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Electronic Spectroscopy Ultraviolet and visible spectroscopy


									Course on Analytical Methods
     Electronic Spectroscopy
Ultraviolet and visible spectroscopy
                    Some applications
             Some features of measurements
             Identification of organic species
            Quantification of Inorganic species
                  Colorimetric analysis
            The origin of the analytical signal
Excitation of atom or molecule by ultra violet or visible radiation
                          190-900 nm
The essential features
• Count the number of photons (intensity)
• Energy analysis
• Analyze other effects (polarizations)
Where in the spectrum are these transitions?
Electronic Excitation by UV/Vis Spectroscopy :
                 UV:                       Radio waves:
 X-ray:                       IR:
                 valance                   Nuclear spin states
 core electron                molecular
 excitation      electronic                (in a magnetic field)
       Ultraviolet (UV) Spectroscopy – Use and Analysis
Of all the forms of radiation that go to make up the electromagnetic
spectrum UV is probably the most familiar to the general public
(after the radiation associated with visible light which is, for the
most part, taken for granted).
UV radiation is widely known as something to be aware of in hot
weather in having a satisfactory effect of tanning the skin but which
also has the capacity to damage skin cells to the extent that skin
cancer is a direct consequence of overexposure to UV radiation.
This damage is associated with the high energy of UV radiation
which is directly related to its high frequency and its low wavelength
(see the equations below).
                 E = energy; c = speed of light;  = wavelength;
                       = frequency; h = Planck‟s constant

      c =         E = h          E = (hc)/               E  1/ 
              Ultraviolet (UV) Spectroscopy – Use and Analysis
 This slide is part automatically animated – if animation does not occur click left hand mouse button.
 When continuous wave radiation is passed through a               When continuous wave radiation passes through a
 prism a diffraction pattern is produced (called a                transparent material (solid or liquid) some of the
 spectrum) made up of all the wavelengths associated              radiation might be absorbed by that material.
 with the incident radiation.
                                                                                                  Spectrum with „gaps‟ in it


                                                                              Transparent material that
                        Diffraction prism
                                                                               absorbs some radiation
   Radiation source

If, having passed through the material, the beam is diffracted by passing through a prism it will produce a light
spectrum that has gaps in it (caused by the absorption of radiation by the transparent material through which is passed).
The effect of absorption of radiation on the transparent material is to change is from a low energy state (called the
ground state) to a higher energy state (called the excited state).
The difference between all the spectroscopic techniques is that they use different wavelength radiation that has
different associated energy which can cause different modes of excitation in a molecule.
For instance, with infra red spectroscopy the low energy radiation simply causes bonds to bend and stretch when a
molecule absorbs the radiation. With high energy UV radiation the absorption of energy causes transition of bonding
electrons from a low energy orbital to a higher energy orbital.
The energy of the „missing‟ parts of the spectrum corresponds exactly to the energy difference between the orbitals
involved in the transition.
         Ultraviolet (UV) Spectroscopy – Use and Analysis
                                    The bonding orbitals with which you are familiar are the -bonding orbitals typified
 Unoccupied                         by simple alkanes. These are low energy (that is, stable).
                                    Next (in terms of increasing energy) are the -bonding orbitals present in all
Energy Levels                       functional groups that contain double and triple bonds (e.g. carbonyl groups and

                Increasing energy
                                    Higher energy still are the non-bonding orbitals present on atoms that have lone
                                    pair(s) of electrons (oxygen, nitrogen, sulfur and halogen containing compounds).
           n                        All of the above 3 kinds of orbitals may be occupied in the ground state.

                                    Two other sort of orbitals, called antibonding orbitals, can only be occupied by an
 Occupied                           electron in an excited state (having absorbed UV for instance). These are the * and
                                   * orbitals (the * denotes antibonding). Although you are not too familiar with the
  Energy                            concept of an antibonding orbital just remember the following – whilst electron
  Levels                            density in a bonding orbital is a stabilising influence it is a destabilising influence
                                   (bond weakening) in an antibonding orbital.
                                    Antibonding orbitals are unoccupied in the ground state

