Absorbance and Fluorescence

Spectroscopy: Determination of
Common Compounds in Energy and
Sports Drinks
Introduction
Research Question(s)
What are the concentrations of dyes and riboflavin (vitamin B2) in common beverages? What
methods can you use to determine the concentration of one or more unknown compounds in a
solution? How can you ensure that your experiment and calculations give reliable data?

Learning Goals
It is often a goal of a chemist to identify and quantify compounds that are present in an unknown.
What compounds are present? How much of each is there? Think of a common analysis, like
trying to determine if a person has enough iron in her blood. The blood sample is a complicated
mixture, and data must be acquired carefully and interpreted consistently to give the patient an
accurate diagnosis. In this experiment you will quantify the food dyes and riboflavin found
commonly in some of the most popular beverages today.
In this experiment you will learn two different techniques for determining the concentration of
species in solution. One technique, ultraviolet-visible (UV/Vis) spectroscopy, relies on the ideas
that many molecules absorb light, and the amount absorbed is proportional to the concentration
of the molecules in solution. The second technique, fluorescence spectroscopy, relies on similar
principles but is more selective of the compounds that can be analyzed. Certain compounds, once
they have absorbed light energy, release that energy at a characteristic wavelength in a process
called emission. Like absorbance, emission can be correlated to compound concentration. In this
lab youll be using absorbance spectroscopy to determine the concentrations of two food dyes in
a sports drink, and youll use fluorescence spectroscopy to determine the concentration of
riboflavin in an energy drink.
You need to understand how the instrument responds to the species present. By generating a
calibration curve, which is a plot of absorbance or emission measurements for samples of a given
compound at known concentrations, you can account for any instrument-based variations that
might be present in your data. You can also use these curves to calculate the concentration of
compounds in unknown solutions. You will then use that calibration curve to determine the
concentration of an unknown solution provided to you.

Absorbance Spectroscopy
When an atom or molecule absorbs a single photon, an electron makes a transition to a higher
energy orbital. Thus, the number of photons a sample of material absorbs is directly related to the
number of particles in the path of the beam light. This important concept means that by
measuring the change in intensity or number of photons (I) of a light beam before (I0) and after
(It) it passes through a solution of molecules, we can measure the number of molecules in the
solution. The intensity change is related to the number of molecules the beam encounters. More
technically, the log of the ratio of I0 to It is called the absorbance (A).

Red
Orange
Yellow
Green
Blue
Indigo
Violet

Red
Orange
Yellow
Green
Blue
Indigo
Violet

Light
Source

I0

It

Red
Orange
Yellow
Green
Blue
Indigo
Violet

It

Red
Orange
Yellow
Green
Blue
Indigo
Violet

Blank

I0

log

Sample

0.35
0.30
Absorbance (a.u.)

0.25
0.20
0.15
0.10
0.05
0.00

Detector
Detector

400

500

600
700
Wavelength (nm)

800

Figure 1: For the Red Dye #3 sample above, all of the red and some of the blue light pass
through the sample, whereas the yellow, orange, and green light is absorbed. Thus, the
absorbance values of yellow, orange, and green light are high and the absorbance values of red
and blue are lower.
Absorbance specifically depends on certain properties of the sample: the wavelength of the
measurement , the molar absorptivity at that wavelength, , the path length of the sample cell, l,
and the concentration of the sample, c. The absorbance, A, of the solution containing a given
species is equal to the molar absorptivity
(in L mol1 cm-1) times the path length l (in cm)
times the concentration c (in mol L-1). If the concentration of the sample is expressed in units
other than molarity, is referred to as the extinction coefficient.

(Beers Law)
(for mixtures)

Notice that there is a linear relationship between absorbance and concentration. The slope of the
line corresponds to the molar absorptivity times the path length of the sample, b. Special
sample cuvettes are used which have a path length of 1.000 cm. The molar absorptivity depends

on the wavelength of the light and is determined by measuring the absorbance of a series of
standard solutions of known concentration. Once and b are known, the concentration of an
unknown solution can be determined by measuring the absorbance of the unknown and using the
Beer-Lambert Law given above.
Alternatively, we can also explain this phenomenon by quantifying the transmission of a sample,
or the number of photons that the sample does not absorb. Transmission, I I , is related to
absorbance:
10
=T
In the example spectrum shown above, the transmission values of red and blue wavelengths of
light would be high but those of orange, green, and yellow would be low. Scientists use both
absorbance and transmission data to report the optical properties of a sample depending on what
information they want to convey.

