Biology Laboratory Manual 11th Edition Vodopich

Solutions Manual

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Biology Laboratory Manual 11th Edition Vodopich Solutions Manual

Exercise 8
SPECTROPHOTOMETRY
Spectrophotometry and its principles represent a technique used in many areas of biology.
Spectrophotometry is as fundamental as titration and pipetting, and students can master its basics
in a short time. The major concepts include the unique nature of a compound's absorption
spectrum and the relationship of light absorbed to concentration of solute. We usually present this
exercise before the lab on cell membranes so students can use their knowledge of
spectrophotometry to quantify the results of the membrane experiments.

SUGGESTED ELEMENTS FOR AN INTRODUCTORY LECTURE

• Spectrophotometry is used to determine the chemical composition and concentration of a
sample.
• A spectrophotometer is an instrument that measures the amount of light absorbed and
transmitted by a sample solution.
• Visible light includes electromagnetic radiation with wavelengths from 380 nm to 700 nm.
• Different molecules absorb and transmit different wavelengths of light.
• The absorbance spectrum of a molecule is unique to that molecule.
• The color of an object we perceive is the combined wavelengths of light that the object’s
molecules do not absorb.
• Standards are solutions of known concentrations that are used to produce a standard
curve.
• A standard curve is a graph that relates absorbance to concentration of a particular
chemical.
• The higher the concentration of a chemical, the more light it will absorb at its wavelength
of peak absorbance.
• Green light is not used by plants because chlorophyll does not absorb light at about 500 nm.

ACTIVITIES
1. Determine the absorption spectrum of CoCl2.
2. Construct a standard curve for cobalt chloride.
3. Determine an unknown concentration of cobalt chloride using the standard curve.
4. Determine the absorption spectrum of chlorophyll.

VOCABULARY
absorbance absorption spectrum blank
filter light source meter
photodetector sample spectrophotometer
spectrophotometry standard standard curve
transmittance

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MATERIALS FOR ALL PROCEDURES
Number of lab sections __________ Total work groups __________
Work groups per section __________ Students per work group __________

TIME LINE FOR LABORATORY PREPARATION

Beginning of the semester:
Determine the number of sections, work groups, and students in the course.
Inventory supplies and, if necessary, re-order supplies.
After the supply of each material is verified, check off the supply in the spaces in the
list(s) below.
Two weeks before lab:
Determine how many work groups you will have.
Verify that the needed quantities of disposable supplies are available.
One–Three days before lab:
Prepare solutions.
Purchase local items.

MATERIALS FOR ALL PROCEDURES

Quantity Needed
√ Materials Total Per group Catalog Number
Equipment
___ spectrophotometer
1 per group _______ ______
___ blender _______ ______ 15 W 2675
Supplies
___ spectrophotometer tubes—
7 per group _______ ______ 14 W 5554
___ spectrophotometer tubes
24 for unknowns, and
chlorophyll extracts _______ ______
___ 1 mL pipet _______ ______ 18 W 2972
___ 5 mL pipet _______ ______ 14 W 3417
___ 125 mL flask—2 per group _______ ______ 17 W 2981
___ wax pencils—1 per group _______ ______ 15 W 1155
___ test tube rack—1 per group _______ ______ 18 W 4214
___ test tube caps _______ ______
Solutions
___ CoCl2 stock solution 100 mg/ml _______ ______ 947 W 6002
___ acetone _______ ______ 950 W 3205
___ 12 CoCl2 unknowns
in spectrophotometer tubes _______ ______
___ 12 spinach-chlorophyll
unknowns in spec. tubes _______ ______
___ spinach or privet leaves,

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 1 bunch or 20–40 g _______ ______

SOLUTION PREPARATION
• Prepare the stock solution of cobalt chloride by adding enough distilled water to 180 g of
CoCl2•6H2O to make one liter of solution.
• Prepare plant extract before class by putting 20–40 g of leaves in a blender. Lawn grass also
makes a good pigment concentration and has little water that mixes with the acetone. Add
200 mL acetone and blend for 15–20 seconds. Let stand for one minute. Pour the slurry into a
paper towel filter or cheesecloth held over a large beaker. Be sure and wear gloves while
handling the acetone. The supernatant can be stored in a capped bottle for many months. To
prepare a sample tube, dilute the plant extract to 2 mL plant extract to 12 mL acetone to get a
solution that is dilute enough to give a good spectrum. Pour the solution into
spectrophotometer tubes and seal with caps. It will last 2–3 days if refrigerated and still give
a good absorption spectrum. Be careful: acetone is combustible and should not be spilled on
the electric blender. The resulting flame does not burn at a very high temperature. So don't
panic, simply cover the entire blender with a metal trash can if a fire extinguisher is not
readily available.

