Spring 2021 CHE 3010 Palmer This work by Alycia Palmer is licensed under CC BY 4.0 1 Spectrophotometric Determination of Fe UVVis spectroscopy of Fe using the method of standard addition Objective Students will determine the concentration of iron (II) in an unknown sample using UV Vis spectroscopy. Standard addition of Fe(phen)3 2+ will serve as the calibration to determine the unknown analyte concentration. Logistics The duration is one week, and students will work individually. Notebook, spreadsheet, and conclusions will be prepared individually and are due before the next lab session. Before lab, prepare your notebook according to the Lab Polici es course document on Canvas . Introduction In this experiment, a sample containing an unknown amount of iron is quantified by converting the aqueous Fe2+ ion into the Fe(phen)3 2+ complex by reacting it with the 1,10 phenanthroline ligand , Figure 1. In chemical literature this structure may be called iron (II) tris phenanthroline. Figure 1. Chemical structures of a) The Fe(phen)3 2+ complex and b) the 1,10phenanthroline ligand
Spring 2021 CHE 3010 Palmer This work by Alycia Palmer is licensed under CC BY 4.0 2 The Fe(phen)3 2+ complex has an intense blood -red color that absorbs light intensely in the visible region. This property enables quantitation using a benchtop spectrophotometer to measure the absorbance of a sample . Spectrophotometric methods of analysis are fast, relatively simple and very widely applied. They rely on the fact that electromagnetic radiation may be absorbed by matter. The extent to which radiation is absorbed is related to the nature and concentration of absorbing material present in a sample as well as the wavelength of the radiation employed. In this experiment max for the Fe(phen)3 2+ complex by acquiring a full spectrum, such as the one shown in Figure 2, and locating the wavelength of maximum absorbance. Figure 2. Absorbance spectrum of a mixture of two dyes, one with max max at 630 nm. Transmittance vs. Absorbance Transmittance (T) through a sample is represented by Equation 1 where Io is the intensity of the incident radiation and It is the intensity of the transmitted radiation (Figure 3). Equation 1 The radiation not transmitted is absorbed by the sample. Absorbance (A) is related to transmittance by Equation 2. In the visible range, the wavelength at which a particular chemical substance absorbs is directly related to the color of the sample. If a wavelength of visible light is absorbed, then the c omplementary color is transmitted . Equation 2
Spring 2021 CHE 3010 Palmer This work by Alycia Palmer is licensed under CC BY 4.0 3 Figure 3. A simple spectrophotometer. Io is the incident radiation. It is the radiation transmitted through the sample. The absorbance of light by the sample is represented by A . The intensity of the light beam as it enters the solution is called the incident intensity and is given the symbol Io. The incident intensity is essentially the number of photons per second that enters the sample solution. As the light traverses the sample some photons may be absorbed by the components of the sample depending on the nature of the components and the wavelength of the light. Absorption of visible and ultraviolet light results in electronic excitations , which lead to changes in the electron distribution in the molecules or ions of the absorbing material. Beerâ€™s Law The relationship between absorbance and concentration can be described by using the Beer Lambert Law, also known as Beerâ€™s Law: Equation 3 where A is absorbance at a particular wavelength ( ), is the distance that radiation travels through the sample (path length), c extinction coefficient (also known as the molar absorptivity or absorption coefficient) with units M1cm1. The extinction coefficient ( ) is a characteristic of the chemical compound being probed, is typically 1.00 cm or 0.100 cm depending on the size of sample holder, and c is the concentration of the sample in mol/L. From Be erâ€™s Law, it is apparent that absorbance is directly proportional to concentration. Thus, taking an absorption spectrum of the same compound at decreasing concentrations will result in decreased absorbance, as shown in Figure 4. Figure 4. Absorbance spectra of the same compound at different concentrations. Sample 1 is the most concentrated and has a maximum absorbance at 630 nm.
Spring 2021 CHE 3010 Palmer This work by Alycia Palmer is licensed under CC BY 4.0 4 Calibration curves The Beer's Law equation may be applied to analyses in a variety of ways. The variation we wi ll use is the standard addition method. A portion of the unknown is diluted with a suitable solvent to some known volume . T he absorbance is measured at an appropriate wavelength. Since this solution has no standard added, we will call it Ao. To a second, equal portion of the unknown is added an additional known amount of the analyte and the volume is adjusted as before. The total analyte concentration then is a sum of the unknown and standard (cunk + cstd). The second solution then has an increased absorba nce compared to Ao due to the added standard. Equation 4 A linear calibration model can be used to determine the concentration of an unknown. For best results, a series of standard solutions are prepared just as in a typical Beer's law analysis, but each standard also contains the same aliquot of the unknown. This method works best when the quantity of added standard (the "spike") is comparable to the quantity of unknown present. The data is analyzed by preparing a calibration curve of a bsorbance vs. standard concentration (Figure 5). Note that the absorbance of the blank has a nonzero absorbance due to the addition of the unknown. Figure 5. P lot of absorbance vs. standard concentration using the method of standard additions graphical procedure with constant volume. The equation of the line is then derived from Equation 4 to give Equation 5. Equation 5 Equation 5 shows that by plotting absorbance on the y-axis and standard concentration on the x-axis, the slope of the line and the y-intercept equals cunk. Thus one can use the absorbance value at the y -intercept (Ao) to solve for the unknown concentration (Equation 6). y = 0.1867x + 0.1452 R = 0.9997 -0.2 0 0.2 0.4 0.6 0.8 1 -2.0000 -1.0000 0.0000 1.0000 2.0000 3.0000 4.0000 5.0000 Absorbance at 512.5 nm Concentration (mg/L)
Spring 2021 CHE 3010 Palmer This work by Alycia Palmer is licensed under CC BY 4.0 5 Equation 6 The method of standard addition is an important alternative to the typical Beer's Law method when the unknown sample contains a complex matrix that influences the sensitivity of the analyte. If the sensitivity of the analyte in the unknown is markedly different than in the standards, serious errors in the interpolated concentration can occur. In the method of standard addition, the samples are prepared to ensure that all of the samples contain the same matrix effects. Thus, all of the solutions are on equal footing as far as matrix effects are concerned. Both methods described above rely on an assumption that Beer's Law accurately describes the absorbance versus concentration behavior of the analyte material under the experimental conditions employed. Howe ver, t here are numerous reasons for deviations from Beer's Law from both instrumental and chemical sources. Preparation of a calibration curve provides the necessary confirmation of Beer's Law . Because 5-8 standards are typically used in preparing the cali bration curves random measurement errors of the standards tend to cancel, at least more so than with the single standard methods. Spectrophotomet ric analyses made by any method rely on some important assumptions. One is that the analyte substance must strongly absorb light of the wavelength employed for measurement. Many substances simply do not absorb light (or absorb only weakly) at any convenient measurement wavelength. Anoth er is that the analyte must be the only substance that absorbs light at the wavelength of measurement. It is also important to recognize that the cuve t t e itself as well as any small impurities present in the color developing reagents may contribute to the absorbance. For this reason, analyses based on measurements of absorbance almost always involve a blank. The blank contains a solution of all of the same substances and at the same concentrations as the sample cuve t t e . The single difference between the bl ank and sample cuvet te is that no iron is added to the blank. In the experiment that follows you will use the same cuvette for each trial, and it should always face the same direction. At the end of all measurements, the blankâ€™s absorbance can be subtracted from each sampleâ€™s absorbance.
