Introduction to spectroscopy

• Spectroscopy is the science dealing with interactions

of electromagnetic radiation with matter.

Electromagnetic Matter
Radiation (light) (sample)

What is light?
1
 Properties of Light
 Light behaves as a wave.

EMR: does not require

supporting media for its
motion

 EMR: electric and magnetic

field that undergo in-phase,
sinusoidal oscillations at right
angles to each other

2
Wave properties of light

 Frequency (): the

number of oscillations
of the field that occur
per second.

 Wavelength (: the

distance between any
two adjacent identical
points of the wave.

 c

3
Electromagnetic radiation (EMR)

4
 Electromagnetic spectrum

Example: What is the frequency of green light of wavelength 5200 Å?

5
Wave nature of light (Refraction)
 Light bends when it passes
through a transparent substance
 The bending occurs due to light = vacuum
having different velocities in the
two media

 Refractive index (n): how

much the light bends
Snell’s law
sin1/sin2 = n2/n1 = v2/v1

Refractive index is measured

with a refractometer.

6
 Wave nature of Light
(Diffraction)

 All EMR spreads as it

passes by a sharp
barrier or through a
narrow opening. This
is called diffraction.
n= BC sin 

7
Wave nature of light
(Interference)
Destructive Interference
Waves may interact with each
other:
 Two waves of the same Wave 1
Wave 2

wavelength that are exactly Result

out of phase will add

together destructively,
destructive interference.
 Two waves of the same
wavelength that are exactly wave 1

in phase will add together

wave 2
result

constructively, constructive
interference.

8
Particle behavior of light
 Light behaves as a stream of
discrete particles called photons.
Each photon has an energy:
hc
E  hv 

 The Photoelectric effect proved
the particle-like behavior of light.

 EMR is a form of energy that

removes electrons from metallic
surfaces

Example: How much energy (in kJ) is carried by a mole of yellow

photons of wavelength 527 nm? (h = 6.63 x 10-34 J.s)
9
Absorption methods
 Consider a beam of power Po traveling a b distance through an
absorbing solution of concentration c.

 Measured after light beam pass through

 Covette + solvent (solvent blank)
 Cuvette + solvent + sample in solution
10
Absorption methods

 Transmittance (T) – the fraction of incident radiation

transmitted through the medium. The ratio of the
output power to the input power.
T = P/Po

 Absorbance (A) – Defined as the –log of the

transmittance.
A = -log T = log Po/P

11
Example

• What value of absorbance corresponds to

100% transmittance?

• What value of absorbance corresponds to 1%

transmittance?

• What value of percent transmittance

corresponds to absorbance of 0.232?

12
Beer-Lambert Law

 Absorbance: depends on three factors

 molar absorptivity coefficient ()
 Path length of the cell (b)
 the concentration c of the analyte (c)

A = bc
 The molar absorptivity is property of a sample that takes in
to account:
 The absorption efficiency of the sample.
 Scattering losses as the light passes through the sample.
 Reflection losses as the light strikes the cell interfaces.

13
 Example 1 0.21
 The following data was 2 0.39
obtained from an optical 5 1.01
absorption instrument. 10 2.02
The cell path length was 1
cm. Find the molar
absorptivity coefficient and
2.0
determine the
concentration of an 1.6

unknown solution that has

Absorbance
1.2

an absorbance of 1.52. 0.8

A = 0.202c - 0.002

R2 = 0.999
0.4

0.0
0 2 4 6 8 10
Conce/ mM

14
Limitations to Beer’s Law

 The law is always true

 But it does not obey either

at high concentrations due
to chemical effect such as
association or dissociation of
analyte or reaction with
solvent or instrumental
effect

15
Possible physical process after absorption

16
Fluorescence and phosphorescence

Fluorescence phosphorescence
17
2. Instrumentation

 Optical spectroscopy is based on

 Absorption
 Fluorescence
 Phosphorescence
 Emission
 Chemiluminescence

 The instrument for each method is different, but

basic components are the same.

1
Components of optical Instruments

 Stable source of radiant energy.

 Device that isolates wavelengths of light for
measurement.
 A transparent container for holding the sample.
 A radiation detector, which converts radiant energy to
electrical signal.
 A signal processor and readout.

Light Wavelength Sample Radiation Signal

Source selector Holder detector processor

NOTE: The wavelength selector can also come after the sample
holder. 2
Absorption measurement

3
Fluorescence measurement

Two wavelength
selectors are needed
in order to isolate the
excitation and
emission wavelengths

4
 Emission measurement

A source of thermal
energy, such as a flame
or plasma, produces an
analyte vapor that emit
radiation isolated by a
wavelength selector

5
Light sources: produce beam with sufficient power
 Continuum sources – emit radiation that changes
intensity only slowly as a function of 

 H2 or D2 lamps give light in the UV region.

 Tungsten filament lamp is very commonly used for visible

light.

 Inert solids heated to 1500-2000K make common IR

sources.

6
 Light sources: produce beam with sufficient power

 Line Sources – emit limited number of lines or band of radiation

that is used in AAS, atomic and molecular fluorescence
 Mercury and sodium vapor lamps provide a few sharp lines in
the UV and Vis regions.
 Hollow cathode lamps

 Laser Sources (Light Amplification by Stimulate Emission of

Radiation)
– Lasers: have high intensities, narrow bandwidths, and
coherent output.

7
 Wavelength Selectors
 Radiation that consists of
narrow, continuous group of
wavelengths is required for
most spectroscopic analyses.

 Narrow bandwidth enhances

the sensitivity of absorbance
measurement.

 Having a narrow band ensures

a linear relationship between
optical signal and
concentration.

