Chapter 10: Spectroscopy

Part 5-Instrumentation: Introduction
to Spectroscopy for Chemical
The Spectrophotometer- Instruments
IMPORTANT: Absorption
FFYI: Double-beam spectrophotometer (better than single beam see previous page):
Light passes alternately through the reference and sample cuvettes. A chopper is a mirror
that rotates in and out of the light path diverting the light between the reference and sample
cuvettes. Routine procedure is to first record a baseline spectrum with two reference
cuvettes. The absorbance of the reference is then subtracted from the absorbance of the
sample to obtain the "true" absorbance at each wavelength.
….Or diode array
Light Sources
FYI: Sources of radiation objects
Any object that is heated emits radiation. Emission from real objects such as a
tungsten filament light bulb emulate blackbody radiation (the emission is a continuous
spectral distribution).
Visible and infrared lamps as light sources approach blackbody radiators. The radiation
from an object's surface expressed as power per unit area is the excitance (emittance), M.
M =  T4
Where  is the Stefan-Boltzmann constant ~ 5.7 X 10-8 W/(m2K4), T = Temperature (K)
Spectral distribution of blackbody radiation
IMPORTANT: Lamps for absorption spectrometers
Typically they are inexpensive and stable.
i) Visible and Near Infrared: Tungsten
Lamp, Xenon lamp
ii) Ultraviolet and Visible: Quartz
Halogen Lamp, Xenon lamp
iii) Ultraviolet: Deuterium Arc Lamp
iv) Infrared: nichrome wire , silicon
carbide (globar)
FYI: Lasers provide ~ single 
Very bright sources for spectroscopy
Properties of Lasers:
· Monochromatic (only one wavelength)
· Collimated (emit in one direction)
· Polarized (only one electric field vector)
· Coherent (electric/magnetic fields in phase)
· Expensive
hHigh maintenance, but some He-Ne, and many solid state lasers may be less
Laser operate on the principle of trapping a large number of physical objects to a
new state in a cavity, and simultaneous release of these objects to a new or old
state with emission
Monochromators and other devices
for separation of radiation objects
Slits +Monochromators
Slits are constructed by machining a sharp edge onto two
metal pieces. These lie in a plane and the spacing between them,
the slit width, can be adjusted. The smaller the slit width, the better
the spectral resolution. (example)
Filters are used to pass on only desired wavelengths of light.
A filter could be colored glass. Most likely they are also based on
constructive or destructive interference of light waves. (example)
Separation of wavelengths on some commercial
instruments. Prisms were used in older instruments, Quartz
or salt crystals.
FYI: selection of colors
Monochromators separate wavelengths of light; they consist of both entrance &
exit slits, mirrors, and diffraction grating or refraction lens/prisms, and filters.
Polychromatic light is collimated (focused) into a beam of parallel rays by a concave mirror
(monochromatic-one wavelength; polychromatic-many wavelengths).
Rays strike the reflection grating (see next figure) and different wavelengths are
diffracted (separated) at different angles.
Diffracted light is focussed by a second concave mirror so that only one wavelength
passes through the exit slit at a time.
Grating Equation: n = d(sinq – sinf)
n = diffraction order (1…n) ; d = groove spacing; f = angle of reflection;
q = angle of incidence
Components of a Grating
diffraction grating is ruled with a series of closely spaced parallel grooves separated by
distance d.
These are often constructed from aluminum metal and coated with a non oxidative
coating applied.
When light is reflected from the grating, each groove behaves as a source of light.
When adjacent rays are in phase, they reinforce each other. When adjacent rays are out of
phase, the partially or completely cancel each other. Thus can be aligned to allow only
certain wavelengths to pass through.
