1. SPECTROPHOTOMETRY
EXPERIMENT NUMBER 10
Written by Honors Student David Teuscher
EXPERIMENT NUMBER 10
PRE-LABORATORY QUESTIONS
Please answer the following questions prior to coming to lab. The questions should be answered in the
lab notebook. The answers will be torn out of the notebook and turned in AT THE BEGINNINGOF
THE LAB PERIOD.
Please read the following lab experiment.
1. Please define the following:
a) spectrophotometry
b) transmittance
c) cuvette
d) wavelength
2. State Beer’s Law and define the terms involved.
3. Describe the Vernier SpectroVis and its function.
4. In your lab notebook, please graph the following data. (Graph the data in your lab book by hand.
Please use at least 2/3 of the page for the graph. Label the graph, the X-axis and the Y-axis.)
Concentration Absorbance
0.222 0.087
0.436 0.179
0.680 0.255
0.900 0.367
1.123 0.500
Now, use this graph to determine the concentration, in [M] that has an absorbance of 0.418. (Use
the method of least squares discussed at the end of this lab to draw the best straight line for this
graph. We want you to do these by hand so you will have a better idea of how to do graphs and
what the spread sheet is doing when it does graphs for you when you get to the post lab
questions.)

2. EXPERIMENTAL PROCEDURE
Spectrophotometry is a long word that is very seldom used in games of Scrabble. It is essentially the
measurement (“metry”) of light particles (“photo”) coming from the electromagnetic spectrum
(“spectro”).
OBJECTIVE: The objective of this experiment is to become familiar with the concept of
spectrophotometry and how it relates to Beer’s Law.
MATERIALS: Riboflavin (C17H20N4O6)
SAFETY: Riboflavin should be carefully as it is normally called Vitamin B2. It plays a key role in
energy metabolism, and for the metabolism of fats, ketone bodies, carbohydrates, and proteins.
Milk, cheese, leafy green vegetables, liver, kidneys, legumes, tomatoes, yeast, mushrooms, and
almonds are good sources of vitamin B2, but That Doesn’t MeanNow is a Good Time to Eat
Your Vitamins!!
PROCEDURE:Read and follow the directions given below.
Before we get to the directions, let’s stop and look at a little theory so we can understand what we are
trying to do in this lab. First, we are dealing with moving electrons around in the outer parts of an atom
or a molecule. When these electrons move from one place to another there is a certain level of energy
involved. This particular level of energy is what we are measuring.
Consider the hypothetical element named Idahoium. Idahoium is an irregular shaped atom of immense
size (compared to other normal atoms). The electrons in Idahoium live in certain places within the atom
(Make up some silly names for these places like Twin Falls, Jerome, Shoshone, and Sun Valley.) When
the electrons travel from one place to another they cannot stop in the middle. (These electrons do not
drink large amounts of coffee before beginning a trip.) When an electron travels from Twin Falls to
Jerome it uses a set amount of energy. When an electron travels from Twin Falls to Shoshone it uses a set
amount of energy also, but the amount of energy it uses is different than the energy used in going from
Twin Falls to Jerome. The trip from Twin Falls to Sun Valley uses a third amount of energy different
from the other two. (In the real world, we could relate the amount of gasoline energy it takes to make
each of these trips.)
Now, assume we know the amount of energy it takes to make each trip. If we look at the energy being
used, we can determine the trip that was made. (Just like your parents could look at the gas gauge on the
car and tell how far you drove on your date last night.)
We are going to be looking at how much energy is involved in moving an electron from one place to
another in a real atom. There are severalrelationships we need to look at next:
E = h E = energy h = a constant = frequency
(the frequency is how many waves of light pass a given point in one second)
 = c/where C is the speed of light and  is the length of one

