Absorbance Experiment 13 Dilutions and Data Handling in a Spreadsheet rev 1/2013 GOAL: This lab experiment will provide practice in making dilutions using pipets and introduce basic spreadsheet skills including graphing and the use of equations and functions to perform calculations. You will use these skills and Excel in several future lab reports. INTRODUCTION: Spreadsheets are as useful for handling numbers as word processors are for handling words. Spreadsheets are like giant data tables that can do all sorts of calculations on that data. Repetitive calculations are handled automatically. If one piece of data is changed, all calculated results are immediately and effortlessly recalculated. Statistical analyses can be performed. Data can be represented in a wide variety of graphical formats. In the exercise you will learn about the spreadsheet Excel. You will complete two short investigations in lab to find the concentration and density of an unknown solution. You will then use an Excel spreadsheet to analyze your data. Determination of unknown concentration One way to find the concentration of an unknown sample is to use spectrophotometric analysis. In this case, we will use UV-Vis spectroscopy. You previously used UV-Vis spectroscopy to determine an unknown concentration in Experiment 9 in the fall. The amount of light that any solution will absorb is proportional to the concentration of that solution. This is expressed mathematically as Beer's Law: A = bc where A is the absorbance of the solution, is the absorptivity constant, b is the thickness of the sample (width of the cuvette), and c is the concentration of the solution. The concentration of any solution can be calculated from its absorbance if we know the values of and b. The value of b is normally 1.00 cm. When we don t know the value of, we can find the concentration of an unknown solution by comparing its absorbance to that of solutions with known concentrations. Typically we will prepare three or more solutions of known concentrations, measure the absorbance of each, and then plot a calibration curve of absorbance vs. concentration, such as the one shown here. After measuring the absorbance of the unknown solution, its concentration can be read from the graph. For example, if the solution of unknown concentration had an absorbance of 0.60, we would consult the calibration curve, and find a concentration of 0.18 M. Calibration Curve for Spec-20 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 0.05 0.1 0.15 0.2 0.25 Concentration, M 1
FORMAT FOR THIS WEEK: In your lab notebook, you will need to record the data from the in-lab portion of this experiment. You do NOT need to make any entries in your lab notebook about the Excel spreadsheet. You will need to use your college email account to email your completed spreadsheet. If you haven t been using it regularly, make sure that you know your password. If you have trouble with any of the software, do not hesitate to get help from computer lab staff or the instructor. LABORATORY REPORT: When you finish this exercise, you should have an Excel spreadsheet. This file will be your lab report this experiment and should be sent by email from your college account to your lab instructor. PROCEDURE: Determination of unknown concentration In this part of the experiment, you will create several solutions with known concentrations from a stock solution and use the resulting calibration curve to determine the concentration of the unknown solution. 1. Take a clean small beaker to the reagent bench. Label this #1 and obtain 15 ml of stock solution. Be sure to note the concentration of this solution in your lab notebook. 2. Choose an unknown sample. Be sure to note the letter of the unknown in your lab notebook. 3. Obtain three 10-mL volumetric flasks and a 5-mL volumetic pipet and bulb. Label the flasks #2- #4. 4. Pipet 5 ml of your stock solution into flask #2. Dilute to the mark with distilled water. Seal with Parafilm and invert several times to mix well. 5. Pipet 5 ml of solution #2 into flask #3. Dilute to the mark with distilled water. Seal with Parafilm and invert several times to mix well. 6. Pipet 5 ml of solution #3 into flask #4. Dilute to the mark with distilled water. Seal with Parafilm and invert several times to mix well. 7. Follow the instructions in the Appendix Use of the Spec-20 to record the absorbance of each solution at the wavelength specified by your instructor. Use distilled water to zero the instrument. Be sure that you do not accidentally dilute any of your solutions by using wet glassware. Do not attempt to dry out the Spec-20 cuvette, but rather rinse the cuvette with a small volume of your solution and then discard this rinse solution down the drain. (These solutions are not hazardous.) You should record the absorbance in order from the most dilute solution to the most concentrated. In other words, start by measuring the absorbance of solution #4 and work backwards to the stock solution. You should have four absorbance readings: each of your dilutions and the stock solution. 8. From the reagent bench, obtain an unknown solution. Record the absorbance of this solution as well. 9. All solutions may be rinsed down the drain when you are finished. Density of unknown solution In this part of the experiment, you will measure the density of your unknown. Take a small dry beaker, your unknown solution, and a 5 ml volumetric pipet and bulb to the balance area. Place the clean, dry beaker on a balance and record the mass of the empty beaker to 0.001 g. Carefully pipet 5 ml of unknown solution into the beaker using a volumetric pipet. Record the mass to 0.001 g. Repeat. You should have at least two measurements of the mass. 2
