Topic 6
Spectrochemistry
Spectrochemical Methods:
the most widely used tools for the elucidation
of molecular structure as well as the
quantitative and qualitative determination of
both inorganic and organic compounds.
Properties of light
1) E  hν  hc 1   hc~ν
λ
2) Electromagnetic Spectrum
3) Absorption vs Emission of light
Properties of light
1
~
E  hν  hc   hcν
λ
Electromagnetic spectrum
Absorption vs Emission of light
• Emission
– Chemiluminescence
– Photoluminescence
• Absorption
Photoluminescence
• fluorescence
• phosphorescence
Absorption of light
P
2) . Transmittance T 
P0
Absorbance A  -logT
When no light is absorbed,
P=P0 and A=0
Beer' s Law : A  εbc



 mol

 M

cm 
 cm  mol 
 

For mix:
Atotal = A1 + A2 + …+ An = 1bc1 + 2bc2 + …+ nbcn
Absorbance is proportional to the
concentration
• A = bc
• : molar absorptivity (M-1cm-1)
Absorption of light
(ex) How effective is sunscreen ?
at the peak absorbance near 300 nm ?
A ~ 0.35
T = 10-A
= 10-0.35
= 0.45
= 45%
 55%
UV-B is absorbed.
Why is a red solution red?
Electronic transitions
An electron moves from one orbital to another.
Vibrational and rotational transitions
• The total energy E associated with a molecule
– E = Eelectronic + Evibrational + Erotational
UV-visible spectra
• Rotate & vibrate freely
in gas phase
• Unable to rotate freely
in nonpolar solvent.
• Collisions &
interactions cause the
spectrum as a single
broad peak in H2O.
Limits to Beer’s Law
• Real deviations
– At concentrations exceeding about 0.01 M,
the average distances between ions or
molecules of the absorbing species are
diminished to the point where each particle
affects the charge distribution and thus the
extent of absorption of its neighbors.
• Instrumental deviations
• Chemical deviations
Chemical deviations
Instrumental deviations
• Polychromatic radiation & stray light
• select λmax to reduce
Emission of electromagnetic radiation
Three types of spectra: line, band, and continuum
Three types of spectra
• The line spectrum consists of a series of sharp,
well-defined spectral lines caused by excitation
of individual atoms that are well separated, as in
a gas.
• The band spectrum, marked bands, is
comprised of several groups of lines so closely
spaced that they are not completely resolved.
• The source of the bands is small molecules or
radicals in the source flame.
• Finally, the continuum spectrum is responsible
for the increase in the background that appears
above about 350 nm.
Energy level diagram
homework
• 24.3, 24.13, 24.18, 24.29
Instruments for optical
spectrometry
A Instrument components
A Instrument components
Optical materials
Sample is usually contained in a cell called a cuvet
Spectroscopic sources
• Continuum sources
• Line sources
Wavelength selectors
Monochromator: disperses light into its component wavelengths and
selects a narrow band of wavelengths to pass through the sample.
a. entrance slit
b. collimating
mirror or lens
c. a prism or
grating
d. focal plane
e. exit slit
Wavelength selectors
Monochromator
Choosing the bandwidth:
exit slit width
Resolution
trade-off
Signal
Detector
A detector produces an electric signal when it is struck
by photons, i.e. convert radiant energy (photons)
into an electrical signal.
An ideal detector :
high sensitivity,
high signal/noise,
constant response for λs,
and fast response time.
Spectrophotometer
a) Single-beam
b) Double-beam
Molecular Absorption
Spectrometry
Ultraviolet and visible molecular absorption
spectroscopy
Absorbing Species: Organic molecules
• Wavelength region
between 180 ~ 780 nm
• Electrons in double and
triple bonds of organic
molecules.
• Chromophores:
unsaturated organic
functional groups that
absorb in the ultraviolet
or visible regions.
Saturated organic compounds containing such heteroatoms as
oxygen, nitrogen, sulfur, or halogens have nonbonding
electrons that can be excited by radiation in the 170 nm ~ 250
nm range.
Absorbing Species: Inorganic Species
Qualitative application of UV/Vis
• Spectrophotometric measurements with UV
radiation are useful for detecting chromophoric
groups.
• Ultraviolet spectra do not have sufficient fine
structure to permit an analyte to be identified
unambiguously.
• UV qualitative data must be supplemented with
other physical or chemical evidence such as
infrared, nuclear magnetic resonance, and mass
spectra as well as solubility and melting- and
boiling-point information.
Solvents
• Usually measured using dilute solutions of the
analyte
• Solvent must be transparent in the region of the
spectrum where the solute absorbs (Table 26.3).
• For qualitative analysis, analyte spectra should thus
be compared to spectra of known compounds
taken in the same solvent.
The Effect of Slit Width
• Small slit widths are used
for qualitative studies to
preserve maximum spectral
detail.
• Peak heights and peak
separation are distorted at
wider bandwidths.
Effect of stray radiation
• Stray radiation
occasionally causes false
peaks to appear when a
spectrophotometer is
being operated at its
wavelength extremes.
Quantitative applications
The important characteristics of spectrophotometric and
photometric methods are:
–
–
–
–
–
Wide applicability
High sensitivity
Moderate to high selectivity
Good accuracy
Ease and convenience
Molecular absorption measurements are applicable in
every area
1. Applications to absorbing species
2. Applications to nonabsorbing species : react
with chromophoric reagents to produce
products that absorb strongly in the ultraviolet and
visible regions.
Chelating reagents
Application in biosystem
• (a) Proteins at 280 nm: tyr, phe, trp.
• (b) A colorimetric reagent to detect phosphate
Application in environment
Ex.: Nitrite in an aquarium
(using a standard curve)
Wavelength selection

