1. Spectroscopy What is Spectroscopy?
The study of the interaction of electro-magnetic radiation with matter.
We use spectroscopy as a tool to look indirectly at molecules.

2. Electromagnetic Radiation
Transports energy.
Electric and magnetic fields oscillate: that’s the “wave”.
Moves at speed of light.
Wavelength, frequency, energy all related.
E = Electric field H E
H = Magnetic field y x z l
Direction of
propagation
Wavelength
Wavelength
Distance between crests
Amplitude
Half the height of trough to crest
Frequency
Number of crests that pass a point in space every second

3. E = h c / l l = c / n n = c / l c = speed of light (3 x 108 m/s)
E = energy
l = wavelength
n = frequency
h = Planck’s constant (4 x eV sec)

4. Energy as wavenumbers (cm-1)
Spectroscopy
Each region
Wavelength (nm)
Energy as wavenumbers (cm-1)
Microwave
Infrared
Visible
Ultraviolet
X-rays
Vibrational
transitions
104
105
103
102 10 1
106
107
Rotational
Electronic
Inner shell
Outer shell
Different
spectroscopic technique
The spectroscopic technique used to study a particular transition will depend on the energy difference between the ground and excited states (DE).

5. electronic excitation IR: molecular vibrations
Different frequencies of light (electromagnetic radiation) interact with different aspects molecular motion (potential and kinetic energy).
UV-Vis: valance
electronic excitation
Radio waves:
Nuclear spin states
(in a magnetic field)
X-ray:
core electron
excitation
IR: molecular vibrations

6. Ultraviolet-Visible Spectroscopy
Transitions between energy levels which involve electrons, electronic transitions, between molecular orbitals.
In UV/Vis spectroscopy we talk in terms of the wavelength (l) of the transition.
Nuc
Level 1
Level 2
Level 3
Gain energy
Move to level 2 or 3
Lose energy
Drop to level 2 or 1
Energy levels of electrons
Transitions occur between
nm in the UV region
nm in the visible region
For a transition to occur the incident radiation must have the correct energy to excite the molecule from the ground state to a higher, ‘excited’ state

7. UV-Vis Absorption Spectroscopy
Molecular Geometry
Energy
S0 (Ground Estate)
S1 (Excited Estate)
rotational levels
vibrational levels hn

8. Important Terms and Symbols in UV-Vis Absorption Spectroscopy
Term and Symbol
Radiant Power (P, P0)
Absorbance (A)
Transmittance (T)
Path length (b)
Absorptivity (a)
Molar absorptivity (e)
Definition
Energy of radiation
Log (P0/P)
P/P0 -
A/lc
Alternative Name and Symbol
Radiation Intensity (I, I0)
Optical density (OD); Extinction (E)
Transmission (T)
l, d
Extintion coefficient (k)
Molar Extintion coefficient

9.  c l Beer-Lambert Law A = = absorbance (no units).
= Molar extintion coefficient (M-1 cm-1).
= Concentration (M).
= pathlength (cm). A  c l

10. This ratio represents the probability of photon capture.
Beer-Lambert Law I0 I S l dx
Block of absorbing matter
A beam of incident intensity I0 passes through length l of an absorbing medium, which decreases the transmitted intensity to I.
Considering a cross-section of area S and infinitesimal thickness dx containing dn molecules, each of which has a cross-sectional surface of photon capture of a.
Based on the probability of photon capture, we can imagine the total area of the molecular photon capturing surface to be dS, and thus the ratio of photon capture surface area to total surface area is dS/S.
This ratio represents the probability of photon capture.

11. Beer-Lambert Law dS S I0 I S l dx
= probability of photon capture.
Now consider the light intensity passing through distance dx.
The intensity entering the section is Ix, proportional to number of photons per square centimeter. Loss of intensity through absorption is given by dIx, which is an absolute quantity; thus the relative loss of power is:
dIx Ix
(minus sign indicates loss)
dIx Ix dS S =
The relative loss of power must be equal to the relative probability for capture.

