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CHEM 403
Exp 8
Experiment 8:
SPECTROSCOPY – USING LIGHT AS A PROBE*
When hydrogen and other gaseous substances are heated, they emit light at a few
characteristic frequencies. Atoms and molecules may also absorb discrete frequencies of light.
Absorption is exactly the opposite of emission. When light is absorbed, an electron undergoes a
transition from a lower (usually the lowest, called the ground state) energy level to a higher energy
level. The atom or molecule gains energy in this process by absorbing a photon of light whose
energy, E, is given by E = ℎν. (where ℎ is Planck’s constant, 6.626 x 10-34 J·s). The energy of the
photon corresponds to the difference in energy between the higher and lower levels. The transition
causes a component of light with a frequency ν to be absorbed while all other frequencies are
transmitted (i.e. not absorbed).
The amount of light absorbed by a substance in solution is determined by the amount of
the substance present, the concentration. The absorption decreases as the concentration decreases.
This statement is expressed mathematically by Beer’s law, A = kc, where A is absorption, c is
concentration, and k is a constant characteristic of the instrument and the substance being studied.
In this experiment, you will receive a solution of cobalt(II) chloride whose concentration
is unknown. Your task is to determine the concentration of this solution.
To begin, the absorbance of a 0.150 M solution of CoCl2 will be measured before and after
a series of dilutions. For each dilution, you will calculate the new concentration of CoCl2 and
prepare a graph plotting the absorbance of each concentration. According to Beer’s Law, the slope
of the best straight line will give you the value of k, the constant from Beer’s Law, under your
experimental conditions.
Your solution of CoCl2 will likely be too concentrated to be reliably measured without
dilution. You must determine the concentration of CoCl2 in this solution from a measurement of
the solution diluted a known amount. It will be up to you to discover the appropriate dilution.
Your measurements will be done on a Spec20™ UV/VIS Spectrophotometer set to a
wavelength of 520 nm. This wavelength is absorbed significantly by CoCl2.
Figure 1. Schematic of a Spectrophotometer
*Adapted from Wentworth, R. A. D. Experiments in General Chemistry.
CHEM 403
Exp 8
When a sample is placed in the spectrophotometer that absorbs at the set wavelength, it
interrupts the transmission of this light from the source to the detector. The detector can read the
difference between the light coming from the source directly and the light coming from the source
that passes through the sample. This difference is given as a readout on the spectrometer.
Procedure
Label seven clean, dry test tubes with numbers 1 to 7. Obtain approximately 30 mL of
0.150 M CoCl2 solution from the instructor. Use a graduated pipet to make the additions of the
CoCl2 solution and distilled water as shown in Table 1. Accurate and consistent pipet work is
crucial for success here. If necessary, practice pipetting exact volumes of water before beginning
to prepare your dilutions. Once the dilutions have been prepared, thoroughly mix the contents of
each tube by shaking it side to side. Do not invert the test tube.
Table 1. Prepared Dilutions of CoCl2
Test tube #
mL 0.150 M CoCl2
mL distilled water
Conc. (M) of CoCl2
1
5.0
0
__________
2
4.0
1.0
__________
3
3.5
1.5
__________
4
3.0
2.0
__________
5
2.5
2.5
__________
6
2.0
3.0
__________
7
1.0
4.0
__________
To calculate the concentration of CoCl2 in each test tube, use Equation 1 to solve for Mdilute
Mconc x Vconc = Mdilute x Vdilute
(1)
where the molarity of the concentrated solution, Mconc, will be 0.150 M for all samples, the volume
of the concentrated solution, Vconc, is the listed volume of the CoCl2 used, the volume of the diluted
solution, Vdilute, for each test tube is 5.0 mL.
To measure the absorbance of your solutions, you want to reduce as much experimental
variation as possible. To that end, you should use the same spectrophotometer for every
measurement you take.
Before measuring one of your solutions, calibrate the photometer. With no cuvette in the
instrument, use the left knob to set the instrument to 0% T (∞A). Place the “blank” cuvette filled
with distilled water in the instrument and line up the mark on the cuvette with the mark on the
photometer. With the “blank” water sample in place, use the right knob to set the instrument to
100% T.
CHEM 403
Exp 8
To measure the absorbance of your sample, fill a cuvette with at least 4 mL of your most
dilute solution (test tube #7). Wipe the outside of the cuvette so that it is free any drops of liquid
or fingerprints. Gently tap the cuvette to remove any bubbles, and place the cuvette in the
instrument so that the mark on the cuvette and the mark on the photometer line up. Record the
absorbance of your sample in your Data & Calculations section.
