Schools' Analyst Irn Bru Practical...Page 1 Royal Society of Chemistry Analytical Division - NE region Schools' Analyst competition heat 2003 Name... School....
Schools' Analyst Irn Bru Practical...Page 2 Health and Safety This is a practical exercise, so normal rules for safety in the laboratory apply. Wear laboratory coats and safety spectacles at all times. Do NOT eat or drink in the laboratory. Always use the pipette fillers provided, and handle glassware carefully to avoid breakages and cuts. Keep long hair under control. IF IN DOUBT ABOUT ANYTHING THEN ASK A DEMONSTRATOR.... Nomenclature: You will find that volumes are given as litres (L) or millilitres (ml). Remember that 1 L = 1 dm 3 and 1 ml = 1 cm 3. There are answer sheets at the back of each of these instruction booklets for your individual use. You may keep this instruction bookelt after the exercise. Each team must complete one answer booklet, attach graphs to it and submit it at the end of the exercise.
Schools' Analyst Irn Bru Practical...Page 3 The Problem "Made in Scotland from girders" the adverts used to proclaim. Now the soft drink Irn-Bru, a Glaswegian delicacy, has gone techno: http://www.irn-bru.co.uk/irnbru.asp What exactly is in it though, and are there likely to any effects on health if large amounts are consumed? Tha label indicates ferric ammonium citrate at 0.002% plus a preservative (E211, sodium benzoate) and caffeine at unspecified concentrations. You will check that the contents do seem to fit the label description, and measure the concentrations of the unquantified ingredients. You will also recommend a maximum allowable consumption in order to stay within guidelines for the ingestion of caffeine. There are three experiments for you to do. Each will take about 2 hours if you work efficiently: i) Measure the amount of iron present in the drink. The method that you will use for this is called colorimetric analysis. ii) Extract some of the organic components of the drink and identify them by thin layer chromatography. iii) Extract the caffeine and determine its concentration by uv spectrophotometry. You need to plan your work carefully to allow completion of the exercise. You should allow at least 30mins at the end for calculations and completion of the questions. What is the best way to organise the work within your team? Show this as a diagram below:
Schools' Analyst Irn Bru Practical...Page 4 Experiment 1 Determination of iron by colorimetry In this experiment you will determine the concentration of iron by complexing with 1,10-phenanthroline. The complex between ferrous ion and 1,10-phenanthroline is red-orange in colour, and its concentration can be measured by colorimetry. In order to determine the total iron content you must first reduce the ferric form to the ferrous form with hydroxylamine, then determine with 1,10-phenanthroline. Equipment Uv/visible spectrophotometer (colorimeter) Plastic 1 cm pathlength cells Pipettes Pipette fillers Pasteur pipettes Volumetric flasks (15 x 100mL plus others) Storage bottles (if insufficient volumetric flasks) Distilled water Stock iron solution (ferrous ammonium sulphate, 2.00 x 10-3 mol L -1 ) 1,10-phenanthroline solution (10.0 x 10-3 mol L -1 in water) Hydroxylammonium chloride solution (1.00 x 10-2 mol L -1 in water) Procedure - colorimetry 1 Prepare a series of standard solutions of the ferrous ammonium sulphate stock solution to cover the range 1.00 to 4.00 x 10-5 mol L -1 iron. Four solutions will be sufficient. Choose dilutions that are evenly spaced and volumes that are easy to make up with the glassware available. 2 In two 100 ml flasks make 2-fold and 4-fold dilutions of Irn-Bru (i.e. pipette 25.0 ml IrnBru into one flask and 50.0 ml into the other), add 50.0 ml of the hydroxyl ammonium chloride solution to each and add distilled water to the mark for the 4-fold dilution. Label them clearly as"2-fold dilution" and "4-fold dilution". 3a) Pipette 5.00 ml of the 1,10-phenanthroline solution into a clean 100mL volumetric flask, add 10 ml hydroxylammonium chloride solution and make up to the mark with water. Label this as "0 x 10-6 mol L -1 Fe". b) Repeat the process in four other flasks to get a set of calibration solutions from your diluted iron standards in step1. Take a clean 100mL volumetric flask and pipette in 25.0mL of one the standard solutions, then 10 ml hydroxylammonium chloride solution then 5.00 ml of the 1,10-phenanthroline solution and make up to 100 ml with water. Label each flask with the standard concentration. (NB if there is insufficient volumetric glassware you can pour your samples into labelled storage bottles, rinse the 100mL volumetric flasks and then reuse them)