543 nm
Application in environment
(toxic when > 1 ppm) NH3
animals & plant
[O]
(toxic when > 1 ppm)
[O]
NO3-
Wavelength selection
Standard Nitrite
ACorrected / Aobserved =Vt / Vi
Calibration curve
from least square
A = 0.1769 [ppm] + 0.0015
ACorrected / Aobserved =Vt / Vi
The multiple-additions method
• Assume that several identical aliquots Vx of the unknown
solution with a concen-tration cx are transferred to
volumetric flasks having a volume Vt.
• To each of these flasks is added a variable volume Vs
mL of a standard solution of the analyte having a known
concentration cs.
As 
bVs c s
Vt

bV x c x
Vt
 kVs c s  kV x c x
As  mVs  b
m  kc s
b  kV x c x
Analysis of a mixture
1) Absorbance of a mixture :
The total absorbance of a
solution at any given
wavelength is equal to the sum
of the absorbances of the
individual components in the
solution.
A1 = M1bcM + N1bcN
A2 = M2bcM + N2bcN
Analysis of a mixture
2) Isosbestic points : for rxn: X  Y, every spectrum
recorded during chemical reaction will cross at the same
point. Good evidence for only two principle species in rxn.
Ex: HIn  In- + H+
Analysis of a mixture
Why isosbestic point?
A
465
ε
465
HIn
HIn
 
