Spectrophotometry (Part I & II)

Spectrophotometry (Part I & II) INTRODUCTION Spectrophotometry (Part I & II) INTRODUCTION In this experiment you will be performing a spectroscopic analysis of known and unknown compounds and mixtures. You will be using the GENESYSTM 20 to find the concentration of an unknown mixture of Bromothymol Blue and Thymol Blue. A Brief Introduction to Spectrophotometry The use of light to measure chemical concentrations is called spectrophotometry. When you pass light through a sample, a certain amount of light is absorbed by the molecules in solution, and the rest passes through. In fact, materials have specific color due to certain wavelengths or colors of the visible spectrum that are being absorbed by the molecules in the material. The color you attribute to the material is actually all the remaining reflected light. If we measure the amount of light that is going into the sample and we measure the amount of light that comes out, we can determine to what extent the sample is absorbing the light. In this experiment you will carefully analyze the concentration of solutions of Bromothymol Blue and Thymol Blue. To do this you will use a spectrophotometer, an instrument which measures the fraction of light (I/Io) that is absorbed by a sample. The sample is placed in a tube called a cuvette and is then irradiated with an incident beam of light (Io) of a specific wavelength. A detector then measures the amount of light that is transmitted through the sample (I). A schematic representation of a spectrophotometer is shown below in Figure 1. Figure 1. A schematic representation of a spectrophotometer. (a) A light source produces a polychromatic beam of light. The source used for this experiment produces light of wavelength ranging from 340-1000 nm. (b) The monochromator selects a particular wavelength for the incident beam of light (Io). (c) This is the sample cell. Treat the Cuvettes with care. (d) The detector measures the intensity of the transmitted light (I). (e) The readout is supplied by a meter. Spectrophotometry (Part I & II) INTRODUCTION For any substance, the amount of light absorbed depends on: 1) the concentration of the absorbing species, 2) the length of solution that the light passes through (commonly called the path length), 3) the wavelength of incident light, and 4) the identity of the absorbing species present. One of the more common ways of expressing the amount of light absorbed is called percent transmittance, %T, which is defined for a particular wavelength as %T = (I/I0) x 100 Since the solvent itself may absorb light of the wavelength selected, the value of Io will be chosen as the intensity of light transmitted when the cuvette is filled with solvent only. Another common method of expressing the amount of light absorbed is called absorbance. Absorbance is defined by A = log10 (Io/I) = log10 (100 / %T) Consider a completely transparent sample. In this case the percent transmittance would be 100 which corresponds to an absorbance of 0. Also note that a percent transmittance of 50 corresponds to an absorbance of 0.301. Although absorbance may at first appear complex, it is a very useful measurement. This is because it is directly related to concentration as is shown in the following equation, called Beer’s Law: A=ℓC where A is the absorbance, c is the molar concentration, l is the path length, and  is the molar absorptivity or molar extinction coefficient. Note that a plot of A vs. c will yield a straight line which has a slope of  ℓ. Be aware that Beer's Law does not necessarily hold for all substances or at all concentrations; for example, it often fails at high concentrations. In this lab you will be finding the concentration of an unknown solution of Bromothymol Blue and Thymol Blue. Knowing that  ℓ is the slope of the line for the graph of A vs. c, then you should be able to find the concentration experimentally. That is, if you find the absorbance of Bromothymol Blue and Thymol Blue at different concentrations and plot this set of data, you should get a linear plot that has a slope of  ℓ. Spectrophotometry (Part I & II) INTRODUCTION Operation of GENESYSTM 20 Spectrophotometer Spectrophotometers all contain some kind of light source (visible instruments often use just a tungsten bulb); various mechanical and optical