Lab Goals - Introduction to the "Spec 20", Graphing, Standard Curves, and Linear Regressions ("Best-Fit Line"). 600
Visible wavelengths Introduction
(Optional; done only in class)
You will have noted that, as you vary the wavelength of light produced by the Spectronic 20, you vary the wavelength of light available for absorption by a material contained within the sample holder.
Measuring the absorbance of a solution - Each type of molecule absorbs each wavelength of light in a characteristic pattern. In general you can make a good guess about a solution's absorbance pattern by looking at the color of the solution. The color it appears is the color that is NOT absorbed. So a blue solution is absorbing all the colors except blue. As you will find out, this does not mean that all blues are identical, nor that the absorbance of other colors is necessarily complete.
Protein Assay - Since the (NaCl/Bradford) components absorb some amount of light themselves, this amount must be accounted for by “subtracting” it from the total absorption for the protein alone. This is done by using the tube containing the Bradford/NaCl only as a "blank" (you did this when "preparing your Spec" on the previous page, see below).
(Once again, this is like the simulation you just did on the previous page) Set the wavelength selector knob to 595 nm. With no spectrophotometer tube in the sample holder but with the sample holder cover closed, use the left-hand, front knob on the Spectronic 20 to adjust to infinite absorbance (0% transmittance). Place the blank (100%NaCl/Bradford) ) into the sample holder, close the sample holder cover and use the right-hand, front knob on the Spectronic 20 to adjust to zero absorbance (100% transmittance). Remove the blank from the sample holder.
Measure the Protein Solutions - Using our virtual Spec 20 below, determine and record (write them down) the absorbance and transmittance values at 595 nm, successively, for each of the different protein concentrations. Using the graph area below, plot the data with Protein concentration on the horizontal axis and absorbance on the vertical axis. (If you haven't done this before here is an Example Graph).
Using data from the the "virtual Spec 20" above, Fill out the below and answer the questions to turn in. Print the data (you will need it) and this page and also blank second copy for use in lab. There is another page to do after you print this one.
Name___________________ Section__________________Lab 1
1. At what protein concentration(s) did you detect maximum absorption? ______
2. At what protein concentration(s) did you detect minimum absorption? ______
3. A 595 nm wavelength corresponds to what color in the spectrum? _________
4. What is the quantitative relationship between concentration and absorbance? _________________________________________________________________________