Spectrophotometry Exam
60 Points
Date________________
Name_____________________________
(4 points each, 16 points total) MULTIPLE CHOICE
__B__ 1. If the concentration of lactase in a protein solution needed to be determined
as accurately as possible using Bradford reagent, what other instrument
would a lab technician need?
A. Thermocycler
B. Spectrophotometer
C. Fermenter
D. Capillary detector
__C___ 2. What is the relationship between absorbance and transmittance of light
through a sample in a spectrophotometer?
A. An increase in transmittance usually results in an increase in absorbance
B. A decrease in transmittance usually results in a decrease in absorbance
C. A decrease in transmittance usually results in an increase in absorbance
D. Transmittance and absorbance should remain equal
__A__ 3. How should the concentration of a colorless sample be determined?
A. Using a UV spectrophotometer
B. Using a VIS spectrophotometer
C. Either a UV or VIS spectrophotometer
D. Using an indicator solution and pH paper
__B__ 4. How should the concentration of a red sample, such as hemoglobin, be
determined?
A. Using UV spectrophotometry
B. Using VIS spectrophotometry
C. Either UV or VIS spectrophotometry
D. With pH paper
SHORT ANSWER
5. (4 Points) List the types of interactions that can occur between light and matter?
Emission, Absorption, Luminescence, Scattering
6. (5 Points) What are the basic parts of any spectrophotometer and what are their
functions?
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Tungsten lamp for white light and or deuterium lamp for UV light
Prism or grating to change wavelength of light being emitted
Calibration buttons or knob to control set the wavelength
Sample holder is dark so no light gets reflected and sample get placed there
Display to see the setting and or read %T or absorbance
7. (5 Points) How would you determine the concentration of an unknown protein sample?
Explain in details.
Use a set of standard proteins with known concentration. Find out their absorbance by
using a spectrophotometer. Plot absorbance on y-axis vs. protein concentration on x-axis
and plot the results. Draw a best fit line and use it to determine the concentration of the
unknown protein after determining their absorbance using the same spectrophotometer
8. (9 Points) Given the following standard curve, answer the following question
0.5
1
1.5
2.0
2.5
3.0
3.5
a. An unknown protein has an absorbance of 0.45, what would its concentration be
in mg?
2.5
b. An unknown protein has an absorbance of 0.20, what would its concentration be
in mg?
1.25
c. An unknown protein has a concentration of 1.60, what would its absorbance be?
0.25
9. (5 Points) What is a Blank? What Purpose does it serve in Spectrophotometry?
In order to effectively use a spectrophotometer we must first zero the machine
Blank contains everything except compound of interest which absorbs light. By
zeroing machine using "the blank," any measured absorbance is due to the presence
of solute of interest
10. (5 Points) Label the axes and the regions on an absorption curve.
Background, Linear, nonlinear
Y= Absorption, X= Concentration
11. (5 Points) What is Lambda max? How is it determined for various samples?
Lambda max is the wavelength at which a molecule absorbs the most light
Absorption spectrum can be determined by measuring proteins light absorbance at
different wavelengths.
Construct a Calibration curve for absorption vs. wavelength
12. (6 Points) What is Bradford reagent? What is it used for? What are the various colors
it has? What does these colors mean?
Way to colorize proteins and use white light spectroscopy
Solution changes from brown to blue when proteins present.
Degree of “blueness” of Bradford-protein mixture can be used to determine
concentration of protein in a solution
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