Tube# concentration(mg/ml) Absorbance A B C D E Concentration vs Absorbance 1 0.9 0.8 Absorbance 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 0.2 0.4 0.6 concentration(mg/ml) 0.8 1 Data Sheet Spectrophotometry Lab Name __________________________ II. Class Data Determining the Best Analytical Wavelength of a Food Dye Dye Color Blue Green Red Yellow Analytical Wavelength (nm) 504.6 Table 1. Analytical wavelengths for the four common food dyes. IV. Beer’s Law Plot Data with Serial Dilutions of Stock Dye Solution Color of Your Dye: Solution A B C D E Unknown A Unknown B Concentration (uL/mL) 504.6 504.6 504.6 504.6 / To be Calculated from Beer’s Law Plot To be Calculated from Beer’s Law Plot Absorbance at ____ 0.217 0.103 0.065 0.037 / 0.251 0.527 Table 2. Concentrations and absorbance values for 1:2 serial dilutions of a stock food dye along with absorbance values for two unknown dye solutions. 1 Spectrophotometry Post-Lab Questions – 15 pts Name ___________________________ 1. Prepare a Beer’s law plot in Excel of the Absorbance versus the concentration (uL/mL) for the dye you analyzed. Follow all of the rules for making a proper graph and report the equation for the line on the graph. If you are unfamiliar with Excel, check out ‘How to Make a Graph in Excel’ which is in the Appendix to the lab. Attach your graph when you submit your post-lab assignment through Blackboard. 5 pts 2. Using the equation from your Beer’s Law plot, calculate the concentration of your dye unknowns. Color of Unknown Dye: Analytical Wavelength: 1 pt Unknown Dye A Conc (uL/mL): 1 pt Unknown Dye B Conc (uL/mL): 1 pt 3. One of your unknowns required a dilution. Which unknown needed the dilution? Describe in detail how you made the dilution. 2 pts 4. Your lab instructor asked you to prepare a series of five standard solutions to be used to make a Beer’s Law plot for copper sulfate which is blue-colored. The first solution should be ½ the concentration of the stock solution which is 0.1 M CuSO4. Each solution should have a total volume of at least 50 mLs. Explain precisely how you would prepare the standard solutions including volumes, glassware, etc. 3 pts 5. The absorbance spectrum at the right shows the maximum absorbance for four dyes – red, blue, yellow and green. Based on this spectrum, determine which of the two-dye combinations on the next page, would give the most accurate data for the dye concentrations at the given analytical wavelengths. HINT: Remember that the analytical wavelength should be sensitive to the dye molecule (show a high absorbance) but also NOT be sensitive to the presence of other dye molecules that absorb at the Figure 1. http://www.vernier.com/innovate/innovativeuse53.html 2 analytical wavelength. Briefly explain your answer. 2 pts o o o Red at 520 nm and yellow at 430 nm Blue at 630 nm and green at 630 nm Red at 520 nm and green at 630 nm 3 Using the Spectrophotometer to Determine the Concentration of Common Food Dyes Terms:       Absorbance Analytical wavelength Beer’s Law Beer’s Law Plot Calibration   Cuvette Molar absorptivity () Path length (c)     Percent transmittance Reference solution/blank Serial dilutions Spectrophotometer  Standard curve Figure 1. An absorbance spectrum showing the visible wavelengths of light absorbed by chlorophylls a & b and carotenoids – the plant pigments responsible for driving photosynthesis. Chlorophylls absorb red and blue wavelengths and reflect green wavelengths which explains why plants they appear green to our eye. http://phototroph.blogspot.com/2006/11/pigments-andIntroduction Spectrophotometry is a absorption-spectra.html technique for measuring the absorbance of light by a molecule in solution when a beam of light passes through the solution. Every compound absorbs or transmits light at certain wavelengths and a spectrophotometer can be used to identify the wavelengths where a given molecule absorbs. Spectrophotometers are widely used across many fields to determine the concentration of a molecule in solution including clinical medicine, chemistry, biology, and physics and engineering. The basic parts of the spectrophotometer are shown in Figure 2. Light emitted by the light source may be from the visible (400 – 700 nm), ultraviolet (~10 – 400 nm) or infrared (IR) (~740 nm – 30 cm) regions of the spectrum. The wavelength that shows the maximum absorbance for a given molecule is used to analyze solutions of that molecule and is called the analytical wavelength. Because the solution is most sensitive to the analytical wavelength, this wavelength gives the most accurate results – assuming there are no other interfering compounds present at the wavelength. Figure 2. Basic structure of a spectrophotometer showing the light source, a lens to focus the light and a monochromator which selects the wavelength of light. After the light passes through the solution a photocell detector measures how much of the incident light was absorbed by the solution. http://chemwiki.ucdavis.edu/@api/deki/fil