elements which are used to create a directed and collimated beam; a wavelength selector of some sort (often either a diffraction grating or a prism); an enclosed cell compartment to hold the absorbing sample; a radiation detector (often a photocell or more commonly now a photodiode); and some kind of readout device (e.g. a needle deflection on a meter). It is important that you understand the operation of the GENESYSTM 20 before you begin this laboratory. The GENESYSTM 20 is an expensive and sensitive instrument and must be operated carefully and intelligently. The light source in a GENESYSTM 20 is an ordinary tungsten lamp whose radiation extends over the entire visible range. The light from the lamp passes through an entrance slit and is dispersed by a diffraction grating. The grating can be rotated so that a small band of selected wavelengths from the dispersed beam passes through an exit slit, and then through the cell (cuvette) containing the sample. The cuvettes used with a GENESYSTM 20 have a path length (internal diameter) of 1.00 cm. The light transmitted through the sample strikes a solid-state silicon detector that generates an electrical signal proportional to the radiant power (light intensity). The signal from the detector drives a meter that can be calibrated to read transmittance or absorbance. The calibration procedure entails setting 0 Absorbance at a given wavelength with a cuvette containing a reference or blank solution. The blank solution is missing the component of interest, but is otherwise as identical as possible to the solution to be analyzed for the component of interest. Typically, the blank solution is just the solvent. This is required since the output of the lamp and the sensitivity of the detector varies with wavelength. The electronics of the instrument automatically sets 100% absorbance. An identical cuvette containing the solution of interest is then inserted into the spectrometer, and the absorbance is read from a meter on the instrument. Both the calibration and the reading must be done at the same wavelength. The reading for the solution then represents the absorbance at the chosen wavelength due to the component of interest. The calibration has accounted for any absorption (or reflection or scattering) of light by the cuvette and other species in the reference solution. Spectrophotometry (Part I & II) INTRODUCTION GENESYSTM 20 Spectrophotometer 1. On / Off switch 2. LCD display 3. Sample compartment hood 4. Keyboard 5. ---6. Lamp compartment door The power switch is located on the bottom left in the back of the instrument. When you turn on your GENESYS 20 spectrophotometer, it performs its power-on sequence. This sequence includes checking the software revision, initializing the filter wheel and the monochromator. The power-up sequence takes about two minutes to complete. Allow the instrument to warm up for 10 minutes before using it. Keyboard Layout of GENESYS 20 spectrophotometer Spectrophotometry (Part I & II) INTRODUCTION Absorbance and %Transmittance Measurements Be sure the cell holder is empty before turning on the instrument. 1. Press A/T/C to select the absorbance or % transmittance mode. The current mode appears on the display. 2. Press nm ▲ or nm ▼ to select the wavelength. Note: Holding either key will cause the wavelength to change more quickly. 3. Insert your blank into the cell holder and close the sample door. NOTE: Position the cell so the light (indicated by the arrow in drawing) passes through the clear walls. 4. Press 0 ABS/100%T to set the blank to 0 A or 100%T. 5. Remove your blank and insert your sample into the cell holder. measurement appears on the LCD display. The sample Spectrophotometry (Part I & II) INTRODUCTION Error Messages This section lists the messages generated to alert you of errors or other abnormal conditions. The instrument recognizes two types of errors. With the first type, the instrument is still functional; with the second, the instrument is not functional until the condition is resolved. 1. Flashing Data Display: The sample has an absorbance or a transmittance value below or above the photometric range of the instrument. The display flashes until the condition is resolved. 