Wavelength selector (monochromator) passes a narrow
bandwidth of radiation (if more narrow , higher
EXPERIMENTAL PARAMETERS : how to get the most
from measurements with absorption spectroscopy
C Choice of the
Wavelength and
Bandwidth –
Effects of monochromator’s
slit: 0.25nm,1.0nm, 2.0nm,
INTERFEROMETERS: from time and length observables to
frequency/energy observables
1) allows for signal averaging
2) allows all wavelengths to be monitored simultaneously
3) mathematical process that converts data obtained in the time domain to be converted
into the frequency domain.
Allows all wavelengths to simultaneously reach the detector
Radiation from source reaches beam splitter, where half of the radiation hits the
moving mirror and half hits the fixed mirror.
The beams reflect and re-combine, the emerging radiation for a wavelength exhibits
constructive or destructive interference.
With constant mirror velocity, the wavelength modulates in a regular sinusoidal
Both the sampling rate of radiation reaching the detector and the mirror velocity is
modulated by a helium-neon laser.
The resulting detector signal typically is stored as a time domain spectrum
Converted to a spectrum in the frequency domain using the mathematical process of
Fourier Transform.
Infra-Red spectroscopy, NMR spectroscopy: FT is a standard technique
•The phototube is used frequently as a detector in UV-Vis spectrometers.
•The cathode consists of a photo-emissive surface.
•Electrons are ejected from the cathode proportional to the radiant power (photons) striking its
•The emitted electrons are attracted to the anode.
•The accompanying voltage is fed to an amplifier and converted to a signal.
•The Photo Multiplier Tube, (PMT) is similar to the photo tube, but is a vast improvement.
•In addition to the cathode and anode, the PMT has dynodes, which produce a cascade effect on the electron
emission production.
•Each photon causes a ~ 107 additional electrons to be produced.
•The PMT possesses high sensitivity, good S/N ratio, and excellent dynamic range.
•PMTs are highly sensitive to visible and UV excitations at extremely low power conditions, (very low
concentrations of analyte).
•Intense light sources (such as daylight or stray light) can destroy and damage PMTs.
•PDAs are a series of silicon photo diodes, with each having a storage capacitor, and a
switch that are combined in a integrated circuit on a silicon chip.
•The number of sensors (silicon photodiodes) in a PDA range from 64 to 4096.
•The slit width of the instrument allows the radiation to be dispersed over the entire
array, allowing the spectral information to be accumulated simultaneously.
•PDAs are not as sensitive nor have the same S/N ratio as the PMT, but one gains the
advantage of gathering multi-channel information (all of spectrum collected
•Advantage of the PDA is recording the entire spectrum in a fraction of the time
required for a conventional scanning spectrometer to scan one wavelength at a time.
An example of PDA use is in atomic emission spectroscopy (AES), UV-vis
spectrophotometry, fluorescence spectrometry, Raman spectrometry.
Eliminating noise: signal averaging
Stray Light, Electrical Noise, Cell PositioningStray light can cause problems:
Stray light arises from two major sources:
1) Misdirected rays coming from the monochromator.
2) Light coming from outside the instrument such as the sample compartment lid
not closed properly.
Other concerns;
•the correct choice of the sample cell (does it require glass or quartz?)
• the alignment of the sample cell (and/or the sample cell holder)
•dust & fingerprints on the cell
Part 5- From VUV to IRIntroduction to Spectroscopy for
Chemical Analysis
absorption, UV/VIS electronic
absorption and emission
Atomic vs. Molecular
• What is same what is different
IMPORTANT: Most atomic spectra are discrete:
IMPORTANT - MOLECULES: Molecular spectra are
broader because of the close electronic and vibrational
FYI: MOLECULES: also electronic molecular orbitals and…..
One must take into
account all molecular
energies from different
“degrees of freedom” :
translational (motion),
electronic and here
vibrational and
rotational motion of
Here are some of the
degrees of freedom of
Harmonic oscillator models
"What Happens When a Molecule Absorbs Light?"
What happens when the absorption process takes place for molecules and compounds?
