INTRODUCTION TO SPECTROPHOTOMETRY Analysis of Dye Mixtures

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1 Chemistry 135 For this experiment: INTRODUCTION TO SPECTROPHOTOMETRY Analysis of Dye Mixtures Complete the Prelab and obtain a stamp before you begin the experiment. Clark College Write your notebook prelab and get it initialed and dated before you begin the experiment. Include all λ and ε data from the prelab handout. Create a Calibration Curve for one dye through a series of dilutions. Analyze a single color unknown using your calibration curve. Analyze an unknown, two-color mixture, using your data and data from a partner. Report all of your unknown data on the spreadsheet in the front of the lab. Turn in the Data Report Sheet, the Stamped Prelab, and calibration curves for both the blue and the yellow dyes. Codes used: A Y is the absorbance at the maximum yellow absorbance, λ Y is the wavelength of the max abs. for yellow, A B is the absorbance at the maximum blue absorbance, λ B is the wavelength of the max abs. for blue. ε refers to the molar absorbtivity of the blue dye at λ Y for yellow. INTRODUCTION You are already familiar with the characteristic blue color of copper solutions and the green color of nickel solutions. The complexes that give rise to the color of the solutions are called chromophores. The color that we visibly see for these chromophores is actually the complement of the color of the wavelengths of light that the species absorbed. For example, the nickel solutions from the qualitative analysis lab appeared green; therefore the nickel complexes actually absorbed red light, as red is directly opposite green on a color wheel and is therefore the complement. Figure 1: The Color Wheel In the following discussion you will learn how the intensity of colors such as these can be used for quantitative analysis. The basic idea is simple. The greater the concentration of a chromophore the more intense the color. For example, the concentration of copper in an unknown solution can be estimated by comparing its color intensity with a series of solutions having known concentrations. One can simply view tubes containing the same amounts of solution on a white background. Such methods are seldom used nowadays, as they can not be quantified. Because the basic principle that gives rise to colored solutions is light absorption, we use an instrument with a photocell for Spectrophotometry Revised Spring 2006 NF Page 1 of 16

2 the analysis. Such an instrument is called a spectrophotometer. The figure below is a block diagram showing the instrument in its simplest form. The technique is called spectrophotometry. As you probably expect, the use of a photocell is much more accurate than the human eye for measurements of light intensity. Another important consequence of photoelectric measurement is that the technique can be extended into the ultraviolet and infrared regions where the human eye does not work. Thus, thousands of substances that appear colorless to the eye can, in fact, be analyzed with a spectrophotometer. The Photocell The current produced by a photocell is called the photocurrent. Equation 1 shows the relationship between light intensity, P, and photocurrent, i. Equation 1 i = k P + d Light Source Monochrometer Photocell Wavelength Analyte solution in absorption cell Figure 2: Block Diagram of a Spectrophotometer Milliammeter In this equation k is a proportionality constant. It is evident that even if the light intensity, P, is zero there is still some current equal to d. Thus, d is called the dark current. Spectrophotometers have an adjustment to cancel out the dark current. When this is done the equation becomes: Equation 2 i = k P Thus, the greater the light intensity the greater the current. Monochromatic Light Of particular importance is the fact that light absorption is a function of the wavelength of light. A solution can be nearly transparent at one wavelength and completely opaque at a shorter or longer wavelength. Consequently, it is essential to use a light beam with a known wavelength. Light having a narrow range of wavelengths is said to be monochromatic. An optical device that accepts a continuous range of wavelengths from the light source and allows only a narrow range to pass is called a monochrometer. Spectrophotometers have a built in monochrometer, usually based on a prism, diffraction grating or interference filter that allows the operator to select any desired wavelength within the range of the instrument. Absorption Cells Some different kinds of cells for holding the solution are shown in the figure below. If measurements are being made with wavelengths of visible light the windows can be made of glass. However, glass is opaque to ultraviolet light so for use in this region windows of either fused silica (SiO 2 ) or some plastics are used. The windows must be polished to be optically flat in order to avoid distortions of the light beam. Spectrophotometry Revised Spring 2006 NF Page 2 of 16

