INTRODUCTION TO
SPECTROPHOTOMETRY
Advancing Science Lab
Gettysburg College
#531, #532, #534
BACKGROUND
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Spectrophotometry is a method of analyzing that
involves how light interacts with the atoms (or
molecules) in a sample of matter.
Visible light is only a small portion of the entire
electromagnetic spectrum and it includes the
colors commonly observed (red, yellow, green,
blue and violet). The visible spectrum consists
of electromagnetic radiation whose wavelengths
range from 400 nm to nearly 800 nm.
BACKGROUND
white light is observed, what is actually seen is a
mixture of all the colors of light
Why do some substances appear colored?
When this light passes through a substance, certain energies (or
colors) of the light are absorbed while other color(s) are allowed to pass
through or are reflected by the substance.
If the substance does not absorb any light, it appears white (all light is
reflected) or colorless (all light is transmitted). A solution appears a
certain color due to the absorbance and transmittance of visible light.
For example, a blue solution appears blue because it is absorbing all of
the colors except blue.
BACKGROUND
A sample may also appear blue if all colors of
light except yellow are transmitted. This is
because blue and yellow are complementary
colors. (See the color wheel above.)
BACKGROUND
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The amount of light absorbed by a solution is
dependent on the ability of the compound to
absorb light (molar absorptivity), the distance
through which the light must pass through the
sample (path length) and the molar
concentration of the compound in the solution.
If the same compound is being used and the
path length is kept constant, then the
absorbance is directly proportional to the
concentration of the sample.
Spectrophotometer
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A spectrophotometer is used to provide a
source of light of certain energy
(wavelength) and to measure the quantity
of the light that is absorbed by the sample.
Light Bulb
Sample
Prism
Detector
Filter
Slit
Spectrophotometer
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The basic operation of the spectrophotometer includes a white light radiation source
that passes through a monochromator. The monochromator is either a prism or a
diffraction grating that separates the white light into all colors of the visible spectrum.
After the light is separated, it passes through a filter (to block out unwanted light,
sometimes light of a different color) and a slit (to narrow the beam of light--making it
form a rectangle). Next the beam of light passes through the sample that is in the
sample holder. The light passes through the sample and the unabsorbed portion
strikes a photodetector that produces an electrical signal which is proportional to the
intensity of the light. The signal is then converted to a readable output that is used in
the analysis of the sample.
Light Bulb
Sample
Prism
Detector
Filter
Slit
Spectrophotometer
The spectrophotometer displays this quantity in one of two ways:
(1) Absorbance -- a number between 0 and 2
(2) Transmittance -- a number between 0 and 100%.
The sample for a spectral analysis is prepared by pouring it into a cuvette
which looks similar to a small test tube. A cuvette is made using a special
optical quality glass that will itself absorb a minimal amount of the light. It is
also marked with an indexing line so that it can be positioned in the light beam
the same way each time to avoid variation due to the differences in the
composition of the glass
Experiment
Viewing the Visible Spectrum
The spectrophotometer is designed to detect absorbances of light at
different wavelengths when the light passes through a solution of some
given concentration. Some compounds absorb more light at one
wavelength than another, so the wavelength must be changed every
time a different compound is being analyzed to achieve optimum results
from a spectrophotometer. The wave-length of light is selected by
adjusting the wavelength dial and read on the wavelength display.
In this lab, the color of light associated with each wavelength will
be observed with the eye. The visible range of light is approximately
400 to 700 nm. The very ends of the visible spectrum will also be
determined in this experiment. Please note that the accepted symbol
for wavelength is the Greek letter lambda ().
Viewing the Visible Spectrum
Objective
To observe the color of light emitted by the spectrophotometer
at various associated wavelengths.
Materials Needed:
A piece of white chalk
approximately 1-2 cm long
Spectrophotometer
A cuvette/cuvette rack
Viewing the Visible Spectrum
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Procedure (Best results are obtained by doing this experiment in a dimly lit room)
Cut or rub one end of the piece of chalk to produce a 45o angle.
Place the piece of chalk in a cuvette with the angle end directed up.
Set the wavelength of the spectrophotometer to 425 nm. Be sure the filter switch is
set to the left.
Place the cuvette in the spectrophotometer so the angle of the chalk faces to the right
of the spectrophotometer.
