INTRODUCTION TO SPECTROPHOTOMETRY

1
INTRODUCTION TO SPECTROPHOTOMETRY

• Advancing Science Lab
• Gettysburg College
• 531, 532, 534

2
BACKGROUND

• 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.

3
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.
4
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.)
5
BACKGROUND

• 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.

6
Spectrophotometer

• 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.

7
Spectrophotometer

• 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.

8
Spectrophotometer

• The spectrophotometer displays this quantity in
  one of two ways
• 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
9
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
(?).
10
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
11
Viewing the Visible Spectrum

• 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.

12
Viewing the Visible Spectrum
13
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.
14
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.
15
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).
16
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.
17
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
(?max) 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
18
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.

19
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).
20
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.
21
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
22
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.

23
PHYSICAL AND CHEMICAL CHANGE
Data Table 1. Absorbance of Food Coloring
Mixture
24
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.
25
PHYSICAL AND CHEMICAL CHANGE
Data Table 2. Absorbances of Juice And
Juice/Acid Mixture
26
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.