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Virtual Drug Screening stream Spring 2013 Lab: Beer’s Law - Absorbance vs. Concentration Objective The purpose of this lab is to explore the relationship between concentration of a substance and the absorbance of light. In this process you will learn how to use a spectrophotometer and to determine the concentration of an unknown solution using Beer’s Law. You will be given a colored solution of known concentration and use a pipettor to make serial dilutions (each one is less concentrated than the one before it). You will take an absorption spectrum to determine the wavelength at which maximum absorbance occurs for your colored solution. Using this maximum wavelength you will measure the absorbance of the dilutions and determine the precision and accuracy of your measurements. From this data you will generate an Absorbance versus Concentration standard curve and determine the concentration of an unknown dilution of the same colored compound. This principle is commonly used in the lab to determine the concentration of protein and DNA samples. - Paraphrase this in your own words for your lab notebook Hypothesis Generate a hypothesis for your lab notebook. Your hypothesis should contain a testable prediction of a dependent and independent variable. Principles Many compounds absorb light in the ultraviolet or visible ranges. The relationship of Absorbance to concentration is summed up by Beer’s Law: A=bc Where: A is absorbance is molar absorptivity (with units L mol-1 cm-1) b is path length of cuvette (usually 1cm) c is concentration (with units mol/L) Abs > 1.5 1 Virtual Drug Screening stream Spring 2013 A typical plot of absorbance versus concentration is shown above. Note that absorbance is not directly proportional to concentration at high concentrations or when the absorbance reading is above 1.5. This is because A = -Log T (T = transmittance or amount of light that is not absorbed by the sample). So if absorbance is high, T is very low and you reach the detection limit of your spectrophotometer (saturation of the spectrophotometer). Thus, it is important to find the linear range of your curve by making dilutions of your colored compound. By plotting absorbance versus concentration you can construct a standard curve for your compound and determines the concentration of a solution based upon its absorbance. This relationship only holds true in the linear region of your plot. The molar absorptivity () is a constant and when related to the graph, would be the slope of the linear portion. A compound with high molar absorptivity is very effective at absorbing light and thus will have higher absorbance reading at lower concentrations when compared to a compound with a low molar absorptivity. Pre-Lab exercises: 1) Calculate the amount in grams of NaCl needed to make 40 ml of a 0.5 M stock solution. Formula weight of NaCl is 58.442 g/mol. Show your calculations in your Lab notebook. 2) How many grams of NaCl would make 40 ml of a 2-fold dilution of the 0.5 M stock solution? What would the final concentration be (in molarity)? (a 2-fold dilution is 1part stock solution: 1part diluent, e.g. H2O). So, your new solution would now be half as concentrated as the stock. Materials needed: 1) Stock solution of chemical 7) compound of known concentration (red or blue) 2) Several > 10 ml reusable, nonsterile, plastic test tubes Spectrophotometer (Visible spectrum) 8) KimWipes® 9) 1ml size pipettor and pipette tips 10) 5ml and 10 ml long pipettes 3) 1 test tube rack 11) Rubber pipette bulb 4) 1 beaker of dH20 12) Labeling tape and marker 5) Parafilm® 13) Water squirt bottle 1cm x 1 cm squares 6) 1 plastic cuvette (round or square) 14) Waster beaker for emptying cuvette Formula weights of chemical compounds: Blue #1 792.84 g/mol Red #3 879.86 g/mol 2 Virtual Drug Screening stream Spring 2013 Experimental Procedures All steps should be recorded in your lab notebook (fill in your lab notebook as you go) 1) Working with your partner, obtain a sample of stock solution from Mentor/TA. 2) Record the name of the chemical. ___________ 3) What is the formula weight of the chemical? ______g/mol 4) If you were making the stock solution yourself, calculate the weight in grams of chemical to make 10 ml of a 0.005 M (mol/L) or 5 mM solution. _____g. Show your work in your lab notebook and your lab report. 