12. dS = adn (concept of absorptivity)
Beer-Lambert Law
Note that dS is the sum of all capture areas for all molecules, and must therefore be proportional to the number of molecules
dS = adn (concept of absorptivity)
where a is the photon capture cross section (molecular absorptivity co-efficient). I0 I S l dx
Integrating over all of the molecules within the entire block (to obtain the total absorbance over an infinite number of slices of thickness dx), we get:
dIx Ix =  Io I
adn S n
which gives: I I0 = an S
-ln I0 I = an
2.303S
log
Now convert to base 10 and invert the fraction to change the sign:

13. Beer-Lambert Law V l S I0 I anl 2.303V log c n mol x 6.02 x 1023 alc
The cross-sectional area S can be expressed as a volume (cm3 or mL) divided by a length (pathlength b in cm), thus: I0 I S l dx = V l S
in cm2
Which leads to: I0 I =
anl
2.303V
log
Finally, since n/V has units of concentration (molecules per cm3), we can convert n/V to moles per liter according to:
1000 cm3/L
V(cm3) = c n
6.02 x 1023
mol x
Combining everything together, we get:
6.02 x 1023 alc
2.303 x 1000 I0 I =
log

14. Beer-Lambert Law  c l  c l 6.02 x 1023 acl 2.303 x 1000 I0 I log AS l dx
6.02 x 1023 acl
2.303 x 1000 I0 I =
log
Finally, collecting all of the constants into a single term, , we get: A
 c l I0 I =
log A
 c l =
= absorbance (no units).
= Molar extintion coefficient (M-1 cm-1).
(wavelength dependent)
= Concentration (M).
= pathlength (cm).  c l

15. Limitations / Beer-Lambert Law
Limitations / Beer-Lambert Law
1) Real Limitations
Beer-Lambert law is derived on the basis of having a dilute solution. At high concentrations, (>0.01 M) intermolecular interactions can occur between molecules (dipole-dipole interactions), directly altering e.
Intermolecular interactions can also occur when secondary species (i.e., salts) interact with charged chromophores, thus altering e.
The value of e is also dependent on refractive index of the medium, thus solvent effects on absorbance can be observed (correct using en/(n2 + 2)2 instead of e).

16. Limitations / Beer-Lambert Law
2) Apparent Chemical Deviations
Arise as a result of chemical reactions involving the absorbing sample (association, dissociation, oxidation, reduction, addition, elimination, etc…).
Classic example is pH sensitive chromophores (indicator dyes). Figure shows the absorbance at 430 nm and 570 nm for a dye that undergoes a protic equilibrium that depends on total dye concentration.
0.00
4.00
8.00
12.00
16.00
0.000
0.200
0.400
0.600
0.800
1.000
Absorbance
Indicator (M x 105)
l = 430 nm
l = 570 nm

17. Limitations / Beer-Lambert Law
3) Instrumental Deviations due to Polychromatic
Radiation
Strict adherence to Beer-Lambert Law requires perfectly monochromatic light.
In cases where the spectral band width of the incoming light is many nanometers, then the sample can have a range of e values.
In practice:
Narrower spectral bandwidth will lead to better linearity.
Taking measurements at the maximum wavelength (at emax) will lead to better linearity.
Absorbance
Wavelength
Concentration
Band B
Band A

18. Limitations / Beer-Lambert Law
4) Instrumental Deviations due to Stray Light
Imperfections in monochromator gratings often lead to – 0.2% stray light.
Differs greatly in wavelength from the value of the principal radiation, and may not even pass through sample (i.e., depends on how light-tight the instrument is).
Observed absorbance in presence of stray light is given by:
2.5
5.0
7.5
10.0
1.0
2.0
Absorbance
Concentration (M x 103)
5 %
1 %
0.2 %
0.0 % Is I0
x 100 % A’
I0 + Is
I + Is
log =
Where Is is the intensity of non-absorbed stray radiation. This leads to a negative deviation in A vs C, as shown in the Figure

19. Reflection losses at interfaces Scattering losses in solution
Measurement of absorbance by molecular samples requires that the sample be placed in some kind of recipient.
Reflection of light occurs at four different interfaces, leading to ~8.5% loss in transmitted light.
Attenuation of light can also occur via scattering or absorbance by species other than the solution.
Reflection losses at interfaces
Incident beam, I0
Emergent beam, I
Scattering losses in solution

20. Instruments for absorption measurements
Instrument components: UV-VIS
signal
processor
optical
source
hn1
sample
hn2
detector
l selector

21. Tungsten filament lamps
Instrument components: UV-VIS
Sources
A source must:
Generate a beam of radiation with sufficient power for easy detection and measurement.
Provide output power that is both stable and intense.
Types of spectroscopic sources
1. continuous sources
Wavelength
Relative energy
H2 and D2 lamp / Xe arc lamp
Tungsten filament lamps
2. lines sources
Intensity
Hollow cathode lamp
Hg vapor lamp / Laser

22. Instrument components: UV-VIS
Sources
6000 K
4000 K
3000 K
2000 K
Xenon arc
Carbon arc
Tungsten lamp
Nerst glower
Relative energy
Wavelength (nm)
500
1000
1500
2000
2500
3000 1 10
102
103
104

23. Instrument components: UV-VIS
Sample containers
Glass nm
Quartz nm
Polystyrene - Visible
Metacrylate - UV

24. Instrument components: UV-VIS
Wavelength selectors
1. Filters
The simplest method for isolating a narrow band of radiation.
1.1 Interference filters
Use constructive and destructive interference to isolate a narrow range of wavelengths.
1.2 Absorption filters
Work by selectively absorbing radiation from a narrow region.