3 1.00 cm Light Beam 1.00 cm 10.0 cm Light Beam Figure 3: Some Different Kinds of Absorption Cells Transmittance (how much of the light goes through the solution) is a fraction and varies from zero to one and is measured by comparing the solution to be measured (analyte) to the reference (the solvent in this case). Often it is expressed as a percentage and given the symbol %T. When a beam of light passes through a solution in a cell, intensity is lost by the following processes: 1. Reflection at each interface. There are four interfaces that the beam of light passes when traveling through the cell: air/glass, glass/solution, solution/glass, and glass/air. Reflection occurs at each of these interfaces resulting in loss of light intensity. 2. Scattering. Imperfections in the window surfaces lead to some light being deflected off in different directions rather than passing straight through the cell. 3. Absorption by cell windows. Neither glass nor fused silica is 100% transparent. The window material itself absorbs some light. In the ultraviolet region glass absorbs so strongly it is opaque. Fused silica often contains impurities that absorb in the ultraviolet. 4. Absorption by the solvent. Water is not 100% transparent and many other solvents are even less transparent than water. 5. Absorption by the solute. In an analysis we wish to measure only absorption due to the solute. The other four factors must be canceled out. Figure 4 on the following page shows how this is accomplished. First the absorption cell is filled with pure solvent and the intensity of light passing through the cell is measured. Next, the cell is filled with a solution containing the analyte and the intensity passing through the cell is measured again. Then, the transmittance, t, is given by: Equation 3 t = i io = k P k Po = P Po In practice it is more convenient to use two matched cells. One, called the reference cell or blank, is filled with all reagents and solvents except the chromophore and is used to measure the initial intensity, P o. The other, called the sample cell, holds the analyte solution. Because the characteristics of the two cells might not be exactly the same a cell correction is applied. This will be described below in more detail. Absorbance One would naturally expect that the amount of light absorbed by a solution will depend on the length of the light path through the absorption cell. The longer the cell the more light is absorbed. Also, Spectrophotometry Revised Spring 2006 NF Page 3 of 16

4 one would expect the amount absorbed to depend on the concentration of chromophores. The relationship between these three factors is non-linear, it is logarithmic: Equation 4 Measurement of Transmittance t = 10 -ε b c Step 1: Measure the photocurrent, i o, with pure solvent in the absorption cell. Step 2: Replace the solvent with the analyte solution and measure the photocurrent, i. In this equation t is the transmittance, ε(α) is a proportionality constant, b is the light path through the cell (the distance between windows) in centimeters, and c is the concentration of the chromophore. Figure 4: Measurement of Transmittance It is therefore convenient to define another quantity called absorbance, A. Equation 5 A = ε b c when c is in mol/l or A = α b c when c is in g/l Spectrophotometry Revised Spring 2006 NF Page 4 of 16

5 The absorbance and transmittance are inversely related: Or, reworking Equation 6: Equation 6 t = 10 -A Equation 7 A = -log t or log (%T/100) Thus, if the transmittance is known, the absorbance can be calculated at the same wavelength. And, of course, if the absorbance is known the transmittance can be calculated. Also, take a close look at the scale on a spectrophotometer and note that one can read either transmittance or absorbance, whichever is desired. Typically, it is often easier to accurately read the %T (linear scale), rather than the Absorbance (log scale). Beer s Law Equation 5, A= εbc, is particularly important. It is called Beer s Law and is the starting point for nearly all spectrophotometric analyses. Thus, a graph of absorbance versus concentration is a straight line that passes through the origin. This is a much easier relationship to utilize than a graph of transmittance against concentration, which is a curve. A graph of absorbance (or transmittance) against concentration is called a calibration curve. Once this graph has been measured one can immediately find an unknown concentration of the analyte simply by measuring its absorbance and reading the concentration off the calibration curve. The linear graph also helps to identify incorrect data that would not be so easily detected in a curved graph. Figure 5: A Sample Calibration Curve The proportionality constant in Beer s Law, ε, is called the molar absorptivity when the concentration of the light absorbing species is given in moles per liter. Often when dealing with unknown compounds or polymers the molecular weight is unknown so it is impossible to calculate a 0.9 Sample Calibration Curve. 0.8 Absorbance (a.u.) y = x Concentration (g/l). molar concentration. In such cases the concentration is expressed in grams per liter and the proportionality constant α is called the gram absorptivity. In this case Beer s Law is written A = α b c. Spectrophotometry Revised Spring 2006 NF Page 5 of 16