                                    A transition of an electron from occupied to an unoccupied energy level can be

                                    caused by UV radiation. Not all transitions are allowed but the definition of which
                                    are and which are not are beyond the scope of this tutorial. For the time being be
                                    aware that commonly seen transitions are  to * which correctly implies that UV is
                                    useful with compounds containing double bonds.
                                    A schematic of the transition of an electron from  to * is shown on the left.
          Ultraviolet (UV) Spectroscopy – The Instrumentation
The instrumentation used to run a UV is shown below. It involves two lamps (one for visible light and one for UV
light) and a series of mirrors and prisms as well as an appropriate detector. The spectrometer effectively varies the
wavelength of the light directed through a sample from high wavelength (low energy) to low wavelength (high
As it does so any chemical dissolved in a sample cell through which the light is passing may undergo electronic
transitions from the ground state to the excited state when the incident radiation energy is exactly the same as the
energy difference between these two states. A recorder is then used to record, on a suitable scale, the absorption of
energy that occurs at each of the wavelengths through which the spectrometer scans.

                                                             The recorder assembly

                                                             The spectrometer itself – this houses the lamps, mirrors,
                                                             prisms and detector. The spectrometer splits the beam of
                                                             radiation into two and passes one through a sample and
                                                             one through a reference solution (that is always made up
                                                             of the solvent in which you have dissolved the sample).
                                                             The detector measures the difference between the sample
                                                             and reference readings and communicates this to the

 The samples are dissolved in a solvent which is transparent to UV light and put into sample cells called cuvettes.
 The cells themselves also have to be transparent to UV light and are accurately made in all dimensions. They are
 normally designed to allow the radiation to pass through the sample over a distance of 1cm.
                  Ultraviolet (UV) Spectroscopy – The Output
The output from a UV scanning spectrometer is not the most informative looking piece of data!! It looks like a series
of broad humps of varying height. An example is shown below.

                                                                                       *Absorbance has no units
                                                                                       – it is actually the
                                                                                       logarithm of the ratio of

                                  Increasing absorbance *
                                                                                       light intensity incident on
                                                                                       the sample divided by the
Beer Lambert Law                                                                       light intensity leaving the

        A = .c.l

                                                             Decreasing wavelength in nm

 There are two particular strengths of UV (i) it is very sensitive (ii) it is very useful in determining the quantity of
 a known compound in a solution of unknown concentration. It is not so useful in determining structure although it
 has been used in this way in the past.
 The concentration of a sample is related to the absorbance according to the Beer Lambert Law which is described
 A = absorbance; c = concentration in moles l-1; l = pathlength in cm ;  = molar absorptivity (also known as
 extinction coefficient) which has units of moles-1 L cm -1.
             Ultraviolet (UV) Spectroscopy – Analysing the Output
                                   Beer Lambert                      Handling samples of known concentration
                                       Law                   If you know the structure of your compound X and you wish
                                                             to acquire UV data you would do the following.
                                     A = .c.l               Prepare a known concentration solution of your sample.
                                                             Run a UV spectrum (typically from 500 down to 220 nm).
0.5                                                          From the spectrum read off the wavelength values for each of
                                                             the maxima of the spectra (see left)
                                                                Read off the absorbance values of each of the maxima (see
0.0                                                             Then using the known concentration (in moles L-1 ) and the
            350                    400                      450 known pathlength (1 cm) calculate the molar absorptivity ()
                                            wavelength (nm)
                                                                for each of the maxima.
    Determining concentration of samples with                   Finally quote the data as follows (for instance for the largest
           known molar absorptivity ().                        peak in the spectrum to the left and assuming a concentration
Having used the calculation in the yellow box to                of 0.0001 moles L-1 ).
work out the molar absorptivity of a compound you                                  max = 487nm A= 0.75
can now use UV to determine the concentration of                         = 0.75 /(0.001 x 1.0) = 7500 moles-1 L cm -1
compound X in other samples (provided that these
sample only contain pure X).
Simply run the UV of the unknown and take the absorbance reading at the maxima for which you have a known value
of . In the case above this is at the peak with the highest wavelength (see above).
Having found the absorbance value and knowing  and l you can calculate c.
This is the basis of your calculation in Experiment 4 of CH199 and also the principle used in many experiments to
determine the concentration of a known compound in a particular test sample – for instance monitoring of drug
metabolites in the urine of drug takers; monitoring biomolecules produced in the body during particular disease states
      UV / visible Spectroscopy

Abs               Abs

         / nm             / nm
UV / visible Spectroscopy
   UV / visible Spectroscopy

    • Electronic transitions involve the
  promotion of electrons from an occupied
      orbital to an unoccupied orbital.