5) Label your tubes using labeling tape: (Stock, Dil 1, Dil 2, Dil 3 etc.) – don’t write on the tubes 6) Make serial dilutions of your stock solution in the following manner: 1ml Stk 1ml Dil 1 + 9ml dH20 + 9ml dH20 mix well mix well 10fold Dil Add 5 ml of dH20 in each “Dilution” tube except for Dilution 1 and 2 tube 10fold Dil 5ml Dil 2 +5ml dH20 mix well 2-fold Dil 5ml Dil 5 5ml Dil 6 5ml Dil 4 5ml Dil 3 +5ml dH20 +5ml dH20 +5ml dH20 +5ml dH20 mix well mix well mix well mix well 2-fold Dil 2-fold Dil 2-fold Dil 2-fold Dil Stock Solution Dilution 1 Dilution 2 Dilution 3 Dilution 4 Dilution 5 Dilution 6 Dilution 7 Conc= 5mM Conc= ____M Conc= ____M Conc= ____M Conc= ____M Conc= ____M Conc= ____M Conc= ____M Use a disposable 10 ml pipette and a pipette bulb to first add the appropriate amount of water to each tube. Then use a 1 ml pipette or a 5 ml long pipette to transfer the colored solution from one dilution tube to the next. Between dilution steps ensure that each dilution is mixed thoroughly. To mix solutions, cover the tubes tightly with Parafilm then invert 5 times with your thumb over the parafilm. Make a similar dilution diagram in your Lab notebook. Remember that you need to record as much detail of your experiment as possible in your Lab notebook as you will be graded on that. Write out the calculation for determining the concentration of each dilution, use the formula: C1 x V1 = C2 x V2 where C1 is the initial concentration of the solution and V1 is the initial volume of the solution. Once you dilute that solution, you will be adding water and therefore increasing the volume to V 2. So, you must take into account the volume of water (Vwater). V2 = V1 + Vwater Now, re-write the first equation in terms of V1. This will lead to a final concentration of C2. Make sure that you are pretty comfortable using this equation – ask a mentor to give you a hand. 3 Virtual Drug Screening stream Spring 2013 If necessary, more dilutions can be made in order to obtain at least 5 absorbance readings within the range of 0.05 – 1.5. This absorbance range will give you a linear curve when you plot absorbance vs. concentration. Note that when your solution is too concentrated and the absorbance reading is above 2, you are saturating the spectrophotometer. If you plot these values on your Standard curve, they plateau out and do not give you a linear curve which is necessary for the correlation between absorbance and concentration. If you don’t have all 5 points beneath 1.5 Absorbance, then you must go back and make 2 more dilutions to get within the range. Maximal Wavelength: Before absorbance readings can be made, you need to determine the wavelength at which your compound absorbs maximally (max). You can then use this wavelength to take the rest of your measurements. Q: Is it absolutely necessary to use the maximal wavelength? Why or why not? Cuvettes Be sure that you grab the cuvettes with the lower Center Height (8.5mm). This will ensure that the light path of the spectrophotometer is passing through your solution in the cuvette. Q: Do you think the material that the cuvette is made of makes a difference? Why? Q: Is it ok to write directly on the cuvette? Are they re-usable? 4 Virtual Drug Screening stream Spring 2013 Vernier Spectrometer Instructions We have several spectrometers for the Beer’s Law experiment. The RedTide spectrophotometers measure wavelength in both the Visible and UV (Ultra violet) ranges of the electromagnetic spectrum. The SpectroVis specs only collect measurements in the visible range but can also do fluorescence measurements with the 500nm and 405 nm excitation source. Any can be used to complete your experiment since we will only be using visible light absorbance here. Diagram of visible spec cuvette Connection for the USB cable Path of the light beam Connecting the equipment The two Red Tide specs must be connected to a power supply. Note the order of connecting that is labeled on the specs. This order is important to prevent malfunctioning of the device. Connect the specs with the power cables and USB cables. (This step must be done before launching the program.) Launch Logger Pro Click on the Logger Pro 3.5.0 icon (or later version): Once the program is opened, it will take a few seconds to recognize the spec. Once it does, you will see the ROYGBIV (colors of the rainbow) spectrum on a graph with absorbance on the y-axis and wavelength on the x-axis. Now you are ready to start your experiment. Calibrating the spec At the headings along the top, go to Experiment → Calibrate → Spectrometer: 1 A small window labeled Calibrate Spectrometer will open and a 60 second countdown will begin as the lamp in the spec warms up. (If the spec has been used right before you, you may skip this step since it will be warmed up already.) Once the countdown finishes, you are instructed to “Place a blank cuvette in the device”. Obtain plastic cuvettes from the mentors (do NOT write on these. We will re-use them. You can use a small piece of