Schools' Analyst Irn Bru Practical...Page 5 c) Repeat the process with each of the diluted Irn-Bru samples from step 2 (i.e. each flask should have 25.0 ml of one of the diluted IrnBru's, 10 ml hydroxylammonium chloride solution, 5.00 ml of 1,10-phenanthroline solution and be made up to 100 ml with water). Label each flask with the iron concentration or IrnBru dilution. d) Also make up two flasks (one from each of the diluted Irn-Bru solutions) without the 1,10-phenanthroline reagent. Label these as "2-fold blank" and "4-fold blank". You should have 9 samples from this step for absorbance readings. 4 ASK FOR A DEMONSTRATION OF THE USE OF THE SPECTROPHOTOMETER 5 Set the spectrophotometer to 515 nm wavelength then zero it with distilled water in the cell(s). 6 Read and record on the result sheets the absorbance of each of the mixtures from step 4. Rinse the cell between each reading. 7 Plot a calibration graph using the absorbances read from each of your standard solutions. 8 Correct for the background absorbance of the colouring material in the drink by subtracting the appropriate "blank" reading from each of the two sample readings (see result sheet). 9 Read off the concentration of iron in each of the diluted IrnBru samples. 10 Correct for dilutions then report the concentration of iron in the original IrnBru drink on the result sheet. 11 Carry out the further calculations on the result sheet.
Schools' Analyst Irn Bru Practical...Page 6 Experiment 2 Extraction and identification of organic components In this experiment you will perform a solvent extraction to remove some of the organic compounds from the Irn-Bru, then separate and identify them by thin layer chromatography. Equipment 100 ml separating funnel and stopper 50 ml measuring cylinder 10mL measuring cylinder Retort stand with ring or clamp 4 mol L -1 sodium chloride 100 ml beaker Dichloromethane Methanol 2 mol L -1 hydrochloric acid Thin layer chromatography plate (silica gel with fluorescent indicator) 4 spotting tubes Chromatography tank with ethyl acetate solvent Standard solution of caffeine (200 mg L -1 ) in methanol Standard solution of benzoic acid (100 mg L -1 ) in methanol Standard solution of citric acid (2000 mg L -1 ) in methanol Ultraviolet plate viewer Procedure We are doing qualitative analysis here, so very accurate volume measurements are not required. This means that you can use measuring cylinders for the volumes (in this experiment only). 1 Place 40 ml of Irn-Bru into the separating funnel then add 5 ml of 2 mol L -1 hydrochloric acid and 5 ml of 4 mol L -1 sodium chloride solution. 2 Add 2 ml dichloromethane (CARE: organic solvent), stopper the funnel and invert a few times. CARE: Hold the funnel upright (stopper at top) and loosen the stopper to relieve gas pressure; dichloromethane is very volatile and pressure will build up inside the funnel. Repeat the process a few times, relieving pressure after each, then shake the flask vigorously for about 20 secs, place on the stand and allow the layers to separate. 3 Run off the lower (dichloromethane) layer into the beaker, being careful not to allow any of the upper Irn-Bru layer to come out.
Schools' Analyst Irn Bru Practical...Page 7 4 Use a pencil to lightly mark an origin line about 1cm from the end of a TLC plate. Now use spotting tubes to place evenly spaced spots of your extract, the benzoic acid, the caffeine and the citric acid on the line. Write in pencil on the other end of the plate (away from the line and spots) what each spot is. You will probably need to make several applications to each spot, allowing time for drying between each application, to get enough on. Allow the spots to dry then place the TLC plate in the developing tank. Remove when the solvent is about 3/4 the way up the plate, and immediately mark the position of the solvent front with a pencil. 5 When the plate is dry, examine under ultraviolet light in a plate viewer and mark the position of each spot with a pencil. 6 Remove the plate from the viewer, measure the disance from the origin to each of the spots and to the solvent front, then calculate the Rf value for each spot (this is the distance travelled by the spot divided by the distance travelled by the solvent front). If your plate is overloaded or underloaded then try again, changing the amount of material spotted onto the plate. 7 Write your results in the table and draw a diagram of your TLC plate in the answer section, labelling it clearly. 8 Is there evidence of all three compounds in the Irn-Bru extract? Explain your answer.