Computer analysis We will use Excel to analyze the data collected in the lab. Excel is available in all college computer labs. You are welcome to use your own version of Excel as well. The graphing directions below are specific to Excel 2010. 1. Open Excel and look around. Look at your empty spreadsheet. It is arranged as a large table with columns lettered alphabetically and numbered rows. Thus, any cell can be identified by designating its column and row, e.g., cell B3 is in the second column and third row. You can use your mouse or the arrow keys to move to any cell in the spreadsheet. This one spreadsheet file can actually contain several related spreadsheets. Near the bottom of your screen, you will see several file tabs labeled Sheet 1, Sheet 2, etc. Clicking on the file tabs will move you among these different sheets. 2. Change the name of the first data sheet To keep things clear, each sheet in a spreadsheet should have a descriptive title in the upper left-hand corner of the sheet. In the first sheet, you will determine the density of your unknown solution. The title of your first sheet, then, should refer to density. Click on cell A1 and add a title and your name. Continue to improve the labeling of your sheet by giving it a more descriptive name than "Sheet 1". To do this, double click on the file tab at the bottom of the screen, labeled Sheet 1. Rename this sheet "Density". 3. Enter a data table including formulas and copying. Use your data and that of classmates with the same unknown to determine the density of your sample. a. A few lines below your title, create the table below in your spreadsheet. Excel does not keep trailing zeros unless it is told to. To keep those trailing zeros, specify the number of decimal places to be retained from the Number menu box. You should have at least 3 digits to the right of the decimal point for each of the mass measurements. Enter the data in the appropriate columns by clicking on the appropriate cell and typing in the value. Unknown number Volume of unknown solution (ml) 5.0 Student 1, trial 1 1, trial 2 2, trial 1 2, trial 2 3, trial 1 3, trial 2 Mass of empty beaker (g) Beaker + solution (g) Mass of solution (g) Average Density = Standard Deviation = Density of solution (g/ml) b. Calculate the mass of the solution for student 1, trial 1. To do this, click on the cell where this mass should go. Then type the formula to do this calculation. Start all formulas with =. If the data you needed to use were in cells C5 and D5, the formula would be =D5-C5. When you press Enter or click on a new cell, the spreadsheet places the calculated result in its cell. 3
c. While we could type in separate formulas for each cell calculation, it is much faster to copy one formula down the full column. Excel will automatically choose data from the correct student for each calculation. Excel considers cell address to be relative address, unless otherwise specified. This allows us to copy formula simply, as Excel automatically adjusts the cells it uses in each calculation. To copy your formula, click on the cell where you have already calculated the solution mass. Use your mouse to point to the lower right corner of this cell. The cursor should turn into a thin + sign. Hold down the left mouse button and drag down the column to highlight all the necessary cells. When you release the mouse button, the new values will be calculated. NOTE: In Excel, the cursor can take on several different forms. Normally the cursor looks like a thick cross. If you wish to designate a block of cells for some reason, click on the top left cell in the block and drag to the bottom right cell. This will highlight the cells. To copy the contents of a cell to one or more other cells, point at the bottom right corner of the first cell. The cursor will turn into a + sign to signal that you are copying. Click and drag to copy the contents of the original cell into one or more adjacent cells. You can also move the contents of cell to a new position. Click on the original cell. Then use the mouse to point to the lower edge of the cell. The cursor should look like an arrow. Click and drag to the new cell. d. To calculate density, each mass must be divided by the volume of the solution. Notice that the same volume was used for every measurement. For this calculation we must specify an absolute address for the cell containing the volume since it doesn t change from one trial to another. Assume that the density to be placed in cell F5 is to be calculated by dividing the mass in cell E5 by the volume in cell C3. We type the formula as =E5/C$3 in cell G5. The dollar sign before the 3 keeps the 3 from changing when we copy the formula down. When this formula is copied, Excel will keep dividing by the volume in cell C3. Calculate the solution density for student 1, trial 1 in your spreadsheet using an absolute address for the volume. e. Copy