Molecular orbitals describe the distribution of electrons in a molecule, just as atomic orbitals describe the
distribution of electrons in an atom. As example; :CO: localized (Lewis) vs delocalized (MO)
In an electronic transition, an electron from one molecular orbital moves to another orbital, with a concomitant increase or
decrease in the energy of a molecule.
Molecular Spectroscopy
The VIS-visible absorption methods rely on complexes or compounds
forming a color and it must be easily distinguishable from other species
The UV methods may be less specific in that typically most compounds
absorb UV radiation; thus the results maybe limited to only
quantitative detection (information). That is, how much is there.
We have a solution containing different proteins, which absorb certain
wavelengths of light (ie 254 nm). But if we require each protein's identification a
more specific technique must be chosen.
On the other hand the IR methods depend upon vibrational and rotational
absorptions. They can give both quantitative,(how much is present) and
qualitative, (compound identification) information.
IMPORTANT - MOLECULES: Absorption of photon:
electrons gain energy (ground to excited state)
Emission of photon: electrons lost energy (excited to ground state)
SSinglet state (S) – when the electron in the excited state is still paired with the ground-state electron. The
spins of the two electrons remains opposed.
Triplet state (T) – when the electron in the excited state becomes unpaired with the ground-state
electron. The spins of the two electrons are now parallel.
Electronic absorption bands are broad due to the large number of vibrational and rotational states
present at each electronic state.
We have discussed the instrumental procedure, components and design or UV-vis spectroscopy.
The visible adsorption methods rely on complexes or compounds forming a color and it must be
easily distinguishable from other species present.
The UV methods may be less specific in that typically most compounds absorb UV radiation; thus
the results maybe limited to only quantitative detection (information). That is, how much is
For example: We have a solution containing different proteins, which absorb certain wavelengths of
light (ie 254 nm). But if we require each protein's identification a more specific technique must be
On the other hand the IR methods depend upon vibrational and rotational absorptions. They can give
both quantitative,(how much is present) and qualitative, (compound identification) information.
At a given wavelength, a solution of a colored compound has a molar absorptivity of 104 M-1cm-1.
Calculate for a 1 cm cell containing a 0.050 mM solution of this compound, at that wavelength: (a) the absorbance,
and (b) the transmittance.
A = ebc = (10000 M-1cm-1)(1 cm)(0.050 mM) = 0.50
A = –log10 T ‹ T = 10–A = 10-0.50 = 0.32 or %T = 32%
At a given wavelength, a cuvet filled with a sample solution has a transmittance of 63.1%. A reference cuvet
filled with the solvent has a transmittance of 94.7% at that same wavelength. What is the corresponding absorbance of
the sample?
A = –log10 T so Asample = –log(0.631) = 0.20
Aref = –log(0.947) = 0.02
Acorrected = Asample – Aref = 0.20 – 0.02 = 0.18
A 0.267 g quantity of a compound with a molecular weight of 337.69 g/mol was dissolved in 100
mL of ethanol. Then 2.000 mL was withdrawn and diluted to 100 mL. The spectrum of this solution exhibited
a maximum absorbance of 0.728 at 438 nm in a 2.000-cm cell. What is e?
Constructing a Calibration Curve - This method is used in other chemical analyses not just
spectrophotometric ones.
A calibration curve is a graph showing how the experimentally measured property depends on the
known concentrations of the standards.
We prefer calibration procedures with a linear response, in which the corrected analyte signal is
proportional to the quantity of analyte.
Procedure for Constructing a Calibration Curve
Step #1.
Prepare known samples of analyte, covering a
convenient range of concentration, and measure the response of the
analytical procedure to these standards.
Step #2.
Subtract the average response of the three blank
samples from each measured responses to obtain the corrected response.
The blank measures the response of the procedure when no analyte is
Step #3.
analyte analyzed.
Make of graph of corrected response vs. quantity of
the following information is obtained during absorbance measurements at 427 nm.