Schools' Analyst Irn Bru Practical...Page 8 Experiment 3 spectrophotometry Determination of caffeine by solid phase extraction and uv In this experiment you will extract caffeine from Ir-Bru and measure how much is present in the drink. To do this you will pass the drink through a solid phase extraction cartridge which will retain the caffeine then, after washing the cartridge you will elute the caffeine and measure its concentration by uv spectrophotometry. Equipment 0.1 & 10mL pipettes (1 of each) Pipette fillers Small beaker (25 or 50 ml) 50 ml volumetric flask Pasteur pipette C 18 solid phase extraction (SPE) cartridge Vacuum filtration assembly (SPE manifold or 50mL Buchner flask with syringe needle through stopper) Dilute ammonium hydroxide (1 mol L -1 ) Dilute hydrochloric acid solution (1 mol L -1 ) Methanol Caffeine solutions 5.00, 10.0, 15.0, 20.0 mg L -1 in 1 mol L -1 hydrochloric acid Ultraviolet scanning spectrophotometer with fused silica UV cell Procedure 1 Pipette a 5.00 ml sample of Irn-Bru into a clean small beaker and add 5 ml of 1 mol L -1 ammonium hydroxide. 2 Condition the SPE cartridge by fitting it to the funnel and flushing through 5 ml of methanol followed by 5 ml of 1 mol L -1 ammonium hydroxide. Try throughout the experiment to get a flow rate of 1-2 drops per second. Turn off the vacuum when the liquid level gets down to the top of the packing and disassemble the apparatus. Discard the liquid in the collector and reassemble. 3 Load some of the alkaline Irn-Bru sample from step 1 and reconnect the vacuum line. Add more when the level nears the top of the packing. 4 After the Irn-Bru, pass 5 ml of 1 mol L -1 ammonium hydroxide through the cartridge to wash the packing (the orange dye will probably come off here). Disconnect the vacuum line when the liquid gets to the top of the packing. 5 Discard the eluate in the flask into the sink, rinse the flask and empty out as much water as possible.
Schools' Analyst Irn Bru Practical...Page 9 6 Reassemble the system and pass through 10.0 ml of methanol, being careful to collect the eluate. Let this pass through until no more liquid is pulled from the cartridge. 7 Disconnect from the vacuum line, remove the bung and transfer the liquid to a 50.0 ml volumetric flask. Make up to the mark with 1 mol L -1 hydrochloric acid, stopper the flask and label it. 8 ASK FOR A DEMONSTRATION OF THE USE OF THE SPECTROMETER YOU MUST USE THE SILICA CELLS FOR THIS EXPERIMENT, NOT PLASTIC ONES. Care - they are expensive and will smash if dropped!! 9 Set the spectrophotometer wavelength to 274 nm then zero it with distilled water in the cell(s). You only need to have the cells 3/4 full. Use a Pasteur pipette for filling the cells. 10 Remove the sample cell, discard the contents, refill with the lowest concentration of the supplied set of standards and record the absorbance on the result sheet. Repeat for the rest of the standard solutions, from low to high concentration. Rinse the cell and Pasteur pipette with water before continuing. 11 Fill the cell with your diluted extract from step 7 and record its absorbance at 274 nm. 12 Rinse the cell, replace the sample with water, set the instrument to scan from 240 nm to 340 nm and store a baseline. 13 Rinse the cell, replace the sample with the highest concentration standard and record its spectrum from 240 to 340 nm Label the spectrum. 14 Rinse the cell, replace the sample with the extract from step 7 and record its spectrum from 240 to 340 nm Label the spectrum. 15 Plot a calibration graph of the standard absorbances at 274 nm against concentration. 16 Read off the concentration of the extract and record on the result sheet. 17 Compare the spectra of the standard solutions with that of the extract. Is there evidence of other compounds being present? Write down your reasoning on the answer sheet and attach your spectra.
