1

Instrumental Analysis
Fundamentals of
Spectroscopy

2
(Absorption) Spectrophotometry
General Stuff:

•Definitions and units

.monochromatic wavelength (cm)
Po.incident radiant power (erg cm -2 s -1 )
P .transmitted radiant power (erg cm -2 s -1
)
b .absorption pathlength(cm)

3
 (Absorption) Spectrophotometry
General Stuff:

•Qualitative: Spectrum (a plot of A vs. ) is

characteristic of a specific species

•Quantitative: Absorbance at a particular can be

related to the amount of absorbing species

4
Qualitative analysis: The spectrum

5
Molecular and Atomic Spectrometry
Spectrometry is the study of
electromagnetic radiation (EMR) and its
applications
To begin to understand the theory and
instrumental application of spectrometry
requires an understanding of the
interaction of EMR (i.e. light) with matter

6
 Questions
What is nature of light?
Are there different types of light?
– How are they the same?
– How are they different?
How does light propagate?

7
 What is Light?
Light is a form of energy
Light travels through space at extremely
high velocities
– The speed of light (c) ~ 3 x 1010 cm/sec or
186,000 miles per second
Light is classified as electromagnetic
radiation (EMR)

8
 Characteristics of Light
Light behaves like a wave.
– That is, it can be modeled or characterized
with wave like properties.

9
 Characteristics of Light
Light also behaves like a particle.
– The photon and photoelectric effect.
Today, we envision light as a self-
contained packet of energy, a photon,
which has both wave and particle like
properties.

10
 Wave Properties of
Electromagnetic Radiation
EMR has both electric (E) and magnetic
(H) components that propagate at right
angles to each other.

11
 EMR Wave Characteristics
Wavelength () is the distance from one wave crest to
the next.
Amplitude is the vertical distance from the midline of a
wave to the peak or trough.
Frequency (v) is the number of waves that pass through
a particular point in 1 second (Hz = 1 cycle/s)

12
 EMR Wave Characteristics
The frequency of a wave is dictated (or fixed) by
its source, it doesn’t change as the wave passes
through different mediums.
The speed of a wave (u), however, can change as
the medium through which it travels changes
umedium = v = c/n
Where n = refractive index
nvacuum = 1
nair = 1.0003 (vair = 0.9997c)
nglass ~1.5 (vgas ~ 0.67c)
Since v is fixed, as  decreases, u must also
decrease
13
The Electromagnetic Spectrum

14
The Electromagnetic Spectrum

15
 The EMR
Spectrum
Different portions of
the EMR spectrum
and different types of
spectroscopy involve
different parts
(quantum states) of
the atom

16
 Particle Properties of EMR

The energy of a photon depends on its

frequency (v)

Ephoton = hv

h = Planck’s constant
h = 6.63 x 10-27 erg sec or 6.63 x 10-34 Js

17
Relationship between Wave and
Particle Properties of EMR
Ephoton = hv ; umedium = v = c/n
With these two relationships, if you know one of
the following, you can calculate the other two
– Energy of photon
– Wavelength of light
– Frequency of light
hc
Ephoton = n
18
 Relationship between Wave and
Particle Properties of EMR

Example: What is the energy of a 500 nm

photon?

 = c/ = (3 x 108 m s-1)/(5.0 x 10-7 m)

 = 6 x 1014 s-1
E = h =(6.626 x 10-34 J•s)(6 x 1014 s-1) = 4 x 10-19 J

19
How Light Interacts with Matter.
Atoms are the basic
blocks of matter.
They consist of heavy
particles (called protons
and neutrons) in the
nucleus, surrounded by
lighter particles called
electrons

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21
How Light Interacts with Matter

An electron will interact with a photon.

An electron that absorbs a photon will
gain energy.
An electron that loses energy must emit a
photon.
The total energy (electron plus photon)
remains constant during this process.

22
Characteristics of Absorption
Absorption is defined as the process by
which EMR is transferred, in the form of
energy, to the medium (s, l, or g) through
which it is traveling
Involves discrete energy transfers
Frequency and wavelength selective
– Ephoton = hv = c/

23
Characteristics of Absorption
Involves transitions from ground state
energy levels to “excited” states
– The reverse process is called emission
For absorption to occur, the energy of the
photon must exactly match an energy level
in the atom (or molecule) it contacts
– Ephoton = Eelectronic transition
We distinguish two types of absorption
– Atomic
– Molecular
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How Light Interacts with Matter.
Electrons bound to
atoms have discrete
energies (i.e. not all
energies are allowed).
Thus, only photons of
certain energy can
interact with the
electrons in a given
atom.

25
How Light Interacts with Matter.
Consider hydrogen, the
simplest atom.
Hydrogen has a specific
line spectrum.
Each atom has its own
specific line spectrum
(atomic fingerprint).

