Chapter 18

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
Non-Instrumental Methods
(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]
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 (): distance (nm, cm, m) from peak to peak
- Frequency (): 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
Properties of Light

 Parameters used to describe light
- Energy (E): the energy of one particle of light (photon) is proportional to its
frequency
E  h
where: E = photon energy (Joules)
= frequency (sec-1)
h = Planck’s constant (6.626x10-34J-s)
As frequency () increases, energy (E) of light increases

Properties of Light

 Relationship between Frequency and Wavelength
  c    c / 
where: c = speed of light (3.0x108 m/s in vacuum))
= frequency (sec-1)
 = wavelength (m)
 Relationship between Energy and Wavelength
hc ~
E  hc

where: ~ = (1/) = wavenumber
As frequency () decreases, energy (E) of light increases

Properties of Light
2.) Types of Light – The Electromagnetic Spectrum

 Note again, energy (E) of light increase as frequency () increases or
wavelength () decreases
Properties of Light
2.) Types of Light – The Electromagnetic Spectrum

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
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:

 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
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 - )
Increasing [Fe2+]
Absorbance is directly proportional to concentration of Fe+2

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
 P 
Percent transmittance %T  100   
 Po 
T has a range of 0 to 1, %T has a range of 0 to 100%

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 )

P
 o
A has a range of 0 to infinity

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  bc
where: A = absorbance (no units)
= molar absorptivity (L/mole-cm)
b = cell pathlength (cm)
c = concentration of analyte (mol/L)
Beer’s Law allows compounds

to be quantified by their ability
to absorb light, Relates directly
to concentration (c)
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

) vs. wavelength for a compound
 The greater the absorbance of a compound at

a given wavelength (high ), the easier it will
be to detect at low concentrations
Absorption of Light
4.) Absorption Spectrum

 By choosing different wavelengths of light (A vs. B) different compounds
can be measured
A B
Spectrophotometer
1.) Basic Design

 An instrument used to make absorbance or transmittance measurements is
known as a spectrophotometer
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) - based on black body radiation:

heat solid filament to glowing, light emitted will
Tungsten Filament be characteristic of temperature more than
nature of solid filament
- Deuterium Lamp: ultraviolet Light (160-375 nm)
In presence of arc, some of the electrical energy is

absorbed by D2 (or H2) which results in the
disassociation of the gas and release of light
D2 + Eelect  D*2  D’ + D’’ + h(light produced)

Excited state
Spectrophotometer
1.) Basic Design

 Wavelength Selector (monochromator): used to select a given
wavelength of light from the light source
- Prism:
- Filter:
Spectrophotometer
1.) Basic Design

 Wavelength Selector (monochromator): used to select a given
wavelength of light from the light source
- Reflection or Diffraction Grating:
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

Spectrophotometer
1.) Basic Design

 Light Detector: measures the amount of light passing through the
sample.
- Usually works by converting light signal into electrical signal
Process:
Photomultiplier tube
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
Spectrophotometer
2.) Types of Spectrophotometers

 Single-Beam Instrument: sample and blank are alternatively
measured in same sample chamber.
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
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
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)

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  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)
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.

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
  *
transition in vacuum UV
n  *
saturated compounds with non-bonding electrons
n  *,   *
requires unsaturated functional groups
(eq. double bonds)
most commonly used, energy good range for UV/Vis
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 (*) in formaldehyde produces
repulsion instead of attraction between Excited State
the carbon and oxygen atom
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

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 asymmetric In-plane scissoring
Out-of-plane twisting In-plane rocking Out-of-plane wagging

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
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)

 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

 Fluorescence – emitting a photon by relaxing from an excited singlet
electronic states to a ground singlet state
S1  S o
 Phosphorescence – emitting a photon by relaxing from an excited triplet

electronic states to a ground singlet state
T 1  So
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

 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

 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

 Also, differences in stability of excited and ground state structure
contribute to energy difference
Chemical Analysis
1.) Excitation and Emission Spectra
Excitation Spectra – measure fluorescence or Emission Spectra – measure fluorescence or

phosphorescence at a fixed wavelength while phosphorescence over a range of wavelengths
varying the excitation wavelength. using a fixed varying excitation wavelength.
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
Chemical Analysis
3.) Example
In formaldehyde, the transition n *(T1) occurs at 397 nm, and the n*(S1)
transition comes at 355 nm. What is the difference in energy (kJ/mol) between
the S1 and T1 states?

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Fundamentals of Spectrophotometry

1.) Particles and Waves

- amplitude (A): height of wave’s electric vector

- Wavelength (): distance (nm, cm, m) from peak to peak

1.) Particles and Waves

As frequency () increases, energy (E) of light increases

1.) Particles and Waves

As frequency () decreases, energy (E) of light increases

2.) Types of Light – The Electromagnetic Spectrum

2.) Types of Light – The Electromagnetic Spectrum

1.) Colors of Visible Light

2.) Ground and Excited State

Energy required of photon

3.) Beer’s Law

Absorbance is directly proportional to concentration of Fe+2

3.) Beer’s Law

T has a range of 0 to 1, %T has a range of 0 to 100%

3.) Beer’s Law

A has a range of 0 to infinity

3.) Beer’s Law

Beer’s Law allows compounds

4.) Absorption Spectrum

- will have different abilities to absorb light at

 Absorption Spectrum – plot of absorbance (or

 The greater the absorbance of a compound at

4.) Absorption Spectrum

1.) Basic Design

1.) Basic Design

Low pressure (vacuum) - based on black body radiation:

- Deuterium Lamp: ultraviolet Light (160-375 nm)

In presence of arc, some of the electrical energy is

D2 + Eelect  D*2  D’ + D’’ + h(light produced)

1.) Basic Design

1.) Basic Design

1.) Basic Design

1. Glass – visible region

3. NaCl, KBr – Infrared region

1.) Basic Design

anode after amplification at 9 dynodes.

2.) Types of Spectrophotometers

2.) Types of Spectrophotometers

 Po is measured with a blank cuvet

 P is measured by placing the sample in the cuvet.

 To accurately measure an unknown concentration,

2.) Limitations in Beer’s Law

 Analyte properties change in different solvents

 Errors in reproducible positioning of cuvet

 Instrument electrical noise

Keep A in range of 0.1 – 1.5 absorbance units (80 -3%T)

3.) Precautions in Quantitative Absorbance Measurements

Bad choice for either

A 3.96x10-4 M solution of compound A exhibited an absorbance of 0.624 at 238

Find the concentration of A in the unknown.

1.) Molecule Promoted to a More

1.) Molecule Promoted to a More Energetic Excited State

Excitation of an electron to the pi antibonding

1.) Molecule Promoted to a More Energetic Excited State

 Singlet to Triplet transition has a very low probability

 Singlet to Singlet Transition are more probable

2.) Infrared and Microwave Radiation