Schools' Analyst Irn Bru Practical...Page 10 Background UV / visible spectrophotometry 1,10-phenanthroline forms a complex with ferrous ion more readily than with ferric ion, and the complex with the ferrous form is more intensely coloured. The intensity of the colour can be used to measure how much of the complex is present. This is the structure of the complex: Caffeine, although forming a colourless solution, absorbs light in the ultraviolet (you will record its absorbance spectrum in this region), and its concentration in solution can be measured most easily by the absorbance at 274nm. Caffeine: H 3 C N O N CH 3 O N N CH 3 Light itself can be split into the spectrum of colours that we see in a rainbow; different colours signify different wavelengths and, therefore, different energies. We call a beam of light of one colour monochromatic. Light will be absorbed by an atom, ion or molecule when the energy of one quantum of a particular wavelength of light matches the energy required to cause an electron in an outer orbital to jump to a higher energy level.
Schools' Analyst Irn Bru Practical...Page 11 Each absorption band is caused by the transition between a given pair of energy levels; because the energy level differences vary with different electronic structures, absorption spectra can often be used to help identify the analyte atom, ion or molecule. The technique of spectrophotometry relies on the absorption of light by the analyte; the intensity of a beam of light is measured in the absence then presence of analyte and the decrease in transmitted intensity is used to determine the analyte concentration. monochromatic light I 0 I detector The Beer-Lambert law expresses the relationship between absorption and concentration: A = bc where A = absorbance, = molar absorptivity (L mol -1 cm -1 ), c = concentration (mol L -1 ) and b = optical pathlength (the distance that light travels through the sample, in cm). If this relationship is valid, then a graph of absobance against concentration for a solution will be a straight line which passes though the origin. This is the basic equation of spectrophotometry. The spectrophotometer (or colorimeter) can only measure the intensity of light, however, so we need an additional relationship linking absorbance to I and I 0. This is A = log (I 0 /I). Thin Layer Chromatography (TLC). In thin layer chromatography a glass or plastic sheet is covered by a uniform thin layer of adsorbent material. Sample analyte, dissolved in a suitable medium, is applied to the thin layer plate, dried and the plate placed in a tank containing solvent mobile phase. The mobile phase moves up the plate carrying the dissolved sample with it. The components of the sample will be retarded relative to mobile phase by interaction with the stationary adsorbent phase, which in this case is silica gel (an acidic polar adsorbent). In general polar adsorbents retain polar compounds whilst non-polar adsorbents retain non-polar compounds (the "like likes like" principle). When the mobile phase almost reaches the top of the plate it is removed and dried. The spots can be visualised by placing the dried playte under an ultraviolet lamp; the silica gel has a fluorescent compound impregnated in it, and the spots appear as dark patches
Schools' Analyst Irn Bru Practical...Page 12 against a greenish background.the position of each spot is marked and the Rf value for each compound is calculated from the ratio: Rf = distance moved by analyte spot distance moved by solvent front The Rf value is used to identify the analyte. This is an example of development chromatography. Solvent-solvent extraction Solvent-solvent extraction, as used in experiment 2, is a classical method of removing organic compounds from aqueous solution. It relies on the use of two immiscible (nonmixing) solvents, one very polar (e.g. water) and one much less polar (e.g. an alkane or, as here, the haloalkane dichloromethane). If the substance that we want can exist in both ionised and neutral forms (as acids and bases can) then by adjustment of the ph we can make the substance dissolve in one or other of the two solvents. For instance, if we make the water solution acid, then organic acids will be protonated to their neutral form. These are more soluble in the less polar solvent, and so the organic acid will move from water to the less polar solvent. On the other hand, if we make the solution alkaline, then the acids will be ionised and stay in the water, while bases will be neutral and accumulate in the organic layer. Solid phase extraction Solid phase extraction, as used in experiment 3, is a modern version of solvent-solvent extraction.the device that you have used here has a very thin layer of an 18-carbon alkane (the less polar solvent) covalently bonded to a solid suport (silica), over which the more ploar solvents are passed. The less polar solvent is "locked" in place and has a large surface area for the substance to interact with, which improves the efficiency of extraction (and also means that you don't have to shake it!). This method reduces the amount of organic solvent that we have to use, so is more environmentally-friendly.