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Energy Transitions and Photons
The energy of photon that can interact with a transition jump
depends on the energy difference between the electronic
levels.

27
 Unique Atomic Signatures
Each atom has a specific set of energy levels, and thus a
unique set of photon wavelengths with which it can interact.

28
Energy Level Diagram
Absorption and emission
for the sodium atom in the
gas phase
Illustrates discrete energy
transfer

ΔEtransition = E1 - E0 = hv = hc/

29
 Molecular Absorption
More complex than atomic absorption
because many more potential transitions
exist
– Electronic energy levels
– Vibrational energy levels
– Rotational energy levels
Emolecule = Eelectronic + Evibrational + Erotational
– Eelectronic > Evibrational > Erotational
Result - complex spectra
30
Energy Level Diagram for
Molecular Absorption

31
Molecular Absorption Spectra of
Benzene in the Gas Phase
Electronic Transition
Vibrational Transition
Superimposed on the
Electronic Transition

Absorption Band –
A series of closely
shaped peaks

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 Molecular
Absorption
Spectra in the
Solution Phase
In solvents the
rotational and
vibrational transitions
are highly restricted
resulting in broad
band absorption
spectra

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40
MEASURMENT OF
ABSORPTION

41
 Beer’s Law
or the Lambert-Beer Law
Pierre Bouguer discovered that
light transmission decreases with
the thickness of a transparent
sample in 1729. This law was
later rediscovered by Lambert, a
mathematician, and then by Beer,
who published in 1852 what is
now known as
the Beer-Lambert-Bouguer law.
Beer's 1852 paper is the one that
is often cited in older textbooks.
Bouguer's contribution
is rarely mentioned and the law is
known as either "Beer's law" or
"the Lambert-Beer law".
 Spectroscopy Terms Describing
Absorption (Beer’s Law)
Consider a beam of light
with an (initial) radiant

Concentration (c)
intensity Po
The light passes through a P0 hv
P
solution of concentration (c)
The thickness of the
solution is “b” cm.
The intensity of the light b
after passage through the
solution (where absorption
occurs) is P
 We Define

Transmittance (T) = P/P0 (units = %)

Absorbance (A) (units = none)
A = log (P0/P)
A = -log (T) = log (1/T)
A = abc (or εbc) <--- Beer’s Law
a = absorptivity (L/g cm)
b = path length (cm)
c = concentration (g/L)
ε = molar absorptivity (L/mol cm)
– Used when concentration is in molar units
 Transmittance
T => transmittance
P
T = -----
Po
b

Po P
 Example

P0 = 10,000 P = 5,000

-b-
P 5000
T    0 .5
P0 10000

A = -log T = -log (0.5) = 0.3010

 Beer’s Law
A = abc = bc

c
 Beer’s Law A =  bc
Path Length Dependence, b
Readout
Absorbance

0.82

Source

Detector
 Beer’s Law A =  bc
Path Length Dependence, b
Readout
Absorbance

0.62

Source
b

Detector Sample
 Beer’s Law A =  bc
Path Length Dependence, b
Readout
Absorbance

0.42

Source

Detector Samples
 Beer’s Law A =  bc
Path Length Dependence, b
Readout
Absorbance

0.22

Source

Detector Samples
 Beer’s Law A = bc
Wavelength Dependence, a
Readout
Absorbance

0.80

Source
b

Detector
 Beer’s Law A = bc
Wavelength Dependence, a
Readout
Absorbance

0.82

Source

Detector
 Beer’s Law A = bc
Wavelength Dependence, a
Readout
Absorbance

0.30

Source
b

Detector
 Beer’s Law A = bc
Wavelength Dependence, a
Readout
Absorbance

0.80

Source
b

Detector
 Non-Absorption Losses

"Reflection and
scattering losses."

AKA
The Guinness Effect
 Limitations to Beer’s Law

Real
– At high concentrations charge distribution effects
occur causing electrostatic interactions between
absorbing species
Chemical
– Analyte dissociates/associates or reacts with solvent
Instrumental
– ε = f(λ); most light sources are polychromatic not
monochromatic (small effect)
– Stray light – comes from reflected radiation in the
monochromator reaching the exit slit.
 Chemical Limitations
A reaction is occurring as you record
Absorbance measurements
Cr2O72- + H2O 2H+ + CrO42-
CrO42-

Cr2O72-
A350 A446

300 400 500 concentration concentration

wavelength
 Instrumental Limitations - ε = f(λ)
How/Why does ε
vary with λ?
Consider a
wavelength scan for
a molecular
compound at two
different wavelength
bands

In reality, a
monochromator can
not isolate a single
wavelength, but
Larger the Bandwidth – larger deviation
rather a small
wavelength band
Applications of SpectrophotometrY
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Red shift of max with increasing conjugation
CH2=CHCH2CH2CH=CH2 max =185 nm

CH2=CHCH=CH2 max =217 nm

vs.