when HIn   In   ε

A 465  ε 465
In
In 

465
HIn
465
ε
 ε 465

In 
 For a mixture :
A
465
ε
465
HIn
 ε 465
b HIn   ε b In 
b  In  HIn  

465
In 


Ex: The metal ion indicator xylenol orange is yellow at pH 6
(max = 439 nm). The spectral chages that occur as VO2+ is
added to the indicator at pH 6 are shown in the figure. The
mole ratio (VO2+)/(xylenol orange) as each point is shown in
the table. Suggest a sequence of chemical reactions to
explain the spectral changes.
Trace
Mole
ratio
Trace
Mole
ratio
0
0
9
0.90
1
0.10
10
1.0
2
0.20
11
1.1
3
0.30
12
1.3
4
0.40
13
1.5
5
0.50
14
2.0
6
0.60
15
3.1
7
0.07
16
4.1
8
0.80
Photometric & Spectrophotometric
Titrations
Titration Curve is a plot of absorbance (corrected for
volume change) as a function of titrant volume.
s:: substance, : p: product, t: titrate
Continuous variations
M + L solutions with identical
analytical concentrations are
mixed such that the total
volume and the total moles of
reactants in each mixture are
constant but the mole ratio of
reactants varies
systematically.
The corrected absorbance is
plotted against the volume
fraction of one reactant, that
is, VM /(VM + VL)
The mole-ratio method
the analytical concentration of one reactant (usually the metal ion) is held constant
while that of the other is varied.
If the formation constant is reasonably favorable, two straight lines of different
slopes that intersect at a mole ratio that corresponds to the combining ratio in the
complex are obtained.
Spectrophotometric Titrations
Spectrophotometric Titrations
Ferric nitrilotriacetate
[used to avoid Fe(OH)3 ]
Spectrophotometric Titrations
Corrected A for the effect of dilution
Corrected A = (Vt / Vi) (observed A) (Beer’s law)
The absorbance measured after adding 125 L of ferric
nitrilotriacetate to 2.000 mL of apotransferrin was 0.260.
Calculate the coorected absorance.
125 μL ferric nitrilotriancetate
2 mL apotransferrin
A corrected = ? 0.276
A = 0.260
Infrared absorption spectroscopy
• A powerful tool for identifying pure organic and
inorganic compounds because almost all molecular
species absorb infrared radiation.
• However, it is a less satisfactory tool for quantitative
analyses than its ultraviolet and visible counterparts
because of lower sensitivity and frequent deviations
from Beer’s law.
• Additionally, infrared absorbance measurements are
considerably less precise.
• The energy of infrared radiation can excite
vibrational and rotational transitions, but it is
insufficient to excite electronic transitions.
Homework (chapter 26)
• 26.1 (b), 26.16, 26.19, 26.20 (a), 26.21(a),
26.22 (A), 26.23
Molecular Fluorescence
What happens when a molecule absorbs
light ?
1) Absorbing species :
M + hν  M* (lifetime : 10-8 ~ 10-9 sec)
Relaxation processes :
a) M*  M + heat (most common)
b) M*  new species (photochemical reaction)
c) M*  M + h (fluorescence, phosphorescence)
A Theory of molecular fluorescence
1. Molecular fluorescence is measured by exciting the
sample at an absorption wave-length, also called the
excitation wavelength, and measuring the emission at a
longer wavelength called the emission or fluorescence
wavelength.
2. Usually photoluminescence emission is measured at right
angles to the incident beam to avoid measuring the
incident radiation.
3. The short-lived emission that occurs is called
fluorescence, while luminescence that is much longer
lasting is called phosphorescence.
4. Fluorescence emission occurs in 10-5 s or less.
5. In contrast, phosphorescence may last for several minutes
or even hours.
Molecular fluorescence
Molecular fluorescence
•
Two most important processes after excited to E1
or E2:
–
–
•
Nonradiative relaxation,
Fluorescence emission.
Two most important nonradiative relaxation:
–
Vibrational relaxation
•
–
transfer of the excess energy of a vibrationally excited species
to molecules of the solvent. This process takes place in less
than 10-15 s and leaves the molecules in the lowest vibrational
state of an electronic excited state.
Internal conversion
•
transfer of the excess energy of a species in the lowest
vibrational level of an excited electronic state to solvent
molecules and conversion of the excited species to a lower
electronic state.
Electron spin states
What happens to absorbed energy
A molecule absorbs light
Luminescence procedures : emission spectrum of
M* provides information for qualitative or
quantitative analysis.
Photoluminescence :
1. Fluorescence : S1  S0, no change in electron
spin. (< 10-5 s)
2. Phosphorescence : T1  S0,
with a change in electron spin. (10-4~102 s)
Chemiluminescence : (not initiated by light)
release energy in the form of light. ex : firefly.
Relationship between Excitation Spectra
& Fluorescence Spectra
This shift to longer wavelength
is called the Stoke’s shift.
Luminescence instrumentation
hνin
 .hνout (photon)
 heat
 breaking a
chemical bond
Luminescence
2) I = kPoC

