Amy Tran 12R
UV-Visible Spectroscopy – Iron in Dietary Supplements Introduction Ultraviolet-visible spectroscopy (UV/Vis) refers to the absorption spectroscopy i n the ultraviolet visible spectral region. Many molecules absorb ultraviolet or visible visible radiation as they move between energy levels. The wavelength of radiation that is absorbed directly affects the perceived colour of the chemicals involved and is related to molecular structure. Different molecules absorb radiation of different wavelengths; therefore an absorption spectrum can be used to qualitatively identify compounds. However, UV/Vis spectroscopy is mainly used in the quantitative analysis of compounds. When a substance absorbs visible light, light, it appears coloured. The human eye does not see the colour that is absorbed by the sample; however, what is seen is the c omplement of the absorbed colour. For example, a solution of copper(II) sulphate appears blue because the absorption energy comes from the orange region of the visible spectrum. Iron is an essential human nutrient, as it has many roles within the body. A lack of iron can lead to the development of iron deficiency anaemia. To combat this, many people use iron dietary supplements in order to help maintain healthy levels of iron. To detect iron with the use of UV/Vis U V/Vis spectroscopy, the 3+ reaction between Fe and thiocyanate irons (SCN ) must occur. This reaction gives an intensely redcoloured product that can be used as a qualitative test to determine the presence presence of Fe3+. However, most of the iron in dietary supplements is in its ferrous form and must be oxidised to the ferric form with the use of hydrogen peroxide (H2O2). This is to ensure that the iron can be sufficiently bonded with the thiocyanate irons, giving the red colour of the solution. Aim To determine the mass of Fe 3+ in a sachet of a dietary supplement using UV-Visible Spectroscopy. Hypothesis It is hypothesised that the mass of Fe 3+ found by using UV-visible spectroscopy is t he same as the mass of Fe3+ stated on the sachet of the t he dietary supplement. Materials 2.000 x 10-4 M Fe3+ standard solution 4 M HNO3 solution 10% KSCN solution 10% H2O2 solution Iron dietary supplement (5mg/25mL) Distilled water 5 x 25 mL volumetric flasks 250 mL volumetric flask 2 x small beakers 6 x Pasteur pipettes 6 x cuvettes Autopipette Spectrophotometer Spectrometer Safety goggles Lab coat
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Method Part 1: Preparation of the calibration curve 1. The beaker labelled “iron standard solution” was rinsed with a small amount of the standard solution and then 40 mL of the solution was placed i nto the beaker. 2. Five 25 mL volumetric flasks were labelled with numbers 1 to 5. 3. An autopipette was used to transfer the amounts of iron standard solution listed in the table to the flasks. 4. The 4M HNO3 and 10% KSCN solutions were added to each of the flasks. 5. The solutions were mixed by stoppering and shaking, then the colour variations i n the flasks were checked by eye. Solution
Fe3+ solution
1 2 3 4 5
0 mL 1 mL 2 mL 3 mL 4 mL
4 M HNO3 solution 2 mL 2 mL 2 mL 2 mL 2 mL
10% KSCN solution 2 mL 2 mL 2 mL 2 mL 2 mL
Distilled water To the line To the line To the line To the line To the line
Fe3+ concentration 0M 0.000008 M 0.000016 M 0.000024 M 0.000032 M
6. 5 cuvettes were filled with each of the solutions using a clean Pasteur pipette each time and were arranged in order of concentration. Part 2: Frequency of light absorbed 1. A spectrophotometer was used to record the spectrum of the solution in flask 4 over a range of approx. 400 – 700 nm. (refer to spectrum included) Part 3: Determining the iron content of the supplement (i) Preparation of the sample solution 1. The contents of the sachet were emptied into a small beaker. 2. An autopipette was used to add 1.0mL of this liquid into a 250 mL volumetric flask. 3. 1 mL of 10% H2O2 solution was added to the flask. 4. 10 mL of 4M HNO3 and 10 mL of 10% KNCS was added to the solution. 5. The flask was made up to the mark with distilled water using a clean plastic pipette. (ii) Analysis of the stock solution 1. The sample was transferred to a cuvette and the a bsorbance was measured at 473.0 nm (the wavelength of maximum absorbance determined in part 2) Part 4: Amount of light absorbed 1. The spectrometer was set to 473.0 nm. 2. The absorbance of each solution was measured. (refer to Table 1) 3. Using this information, a calibration curve was plotted. (refer to Graph 1) Results Table 1: The concentration of Fe 3+ in each solution with the corresponding absorbance Solution Fe3+ concentration (M) Absorbance 1 0 0 2 0.000008 0.026 3 0.000016 0.109 4 0.000024 0.144 5 0.000032 0.205 Sample unknown 0.098
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Graph 1: Calibration curve of the absorbance against c oncentration of Fe3+
Graph 1: Calibration Curve 0.25 0.2
e c n0.15 a b r o 0.1 s b A 0.05 0 0
0.000005 0.00001 0.000015 0.00002 0.000025 0.00003 0.000035
Concentration of Fe 3+ (M) Spectrum 1: Absorbance spectrum (attached) 1.