COMMENTS ON PROCEDURES
• Rather than putting one flask of stock CoCl2 solution in the center of the room, provide each
group with about 50 mL of stock cobalt chloride and 50 mL of water in two 125 mL flasks
with a pipet in each. Pipet bulbs are expensive so we use plastic syringes and attach one to
each pipet with a piece of rubber tubing or aquarium air tubing. They fit well, but remind
students to measure the liquids using the graduated markings on the pipets and not the
markings on the syringes.
• To make 12 unknown solutions of CoCl2, mix two sets of dilutions as directed in Table 7.1.
Then number the unknowns 1–12 to correspond to the dilution numbers. These numbers
allow teaching assistants to easily remember the unknown concentrations.
• Do not mark spectrophotometer tubes such that the wax pencil marks will be in the path of
the light beam.
• After the experiment, we collect from each group leftover cobalt chloride dilutions in 6 large
labeled flasks. To save money, the most concentrated solutions can be reconstituted with
more CoCl2 to make a usable stock solution for next semester.
• Unless otherwise noted, all catalog numbers are Ward’s Natural Science. Comparison
shopping at the following scientific companies might save you money on some supplies:
o Carolina Biological Supply Company, www.carolina.com
o Fisher Scientific, www.fishersci.com
• Safety first: Be sure and cover any safety issues that may be specifically related to this lab
procedure.

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INVESTIGATIVE PROCEDURE
• Inventory/survey class on what supplies are needed for this procedure.

SAFETY
❖ Cobalt chloride is a TOXIC MATERIAL.
❖ Wear PERSONAL PROTECTIVE EQUIPMENT; avoid dust conditions; use under hood.
❖ Avoid contact with: strong oxidizers.
❖ Do not pour cobalt chloride down the sink.
❖ Acetone is a FLAMMABLE LIQUID.
❖ Wear PERSONAL PROTECTIVE EQUIPMENT; avoid sparks and open flames; check
for ignition sources; store in approved containers.
❖ Avoid contact with: chloroform, chromic anhydride, hydrogen peroxide, nitric acid,
acetic acid, sulfuric acid, potassium dichromate.
ANSWERS TO QUESTIONS
1. a. Chlorophyll reflects green light (540–560 nm) and absorbs other wavelengths. What
biologically important molecules other than chlorophyll absorb and reflect certain colors?
Hemoglobin
b. Is the absorbance of light critical to these molecules’ functions, or just a consequence of
their molecular structure? Explain your answer.
In some cases it is critical (chlorophyll), but not in all cases (hemoglobin).
2. Why is it important to clean the sample tubes?
A dirty tube will interfere with the path of light.
3. a. What wavelength is the peak absorbance of CoCl2?
490 nanometers
b. Why is it important to recalibrate with your blank sample often?
To minimize any error in your measurements.
c. Would you expect a curve of the same shape for another molecule such as chlorophyll?
Why or why not?
No, because all molecules have a unique absorption spectrum.
4. Do the plotted data points of your standard curve lie on a straight line?
Not exactly, but close to a straight line of best fit.
5. a. What is the proper blank for determining the absorption of chlorophyll in a plant
extract?
Acetone
b. Which wavelengths are least absorbed by chlorophyll?
Green (mid-range)

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Biology Laboratory Manual 11th Edition Vodopich Solutions Manual

c. Which wavelengths are most absorbed by chlorophyll?

Red and blues (low and high wave lengths of the visible range)

Questions for Further Thought and Study

1. What is the difference between an absorption spectrum and a standard curve?
An absorption spectrum is a plot of the relative absorbance values for a range of wave
lengths passing through a known chemical.
Standard curve is a plot of known absorbance values for a particular wavelength for
each known concentration of a dissolved chemical.
2. Can spectrophotometry be used to determine the concentration of “colorless” solutes such
as salt or sugar? Explain your answer.
Yes. UV or Infrared wavelengths (rather than visible light) may be used in spectroscopy
to detect absorbance in colorless solutions.
3. Why is it important to use standards or to develop a standard curve in spectrophotometry?
The identity of an unknown is determined by comparing its absorption spectrum to that of
known chemicals.
4. Why do leaves of plants appear green? Would plants grow well in greenish-yellow light?
Explain your answer.
The chlorophyll in plant leaves reflects green light, which in turn strikes our eyes. No,
they would not grow well in greenish-yellow light. Those wavelengths are not well
absorbed.
5. How might the basic techniques that you learned about today be used to solve crimes?
Identification of chemicals left behind in a crime scene may help identify suspects.

ADDITIONAL OUTSIDE RESOURCES

• Libraryvideo.com has a small clip describing spectrophotometry for download free:
HTTP://WEBCAST.MEDIAONDEMAND.COM/LIBRARY_VIDEO/20000901/20_SPECTROPH
OTOMETRY_300.ASX

• Carolina Biological Science is on on-line source for plant pigment separation using
chromatography:

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distribution without the prior written consent of McGraw-Hill Education.

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