es/8475/spectrophotometer_structure.pn g 1 After the light passes through the solution containing the molecule (Sample Solution in Fig 2), a photocell on the far side of the solution measures how much of the incident light was absorbed by the solution. Older spectrophotometers reported this quantity as percent transmittance (%T) which equaled the percentage of the incident light on the solution that reached the detector. %T values range between 0% and 100% with a 0 %T reading indicating that none of the incident light reached the detector. More often today spectrophotometers report Absorbance (A) which is related to %T by EQUATION 1. Absorbance is a logarithmic scale and readings vary from 0 to 2.0. Absorbance with its logarithmic scale is preferred to %T because it has a direct linear relationship to the concentration of a molecule in solution as expressed by Beer’s Law. EQUATION 1: Absorbance = 2.000 – log(%T) Beer’s Law Once the analytical wavelength has been identified for a given molecule in solution, three variables influence how much light is actually absorbed by the solution – the concentration of the molecule (c), the path length (b) and the sensitivity of the absorbing molecule to the energy of photons at the analytical wavelength ( See Equation 2 below. Path length is simply the distance which the light travels as it passes through the solution and is dependent on the container used to hold the solution. A very common path length is one centimeter. Molar absorptivity is proportionality constant for a given molecule with the units per mole so it is unaffected the concentration of the molecule. The relationship between absorbance and the three variables is summarized by Beer’s Law (EQUATION 2). It states that the absorbance of a solution is directly proportional to the molar absorptivity times the path length times the concentration of the molecule in solution. Because the path length and molar absorptivity are constants for a given molecule and solution container, Beer’s Law essentially reduces to the statement that for a given molecule, the absorbance of its solution is directly proportional to the concentration of the molecule. EQUATION 2: Beer’s Law: A = bc Using A Beer’s Law Plot to Determine the Concentration of an Unknown Solution Because the relationship between absorbance and concentration is linear, a graph of concentration vs. absorbance produces a plot such as the one seen in Figure 3 for the cobalt thiocyanate ion [Co(SCN)4]2-. In a Beer’s Law plot, concentration is always on the x axis and absorbance on the y axis. The graph of concentration vs absorbance is called a Beer’s Law Plot or a standard curve. The equation it produces can be used to determine the concentration of other cobalt thiocyanate solutions. Just read the absorbance of the unknown solution (same wavelength & container) then use the equation from the Beer’s Law Plot to determine its corresponding concentration. 2 Figure 3. A Beer’s Law plot or standard curve showing that the absorbance for (Co(SCN)4)2-solutions is proportional to the concentration of the molecule. Notice that the color for (Co(SCN)4)2-solutions is violet but the analytical wavelength is 625 nm which is in the orange part of the visible spectrum. The wavelengths which these molecules reflect (don’t absorb) like violet are the color which we see for the molecule. http://www.dartmouth.edu/~chemlab/chem6/cobalt2/full_text/write-up.html http://monitoringsrodowiska.bloog.pl/id,330901017,title,ANALIZA-ANIONOW,index.html Here’s how this works. Let’s say that you have a solution of cobalt thiocyanate and determine that its absorbance is 0.600 at the analytical wavelength. Using the equation from the Beer’s Law Plot (y = 2137.9x - .0179) you find the corresponding concentration by substitution. EXAMPLE: Substitute 0.600 for ‘y’ which is the absorbance value and solve. 0.600 = 2137.9x - .0179 x = 2.89 X 10-4 M One of the easiest ways to prepare the solutions for a Beer’s Law Plot is to do serial dilutions from a stock solution. Serial dilutions are a series of consecutively more dilute solutions. Each solution Figure 4. To prepare serial dilutions of 1:10 from a stock, add 9 mLs of water to four test tubes. Then add 1.00 mL of the stock solution to the first test tube (1:10 dilution) and mix well. Remove 1.00 mL from the 1:10 dilution test tube and add it to the second test tube which would be a 1:100 dilution, http://quizlet.com/19577350/test-study-set-ch1-3-4-6-flash-cards/etc. 3 is progressively more dilute by the same factor from the previous solution. For example, you could prepare four dilutions of 10 mLs each where each is a 1:10 dilution compared to the previous test tube as shown in Figure 4. Serial dilutions are commonly used when extremely accurate but very dilute concentrations are wanted. Complications When Using a Spec The key measurement in spectrophotometry is how much light is absorbed by the solution so anything which might interfere with the transmission of light is a potential