your formula for density down the rest of the column using the copy/drag method. f. Calculate the average density, placing this average beneath the density column. To do this, click on the cell where you wish to put the average. Then type =average( After typing the first half of the parentheses, use your mouse to click/drag highlight the cells containing the individual density values. Add the ending ), and then press Enter. Excel has many similar automatic functions. You can learn more about these by looking on the Formulas tab. Several that you may need in General Chemistry are =average( ) =ln( ) =log10( ) =stdev( ) =sum( ) Takes the average of cells inside the parentheses Takes the natural log of the number inside the parentheses Takes the log base 10 of the number inside the parentheses Returns the standard deviation of the cells inside the parentheses Sums up the numbers inside the parentheses g. Now calculate the standard deviation using the stdev( ) function just as you used the average( ) function. 4
4. Save the spreadsheet. Saving a spreadsheet is like saving any other file, and should be done often. If you are working on the college network, you can save files to a network Z: drive. One advantage of using a network drive is that the IT department regularly backs up these drives and you can t lose the files. Save your file to an appropriate drive, giving it a descriptive name, like Excel lab, and include your initials or name. Any time a file will eventually be sent to an instructor, include your name or initials in the file name. Each time you save this file, save it under the same name, replacing the previous version. 5. Change the name of the second sheet and enter data. A. The second sheet will hold your calibration curve. Enter a descriptive title, your name, and the date at the top of the spreadsheet. Change the name of the sheet to Calibration. B. Leave one or two empty rows at the top of your spreadsheet and then enter the titles to the calibration data, using adjacent columns. There should be three data columns: Sample, Concentration, Absorbance. In these you will enter your calibration data. You will notice that some of the word concentration disappears after you enter it because it is too long for the column width. Adjust your column widths to make your table look better. To widen column A, place your cursor on the line separating the light blue boxes containing the A and B labels at the top of your spreadsheet. The cursor should look like a double headed arrow. Click and drag to adjust the column widths. A B C 1 2 Sample Concentration Absorbance 3 #1 4 #2 5 #3 6 #4 7 8 Enter the concentration of the stock solution in B3. This was provided on the stock bottle in lab. To determine the concentrations of your known solutions, use the equation We can rearrange this equation to solve for M 2. To find the concentration of #2, we will need to know the concentration of the stock solution (M 1 =B3), the volume of stock solution added to #2 (V 1 =5.0 ml), and the final volume of #2 (V 2 =10.0 ml). In cell B4, you should enter =B3*5/10. The remaining solutions were made in the same way, so you can copy your formula down the column using the copy/drag method. 6. Graph the data. We want to prepare a graph of our data which will eventually be used to calculate the concentration of the unknown solution. Follow the general graphing directions below. Highlight the rectangular block of "cells" that contain your data (concentration and absorbance). Do this by pointing the mouse at the upper left cell of your data, holding down the left button as you drag the mouse to the bottom right cell of your data, and then let go. The rectangular block should now be highlighted. A. Click on the Insert tab at the very top of the page. 5
B. Choose the Scatter chart type. (Do NOT choose the line graph!) Then select the top choice in the subtype section, the graph that just shows only data points with no connecting lines. C. This opens a graph on your spreadsheet but it needs to be customized to appear as we will normally want them for a chemistry class. A new set of tabs should have appeared at the top of your screen giving the Chart Tools of Design, Layout, and Format. If these don t appear, click on the graph. At the top of the screen, click on the Layout tab. 1. Add a chart title: Click on the Chart Title button and choose Above Chart. Always use a descriptive title. For this graph, type the title: Exp 13 Calibration Curve. 2. Add axis titles: Click on the Axis Title button and choose Primary Horizontal Axis Title, Title Below Axis. Enter: Concentration (M). Note that you always want to include units on the axis label. 3. Add the vertical axis label by repeating step 2 but choosing Primary Vertical Axis Title, Rotated Title. Enter: Absorbance. Note that absorbance has no units. 4. Usually we turn off the legend. Do this by choosing Layout, Legend, None. 5. Usually we don t want gridlines. Turn these off under the Layout, Gridlines buttons. 6. We often want to add a trendline to our data and use the equation for this line. Do this by clicking on Layout, Trendline, More Trendline Options. Choose a Linear trendline and choose to Display Equation. 7. If the equation for your trendline is too close to the line, click on it and move it to an open area of the chart. 