NAME _________________________________________
SPECTROSCOPY
We know of three types of spectroscopy:
Mass Spectrometry
A mass spectrometer source produces ions. Information about a sample may be obtained by analyzing
the dispersion of ions when they interact with the sample, generally using the mass-to-charge ratio.
Infrared Spectroscopy
The infrared absorption spectrum of a substance is sometimes called its molecular fingerprint. Although
frequently used to identify materials, infrared spectroscopy also may be used to quantify the number of
absorbing molecules.
UV-Vis Spectroscopy
Visible light spectroscopy is the subject of this lab. We will first review “What We Know, In General…
Spectroscopy: What we know thus far:
1. Spectroscopy is the study of the interaction of light with matter (and/or vice versa)
Matter interacts differently with light of different energies. Various methods of spectroscopy use
different wavelengths of light to study different properties of matter.
2. Light can be thought of as having either wave properties or particle properties. (Wave-particle
duality)… meaning that all matter (like an electron) exhibits the properties not only of particles with
mass but also of waves, which transfer energy.
3. We also know that electromagnetic radiation is composed of both electric and magnetic field whose
waves vibrate in mutually perpendicular planes. It is the electric field of light interacting with the
electrons of matter which produces observable properties of a substance (Absorption Spectroscopy and The BeerLambert Law)
In fact, chemical species have a unique spectral fingerprint predicated upon the position of the electrons
relative to the nucleus.
We have had a fair amount of experience with EMISSION SPECTRA, in the honors chemistry course.
There was our work on flame tests and the emission colors of metal ions (as in, fireworks), when we
made Sterno, in Honors Chemistry.
In that course, we also witnessed the phenomenon of light and matter interaction which our use of the
spectroscope and luminous gas samples, such as neon as well as our work in the candle flame lab. In the
candle lab we could identify areas of a flame, relative to the temperature of the system … and we even
saw how changing the conditions affected the burning of the paraffin material.
4. We know now that most of the electromagnetic radiation continuum is invisible to humans – but that we
can perceive portions of the EMS, called visible light. Wavelengths of visible light (ROYGBV) extend
from “red” at about 800 nanometers (8 x 10-9 m) to “violet” at about 400 nanometers (4 x10-9m)
Overall the conclusion to most of the work on emission spectra surrounds the fact that the wavelength (or color)
of light revealed, is due to excited electrons of the matter “relaxing” to a lower energy state (returning to ground
state), by emitting photons (Austin Peay State University Department of Chemistry)
So, if you would pardon the pun, we have focused our work upon the emission of energy. However, there is
also the concept of the absorption of light energy and how this absorption of energy can interact with matter,
and how we use it to our advantage.
Transmittance, Absorbance and the Beer - Lambert Law
Every substance absorbs or transmits certain wavelengths of radiant energy but not other wavelengths. For
example, the nickel(II) ions associated with the protein chlorophyll absorb red and blue/violet light while they
emit (transmit) green light. Our eyes “see” the transmitted or reflected wavelengths, those not absorbed, as the
color green.
But what is of serious interest to chemists is that the specific wavelengths absorbed and transmitted are
charactericstic for a substance, so a spectrum serves as a “fingerprint” of the substance that can help identify
and/or quantify an unknown.
Now, suppose you look at two solutions containing copper(II) ions, one a deeper color than the other. Your
common sense and experience in lab tell you that the more intensely colored solution is the more concentrated
…Hence color can indicate concentration.
We know that when light is absorbed, electrons in the ground state move into what we may term, an excited
state (represented by electrons moving temporarily to higher energy levels).
When light interacts with the electrons of a species, the light can be scattered, reflected, transmitted, or
absorbed. The absorbed light (the focus of this tract) causes changes in the atomic and molecular rotation,
vibrations and electron transitions to higher energy levels.
This results of this absorbance are phenomena such as the release of energy, fluorescence, or the release of
color.
Colored substances, such as dyes or ions of transition metals, absorb light in the visible range of the
electromagnetic spectrum. Often, the color we see in samples containing these species, is the opposite of the
color absorbed. Hence if blue is absorbed we often see reds and oranges.
In a broad absorbance band, a number of wavelengths on both sides of the primary absorbance region, may also
be absorbed … resulting in a released color (a perceived color) which is a mixture of wavelengths not absorbed.
The pigment indigo (as in blue jeans) absorbs maximally in the 500 to 650 nm range. This means that the
wavelengths in the 400 to 500 nm range are NOT absorbed…giving us a blue to violet color.
A green solution would absorb blue, red and yellow … thus we see green.
It is possible to measure the amount of light absorbed by a sample using a spectrophotometer.
First … a little technical vocabulary…
1) Transmittance (T) is the ratio of the amount of light transmitted by or passing through the sample
relative to the amount of light that initially fell on the sample (the incident light)
Transmittance = P/Po where P = intensity of transmitted light and Po is the intensity of
incident light.
Incident Light Po
Transmitted
Light (P)
with lower
intensity
due to
reflection,
scattering
and
absorption
http://pharmaxchange.info/press/2012/04/ultraviolet-visible-uv-vis-spectroscopy-%E2%80%93-derivation-of-beer-lambert-law/
2) Absorbance of a sample is the negative log of Transmittance. … That is, the two values are
inversely related … as absorbance of a solution increase, the transmittance decreases….
Absorbance = -logT or rather… A = -log P/Po
Back to our original solutions containing copper(II) ions we can see that absorbance, A of
a sample increases as the concentration increases … (More light is absorbed as the concentration
of the colored solution increases)
Absorbance is affected also by the length of the path the light must travel through the solution.,
In a wider test tube, the light must travel through more of the solution, an thus more is absorbed.