problem. As a result, special sample holders called cuvettes are used to hold the solution. Cuvettes are a uniform size (same path length) and have special optical qualities which make them good at transmitting light. Whenever you use a spectrophotometer (spec for short), a quick inspection of the cuvettes for fingerprints, scratches or smudges where the light contacts the cuvette is a must. Sometimes other components in a solution may also absorb photons of light at the analytical wavelength. To compensate for this interference, a reference solution or blank is used to zero the spec . The reference solution contains everything in the solution EXCEPT the molecule of interest and is used to show the software what an absorbance of “zero” looks like. When a reference solution is used, any absorbance by a solution containing the molecule being analyzed will be due to the molecule itself not to another component of the solution. The relationship between absorbance and concentration expressed by a Beer’s Law plot does not stretch to infinity covering all possible concentrations of the molecule. Mostly this is simply due to the limitations of the spec. Generally speaking, the greatest sensitivity by a spec to changes in concentration occurs between absorbance values of 0.046 and 1.00 which correspond to % T values of roughly 90%T and 10%T respectively. If the path length remains constant, the concentration values falling within this range of spec limitations depends solely on the molar absorptivity for the given molecule. So what do you do if a solution is too concentrated to be read accurately by the spec and you see that its absorbance value is outside the .046 – 1.00 range of sensitivity? There’s an easy solution; just dilute the sample until its absorbance is within the range. When calculating the concentration for the original solution, don’t forget to account for any dilution which you made. Today you’ll be using the spec to determine the concentrations of two unknown solutions of common food dyes. After you determine the analytical wavelength for the food dye, you’ll prepare serial dilutions from a stock solution of the dye and gather data to make a Beer’s Law Plot. From the Beer’s Law Plot and absorbance values for your unknown solutions, you’ll be able to calculate the concentration of the unknown solutions. Preparing Solutions Like a Pro Since making up solutions is such an important skill in chemistry labs, you’re going to get some practice today preparing solutions. There are really three important parts to preparing a solution accurately: 1) doing accurate calculations; 2) selecting the right glassware and equipment to use; and 3) following the correct procedure in preparing the solution. One word of advice about making solutions, ALWAYS use volumetric flasks to prepare solutions whenever you can. Just add the solid or liquid to the flask and fill the flask with distilled water until the bottom of the meniscus touches the line. Stopper the flask and mix it by inversion numerous times to make sure that everything is thoroughly mixed. 4 Two stations are set up for you to practice making solutions.  At one station you and your lab partner will prepare a 3.5% salt solution using the available glassware. A 1% (mass/vol) solution indicates that 1 gram of salt was dissolved in 100 mL of water or 0.50 grams of salt was dissolved in 50 mL of water. Both solutions are 1% salt solutions – just different total volumes. o The salt concentration of a solution can be measured very accurately using a refractometer – a piece of equipment found on the shelf of every home brewer. The refractometer measures the sugar concentration in the beer so the brewmaster knows when the beer is ready. o Give a sample of your salt solution to your lab instructor. If your solution is accurate, congratulations! If not, think about what you might have done wrong and try again.  At the second station, you’ll take a concentrated food dye stock solution and prepare a more dilute solution, again just using the available glassware. If you’ve forgotten how to use a P1000, now would be a good time to ask. Whenever you need to make a more dilute solution there’s only one equation which you will need and that’s equation 3. EQUATION 3: o o M1V1 = M2V2 where: M1 = Conc. of stock soln V1 = Vol. of stock soln used M2 = Conc. of more dilute soln M1 = Vol. of more dilute soln The accuracy of your dye solution can be measured very precisely with the spectrophotometer. Give a sample of your dye solution to your lab instructor. If your solution is accurate, congratulations! If not, think about what you might have done wrong and try again. The most common mistake when preparing a dye solution is not knowing how to properly use the P1000. 