2. Sample too dark: The instrument has been asked to zero a sample with a high absorbance at a low energy point. The instrument beeps three times to announce the message, the message remains on the display for two seconds, then the normal display returns. 3. Sample too bright: The instrument has been asked to zero a sample while the door of the sample compartment is open. The instrument beeps three times to announce the message, the message remains on the display for two seconds, then the normal display returns. Determining the Blank Cuvette 1. Obtain a pair of cuvettes. One will be used for the blank solution and the other will be used for the standard and unknown samples. 2. Fill both cuvettes with the solvent and wipe the outside with a Kimwipe to make sure it is clean and dry (no fingerprints). Be sure to always add enough solution to reach the to within 1/8 of the triangular mark on the cuvette. This will insure that all incident radiation passes through the solution. It is also critical that the triangular mark on the cuvette be facing you when inserted in the sample holder for all measurements. 3. Insert one filled cuvette into the sample holder that is located under the hood to the left. Close the hood. 4. Calibrate to 0 absorbance by pressing the 0 ABS/100%T key. Remove the cuvette, insert the other cuvette, and read the absorbance for this second cuvette. If the reading is less than 0 absorbance, then this second cuvette will be used as the blank cuvette; if the reading is greater than 0 absorbance, then the first cuvette will be used as the blank cuvette. Spectrophotometry (Part I & II) INTRODUCTION Calibrating the Instrument The calibration procedure must be performed for absorbance measurements taken at each different wavelength. 1. Select the desired wavelength by pressing the up or down “wavelength selector’’ arrow keys of the key pad of the instrument. 2. Insert the “blank” cuvette containing the blank reference solution (which is pH 8.0 phosphate buffer for this experiment) into the sample holder and close the hood. 3. Calibrate to 0 absorbance by pressing the 0 ABS/100%T key. 4. Remove the “blank” cuvette. Do not re-adjust any dials at this point. You are now ready to insert the other cuvette containing the sample of choice for a measurement at this particular wavelength. 5. Repeat steps 1-4 for all measurements that take place at different wavelengths. Spectrophotometry (Part I & II) PART I PROCEDURE Safety: Treat the GENESYSTM 20 spectrophotometers with great care as they are expensive and delicate instruments. Wear your goggles. PART I PROCEDURE You will work in pairs on this experiment. Stock Chemicals Used 1st Chemical Maximum Amount Used Bromothymol Blue Standard 80 mL Thymol Blue Standard 80 mL Conc. pH 8.0 Buffer 50 mL Week: Bromothymol Blue Unknown 2nd Week: Mixture Unknown 4.5 mL 4.5 mL Solution Preparations Preparation of the Bromothymol Blue Solutions 1. Make a diluted stock solution of the pH 8.0 buffer by measuring 50 mL of pH 8.0 buffer into a clean Erlenmeyer flask and dilute with water to the 250 mL graduated mark. (The 5% accuracy of the glassware used is more than adequate. Other solutions will need much greater accuracy.) This reagent will be used as a solvent in preparation of all additional reagents Two Important Notes: Make all your dilutions in steps 3 – 6 using only the two 25 mL volumetric flasks in your locker. Plan your work accordingly. Do not use the stock “Buffers Solutions” at pH 4, 7, and 10 in the boxes at the side 2. Bromothymol Blue (molecular weight 646.38) – 23.2 μM stock solution in a pH 8.0 phosphate buffer will be used to prepare the solutions. (This is standard solution #1) 3. Use a 5 mL volumetric pipet to deliver 5.00 mL of diluted pH 8.0 buffer into a 25 mL volumetric flask. Fill to the mark with 23.2 μM Bromothymol Blue. (This is standard solution #2) 4. Use a 5 mL and a 10 mL volumetric pipet to deliver 15.00 mL of the 23.2 μM Bromothymol Blue into a 25 mL volumetric flask. Fill to the mark with the pH 8.0 buffer. (This is standard solution #3) 5. Use a 10 mL volumetric pipet to deliver 10.00 mL of the 23.2 μM Bromothymol Blue into a 25 mL volumetric flask. Fill to the mark with the pH 8.0 buffer. (This is standard solution #4) Spectrophotometry (Part I & II) Part I PROCEDURE 6. Use a 5 mL volumetric pipet to deliver 5.00 mL of the 23.2 μM Bromothymol Blue into a 25 mL volumetric flask. Fill to the mark with the pH 8.0 buffer. (This is standard solution #5) Preparation of the Thymol Blue Solutions 7. You will use Thymol Blue (molecular weight 488.58) – 61.6 μM stock solution in a pH 8.0 phosphate buffer. 