8
 Monochromators
 Spectroscopic methods: to vary of
radiation continuously (scanning).

 Entrance slit: provides a optical

image.
 A collimating lens or mirror:
produces a parallel beam of
radiation.
 A prism or grating: disperses the
radiation into its component
wavelengths.
 Focusing element: reforms the
image of the entrance slit.
 An exit slit: isolates the desired
spectral band.

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Monochromator slits

 The entrance slit serves as a radiation source.

 The exit slit serves the purpose of selecting the wavelength.

 Moving the exit slit, defines the angle (and consequently
the wavelength) of the reflected (or bent) light.
• Most monochromators have variable slits.
– Need a minimal slit width to resolve narrow absorption or
emission bands.
– Wider slits are needed to let more light in and produce
enough radiative power to be measurable.

10
Transparent sample containers

 Sample containers need to be transparent in the

spectral region of interest.

 Quartz or fused silica is used in the UV region.

 Silicate glass is used between 350 and 2000 nm.

 Crystalline salts (NaCl, KBr) are used for cell windows

in the IR region.
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Transducers (detectors)

 Earliest detector: eye, photographic plate or

films

 Convert radiation energy into an electrical

signal

 An ideal transducer should have

– High sensitivity
– High S/N
– Fast response
12
 Radiation Transducers
• Photomultiplier tubes: are very
sensitive to low radiant power.
 Light strikes a photoemission
cathode.
 The ejected electrons strike a
dynode causing the emission
of several additional
electrons.
 The process is repeated many
times to get a large number of
ejected electrons per photon
of light.
13
Thermal transducers
 Radiation transducers: don’t work well in the IR
because the IR photons lack the energy to cause
photoemission of electrons.

 Thermal transducers: work by absorbing the radiation

to give a temperature increase which is measured.
 Thermistors
 Constructed of a material that exhibits a large change in
resistance as a function of temperature.
 Pyroelectric transducers
 A pyroelectric crystal generates an electrical charge when
heated. 14
Signal processors

 The signal processor is an electronic device that

amplifies the electrical signal from the transducer.

 It may alter the signal from dc to ac or the reverse.

 May perform mathematical operations on signals

such as differentiation, integration, or conversion to
logarithms.

15
 Types of optical Instruments
 Colorimeter
 An instrument for absorption measurements in which the
human eye serves as the detector using one or more color-
comparison standards.
 Spectrometer
 An instrument that provides information about the intensity
of radiation as a function of wavelength or frequency.
 Spectrophotometer
 A spectrometer that is equipped with one or more exit slits
and photoelectric transducers that permit the
determination of the ratio of the power of two beams as a
function of wavelength.
 A spectrophotometer for fluorescence analysis is called a
spectrofluorometer. 16
 Single-beam versus double-beam spectrophotometer
 Single-beam spectrophotometer
 Equipped with a fixed-
wavelength monochromator
 The wavelength is adjusted
manually

 Limitation:
 Not handy and time-consuming
 Accuracy depends on the
stability of light source

17
Double-beam spectrophotometer
 A chopper controls the
radiation’s path, alternating
it between the sample, the
blank and a shutter.
 The signal processor uses
the chopper’s known speed
of rotation to resolve the
signal reaching the
detector into transmission
of the blank, P0, and the
sample, PT
 An opaque surface as a
shutter: continuously
adjust 0% T.
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3. Atomic absorption spectroscopy (AAS)
 To carry out AAS:  Removing the solvent
 The analyte must be converted  Volatilize the analytes
to free atoms  If necessary, dissociate the analyte
into free atoms

 Atomization: the process of Example

converting an analyte to free CuCl2(aq)  CuCl2 (s)  Cu (g) + 2Cl (g)
gaseous atoms

1
Two atomization methods: Flame and electrothermal atomization

1. Flame Atomization
 Sample nebulized

 Mixed with fuel and carried to

flame for atomization

 Burner slot (a): a longer

optical path-length and a
stable flame
 Longer-path-length:
enhance sensitivity
 Stable flame: minimize
uncertainty avoiding
fluctuations of flame

2
Burner slot: adjustable

 Horizontally: to align with the instrument’s optical

path
 Vertically: adjust the height on the flame
 The more time the analyte in the flame the
greater the atomization efficiency

 Competing factor: oxide formation with longer

residence time

 Ag (difficult to oxide): increase free atom

formation with height
 Cr (easily oxidize metal): greater
concentration just above the burner head

3
Flame

 The flame’s temperature: affecting

the efficiency of atomization,
depends on the fuel-oxidant mixture
 Primary combustion zone: rich in
gas combustion products that emit
radiation (not useful for AAS)

 The interzonal region: rich in free

atoms (best for AAS)

 Secondary combustion zone: less

temperature (formation of stable
molecular species)

4
Flame atomization

 Advantage  Disadvantage

 Reproducibility due to  Poor atomization efficiency due to

Production of large size aerosol

 effective sample
droplets (95% of the sample never
introduction
reaches the flame)

 Stable flame formation Large volume of combustion gases

significantly dilutes the sample

5
 2. Electrothermal atomization (graphite furnace)

 Atomization of entire sample in short

period

 Average sample time in optical path is in

seconds
 Evaporation of sample
 Sample ashed at higher temperature
(350-1200oC)
 The sample is atomized rapidly To remove gaseous product
(2000-3000oC)

 High sensitivity for small samples (1000 X

greater sensitive than flame atomization)
6
Atomic Absorption Instrumentation

 Light source
 Use source for element detection
 For Na, use Na vapor lamp
• 3p to 2s transition at 589.6
nm

 Minimize line broadening

 Need a separate light source for

each element

7
Hollow Cathode Lamp
 Ionization of inert gas by potential

 Gas acceleration to cathode

 Atoms on cathode into gas state

 Some are in excited state and

emit radiation characteristic of the
metal (s) from which the cathode
was manufactured