25. Instrument components: UV-VIS
Wavelength selectors
2. Monochromators
2.1 Prism
Quartz or glass cut at an angle (Refraction).
2.2 Gratings
Finely grooved highly reflective surface (Diffraction).
Collimating mirrors
Entrance slit
Exit slit
Grating surface l
>

26. Instrument components: UV-VIS
Wavelength selectors
0.5
1.0
Absorbance
Wavelength
Nominal wavelength
Effective bandwidth
1/2 Peak height

27. Instrument components: UV-VIS
Detectors
The detector changes the radiation transmitted from the UV-Vis Spectrophotometer into a current or voltage.
1. Phototube - + hn e-
Light strikes photocathode (-) that emits electrons.
Electrons are accelerated towards the anode (+).
Current proportional to photons.
2. Photomultiplier
Electrons that are accelerated towards a series of increasingly positive anodes (+).
Electrons are directed towards the collecting anode.
3. Photodiode array
Multi-element detector composed of an array of solid-state detectors.

28. Instrument components: UV-VIS
Signal processors
Amplifies the voltage produced by the detector.
Converts the voltage into absorbance units.

29. Single beam UV-Vis spectrophotometer
Instruments: UV-VIS
Single beam UV-Vis spectrophotometer

30. Double beam UV-Vis spectrophotometer
Instruments: UV-VIS
Double beam UV-Vis spectrophotometer

31. Instruments: UV-VIS

32. Ultraviolet-Visible Spectroscopy
Bonding electrons appear in s and p molecular orbitals
nonbonding in n
Energy s* p* n p s
Antibonding
Nonbonding
Bonding
Electronic transitions can occur between various states.
The energy of the transitions increases in the following order:
(n  p*) < (p  p*) < (n  s*) < (s  s*)

33. Electronic Transitions
Most absorption spectroscopy of organic compounds is based on these transitions.
The absorption peaks for these transitions fall in an experimentally convenient region of the spectrum ( nm).
These transitions need an unsaturated group in the molecule to provide the p electrons.
Excitation from the ground state to the excited state requires EM radiation with a wavelength of 150 nm.
Not useful for routine spectroscopy
The number of organic functional groups with n  s* peaks in the UV region is small.
Excitation requires EM radiation with a wavelength in the range nm.
p  p* band
n  p* band
n  s* band
s  s* band

34. Some Spectroscopic Terminology
Chromophore
Group on molecule responsible for electronic transition
These molecules generally contain conjugated systems.
Adjacent double bonds are conjugated.
The net effect is to bring the ground state and excited states closer in energy.
The longer the chain of conjugation the longer the wavelength of the absorption band.
Bathochromic shift
Hypsochromic shift
Hyperchromic effect
Hypochromic effect
Shift in absorption to a longer wavelength.
Shift in absorption to a shorter wavelength.
An increase in the intensity of absorption.
An decrease in the intensity of absorption.
Some Spectroscopic Terminology

35. Isosbestic Point
When one absorbing species reacts to form another absorbing species, there usually is a point(s) at which the spectra cross. This is the isobestic point.
Example, acid/base indicators.

36. Absorption Spectra of Biopolymers
Most proteins and nucleic acids are colorless in the visible region, however they absorb in the near-UV region. Chromophores in proteins and nucleic acid absorb light only at wavelengths lower than 300 nm. On the other hand, measurements have to be done in wavelengths longer than 170 nm, below which the absorbance of water becomes too high.
UV absorption spectra of proteins
There are 3 classes of protein chromophores
peptide bonds, amino acid side chains and prosthetic groups.
Absorbance
Wavelength (nm)
2.0
1.5
1.0
0.5
0.4
0.3
0.2
0.1
250
300
200
4.5 x 10-7 M
2.25 x 10-6 M
For a typical protein there is a distinctive absorption peak at 280 nm (due to the p  p* transitions in aromatic amino acids), a stronger one at 190 (due to the p  p* transition in the amide group), and a shoulder at nm (due to the weaker n  π* transition in the amide group).