6 Absorption Spectra In the preceding discussion it was emphasized that absorbance depends on the wavelength of light. A graph of %T or A against wavelength is called an absorption spectrum. The figure below is an example, showing the spectrum at three different concentrations. Take special note of the following: a. The shape of the absorption curve is the same, regardless of the concentration. For example, the wavelengths of maximum and minimum absorption are the same in every case. b. At every wavelength the absorption of the middle curve is half that of the top curve. This is required by Beer s Law because the concentrations differ by a factor of two. c. A calibration curve is valid only at the one unique wavelength at which it was measured. Figure 6: An Absorption Spectrum Absorption Spectrum (Yellow) Absorbance (au) Wavelength (nm) Abs. abs 2 abs 3 Spectrophotometry Revised Spring 2006 NF Page 6 of 16

7 Additivity of Absorbance Absorbance values measured at the same wavelength can be added and subtracted. This is important because it allows corrections to be applied and also allows for the analysis of two colored compounds in a mixture Co + Cr Curve c Absorbance Cr Curve b Co 2+ Curve a Wavelength, nanometers Figure 7: An Absorption Spectrum of a Mixture of Co(NO 3 ) 3 and Cr(NO 3 ) 3. Illustration of the Additivity of Absorbances Curve a: M Co(NO 3 ) 2 Curve b: M Cr(NO 3 ) 3 Curve c: A solution that is both M in cobalt (II) and M in chromium (III) nitrates. Note that at every wavelength the absorbances of Curves a and b add to give Curve c. To illustrate the additivity of absorbances consider a solution that contains two chromophores; cobalt and chromium. Chromium (III) compounds are typically green or blue-green in color. Cobalt (II) compounds are typically pink. If we have a solution that contains both cobalt (II) and chromium (III) the observed absorbance at any wavelength is the sum of absorbance to cobalt (II) plus the absorbance due to chromium (III). Thus, we can write: A observed = A cobalt + A chromium This is true at every wavelength and is illustrated in the figure above. Spectrophotometry Revised Spring 2006 NF Page 7 of 16

8 EXPERIMENTAL Absorption and Beer's Law The purpose of this experiment is to learn about absorption spectrophotometry and it s relationship to Beer s Law. In this experiment, you will be using a colorimeter and the Vernier LabPro interface and LoggerPro software. A colorimeter, works on the same principles of spectrophotometry, however the light sources are LEDs, so only a limited number of wavelengths are available. You will need a partner to share graphs and data but do not need to work at the same time. Each of you will use one of the dyes, yellow or blue, for the experiment. General Information 1. Cell Corrections You will use only one sample cell for your various experiments in Parts I and II. To use only one cell, there is no need to clean it between solutions, simply rinse the cell two or three times with the new solution to be analyzed before filling the cell and analyzing. However, as you will need to take measurements at two different wavelengths in Part III, you will need to find two cuvettes that are matched. It is usually more convenient to use two absorption cells; a sample cell and a reference or blank cell. If the cells are perfectly matched they will have the same losses due to reflection, absorption, and scattering at every wavelength. In fact, they seldom do match so perfectly. Consequently it is necessary to apply a cell correction to absorbance measurements. A cell correction is found by using the reference cell, as usual, to calibrate the colorimeter. The absorbance of the sample cell, called A cell, is then measured, while filled with solvent. If the cells are matched at this wavelength A cell will be zero. Frequently it is not zero so a correction must be applied to all absorbance measurements. The corrected absorbance is: A corrected = A observed - A cell Note that it is necessary to measure A cell at each wavelength for which accurate absorbance values are required. If you are able to find cells that match within 2%, you do not need to do a cell correction. To determine cell corrections, fill all cells with water and check each of the Abs at each of the wavelengths to be used. 2. Dilutions Use nominal not calibrated volumes for your pipet, buret and volumetric flasks when you do dilutions. Remember: C 1 V 1 = C 2 V 2. The dilutions are best done with a graduated (Mohr) pipet. Examples : If you dilute a sample by 1:2 then do a 3:4 dilution of the resulting solution, the total dilution is 3:8. A 10 ml sample of dye diluted with 25 ml of water is 10:35 or 2:7 dilution Spectrophotometry Revised Spring 2006 NF Page 8 of 16