 Example
This cathode lamp is made from an alloy
containing Co, Cr, Cu, Fe, Mn, and Ni
8
Hollow cathode lamp
 Each element lamp: provides
several atomic emission lines for
AAS
 The one with higher sensitivity will
be selected
 For example, Cr hollow cathode
lamp: best sensitivity obtained for
wavelength of 357.9 nm

9
Preparation of sample

 Digestion for solid sample: dissolution in mineral acid like HNO3, H2SO4 or
HClO4 using on either a hot-plate or by microwave

 Liquid samples:
 Analysis directly or extracted if the matrix is incompatible with atomization.
For example: Serum sample results difficulty to aspirate in flame atomization
while unacceptable high background absorbance in electrothermal atomization

 Liquid-liquid extraction using an organic solvent and chelating agent is used

for preconcentration
For example: dilute solutions of Cd2+, Co2+, Cu2+, Fe3+, Pb2+, Ni2+, and Zn2+
can be preconcentrated using extraction with pyrrolidine dithiocarbamate in
methyl isobutyl ketone
10
 1. Spectral Interference

 Overlap of analyte signal with other  Prevention

species in sample (or the flame)  Find wavelengths with no
interference (pre-selected in
 Not very common due to narrow line Atomic Absorption software)
widths
 Measure sample at multiple
 If occurs, select different transition wavelengths-if any look strange
throw them out.
 Decrease of intensity of transmitted
radiation due to scattering and
combustion molecular species
absorption.

11
2. Chemical Interference

 Interaction of sample with  Prevention

analyte that prevent
atomization Additives to remove such compounds
 Mask Ca2+ using EDTA
 Formation of compounds
with low volatility  Add something to react with SO42-
andPO43- (like La3+)
 Example:- presence of
SO42- andPO43- hinders  Run to high temperature
atomization Ca2+ analysis

12
3. Ionization Interference

The substance to be analyzed is ionized, this hinders the analysis of atomic

absorption spectroscopy

For example
 The analysis of alkali metal such as sodium, potassium and lithium

 These metals easily ionize because of their small ionic potential-the

energy required to ionize these metals is small.
M  M+ + e-

 To prevent the formation metal ion, cesium metal is added so that it can
easily ionize and results in the formation more electrons. Then the
equilibrium in the above equation favors the reverse reaction

13
Detection limits for atomic emissions

Except for alkali metals, the detection

limit for ICP is much higher than that of
flame emission.

14
Problem
Copper and zinc are isolated from tissue samples by digesting the sample with HNO3 after
first removing any fatty tissue. The concentration of copper and zinc in the supernatant are
determined by absorption using an air-acetylene flame. Copper is analyzed at a wavelength
of 324.8 nm with slit width of 0.5 nm, and zinc is analyzed at 213.9 nm with a slit width of 1.0
nm. Background correction using a D2 lamp is necessary for zinc.
1. Describe the appropriate matrix for the external standards and for the blank?
2. Why is a background correction necessary for the analysis of Zn, but not for the analysis of
Cu?
3. A Cu hollow cathode lamp has several emission lines. Explain why this method uses the line
at 324.8 nm.

15
For the above problem
 A series of standard solutions is prepared
and analyzed, is given in the table.
 After drying and extracting the sample, a
11.23 mg fat free tissue sample gives
absorbance of 0.023. Determine the
amount of copper in the sample in g
Cu/g fat free sample.
(A = 0.0661c(ppm) – 0.0002)

16
4. Atomic Emission Spectroscopy

 Flame test of some alkali metals

 Atomic emission: Occurring when an

excited species rapidly relax back to
lower states, UV-Vis line spectra
arise
 The intensity of an AE line I:
proportional the number of atoms N*,
populating the excited state atoms
I = kN*
Where k is a constant accounting for
efficiency of the transition
1
 Instrumentation

 Similar to AAS, but turning off

the hollow cathode lamp in
the AES

 Plasma: commonly used

atomization and excitation
sources
Plasma Emission Spectroscopy

 Plasma sources have become the most important and most widely
used sources for AES
Advantages
 Lower susceptibility to chemical interferences which is a result of their
higher temperatures
 Good emission spectra results for most elements under a single set of
excitation conditions
 Hence, spectra for dozens of elements can be recorded
simultaneously
 This property is of particular importance for the multi-element analysis
of very small samples
3
Inductive Coupled plasma (ICP)-AES

 A plasma is an electrically conducting gaseous

mixture of cations and electrons

 Argon ions and electrons are the principal

conducting species

 The resistance of the ions and electrons to the

flow charge causes ohmic heating of the plasma

 Such plasmas achieve temperature as high as

10 000 K

4
A typical ICP source
Analyte Atomization and Ionization

 Samples experience temperatures

ranging from 5500 to 8000 K

 Atomization is more complete in

plasmas than in flames and fewer
chemical interference occur

 Advantage of atomization

 Prevent oxide formation

 Avoid self-absorption and self-

reversal effects
5
Sample Supply

 Samples can be introduced into the

ICP by argon flowing at about 1 L/min
through the central quartz tube

 Another method of introducing liquid

and solid samples into a plasma is by
electrothermal vaporization

Device for electrothermal vaporization

6
Multi-Elemental Analysis

 Suitable for multi-elemental

analysis because all analytes
in a sample are excited
simultaneously

 Use scanning monochromator:

program it move rapidly to
analyte desired wavelength

 Sequential analysis with

sampling rate of 3-4 analytes
per minute 7
Application for Metal Analysis

 Used for the analysis of

trace metals in a variety
sample matrices

 Useful for the determination

of over 60 elemental
analysis in the Periodic
Table

8
Detection limits for atomic emissions

Except for alkali metals, the detection

limit for ICP is much higher than that of
flame emission.