Fluorescence Spectroscopy
In absorbance spectroscopy, the measurement is focused on the energies absorbed by the sample.
In emission spectroscopy, the detector focuses on the light emitted by the molecules. Electrons
in molecules are associated with various types of energies. You may be familiar with electronic
energy states, but electrons also occupy rotational and vibrational energy states (see Figure 2).
When a molecule absorbs light, the electrons can be promoted to many different types of excited
states. For example, an electron can be excited from a ground electronic state to a higher
electronic state, i.e. from S0 to S1 (represented by the h1abs transition in Figure 2). It could also
be excited to a different vibrational state, i.e. from the ground vibrational state of S0 to an excited
vibrational state in S2 (represented by h2abs in Figure 2). The molecule can relax back to its
ground state through several different processes of energy emission, one of which is
fluorescence.

Figure 2: Physical processes that occur after a molecule absorbs a photon. When a molecule
absorbs a photon, an electron is promoted to higher energy excited state (e.g. S0 S1). When a
molecule emits a photon, an electron moves to a lower energy state (e.g. S2 S0).
3

If the excited molecule immediately relaxes back to the ground state, light will be emitted at the
same wavelength as the light that was absorbed in a process called resonance fluorescence. In the
figure above, this is represented by h1abs and h1fluor, which are equivalent. But if the molecule
loses some energy to collisions with solvent or loss of heat (a process called internal conversion)
then relaxes back down to the ground state, the energy emitted will be lower than that absorbed
originally. This is true for the absorption/emission pair of h2 abs and h2fluor in the figure, where
h2abs >h2fluor. It is common that excitation occurs with light in the UV range (higher energy)
while emitted light is in the visible range (lower energy), which is part of what makes fluorescent
molecules so fun to work with.
The emission spectrum is often roughly the mirror image of the absorption spectrum. In
absorption, the transition from the ground vibration level to the lowest excited vibrational energy
level is 0. All subsequent transitions are to higher energy vibrational levels (+1, +2, etc.). Thus,
0 is the lowest energy transition and +2 is the highest energy transition.
After absorption, the molecules relax back to the lowest energy excited vibrational state through
internal conversion. This means that for emission, 0 (the transition from the lowest vibration
energy level to the ground vibrational energy level) is the highest energy transitional. All
subsequent transitions are of lower energy. Figure 3 demonstrates the symmetry of absorption
and emission transitions.

Figure 3: Energy-level diagram showing why absorption and emission spectra are roughly
mirror images of each other.
As with absorption spectroscopy, the intensity of emitted light is proportional to the
concentration of the molecules in the sample.

The intensity of light emitted depends on the intensity of light supplied for excitation, I0, and the
concentration, c, of the sample. The constant k is a term that includes the molar absorptivity at
the excitation wavelength and the path length of excitation through the center of the cell.
However, this relationship is only linear at lower concentrations because if the sample gets too
concentrated, the chance of self-absorption of the emitted light is greater. In that case one
molecule might emit light that a neighboring molecule absorbs, and that photon would not be
detected. This is sometimes referred to as fluorescence quenching. For more information about
fluorescence spectroscopy, please consult your Harris textbook or Analytical Chemistry 2.0
(Harvey, 2009).

Absorbance and Fluorescence Measurements

Spectrophotometric determinations based on the Beer-Lambert Law are among the most widely
used analytical procedures. These methods involve the measurement of the fraction of incident
electromagnetic radiation that is absorbed by a sample (Figure 4). To determine the
concentration of a colored species in solution, a cuvette containing the solution is placed in the
spectrometer. Spectrometers consist of a built in light source, which produces a narrow beam of
light that passes through a sample cuvette (I0). The light that passes through the sample (It) is
then split into its component wavelengths by a diffraction grating. A specialized (charge-coupled
device or CCD) detector then measures the amount of light that reaches it at each wavelength. A
computer then converts this into an absorbance reading using the relationship
log .