• Energy differences of 125 - 650 kJ/mole.
UV / visible Spectroscopy

    • Beer-Lambert Law

 A = log(IO/I) = cl
    UV / visible Spectroscopy

               A = log(IO/I) = cl

– A = Absorbance (optical density)
– IO = Intensity of light on the sample cell
– I = Intensity of light leaving the sample cell
– c = molar concentration of solute
– l = length of sample cell (cm)
 = molar absorptivity (molar extinction
 UV / visible Spectroscopy

• The Beer-Lambert Law is rigorously
obeyed when a single species is present
    at relatively low concentrations.
  UV / visible Spectroscopy

• The Beer-Lambert Law is not obeyed:
             – High concentrations

     – Solute and solvent form complexes

 – Thermal equilibria exist between the ground
           state and the excited state

– Fluorescent compounds are present in solution
    UV / visible Spectroscopy

• The size of the absorbing system and the
 probability that the transition will take place
          control the absorptivity ().

• Values above 104 are termed high intensity

 • Values below 1000 indicate low intensity
 absorptions which are forbidden transitions.
UV / visible Spectroscopy

    • Organic Spectroscopy

    • Transitions between
 UV / visible Spectroscopy

• Highest occupied molecular orbital

• Lowest unoccupied molecular orbital
UV / visible Spectroscopy
UV / visible Spectroscopy

• Not all transitions are observed

 • There are restrictions called
       Selection Rules

       • This results in
    Forbidden Transitions
   UV / visible Spectroscopy

• The characteristic energy of a transition
 and the wavelength of radiation absorbed
  are properties of a group of atoms rather
        than of electrons themselves.

• The group of atoms producing such an
          absorption is called a
UV / visible Spectroscopy
UV / visible Spectroscopy
   UV / visible Spectroscopy

• It is often difficult to extract a great deal
   of information from a UV spectrum by

    • Generally you can only pick out
          conjugated systems.
UV / visible Spectroscopy
 UV / visible Spectroscopy

 use in conjunction with
nmr and infrared spectra.
UV / visible Spectroscopy
• As structural changes occur in a
 chromophore it is difficult to predict
exact energy and intensity changes.

    • Use empirical rules.
Woodward-Fieser Rules for dienes
 Woodward’s Rules for enones
UV / visible Spectroscopy
1. Bathochromic shift (red shift)
  – lower energy, longer wavelength
2. Hypsochromic shift (blue shift)
  – higher energy, shorter wavelength.
3. Hyperchromic effect
  – increase in intensity
4. Hypochromic effect
  – decrease in intensity
             Spectroscopic Techniques and
                 Chemistry they Probe
UV-vis                  UV-vis region   bonding electrons

Atomic Absorption       UV-vis region   atomic transitions (val. e-)

FT-IR                   IR/Microwave    vibrations, rotations

Raman                   IR/UV           vibrations

FT-NMR                  Radio waves     nuclear spin states

X-Ray Spectroscopy      X-rays          inner electrons, elemental

X-ray Crystallography   X-rays          3-D structure
        Spectroscopic Techniques and Common Uses
UV-vis                  UV-vis region   analysis/Beer’s Law
                                          Quantitative analysis
Atomic Absorption       UV-vis region        Beer’s Law

FT-IR                   IR/Microwave    Functional Group Analysis
                                          Functional Group
Raman                   IR/UV              Analysis/quant

FT-NMR                  Radio waves      Structure determination

X-Ray Spectroscopy      X-rays             Elemental Analysis

X-ray Crystallography   X-rays          3-D structure Anaylysis
      Different Spectroscopies
• UV-vis – electronic states of valence e/d-orbital
  transitions for solvated transition metals
• Fluorescence – emission of UV/vis by certain
• FT-IR – vibrational transitions of molecules
• FT-NMR – nuclear spin transitions
• X-Ray Spectroscopy – electronic transitions of
  core electrons
    Quantitative Spectroscopy
• Beer’s Law
  Al1 = el1bc
  e is molar absorptivity (unique for a given
  compound at l1)
  b is path length
  c concentration
                Beer’s Law