9
Problem
A sample is prepared by placing a 10-g portion of the salt substitute in 10 mL of 3 M
HCl and 100 mL of distilled water. After the sample has dissolved, it is transferred to
a 250 mL volumetric flask and diluted to volume with distilled water. A series of
standard additions is prepared by placing 35 mL portions of the diluted sample into
50 mL volumetric flasks, spiking each with a known amount of a 10 mg/L standard
solution of Na+, and diluting to volume. A series of standard additions is prepared
using a 10.0 g sample of a salt substitute as shown below. What is the concentration
of Sodium, in g/g, in the salt substitute?
Added Na (g/mL) Ie
0.000 1.79
0.420 2.63
1.051 3.54
2.102 4.94
3.153 6.18
5. UV-Vis Spectroscopy

 Utilizes the ultraviolet (UV) and visible (VIS)

range of electromagnetic radiation
 Causes electronic transition to the analyte (known
as electronic spectroscopy

Visible: 380 – 780 nm

Ultraviolet: 10 - 380 nm

 Below about 200 nm, air absorbs the UV light and

instruments must be operated under a vacuum

1
UV-Vis Chromophore

Chromophore: part of molecule responsible for the color by absorbing a wavelength

in the UV-Vis region

(anti-bonding)
Looks at double bonds of a molecule
(anti-bonding)
Four types of transitions
*
n (non-bonding)
*
n*
n* (bonding)

(bonding)
2
Electronic Transition

  *
 Require the presence of an
unsaturated functional group and
free nonbonding electrons that
can absorb radiation of relatively
low energy (chromophore) to
provide the p orbitals.

 Example:
 alkene, alkyene,

 ketone, aldehydes, phenyl

3
 Conjugation

 Conjugation within a chromophore

causes the electrons to be further
delocalized, lowering the energy of
the level, and causing absorption at
longer wavelengths (red shift).

 Bathochromic shift =
longer wavelength, lower energy,
red shift.

Absorbance
 Hypsochromic shift = shorter
wavelength, higher energy, blue shift.

Wavelength, nm 4
Electron Transition

 Nonbonding or unshared outer electrons that are localized

about the atom.

 These unshared electrons

are designated by the
symbol n.

 Example: Carbonyl
functional group

5
Electron Transition

n  * transitions
 Still rather high in energy. max max
H2O 167 1480

  between 150 and 250 nm. CH3OH 184 150

CH3Cl 173 200
CH3I 258 365
 Not many molecules with
n* transitions in UV/vis
region

6
Absorption by Aromatic Systems

   * transition for aromatic hydrocarbons

 Strong absorption at 184 nm (max = 60,000)

 Weaker band at 204 nm (max = 7900)

 Still weaker band at 256 nm (max = 200)

B band 255–275 nm weak symmetry-restricted    

 Aromatics contain a series of sharp peaks due

to the superposition of vibrational transitions on
the basic electronic transitions.

7
 Most Transition-metal Absorption

The Selection Rule:

For centrosymmetric molecules (those
with an inversion center), transitions
between orbitals of the same
symmetry (parity) are forbidden.
o Allowed: g → u or u → g
transitions (e.g., π → π* in
alkenes).
o Forbidden: g → g or u → u
transitions (e.g., d-d transitions
in octahedral metal complexes).
This rule can be relaxed due to
molecular vibrations.

8
 D-d Transitions: Cu(H2O)62+

 Yellow light is absorbed and

the cu2+ solution is colored in
blue

 The greater , the greater the

E needed to promote the e-,
and the shorter 

  depends on the nature of

ligand, NH3 >  H2O

9
Transitions Metal Solution are colored? Why?

d-d transitions: max = 1 – 100 L mol-1 cm-1, weak. Why?

10
Inorganic –Charge Transfer

 Inorganic complexes – metal ions

with surrounding ligands

 They undergo absorption processes

where the electron jumps mostly
from an orbital the ligand to an
orbital the metal ion

Wavelength, nm
11
UV-Vis Spectrophotometer

In a double-beam spectrophotometer,
light coming from the light source
splits into two beams: one passes
through the sample, and the other
only passes through the reference.

12
Light Source

A combination of tungsten/halogen and

deuterium(D2) lamps

 D2 gas is discharged by contact with a high

voltage tungsten cathode, which is a
continuous spectrum from ~150 nm - ~370 nm

 Usually used in conjunction with a

tungsten/halogen source, which handles the
visible spectrum

D2 Lamp
13
 Sample Holder

 Must use quartz cuvettes in the UV region.

 These are also transparent in the visible.
 They are expensive

 Silicate glass cuvettes can be used in the Quartz

visible only.
 Much cheaper.

 Plastic (disposable) cuvettes can also be used

in the visible region.

Glass
14
Quantitative Application

 Beer’s Law:  For quantitative measurement

A = bc  Calibration curve technique
 If there is a strong matrix
 The total absorbance of a solution: equal interference, standard addition
to the sum of the absorbance of the method is used,
individual components.

A1csVs
A = x b[X] + y b[Y] + z b[Z]+… cx 
( A2  A1 )Vx

15
Problem 1
The concentrations of Fe3+ and Cu2+ in a mixture can be determined following their
reaction with hexacyanoruthenate (II), Ru(CN)64–, which forms a purple-blue complex
with Fe3+ (max = 550 nm) and a pale-green complex with Cu2+ (max = 396 nm). The
molar absorptivities (M–1 cm–1) for the metal complexes at the two wavelengths are
summarized in the following table.
550 396
Fe3+ 9970 84
Cu2+ 34 856
When a sample containing Fe3+ and Cu2+ is analyzed in a cell with a pathlength of
1.00 cm, the absorbance at 550 nm is 0.183 and the absorbance at 396 nm is 0.109.
What are the molar concentrations of Fe3+ and Cu2+ in the sample?

