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Fundamentals of Spectrophotometry

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Title: Fundamentals of Spectrophotometry


1
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
2
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
3
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

4
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
5
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 wavelength (l) decreases, energy (E) of light
increases
6
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

7
Fundamentals of Spectrophotometry
  • Properties of Light
  • 2.) Types of Light The Electromagnetic Spectrum

8
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

9
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
10
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
11
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
12
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
13
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)
14
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

15
Fundamentals of Spectrophotometry
  • Absorption of Light
  • 4.) Absorption Spectrum
  • By choosing different wavelengths of light (lA
    vs. lB) different compounds can be measured

16
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

17
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)
18
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)
19
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.
20
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

21
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

10-4 to 10 s
Life-times
10-5 to 10-8 s
22
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
23
symmetric
asymmetric
In-plane scissoring
Out-of-plane wagging
In-plane rocking
Out-of-plane twisting
24
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
25
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)

26
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

27
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

28
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

29
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
30
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

31
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.
32
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)
33
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?
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