Part 5-Instrumentation: Introduction to Spectroscopy for Chemical Analysis

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Part 5-Instrumentation Introduction to
Spectroscopy for Chemical Analysis

• KR
• LSU

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The Spectrophotometer- Instruments
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IMPORTANT Absorption spectrophotometer
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(a)
(b)
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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.
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FYI .Or diode array spectrophotometer

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Light Sources
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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
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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) v) VIS and UV Atomic absorption
Hollow Cathode Lamps !!! (see later)
Examples
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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 expensive
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
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Monochromators and other devices for separation
of radiation objects
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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 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) Prisms Separation of wavelengths on
some commercial instruments.     Prisms were used
in older instruments, Quartz or salt crystals.
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FYI selection of colors
 
II)       Monochromators separate wavelengths of
light they consist of both entrance exit
slits, mirrors, and diffraction grating or
refraction lens/prisms, and filters.
Gratingmonochromators 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 nl d(sinq sinf)  
n diffraction order (1n) d groove
spacing ? angle of reflection
  ? angle of incidence
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Components of a Grating Spectrophotometer   i
  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.
 
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Wavelength selector (monochromator) passes a
narrow bandwidth of radiation (if more narrow ,
higher resolution!!)
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EXPERIMENTAL PARAMETERS how to get the most
from measurements with absorption spectroscopy
C Choice of the Wavelength and Bandwidth  
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Effects of monochromators slit 0.25nm,1.0nm,
2.0nm, 4.0nm
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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.
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        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 manner.         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
(interferogram). 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
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Detectors
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-detectors

• 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 surface.
• The emitted electrons are attracted to the anode.
  
• The accompanying voltage is fed to an amplifier
  and converted to a signal.

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• 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.

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FYI PHOTO DIODE ARRAY diode
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• 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 simultaneously).
• 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.

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CCD detectors read textbook FTIR detectors
read textbook
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Noise
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Eliminating noise signal averaging
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Stray Light, Electrical Noise, Cell Positioning-
  Stray 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
•  

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Part 5- From VUV to IR-Introduction to
Spectroscopy for Chemical Analysis

• KR
• LSU

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MOLECULAR SPECTROSCOPY IR absorption, UV/VIS
electronic absorption and emission
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Atomic vs. Molecular

• What is same what is different

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IMPORTANT Most atomic spectra are discrete
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IMPORTANT - MOLECULES Molecular spectra are
broader because of the close electronic and
vibrational energies
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One must take into account all molecular energies
from different degrees of freedom
translational (motion), electronic and here
vibrational and rotational motion of nucleiHere
are some of the degrees of freedom of H20
FYI MOLECULES also electronic molecular
orbitals and..
Harmonic oscillator models
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"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.
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Molecular Spectroscopy  
The VIS-visible absorption 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 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.
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IMPORTANT - MOLECULES Absorption of photon
electrons gain energy (ground to excited state)
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IMPORTANT Emission of photon electrons lost
energy (excited to ground state)
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EXAMPLES ABSORPTION IR and UV., atoms and
molecules
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Absorption/excitation Emission/Luminescence
(fluorescence and phosphorescence)
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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 (Luminescence)   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

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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 there.
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 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.
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(a)
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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)
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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).  
lem gt lab
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Luminescence Rrefers to emission of light by
any mechanism from any type of molecule.   LLumine
scence 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 Al3
-Alizarin garnet R complex.
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Principle Components of a Fluorescence
Spectrometer
i)Light sources mercury arc lamp, xenon arc
lamp, lasers 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
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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. Since   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.
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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.
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Example 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 10A 10-0.50 0.32 or
T 32  
Example 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
Example 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?
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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 present.   Step 3. Make of graph of
corrected response vs. quantity of analyte
analyzed.  
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See examples under linear calibration previous
material and Excel




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IR spectroscopy-methods, instruments
(including ATR)-scales, physics-applications
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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)
(After absorption)   Ylt---- M
----gt X and Y lt---- M ----gt X
Y lt---- M ----gt X E1
E2 E2
- E1 ?E h? or ?E hc/l hcs l
is usually in µm   s (wave numbers) 1/l (in
cm) s is in cm-1   cm-1 1/µm 104 or µm
1/(cm-1) 104  
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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 n (1/2p)
(ƒ/µ)1/2 where µ (m1m2)/(m1 m2) (Reduced
Mass)    
s (1/2pc) (ƒ/µ)1/2 s 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
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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   s (5.3
x 10-12) (1 x 106)/(1.1 x 10-23)1/2 s 1.6
x 103 cm-1
1 Newton 105 dyne
Experimentally found in the 1500 ----gt 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.  
NOTE small mass higher energy, vibrational
frequency
NOTE NO dipole, NO IR transitions !!!
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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.  
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C) IR spectra are often displayed as
Transmission verses frequency units
(cm-1) wavenumber rather than (nm) wavelength
units.
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• 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 photocell
• 4) Pyroelectric crystal - solid state circuit -
  crystal polarization change
• Photoconductive - semiconductor, IR radiation
  changes conductance
• 4) deuterated triglycine sulfate (DTGS)

iii) FTIR optics(on dispersive Instruments) use
filters and diffraction grating, BUT ARE NOT USED
MUCH IN AC iv) Sample cells for typical
applications are salt plates(NaCl, KBr, etc. or
CaF2) for liquids, ATR or salt pellets for
solids, and special gas cells for gases.
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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
other thermal detectors is slow.
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ATR -Attenuated total reflection (ATR)
ATR based on total internal reflection that can
form an evanescent wave IR light is passed
through the ATR crystal in such a way that it
reflects at least once off the internal surface
which is in contact with the sample. This
reflection forms the evanescent wave which
extends into the sample. The penetration depth
into the sample is typically between 0.5 and 2
(angle, wavelength dependent and n-s).
Reflected IR
Incident IR
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Both qualitative and quantitative analysis with
FTIR.
Some mixtures can be analyzed, more complex
process.
CO measurements, see textbook.
ATR
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Additional Scattering-non-resonant
scattering-active transitions (virtual vs.
IR)-microscopies