16
Problem 2

A 25.0 mL aliquot of an aqueous quinine solution was diluted to 50 mL

and found to have an absorbance of 0.656 at 348 nm when measured in
a 2.50 cm cell. A second 25.0 mL aliquot was mixed with 10.0 mL of a
solution containing 25.7 ppm of quinine after dilution to 50.0 mL, this
solution had an absorbance of 0.976 (2.50 cm cell). Calculate the
concentration of quinine in parts per million in the sample.

17
6. Photoluminescence

 Light emission from a substance after

photon absorption.

M + hv  M*

 Fluorescence: the electronic energy

transition responsible for fluorescence
does not involve a change in electronic
spin.
M*  M + hv

 Phosphorescence: involving a change

in electronic spin
Electronic Transition
 Fluorescence is short lived (10-5 -10-8s) and phosphorescence is long lived
(several seconds).

When a solution is exposed to

UV radiation, many substances
will be re-emitted with colored
 All paired  singlet state. visible light
 Two unpaired electrons  triplet state.
 Jablonski Energy diagram

Molecular fluorescence bands are

centered at  that are longer than the
absorption band peak. This shift towards
longer  is known as Stokes shift

Photoluminescence emission is longer wavelength than absorption

 Quantum Yield ()
 Quantum yield or quantum efficiency: the
ratio of photons (quanta) emitted to
photons (quanta) absorbed by the system
number of excited molecules.

𝑃ℎ𝑜𝑡𝑜𝑛𝑠 𝑒𝑚𝑖𝑡𝑡𝑒𝑑
∅=
𝑃ℎ𝑜𝑡𝑜𝑛𝑠 𝑎𝑏𝑜𝑟𝑏𝑒𝑑
 A larger  of a species: indicating a
greater intensity of fluorescence emission

High quantum yield: 0.90.

90% of absorbed incident photons show

up as fluorescent photons.
Fluorophores
 Fluorescence generally occurs due to
either *   or *  n transition.
 Fluorescence most often is *  
since these transitions have larger
transition probability and low energy.

Chlorophyll a
Fluorescence Instrumentation

Schematic diagram for measuring

fluorescence showing the placement of the
wavelength selectors doe excitation and
emission

 When a filter is used the instrument is a

fluorimeter

 When a monochromator is used the

instrument is a spectrofluorometer
Factors

Enhancing Fluorescence Detrimental to Fluorescence

 Presence of heavy atoms in fluorophore or
 High probability   * in solvent (spin-orbit coupling enhances
absorption transitions intersystem crossing)

 Presence of charged groups, mediated by

 Low intersystem crossing pH change

 Aromaticity and/or rigidity (low  Presence of molecular oxygen or other

paramagnetic species (enhanced
internal conversion)
intersystem crossing)
Molecular Structural Features

 The most intense and useful fluorescence is found in compounds containing

aromatic rings with low * →  transition levels.

 Groups with non-bonding electrons enhance fluorescence

-NH2, -OH, -OCH3, -NHR and –NR2

 Groups with electron withdrawing groups quench fluorescence

-COOH, -NO2, -NH-CO-CH3, -Cl, -Br, -I
 Molecular Structure Features

These fused ring structure fluoresce. Simple Hetrocycles, they don’t fluoresce.
Structural Rigidity
 Molecules with rigid structures tend to have greater fluorescence yields.
 Fluorene exhibits 5 times higher quantum yield than that of biphenyl

CH2
Biphenyl Fluorene
 A number of organic chelating agents do not fluoresce by themselves, but
when they are bound to a metal atom, the fluorescence is markedly increased.
Fluorescence and Concentration
 Fluorescent power is proportional to the power of the incident excitation
source.
F  K' P0  P
 The absorption of the incident power is modeled as Beer’s Law.
P  bc bc
 10 P  P0 10
P0
 Substitution of this term in the above equation gives:

F = K’Po(1 – 10-bc)

 A Maclaurinexpansion of the exponential, and dropping higher order terms,

demonstrate the linearity with concentration
F  2.3 K' P0  bc  K c
 Deviation from Linearity in Fluorescence

Cause: Inner Filter Effect Example:

 Quinine sulfate shows linearity at low
concentration
 Occurs at high concentration
 Deviates at higher concentration due to
 Sample absorbs excitation/emission light self-absorption

 Two types: How to Minimize:

 Primary: excitation light absorbed
• Use dilute solutions (<10⁻4 M)
 Secondary: emitted light reabsorbed
• Use shorter path length
• Proper calibration with standards
 Fluorescence intensity decreases →
nonlinearity
Applications of Fluorescence
1. Using complexing agent

2. Naturally photoluminescent organic analytes

Applications of Fluorescence
 Problem
Quinine is an alkaloid used in treating malaria. It is a strongly fluorescent
compound in dilute solutions of H2SO4 ( = 0.55). Quinine’s excitation spectrum
has absorption bands at 250 nm and 350 nm and its emission spectrum has a
single emission band at 450 nm. Quinine is rapidly excreted from the body in
urine and is determined by measuring its fluorescence following extraction from
the urine sample. After ingesting 10.0 mg of quinine, a volunteer provided a
urine sample 24 h later. Analysis of the urine sample gives an emission
intensity of 28.16. Determine the concentration of quinine in the sample
120
Fluorescent Intensity

100

80

60

40

20 I = 9.9781C + 0.1218
0
0 2 4 6 8 10 12
Concentration (g/mL)
Infrared spectroscopy

 It uses light from infrared region of electromagnetic radiation

 The absorption IR resulting in absorption band in the IR spectrum

 The x-axis of the spectrum: wave number (𝜐̅ )

10
𝜐̅ = = 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑤𝑎𝑣𝑒𝑙𝑒𝑛𝑔𝑡ℎ 𝑝𝑒𝑟 𝑐𝑚
𝜆(𝜇𝑚)

 Wave number is directly proportional to the energy

Infrared spectrum
 Used to determine the functional groups present (or absent) in a molecule

2
Infrared spectroscopy
 The covalent bond between two atoms acts like a spring, allowing the atoms to
vibrate (stretch and bend) relative to each other.

