Fundamentals of Spectrophotometry

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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 solutions 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 samples ability or inability to absorb light
  at a certain wavelength.

Colorimetry
Instrumental Methods (spectrophotometry)
Non-Instrumental Methods
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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
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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 waves 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

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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

where E photon energy (Joules) n frequency
(sec-1) h Plancks constant (6.626x10-34J-s)
As frequency (n) increases, energy (E) of light
increases
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Fundamentals of Spectrophotometry

• Properties of Light
• 1.) Particles and Waves
• Relationship between Frequency and Wavelength
• Relationship between Energy and Wavelength

where c speed of light (3.0x108 m/s in
vacuum)) n frequency (sec-1) l wavelength
(m)
where (1/l) wavenumber
As frequency (l) decreases, energy (E) of light
increases
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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

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Fundamentals of Spectrophotometry

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

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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

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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)
• 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

Energy required of photon to give this
transition DE E1 - Eo
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Fundamentals of Spectrophotometry

• Absorption of Light
• 3.) Beers 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 Fe2
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Fundamentals of Spectrophotometry

• Absorption of Light
• 3.) Beers Law
• The relative amount of light making it through
  the sample (P/Po) is known as the transmittance
  (T)

Percent transmittance
T has a range of 0 to 1, T has a range of 0 to
100
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Fundamentals of Spectrophotometry

• Absorption of Light
• 3.) Beers 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)

A has a range of 0 to infinity
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Fundamentals of Spectrophotometry

• Absorption of Light
• 3.) Beers Law
• Absorbance is useful since it is directly related
  to the analyte concentration, cell pathlength and
  molar absorptivity.
• This relationship is known as Beers Law

where A absorbance (no units) e molar
absorptivity (L/mole-cm) b cell pathlength
(cm) c concentration of analyte (mol/L)
Beers Law allows compounds to be quantified by
their ability to absorb light, Relates directly
to concentration (c)
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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

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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
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Fundamentals of Spectrophotometry

• Spectrophotometer
• 1.) Basic Design
• An instrument used to make absorbance or
  transmittance measurements is known as a
  spectrophotometer

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

- based on black body radiation heat solid
filament to glowing, light emitted will be
characteristic of temperature more than nature of
solid filament
Low pressure (vacuum)
Tungsten Filament
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 ? D2 ? D D hn
(light produced) Excited state
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Fundamentals of Spectrophotometry

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

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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

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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
• Glass visible region
• Quartz ultraviolet
• NaCl, KBr Infrared region

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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

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
Photomultiplier tube
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Fundamentals of Spectrophotometry

• Spectrophotometer
• 2.) Types of Spectrophotometers
• Single-Beam Instrument sample and blank are
  alternatively measured in same sample chamber.

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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

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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

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Fundamentals of Spectrophotometry

• Chemical Analysis
• 2.) Limitations in Beers 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
-3T)
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Fundamentals of Spectrophotometry

• Chemical Analysis
• 3.) Precautions in Quantitative Absorbance
  Measurements
• Choice of Wavelength
• Choose a wavelength at an absorption maximum
• Minimizes deviations from Beers 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 compound (a) or (b)
Best choice compound (b)
Best choice compound (a)
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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.
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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

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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
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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

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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 gtgt Vibrational gt Rotational
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symmetric
asymmetric
In-plane scissoring
Out-of-plane wagging
In-plane rocking
Out-of-plane twisting
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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
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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)

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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

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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

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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

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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
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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

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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

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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.
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Fundamentals of Spectrophotometry

• Chemical Analysis
• 2.) Fluorescence and Phosphorescence Intensity
• At low concentration, emission intensity is
  proportional to analyte concentration
• Related to Beers law
• 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

where k constant Po light intensity c
concentration of analyte (mol/L)
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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?