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Instrumental Test 2b
Terms in this set (67)
A molecule is made up of what types of energy
electronic, vibrational, rotational, translational
molecular spectroscopic methods include:
UV-Visible , Vibrational (Raman and Infrared) , Nuclear Magnetic Resonance
what is the wavelength range and types of transition for UV-Visible spectroscopy
Wavelength: 180-800 nm
Types of transition: Bonding and valence
what is the wavelength range and types of transition for Vibrational (Raman and Infrared) spectroscopy
Wavelength Range: 0.8-300 μm
Types of transition: Molecular rotation and
what is the wavelength range and types of transition for Nuclear Magnetic Resonance spectroscopy
Wavelength Range: 0.5-10 m
Spin of nuclei in a magnetic field
what are the most important transitions and why
and π→ π
They are the more probable transitions.
They have large ε
They involve functional groups that can be
used for analyte identification, and occur in
easily accessible wavelength region.
a molecule must contain π bonds or atoms with a
lone pair of electrons for absorption where
in the region 200- 800 nm
types of UV-Visible measurements
chromphore, bathochromic shift or red shift, and Hypsochromic shift or blue shift, auxochrome
A functional group or part of a molecule
producing typical absorption of ultraviolet or
visible radiation of a particular wavelength of
Bathochromic shift or red shift
A shift to lower energy or longer wavelength.
Hypsochromic shift or blue shift.
A shift to higher energy or shorter
nature of absorption spectra change in bathochromic (red shift)
shift to longer wavelength
nature of absorption spectra change in hypsochromic(blue shift)
shift to shorter wavelength
nature of absorption spectra change in hyperchromism
increase in molar absorptivity
nature of absorption spectra change in hypochromism
decrease in molar absorptivity
A substituent in a molecule that can increase
the intensity of absorption, and possibly shift
the wavelength of absorption when attached
to a chromophore. It contains unshared
(nonbonding) electron pairs.
examples of auxochrome
• Hydroxyl groups
• Amino groups.
auxochrome interacts with
the π electrons in
chromophore via n→π conjugation.
solvents for UV - Visible measurements include:
water, ethanol, hexane, cyclohexane, carbon tetrachloride, diethyl ether, acetone, dioxane, cellosolve
The solvent used must be
transparent in the
region being analyzed. Careful selection of
solvent is very crucial.
if the polarity of solvent is increased:
More polar n electrons decrease in energy
n→ π* transition are shifted to shorter
wavelength (blue or hypsochromic shift)
Changing the pH of an aqueous solvent may
cause change in
the nature of the acidic or basic species.
In acidic solution phenol exist as protonated
species (ROH), which absorbs
at 270 nm
in alkaline solution phenol exist as deprotonated species (RO-
), which absorbs at
Absorption of Ultraviolet or Visible Radiation - Inorganic Compounds
Lanthanide and actinide ions, metals, Metal chelates , Coordination of transition metals
Lanthanide and actinide ions
o Undergo absorption due to f-f transitions.
o Absorption bands are narrow and relatively
unaffected by the nature of the solvent or the
species bonded by the outer electrons.
form highly colored complexes with
many organic ligands called chelates
o Produce bands often extremely intense with
very large ε (> 10, 000) due to charge
Ligand → metal charge transfer.
Metal→ ligand charge transfer.
example of metal chelates
Thiocyanate complex of iron(III)
Phenolic complex of iron(III)
1,10-phenanthroline complex of iron (II), etc.
Coordination of transition metals
Cause splitting of the d-orbital energies.
The magnitude of the splitting depends on
the ligand field strength of the complexing
The Lambert-Beer Law
Absorbance of a band in a UV-Visible spectrum is directly proportional to the sample path length and concentration
The Lambert-Beer Law Quantitatively
A = log10 x Io/I = abc = ecl
The Lambert-Beer Law: A =
The Lambert-Beer Law: Io =
radiation incident on the sample
The Lambert-Beer Law: I =
radiation transmitted from the sample
The Lambert-Beer Law: l =
The Lambert-Beer Law:l =
path length (cm)
The Lambert-Beer Law: a
The Lambert-Beer Law: e =
molar absorptivity (M-1 cm-1)
A system that conforms t Lambert Beer's yields
a linear plot of absorbance against concentration and the slope is equal to ab or e
Deviation from the Lambert-Beer Law Concentration Effect:
High concentration effect can be due to
Self-association (dimer as well as monomer
formation e.g. methylene blue)
Dilution of a solution during measurement must
not lead to
chemical reactions such as hydrolysis,
association, or polymerization.
pH effect is caused by
Protonation/deprotonation of species.
