Fundamentals of Spectrophotometry
Introduction
1.) Colorimetry

An analytical technique in which the concentration of an analyte is measured
by its ability to produce or change the color of a solution
-
Changes the solution’s ability to absorb light
2.) Spectrophotometry


Any technique that uses light to measure chemical concentrations
A colorimetric method where an instrument is used to determine the amount of
analyte in a sample by the sample’s ability or inability to absorb light at a
certain wavelength.
Colorimetry
Instrumental Methods
(spectrophotometry)
Non-Instrumental Methods
Fundamentals of Spectrophotometry
Introduction
3.) Illustration

Measurement of Ozone (O3) Above South Pole
-
O3 provides protection from ultraviolet radiation
Seasonal depletion due to chlorofluorocarbons
O3 cycle
Chain Reaction Depletion of O3
Spectra analysis
of [O3]
Fundamentals of Spectrophotometry
Properties of Light
1.) Particles and Waves


Light waves consist of perpendicular, oscillating electric and magnetic fields
Parameters used to describe light
-
amplitude (A): height of wave’s electric vector
-
Wavelength (l): distance (nm, cm, m) from peak to peak
-
Frequency (n): number of complete oscillations that the waves makes each
second

Hertz (Hz): unit of frequency, second-1 (s-1)

1 megahertz (MHz) = 106s-1 = 106Hz
Fundamentals of Spectrophotometry
Properties of Light
1.) Particles and Waves

Parameters used to describe light
-
Energy (E): the energy of one particle of light (photon) is proportional to its
frequency
E  hn
where:
E = photon energy (Joules)
n = frequency (sec-1)
h = Planck’s constant (6.626x10-34J-s)
As frequency (n) increases, energy (E) of light increases
Fundamentals of Spectrophotometry
Properties of Light
1.) Particles and Waves

Relationship between Frequency and Wavelength
ln  c  n  c / l
where:

c = speed of light (3.0x108 m/s in vacuum))
n = frequency (sec-1)
l = wavelength (m)
Relationship between Energy and Wavelength
E
where:
hc
l
~
 hcn
n~ = (1/l) = wavenumber
As frequency (l) decreases, energy (E) of light increases
Fundamentals of Spectrophotometry
Properties of Light
2.) Types of Light – The Electromagnetic Spectrum

Note again, energy (E) of light increase as frequency (n) increases or
wavelength (l) decreases
Fundamentals of Spectrophotometry
Properties of Light
2.) Types of Light – The Electromagnetic Spectrum
Fundamentals of Spectrophotometry
Absorption of Light
1.) Colors of Visible Light

Many Types of Chemicals Absorb Various Forms of Light

The Color of Light Absorbed and Observed passing through the Compound are
Complimentary
Fundamentals of Spectrophotometry
Absorption of Light
2.) Ground and Excited State

When a chemical absorbs light, it goes from a low energy state (ground state)
to a higher energy state (excited state)
Energy required of photon
to give this transition:
DE  E1 - Eo

Only photons with energies exactly equal to the energy difference between the
two electron states will be absorbed

Since different chemicals have different electron shells which are filled, they
will each absorb their own particular type of light
-
Different electron ground states and excited states
Fundamentals of Spectrophotometry
Absorption of Light
3.) Beer’s Law

The relative amount of a certain wavelength of light absorbed (A) that passes
through a sample is dependent on:
distance the light must pass through the sample (cell path length - b)
amount of absorbing chemicals in the sample (analyte concentration – c)
ability of the sample to absorb light (molar absorptivity - e)
Increasing [Fe2+]
Absorbance is directly proportional to concentration of Fe+2
Fundamentals of Spectrophotometry
Absorption of Light
3.) Beer’s Law

The relative amount of light making it through the sample (P/Po) is known as
the transmittance (T)
P
T
Po
Percent transmittance
 P 

%T  100  
 Po 
T has a range of 0 to 1, %T has a range of 0 to 100%
Fundamentals of Spectrophotometry
Absorption of Light
3.) Beer’s Law

Absorbance (A) is the relative amount of light absorbed by the sample and is
related to transmittance (T)
-
Absorbance is sometimes called optical density (OD)
P 
A  - log    - log (T )  - log (%T / 100 )
 Po 
A has a range of 0 to infinity
Fundamentals of Spectrophotometry
Absorption of Light
3.) Beer’s Law


Absorbance is useful since it is directly related to the analyte concentration,
cell pathlength and molar absorptivity.
This relationship is known as Beer’s Law
A  ebc
where:
Beer’s Law allows compounds
to be quantified by their ability
to absorb light, Relates directly
to concentration (c)
A = absorbance (no units)
e = molar absorptivity (L/mole-cm)
b = cell pathlength (cm)
c = concentration of analyte (mol/L)
Fundamentals of Spectrophotometry
Absorption of Light
4.) Absorption Spectrum