Red shift of max with # of rings

Benzene max =204 nm
Naphthalene max =286 nm
Polar solvents more likely to shift absorption maxima
Shifts of max with solvent polarity
n* hypsochromic/blue shift
* bathochromic/red shift
 O
Hypsochromic shift

methanol heptane
 Generally, extending conjugation
leads to red shift
“particle in a box” QM theory; bigger box
Substituents attached to a chromophore that cause a red shift are called
“auxochromes”
Strain has an effect…

max 253 239 256 248

 Generally, extending conjugation
leads to red shift
“particle in a box” QM theory; bigger box
Bathocromic and hypsochromic shifting of Ni-complex

74
 UV Spectroscopy and Qualitative Analysis

1) UV-vis spectroscopy is usually not

very useful for qualitative analysis
because there are few absorption
maxima and minima
2) Solvents:
a. Must be transparent in region of
interest
b. Should not interfere with absorbing
species (but usually it does). Polar
solvents tend to obliterate fine
structural detail in molecular spectra
 UV Spectroscopy and Qualitative Analysis
3) UV-vis spectroscopy does provide some
information on functional groups
Saturated organic compounds containing such heteroatoms as oxygen,
nitrogen,
sulfur, or halogens have nonbonding electrons that can be excited by
radiation in the 170- to 250-nm range.
Absorption occurs when electrons make
transitions between filled and unfilled d-
orbitals with energies that depend on the
ligands bonded to the metal ions.

The energy differences between these d-

orbitals
(and thus the position of the corresponding
absorption maxima) depend on the position
of
the element in the periodic table, its
oxidation
state, and the nature of the ligand bonded to
it.
Charge-transfer absorption is important for
quantitative analysis because
molar absorptivities are unusually large
(e > 10,000 L mol 1 cm 1), which leads to
high
sensitivity.

Many inorganic and organic complexes

exhibit this type of absorption and are
therefore called charge-transfer
complexes.

A charge-transfer complex consists of an

electron-donor group bonded to an
electron acceptor.

In most charge-transfer complexes

containing a metal ion, the metal serves as
the
electron acceptor.
Qualitative Applications of Ultraviolet/ Visible Spectroscopy

Spectrophotometric measurements with UV radiation are useful for detecting

chromophoric groups.

However, 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.
 UV-vis spectroscopy and
Quantitative Analysis
1)Scope is huge
a.)95% of all quantitative analyses in
health care field are done by UV-vis
spectroscopy
b.) wide applicability to organic and
inorganic analyses
c.) even non-absorbing species can
be used by doing colorimetric
reactions (reactions must go near to
completion)
Procedural Details

Wavelength Selection: To realize maximum sensitivity, spectrophotometric

absorbance measurements are usually made at the wavelength of maximum
absorption.

Variables that influence absorption: solvent, the pH of the solution,

temperature, high electrolyte concentrations, and the presence of interfering
substances.

The relationship between absorbance and concentration: The calibration

standards should approximate as closely as possible the overall composition
of the actual samples and should encompass a reasonable range of analyte
concentrations.

The Standard Addition Method: The composition of calibration standards

should approximate the composition of the samples to be analyzed.
 Construction of Calibration Curves is often done
on the absorption max.
For example: β-Carotene analysis in a sample
 is max.
value

Uv/vis. Spectrum is compared with pura

compound at certain concentration in C=10-3-10-7 M
the same type of solvent

Equal conditions

C 2C 3C 4C 5C
 Beer’s Law A = bc

?
Readout ?
Absorbance
Sample
0.80

Source
b

=450 nm

Detector = constant
b= constant
 The ultraviolet absorbances
Example of a series of 9 standards
having different nitrate
concentrations were
determined at 220nm
using a 1.0cm cell; 8
samples of river water
were taken downstream
from a chemical plant,
avg. absorbance 0.642.
What is the nitrate content
of the river in mg/mL?

NO3 0 .004 .015 .025 .035 .04 .05 .06 .07

(mg/
mL

Abs. .003 .10 .211 .350 .453 .556 .623 .671 .691
Determination of Concentration – Multicomponent System

C
B

1 2 Wavelength (nm)

C+B

1 2 Wavelength (nm)
Determination of Concentration – Multicomponent System

C
B

1 2 Wavelength (nm)

(1) Measure B and C at 1 and 2 (pure substances)

Determination of Concentration – Multicomponent System

(2) Measure Aat 1 and 2 (mixture)

C+B

1 2 Wavelength (nm)
Determination of Concentration – Multicomponent System

A1 = xB + yC
1 1

A2 = xB2 + yC2

x = molarity B
y = molarity C

C+B

1 2 Wavelength (nm)
 Isobestic Points

HIn (acid form) In- (base form)