9 Procedure NOTE: The dye solutions are made with a food-grade dye and can be disposed safely down the drain. Part I Creating the Calibration Curve 1. Obtain and wear goggles. 2. Obtain approximately 30 ml of the stock dye solution of the appropriate color in a small beaker. Pour approximately 30 ml of distilled water into another small beaker. 3. Prepare 4 solutions of the dye by appropriately diluting the original stock solutions. Be sure to label your dilutions, and record your method and data in your notebook. You will only need 10 ml of each solution (this is generous!)! You now have five solutions: the original stock solution and the four that you have made. 4. You are now ready to create your calibration curve. Take your five solutions, along with a wash bottle and a waste beaker to a computer with a colorimeter. Obtain one plastic cuvette. Make sure that your cuvette is clean and free of scratches. 5. Open the file Exp 11 Beer s Law in Chemistry with Computers. The vertical axis has absorbance and should be scaled from 0 to 1.0. The horizontal axis has concentration scaled from 0 to 0.5 mol/l. 6. Prepare for data collection. You will need to rescale the x-axis. Choose Data column options concentration. Change the units to g/l, increase the displayed precision to 5 digits. 7. You are now ready to calibrate the Colorimeter. Prepare a blank by filling a cuvette 3/4 full with distilled water. To correctly use a Colorimeter cuvette, remember: All cuvettes should be wiped clean and dry on the outside with a Kimwipe. Handle cuvettes only by the top edge of the ribbed sides. All solutions should be free of bubbles. The cuvette cap should be securely snapped on to the cuvette. Always position the cuvette with its clear sides facing the white mark inside the sample holder. 8. Calibrate the colorimeter. a. Holding the cuvette by the upper edges, place it in the cuvette slot of the Colorimeter and close the lid. b. Set the wavelength on the Colorimeter to the appropriate wavelength for your dye, press the CAL button, and wait for the light to stop flashing. The colorimeter is now calibrated. 9. You are now ready to collect absorbance data for the five standard solutions. Click Collect. Empty the water from the cuvette. Using the most dilute solution first, rinse the cuvette twice with ~1-mL amounts of the solution and then fill it 3/4 full. Dislodge any bubbles in the solution and cap the cuvette. Wipe the outside with a kimwipe and place it in the Colorimeter. After closing the lid, wait for the absorbance value displayed on the monitor to stabilize. Then click Keep, type in the concentration in the edit box, and press the ENTER key. The data pair you just collected should now be plotted on the graph. 10. Discard the cuvette contents into your waste beaker. Repeat the process of rinsing and filling the cuvette in order of increasing concentration of your dye solutions. measure the absorbance of each solution in a similar fashion, entering the appropriate concentration with each kept data point. 11. When you have finished collecting data for all of your known solutions, click Stop. Spectrophotometry Revised Spring 2006 NF Page 9 of 16

10 12. Record the absorbance and concentration data pairs that are displayed in the Table window in your notebook. Save your calibration curve in the 135 folder on the desktop, using an approriate file name. 13. Examine the graph of absorbance vs. concentration. To see if the curve represents a direct relationship between these two variables, click the Linear Regression button,. A best-fit linear regression line will be shown for your five data points. This line should pass near or through the data points and the origin of the graph. 14. Title and rescale the graph appropriately (Absorbance vs. Concentration is not an appropriate title). Print a graph of absorbance vs. concentration, with a regression line and the equation of the regression line displayed. Print two copies of the Graph window, one for you and one to share with someone from the other color group. Part II Determining the concentration of an unknown dye solution 1. Obtain a 10 ml sample of the appropriate single-color unknown. Read its absorbance using the colorimeter, and record this value in your notebook. 2. With the linear regression curve still displayed on your graph, choose Interpolate from the Analyze menu. A vertical cursor now appears on the graph. The cursor s x and y coordinates are displayed at the bottom of the floating box. Move the cursor along the regression line until the absorbance (y) value is approximately the same as the absorbance value you recorded for your unknown. The corresponding x value is the concentration of the unknown solution, in g/l., record this concentration. 3. Repeat this process with 3 other different samples of the unknown dye. Do not use the same sample empty the cuvette, rinse and refill it for each sample. Record the concentration of each unknown trial. Spectrophotometry Revised Spring 2006 NF Page 10 of 16