Figure 4: Schematic of an absorbance measurement using a spectrometer (top view).

Fluorescence measurements are very similar to absorbance. However, the molecules in the
sample are excited by a specific wavelength of light (which can be done for absorbance
spectroscopy but is not in this lab), and the emission of light is detected at 90 relative from the
excitation (Figure 5).

Figure 5: Schematic of a fluorescence measurement using a spectrometer (top view)

Serial Dilutions and Calibration

No instrument will automatically determine the concentration of a sample. Instead you must
calibrate the instrument using a known solution and compare the response of an unknown. By
measuring the absorbance of a set of standard solutions of known concentrations, you can create
a calibration curve that allows you to determine the concentration of an unknown of the same
species. This calibration curve correlates to how the spectrometer responds to various
concentrations of standard solutions. Beers Law may fail if a solution is too concentrated or too
dilute. This means that you can only accurately determine the concentration of an unknown if its
absorbance falls within the linear range of the dataset.
An example for determining the linear range of a dataset using Red Dye #3 is shown below.
Figure 1 (previous page) shows that the maximum absorbance for Red Dye #3 is at 526 nm.
Figure 6 shows the absorbance measurements for a series of eight standard solutions of Red Dye
#3 at 526 nm. Notice that at high concentrations the data no longer appears linear and the R2
value is only 0.9691. For a perfectly linear response, the R2 value is 1. The closer the R2 value is
to 1, the more reliable your data is. A linear regression of the five most dilute samples gives a
much more linear response, R2 = 0.9995. The linear response range for a compound must be
experimentally determined at the wavelength of interest.
The extinction coefficient for Red Dye #3 at 526 nm can be determined from the slope of the
calibration curve using the Beers Law data.
6

m x

A plot of absorbance versus concentration will have a slope of . For Red Dye #3 the equation
for the calibration curve is y = 0.0805x 0.0791 as shown in Figure 2. Most sample holders (l)
are 1 cm.
0.0805 /


0.0805

2.500

Absorbance at 526 nm

Absorbance at 526 nm

An unknown solution with absorbance of 0.9054 would correspond to a concentration of 12.23

mg/L of Red Dye #3.

2.000
1.500
1.000
y = 0.0644x + 0.088
R = 0.9691

0.500
0.000
0.00

10.00

20.00

30.00

40.00

Concentration of Red#3 (mg/L)

2.000
1.500
1.000
0.500
0.000
0.00

y = 0.0805x - 0.0791
R = 0.9995
5.00

10.00

15.00

20.00

25.00

Concentration of Red#3 (mg/L)

Figure 6: Beers law plot for Red Dye #3 using full and linear portion data set at 526 nm
Rather than trying to analyze really concentrated solutions, it is considered good lab practice to
quantitatively dilute solutions to concentrations that give absorbance values below 1.5
absorbance units. Quantitative dilution is usually achieved through a technique called serial
dilution. This is best explained by example. To begin, you would make a standard solution of the
highest concentration you want to test (1 M, for this example). A serial dilution involves taking a
small portion of this solution and diluting it incrementally to make several solutions of lower
concentration. For instance, you could take 10 mL of the 1 M solution and dilute it to 100 mL
total volume to create a 0.1 M solution. (Note: do you remember which equation helps you with
this calculation? See Analytical Chemistry 2.0 Chapter 2E or Harris Chapter 1.3) In the next step,
you take 10 mL of this 0.1 M solution and repeat the process to create a 0.01 M solution. This is
done repeatedly until youve created a sufficient number of solutions for your experiment. Serial
dilutions are usually executed using volumetric flasks and pipets to achieve the greatest accuracy
and precision possible. They are also the easiest way to prepare solutions over a very wide
concentration range with minimal waste.

Simultaneous Determination of Two Dyes

M+N
N

Absorbance

You will determine the

concentrations of two dyes
simultaneously by
spectrophotometric analysis. Since
the total absorbance of the solution
at a given wavelength is equal to the
sum of the absorbances of the
individual components, it is possible
to analyze the individual
components of a mixture even when
their spectra overlap. Consider the
spectra of M and N given in Figure
7.