7. Calculate unknown concentration Use the functions slope() and intercept() to find the numerical values of these properties for your data. Do not copy the numbers from the graph; instead, use the functions as you did for average and standard deviation in Sheet 1. For slope and intercept, you must select the appropriate data ranges in the correct order. If your concentration data were in cells B3-B6 and the absorbance data were in cells C3-C6, the correct use of the slope formula would be =slope(c3:c6,b3:b6). Note that the y-axis data is listed first followed by the x-axis data, with a comma separating the two data ranges. A general linear trend line has the formula y = m*x + b. In this case, y = absorbance and x = concentration. The slope and intercept are represented by m and b, respectively. We can rewrite this equation as follows: To find the concentration of the unknown solution, you need to rearrange this equation to solve for concentration in terms of absorbance. Add an additional line to your data table for the unknown. Label this unknown in the first column. Use the rearranged equation above and your calculated values for slope and intercept to find concentration. Be sure to enter equations into your spreadsheet where appropriate. 8. Save the spreadsheet. Save your spreadsheet again, using the same filename used before. Exit from Excel. 9. E-Mailing Your Spreadsheet File. Send an e-mail to your instructor with your Excel file attached. You should also send a carbon copy of this email to yourself so that you will be sure that is was done correctly. This and your yellow sheets are the complete lab report for this week. Don t forget to turn in your yellow sheets before you leave. 6
APPENDIX: USE OF THE SPEC-20 The Spec-20 is a spectrophotometer, which provides quick, simple measurement of the fraction of visible light that a sample absorbs. A schematic diagram of a simple spectrophotometer like a Spec-20 is shown in Figure 1. Light Monochrometer Sample Source Cell Figure 1: Schematic Diagram of a Spectrophotometer Detector Output meter The light source in a Spec-20 is basically a light bulb that produces light across the entire visible region. Some of this light passes through a slit to the monochrometer, which selects the single wavelength of light at which the measurement will be made and allows only this light to pass on to the sample. While the schematic diagram above shows a prism as the monochrometer, most modern instruments, including our Spec-20s, use a diffraction grating to separate the light into its component wavelengths. The sample cell contains the solution being studied. The sample cell in a Spec-20 is a cuvette. Some cuvettes look very much like test tubes, while others are plastic and have flat sides. The width of the sample cell is the pathlength, b, referred to in Beer's Law and is a constant of usually 1 cm. The detector measures the amount of the light that passes through the sample, that is, the light that doesn t get absorbed, and then sends an electrical signal to the output meter. The Spec-20 can be read in two different units, either absorbance or percent transmission. On digital models, there is a button for setting the units. On analog models, both scales are on the same dial. We normally record absorbance because this can be put directly into Beer's Law, which says A = bc, where A is the absorbance of a sample, is the absorptivity constant for the compound, b is the pathlength of the light, and c is the concentration of the solution. Operating Instructions for the Digital Spectronic 20 Genesys 1. Turn on power switch on back of instrument. The initialization sequence takes about 2 minutes. Wait until this is complete. A longer (e.g. 30 minute) warm up period is recommended. The display will show the wavelength at which the instrument is set and the units in which it is set to read (absorbance, %T, or concentration). 2. Adjust the wavelength using the nm up and nm down buttons. 3. Use the A/T/C button to read in the desired units. We normally read absorbance. 7
4. Fill a cuvette about two-thirds full with distilled water (or solvent blank) and wipe the cuvette with a tissue. Insert the cuvette into the sample chamber and close the cover. Press the 0 Abs/100%T button to zero the instrument. Light travels front to back in this instrument, so be sure that the cuvette is inserted with clear glass front and back. If the sample to be studied isn't simply dissolved in water, the solvent blank should contain the appropriate solvent and any other reagents that may be in solution which could absorb. This instrument can take square or round cuvettes. See your instructor for help if needed. 5. Fill a cuvette about two-thirds full with the sample to be studied, wipe the cuvette, insert it into sample chamber, close the cover, and read the absorbance (or %T) from the meter. Helpful Hints. If the absorbances of several solutions are to be measured, simply measure the first one, discard it, then rinse the cuvette several times with small amounts of the next solution to be studied. Any dirt on the outside of the cuvette, or bubbles or particles in the tube can distort your reading. Turn the Spec-20 on at the beginning of the lab period and leave it on. Do make sure that it is turned off at the end of the day. 8