MOLECULAR ABSORPTION

SPECTROMETRY

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 Introduction
• Molecular spectroscopy:
– Quantitative and qualitative analyses
• UV/Vis spectroscopy • IR Spectroscopy

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 Ultraviolet and Visible Instrument
• Single-Beam Instruments
– The light source is imaged upon the sample
– A fraction of the light is transmitted or reflected
from the sample
– The light from the sample is imaged upon the
entrance slit of the monochromator
– The monochromator separates the wavelengths of
light and focuses each of them onto the
photodetector sequentially.

Fixed wavelength light source

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 Ultraviolet and Visible Instrument
• Single-Beam Instruments
– two sources are used, a tungsten halogen and a
deuterium lamp
– mechanism for source selection should be available.
– The wavelength selector is a grating or prism
monochromators and
– the detector is usually a vacuum phototube or a
photomultiplier tube in higher cost instruments.
Continuous source

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 Ultraviolet and Visible Instrument
– Double-Beam Instruments
• Schematic diagram of a double-beam UV-Vis.
Spectrophotometer
–Sources (UV and visible):
–Wavelength selector (monochromator)
–Sample containers
–Detector
–Signal processor and readout

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 Ultraviolet and Visible Instrument
– Double-Beam Instruments

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 Ultraviolet and Visible Instrument
Monochromator Detector>> PMT
Concave
mirror

– It is a device that contains an – Types of photon detectors

entrance and exit slits
– Wavelength selector
– Exit slit is for isolation of a small
band of wavelengths
– Polychromator has multiple exit
slits
• Phototube and photomultiplier tube
(PMT)
• Photoconductive cells
• Silicon photodiodes and
photo/diode arrays
• Charge transfer device 7
 Photomultiplier tube (PMT)
• PMT is a commonly used detector in UV-Vis
spectroscopy
• It consists of a photoemissive cathode, several dynodes
and an anode.
• A photon of radiation entering the tube strikes the
cathode, causing the emission of several electrons.
• The electrons strike the dynode, causing the emission
of several electrons for each incident electron.
• Each original photon has produced 106 - 107 electrons.
• The resulting current is amplified and measured.

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 Ultraviolet and Visible Spectroscopy
• Visible Spectroscopy
– Sunlight is white light and covers a wavelength
range of 380-750nm.
– A simple physics experiment shows that white light
is actually a composition of a range of colours i.e.,
light of different energies and hence wavelengths.

Red
Orange
WHITE
Yellow
LIGHT
Green
Blue
Indigo
Violet

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 Ultraviolet and Visible Spectroscopy
• When a sample only absorbs light of a single
wavelength the eye sees COMPLEMENTARY colours.
Colour
Wavelength Range Absorbed Absorbed Colour Seen By Eye
380 - 430 Violet Yellow - Green
430 - 480 Blue Yellow
480 - 490 Green - Blue Orange
490 - 500 Blue - Green Red
500 - 560 Green Purple
560 - 580 Yellow - Green Violet
580 - 590 Yellow Blue
590 - 610 Orange Green - Blue
610 - 750 Red Blue - Green

LOW ENERGY HIGH 10

 UV/Vis Absorption process
• The absorption of UV or visible radiation corresponds
to the excitation of outer electrons.
– There are three types of electronic transition which
can be considered
• Transitions involving p, s, and n electrons
• Transitions involving charge-transfer electrons
• Transitions involving d and f electrons

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 UV/Vis Absorption process
• σ → σ* and σ → π* transitions: high-energy,
accessible in vacuum UV (λmax <150 nm). Not usually
observed in molecular UV-Vis.
• n → σ* and π → σ* transitions: non-bonding
electrons (lone pairs), wavelength (λmax) in the 150-
250 nm region.

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 UV/Vis Absorption process
• n → π* and π → π* transitions: most common
transitions observed in organic molecular UV-Vis,
observed in compounds with lone pairs and multiple
bonds with λmax = 200-600 nm.

• Any of these require that incoming photons match in

energy gap corresponding to a transition from ground
to excited state.