5 Protocol: I. Setting up & Calibrating the Spectrophotometer 1. Turn on the laptop, sign in and open the Logger Pro software. Connect the SpectroVis Plus Spectrophotometer to the USB port of the laptop. From the File Menu, select ‘New’. If you receive a message that the Logger Pro software is not working when you first open it, here’s what to do. Unplug the spec at the USB port and plug it into a different USB port. Then, close the software and reopen it. This usually takes care of the problem but if it doesn’t, check that all the connections are secure. 2. Retrieve two empty cuvettes, being careful NOT to touch the clear sides of the cuvettes where the light must pass through. Hold the cuvette up to the light and look for fingerprints, scratches, dust or drops of dried liquid which might interfere with the passage of light. Clean the cuvette by rinsing it with distilled water from the squirt bottle twice. 3. Prepare the blank or reference solution by filling one cuvette ~3/4 full with distilled water. Place it in the Spec so that one of the clear sides is facing the white arrow. A reference or blank solution in spectrophotometry includes everything EXCEPT the molecule which you will be measuring. Today the reference solution is just distilled water because the food dyes are dissolved in pure water. 4. From the Experiment menu choose ‘Calibrate’ then ‘Spectrometer 1’. Once the warm-up period is complete, select ‘Finish Calibration’, and OK. You only need to calibrate these specs once when you first turn them on. II. Determining the Best Analytical Wavelength for a Dye. The best analytical wavelength for a molecule is usually the wavelength which shows the maximum absorbance for the molecule simply because this wavelength is the most sensitive to changes in concentration. A different wavelength may be used for analysis however if other molecules absorb strongly near the wavelength of maximum absorbance. 5. Retrieve ~5 mLs of your stock dye solution in a clean and dry test tube labeled ‘Stock’. Rinse your second cuvette twice with a small portion of your stock dye solution. Then fill it ~3/4 full with the stock dye and place it in the Spec with one of its clear sides facing the light. 6. Click on the green ‘Collect’ button. A full spectrum graph of ‘Absorbance vs Wavelength’ should appear. Select ‘Stop’ because the data collection is complete. To identify the wavelengths of maximum absorbance, choose ‘Examine’ from the Analyze menu. Move your cursor to the point with the highest absorbance on the graph and you should see the corresponding wavelength in the pop-up box. You may need to drag the pop-up box out of the way if it’s on 6 top of the maximum-absorbing wavelengths. Record the absorbance values for any other significant peaks in your dye. 7. In order to save the data, move the cursor to the data box and right click. Select ‘Copy’ then paste the data into an Excel file and save it to ‘My Documents’. Be sure to give the file a unique name, so that you will be able find it again. Copy and paste the graph into the same Excel file. At the end of lab email any files to yourself and your lab partner because you’ll need them later. 8. Write the analytical wavelength for your dye on the board and copy down the analytical wavelengths determined by groups using the other food dyes. III. Making Serial Dilutions for the Beer’s Law Plot 9. Assemble 5 clean and dry test tubes in a rack and label them A – E. Using either a volumetric pipette or a P1000, add 3 mL of distilled water to each of the test tubes A to E.  If you’re using a P1000 and have forgotten how to use it correctly, ask your TA for a quick refresher.  If you’re using a volumetric pipette, first rinse the pipette with water by drawing ~1 mL of the deionized water into the pipette with a pipette pump. Lay the pipette on its side and remove the pipette pump. Rotate the pipette so that the water contacts as much of the inside surface of the pipette as possible. Drain the water into the sink and repeat the rinsing procedure one more time. Whenever you are using a different solution with the pipette you should following this rinsing procedure to avoid contamination. 10. Using a P1000 transfer 3.0 mLs of the stock dye into test tube A. See Figure 5 below. Thoroughly mix the contents of A then transfer 3.0 mLs from A into test tube B. Thoroughly mix test tube B, and repeat the procedure for the remaining tubes. Be certain that the contents of each test tube are thoroughly mixed BEFORE you transfer an aliquot to the next test tube. 3.0 mL Tube H2O (mL) Final Dilution Stock ----- 3.0 mL A 3.0 1/2 3.0 mL B 3.0 1/4 3.0 mL C 3.0 1/8 3.0 mL D 3.0 1/16 E 3.0 1/32 Figure 5. How to prepare serial 1:2 dilutions from a stock solution. In this example, 3.0 mLs of deionized water was added to test tubes A – E. First, 3.0 mLs of the stock solution was added to test tube A, and thoroughly mixed. Next, 3.0 mLs was removed from tube A and added to tube B and mixed well, and so on. Each test tube is a 1:2 dilution of the previous test tube. 7 IV. Preparing a Beer’s Law Plot for the Food Dye & Analyzing the Two Unknowns 11. Rinse a cuvette three times with small amounts of Solution E – your most dilute solution. Fill the cuvette with Solution E and record the absorbance at the analytical wavelength. Repeat this procedure until you have analyzed all five solutions (A – E) needed to create your Beer’s Law Plot.  