M
1

2
Wavelength (nm)

There is obviously no wavelength at

Figure 7: Absorbance spectra of components M,
which the absorbance of this mixture
N and the spectra of both components together.
is due simply to one of the
components; thus an analysis for
either M or N by a single measurement is not possible. However, the absorbances of the mixture
at the wavelengths 1 and 2 may be expressed as follows:
At 1 : A1 1MbcM 1N bcN
At 2 : A2 M2 bcM N2 bcN
The four molar absorptivities, 1M , M2 , 1N , and N2 can be evaluated from standard solutions
containing only M or N. Then, if the absorbances of the mixture are measured at 1 and 2, the
concentrations of the individual components can be calculated by solving the two equations
given above simultaneously. The best accuracy in an analysis of this sort is obtained by
choosing wavelengths at which the differences in molar absorptivities between the two species is
large. For your unknown, you will use the absorption spectra of yellow and red dyes to choose
two wavelengths, 1, and 2, for analysis. For each dye what is the wavelength that corresponds
to the highest absorption measurement? This value is called max and is the wavelength at which
the instrument is most sensitive to the species being analyzed.

Additional Reading
Quantitative Chemical Analysis (Harris, 8th ed.):
Chapter 2.4-2.6 (Burets, Volumetric Flasks, Pipets
and Syringes)
Chapter 2.10-2.11 (Graphing with Microsoft Excel)
Chapter 17 (Fundamentals of Spectrophotometry)
Chapter 18.1 (Analysis of a Mixture)

Analytical Chemistry 2.0 (Harvey, 2009):

Chapter 2D.2 Equipment for Measuring Volume
Chapter 5D.1 Linear Regression of Calibration
Curves
Chapter 5F.1 Using Excel for Regression Analysis
Chapter 10A 10C Spectroscopy
Chapter 10F Photoluminescence Spectroscopy

Pre-Lab Exercises
1. Imagine that you and your lab partner made solutions according to the scheme below using a
buret to dispense dye into a volumetric flask. Calculate the exact concentration of the final
solutions in the last column. Show one example calculation.

Standard
#1
#2
#3
#4

Initial
concentration
of dye
(M)
5.00 10-5
5.00 10-5
5.00 10-5
5.00 10-5

Target
volume of
dye
(mL)
0.5
1.0
1.5
2.0

Actual
volume of dye
dispensed
(mL)
0.48
1.10
1.51
2.05

Total solution
volume*
(mL)

Final
concentration
of dye
(M)

5.00
5.00
5.00
5.00

2. For the solutions shown above, you and your partner did not dispense exactly the target
volume from the buret. The target volume of dye was 3.5 mL, and you dispensed 3.48 mL.
Does this mean the solutions have to be made over again? Why or why not?
3. Go to the PhET interactive simulation site: http://phet.colorado.edu/sims/html/beers-lawlab/latest/beers-law-lab_en.html. Double click on the Beers Law icon to begin the
simulation.
a. Adjust the concentration of the red drink to 50mM and record %T and absorbance.
b. Now double the concentration to 100mM and record %T and absorbance. Repeat for
200mM concentration. What happens to the ability to detect the solution at high
concentrations?
c. Set the wavelength of the light to variable. What colors does the red drink absorb
well? What colors are transmitted? How does the absorbance and transmittance
correspond to the color of the solution?
4. In fluorescence spectroscopy, the light detected is 90 from the incident light. What is the
reason for this? What would happen if the detector was 180 (directly across) the incident
light like it is in absorbance spectroscopy?

Experimental
Work in groups of three to complete this experiment. Student #1 should prepare the Red #40
calibration curve, and Student #2 should prepare the Yellow #5 calibration curve (Part 1).
Student #3 should prepare the riboflavin calibration curve (Part 2). All three students should
work together to measure the absorbance of a mixture of dyes in a sports drink and the
fluorescence of riboflavin an energy drink (Rockstar).

Part 1: Absorbance of food dyes

Goal
Divide work amongst your group to create calibration curves for Red #40 and Yellow #5 dye.
Use these calibration curves for the simultaneous determination of red and yellow food dye in a
sports drink.