Hence, absorbance increases with path length.
The study of the effects of absorbance of light in is best studied in our class, with solutions containing colored
species. Clear solutions absorb in the UV or IR regions of the spectrum, and we just don’t have the equipment.
IR spectroscopy helps to determine qualities of the bonds between atoms. (Your aged teacher was a bit of a
whiz-bang at IR spectroscopy …but that was so long ago, light had just been created….)
A spectrophotometer separates electromagnetic radiation into wavelengths and passes these wavelengths
through a sample, detecting the intensity of the transmitted light.
In analyzing a new sample, a chemist would probably first determine an absorption spectrum by plotting
absorbance as a function of wavelength … showing how the absorbance of light depends upon the wavelength
of light.
There is, more often than not a wavelength of maximum absorption (λmax), at which absorbance is greatest. In
theory, we could choose any wavelength for quantitative analysis of concentration. However, the magnitude of
the absorbance is important, when very small amounts are available. Thus, finding λmax is quite important.
Determining λmax (lambda max) gives us the highest sensitivity of the species to a particular region of
light … helping us to minimize variations with Beers-Lambert Law.
It is also a unique characteristic of the species … which in turn may help provide insight as to the electronic
structure of the species.
We shall use an instrument called a Spec 20 and the Beer’s-Lambert Law: A = ɛ x ℓ x c
Where: A = absorbance (a dimensionless number)
ℓ = path length with the unit of cm
c = concentration with units of mol/L
ɛ = molar absorptivity …a constant for the species being tested at constant temperature and
wavelength. Its units are L/mol•cm
Hence the equation suggests that: A α ℓ x c
Every Spec 20 has a source of radiant (light) energy, a prism or grating to isolate radiant energy to narrow
wavelength regions (a monochromator), a cradle for holding a test solution and a detector for measuring the
light intensity of transmitted light.
And this brings us to two good points to get clarified.
First, we shall assess the absorbance of light, by measuring the transmitted light intensity …. Any light that
does not come “through” the solution, using the Beer-Lambert Law.
Secondly, since we are dealing with light passing through a test tube holding a solution, certain issues become
wildly important. The first is cleanliness … Any water droplets or fingerprints on the outside of the sample test
tube (called a cuvette) will hamper readings. Guess what?
Water droplets and fingerprints can cause transmission interference by refracting light. Fingerprints might even
mess with the absorbance … So keep it clean.
When we apply the Beer’s-Lambert equation we will see that other factors such as molar absorptivity, the
length of the pathway and solution concentration also matter. But, keeping it clean is paramount!!!!
Using Spectrophotometry in Chemical Analysis
Very often in a spectrophotometry lab we would prepare a calibration plot of Absorbance as a function of
Molarity (Concentraiton).
Thus once we know λmax we can test a series of solutions with known concentrations. Due to the linear
relationship between concentration and absorbance (at a given wavelength and path length), we can get a
straight line with a positive slope. We can then test our unknown sample and use the calibration plot (or its
equation of the straight line) to determine the concentration.
Example: A solution of KMnO4 has an absorbance of 0.539 when measured at 540 nm in a 1.0 cm cell. What
is the concentration of the unknown solution of KMnO4?
Prior to determining the absorbance for the unknown solution, the following calibration data were collected for
the spectrophotometer. (Kotz p 192)
Concentration of Absorbance
KMnO4 (M)
0.0300
0.162
0.0600
0.330
0.0900
0.499
0.120
0.670
0.150
0.840
Calibration Plot:
Absorbance as a funciton of [KMnO4]
0.9
0.84
0.8
Absorbance
0.7
0.67
0.6
0.5
0.499
0.4
0.33
0.3
0.2
0.162
0.1
0
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
Concnetration (M)
If you estimate by just reading the calibration curve, what is the concentration of the unknown solution,
at an absorbance of 0.539? *approx. 0.09
If calculated …. using Excel to determine the straight line we get: y = 5.653x – 0.009
Where y = absorbance and x = concentration
Plug in the absorbance for the unknown solution: 0.539 = 5.653(concentration) -0.009
Unknown concentration = 0.0969 M
Questions: All of the answers may be found in the reading or extended from the reading….
T/F
1) ____ As absorbance increases, transmission must decrease.
2) ____When a solution is diluted to ½ the original concentration, the absorbance of light will increase.
3) ____Colorless solutions are best tested using UV or IR spectroscopy
4) ____Solutions with colored ions can be analyzed using a Spec-20
5) ____It does not matter what the width of the test tube is, when using a Spec -20
6) ____When comparing two colored aqueous solutions of the same compounds, under the same conditions of
test tube, temperature, volume etc… the darker solution is more concentrated.
7) ____ As path length increases, absorbance increases.
8) ____ There is an inverse relationship between the absorbance and transmission of light through a colored
solution.
9) ____ Every substance absorbs or transmits the exact same wavelengths of radiant energy, making all
solutions identical.
10) ____ Lambda x (λmax) is the wavelength at which a specific colored solution, demonstrates a maximum
absorbance of light.
11) ____ The amount of light that is absorbed by the solution depends on the concentration of the absorbing
species in the solution.
12) ____ The intensity of the light shined on the sample is greater than the intensity of light that exists the
sample, if the sample absorbs any of the light.
Answers:
1) T
2) F
3) T
4) T
5) F
6) T
7) T
8) T
9) F
10) T
11) T
12) T