Description of the test QUICK STARTER: Can you complete the summary table on the practical biochemistry tests for the presence of carbohydrates? Test for Description of the test Expected result Starch Reducing sugars Non-reducing sugars

Description of the test Test for Description of the test Expected result Starch Add a few drops of iodine solution Brown to blue-black Reducing sugars Add Benedict’s solution, heat to 80°C in a water bath Blue to orange-red Non-reducing sugars (If reducing sugar test is negative) boil with hydrochloric acid, cool and neutralise with sodium carbonate solution, repeat Benedict’s test Initially no change, repeated Benedict’s test will turn blue to orange-red

Benedicts test Testing for reducing sugars: A reducing sugar is a monosaccharide or a disaccharide. Reducing: A molecule of the sugar can react with other molecules giving electrons to them (OIL RIG) Benedict’s solution is a turquoise/blue solution containing copper ions and sodium hydroxide; the copper ions exist as Cu2+in this reagent If a sugar is a reducing sugar then the Cu2+ ions are reduced to Cu+ which, in the presence of alkaline sodium hydroxide, form copper oxide Copper oxide is insoluble and precipitates out of the solution as a brick-red precipitate

When a reducing sugar is heated with Benedict’s solution (alkaline copper sulphate) the solution will change from blue to an orange red (Benedict’s test) The orange red is described as a precipitate because it has come out of solution and forms suspended dispersed solid particles The result is positive if there is a colour change and negative if not. The colour scale below is used as a “results range” for the test which is used to describe the amount of reducing sugar in a sample based on the strength of the colour and the colour change: (nothing) blue → green → yellow → orange → red (lots) BUT We call these tests semi-qualitative because they don’t produce quantifiable results.

Semi quantifiable: Using the Benedict’s test reveals the presence of reducing sugars, resulting in an orange-red precipitate and the more reducing sugar there is the more copper sulphate (Benedict’s solution) will have been used up, so the precipitate can be filtered and the concentration of the remaining solution measured telling us how much Benedict’s solution has been used up allowing an estimate of the concentration of reducing sugar in the original sample You would then look at the degree of the colour change (nothing) blue → green → yellow → orange → red (lots) Could have a known standard solution which you could compare to

Obtaining quantifiable results: Colorimeter : a device which shines a beam of light through a sample calculating percentage light transmission; The sample is placed into a cuvette which goes into the colorimeter, and then a photoelectric cell picks up on the amount of light transmitted, and the reading gives a measure of the amount of reducing sugar, based on the principle that the more copper sulphate that has been used up the less light will be blocked out

Operating instructions for a typical colorimeter Switch on the instrument at least 5 minutes before use to allow it to stabilise. Select the most appropriate filter for the analysis and insert it in the light path Colour choice of filter: Why red on a blue/green solution: The blue solution appears blue as all other colours have been preferentially absorbed. The red is the most strongly absorbed Place the reagent blank solution/or water in the cuvette and zero the instrument (consult your manufacturers instruction about how to do this.) Place the sample in the colorimeter and read the absorbance of the solution. If the absorbance is "over range" (usually > 2.0) then the sample must be diluted to yield a value within the limits of the instrument. At intervals, recheck the reagent blank to ensure that there is no drift in the zero value

Less transmission=less sugar Medium transmission= medium sugar Sugar solution + Benedicts HEAT Precipitate in solution Filter precipitate out Filtrate DARK BLUE MEDIUM BLUE COLOURLESS Transmission in colorimeter Less transmission=less sugar Medium transmission= medium sugar Highest transmission= Highest sugar Higher concentration of reducing sugar, the more blue Benedicts solution will be used, more precipitate formed SO the filtrate will be clearer so more transmission

Benedicts test results Plot onto a graph Concentration of glucose /g dm³ % Transmission 10 98 5 78 2 36 1 12 0.1

Quantify: Calibration curve Whilst using a colorimeter alone will provide a measure, it doesn’t specify an exact amount: in order to quantify the amount, a calibration curve must be made… Take a range of known concentrations of reducing sugar, carry out a Benedict’s test on each one, then filter out the solution; use a colorimeter to give readings of the amount of light passing through the solutions (transmitted) Plot the readings in a graph to show the amount of light getting through (transmission) against reducing sugar concentration Then you can take the reading of an unknown concentration – use the graph to make a precise measurement This is known as an assay.