8. Using the stock solution of Thymol Blue, prepare a series Thymol Blue standard solutions using the same method outlined for Bromothymol Blue in steps 3 – 6. 9. You have now made a total of 10 samples. Calculate the concentration of each and record the data in your lab notebook. Your TA will check off your concentrations before you leave. Spectral Scans of Bromothymol Blue and Thymol Blue 10. Calibrate the GENESYS 20 spectrophotometer as described in the discussion above. 11. Set the wavelength to 370 nm. 12. Fill a cuvette with pH 8.0 phosphate buffer as the blank. 13. Fill a second cuvette with 23.2 μM Bromothymol Blue and measure the absorbance 14. Fill a third cuvette with 61.6 μM Thymol Blue and measure the absorbance. 15. Use the same three samples to measure the absorbance at increasing wavelengths. For each measurement increase the wavelength increment by 10 nm from 370 nm to 670 nm (i.e., 370 nm, 380 nm, 390 nm, … , 670 nm). Also, measure the absorbance at 434 and 618 nm during this data collection. 16. Make a rough plot during the laboratory period of Bromothymol Blue vs. wavelength and Thymol Blue vs. wavelength. For each graph, add the absorbance values at each particular wavelength to create a new graph of absorbance (sum of Bromothymol Blue and Thymol Blue) vs. wavelength. After the laboratory, make a clear plot using Excel. Question: What does each graph represent? The answer to this question will prepare you for Part II of this laboratory next week. Include an answer of this question in your Post-laboratory conclusion statement. Spectrophotometry (Part I & II) PART I DATA ANALYSIS Measuring Absorbances of the Bromothymol Blue Solutions 17. Calibrate the GENESYS 20 spectrophotometer as described in the discussion above. Remember to use the pH 8.0 phosphate buffer for the blank. 18. Using the reference standards (steps 2 – 6) for Bromothymol Blue, measure the absorbances at 618 nm (the absorption maximum of Bromothymol Blue). Record all five absorbance readings in your notebook. 19. Obtain a Bromothymol Blue solution with unknown concentration from the TA. Record the unknown letter in your notebook. Measure the absorbance at 618 nm using the GENESYS 20 and record the data. Making your Beer’s Law Plot for Bromothymol Blue 20. Make a Beer’s law plot of Absorbance vs. Concentration using the data from the data collected in step 18. Make a rough plot during the laboratory period to see if the graph is linear. If not, re-measure a point that does not fit the line. After the laboratory, make a clear plot using Excel. Include the best-fit line for your data that goes through the origin. Find the slope of your graph. Using 1.0 cm for l, calculate the value of  in units of μM-1cm-1. 21. Determine the concentration of the unknown solution of Bromothymol Blue by using the Beer’s law plot and the measured concentration. SAVE YOUR 10 SAMPLES FOR PART II PART I DATA ANALYSIS Why do we allow electronic instruments to warm-up before use? Why is it important not to have fingerprints on the cuvette? Why is it important to align the cuvette in the sample holder the same way each time? Report the precise concentration of the undiluted stock solution #1 of the Bromothymol blue in micromoles per liter. This is your most concentrated (undiluted) standard solution for which you measured the absorbance. Report the precise volume of the Bromothymol blue standard solution #1 in milliliters used to prepare standard solution #2. You diluted a portion of the Bromothymol blue standard solution #1 with the pH 8.0 buffer to make standard solution #2. Report the total volume of the resulting Bromothymol blue standard solution #2 you prepared. Calculate the molarity of Bromothymol blue standard solution #2. Report the precise volume of the Bromothymol blue standard solution #1 in milliliters used to prepare standard solution #3. Spectrophotometry (Part I & II) Part I DATA ANALYSIS You diluted a portion of the Bromothymol blue standard solution #1 with the pH 8.0 buffer to make standard solution #3. Report the total volume of the resulting Bromothymol blue standard solution #3 you