.50
1.00
2.00
10 of 4.00%
4.00
Textbook pp

10. Making Glucose Concentrations.
Volume of
de-ionised water
(cm3)
Volume and conc. of glucose solution
Total volume (cm3)
Concentration of glucose solution (%) 10
0.00 5
0.25
0.50
1.00
2.00
10 of 4.00%
4.00
Control
To show that....
You are given a stock solution of 4% glucose.
Complete this row first, then think about diluting the solution you have just made to get the row above
Textbook pp

11. Volume of
de-ionised water
(cm3)
Volume and conc. of glucose solution
Total volume (cm3)
Concentration of glucose solution (%) 10
0.00 5
5 of 0.5%
0.25
5 of 1%
0.50
5 of 2%
1.00
5 of 4%
2.00
10 of 4%
4.00
Why 4.00, not 4?
Textbook pp

12. How can we display this data?
Is this data quantitative or qualitative?
What is the independent variable?
What is the dependent variable?
Where should these be positioned in a results table?
Independent Variable /units
Dependent Variable Trial 1/ units
Dependent Variable Trial 2/ units
Dependent Variable Trial 3 / units
Mean result / units

13. Results table 0.25 0.50 1.00 2.00 4.00 Final colour of mixture
Concentration of glucose solution (%)
Final colour of mixture
Final cloudiness of tube (amount of precipitate)
0.25
0.50
1.00
2.00
4.00

14. Unknown solution Final colour of tube ………………………..
Final cloudiness of tube ………………….
Concentration of reducing sugar is ………………………………………………..
Hmmm – we can’t be precise about the value – this is why this method is semi-quantitative
The answers here relate to the skilful practice assessment of coursework

15. Benedict’s test Benedict’s reagent contains copper (II) sulphate
Heat a reducing sugar with Benedict’s reagent and an insoluble precipitate is formed
The copper ions are reduced by the sugar and insoluble copper (I) oxide produced
(OILRIG: Reduction is gain: Cu2+ gains an electron and becomes Cu+)
The solution is clear blue initially and changes colour depending on the quantity of reducing sugar present

16. Learning Objectives
You will have been successful if by the end of the lesson you can:
Explain how to test for reducing sugars
Demonstrate skilful and safe practical techniques using suitable qualitative/quantitative method
Follow instructions to practise the preparation of a serial dilution
Make and record valid observations.
Use the Benedict’s Test to carry out semi-quantitative method of determining concentration of a reducing sugar and explain the results.

17. Learning Objectives
You will have been successful if by the end of the lesson you can:
Evaluate data and practical skills by:
Identifying and explaining the main limitations of the data collection strategy.
Suggesting and giving reasons for simple improvements to the experiment;
Commenting upon the reliability of the data collected; and discussing the validity of the conclusions.

18. What are the limitations of this method?
Semi quantitative = subjective
Difficult to describe the colour change and cloudiness.

19. How to make the Benedict’s test quantitative?
We need to know exactly how much of the Cu2+ has been reduced
Measure the cloudiness of the solution (indicating the amount of precipitate) with a colorimeter
Measure the ‘blueness’ of the remaining copper sulphate solution using a colorimeter
Measure the mass of precipitate (will have to be fully dry)

20. Another Serial Dilution
The test tubes contain a series of solutions, each one more dilute than before
9cm3 of de-ionised water +
1cm3 of 10% glucose solution
1cm3 of 10% glucose in 10 cm3 = 1/10 of original concentration
=0.1% glucose solution
1cm3
9cm3 water
10% glucose solution
= 1% glucose solution
Benedict’s test results?
0.01%
0.001% 1%
0.1%
10%

21. Look – more red precipitate, and the solution is less blue

22. Using a colorimeter
Choose a filter with a complementary colour to your standard solution
The deepest colour should absorb the most – and give the highest absorbance reading
Interactive
colourimeter
A graph can be plotted and used to determine the concentration of glucose in an unknown solution
Textbook pp

23. Calibration curve
Use solutions of known concentrations to produce a set of values.
Plot a calibration curve
Find a value for the unknown concentration
Draw intercepts on the graph to determine the concentration of the unknown solution
Textbook pp

24. sample results
Concentration of reducing sugar solution (%)
Colorimeter reading (Absorbance)
4.00
0.05
2.00
0.25
1.00
0.65
0.50
0.96
1.15
Orange juice
0.12
Textbook pp

25. Colorimeter reading
(Absorbance)
Textbook pp

26. Calibration curve 2 Mass of precipitate
The unknown solution gives a value of 11g
Using the calibration curve this gives a concentration of 2.75%
Textbook pp

27. Learning Objectives
You will have been successful if by the end of the lesson you can:
Evaluate data and practical skills by:
Identifying and explaining the main limitations of the data collection strategy.
Suggesting and giving reasons for simple improvements to the experiment;
Commenting upon the reliability of the data collected; and discussing the validity of the conclusions.

28. Match up! A ….make an experiment more reliable 1. A colorimeter
B …..make an experiment more valid
2. Calibration curve
C …..gives quantitative objective data
3. Repeats used to calculate a mean
D ……make an experiment more precise
E …..gives qualitative subjective data
4. Controlling all the variables, and being accurate
F …….allows the concentration of an unknown solution be found from a set of samples of known concentration
5. A control, with no glucose present,
G …..gives a colour standard to compare against

29. Match up! 1 C 2 F 3 A 4 B 5 G A ….make an experiment more reliable
1. A colorimeter
1 C
2 F
3 A
4 B
5 G
B …..make an experiment more valid
2. Calibration curve
C …..gives quantitative objective data
3. Repeats used to calculate a mean
D ……make an experiment more precise
E …..gives qualitative subjective data
4. Controlling all the variables, and being accurate
F …….allows the concentration of an unknown solution be found from a set of samples of known concentration
5. A control, with no glucose present,
G …..gives a colour standard to compare against

30. Answers to practical questions
stirred both / left to settle;
compared colours;
compared, amount of precipitate / opacity; max 2

31. Answers to practical questions
2 not a precise match;
subject to colour judgement;
intermediate values;
colour of juice may impair;
difficulty in matching cloudiness by eye;
use of a background;
restirring to overcome settling;
take into account cloudiness or precipitate;
AVP; max 6

32. Answers to practical questions
3 mark one method only
filter / settle;
colorimeter;
ref transmission / absorbance;
use more intermediates of colour comparisons;
filter;
dry;
take mass;
max 3

33. Answers to practical questions
4 five concentrations used gives a (wide) range;
different results obtained for each % of glucose;
ref to anomalous results;
range of standards encompasses that of juices;
standardised (boiling) time;
long enough for reduction of, Benedict's / Cu;
volume of Benedict’s to test solution gives excess Cu ions / AW;
timing of stirring consistent;
standardised volumes;
measure accurately with syringes;
clean syringes;
clean dry rod;
reason for clean syringes or rod / non-contamination;
using cloudiness and colour enables two ways to make a comparison;
results did not have to be collected while test tubes were in water bath;
results did not have to be taken at same time;
therefore results more likely to be accurate;
AVP; max 9

34. Answers to practical questions
5 other reducing sugars may be present;