Procedure
Group work: Student #1 and #2 work together to create dye stock solutions.
Obtain approximately 2-3 mL of Yellow #5 and Red #40 dyes (approximately 2.5 10-3 M).
Record the exact concentration of the stock solution for both dyes.
You will need to create dilutions in order to measure the molar absorptivity.
What is the difference between molar absorptivity and the extinction coefficient?
For each dye, dilute your solution to a final concentration of 5.00 10-5 M using volumetric
glassware. You only need 50-mL of each solution. Your GSI will guide you through this
process.
How does your final concentration compare to your target concentration of 5.00 10-5
M? This will be the stock solution you use for your red and yellow dye calibration
curves.
You will need to create dilutions of each stock solution in order to measure the constant

for each dye.

10

in

Buret and volumetric flask

Group work: Student #1 - Red #40, Student #2 - Yellow #5.
Work together to choose two wavelengths that will produce the greatest accuracy in determining
the concentration of mixture of red and yellow dye. Use the same spectrometer for both dyes
(share between Students #1 and #2).
Using the buret and flask method (Appendix A), prepare one standard solution at a time,
transferring each solution to a cuvette for analysis. Follow the scheme shown in the table below.
Dilute to the 5.00 mL mark with distilled water.
When reusing the same glassware or cuvettes, it is good analytical practice to proceed
from low to high concentration. Why?
Record the exact volumes you actually dispense and use these to calculate your final dye
concentration.
Tip: 5.00 mL volumetric flasks easily tip over. Place your flask in a small beaker to stabilize it.

Standard

Initial
concentration
of dye
(~5 10-5 M)

#1
#2
#3
#4
#5
#6
#7
#8

Target
volume of
dye
(mL)
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0

Actual
volume of dye
dispensed
(mL)

Total solution
volume*
(mL)

Final
concentration
of dye
(M)

5.00
5.00
5.00
5.00
5.00
5.00
5.00
5.00

Choosing wavelengths
Produce an absorbance versus wavelength spectrum for both the yellow dye AND red dye
solutions using the same spectrophotometer for both dyes (Appendix B). From these two
spectrums, select the two wavelengths (one for red and one for yellow dye) that will produce the
greatest accuracy in determining the concentration of yellow and red dye in a mixture.
What do you need to consider when choosing these wavelengths? Consider the influence
of concentration. Check these values with your GSI before you proceed.
Record the absorbance values for BOTH wavelengths in your lab notebook.

11

Calibration curve
Measure the absorbance spectra of each standard solution (#1 - #8) at BOTH wavelengths chosen
above.
Record the absorbance values in your lab notebook as shown below. 1 is the
wavelength of maximum absorbance for the red dye, 2 is the same for the yellow dye.
Standard

Absorbance of Red Dye

Absorbance of Yellow Dye

#1
#2
#3
#4
#5
#6
#7
#8
Analysis of an unknown sports drink
Group work: Full group (students #1, #2, #3)
Obtain a vial of your assigned unknown orange sports drink solution. Most sports drinks contain
food coloring, and Red #40 and Yellow #5 are some of the most common ones.
Record the unknown number in your lab notebook.
Collect a spectrum of your unknown that contains your chosen wavelengths for Yellow #5 and
Red #40 (see Appendix B for UV-Vis spectrometer instructions). Dilute quantitatively if the
solution absorbance is above 1. Ask your GSI for help with this step if needed.
Record the absorbance values at BOTH relevant wavelengths ( 1 and 2 ). Use this data
to calculate the concentrations of Red #40 and Yellow #5 in the sports drink.

12

Part 2: Fluorescence of riboflavin

Goal
Divide work amongst your group to create a calibration curve for riboflavin. Use the calibration
curve to determine the concentration of riboflavin in Rockstar energy drink.

Procedure
Obtain approximately 50 mL of riboflavin (approximately 25.00 mg/L).
Record the exact concentration of the stock solution.
You will need to create dilutions of this stock solution in order to measure the constant k in
.
Buret and flask
Group work: Student #3 - Riboflavin
Using the buret and flask method (Appendix A), prepare one standard solution at a time,
transferring each solution to a cuvette for analysis. Use the table below as a guide for solution
preparations. Use %1 acetic acid to dilute your solutions to total volume of 5-mL.
Record the exact volumes you actually dispense and use these to calculate your final
riboflavin concentration.
When reusing the same glassware or cuvettes, it is good analytical practice to proceed
from low to high concentration. Why?