What is the iron concentration in the sachet solution (in the 250 mL flask)? 16 x 10-6 M (according to the graph)
2. Assuming the sachet contains exactly 25.00 mL of iron supplement, what is the mass of iron in the sachet?
3. Calculate the mass of Fe 3(PO4 )2 in the sachet.
Amy Tran 12R
Discussion 1. Briefly describe the method used in your analysis. A diagram illustrating the various steps involved might be helpful. Standard solutions were prepared with varying concentrations of Fe3+.
Using the calibration curve, the concentration of Fe3+ in the dietary supplement was found.
Sample solution was prepared.
A spectrometer was used to find the absorption of each of the samples. From these results, a calibration curve was determined.
Sample and standard solutions were transferred into cuvettes.
A spectrophotometer was used to find the absorption spectrum.
2. Briefly describe the principles of operation and the major components of the spectrometer you used. A labelled diagram might be helpful.
3. Are there other parts of the instrument (aside from the sample) that could absorb light from your light source? Has this affected your results? Why/why not? Aside from the sample, no other parts of the instrument could absorb light from the light source. If there was a part of the instrument that could absorb light, then th e end results would be different, making the amount of iron determined using UV/Vis would be different to what was stated by the manufacturer. 4. Was the analytical procedure quantitative or qualitative, or both? Explain fully. The analytical procedure was quantitative because it was used to determine the concentration of Fe3+ in the dietary supplement in terms of molarity. 5.
What is the iron content as stated by the manufacturer? The iron content as stated by the manufacturer was 5mg/25mL.
Amy Tran 12R
6. What uncertainties (or errors) were involved in the procedure? The nitric acid and potassium thiocyanate were not added to the flask with the 0 M Fe3+ (flask 1). The contents of this flask were used to cali brate the spectrometer to 0 M concentration. Fortunately, the results of the procedure were unaffected despite the nitric acid a nd potassium thiocyanate not being present and the calibration curve produced was as expected. 7.
Were there any unexpected results? All the results were as expected and there were no unexpected results.
8. What are some other applications or uses of UV-visible spectroscopy that you investigated? UV/Vis can be used in clinical analysis, measuring the concentrations of specific substances in body fluids such as urine or blood. For example, the haemoglobin content and sugar levels in blood can be found by using UV/Vis. In addition to this, like the experiment conducted, UV/Vis can be utilised to identify the presence of metal ions; even if the metal ion itself is not coloured, it is possible to be analysed if it is converted into a coloured compound. For example, finding the amount of calcium in urine can be found using UV-Vis if an organic complexing agent (eg. arsenazo III) is reacted with it to form a highly coloured liquid. 9. Why is iron particularly suited to this form of analysis? Do you think it would be detected by the other instruments used in this workshop? Iron is particularly suited to this form of analysis because UV/Vis is routinely used in analytical chemistry for the quantitative determination of different substances like transition metal ions. They can be detected because the solutions of these metal ions are often coloured. The detection of iron could also be done by Atomic Absorption Spectrosc opy (AAS) because it is also a type of spectroscopy therefore meaning it is able to detect iron ions in a solution. Conclusion The amount of iron in a 25mL sachet of iron dietary supplement acc ording the manufacturer is 5mg. By using UV/Vis spectroscopy, the dietary supplement was tested and it was determined that there is 5.44mg of iron in the supplement. This result therefore supports the hypothesis that the mass of iron found by using UV-Vis is the same as the mass of iron as stated on the sachet of the dietary supplement. Five standard solutions were made with increasing concentrations of Fe 3+ with the addition of nitric acid and potassium thiocyanate in order to make the solution a re d colour. Then the iron from the dietary supplement was prepared by o xidising the Fe2+ ions to Fe3+ using hydrogen peroxide, then nitric acid and potassium t hiocyanate were added to make this solution red as well. The standard solutions and sample were then transferred into cuvettes, and an absorption spectrum was found using a spectrophotometer and the solution in cuvette 4. The absorption spectrum clearly showed the wavelength where the maximum amount was absorbed (473.0 nm), a llowing us to use the spectrometer to find the absorbance of each of the stan dard solutions and the sample. From these results, a calibration curve was made and the concentrat ion of the sample was able to be found. The reason as to why there was a 0.44mg difference between the final result and the mass stat ed on the sachet is likely to be because the manufacturer found it unnecessary to have the mass of iron in the sachet to have a decimal point when their users are likely not to mind the miniscule difference. Alternatively, another reason as to who there was a difference is because of inaccurate results. The accuracy of the results could be improved if there weren’t any time restrictions, th erefore allowing more time to prepare more standard solutions and, in turn, a more accurate calibration curve. In conclusion, it was learned that UV/Vis has many uses, i.e. finding the concentration of metal ions in a compound. Finally, a greater understanding of the principles and a pplications of UV/Vis was gained from the outcome of this experiment.