tape to label them). Then using a 1 ml 5 Virtual Drug Screening stream Spring 2013 pipettor, load a tip on the pipettor and transfer 1 ml of dH 2O into the plastic cuvette. To eliminate fingerprints or any smudges on the cuvette, wipe off the sides with a kimwipe There are two ways to insert the cuvette. The path length will either be 1 cm or 0.4 cm depending on which way you have it turned. Be sure to note which way you have it. Make sure you set it up the same way each time. Sometimes there is an arrow that helps to orient the cuvette. Make sure that the liquid level is above the light beam in the spectrophotometer. Take a reading to and select ‘Calibrate’ on the spectrophotometer, press Finish Calibration and then ‘OK’. this will serve as your blank. This is essentially a ‘baseline’ reading that lets the computer know what the lowest absorbance can be. All other measurements are then based off of this reading. Remove the cuvette containing your solvent and pour it out. Rinse with water from the squirt bottle and pout out again. Then try to tap out all remaining liquid from the cuvette by inverting and tapping onto a clean paper towel on the bench top. You are now ready to find the wavelength of maximum absorbance (max) for your sample. Collecting Data Finding the max To find the max of your solution, fill the cuvette a little over halfway full with a portion of your Dilution 2 sample. Insert the cuvette into the spec compartment. Press the green Collect button to start taking absorbance data. You will observe a red line tracing absorbance of the solution along the bottom. Press the blue A figure at the top near the menus to scale the spectral data. Alternatively, right click near the axes then select ‘Autoscale’ Press the red Stop button at the top. Next, go to ‘Analyze’ menu then ‘Examine’ button at the top of your screen. This will allow you to use your cursor to find values along the spectrum by placing a crosshair on your graph. Place the crosshair over the point in the spectrum that represents your maximum absorbance. Record this wavelength and absorbance in your lab notebook. To verify that this is correct, go to the ‘Analyze’ menu then select ‘Statistics’. This will pop-up a window (Statistics Box) that has the Maximum, Minimum, Mean, Median and Standard Deviation of your spectra. The ‘max’ wavelength here is the max for your sample. The max for your compound is ______nm. 6 Virtual Drug Screening stream Spring 2013 Print a copy of the spectrum with the Statistics box and tape it into your Lab notebook and save the graph electronically to show in your report. Make sure that both axes are labeled. (You should also include a sentence to describe the spectrum and what it tells you.) NOTE: print this now – because you won’t have a copy of Logger Pro at home. From this point on, you will use this wavelength to measure absorbance values from all other dilutions. Taking absorbance readings for the Standard Curve Remove the cuvette and properly clean and dry as before. Starting with your most dilute sample (Dilution 8), pour the solution into the cuvette, insert the cuvette into the spec, press the Collect button, after the reading has stabilized, hit STOP. Place the crosshair on the absorbance peak at the same wavelength (max) you found previously (or look up in the table). - Record the absorbance reading Repeat this process three (3) times for each dilution and record the absorbance values for each dilution in your lab notebook. This means emptying the cuvette into the waste beaker, reloading it with solution and then taking another measurement. NOTE: for each successive reading, choose to ‘Store Latest Run’ From this data, you will later be able to construct a standard curve. You should save ALL of you raw data from experiments – i.e. this spreadsheet in .CMBL format. Give your file a name and save it to the VDS folder. Later save it to the ‘cloud’ Proper filename format: UTEID_VDS_BeersLawLab_DATE.cmbl Print a copy of the raw data of all of the spectra combined. You will put this in your Lab Notebook – but it is too ‘busy’ for the lab report. Dil 1 Dil 2 Dil 3 Dil 4 Dil 5 Dil 6 Dil 7 Dil 8 UNKNOWN Conc. (known) Absorbance Trial 1 Absorbance Trial 2 Absorbance Trial 3 Average Abs Standard Dev. After your standards, Measure the absorbance of your unknown sample From the mentors, obtain an unknown sample of your same dye, i.e. if you have been taking measurement of Red #3 – get red. But if you used Blue #1 – then get a blue unknown. Record the unknown sample number in your lab notebook and on your lab report. _________ If we don’t know your sample #, we can’t compare your answer to the key, which makes this lab pointless… 7 