Non-symmetrical chemical equilibrium.
Lambert-Beer Law Solvent Effect:
Solvent and solute interaction.
Lambert-Beer Law Instrumentation Effect:
Instrumentation effect can be caused by
Incident light (Photochemical reaction).
Use of polychromatic radiation instead of
Instrumentation effect (IE) can also be due to
Stray light reaching the detector.
Power fluctuations of light source and
detector amplification system.
Types of UV/Vis Instruments
Filter photometer, Spectrophotometer, Monochromators
Simplest instrument for measuring UV/Vis
Uses an absorption or interference filter to
select particular wavelengths for analysis.
Filters provide low-resolution wavelength
selection for quantitative analysis. They can
not be varied continuously during
Uses a monochromator to provide narrow
band of radiation for UV/Visible absorption
Monochromators provide high resolution
wavelength selection for qualitative and
Can be used to obtain entire spectra.
Designs of the spectrophotometer
a. Single-beam ( e.g. Spectronic 20 or 21)
b. Dual or double beam (e.g. Lambda 25 or
Devices that use diffraction grating or prisms
as dispersing elements to isolate a narrow band
Can be classified as fixed-wavelength or
components of monochromator
o Entrance slit
o Collimating lens or mirror
o Dispersion grating or prism
o Focusing mirror
o Exit slit
Ideal transducer should possess
o High sensitivity.
o High signal-to-noise ratio.
o Constant response over a wide range of
o Electrical signal produced be directly
proportional to the beam power, P.
o It is a device that consists of a photo emissive
cathode and an anode. The cathode emits electrons
when struck by visible light or UV radiation.
o It has a wavelength dependent response.
o It is commonly used in the UV and visible region.
o It does not respond to all photons equally and
produce a small amount of current in the absence of
radiation from the source.
Photomultiplier tube (PMT)
o PMT is more sensitive than a phototube for
detecting UV and visible radiation.
o It consists of a photo emissive cathode and series of
electrodes called dynodes. The dynodes are
prepared from materials that hold their electrons
o Each photon reaching the tube undergoes up to 10
stages of amplification and thus the anode receives ~
107 electrons for each photon.
Silicon photodiode and photodiode arrays
o It uses semiconductor pn-junction diodes as
the photosensitive surface. When photon
strikes the semiconductor, electron-hole
pairs are formed, producing a measurable
o It can detect radiation (several wavelength)
simultaneously from UV to near-IR.
Short-lived emission (10-5 or less) of photon
during a transition between states with the
same spin quantum number (S1→ S0).
Long lasting (10-4
to 10 s or more) emission
of photon during a transition between states
with different spin quantum number (T1→S0).
Instrumentation for fluorescence measurements
A radiation or light source to the sample, A primary filter or excitation monochromator, A sample cell, A secondary filter or emission monochromator, A detector
the radiation or light source used in the fluorescence measurements is
Mercury or xenon arc lamps are used. Laser
light sources are ideal due to their high
the sample cell used in fluorescence measurements
Quartz or glass cuvet with four optical
A secondary filter or emission monochromator used in fluorescence instrumentation
filter or monochromator is placed at 90°
to the incident beam to eliminate background
light from the light source.
the detector used in fluorescence instrumentation
PMT (Most common)
Diode array (in order to collect entire the entire
spectrum at once)
CCD (another alternative to PMT)
molecular fluorescence measurements: Excitation spectrum
o Graph of emission intensity as a function of
o Vary the excitation wavelength ( λex) and
measure the emitted light at fixed emission
wavelength (λem ).
o Looks like absorption spectrum.
molecular fluorescence measurements: Emission spectrum
o Graph of emission intensity as a function of
o Vary the emission wavelength ( λem) and
measure the emitted light at fixed excitation
wavelength (λex ).
concentration and fluorescence intensity relationship F = K (Po- P )
P = the power of the incident radiation
P = the power after it passes through a length b of the medium
K = a constant (depends on the quantum efficiency of the fluorescence process).
concentration and fluorescence intensity relationship From Beer's law
P/Po = 10^-ebc
If the absorbance of a solution is not toohigh ( 0.05),
then thefluoresence power (often called intensity)
is given by
A plot of F as a function of c is linear at low concentration
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