incident radiation
sensitivity  by P0  or C 
3) more sensitive than Absorption
Luminescence: application 1
Fluorimetric Assay of Selenium in Brazil Nuts
– Se is a trace element essential to life: destruct
ROOH (free radical)
– Derivatized:
– Self-absorption: quench
why is the figure curved?
why does it reach a maximum?
Luminescence: application 2
Immunoassarys
 employ anitbody
to detect analyte.
Ex: ELISA
(enzyme-linked
immunosorbent
assay)
Luminescence
a. pregnancy test. sensitive to < 1 ng of analyte
b. Enviromental Analysis. (ppm) or (ppt)
pesticides, industrial chemicals, &
microbialtoxins.
homework
• 27-3, 27-4, 27-6
Mass Spectrometry
Principles of mass spectrometry
The ions formed are separated on the basis of their mass-tocharge ratio (m/z) and directed to a transducer that converts
the number of ions (abundance) into an electrical signal.
a mass spectrum
Mass spectrometers
An instrument that produces ions, separates them according to
their m/z values, detects them, and plots the mass spectrum.
magnetic sector analyzer
Quadrupole analyzers
Quadrupole analyzers are mass filters that only
allow ions of a certain m/z to pass.
Atomic mass spectrometry
Interferences effects
Interference effects:
1. spectroscopic interferences: an ionic species
in the plasma has the same m/z value as an
analyte ion.
2. matrix effects become noticeable when the
concentrations of matrix species ex-ceed
about 500 to 1000 µg/mL.
These effects cause a reduction in the analyte
signal, although enhancements are sometimes
observed.
Molecular mass spectrometry
Ethyl benzene (C6H5-CH2-CH3)
Ion Sources
A gas-phase source, the sample is first vaporized
and then ionized.
A desorption source, the sample in a solid or liquid
state is converted directly into gaseous ions.
Desorption sources are applicable to nonvolatile and
thermally unstable samples.
Ionization
•
•
Electron ionization
Chemical ionization
1) Electron ionization
M + e -  M + + e - + e70 eV
Molecular ion
break into
fragments.
Base peak:
most intense
peak.
-55 eV
0.1eV
2) Chemical ionization
CH4 + e-  CH4+ + 2eCH4+ + CH4  CH5+ + CH3
CH5+ + M  CH4 + MH+
CH4+  CH3+ + H
CH3+ + CH4  C2H5+ + H2
• Total ion Chromatograms
21-16a is a reconstructed total ion
chromatogram showing all ions from
seven opium alkaloids found in street
heroin.
• Selected ion Chromatograms:
– Simplify analysis
– improve S/N
Information in a Mass Spectrum
Nominal Mass : C4H9Br is 136
Information in a mass spectrum
Rxn : CH3(CH2)2CH2–OH + Br-  CH3(CH2)2CH2–Br
1–Butanol
1–Bromobutane
Information in a mass spectrum
Fragmentation Patterns
CH3 15
CH2 14
Br
79
C4H979Br+ 50.0%
C4H981Br+
Information in a mass spectrum
Isotope Patterns
CnHxOyNz
12C/13C
Intensity = n x 1.1%
Ex: C6H6
(M+1)/M+ = 6 x 1.1 %
Nitrogen Rule:
A compound: odd nominal mass / odd number of N atoms;
even nominal mass/ even number of N atoms

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