Standard
#9
#10
#11
#12
#13
#14
#15
#16

Initial
concentration
of riboflavin
(~25 mg/L)

Actual
volume of
riboflavin
dispensed
(mL)

Target
volume of
riboflavin
(mL)
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0

Total solution
volume*
(mL)
5.00
5.00
5.00
5.00
5.00
5.00
5.00
5.00

13

Final
concentration
of riboflavin
(mg/L)

Using a fluorescence spectrometer, measure the intensity versus wavelength (Appendix C) for
each standard solution.
Note: Use an integration time of 500 ms for each measurement (see Appendix C for detailed
instructions).
For fluorescence measurements no blank is required. Why is this?
Record the intensity at the wavelength of maximum fluorescence emission for each
standard solution (#10 - #16).
Analysis of an unknown energy drink
Group work: Full group (students #1, #2, #3)
Obtain a sample of Rockstar energy drink and place into a cuvette (remember, you must use a
cuvette that is clear on all four sides).
Record the fluorescence of the energy drink using the same spectrometer you used
previously. See Appendix C for fluorescence spectrometer instructions.
Graph your data, and use the calibration curve to determine the concentration of
riboflavin present in Rockstar. Compare this the concentration of riboflavin reported on
the back of the can.

14

Lab Report
1. Calculate the actual concentrations of all the dye standards and riboflavin standards you
made.
2. Construct calibration plots of absorbance vs. concentration or fluorescence vs. concentration,
and determine the extinction coefficient or it fluorescence counterpart.
a. Dye Standards:
i. Use concentration and the absorbance data to construct Beers Law Plots for
all your samples.
ii. Using the techniques of linear regression, determine the slopes (b) of these
curves.
iii. Calculate the extinction coefficients for each dye studied. Report the
wavelength used for each value.
b. Riboflavin Standards:
i. Use concentration and the fluorescence data to construct a linear calibration
curve for all your samples. Do this for your data from both Method 1 and
Method 2 of standard solutions preparations.
ii. Using the techniques of linear regression, determine the slopes of these
curves. For fluorescence, what does the slope represent?
3. When you made your stock 5.00 10-5 M stock solution, you assumed that the 2.50 10-3 M
solution was exactly 2.50 10-3 M. We will now evaluate this more realistically, and assume
that the uncertainty in the 2.50 10-3 M solution was 0.01 10-3.
a. Write an expression that describes the dilution of the 2.50 10-3 M solution. It
should include the volume of the pipet, volume of the volumetric flask, and the initial
concentration.
b. Use the rules of random error propagation (see Analytical Chemistry 2.0 Chapter 4C
or Harris Chapter 3.4) to calculate the uncertainty in the 5.00 10-5 M. Use the values
of uncertainty for the pipet and volumetric flask given in Table 4.2 in Analytical
Chemistry 2.0 or Chapter 2 of Harris.
4. Critically examine the data you collected. Examine the data acquired for all your calibration
curves.
a. Were you able to effectively read the meniscus in the buret to two decimal places?
b. What assumptions are you making about the total volume in the calculation of
concentration for Method 1? Do you think this assumption is valid? Why or why not?
c. How many significant figures does the concentration data have from Method 2
(buret)?
d. Which method provided you the better data? How can you tell?
5. For the calibration curves from the method you found most reliable, determine the
concentrations of Yellow #5 and Red #40 in your unknown. You will need to use data from
both dyes at both wavelengths explored.
6. For the calibration curves from the method you found most reliable, determine the
concentrations of riboflavin in Rockstar.
Attach all tables and plots needed to the answers to these questions.

15

Appendix A: Calibration Curve Methods

Prepare your buret by rinsing with 2-3mL of the dye or riboflavin solution. To rinse tilt the buret
gently to the side to make sure the dye or riboflavin coats the glass, then open the stopcock to let
the solution rinse through. Repeat with another 2-3mL of solution.
Note: It is not necessary and wasteful to completely fill a buret to rinse it.
Fill your buret to approximately 30 mL (ensure the tip of the buret is filled). Record the exact
initial buret reading to the nearest 0.01mL. You will have to estimate the last digit. Remember
that burets can and should be read to two decimal places.
Using a buret and a 5.00 mL volumetric flask, prepare eight standard solutions of each dye or
riboflavin solution. The choice of concentrations does not matter, as long as you know the exact
concentration and cover the range needed for calibration.

