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)
Absorbance specifically depends on certain properties of the sample: the wavelength of the
measurement , the extinction coefficient , the path length of the sample cell, l, and the concentration
of the sample c.
(Beers Law)
(for mixtures)
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
t
/I
0
, is related to absorbance:
-
(
)
In the example above, the transmission values of red and blue 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.
During this laboratory experiment, think about how altering , l, and c would change the absorbance
and transmission of the samples that you will observe. You will also be using a spectroscope and some
filters to explore light and absorbance.
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
A
b
s
o
r
b
a
n
c
e
(
a
.
u
.
)
800 700 600 500 400
Wavelength (nm)
Red
Orange
Yellow
Green
Blue
Indigo
Violet
Sample
I
O
I
t
For the red colored 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.
Red
Orange
Yellow
Green
Blue
Indigo
Violet
127
Exp 11 Prelab Exercise, Yellow Dye Example
The absorption spectrum on a unknown yellow solution is shown below. Spectra of three known
solutions (Yellow #5, #6 and turmeric) are also shown.
1) Which dye is present in the unknown?
2) How can you tell? (Explain your reasoning.)
0.00
0.10
0.20
0.30
0.40
0.50
3
0
0
3
4
0
3
8
0
4
2
0
4
6
0
5
0
0
5
4
0
5
8
0
6
2
0
6
6
0
7
0
0
A
b
s
o
r
b
a
n
c
e
Wavelength (nm)
Absorption Spectra for
Unknown
0.00
0.50
1.00
1.50
3
0
0
3
4
0
3
8
0
4
2
0
4
6
0
5
0
0
5
4
0
5
8
0
6
2
0
6
6
0
7
0
0
A
b
s
o
r
b
a
n
c
e
Wavelength (nm)
Absorption Spectra for Yellow
Dye #6
0.00
0.20
0.40
0.60
0.80
1.00
1.20
3
0
0
3
4
0
3
8
0
4
2
0
4
6
0
5
0
0
5
4
0
5
8
0
6
2
0
6
6
0
7
0
0
A
b
s
o
r
b
a
n
c
e
Wavelength (nm)
Absorption Spectra for Yellow
Dye #5
0.00
0.20
0.40
0.60
0.80
1.00
3
0
0
3
4
0
3
8
0
4
2
0
4
6
0
5
0
0
5
4
0
5
8
0
6
2
0
6
6
0
7
0
0
A
b
s
o
r
b
a
n
c
e
Wavelength (nm)
Absorption Spectra for
Turmeric (a natural food dye)
128
3) Sketch a Beers Law Plot using the data provided. Estimate the slope of the line.
4) What is the concentration of the dye present in the unknown shown on page 1?
0.00
0.20
0.40
0.60
0.80
1.00
1.20
300 340 380 420 460 500 540 580 620 660 700
A
b
s
o
r
b
a
n
c
e
Wavelength (nm)
Absorption Spectra for Yellow Dye #5
0.0025 g/L
0.005 g/L
0.01 g/L
0.015 g/L
0.02 g/L
0.000
0.200
0.400
0.600
0.800
1.000
1.200
0.0000 0.0050 0.0100 0.0150 0.0200 0.0250
A
b
s
o
r
b
a
n
c
e
Concentration (g/L)
Beer's Law for Yellow #5
129
Absorbance of Filters
Explore light, transmittance and absorbance using your own eyes as the detector.
A spectroscope contains a diffraction grating that separates light into its component wavelengths. For
example, when you look at fluorescent light without a spectroscope, it looks white, but when you look
through the spectroscope, you can see the colors that the light is composed of.
To use the spectroscope, make sure that the slit is aligned with the light source. Then look at the scale
to the left without moving the spectroscope. You should see white light spread into its rainbow of
colors.
Schematic of aligning the spectroscope.
1. Take orange, blue, red, and purple filters from the supply shelves, their corresponding
transmission spectra, and spectroscope for each individual in your group.
2. Use the spectroscope to look at the reflected sunlight through the window. What do you
observe? What colors do you see? Draw the transmission spectrum in your lab notebook using
the format shown below.
I
n
t
e
n
s
i
t
y
o
f
T
r
a
n
s
m
i
t
t
e
d
L
i
g
h
t
Red Orange Yellow Green Blue Indigo Violet
Now look through the spectroscope with the orange filter placed over the slit. What do you
observe? What colors do you see? Draw the transmission spectrum in your lab notebook. How
does this spectrum compare to the outdoor or room light?