Abs readings readings readings
Beer's cal plot
y = 9.0088x - 0.0015
-0.2 0
W What is the fate of the absorbed electronic energy associated with UV-vis
spectrometry? Sometimes it results in the emission of another photon of light
Excess energy is dissipated by the excited molecule through;
 collisions with other molecules (solvent)
 vibrations
 rotations
 heat
 produce a photon and relax back to the ground state. This emission process is termed luminescence.
What kinds of molecules typically exhibit luminescence?
The emission spectrum typically resembles the mirror image of the absorption spectrum, but is shifted to longer wavelengths.
•Highly degree of conjugation (multiple double bonds)
•Aromatic molecules
• Molecules with atoms, which have unpaired nonbonding valence electrons
•Molecules with molecular rigidity (polycyclic)
• Metal chelates
What can happen when a molecule absorbs light and an electron is promoted from the
ground state, So, to a vibrationally and rotationally excited level of the excited
electronic state S1?
vibrational relaxation is a radiationless (does not produce photon) transfer of energy to
other molecules (typically the solvent) by collisions – manifested as heat
internal conversion is a radiationless transition between states with the same spin
quantum numbers (e.g., S1  S0)
intersystem crossing is a radiationless transition between states with different spin
quantum numbers (e.g., T1  S0)
fluorescence is a radiational transition between states with the same spin quantum
numbers (e.g., S1  S0)
phosphorescence is a radiational transition between states with different spin quantum numbers (e.g., T1  S0)
phosphorescence is a radiational transition between states with different spin quantum numbers (e.g., T1  S0)
In general, fluorescence and phosphorescence are observed at a lower energy than that of the absorbed
radiation (the excitation energy).
em > ab
Rrefers to emission of light by any mechanism from any type of molecule.
LLuminescence measurements are inherently more sensitive than absorption measurements.
It is much easier to detect and measure a small signal rather than the difference in changes of
signals associated with absorption.
Luminescence (light emission detection methods) instrumentation has been developed around
fluorescence methods rather than phosphorescence methods since fluorescence is much more sensitive.
AApplications of Fluorescence spectroscopy
i) determination of biomolecules: enzymes, steroids, drugs: for example, as little as 1 ng/L (pp
trillion) of the drug LSD in 5 mL blood sample.
ii) determine trace contaminants; ppb of benzopyrene in air pollution samples
iii) "Electronic dog at airports", check air samples that contain TNT explosive; detection in the
600 ng/mL range.
iv) determination of the Fluoride ion, [F-], indirectly by its ability to quench(inhibit) fluorescence
in the Al+3 -Alizarin garnet R complex.
Principle Components of a Fluorescence Spectrometer
i)Light sources: mercury arc lamp, xenon arc lamp,
ii) absorption and interference filters
iii) Grating monochromators: excitation and
emission (90geometry) (remember, the UV-vis
experiment is 180o configuration)
iv) Detectors - PMT, photodiode arrays
v) sample cells - silica glass
vi) amplifier and read out
As we see in the instrumental diagram: two optical units are required. The excitation monochromator selects the wavelength from the source and
directs it onto the sample cell. The emitted luminescence is directed through the emission monochromator to the detector (As seen in the diagram,
they are at 90 degrees to the lamp source).
In emission spectroscopy, we measure the intensity of emitted radiation, not the
fraction of radiant power striking the detector as we do in absorption methods.
Emission Intensity: I = k P0 c
We can decide the excitation monochromator and the emission monochromator
settings by looking at:
Emission spectrum: constant ex and variable em
Excitation spectrum: constant em and variable ex
As shown above right, a standard curve can
be constructed, similar to a Beer's law plot
for an absorption measurement. The points
represent readings at different
concentrations. At higher concentrations
the curve becomes nonlinear.
At low concentrations, the absorbance effects are small and the emission intensity (I) is
directly proportional to the sample concentration (c) and to the incident radiant power (P0);
where the constant k depends on the quantum efficiency of the fluorescence process, cell
path length, etc.