Step 1. Add a
known amount of
standard to the
volumetric flask
from the buret.

Step 2. Fill the

volumetric flask
to about full
with solvent.
Swirl gently to
mix.

Step 3. Add more

solvent until the
meniscus aligns with
the etched line on the
flask. Add the last
drops by pipet.

Step 4. Cap the

flask and mix
the solution
gently by
inverting until
homogenous.

Figure 6. Preparing standard solutions using a buret and a volumetric flask

16

Step 5.
Remove an
aliquot of
sample and
analyze by
UV-Vis.

Appendix B: UV-Vis Spectrometer Instructions

Blanking the spectrometer
1. If the Stellar Net software is not open, double click on the desktop icon
SpectraWiz (left) to open the application.
2. In the top panel, click the Scope mode. This will report the number of counts (photons) the
detector receives.

3. Remove the cuvette holder cap. Place your blank into the black metal case (cuvette holder)
attached to the orange light source. The arrows below show the cuvette holder and other parts
of the spectrometer.

Light Source

Computer with
Software

Spectrometer

Cuvette
Holder
Fiber Optic

4. Make sure the cuvette is sitting down in the cuvette holder. Wiggle the cuvette to make sure
it is all the way down. Replace the metal lid on the cuvette holder it should sit snugly on
the holder.

17

5. Integration Time
1. You can change the integration by either using the sliding scale or clicking on the
Integration Time clock icon (shown below) and typing in the integration time you want
(value between 1 and 1000 ms).

NOTE: A textbox may appear and prompt you to take new references select OK.
2. Make sure the Integration Time is set such that your spectrum is not saturated but as close
to 65,000 counts as possible. The spectrum below is saturated (i.e. above 65,536
counts):

3. This means you will want to be able to see the WHOLE spectrum of your blank within
the spectral area. In the picture below, the spectrum is correctly optimized to be right
below the saturation limit (65,536 counts):

Saturation Limit
(65,536 Counts)

Spectrum
(water blank)

18

6. Blank the spectrometer, click on the yellow light bulb icon. Make sure that your blank
cuvette is in the cuvette holder and the cap is on the cuvette holder. The blank is also
referred to as a (light) reference spectrum.

7. Zero the spectrometer (or take a dark reference spectrum):

a. FIRST, hold the shutter button for at least 3 seconds. The red button below is the
shutter button.

Shutter
(red circular
button)

b. You should note that the spectra fall flat often nearly to zero everywhere on your graph:

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c. THEN, while STILL holding the shutter button, click on the dark light bulb icon
you will need to hold the shutter button the whole time (you should see the flat
spectrum shown above for the whole process).

The baseline will drop to zero:

d. Once you have clicked on the dark light bulb release the shutter button. You should see
the spectrum return to its original profile.
NOTE: You must retake the dark scan if you make changes to any setting in the SpectraWiz
application (i.e. integration time, scans to average, or pixel smoothing) between measurements.
8. Now that you have properly taken a blank and dark spectrum, click on the AU (absorbance)
mode from the tray.

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9. If you have properly taken your blank and zero spectra you should see a nearly flat
absorbance spectrum once you switch to AU mode (the noise below 300 nm in the spectrum
below is from using a cuvette that absorbs in the UV region).

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Taking an absorbance sample

10. Replace your blank sample with your actual sample. The instrument will now display the
absorbance in real time. Depending on the specifics of the instrument, it will update quickly
or slowly.
11. It is good technique to re-blank and zero between separate trials, or if you have let the
instrument sit for more than 5 minutes between sample.
12. To find a peak, use the mouse to right click on either of the following icons:

Clicking the arrow pointing to the left will find a peak to the left of the vertical line in the
spectra. Clicking the arrow pointing to the right will find a peak to the right of the vertical
line in the spectra.