• Energies correspond to a 1-photon of 300 nm light are

ca. 95 kcal/mol
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Absorbing species containing p, s,
and n electrons

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 d-d or f-f electronic transitions
• Four types of transitions:
(i) Within the same atom e.g. d-d or f-f transition
(ii) To adjacent atom (charge transfer)
(iii) To a delocalized energy band (photoconductivity)
(iv) Promotion of an electron from valence band to
conduction band (band-gap in semiconductors)

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 Charge - Transfer Absorption
• Many inorganic species show charge-transfer absorption
and are called charge-transfer complexes.
• For a complex to demonstrate charge-transfer
behaviour, one of its components must have electron
donating properties and another component must be
able to accept electrons.
• Absorption of radiation then involves the transfer of an
electron from the donor to an orbital associated with the
acceptor.
• Molar absorptivities from charge-transfer absorption are
large (greater than 10,000 L mol-1 cm-1).
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 UV / Visible Spectroscopy - Theory
• Sample can absorb some of the radiation then the
transmitted light intensity (It ) will be less than the
incident light intensity (Io). It< Io
INCIDENT LIGHT TRANSMITTED LIGHT
254nm 254nm
SAMPLE
Intensity (I o ) Intensity (I t )

• The amount of light transmitted with respect to

the incident light is called TRANSMITTANCE (T)
ie., >>>>Fraction of incident radiation
transmitted by the solution

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 UV / Visible Spectroscopy - Theory
• Transmittance: T=
It

Io

It
% T= X 100
Io

• ABSORBANCE: A = - log10 T

It 2
A = - log10
B
Io
A
A = log10 Io 0
It 220 Wavelength(nm) 380
For of %T = 0 and 100 the corresponding absorbance
values will be 0 and 2, respectively
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 The Laws of Spectrophotometry
• There are two very important basic laws and a
third one which is a combination of the two:

– LAMBERTS LAW – ABSORBANCE (A) is

proportional to the PATH LENGTH (l) of the
absorbing medium.

– BEERS LAW - ABSORBANCE (A) is

proportional to the CONCENTRATION (c) of the
sample.

– BEER- LAMBERT LAW - ABSORBANCE (A) is

proportional to c x l
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 Beer-Lambert Law
– The Beer-Lambert law (or Beer's law) is the linear
relationship between absorbance and concentration of an
absorbing species.
– The general Beer-Lambert law is usually written as:
A = abc
where A is the measured absorbance, a is a wavelength-
dependent absorptivity coefficient, b is the path length,
and c is the analyte concentration.
– When working in concentration units of molarity, the
Beer-Lambert law is written as:
A = εbc
where ε is the wavelength-dependent molar absorptivity
coefficient with units of M-1 cm-1. 20
http://www.chemistry.adelaide.edu.au/external/soc-rel/content/beerslaw.htm
 Beer-Lambert Law
• UNITS OF THE MOLAR EXTINCTION COEFFICIENT
– CONCENTRATION (c) - Moles litre-1
– PATHLENGTH (l) - cm

A = εcl Hence ε =A
cl

E = 1 ˛

mole litre-1 x cm
E = mole-1 litre x cm -1
But 1 litre = 1000cm3
E = 1000 mole -1 cm3 x cm -1
Hence Units of ε= 1000 cm2 mole -1 or L/mol/cm
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 Importance of the Beer-Lambert Law
• A = εcl but if ε and l are constant

• ABSORBANCE ∝ CONCENTRATION and should be linear relationship

– Prepare standards of the analyte to be quantified at known concentrations
– and measure absorbance at a specified wavelength.

– Prepare calibration curve.

• ABSORBANCE AT 300nm
• x
– From measuring absorbance of sample
• x
– Concentration of analyte in sample • x
– can be obtained from the calibration curve • x
– Linear regression line (y=mx+c)
– ε can be obtained from the slope of the • x
• CONCENTRATION (moles litre-1 )
– calibration curve for a given wavelength (λ)

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 Importance of the Beer-Lambert Law
RULES FOR QUANTITATIVE ANALYSES
x

ABSORBANCE AT 300nm
At high concentrations the calibration
curve may deviate from linearity x
– Always ensure your concentration of x
the sample falls within the linear range x
– if necessary dilute sample x
CONCENTRATION (moles litre-1 )

Absorbance not to exceed 1 to reduce

error*

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 Importance of the Beer-Lambert Law

ABSORBANCE AT 300nm
CHOOSE CORRECT WAVELENGTH
An analyte may give more than one absorbance x
maxima (λmax) value.
x
Many compounds absorb at 220-230nm hence do x
not use A
x
CONCENTRATION (moles litre-1 )
Need to choose wavelength more specific
A
to compound (SELECTIVITY) and if more C λmax
0.6
B
than one select one with highest absorbance
as this gives less error – hence use C

0
220 Wavelength (nm) 380
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 Example 1: Using Beers’ Law
• A 7.25 x 10-5 M solution of light emitting dye (LED) for LCD
display applications has a transmittance of 44.1% when
measured in 2.10 cm cell at a 525 nm. Calculate (a) absorbance
of this solution; and (b) the molar absorptivity of LED.

