Absorbance Spectrophotometry: Analysis of FD&C Red Food Dye #40 Calibration Curve Procedure


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1 Absorbance Spectrophotometry: Analysis of FD&C Red Food Dye #40 Calibration Curve Procedure Note: there is a second document that goes with this one! Absorbance Spectrophotometry. Make sure you get a copy of that document! It has the discussion, procedure, data page and advanced study for the experiment. Note: You should look this document over before you print it! You may not need to print the entire document; if you are comfortable using a computer or calculator to calculate slopes and intercepts for lines, you probably don t need to print this document! Part C: Plotting the Calibration curve (between week 1 and week 2). Note: you should plot your calibration curve and have it ready when you arrive for week 2 of the lab! Now that you have acquired data for your four standards, you need to plot the data (which should be linear) and determine the slope of your calibration curve. There are several ways of determining the slope choose the one that is easiest for you: Using Microsoft Excel to plot calibration curve: Note: there are many different versions of MS Excel, and these instructions may vary slightly in different versions! 1. The first step in creating a graph using Microsoft Excel is entering the data. The data should be in two adjacent columns with the x data in the left column. In the figure below, I have labeled the columns but this is not necessary to create the graph. Note that your absorbance data should include the origin {concentration = zero, absorbance = zero}:
2 2. Position the cursor on the first X value (i.e., at the top of the column containing the x values, or "Concentration values), hold down the left mouse button and drag the mouse cursor to the bottom Y value (i.e., at the bottom of the column containing the y values, or "Absorbance" values). All of the XY values should now be highlighted (do NOT highlight the labels!): 3. Click on the Insert tab at the top of the toolbar. 4. Under the Chart section, choose Line. At the bottom of this menu, choose All Chart Types.
3 lines). 5. Under Chart Type choose XY (Scatter), and pick the first option, (without (On older versions of Excel, you will have to choose the chart subtype choose the option that plots the points without lines). 6. After you click OK, a reduced version of your graph will appear; it should be a set of points that makes a straight line with a positive slope.
4 7. If you wish, you may label your graph (ie, put a title at the top of the graph). You may wish to label the yaxis absorbance and the xaxis concentration. You may also modify the style of line, the legend, the data markers, and other options if you wish, although this is not necessary. 8. Next you want to determine the slope and intercept of your line. Move the mouse cursor to any data point and press the left mouse button. All of the data points should now be highlighted. Now, while the mouse cursor is still on any one of the highlighted data points, press the right mouse button, and click on Add Trendline from the menu that appears. 9. From within the "Trendline" window, select the type of Trend / Regression Type you want for a Beer s Law plot the function should be Linear. 10. At the bottom of the same menu, choose the option Display Equation on Chart. 11. Click OK. A line and an equation should appear on the graph, as shown below. Notice that this equation is in the format {y = mx + b}, and numerical values are provided for the slope and intercept. The value of the intercept should be close to zero, a small number, but it may not be exactly zero. In my example, the slope is and the intercept is :
5 Using other software to plot calibration curve: If you don t have MS Excel (or access to this program), there are several free programs available that will serve the purpose. One of the easiest to use for this application is Graph 4.3, a free program which you can download at (Prof. Rubini can provide a copy if you cannot download from this site). You will have to download the installation program and install Graph before you continue. Once you have Graph running, it is simple to plot a linear function and determine the slope and intercept. Select the menu Function and click on the choice Insert point series. A dialog box will open, allowing you to enter the x and y data points use the Enter key after each datum to advance to the next one. Enter your five data points (don t forget that the origin, {0, 0} is one of your points!), and click OK when you are finished. You can also cut and paste data from other applications such as Excel. The data points may be difficult to see on the standard axes that Graph provides. To zoom in on the data, select the menu Zoom and click on the choice Fit ; this will rescale your graph so that all the data points fit nicely on the screen and can be easily read. Now you want to determine the slope and intercept of your line. Select the Function menu and click on the choice Insert Trendline. Several choices will appear (linear, logarithmic, polynomial, exponential, etc.); choose the linear fit and click OK. When your graph reappears, you will see a line that fits your data, and the function appears in the upper left corner of the screen, like this: f(x) = mx + b where, of course, m is the slope and b is the intercept. The program will provide numerical values for m and b; the value of the intercept should be close to zero, a small number, but it may not be exactly zero. You may print the graph, or save it as a bitmap or jpg image, by selecting the appropriate choice from the File menu. You may also save the data set, from the File menu, so you can recall it later without entering the numbers again. Using a graphing calculator to plot calibration curve: Almost all scientific calculators (e.g. TI83, TI84, TI89) will calculate slopes and yintercepts for linear data sets. Please note that you don t necessarily need a graphing calculator; almost any scientific calculator will calculate slope and intercept for a data set of x,y data. Refer to the documentation for your calculator if you want to determine your slope and intercept directly, using your calculator. (This will require you to plot and print the calibration curve separately).
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