Schools' Analyst Irn Bru Practical...Page 13 Results Experiment 1 Determination of iron by colorimetry What is the iron-containing compound added to Irn Bru?: What is the concentration specified on the bottle?: Absorbance of standard solutions: Standard iron concentration: Absorbance: Absorbance of Irn-Bru samples: Sample: 2-fold dilution 4-fold dilution Absorbance with phenthroline Absorbance without phenanthroline (blank) Difference (corrected absorbance) Attach calibration graph Readings from graph: (keep to a realistic number of significant figures) Concentration of iron in 4-fold dilution: mol L -1 Concentration of iron in 2-fold dilution: mol L -1
Schools' Analyst Irn Bru Practical...Page 14 Calculations: Explain each step of your calculation in words, write the relevant numbers in an equation and include units at each stage Correct for the dilutions of each sample: Convert the molar concentrations to mass concentrations (g L -1 ) of iron: (relative molar mass Fe = 55.85, i.e. one mole of iron weighs 55.85 g): Explain your calculation. Now convert to mg L -1 (or parts per million, ppm). Remember that there are 1000 mg in a g: Final answers: The concentration of iron in Irn Bru is: mol L -1 or g L -1 or mg L -1
Schools' Analyst Irn Bru Practical...Page 15 Experiment 2 components Extraction and identification of organic Draw a diagram of your TLC results here: Distance moved by solvent front = Spot identity: Distance moved: Rf value calculations: Explain the calculation in words and show the equation for each sample: Conclusions from TLC data:
Schools' Analyst Irn Bru Practical...Page 16 Experiment 3 spectrophotometry Determination of caffeine by solid phase extraction and uv Standard solutions of caffeine Concentration: Absorbance: Absorbance of Irn-Bru extract = (attach calibration graph) Concentration of caffeine in extract (from graph): mg L -1 (keep to a realistic number of significant figures) Calculations: Explain each step of your calculation in words, write the relevant numbers in an equation and include units at each stage Correct for dilution of the sample: (the extract was in 50.0mL which contained all the caffeine from 5.0mL of Irn-Bru, so you should multiply the value from the graph by an appropriate factor (what will it be??) to get the concentration of caffeine in the original Irn-Bru sample) Convert the caffeine concentration from mg L -1 to g L -1 (remember that there are 1000 mg in one g): Convert the caffeine concentration from g L -1 to mol L -1 (the relative molar mass of caffeine is 194.2, i.e. one mole of caffeine weighs 194.2 g). Explain your calculation in words and show the numbers clearly:
Schools' Analyst Irn Bru Practical...Page 17 Deductions Compare the uv spectrum of pure caffeine with that of your extract. Is there evidence of other compounds being present in this extract? Attach your spectra (label each clearly!) and explain your answer.
Schools' Analyst Irn Bru Practical...Page 18 Questions 1 What are the oxidation states of iron in: i) the ferrous form (e.g. in FeCl 2 ): ii) the ferric form (e.g. in FeCl 3 ) 2 Draw the structure of benzoic acid (C 6 H 5 COOH) in both its neutral and ionised forms: 3 Why did we need the "blank" IrnBru samples (without phenanthroline reagent) in the first experiment? 4 How many significant figures did you express your answers to, and why did you think this number to be "realistic"?
Schools' Analyst Irn Bru Practical...Page 19 5 The following symptoms may be exhibited when amounts of caffeine greater than 250mg have been ingested (from Desk Reference to the Diagnostic Criteria from DSM- 3-R (American Psychiatric Association, 1987): restlessness, nervousness, excitement, insomnia, flushed face, diuresis, gastrointestinal disturbance, muscle twitching, rambling flow of thought and speech, tachycardia or cardiac arrhythmia, periods of inexhaustibility, psychomotor agitation. How much Irn-Bru would you have to drink to exhibit such symptoms? (show your working):