3. wave of the light. We call it the wavelength.
If we combine these two, we get E = hc/
What is important here is that energy is directly related to the wavelength of light. So if we monitor the
wavelength of light we can know something about the energy involved. Specific amounts of energy (like
going to Jerome or Sun Valley) have specific wavelengths associated with them.
There is one other key relationship we need to discuss before beginning the lab. It is called Lambert-
Beer’s Law or simply Beer’s Law. (No it is not “tastes great, less filling”.)
The law is A = abc
Where A = absorbance,which is a function of how much light is absorbed by the atoms of
interest (in effect how much energy is used by the sample when we make the electrons
go from Twin Falls to Shoshone.)
a = Absorption coefficient which is how efficiently the atoms absorb the energy (It is
a constant for each atom for each different trip it can be related to the miles per
gallon of your vehicle.)
b = The path length of light through the sample. The longer the path through the
sample the more likely the light will interact with an atom.
c = The concentration of the atom or molecule of interest in the sample which is
going to be interacting with the light. The concentration is usually given in terms
of Molarity, moles of solute per liter of solution.
One final term we need to look at before beginning the lab is the concept of transmittance. Transmittance
is related to absorbance. Transmittance,however, is not linearly related to the concentration so it is
harder to work with. It is easy to mathematically relate the two.
A = -logT In English, this says the absorbance of a sample is equal to the negative
logarithm of the transmittance. (NOTE – most instruments will report the
percent transmittance, so you must divide the percent transmittance by 100 to get
transmittance before mathematically converting to absorbance to use Beer’s
Law.)
So we are going to pick a wavelength [energy corresponding to a given trip of the electrons, officially
called an electron transition] and then measure how often the atoms absorb this amount of energy. Using
Beer’s Law we can then determine the concentration of the atom or molecule in the sample.
Now if you understand the preceding three pages of theoretical stuff we are ready to begin. If you don’t
understand the preceding pages, then we are going to begin anyway so we hope you catch on quickly.
DIRECTIONS:
See the USE OF A Vernier SpectroVis Spectrophotometer at the end of this lab for
directions on how to use the instrument. The short time that you spend before lab reading
how to use this machine could get you finished within the first hour of LAB!!!
1. Prepare a 500 mL of a .10 M NaCl Solution in a large flask (It probably been a little while since
you calculated molarities hint---Use Dimensional Analysis… moles/liter etc…to calculate how

4. many grams you will need before you make any mixtures. Then make the solution. When making
the solution put NaCl in flask and then fill approximately half to ¾ full and use a pipet and pipet
pump to accurately achieve proper Molarity when adding distilled water. ~~~Remember to rinse
and condition the pipet because other solutions could interfere with results
2. Calculate how many grams you will need to make a 250 mL riboflavin solution in a volumetric
flask containing 40 mg/L. You will add the milligrams of riboflavin to the empty flask and then
add the .10 M NaCl solution, remember to condition the pipet once again except this time with
the NaClsolution. Take your Time, or You Will Have to Repeat Previous Steps to Get
Accurate Results! (Hint—At this point you should have a flask just .10 M of NaCl solution and a
flask with riboflavin with .10 M NaCl solution)
3. Calculate the Molaritiy of the Riboflavin solution as you will need it later when doing Beer’s Law
with the Vernier SpectroVis.
4. Now that the molarity of the riboflavin solution at 40 mg/L has been obtained, we need to prepare
a series of four dilutions to obtain samples of 5, 10, 20, and 30 mg/L of riboflavin using a solution
matrix of the .10 M NaCl. See table below:
Dilution Calculation Equation: M1V1=M2V2
M2=M1V1 / V2
SOLUTION #1 mL of Your Stock
Solution (riboflavin)
mL of NaCl Solution Total Volume of
Solution
1 .75 5.25 6
2 1.5 4.5 6
3 3 3 6
4 4.5 1.5 6
5 6 0 6
5. Now comes the fun part, calculate the molarity of riboflavin in the first four solutions. If you’re
wondering what about the fifth, it will be the first sample tested in the SpectroVis, see step 9
6. Make sure all your power, USB cables are plugged in and then open Logger Pro in the upper left-
hand corner. With Luck, the application will open and you will see colors like you flashed back to
the 70’s. If you don’t it could be a malfunction. If your neighbors have the proper screen and you
don’t, consult the instructor.

5. Should look like this except no lines, just colors.
7. Follow instructions below to calibrate:
Calibrate
1. To calibrate the Vernier SpectroVis, choose Calibrate ► Spectrometer from the Experiment
menu.
2. Fill a cuvette about 3/4 full with .10M NaCl Solution and place it in the cuvette holder.
3. Follow the instructions in the dialog box to complete the calibration which should consist ofa
warm up,and then Click OK .
8. The absorbance should be between 0 and 1.2 during this part of the experiment. If the absorbance
exceeds 1.2 see your instructor about how to dilute your solution.
9. Measurement vs. Wavelength (Generate a Spectrum)
Fill a cuvette about 3/4 full of the fifth solution above in the table to be tested.Place the sample in the cuvette
holder of the SpectroVis.
Figure 1: Typical absorbance spectrum
10. Click to generate a spectrum. Click to end data collection.
11. COPY AND PASTE all of the data generated in the left had side. Should be two columns
of 102 values or so when pasted into excel. You will need to make a graph as indicated in
the end of this lab. You will only do this once.
12. The next part will use the Vernier SpectroVis to run Beer’s Law experiment for
riboflavin are as follows:
Measurement vs. Concentration (Beer’s LawExperiment)
1. Collect a spectrum as described above.