37. Chromophores in proteins
The peptide bond

38. Amino acid side chains
A number of amino acids – Asp, Glu, Asn, Gln, Arg & His have electronic transitions around 210 nm. Usually these cannot be observed in proteins because they are masked by Intense peptide bond absorption.
The most useful range for proteins is above 230nm where there are absorptions from the aromatic side chains of Phe, Tyr and Trp.
The absorption of Phe is low and if Tyr & Trp are present in a protein it contributes little to the absorption above 230nm. The disulphide group of Cys also has a weak absorption ~250 nm which can be important in protein optical activity.
The absorption spectra of Tyr & Trp contain contributions from at least three electronic transitions. Assignment of individual transitions in proteins containing many Tyr and Trp in a variety of environments is not possible.

39. e - Molar absorptivity (M-1 cm-1)
Amino acid side chains
The aromatic Trp, Tyr, and Phe are the only residues absorbing significantly at wavelengths higher than 230 nm. The maximum at 280 nm is due to Trp and Tyr.
e - Molar absorptivity (M-1 cm-1)
Tyrosine
Phenylalanine
Tryptophan
200
220
240
260
280
300
320
10000
5000
2000
1000 10 20 50
100
500
40000
20000
4000
3000
190
210
230
Alanine
Lysine HCl
Methionine
Wavelength (nm)

40. Absorption Spectra of Biopolymers
SECONDARY STRUCTURE
The local environment of the amino acids in a native protein depends on the electric properties of the peptide chain near by and the associated solvent. The resulting change in spectra is sufficient to diagnose whether a protein is folded or not, but CD is more informative.
UV spectra of poly-L-lysine hydrochloride (random coil) changes upon formation of secondary structure (raising pH induces helix formation - reducing the net positive charge of the lysine side chain -; raising the temperature induces beta sheet).

41. Absorption Spectra of Biopolymers
Many proteins include prosthetic groups such as iron porphyrins in hemeproteins, flavin in flavoproteins, or phosphate groups, that have strong absorption in the visible and near UV. Because these bands are sensitive to the environment, they can be used in the study of enzymatic reactions.
e x 10-4

42. Chromophores in genetic material

43. Absorption Spectra of Biopolymers
UV absorption spectra of nucleic acids
The strong near-UV absorption of nucleic acids is due to the purine and pyrimidine bases. The electronic states of these bases are much more complex than those of protein chromophores, and apparent gaussians in the spectra are really composites of several electronic transitions.
180
200
220
240
260
280
300
0.2
0.4
0.6
0.8
1.0
E. coli DNA 82°C
Enzymatic digest 25°C
Native 25°C
l (nm)
Absorbance
The nucleotides all have similar absorption spectra, dominated by an absorption peak at 260 nm and a shoulder at 200 nm.
The free base, the nucleoside (attached to sugar), the nucleotide (attached to a sugar phosphate), and the denatured polynucleotide all have similar spectrum.
In general polynucleotides and nucleic acids absorb less per nucleotide that their constituent nucleotides. Also, native double-stranded DNA absorbs less per nucleotide than denatured (melted strands). This results from the interactions between adjacent bases. Stacked base pairs in a double helix absorb less than partially stacked bases in a single strand, which absorb less than the mononucleotides.

44. What determines absorbance?
The absorbance spectrum for a chromophore under standard conditions is only partly determined by its chemical structure.
The environment of the chromophore also affects the precise spectrum obtained. pH
Solvent polarity
Orientation effects
Chromophores can act as reporter molecules which can give information about their immediate environment.
Defined conditions (pH and solvent).
Protonation/deprotonation effects resulting from pH changes or oxidation/reduction effect electron distribution in chromophores.
Solvent polarity –Altered spectra in different solvents (DMSO, dioxane, ethylene glycol, glycerol and sucrose) compared to water = Solvent perturbation.
Orientation effects result from the relative geometry of neighboring chromophore molecules. E.g. hyperchromicity of nucleic acids.

45. Applications of absorption spectra
Quantitative
Reaction rate calculation
Standard curve
Structural studies
Enzymatic
Identification
Beer-Lambert law to determine concentration.
Continuous or stopped.
measure concentration.
folding, assembly, denaturation, ligand binding
pathways, reaction intermediates
of compounds (vitamins, hormones) not much use for proteins/nucleic acids, unless they contain groups with absorption in the visible region of spectrum.
Immunodetection
commercial detection kits.