Experiment Seven Determination Of The Composition Of A Multi-Component Mixture By Spectrophotometric Analysis

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1 CHEM 3281 Experiment Seven Determination Of The Composition Of A Multi-Component Mixture By Spectrophotometric Analysis Objectives: 1.Solid sample handling 2.The composition of a three-component mixture will be assayed by dissolution of the soluble components in water and measurement of their individual concentrations by visible spectrophotometry. Text Reference: Handbook of Instrumental Techniques for Analytical Chemistry, Frank Settle, editor. Prentice Hall,, Upper Saddle River, NJ 1997, Ch 25. H.H. Willard, et. al. Instrumental Methods of Analysis, 7th edition, Wadsworth Publishing Co.:Belmont, CA 1988, p Skoog, Holler and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing, Fort Worth, TX 1998, Ch 13 & 14D. Introduction: The Beer-Lambert law states that the log of the ratio of the power of a beam of monochromatic light incident on the sample (I o ) over the power passed through the sample (I) is defined as the sample absorbance (ABS). The absorbance of a solution containing only one chromophore will be proportional to the concentration of the chromophore, C, the absorptivity, a or ε, and the optical path length, b. ABS = log (I o /I) = εbc or abc at any one wavelength ε = molar absorptivity (L/mole-cm) a = absorptivity (L/g-cm) b = path length (cm) C = concentration (moles/l or g/l) (depends on whether you are using ε or a to represent absorptivity) The absorbance of a solution containing more than one chromophore will be equal to the sum of the absorbances of each of the components at any one wavelength. ABS total = ABS 1 + ABS ABS n

2 where the subscripts refer to the individual chromophoric components 1, 2... n. For a solution containing n components, if the optical pathlength and the molar absorptivities for each component are known, the concentration of each chromophore can be determined by measuring the total absorbance of the solution at n wavelengths (selected where a 1 /= a 2 /=... a n ) and solving the simultaneous equations. In this experiment, the optical path length is set by the physical dimensions of the sample compartment or cuvette. The width of the cuvette is 1.00 cm. The proportionality constants (molar or mass absorptivities) for each chromophore are determined by measuring the absorbance of solutions of known concentration and fitting the Beer-Lambert law to the data. The unknowns provided may contain an insoluble or non-absorbing component. If your particular unknown has an insolube component, dissolution in water will afford the separation of one component from the other two. Note that best results will be obtained when each aliquot prepared for analysis is representative of the sample. Procedure: 1. Turn on the tungsten power supply (switch on small blue box) and boot up the spectrometer software, OOIBase Obtain the stock solutions and an unknown sample mixture from the teaching assistant. Decide on how to sample your solid reliably. Weigh (to the nearest 0.1 mg) a representative sample of approximately 0.50 g from your unknown mixture into a 25 ml volumetric flask and dilute to the mark with distilled, deionized water. Ask your T.A. how to prepare and transfer samples quantitatively so you will not lose any moles of sample if you do not know how. Prepare three sample solutions in this manner from the same unknown mixture. Observe whether or not your unknown contains insoluble components and record the unknown number. 3. For each stock solution provided, prepare a series of standard solutions of known concentration by precisely delivering (via analytical pipette) 5.00, 10.00, and ml aliquots of the standard to four separate and appropriately labeled ml volumetric flasks. Dilute each to the mark with distilled, deionized water. Once again if you are unsure of how to pipette analytically so that no moles are lost in transfer, ask the T.A. Be sure you have the correct molecular weights and stock solution concentrations for each of the standards from lab. 4. Learn how to operate the spectrometer by following instructions below.

3 GENERAL INSTRUCTIONS FOR RUNING OCEAN OPTICS FIBER OPTIC VISIBLE DIODE ARRAY SPECTROMETER Turn on the tungsten power supply (switch on small blue box) and boot up the spectrometer software, OOIBase32. The spectrometer, which is mounted on a half card in the computer, will come up in the SCOPE mode; INTEGRATION TIME=100ms; AVERAGE=1; BOXCAR=0. The real time output of the linear diode array will be displayed. The power spectrum of a tungsten lamp should be roughly a gaussian distribution. If it is a straight line then the diodes are being saturated with too much light. - Lower the integration time until the light hitting the detectors is as gaussian as possible. A neutral density filter can be used in the cuvette slot if available. - Put a cuvette with distilled water in it into the sample holder. Click on STORE REFERENCE (yellow light bulb). Wait a second or two. - Block the source beam by placing something opaque in the cuvette holder. Click on STORE DARK (gray light bulb). Wait a second or two. - Put a chromophore (colored solution) into the cuvette holder and click ABSORBANCE MODE. Rescale the X-axis to 350 nm to 800nm and the Y-axis to about 1 absorbance unit. WHY IS THERE NOISE FROM nm?? - While the spectrometer is acquiring data change the AVERAGE & BOXCAR data handling features to suit your needs. - ANYTIME YOU CHANGE INTEG TIME OR AVERAGE OR BOXCAR YOU MUST REDO REFERENCE & DARK AND THEN REDO YOUR ABSORBANCE SPECTRUM TO OBTAIN A WELL-CORRECTED SPECTRUM - Bring up a cursor and locate spectral features of interest like λ max - Click on SNAPSHOT to freeze data and print it. - To acquire data at specific wavelengths: o click TIME ACQUISITION/ CONFIGURE / CONFIGURE ACQUISITION o click SHOW VALUES IN STATUS BAR pick a frequency of ~1 sec & duration ~20 secs. (For non-kinetic samples!) o click TIME ACQUISITION / CONFIGURE / CONFIGURE TIME CHANNELS o Input the first wavelength in channel A, second wavelength in channel B, etc. up to six λ s. Be sure to leave the master checked (Computer software can run 1 master and 4 slave spectrometers simultaneously!) o Put sample in cuvette holder and click on stopwatch & green light to start data acquisition. The data at each λ of interest will be displayed in the bottom status bar. It will come up fast and noisy depending on your INTEGRATION TIME, AVERAGE & BOXCAR setting! - Remember this is a single beam instrument and the source will drift as it warms up with time. You decide how often to redo REFERENCE and DARK current! WHY DON T YOU HAVE TO EXCLUDE ROOM LIGHT? Turn off light source and clean up. Close OOIBASE32 software or it will keep reading data in