Different chemicals have different energy
levels
-
different ground vs. excited electron states
-
will have different abilities to absorb light at
any given wavelength
Absorption Spectrum – plot of absorbance (or
e) vs. wavelength for a compound
The greater the absorbance of a compound at
a given wavelength (high e), the easier it will
be to detect at low concentrations
Fundamentals of Spectrophotometry
Absorption of Light
4.) Absorption Spectrum

By choosing different wavelengths of light (lA vs. lB) different compounds
can be measured
lA
lB
Fundamentals of Spectrophotometry
Spectrophotometer
1.) Basic Design

An instrument used to make absorbance or transmittance measurements is
known as a spectrophotometer
Fundamentals of Spectrophotometry
Spectrophotometer
1.) Basic Design
Light Source: provides the light to be passed through the sample
Tungsten Lamp: visible light (320-2500 nm)

Low pressure (vacuum)
Tungsten Filament
-
- based on black body radiation:
heat solid filament to glowing, light emitted will
be characteristic of temperature more than
nature of solid filament
Deuterium Lamp: ultraviolet Light (160-375 nm)
D2 or H2 Gas
Filament
40V
Electric Arc
In presence of arc, some of the electrical energy is
absorbed by D2 (or H2) which results in the
Sealed Quartz
Tube
disassociation
of the gas and release of light
Electrode
D2 + Eelect  D*2  D’ + D’’ + hn (light produced)
Excited state
Fundamentals of Spectrophotometry
Spectrophotometer
1.) Basic Design

Wavelength Selector (monochromator): used to select a given
wavelength of light from the light source
Prism:
-
Filter:
Fundamentals of Spectrophotometry
Spectrophotometer
1.) Basic Design

Wavelength Selector (monochromator): used to select a given
wavelength of light from the light source
Reflection or Diffraction Grating:
Fundamentals of Spectrophotometry
Spectrophotometer
1.) Basic Design

Sample Cell: sample container of fixed length (b).
-
Usually round or square cuvet
Made of material that does not absorb light in the wavelength
range of interest
1.
Glass – visible region
2.
Quartz – ultraviolet
3.
NaCl, KBr – Infrared region
Fundamentals of Spectrophotometry
Spectrophotometer
1.) Basic Design

Light Detector: measures the amount of light passing through the
sample.
-
Usually works by converting light signal into electrical signal
Photomultiplier tube
Process:
a) light hits photoemissive cathode and e- is emitted.
b) an emitted e- is attracted to electrode #1
(dynode 1), which is 90V more positive.
Causes several more e- to be emitted.
c) these e- are attracted to dynode 2, which is
90V more positive then dynode 1, emitting
more e-.
d) process continues until e- are collected at
anode after amplification at 9 dynodes.
e) overall voltage between anode and cathode
is 900V.
f) one photon produces 106 – 107 electrons.
g) current is amplified and measured
Fundamentals of Spectrophotometry
Spectrophotometer
2.) Types of Spectrophotometers

Single-Beam Instrument: sample and blank are alternatively
measured in same sample chamber.
Fundamentals of Spectrophotometry
Spectrophotometer
2.) Types of Spectrophotometers

Double-Beam Instrument
-
Continuously compares sample and blank
Automatically corrects for changes in
electronic signal or light intensity of source
Fundamentals of Spectrophotometry
Chemical Analysis
1.) Calibration

To measure the absorbance of a sample, it is
necessary to measure Po and P ratio
-

Po – the amount of light passing through the
system with no sample present
P – the intensity of light when the sample is
present
Po is measured with a blank cuvet
-
Cuvet contains all components in the sample
solution except the analyte of interest

P is measured by placing the sample in the cuvet.

To accurately measure an unknown concentration,
obtain a calibration curve using a range of known
concentrations for the analyte
Fundamentals of Spectrophotometry
Chemical Analysis
2.) Limitations in Beer’s Law


Results in non-linear calibration curve
At high concentrations, solute molecules
influence one another because of their proximity
-
Molar absorptivity changes
Affect on equilibrium, (HA and A- have
difference absorption)

Analyte properties change in different solvents

Errors in reproducible positioning of cuvet
-

Also problems with dirt & fingerprints
Instrument electrical noise
Keep A in range of 0.1 – 1.5 absorbance units (80 -3%T)
Fundamentals of Spectrophotometry
Chemical Analysis
3.) Precautions in Quantitative Absorbance Measurements

Choice of Wavelength
-
Choose a wavelength at an absorption maximum

-
Minimizes deviations from Beer’s law, which assumes e is constant
Pick peak in absorption spectrum where analyte is only compound
absorbing light
Or choose a wavelength where the analyte has the largest difference in its
absorbance relative to other sample components
Bad choice for either
Best choice compound (b)
(a)
compound (a) or (b)
Fundamentals of Spectrophotometry
Chemical Analysis
4.) Example:
A 3.96x10-4 M solution of compound A exhibited an absorbance of 0.624 at 238
nm in a 1.000 cm cuvet. A blank had an absorbance of 0.029. The absorbance
of an unknown solution of compound A was 0.375.
Find the concentration of A in the unknown.
Fundamentals of Spectrophotometry
What Happens When a Molecule
Absorbs Light?
1.) Molecule Promoted to a More
Energetic Excited State