prepared. Calculate the molarity of Bromothymol blue standard solution #3. Report the precise volume of the Bromothymol blue standard solution #1 in milliliters used to prepare standard solution #4. You diluted a portion of the Bromothymol blue standard solution #1 with the pH 8.0 buffer to make standard solution #4. Report the total volume of the resulting Bromothymol blue standard solution #4 you prepared. Calculate the molarity of Bromothymol blue standard solution #4. Report the precise volume of the Bromothymol blue standard solution #1 in milliliters used to prepare standard solution #5. You diluted a portion of the Bromothymol blue standard solution #1 with the pH 8.0 buffer to make standard solution #5. Report the total volume of the resulting Bromothymol blue standard solution #5 you prepared. Calculate the molarity of Bromothymol blue standard solution #5. Report the precise concentration of undiluted stock standard solution #1 for Thymol blue in micromoles per liter. This is your most concentrated undiluted standard solution for which you measured the absorbance. Report the precise volume of the Thymol blue standard solution #1 in milliliters used to prepare standard solution #2. You diluted a portion of the Thymol blue standard solution #1 with the pH 8.0 buffer to make standard solution #2. Report the total volume of the resulting Thymol blue standard solution #2 you prepared. Calculate the molarity of Thymol blue standard solution #2. Report the precise volume of the Thymol blue standard solution #1 in milliliters used to prepare standard solution #3. You diluted a portion of the Thymol blue standard solution #1 with the pH 8.0 buffer to make standard solution #3. Report the total volume of the resulting Thymol blue standard solution #3 you prepared. Calculate the molarity of Thymol blue standard solution #3. Report the precise volume of the Thymol blue standard solution #1 in milliliters used to prepare standard solution #4. Spectrophotometry (Part I & II) PART I DATA ANALYSIS You diluted a portion of the Thymol blue standard solution #1 with the pH 8.0 buffer to make standard solution #4. Report the total volume of the resulting Thymol blue standard solution #4 you prepared. Calculate and enter the molarity of Thymol blue standard solution #4. Report the precise volume of the Thymol blue standard solution #1 in milliliters used to prepare standard solution #5. You diluted a portion of the Thymol blue standard solution #1 with the pH 8.0 buffer to make standard solution #5. Report the total volume of the resulting Thymol blue standard solution #5 you prepared. Calculate the molarity of Thymol blue standard solution #5. Report your absorbance readings for the five Bromothymol blue standard solutions, beginning with the most concentrated solution. Plot absorbance versus concentration in µmol/L of the Bromothymol blue at the wavelength of its maximum absorbance on a spreadsheet or a programmable calculator, and carefully determine the slope of the best straight line fit through the data. Report the letter / number of the sample of unknown concentration that was assigned to you by your TA. Report the absorbance reading you measured for the Bromothymol blue solution of unknown concentration at the wavelength of its maximum absorbance. Report the value you calculated for the concentration of your unknown sample of Bromothymol blue. Determine the percent error in your analysis of the Bromothymol blue solution of unknown concentration. Briefly discuss interpretations of your observations and results. Include in your discussion, any conclusions drawn from the results and any sources of error in the experiment. Spectrophotometry (Part I & II) Part II. Procedures PART II. PROCEDURES Spectral Assignments Using Beer’s Law Plots to Determine the Concentrations of Unknown Mixtures 1. Calibrate the GENESYS 20 spectrophotometer as described in the discussion above. Remember to use the pH 8.0 phosphate buffer for the blank. 2. Using the five reference standards for Thymol Blue, and measure the absorbances at 618 nm (the absorption maximum of Bromothymol Blue). Record the five measured absorbances in your notebook. The five Bromothymol Blue values were already measured at 618 nm in Part I. 3. Repeat step 2 for Thymol Blue and Bromothymol Blue at 434 nm (the absorption maximum of Thymol Blue). 