•   A = -logT = log(P0/P) = ebc
•   T = Psolution/Psolvent = P/P0
•   Works for monochromatic light
•   Compound x has a unique e at different
           Characteristics of
           Beer’s Law Plots
• One wavelength
• Good plots have a range of absorbances
  from 0.010 to 1.000
• Absorbances over 1.000 are not that valid
  and should be avoided
• 2 orders of magnitude
            Standard Practice
•   Prepare standards of known concentration
•   Measure absorbance at max
•   Plot A vs. concentration
•   Obtain slope
•   Use slope (and intercept) to determine the
    concentration of the analyte in the
Typical Beer’s Law Plot

                y = 0.02x

          0.0          20.0      40.0     60.0
                     concentration (uM)
        UV-Vis Spectroscopy
• UV- organic molecules
  – Outer electron bonding transitions
  – conjugation
• Visible – metal/ligands in solution
  – d-orbital transitions
• Instrumentation
  Characteristics of UV-Vis spectra of
         Organic Molecules
• Absorb mostly in UV unless highly
• Spectra are broad, usually to broad for
  qualitative identification purposes
• Excellent for quantitative Beer’s Law-type
• The most common detector for an HPLC
Molecules have quantized energy levels:
  ex. electronic energy levels.


                                                         = hv
   Q: Where do these quantized energy levels come from?
   A: The electronic configurations associated with bonding.

    Each electronic energy level
    (configuration) has
    associated with it the many
    vibrational energy levels we
    examined with IR.
            Broad spectra

• Overlapping vibrational and rotational
• Solvent effects
     Molecular Orbital Theory
• Fig 18-10

 2p              2p


2s              2s

Ethane                             C C

                         *                                *

                                                           

                                        *
      H         H
                     max = 135 nm (a high energy transition)
          C C
      H         H

 Absorptions having max < 200 nm are difficult to observe because
 everything (including quartz glass and air) absorbs in this spectral
                   *                      *
C C               *                      *
                                                   = hv
                                         
                                             
                                  *
      Example: ethylene absorbs at longer wavelengths:
                   max = 165 nm = 10,000
                             *                            *
                            *                            *
                 n                      hv

                                                         
                                                             
                                    n        *
      The n to pi* transition is at even lower wavelengths but is not
      as strong as pi to pi* transitions. It is said to be “forbidden.”
              Acetone:         n* max = 188 nm ; = 1860
                               n* max = 279 nm ; = 15
C C         *            135 nm

C C         *            165 nm
C O         n*            183 nm    weak

C O         *            150 nm
            n*            188 nm
            n*            279 nm    weak

                      180 nm

      C O
                                279 nm

Conjugated systems:
      C   C



Preferred transition is between Highest Occupied Molecular Orbital
(HOMO) and Lowest Unoccupied Molecular Orbital (LUMO).
 Note: Additional conjugation (double bonds) lowers the HOMO-
 LUMO energy gap:
        1,3 butadiene:      max = 217 nm ; = 21,000
       1,3,5-hexatriene      max = 258 nm ; = 35,000
Similar structures have similar UV spectra:



max = 238, 305 nm    max = 240, 311 nm      max = 173, 192 nm

   max = 114 + 5(8) + 11*(48.0-1.7*11) = 476 nm
   max(Actual) = 474.
      Metal ion transitions


D-orbitals        D-orbitals
of naked Co       of hydrated Co2+

                  Octahedral Configuration
 Octahedral Geometry


• Fixed wavelength instruments
• Scanning instruments
• Diode Array Instruments
  Fixed Wavelength Instrument
• LED serve as source
• Pseudo-monochromatic light source
• No monochrometer necessary/ wavelength selection
  occurs by turning on the appropriate LED
• 4 LEDs to choose from

                                beam of light

       LEDs                          photodyode
                        Scanning Instrument
  Scanning Instrument

Tungsten         slit
Filament (vis)