I
n
t
e
n
s
i
t
y
o
f
T
r
a
n
s
m
i
t
t
e
d
L
i
g
h
t
Red Orange Yellow Green Blue Indigo Violet
130
Repeat with the blue, red, and purple filters.
I
n
t
e
n
s
i
t
y
o
f
T
r
a
n
s
m
i
t
t
e
d
L
i
g
h
t
Red Orange Yellow Green Blue Indigo Violet
I
n
t
e
n
s
i
t
y
o
f
T
r
a
n
s
m
i
t
t
e
d
L
i
g
h
t
Red Orange Yellow Green Blue Indigo Violet
I
n
t
e
n
s
i
t
y
o
f
T
r
a
n
s
m
i
t
t
e
d
L
i
g
h
t
Red Orange Yellow Green Blue Indigo Violet
3. Does the transmission spectrum observed from the purple filter correspond to a spectrum
observed when the blue and red filters are overlapped? Draw the transmission spectrum
observed when the blue and red filters are overlapped.
I
n
t
e
n
s
i
t
y
o
f
T
r
a
n
s
m
i
t
t
e
d
L
i
g
h
t
Red Orange Yellow Green Blue Indigo Violet
4. You are provided with the absorbance spectra of these filters. How do these graphs correspond
to your observations?
131
Experimental
Investigation 1: Dye Extraction
The Problem
Create a procedure to extract natural dyes from carrots and turmeric.
The Approach
Part 1: Design your own experiment:
1) Address each of the following issues to help you design your experiment.
I. Based on the structure of beta-carotene and curcumin (the dye molecules in carrots and
turmeric respectively), which solvent will you use: ethanol or water?
II. Besides solvent, there are three other variables for you to consider: temperature, time, and
sample preparation. Design a set of three experiments that tests one of these variables. In other
words, if you want to test the effect of temperature then decide on three different temperature
and use the same time and sample preparation. Or you can vary the time that you run the
extraction and keep the sample preparation and temperature the same. Record which variable
you are going to test and what you will change. You may test a different variable for the carrots
and the turmeric.
III. For all of your experiments you will be using 5 g of the plant material and 50 mL of solvent. In
total you will be running 6 extraction experiments, three with carrot and three with turmeric.
2) Write up a procedure and show it to your GSI who will help you locate all of your materials.
3) Follow your procedure and isolate your dye solutions. Be sure to record all of the materials and
amounts of chemicals that you used and isolated during your extraction process.
132
Investigation 2: Dye Quantification
The Problem
Determine the concentration of a natural dye molecule from an extract.
The Approach
1. Make a Beer's law plot of your peak absorption data vs. the concentration in grams per milliliter
based on the data provided for both curcumin and beta-carotene. You may use the origin (0, 0)
as a data point. This is equivalent to zeroing the spectrometer. Instructions for making linear
graphs in Excel can be found in the appendix of this manual.
2. Determine the slope of the line using a linear regression routine on a calculator. Be careful with
units. Record your value for (the slope); you will use this value to calculate the amount of dye
in each of your solutions from Investigation 1.
3. Using your Beers law plot, determine the amount of dye in each of your solutions from
Investigation 1.
a. Are there any trends based on the amount variable that you tested? Did temperature,
time, or sample preparation effect the amount of dye that you extracted from the carrot
and curcumin)?
4. Estimate the total amount of dye that you extracted from the carrot and turmeric in your best
procedure. What was the highest total amount of dye that you extracted from each starting
material? Record this value on the board.
133
Experiment 11- Dyes, Molecules and Light Your Name___________________________
Report Sheet Partners_____________________________
_____________________________________
GSIs Name___________________________
1) Construct a Beers Law plot (Absorbance vs. concentration) for both beta-carotene and curcumin
from the data provided. Fit the data to a straight line and calculate a value for R
2
. Attach the graphs to
your report and report the equation for the line on the graph.
2) Calculate the extinction coefficient for your natural dye.
3) Calculate the concentration of natural dye (g/mL) that you isolated based on your Beers Law plot for
each of your extractions.
4) Calculate how many milligrams (mg) of dye you extracted from the plant material for each extraction.
134
5) Briefly describe your best extraction procedure, the one that yielded the most dye.
6) How do your best results compare to other groups? Suggest ways to make your experiment either
more efficient or greener.
7) How is using a spectrometer different from using a spectroscope? Describe the benefits of using a
spectrometer rather than a spectroscope for the quantitation.