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Unformatted text preview: not mouth pipette; handle the solutions with care and wash your
hands thoroughly after handling
o Also, when you have completed your determination, dispose of your
solutions in the manner described by your TA Biology 05LA – Fall Quarter 2012 Lab 2 – page 4 Making an absorption spectrum of potassium ferricyanide – (work in groups of 4 students).
1) Obtain 5 ml of 0.75 mM potassium ferricyanide in a 10 ml disposable test tube.
2) Use 5 ml of deionized water as the blank to zero the spectrophotometer, and take readings of
the K3Fe(CN)6 solution every 10 nm over the 380 nm to 500 nm range. (13 total readings)
3) Graph the absorbance as a function of wavelength. Do this now. Be certain that your graph
shows the wavelength where absorbance is greatest (the max) and the absorbance value at
that wavelength – you will need this information later.
Quantitative Spectrophotometry
Measuring the amount of light that a sample absorbs allows the quantification of how much
material is present in the sample. Although you may have had a slow and tedious time taking readings
for an absorption spectrum, you gathered a large amount of data so quickly that it is already entered on
a graph. As you will see, these absorption values can be easily converted to molar concentrations.
The relationship between the amount of light absorbed by a solution, and the concentration of the
material in the solution is determined with the use of Beer's Law. Beer's Law describes the
relationship between the absorbance of a given solution and the following parameters:
“c”  the concentration (in moles per liter) of the solution;
“l”  the path length; this is the distance (in cm) that light must travel to pass completely through
the sample. For our labs, a test tube with an inside diameter 1 cm is used giving an “l”
value of 1 cm.
“E”  the molar extinction coefficient; this is equal to the absorbance, at a specified wavelength,
of a 1.0 M solution of a particular chemical measured with a path length of one centimeter.
With reference to the above parameters, Beer's Law appears as follows: Absorbance (A) = Ecl.
Reference texts that describe the properties of solutions of different molecules will often give the
molar extinction coefficient (E) values, but only for chemicals that are colored, or absorb wavelengths
that can be monitored by readilyavailable spectrophotometers. For example, the E value of pnitrophenol at 410 nm is 1.83 x 104 liters per mole x cm.
An exercise in practical quantitative spectrophotometry.
In order to familiarize you with the practice of quantitative spectrophotometry, your group will
be supplied with a numbered vial containing a solution of potassium ferricyanide K3Fe(CN)6 at a
concentration that is known only by your TA. Before you proceed, make sure you record the number
of your sample in your lab notebook. The goal of this exercise is to use your knowledge of Beer's Law
and the spectrophotometer to determine the concentration of K3Fe(CN)6 in your sample.
GENERAL COMMENTS
Before you begin this exercise, a few bits of information must be given. The first concerns the
absorption spectrum of K3Fe(CN)6. As you should know, aqueous solutions of this compound have a
simple absorption spectrum, composed of a single discrete peak that corresponds with the wavelength
of light absorbed maximally by the solution. [This is the " max" for K3Fe(CN)6.] The first task in
the completion of this exercise will be to produce an abbreviated absorption spectrum for K3Fe(CN)6
that shows the location of this peak and a usable absorption reading at the max. Producing an
absorption spectrum that meets these criteria is complicated by the fact that our spectrophotometers
can only read an absorbance of less than TWO! Thus, if the concentration of your sample is too high,
you will not be able to read its absorbance! In which case, it will be necessary to dilute your sample in Biology 05LA – Fall Quarter 2012 Lab 2 – page 5 order to read its absorbance. Because we are trying to accurately determine the concentration of a
sample, the required dilutions must be made in precise way.
Making accurate dilutions. A formula for making accurate dilutions is as follows:
C1V1 = C2V2 where: C1 = the concentration of the solution to be diluted;
V1 = the volume of C1 required for the dilution;
C2 = the concentration of the solution after dilution;
V2 = the final volume of the diluted solution.
In order to show you how this works, let's pose the following problem: Suppose you have a 1.0
M solution of sucrose and you want to make 100 ml of 0.2 M sucrose. (Another way to state the
problem would be to ask – what volume (V1) of 1.0 M sucrose (C1) is needed to make 100 ml (V2) of
0.2 M sucrose (C2 )? The solution is easy: realize that V1 is the unknown value; plug in the above
known values into the formula; and solve for V1. After doing so, you find that V1 = 0.02 liters or 20
ml. What do you do with this value? Measure out 20 ml of 1 M sucrose and then add 80 ml of water
to make u...
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 Fall '12
 AbbottL
 Biology

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