After measuring this solution, pour the solution back into the appropriate test tube, and
rinse the cuvette with a few drops of your next most dilute solution (test tube #6). Discard this rinse
into a waste beaker, then fill the cuvette with 4 mL of the solution and follow the same steps to
measure the absorbance of this sample.
You do not need to recalibrate the photometer with the “blank” between every
measurement, but should check the calibration after every 3-5 measurements. Each of your
absorbance readings for your solutions should fall between 0.1 and 1.2. See the lab instructor if
your readings are outside of this range.
When you have completed your absorbance readings for all seven of your samples, you
can prepare your graph. Label a piece of graph paper so that absorbance (unitless) appears on the
vertical (y) axis and concentration of CoCl2 (M) appears on the horizontal (x) axis. Use the whole
graph paper – do not squeeze your data points into one corner.
Enter each data point on the graph. The data should correspond roughly to a straight line.
Find the slope, k, by placing a ruler at the origin (x = 0, y = 0) and pivoting until the ruler passes
as close to each point as possible. If there is a significant outlier from the straight line, either ignore
the point, or remeasure the absorbance of that solution. Draw your “best fit” line, and calculate its
slope, k = Δy/Δx, by choosing two points on the line.
Once you have prepared your absorbance versus concentration graph, obtain an unknown
from the instructor. A portion of this solution must be diluted until the absorbance lies within the
absorbance range of your standard solutions run previously. To establish the correct dilution,
measure the absorbance of the undiluted unknown. Use your standard curve to estimate an
appropriate dilution. Using your pipet, prepare that dilution and measure its absorbance. If your
diluted solution is still outside of your known range, return to your concentrated sample, prepare a
more dilute solution, and measure its absorbance.
Calculate the concentration of CoCl2 in the diluted unknown solution using the absorbance
and the “best fit” line on your graph and equation using your calculated k. Then, working
backwards from your diluted concentration, calculate the concentration of CoCl2 in the original
(undiluted) solution.
CHEM 403 – Exp 8
Name:______________________________ Section: _________
Data & Calculations
Preparation of Beer’s Law graph
For your concentrations in Table 1, show one example calculation (for a test tube 2-7):
Absorbances of solutions of known concentrations:
Test tube #
Conc. (M) of CoCl2
Absorbance
1
__________
__________
2
__________
__________
3
__________
__________
4
__________
__________
5
__________
__________
6
__________
__________
7
__________
__________
Select two points that fall close to your line of “best fit” on your prepared graph, and use the
following equation to calculate the slope of your line, which is k in the equation A = kc.
slope, k =
∆y
∆x
k = ________________
(give correct units)
Determination of unknown concentration
Unknown number ______
Trial dilutions (you may not need three trials to obtain an appropriate absorbance)
mL unknown
mL water
Absorbance
___ 5.0____
___ 0.0____
__________
__________
__________
__________
__________
__________
__________
__________
__________
__________
For one of your unknown trials:
Volume of undiluted unknown
__________ mL
Volume of water added
__________ mL
Absorbance = __________
Concentration = __________
You can determine your diluted sample’s concentration two ways. Below, show your calculation
using A = kc, and report the concentration found from the line of best fit on your graph. Do these
values match?
Calculate the undiluted concentration from the dilute concentration:
Undiluted solution concentration = __________
CHEM 403 – Exp 8
Name:______________________________ Section: _________
Prelab Assignment
The following data were collected when the absorbance of a series of solutions containing NiCl 2
were measured.
Test tube #
1
2
3
4
5
5.0
4.0
3.0
2.0
1.0
0.0
1.0
2.0
3.0
4.0
Concentration
_____
_____
_____
_____
_____
Absorbance
1.090
0.855
0.647
0.428
0.215
Volume of 0.390 M NiCl2
solution (mL)
Volume of water (mL)
1. Complete the table by calculating the concentration of NiCl2 in each solution (use Equation 1).
Show one of your calculations below.
2. Select two concentrations and their absorbances, and use the following equation to calculate k
in the equation A = kc. Noting that absorbance is unitless and concentration is in units of M, give
the correct units for k in your answer.
k=
Absorbance1 – Absorbance2
Concentration1 – Concentration2
k = ________________
(give correct units)
3. The absorbance of a solution of NiCl2 of unknown concentration is found to be 0.555. What is
the concentration of this solution based on the equation A = kc?
4. Does it make sense that when preparing a best-fit line for a graph of absorbance versus
concentration, the line should go through the origin, (0,0)? Briefly explain.

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