Spring 2021 CHE 3010 Palmer This work by Alycia Palmer is licensed under CC BY 4.0 6 Procedure Provide your instructor with a clean 100 mL volumetric flas k for your unknown sample. The instructor will provide a centrifuge tube with a small volume of unknown Fe2+ sample and will assign a sample number. Be sure to record your unknown number in your lab notebook! A. Preparation of the iron solutions Obtain ~ 20 mL of the standard s olution that contains Fe(NH4)2(SO4)2â€¢6H2O . You will use this to create the calibration standards. From the information on the label, calculate the Fe2+ concentration of this solution and express it in milligrams per liter (mg/L). Obtain 3 additional 100 mL volumetric flasks from the equipment cabinet to add to the 4 from your drawer. In these volumetric flasks prepare the following solutions. Do not add water past the 100.00 mL marking, or you will need to discard the solution! Standard Unknown soln. Fe 2+ standard soln. Citrate hydroquinone ophen [Fe 2+ ] std Blank. 0.00 mL 0.00ml 3 drops 2.00mL 10.00 mL 0 mg/L A 2.00 mL 0.00ml 3 drops 2.00mL 10.00 mL B 2.00 mL 0.50ml 3 drops 2.00mL 10.00 mL C 2.00 mL 1.00ml 3 drops 2.00mL 10.00 mL D 2.00 mL 2.00ml 3 drops 2.00mL 10.00 mL E 2.00 mL 5.00 ml 30 drops 2.00mL 10.00 mL F 2.00 mL 10.00ml 30 drops 2.00mL 10.00 mL Table 1 . Volumes of each reagent to be added to the 100 mL volumetric flasks. In your notebook, calculate the concentration of Fe2+ standard in solutions A F after the dilution. B. Measuring the absorbance We will be using the spectrophotometers connected to the Vernier Logger Pro systems. Get instruction on t heir use from the instructor or teaching assistant. Include in your Data section a coherent description of how to use the Vernier software and procedures you followed/devel oped. Perform a blank calibration by choosing the Experiment drop-down menu and selecting calibrate. Then t ake a full spectrum of each of the standards A -F, making sure to rinse the cuvette with each sample before filling the cuvette for analysis . It is good practice to analyze dilute solutions first, and more concentrated solutions last. Always check the cuvette so make sure it is oriented in the same, correct direction. max, and record the absorbance of each standard at that sample value. These absorbances will be used to make your calibration curve. Have the instructor check your graphs before le aving. Clean up Thoroughly clean the volumetric flasks that you borrowed, rinse with DI water, and return to the shelf. Discard of all solution in the iron waste container.
Spring 2021 CHE 3010 Palmer This work by Alycia Palmer is licensed under CC BY 4.0 7 Post lab Assignment The following should be placed after the data section in your notebook. Remember that graphs inserted should have your signature centered over the edge so that it contacts the graph and notebook paper. 1. Spreadsheet analysis a. P lot the absorbances of the samples A F on the same graph, similar to Figure 4. Be sure to lab el the value of the max that you used to record your absorbance values. b. Use excel to construct a linear calibration plot of the corrected absorbances at max vs. [Fe2+]std. Include the equation of the line and R2 value. Be sure to format your x axis to include the x intercept. c. Submit your spreadsheet us ing the submission link on Canvas and tape the two graphs into your notebook. 2. In notebook calculations a. 3 2+ using the equation of your line. Be sure it has units of M1cm1. (Recall that you r graph uses mg/L, not molarity.) b. Determine the concentration of Fe2+ in your unknown using the process outlined in the introduction . Remember that you diluted your unknown to prepare your sample! The concentration of Fe2+ should be reported in mg/L. Conclusion Your conclusion should be printed and submitted with your lab manual during the following lab period. Format your typed conclusion t o discuss the following points. Tell the purpose of the lab and the main result. Be sure to tell if you tested unknown A or B and the [Fe2+] in mg/L in the original solution. Compare your determined value of 3 2+ with the literature value. Be sure to match the solvent used. *Any references should be cited at the end of your conclusion using ACS specifications.