6. 2. Click on the Configure Spectrometer Data-Collection button .
3. Select Absorbance vs. Concentration as the data-collection mode. The wavelength with the
maximum value from the spectrum (λ max) will be automatically selected,Double check to make
sure, press OK.
4. Place your 1st Solution in the cuvette slot of the SpectroVis Plus. Click and then
click . Enter the concentration of the sample and click OK .
(You will use the same cuvette for the rest of the samples dump tested solution into a waste container
and use the same cuvette to get the most accurate results.)
5. Place your second sample in the cuvette slot. After the readings stabilize, click .
Enter the concentration of the second sample and click OK.
6. Repeat Step 6 for the remaining samples. When finished, click to end the
data collection.
13. Record all information into Excel, save, and then send it to your email.
14.Wash everything up and return the cuvettes to the instructor.

7. EXPERIMENT NUMBER 10
POST-LABORATORY QUESTIONS
Please answer the following questions in your notebook. The answers should be at the end of the
experiment. The answers to these questions will be turned in as part of the experiment. Perform all math
calculations using spread sheet formats. Let the spread sheet make your graphs for you. The graphs
will be made on the computer and attached to the lab.
Note – Use the XY (scatter) graph. After you have graphed each of the three graphs, you can let the
spread sheet draw the line for you. Put the cursor on one of the data points and the (x,y) coordinates
should appear. Use the right mouse button to right click on that data point. At this point you should get a
box listing severaloptions. The one you want is ADD TREND LINE. For the graphs of absorbance
versus wavelength you will choose the MOVINGAVERAGE trend line. For the graph of concentration
versus absorbance,you will choose the LINEAR trend line. For the linear trend line there is one
additional step you will need to do. Go to OPTIONS and look near the bottom of the dialog box. (Check
the boxes for SET INTERCEPT and DISPLAY EQUATION ON CHART.)
1. Calculate the concentration of your stock solution. (This is also solution #6.)
2. Based on the dilutions performed in Step #12, calculate the concentration of potassium
permanganate in each of the 5 solutions in Step #12. You may want to review dilutions in the
textbook if you are uncertain about how to do these calculations. Look for an equation something
like M
1
V
1
= M
2
V
2
.
3. Graph the results of the calculation of the concentrations performed in Question #1 of the post lab
putting absorbance on the ordinate and the concentration on the abscissa. If you wish to receive
full credit, you should be sure everything is labeled correctly and completely.
4. Use the functions of the spread sheet as described at the beginning of this section to determine the
equation for the straight line for the absorbance versus concentration graph.
5. Use the equation provided by the spread sheet along with the “y” intercept to determine the
concentration of the unknown. Remember,for a straight line the equation is: y = mx + b. Place
your value of the absorbance in for the “y” term in this equation and solve for “x”.
6. Please give the number or letter designation of the unknown you analyzed and the concentration
of the unknown which was in the original bottle. (If you made any dilutions of the unknown,
please adjust the reported concentration appropriately.)
7. In using the Vernier SpectroVis:
a) Why did you always use the same cuvette for your sample?
b) Why was it important to wipe your finger prints off the cuvette after handling it?
c) Why did you have to recalibrate the instrument every time you changed the wavelength?
8. The concentration of the unknown was determined using a calibration curve. A calibration curve
is a graph produced by plotting the known concentrations of a series of solution versus the
absorbance of those solutions. Why did we use a calibration curve to determine the unknown?
(To put this question another way, why didn’t we just measure the