Virtual Drug Screening stream Spring 2013 Add 1 ml of the unknown sample into your cleaned cuvette and measure its absorbance at the max you found earlier. Record the absorbance value in your lab notebook. Repeat twice to get n=3 to fill in the table. You now have enough data to analyze and complete this lab. Save the data as an experiment file (.cmbl) on the desktop in the VDS folder and to the ‘cloud’ – e.g. UT Webspace, to back up your data. Close the program when done. Input your data into Microsoft Excel® or OpenOffice Calc ®. Calculate the Average and Standard Deviation. Which standard deviation should you use? Be sure to use the correct number of Significant Figures. Plot your data Standard curve (absorbance on the y-axis and concentration on the x-axis) using values obtained in your graph. Include Axes Titles that show the Units. Make Error Bars that are the Standard Deviations – see end of this handout if not sure. Then create a Linear Best Fit line to the Standard Curve (i.e. the known concentration values). see ‘Data analysis for Beer’s Law lab Using Excel‘. Show the equation on your graph. As a separate series within the same graph, plot you single unknown as a data point. Print out this graph for your lab notebook and save an electronic version for your report. From the resulting equation of the best fit line to the Standard Curve, determine the concentration of the unknown sample. Show your work. Concentration of Unknown: ______M What is the molar absorptivity () of your chemical compound? _______L mol-1cm-1 if the path length (c) of the cuvette is either (1 cm or 0.4 cm depending on which way you had the cuvette turned). Show your work. Clean Up, Clean Up, everybody Clean Up: Clean your station, rinse tubes and beaker – hang to dry, unplug spectrophotometer. If you are the last person using the spec, you should replace it back into the box and put in the drawer. The cuvette should be washed thoroughly with Alconox (soap) and some water, inverted to dry and saved with other Used Cuvettes. Pipette tips can be thrown in the trash. Solutions can go down the drain with water. Clean and dry your test tubes and replace them where you found them. Return other supplies to their location. (mention clean up briefly in your lab notebook – especially proper disposal of reagents) 8 Virtual Drug Screening stream Spring 2013 LAB REPORT See the Wikispaces page for Due Date of this lab report. See the “Lab notebook versus Lab report.doc” on the Black Board site for guidelines. Length 1,250 words Introduction – should include background information on spectroscopy and how it is used in research Objective – put at end of the Intro paragraphs • Hypothesis – 1-2 sentences Materials & Methods – describe your procedures. Balance necessary items vs. being overly detailed. Significant equipment should include name (company, location). Results & Discussion - Embed your Maximal wavelength spectrum, your data table and final X-Y scatter plot graph into a Word or Writer document for your lab report. (These should not be sent as separate files). Include captions. There should also be paragraphs of text describing your results and then analyzing them . Conclusions – summary of what you did and commentary on what the next step would be and/or how this fits in the big picture of what VDS does. References: for any sources you used in your Introduction. But only need a few. Use ACS format and Number your Bibliography PEER REVIEW Your report will be subjected to a review by one or more of your classmates. We will make this a double-blind review so that you don’t know who will review your paper and the review does not know which paper they received. Once you have submitted your original document. You will receive more information on the peer review assignment where you then make revisions to your own paper based upon the reviewer’s comments. This final version will be turned in as well. SUBMITTING Since we are doing a blinded peer review, you will need to submit your paper as a WORD® document – not as a PDF this time! Include the following info in the header of your WORD document (We will go in and remove this later): UTEID and Name VDS Spring 2013 Beer’s Law Lab Report - Original Include the page number at the ‘footer’ – i.e. bottom of page. See the header and footer of this document for examples. Title your filename appropriately – using the standard format that we have been using. Format for filename of submitted assignments: UTEID_Name _Date_ Assignment.docx e.g. JDS297_JonasSalk_021313_BeersLawReportOriginal.docx Upload to the DROPitTOme site. Passwerd: virtual214 http://www.dropitto.me/vdsclass Tip of the Day: to display characters in Word® as