4. When finished, you should have collected a total of 20 readings. 5. The TA will provide an unknown. The unknown is a mixture of Bromothymol Blue and Thymol Blue. Subsequent data analysis will be used to determine the concentration of each substance in the mixture. Scan the Unknown Mixture 6. Starting at 370 nm, measure and record the absorbance of the unknown mixture. 7. Increase the wavelength by 10 nm and measure and record the absorbance again. Remember to blank at each change in wavelength. 8. Repeat steps 6 and 7 until reaching 670 nm. In addition, measure the absorbance at 618 nm and 434 nm (Bromothymol Blue and Thymol Blue absorption maximum, respectively). Record all 33 measured values. 9. Plot Absorbance vs. Wavelength of the unknown mixture. Two peaks will be observed. Make a rough plot during the laboratory period. After the laboratory, make a clear plot using Excel. Question: What do the two peaks in the plot of Absorbance vs. Wavelength represent? Include an answer of this question in your Post-laboratory conclusion statement. Spectrophotometry (Part I & II) PART II DATA ANALYSIS PART II DATA ANALYSIS Analysis using Beer's Law Plots The data collected in Part II can be used to obtain the values of effective molar absorptivity, symbolized by *, for both the Bromothymol Blue and Thymol Blue at the two wavelengths of interest here. Using that information you will calculate the concentration of each in the unknown. We can combine the molecular property  and the effective path length of the cell being used for all the measurements into a new constant, *, whose magnitude is valid only for the particular system (absorber and cuvette) under study. Beer's Law then takes the simpler form A = *C. (Note that the way it is defined here * must have dimensions M-1 because absorbance is dimensionless.) We will distinguish between Bromothymol Blue and Thymol Blue using the following symbols in the equations: BTB = Bromothymol Blue TB = Thymol Blue We shall henceforth distinguish the four different * values by the subscripts listed below: BTB,618 = Bromothymol Blue solution examined at the wavelength of the Bromothymol Blue absorption maximum (618 nm) BTB,434 = Bromothymol Blue solution examined at the wavelength of the Thymol Blue absorption maximum (434 nm) TB,618 = Thymol Blue solution examined at the wavelength of the Bromothymol Blue absorption maximum (618 nm) TB,434 = Thymol Blue solution examined at the wavelength of the Thymol Blue absorption maximum (434 nm) Determining Effective Molar Absorptivity Calculate the molar concentrations of the Bromothymol Blue and Thymol Blue solutions you studied in Part I. Prepare four graphs of absorbance vs. concentration. You must plot the data on an Excel spreadsheet. From these plots determine the four pertinent values of * (slope of the graph). Spectrophotometry (Part I & II) PART II DATA ANALYSIS Spectrophotometric Analysis of an Unknown Mixture of the Two Complexes The absorbance of a mixture of absorbing species that do not interact with each other in any way is simply the sum of the absorbances of the individual species. This means that in a solution that contains both the Bromothymol Blue and Thymol Blue compounds in significant amounts the absorbance at 618 nm (max of Bromothymol Blue) will be given by the equation A618 = *BTB,618 [CBTB] + *TB,618 [CTB] and the absorbance at 434 nm (max of Thymol Blue) will be given by the equation A434 = *BTB,434 [CBTB] + *TB,434 [CTB] where [Cx] is the molar concentration of X = TB, BTB. It follows from this that if the four * values are all known, the individual concentrations in a mixture of the two species can be obtained by measuring its absorbance at the two maximal wavelengths and solving the pair of simultaneous equations given just above. (Students who know some linear algebra will recognize that these equations can be compacted into the matrix equation.) Calculate the concentrations of the Bromothymol Blue and Thymol Blue in the mixture. Scan Adjustments Using the spectral scans of Bromothymol Blue and Thymol Blue from Part I (steps 15 & 16), adjust the measured absorbances to compare to the scan of the unknown mixture. An adjustment can easily be using the equation: A2 = (c2/c1) A1, where c2 is the concentration of either Bromothymol Blue or Thymol