                           slit                   tube

   Deuterium lamp
   Filament (UV)
• Tungten lamp (350-2500 nm)
• Deuterium (200-400 nm)
• Xenon Arc lamps (200-1000 nm)
• Braggs law, nl = d(sin i + sin r)
• Angular dispersion, dr/d = n / d(cos r)
• Resolution, R = /=nN, resolution is
  extended by concave mirrors to refocus
  the divergent beam at the exit slit
             Sample holder
• Visible; can be plastic or glass
• UV; you must use quartz
 Single beam vs. double beam
• Source flicker
                 Diode array Instrument
                                                      Diode array detector
                                                      328 individual detectors
Filament (vis)


Deuterium lamp
Filament (UV)

• Scanning instrument
  – High spectral resolution (63000), /
  – Long data acquisition time (several
  – Low throughput
• Diode array
  – Fast acquisition time (a couple of
    seconds), compatible with on-line
  – High throughput (no slits)
  – Low resolution (2 nm)

                  6-port    HPLC
Mobile   Sample             column
phase    loop

        UV / visible Spectroscopy
• The radiation which is absorbed has an energy
  which exactly matches the energy difference
  between the ground state and the excited state.

• These absorptions correspond to electronic
                Why should we learn this stuff?
   After all, nobody solves structures with UV any longer!

Many organic molecules have chromophores that absorb UV

UV absorbance is about 1000 x easier to detect per mole than NMR

Still used in following reactions where the chromophore changes. Useful
because timescale is so fast, and sensitivity so high. Kinetics, esp. in
biochemistry, enzymology.

Most quantitative Analytical chemistry in organic chemistry is conducted
using HPLC with UV detectors

One wavelength may not be the best for all compound in a mixture.
Affects quantitative interpretation of HPLC peak heights
     Uses for UV another aspect
Knowing UV can help you know when to be skeptical of quant results. Need
    to calibrate response factors
Assessing purity of a major peak in HPLC is improved by “diode array” data,
    taking UV spectra at time points across a peak. Any differences could
    suggest a unresolved component. “Peak Homogeneity” is key for purity
Sensitivity makes HPLC sensitive
e.g. validation of cleaning procedure for a production vessel
But you would need to know what compounds could and could not be detected
    by UV detector! (Structure!!!)
One of the best ways for identifying the presence of acidic or basic groups, due
    to big shifts in  for a chromophore containing a phenol, carboxylic acid,

              “hypsochromic” shift        “bathochromic” shift

      The UV Absorption process
–   * and   * transitions: high-energy, accessible in
  vacuum UV (max <150 nm). Not usually observed in molecular
– n  * and   * transitions: non-bonding electrons (lone
  pairs), wavelength (max) in the 150-250 nm region.
– n  * and   * transitions: most common transitions
  observed in organic molecular UV-Vis, observed in compounds
  with lone pairs and multiple bonds with max = 200-600 nm.
– Any of these require that incoming photons match in energy the
  gap corrresponding to a transition from ground to excited state.
– Energies correspond to a 1-photon of 300 nm light are ca. 95
    What are the nature of these absorptions?

                                           π*            π*        π*       Example
 Example:   * transitions
 responsible for ethylene                  π*            π*        π*       for a
 UV absorption at ~170 nm calculated       π*            π*        π*       enone
 with ZINDO semi-empirical excited-        n             n         n
 states methods (Gaussian 03W):
                                           π             π         π
                                           π             π         π
                                       -*; max=218          n-*; max=320
                                             =11,000                =100

                                       h 170nm photon

HOMO u bonding molecular orbital              LUMO g antibonding molecular orbital
                  How Do UV spectrometers work?

                                      Matched quartz cuvettes
to achieve scan
                                      Sample in solution at ca. 10-5 M.
                                      System protects PM tube
                                      from stray light
                                      D2 lamp-UV
                                      Tungsten lamp-Vis
                                      Double Beam makes
                                      it a difference technique
                   Experimental details
What compounds show UV spectra?

Generally think of any unsaturated compounds as good candidates.
   Conjugated double bonds are strong absorbers
Just heteroatoms are not enough but C=O are reliable

Most compounds have “end absorbance” at lower frequency.
 Unfortunately solvent cutoffs preclude observation.