 Masses of the atoms in a bond

AW Freq

 Strength of the bond or bond order

BO Freq
3
Molecular vibrations
This oscillating dipole interacts with the oscillating E-M field of the photon, leading
to absorption.

Stretching vibration Bending vibration

Symmetric anti-symmetric rocking scissoring twisting wagging

+ – +

–
In-plane Out-of-plane
Changes in bond length Changes in bond angle
 Energy level in vibrational quantum

Harmonic oscillation

By comparing Newton’s Second

Law with Hooke’s Law, we obtain a
differential equation.
Solution of differential equation
Molecular motion is quantized;
k k vibrational quantum levels (quantum
x(t)  A cos2  t    number “v”)
m 

m1m2

m1  m2

 Energy absorbed is energy difference

where is called the angular frequency.
between two levels
It is related to the frequency n by 2πn.
 For SHO, spacing is same between
The µ is reduced mass and is used when all adjacent levels
both ends of the bond can move
(always).
Selection rules: ∆v = ±1 for electric dipole transitions (i.e. infrared transitions)
Instrumentation

Wavelength selectors
 Interference filters
 Prisms
 NaCl (2.5-20 mm) mid-IR
 KBr, (15-40 mm) far-IR

 Reflection gratings
Nernst glower
Globar Silicon carbide rod, 6 x 50 mm
Nichrome coil Electrically heated to 1600K, emits mid-
Mercury arc lamp IR; better than glower at <5 mm

7
Sample handling and cell

 Mulls (pastes)
 Finely ground solid and mulling agent N aC l D isc
S am ple C alled a
fashioned into a thin film S alt P late
 Mineral oil, Nujol, Fluorolubes used as
mulling agents
In ciden t T ran sm itted
IR B eam IR b eam
IR D etector
 Pellets S o u rce Io I
 Finely ground solid (1-100 mg) + KBr I o = Inciden t In ten sity
 Pressed into a transparent disk at I = T ransm itted In ten sity
60,000 to 100,000 psi
I < I o is an a b so rp tio n
Factors affecting intensity of IR absorption

 The polarity of a bond has a significant impact on the intensity of an IR

absorption band.

 Vibrations that cause a significant change in the dipole moment of a chemical

bond results in a strong absorption bands.

 Vibrations that result in no change/very little change in dipole moment leads

to very weak or no absorption band.

 Symmetrical bonds often exhibit very weak or no absorption band.

9
 IR spectrum interpretation

 IR spectrum results from a combination of all possible stretching and/or

bending vibrations of the individual bonds and the whole molecule.

 Simple stretching: ~1600-4000 cm-1 used to identify functional groups that are
present (or absent)

 Complex vibrations: 600-1400 cm-1, called the “fingerprint region.”

10
 Carbon-carbon bonds
 Increasing bond order leads to  Conjugation lowers the frequency:
higher frequencies:
 isolated C=C 1640-1680 cm-1
 C-C 1200 cm-1 (fingerprint region)
 conjugated C=C 1620-1640 cm-1
 C=C 1600 - 1680 cm-1
 aromatic C=C approx. 1600 cm-1
 CC 2200 cm-1 (weak intensity)

11
Carbon-hydrogen bonds
 Bonds with more s character absorb at a higher frequency.

 sp3 (alkane) C-H - just below 3000 cm-1 (to the right)

 sp2 (alkene or aromatic hydrocarbon) C-H -just above 3000 cm-1

(to the left)

 sp (alkyne) C-H at 3300 cm-1

12
13
14
O-H and N-H bonds
Both bond stretching appear around 3300 cm-1, but they look different.
 Alcohol O-H
broad with rounded tip when hydrogen bonding is present (sharp in the
absence of hydrogen bonding)

 Secondary amine (R2NH)

Broad (usually) with one sharp spike

 Primary amine (RNH2)

Broad (usually) with two sharp spikes.

 No signal for a tertiary amine (R3N)

15
16
 N-H stretch and bend
A broad, round peak may be observed around 1600 cm-1 for the N – H bend,
especially with primary amines.

NH2
stretch
N-H bend has a
N-H different shape than
bend an aromatic ring or
C=C

17
Carbonyl stretching
 Carbonyl stretches are generally strong:
-1
1745 cm
Aldehyde ~1710 cm-1
Ketone ~1710 cm-1
O
Carboxylic acid ~1710 cm-1
H3C
Ester ~1730 - 1740 cm-1
Amide ~1640-1680 cm-1

 Conjugation shifts all carbonyls to lower frequencies.

 Ring strain shifts carbonyls to higher frequencies

18
Aldehyde

19
Carboxylic acid

20
Ketone

21
Example 1
Which one of the following compounds is the most reasonable structure for
the IR spectrum shown below?
O
O
OCH3 OH

O O
A B C D

1721

22
Example 2

Which of the following compounds is the most reasonable structure for the IR spectrum
shown below?
O
O OH
OH
OH OH
NH2 O
O

A B C D E

1603

1689

23
Nuclear Magnetic Resonance (NMR) spectroscopy

An NMI instrument: useful for A. Spinal cord compression

visualizing soft tissue B. Spinal cord (wouldn’t visualized with1 x-rays)
NMR spectroscopy

 A proton spins on its axis, it creates a magnetic field,

(considered as a tiny bar magnet.)

 These tiny bar magnets are randomly oriented in

space.

 Presence of an external magnetic field B0, they are

oriented with or against this applied field.
2
Energy levels in a magnetic field

Only nuclei that contain

odd mass numbers (such
as 1H, 13C, 19F and 31P)

or odd atomic numbers

(such as 2H and 14N) give
rise to NMR signals.