Amy Tran 12R
Risk Assessment Preparation/Provision of Nitric acid
Potassium thiocyanate
Acute Hazards
Control Measures
First Aid
It is not combustible, Ensure the acid is well In case of fire, do not but can enhance away from flammable use foam. Keep cool combustion of other substances and any by spraying with substances. combustibles or water. Gives off organic chemicals. If inhalation occurs, irritating/toxic fumes. Keep in a welltreat with fresh air If inhaled, it can cause ventilated room that is and rest in a halfsore throat, coughing, cool and dry. upright position. burning sensation, Wear safety gear (lab Artificial respiration headache, shortness of coat, gloves, protective may be needed. Refer breath and labored goggles) and do not for medical attention. breathing. eat, smoke or drink If there is exposure to Can cause serious skin during work. skin, remove burns with yellow contaminated clothing discolouration. and rinse skin with If it comes into plenty of water. Refer contact with eyes, it for medical attention. can cause redness, If there is contact with pain and burns. eyes, rise with plenty If ingested, it can of water for several cause abdominal pain, minutes and refer for sore throat, burning medical attention. sensation, shock or If ingested, do not collapse and vomiting. induce vomiting. Rest and give one or two glasses of water to drink. Refer for medical attention.
It is not combustible, but gives off irritating fumes/gases in a fire. If inhaled, it can cause coughing. If ingested, it can cause confusion, convulsions, nausea, vomiting and weakness.
Keep separated from strong oxidants in a dry, well-closed area. Wear safety gear (lab coat, gloves, protective goggles) and do not eat, smoke or drink during work.
In case of fire, use the appropriate extinguishing medium. If inhaled, treat with fresh air and rest. If there is exposure to skin, remove contaminated clothing and rinse skin with plenty of water. If there is contact with eyes, rise with plenty of water for several minutes and refer for medical attention. If ingested, rinse mouth. Give a slurry of activated charcoal in water to drink. Refer for medical
Amy Tran 12R
Hydrogen peroxide
It is not combustible, but it can ignite combustible materials. There is a risk of fire on contact with heat or metal catalysts. Inhalation can cause sore throat, cough dizziness, headache, nausea and shortness of breath. If it comes into contact with skin, it is corrosive and causes white spots, redness, skin burns and pain, If it comes into contact with eyes, it is corrosive and causes redness, pain, blurred vision and severe deep burns. If ingested, it can cause abdominal pain, abdominal distention, nausea and vomiting.
Ensure the acid is well away from combustibles, reducing agents and hot surfaces. Store in a cool, dark area in vented containers separated from combustible and reducing agents, food, strong bases and metals. Wear safety gear (lab coat, gloves, protective goggles) and do not eat, smoke or drink during work.
attention. In case of fire, use water in large amounts (water spray). If inhalation occurs, treat with fresh air and rest in a halfupright position. Refer for medical attention. If there is exposure to skin, remove contaminated clothing and rinse skin with plenty of water and rinse again. Refer for medical attention. If there is contact with eyes, rise with plenty of water for several minutes and refer for medical attention. If ingested, rinse mouth, do not induce vomiting and refer for medical attention.