You will find molar absorbtivities  in L•cm/mol, tabulated.
Transition metal complexes, inorganics

Solvent must be UV grade (great sensitivity to impurities with double

The NIST databases have UV spectra for many compounds
             An Electronic Spectrum
                                                      Make solution of
                                                      concentration low
                                                      enough that A≤ 1
1.0                                                   (Ensures Linear Beer’s
                                                      law behavior)
                                     UV     Visible   Even though a dual
                                                      beam goes through a
                                                      solvent blank, choose
                                                      solvents that are UV

                                                      Can extract the  value
                                                      if conc. (M) and b (cm)
                                                      are known
                                                      UV bands are much
                                                      broader than the
                                                      photonic transition
0.0                                                   event. This is because
  200                                     400                     800
                                                      vibration levels are
                                                      superimposed on UV
             Wavelength, , generally in nanometers (nm)
     Solvents for UV (showing high
            energy cutoffs)
•   Water     205   •   THF       220
•   CH3CN    210   •   CH2Cl2    235
•   C6H12     210   •   CHCl3     245
•   Ether     210   •   CCl4      265
•   EtOH      210   •   benzene   280
•   Hexane    210   •   Acetone   300
•   MeOH      210
•   Dioxane   220
Organic compounds (many of
   them) have UV spectra
             • One thing is clear
             • Uvs can be very non-
             • Its hard to interpret except at
               a cursory level, and to say
               that the spectrum is
               consistent with the structure
             • Each band can be a
               superposition of many
             • Generally we don‟t assign
               the particular transitions.

               From Skoog and West et al. Ch 14
               The Quantitative Picture
• Transmittance:
                                                           P0                      P
                                    T = P/P0               (power in)          (power out)

• Absorbance:                                              B(path through sample)
A = -log10 T = log10 P0/P

•The Beer-Lambert Law (a.k.a. Beer‟s Law):
                                   A = ebc
   Where the absorbance A has no units, since A = log10 P0 / P
   e is the molar absorbtivity with units of L mol-1 cm-1
   b is the path length of the sample in cm
   c is the concentration of the compound in solution, expressed in mol L-1 (or M, molarity)
           Beer-Lambert Law
Linear absorbance with increased concentration--
  directly proportional
Makes UV useful for quantitative analysis and in
  HPLC detectors
Above a certain concentration the linearity
  curves down, loses direct proportionality--Due
  to molecular associations at higher
  concentrations. Must demonstrate linearity in
  validating response in an analytical procedure
    Polyenes, and Unsaturated Carbonyl groups;
               an Empirical triumph
R.B. Woodward, L.F. Fieser and others
Predict max for π* in extended conjugation
  systems to within ca. 2-3 nm.
                                        Attached group increment, nm
                                        Extend conjugation     +30
                                        Addn exocyclic DB      +5
            Homoannular, base 253 nm    Alkyl                  +5
                                        O-Acyl                 0
                                        S-alkyl               +30
            Acyclic, base 217 nm
                                        O-alkyl                +6
                                        NR2                    +60
                                        Cl, Br                 +5
           heteroannular, base 214 nm
       Interpretation of UV-Visible Spectra
   •Transition metal complexes;
   d, f electrons.

   •Lanthanide complexes –
   sharp lines caused by
   “screening” of the f electrons
   by other orbitals

   • One advantage of this is the
   use of holmium oxide filters
   (sharp lines) for wavelength
   calibration of UV

See Shriver et al. Inorganic Chemistry, 2nd Ed. Ch. 14
Quantitative analysis
                                   Great for non-aqueous
                                   Example here gives detn
                                   of endpoint for
                                   bromcresol green
                                   Binding studies
                                   Form I to form II

       Isosbestic points

    Single clear point, can exclude
    intermediate state, exclude light scattering
    and Beer’s law applies
   More Complex Electronic Processes
• Fluorescence: absorption of
  radiation to an excited state,
  followed by emission of radiation
  to a lower state of the same
• Phosphorescence: absorption of
  radiation to an excited state,
  followed by emission of radiation
  to a lower state of different
• Singlet state: spins are paired, no
  net angular momentum (and no
  net magnetic field)
• Triplet state: spins are unpaired,
  net angular momentum (and net
  magnetic field)

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