11 Part III Analysis of a Mixture 1. Get together with your partner that chose the other color dye in Part IIA so that you have a calibration curve for two different dyes. (Or you can do a Beer's law plot for a second dye if you want to.) 2. Obtain a 10 ml sample of an unknown dye mixture. Check to see if the absorbance is between 0.1 and 0.9. You may have to dilute your mixture to get a good reading for absorbance (0.1 to 0.9). Record any dilution measurements precisely. 3. Remembering that Absorbances are additive for mixtures, measure the absorbance for the mixture at the two wavelengths corresponding to the wavelengths that give maximum absorbance, λ max, for each of the two dyes. Be sure to recalibrate with a blank when you change wavelengths. Record all data carefully. Repeat this process with 3 other samples of your unknown mixture. 4. Calculate the concentrations of each dye in the unknown mixture using two equations and two unknowns, and using each set of absorbance data. You have αb at each maximum wavelength from each of your graphs. αb is different for each dye at each wavelength and can be calculated at the second wavelength for your dye using data obtained. 5. For our purposes, we will assume αb at the second wavelength is small and we can calculate the value. In the prelab you did this calculation. Record all four αb s, blue at λ 1 and λ 2 as well as for yellow at both wavelengths in your data table. 6. Calculate the original concentration of each component in the mixture. The are shown below for a 2 component system. As in algebra, you will need to set up both equations and will find you have two unknowns, C Y and C B. You will need to pick one, solve one of the equations for the concentration of one dye in terms of the other, then substitute back into the second equation. Abs of the mixture at λ Y : A Ymix = α bc Y + α bc B Abs of the mixture at λ B : A Bmix = α bc Y + α bc B Spectrophotometry Revised Spring 2006 NF Page 11 of 16

12 Spectrophotometry Revised Spring 2006 NF Page 12 of 16

13 Chem 135 Spectrophotometry DATA REPORT SHEET Be sure to include all of your graphs for credit! Part I Calibration Curve Name Color = λ = α = Trial Concentration (g/l) Absorbance Sample Calculation for dilution and α: Part II Unknown Single Dye Color = λ = Trial Absorbance Concentration (g/l) Average Concentration Standard Deviation 4 RSD Spectrophotometry Revised Spring 2006 NF Page 13 of 16

14 Part III Unknown Mixture Codes used: A Y is the absorbance at the maximum yellow absorbance, λ Y is the wavelength of the max abs. for yellow., A B is the absorbance at the maximum blue absorbance, λ B is the wavelength of the max abs. for blue. ε refers to the molar absorbtivity of the blue dye at λ Y for yellow. Unknown ID Notebook Page Number for Calculations λ Y = λ B = α = α = α = α = *From the Graph and question 3 in the Prelab Trial Abs, λ Y Abs, λ B Trial Conc, Yellow (g/l) Conc, Blue (g/l) Avg S RSD Record your values into the class spreadsheet in the lab. Attach all graphs to the final report. Include a copy of your partner s graphs as well. Spectrophotometry Revised Spring 2006 NF Page 14 of 16

15 Chem 135 Introduction to Spectrophotometry PRELAB A= αbc Beer s Law where α is in L/(g cm). Codes are in the experiment handout. Using the attached graph, answer the following questions. 1. Sketch in a smooth curve for the data points on the graph. Name 2. Find the wavelength on the graph that gives the maximum absorbance for each color and list it below. a. Yellow = λ Y b. Blue = λ B 3. For the graph on the back of the page, the concentration for the yellow, C Y, is given as 0.55 g/l and for blue, C b, it is 0.20 g/l.. a. Use the graph find A and calculate α for yellow at the wavelength for blue b. Use the graph to find A and calculate α for blue at the wavelength for yellow. They will be small values. You will use these values later for calculations. (Note that these are not the same concentrations that you will use for the lab but the constants will remain the same at those wavelengths.) A = αbc, where b = 1.00 cm. Don t forget your units! C Y = 0.55 g/l C B = 0.20 g/l a. A = A = b. α b= α b= 4. Using the concept of the additivity of absorbances at a given wavelength, sketch the sum of the two curves on the graph with a black blue or pencil line for the yellow and blue dyes similar to curve c in the Cr +3 and Co +2 example. Note that since absorbance is additive and it is easy to chose a wavelength, add the A values of each line, and plot the new point at that given wavelength. Do this at enough wavelengths to give a smooth curve. 5. Look up the equation for converting %T to absorbance. Calculate the absorbance of a sample if the % transmittance is 84.2%. Abs = Spectrophotometry Revised Spring 2006 NF Page 15 of 16

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