Colorimeters Can also measure absorption Colorimetry also works because of light absorbance. A substance in a solution absorbs light, which decreases the amount of light that passes through the solution. The amount of light absorbed by a solution is related to the concentration of a substance in that solution. What would your results be if you were measuring absorption? Less absorbance= more sugar To calculate % absorbance from a transmission reading: = 100% – transmission % = absorbance % How else could quantifiable results be obtained??? THINK?SUGGEST?

Quantifiable results: Filter the precipitate and weigh it Greater mass= more sugar present Could compare to a standard curve Centrifuge: look at the size of the pellet produced/ colour of the liquid, to indicate the concentration

Exam question: 6 marks The activity of an enzyme can be measured by testing for the concentration of its product at regular intervals. Describe how the concentration of a reducing sugar can be measured using a colorimeter.

5 Marks

PAG 5.2: Determining glucose concentration So.. You have already done this at AS you just need to understand how you could make your own calibration curve Aim: To determine the concentration of glucose in a solution of unknown concentration Aim: To create a calibration curve and use this to determine (quantitatively) the glucose concentration of a solution of unknown concentration using colorimetry.

Practical skills 1.2.1 (b) Safely and correctly use a range of practical equipment and materials 1.2.1 (c) Follow written instructions 1.2.1 (d) Make and record observations/measurements 1.2.1 (e) Keep appropriate records of experimental activities 1.2.1 (f) Present information and data in a scientific way 1.2.1 (j) Use a wide range of experimental and practical instruments, equipment and techniques appropriate to the knowledge and understanding included in the specification 1.2.2 (a) Use of appropriate apparatus to record a range of quantitative measurements 1.2.2 (b) Use of appropriate instrumentation to record quantitative measurements, such as a colorimeter 1.2.2 (c) Use of laboratory glassware apparatus for a variety of experimental techniques to include serial dilutions

Mathematical Skills M0.1 Recognise and make use of appropriate units in calculations M0.2 Recognise and use expressions in decimal & standard form M1.1 Use an appropriate number of significant figures M3.1 Translate information between graphical, numerical and algebraic forms M3.2 Plot two variables from experimental or other data M3.4 Determine the intercept of a graph

Preparing dilutions of glucose You will make up using serial dilutions glucose at a selection of concentrations: 1%,0.50%,0.25%,0.13%, 0.06% Plus one at 0.00% and 1 unknown (7 tubes) You put 1cm³ of glucose solution into each test tube plus 10cm³ of Benedict's reagent Water bath for 15 minutes Allow to cool Pipette 2cm³ of each solution into centrifuge tube: centrifuge, pipette supernatant into clean cuvettes Set colorimeter to red filter Calibrate with distilled water to 100% transmission With the results create a calibration curve Use calibration curve to find glucose concentration of unknown substance Can leave the tubes overnight, why? Instead of using a centrifuge it gives the precipitate time to settle out

How are you going to record the data? % Transmission Transmission data and the concentration of glucose SKETCH OUT THE GRAPH Sketch it for absorbance! Glucose concentration g dm3

Think: Q: Explain why the transmission of red light increases as the glucose concentration in the sample rises. Q: Why is it necessary to centrifuge the sample before taking colorimeter readings? Q: 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?

Think: Q: Explain why the transmission of red light increases as the glucose concentration in the sample rises. More glucose means more reduction of Cu2+ to Cu1+ which means less dissolved Cu2+. This means less intensity of blue colouration which in turn indicates that more red light is transmitted. Q: Why is it necessary to centrifuge the sample before taking colorimeter readings? The precipitate must be removed from the sample before colorimetry because any suspended particles will scatter light creating a false low transmission reading. Q: 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? There are several possibilities 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. Alternatively creating an extended calibration curve using higher concentrations of known samples would also work.