4 real time!

5 Transport all solutions to the spectrometer bench. You may use the same cuvette throughout the experiment (different cuvettes will result in slightly different absorbance measurements) or use different cuvettes to speed up your analysis with concomitant increase in error. (how are you to estimate the error in using different cuvettes??) 5. Put one of your strongest standards into the cuvette holder and click on ABSORBANCE MODE. Turn on the cursor and chose λ max for your strongest standard. Click on SNAPSHOT to freeze data acquisition and then print your output. - Repeat this procedure with your other chromophore. Be sure and use the same wavelength range so you can compare them. Analyze the spectral data and identify the wavelengths that provide the highest sensitivity for EACH stock solution. You want wavelength(s) where the absorbance is high for each compound and you would like a or ε not to vary much at the wavelength(s) used. Usually this is λ max for each compound-the wavelength of maximum absorbance. Pick two wavelengths to run your standards and unknown at.(1-λ max for Co & 1-λ max for Cr) 6. After picking the wavelengths, setup the TIME ACQUISITION to acquire data at the two wavelengths you have chosen. Next, measure the absorbance of each of the 5 solutions prepared in part 3 from stock solution A, as well as each of the 5 solutions from stock solution B. To minimize error, measure the absorbance of the lowest concentration first and rinse the cuvette with distilled, deionized water in-between standards. Record the average absorbance for each wavelength used. 7. Measure the absorbance for each unknown solution at each of the two designated (maximum) wavelengths. Determine the repeatability of the absorbance measurement for each unknown solution by refilling the cuvette with the same unknown solution and remeasuring the absorbance at least twice. Calculations: 1. Plot Absorbance vs. Concentration ( can use g/l) at each wavelength for both Co and Cr standards. Plot them all together on one graph (4 plots). Using a spreadsheet is the easiest way to accomplish this. Determine the absorptivity of each standard by performing a linear regression analysis to the data. Remember, if the solution obeys Beer s Law, Absorbance should be linear with respect to Concentration. (What is the slope equal to?) Repeat this procedure for each standard series of solutions and at each wavelength selected. Can calculate absorptivity in units of L/g-cm instead of 1/molarity for ease in calculating total mass later. 2. Calculate the concentration of each component in your unknown solutions by solving simultaneous equations. You only need two equations to solve for two unknowns. Because total absorbance is additive at any one wavelength you can write for each pair of wavelengths: A λ1 = a Cr at λ1 b c Cr + a Co at λ1 b c Co A λ2 = a Cr at λ2 b c Cr + a Co at λ2 b c Co Normally you pick wavelength pairs where one chromphore is absorbing maximally and

6 the other chromophore is absorbing minimally so as to introduce as little error as possible into the calculation 3. Compute the percent composition by weight of the components in your unknown mixture for each sample taken. Determining the percentage composition of the sample must include the non-colored component if you had one. Using the Beer-Lambert law and simultaneous equations from above, the individual concentrations of the soluble (colored) components can be determined. These concentrations can be converted into the mass of component in the aliquot analyzed. (Remember you only took a portion of the solid to makeup your standards!) The mass of the insoluble component can then be deduced by difference. Determine the mean value and standard deviation for % composition in your unknown sample. Report these values in a table of results. Reporting the Results: The report should consist of a brief description of UV-visible spectrophotometry, labeled diagram of your instrument, instrumental parameters used, raw data-labeled, calculated data -in tables, please), sample calculations for anything calculated, all plots and a short discussion of the results. Organize all results into tables; Table 1 - Co solutions Table 2 - Cr solutions Table 3 - absorptivities of the Co and Cr solutions at 2 wavelengths Table 4 - unknown solutions (wavelength, absorbance, conc. of each component, % weight of each component, mean values, standard deviations, etc.) In addition, the discussion section should contain answers to the following questions: Questions: 1. Was your instrument single-beam or double beam? 2. Assess the precision of your result, the sources of any inaccuracies and suggest modifications to improve on both. What sample handling methodology did you use? 3. Did the weight percent of each of the three samples vary? Why or why not? 4. If you used more than 2 cuvettes, how do you think that contributed to the overall error in your analysis. Did you use any other sample container handling techniques to try and reduce error?

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