Absorption of UV-vis light results in
an electron promoted to a higher
energy molecular orbital
s  s*
transition in vacuum UV
n  s*
saturated compounds with non-bonding electrons
n  p*, p  p*
requires unsaturated functional groups
(eq. double bonds)
most commonly used, energy good range for UV/Vis
Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
1.) Molecule Promoted to a More Energetic Excited State

Geometrical Structure of the Excited State will Differ from the Ground State
Ground State
Excitation of an electron to the pi antibonding
orbital (p*) in formaldehyde produces
repulsion instead of attraction between
the carbon and oxygen atom
Excited State
Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
1.) Molecule Promoted to a More Energetic Excited State

Two Possible Transitions in Excited State
-
Single state – electron spins opposed
Triplet state – electron spins are parallel

In general, triplet state has lower energy than singlet state

Singlet to Triplet transition has a very low probability

Singlet to Singlet Transition are more probable
Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
2.) Infrared and Microwave Radiation

Not energetic enough to induce electronic transition

Change vibrational, translational and rotational motion of the molecule
-
The entire molecule and each atom can move along the x, y, z-axis
When correct wavelength is absorbed,

Oscillations of the atom vibration is increased in amplitude

Molecule rotates or moves (translation) faster
Vibrational States of Formaldehyde
Energy: Electronic >> Vibrational > Rotational
symmetric
Out-of-plane twisting
asymmetric
In-plane rocking
In-plane scissoring
Out-of-plane wagging
Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
3.) Combined Electronic, Vibrational and Rotational Transitions


Absorption of photon with sufficient energy to excite an electron will also
cause vibrational and rotational transitions
There are multiple vibrational and rotational energy levels associated with
each electronic state
-

Excited vibrational and rotational states are lower energy than electronic
state
Therefore, transition between electronic states can occur between different
vibrational and rotational states
Vibrational and rotational states
associated with an electronic state
Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
4.) Relaxation Processes from Excited State


There are multiple possible relaxation pathways
Vibrational, Rotational relaxation occurs through collision with solvent or
other molecules
-

energy is converted to heat (radiationless transition)
Electronic relaxation occurs through the release of a photon (light)
Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
4.) Relaxation Processes from Excited State


Internal conversion – transition between singlet electronic states through
overlapping vibrational states
Intersystem crossing – transition between a singlet electronic state to a
triplet electronic state by overlapping vibrational states
Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
4.) Relaxation Processes from Excited State

Fluorescence – emitting a photon by relaxing from an excited singlet
electronic states to a ground singlet state
S1  So

Phosphorescence – emitting a photon by relaxing from an excited triplet
electronic states to a ground singlet state
T1  So
Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
5.) Fluorescence and Phosphorescence




Relative rates of relaxation depends on the molecule, the solvent,
temperature, pressure, etc.
Energy of Phosphorescence is less than the energy of fluorescence
Phosphorescence occurs at a longer wavelengths than fluorescence
Lifetime of Fluorescence (10-8 to 10-4 s) is very short compared to
phosphorescence (10-4 to 102 s)
Fluorescence and phosphorescence are relatively rare
Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
5.) Fluorescence and Phosphorescence


Fluorescence and phosphorescence come at lower energy than absorbance
Emission spectrum is roughly mirror image of absorption spectrum
Color Change Due to Fluorescence at
Higher Wavelength
Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
5.) Fluorescence and Phosphorescence

Emission spectrum are of lower energy or
higher wavelength because of the
efficiency of vibrational relaxation
-
Absorption to an excited vibrational
state will relax quickly to a ground
vibrational state before the electronic
relaxation
Fundamentals of Spectrophotometry
What Happens When a Molecule Absorbs Light?
5.) Fluorescence and Phosphorescence

Also, differences in stability of excited and ground state structure
contribute to energy difference
Fundamentals of Spectrophotometry
Chemical Analysis
1.)
Excitation and Emission Spectra
Excitation Spectra – measure fluorescence or
phosphorescence at a fixed wavelength while
varying the excitation wavelength.
Emission Spectra – measure fluorescence or
phosphorescence over a range of wavelengths
using a fixed varying excitation wavelength.
Fundamentals of Spectrophotometry
Chemical Analysis
2.)
Fluorescence and Phosphorescence Intensity

At low concentration, emission intensity is proportional to analyte
concentration
-
Related to Beer’s law
I  kPo c
where:

k = constant
Po = light intensity
c = concentration of analyte (mol/L)
At high concentrations, deviation from linearity occurs
-
Emission decreases because absorption increases more rapidly
Emission is quenched  absorption of excitation or emission energy by
analyte molecules in solution
Fundamentals of Spectrophotometry
Chemical Analysis
3.)
Example
In formaldehyde, the transition n p*(T1) occurs at 397 nm, and the np*(S1)
transition comes at 355 nm. What is the difference in energy (kJ/mol) between
the S1 and T1 states?