– (a) A = -log T
= -log (0.441)
= 0.355

– (b) A=εcl or A=εbc

ε= A/cl
= 0.355/ (2.10 cm x 7.25 x 10-5 mol/L)
=2.33 x 103 L/mol/cm

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 Example 2: Using Beers’ Law
• At 580 nm, the wavelength of its maximum absorption in a 1.00
cm cell, the complex FeSCN2+ has a molar absorptivity of 7.00
x 103 L/cm/mol.
Calculate:
(a) the absorbance of a 7.25 x 10-5 M this solution; and
(b) when the concentration is twice that in (a).
(c) the transmittance of the solutions described in (a) and (b).
(d) the absorbance of a solution that has half the transmittance of
that described in (a).

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 Example 3: Using Beers’ Law
• Given the following set of data for a compound C: (a) Calculate
molar extinction coefficient; and (b) What is the concentration
of C when we obtain an Absorbance of 0.3321?

Conc (M) Abs

0.7
0.1 0.2322 0.6 y = 1.0137x + 0.1378

0.2 0.3456 0.5 R2 = 0.997

0.3 0.4532 0.4

Abs
0.3
0.4 0.5331
0.2
0.5 0.6453 0.1
0
Is the fitting of the curve to the
0 0.2 0.4 0.6
equation acceptable? How can you conc
tell?
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eprints.lincoln.ac.uk/2366/22/Chem_Physical_spectroscopy-_basics.pptx
 Example 3: Using Beers’ Law
• Given the following set of data for a compound C: (a) Calculate
molar extinction coefficient; and (b) What is the concentration
of C when we obtain an Absorbance of 0.3321?

Method 1: The concentration is: Abs= 1.0137 Conc + 0.1378

Abs= 0.3321 – Abs blank

= 0.3321- 0.13800
= 0.1941

Conc= Abs = 0.1941 = 0.2 M

1.0137 1.0137

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 Example 3: Using Beers’ Law

Method 2
The concentration is: Abs= 1.0137 Conc + 0.1378
Abs= 0.3321
Conc= 0.3321-0.1378 = 0.1941 = 0.2 M
1.0137 1.0137

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 Example 3: Using Beers’ Law
Method 3
ε=slope=y2-y1 =1.003
x2-x1

Abs
Conc (M)
Corr
0.1 0.2322 0.0944
0.2 0.3456 0.2078
0.3 0.4532 0.3154
0.4 0.5331 0.3953
0.5 0.6453 0.5075
unknown
C 0.3321 0.1943 Therefore unknown [C]= 0.2 M

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Limitations and deviations from Beer’s Law
• Real limitations
– Non-linearities due to intermolecular interactions
• Self aggregation effects and electrolyte effects
• Apparent
– Dynamic dissociation or association of analyte
• Instrumental
– Polychromatic radiation
• Different molar absorptivities at different
wavelength leads to non-linearities in Beer’s
Law
– Stray radiation and Mismatched cells
• Non-zero intercept in calibration curve
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http://www.monzirpal.net/Instrumental%20Analysis/Contents/UV_Vis1.htm#_1._Single_Beam_Instruments
 Deviations from Beer’s Law

I r (η 2 − η1 ) 2
=
I 0 (η 2 + η1 ) 2
Successful at low analyte concentrations (0.01M)!
High concentrations of other species may also affect 32
 Tutorial Questions
1. Why is a solution of metal free phthalocyanine dye blue? (2
marks)

2. Identify factors that cause the Beer’s law relationship to depart

from linearity. (3 marks)

3. A solution that was 3. 78 x 10-3 M in X had a transmittance of

0.212 when measured in a 2.00-cm cell. What concentration of
X would be required for the transmittance to be increases by a
factor of 3 when a 1.00-cm cell was used? (5 marks)

4. Describe the following terms: Photomultiplier tube and

Monochromator. (5 marks)

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