Testing for Reducing sugars Benedict’s Test

1. Testing for Reducing sugarsBenedict’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 pp112-114

3. Benedict test for reducing sugars – risk assessment

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 pp112-114

6. Concentration of sugar

7. One method for creating a range of dilutions

8. Serial dilutionMaking progressively more dilute solutions from the previous concentration +5cm3 de-ionised water 5cm3 More dilute solution More concentrated solution 4% glucose solution = 4g glucose in 100cm3 de-ionised water We can produce a 2% solution from a 4% solution : 5cm3 of4% 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

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 Textbook pp112-114

10. Making Glucose Concentrations. 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 pp112-114

11. Why 4.00, not 4? Textbook pp112-114

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?

13. Results table

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. 0.001% 0.01% 0.1% 1% 10% 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?

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 pp113-114

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 pp113-114

24. sample results Textbook pp113-114

25. Colorimeter reading (Absorbance) Textbook pp113-114

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 pp113-114

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. 2. Calibration curve F …….allows the concentration of an unknown solution be found from a set of samples of known concentration Match up! A ….make an experiment more reliable 1. A colorimeter B …..make an experiment more valid 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 5. A control, with no glucose present, G …..gives a colour standard to compare against

29. 2. Calibration curve F …….allows the concentration of an unknown solution be found from a set of samples of known concentration Match up! A ….make an experiment more reliable 1 C 2 F 3 A 4 B 5 G 1. A colorimeter B …..make an experiment more valid 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 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; 1