Where  is the magnetogyric constant and m is magnetic quantum states

(+1/2 or -1/2)
3
NMR spectroscopy
Absorbing radio frequency radiation
causes the nucleus to spin flip

Higher energy state

Lower energy state

 Radio frequency radiation (h) that matches the energy difference (E) between
these two states is applied
 Energy is absorbed, causing the nucleus to “spin flip” from one orientation to
another. 4
The NMR spectrometer

Modern NMR spectrometers use a constant magnetic field strength B0

 1H NMR spectrum

 An NMR spectrum is a plot of the

intensity of a peak against its
chemical shift, measured in parts
per million (ppm).

 NMR absorptions are measured

relative to the position of a
reference peak at 0 ppm on the 
scale due to tetramethylsilane
(TMS).

 The frequency at which a particular

proton absorbs is determined by its
electronic environment. 6
1H NMR spectrum

 The chemical shift of the x axis gives the position of an NMR signal, measured in
ppm, according to the following equation:

Chemical shift (, ppm) =

 ( )

 Four different features of a 1H NMR spectrum provide information about a

compound’s structure:
[Link] of signals
[Link] of signals
[Link] of signals.
[Link]-spin splitting of signals.

7
Number of signals

 The number of NMR signals: number of different types of protons in a compound.

Equivalent protons Protons in different environments give different

give the same NMR NMR signals
signal.

To determine equivalent protons in cycloalkanes and alkenes, always draw all

bonds to hydrogen.

8
Number of signals
In comparing two H atoms on a ring or double bond, two protons are equivalent only
if they are cis (or trans) to the same groups.

1,1-dichloroethyene 1-bromo-1-chloroethyene chloroethyene

One type Two types Three types

one signal two signals three signals

9
Position of signals
Isolated proton

The nucleus feels Bo only

Isolated proton surrounded by  The magnetic field generated by the

electron density circulating electron decreases the
external magnetic field that the proton
“feels”.

 Lower frequency is to the right in an

NMR spectrum, toward a lower chemical
The induced field decreases the strength shift, so shielding shifts the absorption
of the magnetic field felt by the nucleus upfield. 10
Chemical shift positions
The chemical shift of a C—H bond increases with increasing alkyl substitution.

 The less shielded the nucleus becomes, the more of the applied magnetic field
(B0) it feels.

 Higher frequency is to the left in an NMR spectrum, toward higher chemical

shift—so deshielding shifts an absorption downfield.
11
Chemical shift positions
Shielding effects
 An electron shields the nucleus

 The absorption shifts upfield

Deshielding effects
 Decrease electron density deshields a
nucleus

 The absorption shifts downfield

12
Shielding and deshielding effects

As the electron density around the nucleus

increases, the nucleus feels a smaller
resultant magnetic field, so a lower frequency
is needed to achieve resonance.
(Shielded)

As the electron density around the nucleus

decreases, the nucleus feels a larger resultant
magnetic field, so a higher frequency is
needed to achieve resonance. (Deshielded)

13
Examples

 The Hb protons are deshielded because they are closer to the electronegative
Cl atom, so they absorb downfield from Ha

 Because F is more electronegative than Br, the Hb protons are more deshielded
than the Ha protons and absorb farther downfield

 The larger number of electronegative Cl atoms (two versus one) deshields Hb

more than Ha, so it absorbs downfield from Ha.

14
Regions in the proton NMR spectrum

 Shielded protons absorb at lower chemical shift (to the right)

 Deshielded protons absorb at higher chemical shift (to the left) 15

Chemical shift: magnetic anisotropic

Circulation of  electrons
Induced magnetic field

Inner hydrogen: 1.8 ppm

Paramagnetic zone Diamagnetic zone
Deshielded nuclei Shielded nuclei Outer hydrogen: 8.9 ppm
16
 Chemical shift position: Aromatic ring

 The six  electrons in benzene circulate around the ring creating a ring current.

 The protons thus feel a stronger magnetic field and a higher frequency is needed for
resonance. Thus, they are deshielded and absorb downfield.

The circulating  electrons

create a ring current
1.4 ppm
The magnetic field induced by
these moving electrons
reinforces the applied
magnetic field in the vicinity of
the protons.
6.5 - 8.0 ppm
17
Chemical shift position: Alkene

 In a magnetic field, the loosely held  electrons of the double bond create a
magnetic field that reinforces the applied field in the vicinity of the protons.

 The protons now feel a stronger magnetic field, and require a higher frequency for
resonance. Thus, the protons are deshielded and the absorption is downfield.

The induced magnetic field reinforces the

external field Bo in the vicinity of the protons

The protons are deshielded. The absorption

is downfield at 4.5 – 6 ppm 18
Chemical shift position: Alkyne
The induced magnetic fields opposes the
external field Bo in the vicinity of the proton

The proton is shielded and the absorption is

upfield at 2.5 ppm

 The  electrons of a carbon-carbon triple bond are induced to circulate, but in this
case the induced magnetic field opposes the applied magnetic field (B0).
 Thus, the proton feels a weaker magnetic field, so a lower frequency is needed
for resonance. The nucleus is shielded and the absorption is upfield. 19
 Intensity of signals
 An NMR spectrometer
automatically integrates the area
under the peaks, and prints out a
stepped curve (integral) on the
spectrum.
 The height of each step is
proportional to the area under the
peak, which in turn is proportional
to the number of absorbing protons.

 The ratio of integrals to one another

gives the ratio of absorbing protons
in a spectrum.
Chemical shift, ppm
20
Problem
A compound of molecular formula C9H10O2 gives the following integrated spectrum.
How many protons give rise to each signal?

21
Spin-spin splitting
Spin-spin splitting occurs only between nonequivalent protons on the same carbon
or adjacent carbons.