Question: Why is it better to start with the most dilute solution instead of the most concentrated?  Once you have created your Beer’s law graph in Excel or similar graphing software, the equation for the line will give you the quantitative relationship between the dye concentration and absorbance. See the Appendix of this lab for help in creating a graph in Excel. 12. Retrieve a ~2 mL sample for each of the unknowns associated with your dye. Rinse a cuvette X3 with the first unknown solution and record its absorbance in your lab notebook. Do the same for the second unknown. Once you’ve determined the equation for your Beer’s Law Plot, you can use it to find the concentration of the unknowns.  If either of the unknowns has a concentration greater than the spec can reliably measure, prepare a dilution for that unknown dye. Why? The Beer’s Law Plot becomes nonlinear at some point and it can only be used to determine the relationship between absorbance and concentration IF the absorbance is in the linear portion of the Beer’s Law Plot.  If you dilute an unknown, be sure to record how you diluted the unknown. You’ll need this information when calculating its final concentration.  The stock concentrations for the four dyes are: Stock Dye Color Blue Green Red Yellow Conc. of Stock Dye (uL/mL) 0.440 0.540 0.600 0.400  Report the concentrations of your unknowns in microliters/mL. 13. All dye solutions can be poured down the drain. Rinse your cuvettes X3 with distilled water and upend them on a paper towel to dry. Wash your test tubes with warm soapy water, thoroughly rinse them and return them to your tub. 8 Appendix: How to Create a Graph in Excel with a Best Fit Line* Here’s an example using some sample data to show you how to make a proper graph in Excel. Remember all good graphs have the independent variable on the ‘x’ axis and the dependent variable on the ‘y’ axis. Also, each graph should be numbered, have a descriptive title, with labeled axes including the units and most importantly have a legend. Legends are often the hardest thing for students to remember to include. A legend is not a ‘key’ to colors or shapes in the graph, it’s an explanation of what the reader should see and understand from the graph. For an example, look at the legend beneath the graph below. To create a graph, open an Excel spreadsheet. In the first column put the independent variable which will go on the ‘x’ axis and label it appropriately e.g., ‘Red Dye Concentration (mg/mL)’ for this example. In the second column, put the dependent variable for the ‘y’ axis and label it ‘Absorbance’. (Absorbance is one of those odd variables with no units). Enter the values you recorded for each set of data points. Look at the data below. To create a chart, move the cursor to an appropriate cell, then highlight both columns and under “Insert” select the chart option. To do a straight line plot, choose the “XY (Scatter plot)” without any lines. You should now have a graph which shows only your data points. Next, right click on any one of the points on the graph and select “Add Trendline” and then ‘Linear” and “Display Equation on Chart” (which is near the bottom) and ‘Close’. Viola! You have a best fit line. You can use this equation to convert any y value into an x value and vice versa. For example, any absorbance value can be converted into a corresponding dye concentration. Just substitute the absorbance into the equation for y and solve for x – the dye concentration. Finally, when calculating a concentration from the graph, remember to account for any dilutions which you made to your sample before reading its absorbance. To add a title and labels for the axes, under “Layout” select ‘Chart Title” and how you want the title displayed. Make your chart title an informative one so that the reader knows what the graph is all about. Under ‘Layout’, you can also create labels and units for your axes and include a legend for your graph. A legend should describe what the reader should see from the graph. Finally, to copy the chart into Word, just select the chart, copy it and then ‘Paste Special’ to insert it into the desired location of your text. Dye Conc. mg/ml 0.0 2.0 4.0 6.0 8.0 10.0 Absorbance 0 0.15 0.32 0.43 0.63 0.82 Absorbance at 540 nm Red Dye Standard Curve 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 y = 0.0807x - 0.0119 0.0 5.0 10.0 15.0 Red Dye Conc. (mg/ml) *Different versions of Excel may show some variation from the described procedure. Figure 1. Standard curve for red dye showing that as the concentration of the dye increases, the absorbance at 540 nm increases linearly also. 9 Unknown 504.6 -3 0.251 Solution D 0.036 m Beer's law,-) A = Em CI Solution Concentration Abs. 5 504.6 Stock 0.600 А A B 103 с 0.075 .064 "D" 0.0315 .037 .321 .217 0.300 0.150 3 443 682 4974
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