Emission Intensity: I = k P0 c
Factors that inhibit fluorescence:
i) temperature: increased temperature increases collisions
ii) solvent: halogen compounds, pH, dissolved oxygen
iii) concentration dependent (too much)
Some analytes are naturally fluorescent and can be analyzed directly.
For example: Vitamin B2 , riboflavin
Most compounds are not naturally luminescent enough to be analyzed directly. However,
coupling a fluorescent moiety provides an easy route to this sensitive analysis.
IR spectroscopy
Infrared Spectroscopy (looking at vibrational transitions):
Most organic as well as inorganic compounds, which are covalently bonded and exhibit a dipole moment absorb infrared
radiation. The absorption process is quantized and as opposed to UV-vis, results in the excitation of a vibrational
process in the molecule, not an electronic one. An absorbed energy matches the energy for a vibrational mode of a
molecular bond. Several vibrational modes are possible: stretching, bending, twisting, etc.
A) IR active compounds have a dipole moment and are not symmetric. A dipole is merely an unequal distribution of
charge (e-) due to the nature of the atoms in the molecule (electronegativity).
(+)  (-)
HCl e- density "pulled" towards Cl
Molecules like N2, O2, or Cl2 do not have a net dipole change when they vibrate or rotate. They are IR inactive.
CO2 does have a dipole change though.
Background on vibrations in molecular species
(Normally vibrating in the environment)
Y<---- M ----> X and Y <---- M ----> X
E2 - E1 = ∆E = h
(After absorption)
Y <---- M ----> X
∆E = hc/ = hc
 is usually in µm
 (wave numbers) = 1/ (in cm)  is in cm-1
cm-1 = [1/µm] * 104 or µm = [1/(cm-1)] * 104
We can treat the two portions of the molecule like two masses connected by a spring with a force constant ƒ.
Hook's Law states: F = -ƒx
F is the restoring force, x is ∆r, the distance from eq. position
If we integrate these, we get the Potential Energy:
E = 1/2 ƒx2
The frequency at which a molecule vibrates is
 = (1/2π) (ƒ/µ)1/2
µ = (m1m2)/(m1 + m2) (Reduced Mass)
 = (1/2πc) * (ƒ/µ)1/2
 = 5.3 x 10-12 * (ƒ/µ)1/2
So we can calculate an approximate wavelength for an absorption in IR!
mC =
12 g/mole
= 1.1 x 10-23 g/atom
6.02 x 1023 atom/mole
mO = 2.7 x 10-23 g/atom
µ = (2.0 x 10-23) (2.7 x 10-23) = 1.1 x 10-23
4.7 x 10-23
A double bond has a force constant ƒ ≈ 1 x 106 dynes/cm
 = (5.3 x 10-12) [(1 x 106)/(1.1 x 10-23)]1/2
 = 1.6 x 103 cm-1
Experimentally found in the 1500 ----> 1900 cm-1 region
Single bonds, ƒ ≈ 5 x 105 dynes/cm
Thus, these are very characteristic of the environment of the chemical bonds!
We can use these very characteristic stretching frequencies to deduce structures of molecules.
B) Since different bonds possess different vibration modes, different compounds should not have identical IR spectra.
Thus, each IR active compound has its own unique IR fingerprint spectrum.
 (cm-1)
C) IR spectra are often displayed as % Transmission verses frequency units
(cm-1) rather than verses wavelength units.