Let us consider how the doublet due to the CH2 group on BrCH2CHBr2 occurs:

 When placed in an applied With no adjacent H’s: the

electric field, (B0), the absorbing H feel only one
adjacent proton (CHBr2) magnetic field (singlet)
can be aligned with () or
against () B0.
With one adjacent H: the
absorbing H feel two
magnetic field (doublet)

22
 Triplet and doublet spin-spin splitting

Consider the spectrum BrCH2CHBr2

When placed in an applied magnetic field (B0), the adjacent protons Ha and Hb can
each be aligned with () or against () B0.
23
 Triplet spin-spin splitting
 Absorbing proton feels three slightly different magnetic fields—one slightly larger
than B0, one slightly smaller than B0, and one the same strength as B0.
Consider how a triplet arises:
 Because there are two different ways to align
one proton with B0, and one proton against
B0—that is, ab and ab—the middle peak of
the triplet is twice as intense as the two outer
peaks, making the ratio of the areas under the
three peaks 1:2:1.
 The spacing between peaks in a split NMR
signal, measured by the coupling constant (J)
value

With two adjacent Hs: the NMR signal

24
split
in to three (triplet)
General rule for neighboring proton (n)
Three general rules describe the splitting patterns commonly seen in the 1H NMR
spectra of organic compounds.

 Equivalent protons do not split each other’s signals.

 A set of n nonequivalent protons splits the signal of a nearby proton into n + 1
peaks.
 Splitting is observed for nonequivalent protons on the same carbon or adjacent
carbons.
If Ha and Hb are not equivalent, splitting is observed when:

Ha and Hb: adjacent carbons (three

 bonds separation Ha and Hb: no spin splitting (four 
bonds separation) 25
Examples of Multiplicity

Pascal diagram

26
 Names based on splitting

Adjacent equivalent protons (Ha for Hb)

use the n + 1 rule to determine the
splitting pattern

Example:
 Ha doublet
 Hb septet
27
Different adjacent proton splitting
When two sets of adjacent protons are different from each other (n protons on one
adjacent carbon and m protons on the other), the number of peaks in an NMR signal
= (n + 1)(m + 1).

CH3CH2CH2-Br

Ha Hb Hc
Three Ha protons split the Hb signal
into 3+1 = 4 peaks

Two Hc protons split the Hb signal

into 2+1 = 3 peaks

The Hb signal is split into 12 peaks, a quartet of triplets. When Jab and Jbc are
similar in magnitude, peaks overlap and fewer lines are observed
28
 Example
Draw proton NMR spectrum for ethanol (CH3CH2OH)

 Protons on electronegative atoms rapidly exchange between molecules in the presence of

trace amounts of acid or base.

 Thus, the CH2 group of ethanol never “feels” the presence of the OH proton, because the OH
proton is rapidly moving from one molecule to another. This phenomenon usually occurs with
NH and OH protons.
29
Problem
Using its 1H NMR spectrum, determine the structure of an unknown compound X that
has molecular formula C4H8O2 and contains a C=O absorption in its IR spectrum

30
Spin-spin splitting: double bond

 Protons on carbon-carbon double bonds often give characteristic splitting patterns.

 A disubstituted double bond can have two geminal protons, two cis protons, or two trans
protons.
 When these protons are different, each proton splits the NMR signal of the other so that
each proton appears as a doublet.
 The magnitude of the coupling constant J for these doublets depends on the arrangement
of hydrogen atoms.

31
Example
Splitting protons in vinyl acetate are shown below.

Note that each pattern is different in appearance because the magnitude of the
coupling constants forming them is different.
32
Splitting cis-trans isomers

(E)- and (Z)-3-chloropropenoic acid show two doublets in their 1H NMR spectra
for their alkenyl protons, Jtrans > Jcis. This helps one differentiate between cis and
trans-isomers using NMR.
33
Problem
Draw the proton NMR spectrum for vinyl acetate

34
Proton NMR in benzene
 Benzene has six equivalent deshielded protons and exhibits a single peak in its 1H
NMR spectrum at 7.27 ppm.
 Monosubstituted benzenes contain five deshielded protons that are no longer
equivalent, and the appearance of these signals is highly variable, depending on
the identity of Z.

The appearance of the signals in the 6.5-8 ppm region of the 1H NMR spectrum
depends on the identity of Z in C6H5Z.

What about para-substituted aromatic ring? 35

13C NMR spectroscopy
 The lack of splitting in a 13C spectrum is a consequence of the low natural
abundance of 13C.

 This 1H-13C splitting is usually eliminated from the spectrum by decoupling the
proton-carbon interactions, so that every peak in a 13C NMR spectrum appears as
a singlet.

 The two features of a 13C NMR spectrum that provide the most structural
information:
 Number of signals
 The chemical shifts

36
13C NMR spectroscopy
13CSpectra are easier to analyze than 1H spectra because the signals are not split
Each type of carbon atom appears as a single peak.

37
13C NMR-number of signals
 The number of signals in a 13C spectrum gives the number of different types of
carbon atoms in a molecule.
 Because 13C NMR signals are not split, the number of signals equals the number
of lines in the 13C spectrum.

Unlike the 1H NMR situation, peak intensity is not proportional to the number of
absorbing carbons, so 13C NMR signals are not integrated.
38
13C NMR-position of signals
 In contrast to the small range of chemical shifts in 1H NMR (1-10 ppm usually), 13C NMR
absorptions occur over a much broader range (0-220 ppm).
 The chemical shifts of carbon atoms in 13C NMR depend on the same effects as the chemical
shifts of protons in 1H NMR.

13C NMR Chemical shifts

39
Example
 The three types of C’s in 1-propanol give rise to three 13C NMR signals

 Deshielding increases with increasing proximity to the electronegative O atom,

and the absorption shifts downfield

40
Example

Draw 13C NMR for Methyl acetate

41
Problem

Differentiate these molecules by showing 13C NMR spectra

42