To complete this laboratory, you must construct two graphs. The first graph is constructed from data taken to find the wavelength of maximum absorbance for bromphenol blue. This is the wavelength of light most readily absorbed by bromphenol blue. Determination of this wavelength is important because this is the wavelength of light most sensitive to subtle changes in concentration.
To construct this graph, simply plot the wavelength on the X-axis (the independent variable) and the absorbance values on the Y-axis. When you are complete, the graph should look like this:
To construct the standard curve for bromphenol blue, you must first prepare a series of dilutions for bromphenol blue. The instructions for this are found on page *** of the lab manual. Before you prepare the dilutions, you must determine what the final concentration of each dilution will be. To do so, you simply use the following formula:
In this formula:
For example, the first dilution you prepared had 1.0 ml of bromphenol blue and 7.0 ml of water, for a total volume of 8.0 ml.
Solving for C2, you get:
Now use this formula to solve for the remaining concentrations in the dilution series.
Once you have the concentrations calculated, you can now prepare the standard curve. You should have a data set consisting of concentrations of bromphenol blue and absorbance values. Prepare the graph by plotting concentration of the X-axis and absorbance on the Y-axis. After plotting these values, determine the BEST STRAIGHT LINE for your data. Do not simply connect the dots! Then have the spreadsheet program generate the equation of the best-fit line. When you are done, your graph should look like this:
To determine the concentration of your unknown (remember, this was the purpose of this exercise...) simply take the absorbance value of your unknown, substitute this value for y in the equation of the line, and solve for x. For example, if the absorbance of your unknown was 0.855 and the equation of your line was y = 101.65x - 0.0849, the concentration of the unknown would be:
0.855 = 101.65x - 0.0849
0.940 = 101.65x
x = .00924 mg/ml