8. absorbance of the unknown and figure out the concentration without going through the first 12
steps in the experiment? It would have been a much shorter lab to use Beer’s Law directly.)
9. Compare Experiment Number 4 (which was the Group I experiment) and this experiment (using
the Spectrophotometer) with regards to the following:
(a) Which of these two experiments is best suited for the qualitative analysis of chemicals?
(Qualitative means which elements or compounds are present. Qualitative is usually a
yes/no or present/absent type answer.)
(b) Which of these two experiments is best suited for the quantitative analysis of chemicals?
(Quantitative means how much of something is present. Quantitative always has a
number for the answer.)
(c) Which of these two experiments do you think is best suited for the determination of the
concentration of chemicals at a very low level?
10. Describe a way in which the Vernier SpectroVis might have been used in Experiment #4 to
facilitate the determination of the unknown? (Hint—think about the first graph you did very
early in this lab.)
GRAPHING
Chemists frequently use graphing in the experimental procedure to assist in data interpretation. Based on
our experience, many of the students are somewhat weak in the basic skills of graphing. Consequently,
we are going to engage in a detailed, everything you wanted to know about graphing, exercise in the
following couple of pages. If you are not familiar with graphing then it is time you learned. If you have
seen it before and forgotten everything, then hopefully this will be an adequate review. If you are an
expert and know everything, then just go along with us for the fun of it.
There are basically two kinds of mathematical data:
CONTINUOUS DATA: Continuous data can have any possible value within the limits of the
measurement process. An example of this type of data would be the height of the people in the
class. There are no restrictions within the normal limits of human growth. Most of what we do in
chemistry fits this category.
NON-CONTINUOUS DATA: An example of non-continuous data would be the number of
stories in a building. Here the data is limited to specific integer values since buildings don’t have
fractional stories (unless under construction or destruction). We don’t use this type of data very
often in chemistry unless we are reporting grades or something else of little interest to students.
Here are a few other points to remember when graphing. If you don’t remember them later when we
actually graph things, then the ever present red pen will be forced to make nasty marks on your lab
resulting in less than superb grades.
 Use graph paper
 Use as much ofthe graph paper as possible. The graphs will look better and will give more
precise results.
 If more than one graph is to be on the same sheet ofgraph paper, use different colored lines or

9. some other trick to differentiate the multiple graphs. Be sure to include a LEGEND so
everyone will be able to interpret the graph correctly.
 All graphs have a title such as “Height ofStudents in Chem 111 Fall Semester 1996 born on or
before 1910.”
 Be sure to label the axesfor the graph. The horizontal axis is called the abscissa while the
vertical axis is called the ordinate.
 Place the proper scale at reasonable intervals along each axis to facilitate the interpretation of
the data.
 You do not need to start at “0” for the ordinate or abscissa. Graph only that part of the
experiment that contains the data ofinterest in the experiment. This will facilitate accurate
interpretation ofthe graph. As an example,ifyou were to graph the height of the people in the
lab room, you would probably graph from 5 feet to 6.5 feet. Very fewpeople in this lab are
below3 feet tall or above 8 feet tall so why waste graph paper for these situations that are not
practical. (NOTE: sometimeswhen using the least squares method,you need to use the origin
so then you would include it in your graph for that purpose.)
Look at the graphs on the following pages. The first page of graphs shows two graphs containing the
same information. The top graph is done properly. The bottom graph is not done well because the axes
were not chosen properly. The second page of graphs shows two graphs that also contain the same
information. The top graph shows how to correctly draw a straight line through the data points (notice the
line may not go through every point). The bottom graph shows how not to connect the dots. Straight line
graphs (such as this one should be) do not have bends in them.
TITRATION OF UNKNOWN ACID
4
5
6
7
8
9
10
21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 25.0 25.5
pHofsolution
Volume of acid added, mL
Series 1

10. TITRATIONOFUNKNOWNACID
0
2
4
6
8
10
12
14
0 5 10 15 20 25 30 35 40 45 50
pHofSolution
Volumeofacidadded, mL
Series1
Which of the two graphs above would you rather use to determine a given volume to the nearest 0.10 mL?
Both graphs contain exactly the same information, but only one of the graphs has the information
presented in such a way that it is really useful.
The two graphs above are identical. The top graph tries to make the best straight line through the data
points. The bottom graph connects the dots. We don’t do much connecting dots in chemistry. The top
graph is useful, the bottom graph is not.
Here are some example graphs that yours should resemble. If your graphs are wacky compared to yours,
seek help.
y = 51177x
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 0.000005 0.00001 0.000015 0.00002 0.000025 0.00003
Absorbance
Concentration
Absorbancevs. Concentration
Series1
Linear (Series1)

11. -0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
400 500 600 700 800
Absorbance
Wavelength
Wavelength Determination
Series1