Open the light slit by turning the transmittance adjustment knob (right knob)
clockwise.
Look into the sample compartment and record on the data sheet the color of the light
striking the chalk.
Repeat Step 5 increasing the wavelength by 25 nm each time. Continue the process
until reaching 675 nm. At 600 nm, move the filter lever (#11 in the diagram) to the
right.
While looking at the piece of chalk, slowly increase the wavelength to the point where
the color is no longer seen. This is one end of the visible spectrum. Record this
wavelength value.
Adjust the wavelength back to 425 nm. While looking at the piece of chalk, slowly
decrease the wavelength to the point where the color is no longer visible. This is the
other end of the visible spectrum. Record this wavelength value.
Viewing the Visible Spectrum
Frequency
(Hertz)
Wavelength (nm)
Observed Color
End of Spectrum
7.1 x 1014
425
6.7 x 1014
450
6.3 x 1014
475
6.0 x 1014
500
5.7 x 1014
525
5.5 x 1014
550
5.2 x 1014
575
5.0 x 1014
600
4.8 x 1014
625
Notes
Spectral Curve of Food Coloring
Background Information:
A visible spectrophotometer can be used to learn why colored
solutions appear a particular color. For example, WHY does blue food
coloring appear blue? Simply put, the solution is blue because it
transmits (and reflects) blue visible light more than it transmits other
colors of visible light. In other words, blue food coloring absorbs blue
visible light the least and absorbs other colors of light more.
When white light is observed, what is actually being seen is a
mixture of all the colors of light. When this light passes through a
substance, certain energies (or colors) the light are absorbed while
other color(s) are allowed to pass through or are reflected by the
substance. This is why some substances appear colored. The color
that we see is the combination of energies of visible light which are not
absorbed by the sample. If the substance does not absorb any light, it
appears white or colorless.
2. Spectral Curve of Food
Coloring
A solution appears a certain color due to the absorbance and
transmittance of visible light. For example, the blue solution
appears blue because it is absorbing all of the colors except
blue. A sample may also appear blue if all colors of light except
yellow are transmitted (yellow is absorbed). This is because
blue and yellow are complementary colors. Any two colors
opposite each other on the color wheel (see figure above) are
said to be complementary. The wavelength (numbers outside
the wheel) associated with the complementary color is known as
the wavelength of maximum absorbance. This is because in a
colored solution the maximum amount of light is absorbed by the
complementary color. Note: cyan = green.
Spectral Curve of Food Coloring
How a Spectrophotometer works: Inside the spectrophotometer
[See Figure 1] is a light bulb which produces white light. The white
light is separated into the different colors of light by either a prism or a
diffraction grating (An example of a grating is a CD ROM surface. The
reflective surface has tiny grooves etched into it, which separate white
light, in a manner similar to light passing through a prism). After the
light is separated, it passes through a filter (to block out unwanted
light, such as light of a different color) and a slit (to narrow the beam of
light--making it in the shape of a rectangle). Next the beam of light
passes through the sample that is in the sample holder. The amount
of light that passes through the sample is measured and the
spectrophotometer displays this quantity in one of two ways:
Absorbance -- a number between 0 and 2. This is a measure of how
much light is absorbed by the solution, in other words, now much does
not pass through.
Transmittance -- a number between 0 % and 100 %. This is a
measure of how much light passes through the solution (this is
transmitted light).
Spectral Curve of Food Coloring
The spectrophotometer is designed to detect the
absorbance of light at different wavelengths (different
colors) when the light passes through a solution of some
given concentration. Some compounds absorb more light
at one wavelength than another, so the wavelength must
be changed every time a different compound is being
analyzed to achieve optimum results from a
spectrophotometer. The wavelength of light is selected by
adjusting the wavelength dial and read on the wavelength
display.
Spectral Curve of Food Coloring
Objectives:
To measure the wavelengths of visible light that various colored
solutions absorb.
To plot a graph of wavelength versus absorbance and determine
the maximum wavelength (λmax) for each sample.
Using this information we will reason as to why each solution
appears a particular color.