Brocresol green

When one absorbing species is converted to another, it is apparent in the

absorption spectrum.
 Isobestic Points

pH
I see pH [OH-]
an [H+]
isobesti
c
point!!!
 Isobestic Points

HIn (acid form) In- (base form)

The Total concentration of Bromocresol Green is constant throughout the reaction

[Hin] + [In-] = F bromocresol green

Cx + Cy =C
A = b (xCx + yCy)
But at the isosbestic point both molar absorptivities are the
same!
x + y = 
 Isobestic Points

HIn (acid form) In- (base form)

x + y = 
Therefore, the absorbance does not depend on the extent of reaction
(i.e. on the particular concentrations of x and y)

A = b (xCx + yCy) = b Cx + Cy) = b C

An isobestic point is good evidence

that only two principal species
are present in a reaction.
 Isobestic Points - Application
Oximetry
 O2
Hb HbO2
deoxyhemoglobin Oxyhemoglobin

100 % O2

0 % O2
 Measuring the equilibrium constant
(The Scatchard Plot)
Biochemistry example: The cellular action of a hormone begins when the hormone (L)
Binds to it’s receptor protein (R) in a tight and specific way.
The binding thus gives rise to conformational changes which change the biological activity
of the receptor (an enzyme, an enzyme regulator, an ion channel,
or a regulator of gene expression.

R + L RL

The binding depends of the concentration of the concentration of the components.

[ RL] k f 1
Ka   
[ R][ L] k r K d

Ka is the association constant and Kd is the dissociation constant

 Measuring the equilibrium constant
(The Scatchard Plot)
When binding has reached equilibrium, the total number of binding sites, Bmax = [R]
+ [RL]
whereas, the number of unbound sites would be [R] = Bmax – [RL].
[ RL]
The equilibrium constant would then be Ka 
[ L]( Bmax  [ RL])

This expression can then be rearranged to find the ratio of bound to unbound (free) hormon

[bound ] [ RL] 1
  K a ( Bmax  [ RL])  ( Bmax  [ RL])
[ free ] [ L] Kd
Measuring the equilibrium constant
(The Scatchard Plot)

[bound ] [ RL] 1
  K a ( Bmax  [ RL])  ( Bmax  [ RL])
[ free ] [ L] Kd
The method of continuous variation (for isomolar
solution)
(Job’s method)
A + nB ABn
Call this C

Allows for the determination

of the stoichiometry of the
Predominant product.

0 1
Mole fraction ( = nb / na + nb)
The method of continuous variation (for isomolar
solution)
(Job’s method)
Requirements:

The system must conform to Beer's law.

Only single equilibrium

Equimolar solutions MA = MB

Constant volume 1 = VT = VA + VB

K reasonably greater than 1

pH and ionic strength must be maintained constant

The method of continuous variation (for isomolar
solution)
(Job’s method)
A + nB C

Start M(1-x) Mx 0

Change -C -nC +C

Equil. M(1-x)-C Mx-nC C

M (1  x) MVA
M(1-x) is a concentration 
1 VT
 A3X AX AX2

0 0.25 0.5 0.66 1

Mole fraction ( = nb / na)

Photometric and Spectrophotometric Titrations

Titration Curves

A photometric titration curve is a plot of absorbance (corrected for volume

change)
as a function of titrant volume.
Instrumentation

Photometric titrations are usually performed with a spectrophotometer or a

photometer that has been modified so that the titration vessel is held
stationary in the light path.

Application

Photometric titrations often provide more accurate results 
than a direct photometric determination because the data 
from several measurements are used to determine the end 
point.

The presence of other absorbing species may not interfere 
since only a change in absorbance is being measured.
The photometric end point has also been used to great 
advantage in titrations with EDTA and other complexing 
agents.
Instrument
 FLORESANS
SPECTROSCOPY
Molecular Absorption Spectrophotometer

Signal Processor
Source Sample Wavelength Selector Detector Readout

Molecular Fluorescence and/or

Scattering Spectrophotometer
Signal Processor
Sample Wavelength Selector Detector Readout
0-90o

Source
 Results from
thermal energy
Stokes Shift losses
 Molecules see more
non-resonance
fluorescence
 Not all excited energy
is transmitted as
radiation (some is
non-radiative)
 Makes emission
spectrum look like
mirror image of
excitation spectrum
 This shift in the
spectrum toward
longer wavelengths is
called Stokes Shift
More about fluorescence
 Why do some
molecules fluoresce
and others don’t?
 Want as fast of way as
possible to get down to the
ground state, generally
non-radiative internal
conversion is fastest but
sometimes due to
molecules configuration,
fluorescence may be faster