13. To find the absorbance of a specific point, right click directly onto the spectrum.
14. To find the specific wavelength your cursor is at, look to the bottom of the spectra at the
following bar. Wave tells you the specific wavelength. Val tell you the absorbance.

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Appendix C:
Fluorescence Spectrometer Instructions
Zeroing the spectrometer
1. If the Stellar Net software is not open, double click on the desktop icon
SpectraWiz (left) to open the application.
2. In the top panel, click the Scope mode. This will report the number of counts (photons) the
detector receives.

3. Make sure the Integration time is set to 1 sec (1000ms). You can change the integration by
either using the sliding scale or clicking on the Integration Time clock icon (shown below).
Either slide the bar all the way to the right or select the clock icon to the left of the sliding
bar and set the number to 1000.

NOTE: A textbox may appear and prompt you to take new references select OK.
4. Zero the spectrometer (or take a dark reference spectrum):
a. Gently press the red button on the back of the light source (orange box) to turn the
LED lamp off. The button on the back of the light source will not be pressed in if the
light is off.
NOTE: Do not move the light source while taking your measurements.
Spectrometer

Light Source

Cuvette
Holder

Optical Fiber

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b.

After several minutes the baseline will settle:

c. Once the baseline has settled, click the dark light bulb icon to take a dark reference
spectrum. (Note: make sure the light source is still off.)

d. The baseline will drop to zero:

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Taking a fluorescence measurement

5. After you have zeroed the instrument, you can take fluorescent measurements of your
samples:
NOTE: You do not blank the spectrometer (take a light reference spectrum) for fluorescence
spectroscopy. Why is this?
a. Turn on LED light source. Gently press the red button on the back of the light
source (orange box) to turn the LED lamp on. The button on the back of the light
source will be pressed in if the light is on.
b. Remove the cuvette holder cap. Place one of your sample cuvettes into the black
metal case (cuvette holder) attached to the orange light source.
NOTE: The cuvettes used for fluorescence are clear on all four sides. They are not
interchangeable with the standard cuvettes used for UV-Vis spectroscopy. Why is this
necessary?
c. Make sure the cuvette is sitting down in the cuvette holder. Wiggle the cuvette to
make sure it is all the way down. Replace the metal lid on the cuvette holder it
should sit snugly on the holder.
d. Remain in scope mode. The instrument will now display the photon count in real
time. Depending on the specifics of the instrument, it will update quickly or slowly.
6. It is good technique to re-zero between separate trials, or if you have let the instrument sit for
more than 5 minutes between samples.
7. To find a peak, use the mouse to right click on either of the following icons:

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Clicking the arrow pointing to the left will find a peak to the left of the vertical line in the
spectra. Clicking the arrow pointing to the right will find a peak to the right of the vertical
line in the spectra.

8. To find the photon count of a specific point, right click directly onto the spectrum.
9. To find the specific wavelength your cursor is at, look to the bottom of the spectra at the
following bar. Wave tells you the specific wavelength. Val tell you the photon count.

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Appendix D: Helpful Spectrometer Tips

Adjustments you can make include changing the number of scans the instrument averages or the
integration time.
NOTE: If you change the integration time or the number of scans to average you will need to reblank and zero your spectrometer.

Adjusting Number of Scans Averaged:

To adjust the number of scans the instrument averages, click on the Setup icon in the top left
of the computer. Click on scans to average and input a number. The more scans you average,
the longer it will take before the instrument updates the on-screen spectra.

Troubleshooting:
If your spectra is constantly fluxing/changing, or if you have a very noisy baseline:
Adjust your scans to average. Increasing the number of scans averaged will reduce noise in your
baseline and spectra by the square root of number of scans.
If your spectra shows very small peaks, or if the peaks are too high (saturation):
This often indicates the instrument needs to have its integration time adjusted. Increasing the
integration time will allow the instrument to collect more photons within each given scan,
increasing the height of your peaks. You will still want to make sure the blank does not saturate
when in scope mode.
If your spectrum is saturated, you can decrease the integration time to reduce the height of the
peaks. Alternatively, you can make a dilution of your sample. Making a dilution might be the
best option, since the sample is likely in the non-linear region if it displays an absorbance above
1.5 or 2.0 absorbance units.

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