Materials:
Visible spectrophotometer
Distilled water in squeeze bottle
Food coloring (red, blue, yellow and green) Tissues or Kimwipes
Cuvettes
Excel graphing program
Cuvette rack
Plastic wrap
Spectral Curve of Food Coloring
After you collect the data: handout-you will
do the following data analysis
•Data Analysis: Now you will plot your data
on a graph. (See separate instructions for
Excel if you are using a computer to plot your
data. Create a scatter graph and then choose
the option that connects the dots to draw the
curve). Wavelength is plotted on the x-axis and
Absorbance is plotted on the y-axis.
Remember to put units on your axes and to
give your graph a title that tells the purpose of
the graph.
3. PHYSICAL AND CHEMICAL
CHANGE
BACKGROUND:
Physical change - a change to a substance, such as boiling, freezing, or breaking,
that does not result in the formation of a new substance.
Chemical change - a change to a substance, such as burning or rusting, that
results in the formation of a new substance.
Color change, bubbles and production of gas, heat and/or light given off, the
formation of a precipitate, and odor changes are often observed as chemicals
undergo change. Unfortunately, many of these clues may be observed for both
chemical and physical changes. The best indication of a chemical change is
evidence that a new substance, with different properties from the original
substance, has formed, but even this is not always directly observable.
One of the properties of most materials is the wavelength(s) of light that they will
absorb. In this lab, you will measure the absorbance by obtaining a spectral curve of
the materials before they are mixed and again after the mixture has been formed.
The spectrophotometer will help you determine whether the color change shows
formation of a completely new substance (a chemical change), or simple physical
combination of two substances (a physical change).
PHYSICAL AND CHEMICAL
CHANGE
OBJECTIVES:
To produce spectral curves of solutions before they are
combined and after they are combined.
To determine, by using the spectral curve, whether the color
observed is a new product or a mixture of the original
solutions that were combined.
To determine whether changes resulted in a chemical
change or a physical change.
PHYSICAL AND CHEMICAL
CHANGE
MATERIALS:
Spectronic Spec 20D spectrophotometer
Computer with printer
Pipettes
Graphing software
KimWipes
Distilled water
Red and blue food color
1.0 M Hydrochloric Acid
Red cabbage or black bean juice
Graduated cylinder
Cuvettes
Cuvette rack
Spectral Curve for Red Food Color
Spectral Curve for Blue Food Color
PHYSICAL AND CHEMICAL
CHANGE
PROCEDURE:
Mix 5 drops of red and 5 drops of blue food coloring into a cuvette
and then dilute with about 5 ml of distilled water. Mix the
solution by carefully tapping the bottom of the cuvette as
demonstrated by your teacher.
Fill a second cuvette about halfway with distilled water. This will
serve as a “blank”.
Obtain a spectral curve for the food coloring mixture from a
wavelength of 350 nm to a wavelength of 675 nm and record
the absorbance values in Data Table 1.
PHYSICAL AND CHEMICAL
CHANGE
Data Table 1. Absorbance of Food Coloring Mixture
Wavelength (nm)
Absorbance
Wavelength (nm)
350
525
375
550
400
575
425
600
450
625
475
650
500
675
Absorbance
PHYSICAL AND CHEMICAL
CHANGE
Add ______ (determined by the instructor) drops of red cabbage
or black bean juice to a cuvette and dilute with about 5 ml of
distilled water.
Prepare another cuvette using ______ drops of red cabbage or
black bean juice, dilute with about 5 ml of distilled water, and add a
few drops of acid to the juice until you see a color change. Mix the
solution by carefully tapping the bottom of the cuvette.
Obtain spectral curves for the juice, and juice/acid mixture and
record them in Data Table 2.
PHYSICAL AND CHEMICAL
CHANGE
Data Table 2. Absorbances of Juice And Juice/Acid Mixture
Wavelength (nm)
Absorbance Juice
Absorbance
Juice/Acid
Wavelength (nm)
350
525
375
550
400
575
425
600
450
625
475
650
500
675
Absorbance Juice
Absorbance
Juice/Acid
PHYSICAL AND CHEMICAL
CHANGE
Make scatter type graphs of your data, using
graphing software or by hand, plotting
wavelength on the X axis and absorbance on
the Y axis. Compare the spectral curve of the
mixture to the spectral curve(s) of unmixed
substances to answer the following questions.
(If using graphing software, use overlay
techniques to compare the graphs.
Instructions for Excel graphing are attached to
this lab.)
Answer questions on question sheet with your handout.