Key Terms Starter Here are some terms we have discussed in previous practical work. What does each one mean? Validity Controlled variable Independent variable Dependent variable Subjectivity Limitation

Lesson Focus Key terminology frequently required for coursework/experimental questions Further development of skills needed for Unit F213 (Coursework)

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. Objectives 2,and 3 as described by the OCR Specification for Unit F213 Textbook pp112-114

Learning Objectives You will have been successful if by the end of the lesson you can: 4. 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.

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

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

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 Textbook pp112-114

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. Textbook pp112-114

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

Concentration of sugar

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

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 12

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 pp112-114

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 pp112-114

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 pp112-114

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

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

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

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 Textbook pp112-114

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. Textbook pp112-114

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.

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

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) Textbook pp112-114

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%

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

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

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

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 pp113-114

Colorimeter reading (Absorbance) Textbook pp113-114

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

Will results produced through a quantitative method be more: Valid Reliable Accurate Precise Yes. Other variables are being controlled and measured No. Without replicates, we cannot judge reliability No. This assesses how true to the real value the result are and this reflects the design and execution of the method No as the units of measurement have not changed.

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.

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

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

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

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

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

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

Answers to practical questions 5 other reducing sugars may be present; 1