D) Two types of IR spectrometers: the dispersive instrument and the nondispersive (Fourier Transform) instrument
i) Common light sources for both types of instruments are :
1) Nernst Glower - rod consisting of fused mixture of Zr, Y, & Th
2) Globar - rod consisting of silicon-carbide
3) nichrome wire coil
ii) Common detectors:
1) Thermocouple - junction potential between two conductors changes with temperature
2) Thermistor, (bolometer) - consists of metal oxide flakes whose resistance changes with temperature
3) Golay cell - thermal expansion of gas changes a flexible mirror (this changes radiant power reference reaching a
4) Pyroelectric crystal - solid state circuit - crystal polarization change
5) Photoconductive - semiconductor, IR radiation changes conductance
E) The dispersive instrument shown in the notes is similar to the instrumental setup for UV-vis spectrometers we have
discussed. Most are double beam where the source beam is split into two identical radiant power beams.
iii) Monochromators (on dispersive Instruments): use filters and diffraction grating
iv) Sample cells for typical applications are: salt plates(NaCl, KBr, etc.) for liquids, salt pellets for solids, and special gas cells
for gases.
F) Fourier Transform (nondispersive) instrument, see the notes, is different from the conventional dispersive instrument in
that it has an interferometer. The radiation exiting the interferometer is a complex mixture of modulation frequencies,
which after passing through the sample, are focused onto the detector. The resulting signal, an interferogram, is either
stored or it is transformed using the Fourier algorithm to produce the spectrum. The Pyroelectric crystal and the
Photoconductive detectors are used since the response time of the thermal detectors is slow.
I m p o rta nt U se s fo r I R S p e c tro sc o p y
i) S t ru c t u ra l in form a t ion a n d c om p ou n d id e n t ific a t ion a re t w o im p ort a n t
u se s for I R sp e c t rosc op y .
ii) I R sp e c t rom e t e rs h a ve b e e n c om b in e d w it h se p a ra t ion t e c h n iq u e s su c h a s
G C (g a s liq u id c h rom a t og ra p h y ), H P L C (H ig h P e rform a n c e c h rom a t og ra p h y )
a n d S F C (S u p e rC rit ic a l F lu id C h rom a t og ra p h y ). I n t h e se in st a n c e s, t h e I R
a c t s a s t h e d e t e c t or t o id e n t ify t h e se p a ra t e d c om p ou n d s. T h e a d va n t a g e s
of a n a ly t e se p a ra t ion a n d t h e ir p e a k id e n t ific a t ion w ill b e ob viou s w h e n w e
d isc u ss t h e se se p a ra t ion m e t h od s. R e m e m b e r, I R c a n n ot a n a ly z e
m ix t u re s, on ly p u re c om p o u n d s.
iii) A s G ove rn m e n t a t m osp h e ric re g u la t ion s p rolife ra t e , I R se e m s t o b e on e
m e t h od su m m on e d t o m e e t t h e g oa ls for t h e ra p id m on it orin g of a t m osp h e ric
c on t a m in a n t s.
iv) A n I R t e c h n iq u e w it h a d iffe re n t a p p roa c h for e x t ra c t in g in form a t ion
a b ou t d iff e re n t t y p e s of solid s (su c h a s p oly m e r film s, p a st e s, p ow d e rs,
p a p e rs, a d h e sive s, t h re a d s, e t c .) is I n t e rn a l- R e fle c t ion I R sp e c t rosc op y .
U se fu l in form a t ion t o a m a n u fa c t u rin g p roc e ss c a n b e a c c om p lish e d u sin g
t h is t e c h n iq u e . I t d e a ls w it h t h e a n g le of re fle c t ion , t h e a n g le of t h e
in t e rfa c e , t h e p e n e t ra t ion of t h e ra d ia t ion b e a m a n d t h e in d e x of re fra c t ion
of t h e m a t e ria l.
v ) F T IR h as b een co m b in ed w ith o p tical m icro sco p y to an aly ze sam p les w ith
sm all size. So m e ap p licatio n s: im p u rities fo u n d in p o ly m er film s, p ap er, &
sem ico n d u cto rs; fo ren sic ex am in atio n o f p ain t ch ip s, d ru g s, ex p lo siv es; an d
stu d y in g b io lo g ical sam p les (p lan t leav es, etc).

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