Blue in the unknown mixture, c1 is the concentration of either Bromothymol Blue or Thymol Blue from the spectral scans of Part I, and A1 is the absorbance of either Bromothymol Blue or Thymol Blue from the spectral scans of Part I. After making an adjustment to the spectral scans of Bromothymol Blue and Thymol Blue from Part I, then add the absorbance values at each particular wavelength to create a new graph. The graph is a plot of absorbance (sum of Bromothymol Blue and Thymol Blue) vs. wavelength. Plot this graph together with the graph created on step 8 of Part II. Discuss any questions about these graphs with your TA before leaving lab or at office hours. Post-laboratory Questions Report the 5 absorbance readings in corresponding order of solution concentration (high concentration to low concentration; values of absorbance will be running in the same sense, high absorbance to low absorbance). Plot absorbance versus concentration in μmol/L of the Bromothymol blue at the wavelength of the maximum absorbance of Thymol blue, 434 nm, on a spreadsheet or a Spectrophotometry (Part I & II) PART II DATA ANALYSIS programmable calculator, and carefully determine the slope of the best straight line fit through the data. Using this Beer's Law plot, you will determine the value of ε*BT. Report the 5 absorbance readings in corresponding order of solution concentration (high concentration to low concentration; values of absorbance will be running in the same sense, high absorbance to low absorbance). Plot absorbance versus concentration in μmol/L of the Thymol blue at the wavelength of the maximum absorbance of Thymol blue, 434 nm, on a spreadsheet or a programmable calculator, and carefully determine the slope of the best straight line fit through the data. Using this Beer's Law plot, you will determine the value of ε*TT. Report the 5 absorbance readings in corresponding order of solution concentration (high concentration to low concentration; values of absorbance will be running in the same sense, high absorbance to low absorbance). Plot absorbance versus concentration in μmol/L of the Thymol blue at the wavelength of the maximum absorbance of Bromothymol blue, 618 nm, on a spreadsheet or a programmable calculator, and carefully determine the slope of the best straight line fit through the data. Using this Beer's Law plot, you will determine the value of ε*TB. Report the value of AT, the absorbance reading you measured for the mixture at the wavelength of maximum absorbance of Thymol blue, 434 nm. Please enter the value of AB, the absorbance reading you measured for the mixture at the wavelength of maximum absorbance of Bromothymol blue, 618 nm. Using the values of the effective molar absorptivities you determined from your slopes and the values of the absorption you obtained for the mixture at the spectral maxima of 434 nm and 618 nm, solve the 2x2 simultaneous linear equations for the concentrations of Bromothymol blue and Thymol blue. Report the values you calculated for the concentrations of Bromothymol blue and Thymol in the mixture. Report the letter of the mixture sample that was assigned to you by your TA. Determine the percent error in your analysis of the first dye. Your percent error is determined by comparing your calculated concentrations of the sample to the known values. Determine the percent error in your analysis of the second dye. Your percent error is determined by comparing your calculated concentrations of the sample to the known values. Plot your recorded absorbances of the mixture versus wavelength using a spreadsheet such as Excel or a programmable calculator. Be sure to turn in this graph along with the four Beer's Law plots to your TA at the beginning of the next laboratory class. Spectrophotometry (Part I & II) PART II DATA ANALYSIS Look at the absorption spectrum of your mixture. How many peaks do you see in the spectrum? Which species is responsible for the peaks near 436 nm and 618 nm, respectively? Briefly discuss interpretations of your observations and results. Include in your discussion, any conclusions drawn from the results and any sources of error in the experiment. What similarities are observed between the plots? What is the purpose of doing a scan adjustment as instructed earlier? Where does the equation A2 = (c2/c1) A1 come from?