Superscript or Subscript – use “Ctrl + Shift + =” or “Ctrl + =” 9 Virtual Drug Screening stream Spring 2013 Data analysis for Beer’s Law lab Using Excel Start Excel and put your names in the top line, the stream name and date, and what experiment you are doing. NOTE: A slick way enter the Date in Excel spreadsheets is to hit: ‘Ctrl’ + ‘;’ the control and semicolon keys together. While, this puts the current time into the cell that your cursor is in: ‘Ctrl’+ ’Shift’ + ‘;’ Enter the Labels in one row, then the known concentrations of each tube in the next row. Then enter each of the three absorbance readings you did for each solution. In the bottom row we will calculate the average of all three. A B 1 2 C D Dil 1 Conc (known) E Dil 3 F G Dil 4 H Dil 5 I Dil 6 0.5 0.005 0.025 0.0125 0.00625 0.003125 0.001563 Abs 1 1.92 1.483 0.881 0.486 0.249 0.132 0.086 4 Abs 2 1.938 1.482 0.882 0.483 0.25 0.131 0.087 5 Abs 3 1.956 1.481 0.883 0.48 0.251 0.13 0.088 y-axis Average Abs Error Std. Deviation J Dil 7 3 6 x-axis Dil 2 milliMolar To calculate the average you will enter this text into the empty cell C6: ‘=AVERAGE(C3:C5)’ ………..… and then hit ‘enter’ This tells Excel to average the values in cells C3 to C5. Do this for each Average Abs value. For example, the next one over would be: =AVERAGE(D3:D5) For Standard Deviation: use =STDEV(D3:D5) or STDEVP(D3:D5) To use Excel to generate your standard curve: Go to the top menu ‘Insert’ and select ‘Scatter’ Choose ‘Scatter with only markers’ This is an ‘XY’ scatter plot – because Y is dependent on X Under ‘Chart Tools’ click ‘Select Data’ (or right click on the chart) Ignore the Data Range page and Click on the Series Tab ‘Add’ ‘Series Name’ – could be called ‘Standard Curve’ Hit ‘Add’ for the ‘X Values’ hit the red arrow box and then select your Concentrations row by clicking and dragging (for example: the shaded row on top in the table above). For the ‘Y-Values’ select the Average Abs row (for example: the bottom shaded row in the table above). Under ‘Chart Tools’ and ‘Chart Layouts’ – select the one with an X-axis title, a Y-axis title and a Legend. (Layout 1) X-axis is named: ‘Concentration [mM]’ Y-axis is named: ‘Absorbance [unitless]’ Usually we don’t put the title on the actual chart. Instead the chart title is incorporated into the caption. So, delete the title from the graph. 10 Virtual Drug Screening stream Spring 2013 Error Bars: In order to add the standard deviation as error bars – click on the chart, go to ‘Chart Tools’ then ‘Layout’ then select ‘Error Bars’. DO NOT – do the default ones – instead go to ‘More Options’ From there select ‘Vertical Error Bars’ with ‘Both’ and then ‘Custom’ Click on ‘Specify Value’ then for ‘Positive Error Value’ click and drag on your Standard Deviation values that you calculated. Do the same for the ‘Negative Error Value’ If it places Horizontal Error Bars on the graph – just click and delete them Finish Add a caption to the chart when you place it in your Lab Notebook or in your report. This caption should be descriptive of what was done for the figure: Fig. Error bars in Excel Fig 1: Spectrophotometric determination of Beer’s law by measuring absorbance of blue dye vs concentration at X nm wavelength. Error bars as Standard Deviation To calculate the Linear Best Fit Line Right click on one of the data point on your graph and select ‘Add Trendline’ Select the ‘Linear’, then hit Options tab, and check all three boxes at the bottom: ‘Set Intercept = 0’, ‘Display Equation on Chart’, ‘Display R-squared value on chart’ We know we can set the Intercept = 0 because if there were Zero dye in our solution, we would get Zero absorbance. We can’t always do this in experiments though. A line will pop up on the graph with the equation and the R2 value. This last term is a measure of how well the Linear Best Fit Line actually matches the data. If the line does not match well that probably means that one of your data points if too high. Usually the most concentrated solution Dil 1 will be out of the linear range of the data. So, we would need to go back and remove this data point from the calculation. You can change the data values by right clicking on the graph and selecting ‘Source Data’. Then reselect only the values you want to keep. Once you have a good R2 that is close to 1, then you can use the equation for your line (in the form of y = mx, in this case y is absorbance, x is concentration, and m is the slope of your line). Using the Absorbance for the unknown solution that you did as the last reading in the lab, you can figure out the concentration. Print the graph and table to tape into your lab notebook and also insert them electronically into your lab report. 11