SBC 470: Principles of Organic Spectroscopy

Electromagnetic spectrum, Relationship between energy, frequency and wavelength. Absorption and
emission of electromagnetic radiation, electric dipole and magnetic dipole transition moments, Einstein
coefficients, Beer-Lambert Law, Chemical and physical properties of biomacromolecules, The
interrelationship of molecular interactions and thermodynamic properties as determinants of higher order
structure. The use of UV, IR, NMR and crystallography in determining macromolecular structure.

References

1. Allan Cooper (2004). Biophysical Chemistry, Tutorial Chemical Texts, Royal Society of
Chemistry, Cambridge, UK.
2. Rosaleen J.A., David J.B. and Paul W.G. (2004). Organic Spectroscopic Analysis, Tutorial
Chemical Texts, Royal Society of Chemistry, Cambridge, UK.
3. William Kemp (1993). Organic Spectroscopy, 3rd Edition, ELBS, The Macmillian Press Ltd,
Hamshire
4. Silverstein R.M., Bassler G.C. and Morrill T.C. (1991). Spectroscopic Identification of Organic
Compounds, 5th Edition, John Wiley and Sons, Inc., New York
5. Milton H.W. (2014). Nuclear Magnetic Resonance (NMR) Spectroscopy: Structural Analysis of
Proteins and Nucleic Acids. Encyclopedia of Life Sciences, www.els.net.
6. Chemistry is the Logic of Biological Phenomena,
www.cengage.com/resource_uploads/downloads/1133106293_352206.p.
7. Kalsi P.S. (1995). Spectroscopy of Organic Compounds, New Age International Limited
Publishers, India
8. Wienk, H. And Wechselberger, R. (2011). NMR Spectroscopy in Structural Analysis.
www.nmr.chem.uu.nl/Education/Files/Dictaat_2011.pdf.
9. Three-dimensional Models for Biochemical Compounds. http://www.biocheminfo.org/klotho/
10. Spectral Database System (SDBS). http://www.aist.go.jp/R10DB/SDBS/menu-e.html
11. Searches for Physical and Chemical Properties as well as links to useful biochemical and
structural sites for biomolecules. http://chemfinder.com

Spectroscopy

Interaction of electromagnetic radiation with matter

(i) Absorption Spectroscopy  Can refer to the absorption of any frequency of radiation. Most
common are:
 UV – Visible absorption (electronic).
 IR absorption (vibrational)
 Microwave absorption (rational)
 NMR

Energy of the radiation ≅ energy of transition.

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 (ii) Emission Spectroscopy  Emission of any frequency of radiation.
 concerned with the properties of emitted photons.
 Uv-Vis-NIR (electronic transitions)  Fluorescence, phosphorescence,
chemiluminescence, photoluminescence.

Fluorescence  important in biology  based on chemistry and physics.

(iii) Scattering spectroscopy  look at how light scatters from molecules.  can use neutrons,
x-rays etc.
Most important is Raman spectroscopy.
 Molecular technique
 Great for forensics etc.

Electromagnetic Spectrum

 Propagation of light by light waves involves both electronic and magnetic forces.

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NB:

1. Nuclear Magnetic Resonance transitions (NMR)  smallest gap between energy levels.
2. Ultraviolet – visible  (UV – Vis)  largest gap between transition levels.
3. X – Rays  high energy capable of ionizing single – crystal X-ray diffraction.
4. Microwave (rotational) spectra are very complex and give little useful information on organic
molecules which are relatively large.
5. Rotational transitions are often responsible for the broadness of IR bands since each vibrational
transition has a number of rational transitions associated with it.
6. Electronic transitions accompanied by vibrational and rotational transitions  give UV-Vis as
broad absorption bands.
7. Not all transitions are allowed.
 Transitions in UV – Vis, IP and NMR are governed by selection rules that state which
transitions are “allowed” and which are “forbidden”.

Wavelength units are m, m or nm.

Wave number (ῡ) units are m-1 or cm-1
Frequency units are s-1 or Hz (1 MHz = 1,000,000 Hz)
1nm = 10-7 cm = 10-9 m
1m = 10-6 m
For example:

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Molecular energy levels

Typically ΔEel  ΔEvib  ΔErot

Transitions at λ  500 – 100nm (electronic) used in UV, 100 - 2m (vibrational) used IR and 10cm –
1mm (rotational) used in microwave.

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The interaction of electromagnetic Radiation with Molecules

 Energy levels of atoms and molecules are quantized i.e. there are discrete energy levels in
atoms and molecules.
 electronic, vibrational, rotational.
 Spectra reflect these defined changes (band structure).

E corresponds to certain frequency () or wavelength () of electromagnetic radiation. This will depend
upon the type of transition and hence separation between the energy levels.

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Emission Spectra

Plot of emission intensity versus  or λ is called emission spectrum.

Two types of molecular emission spectra (luminescence)

Fluorescence  energy emitted can be the same or smaller than the corresponding molecular
absorption spectra (if heat is released before radiation).
e.g. absorption in UV region and emission in UV or visible region (the
wavelength of visible region is longer than that of UV thus less energy).
 complete after ≅ 10-5 seconds or less from the time of the excitation (short – lived)
 Study of fluorescence from Biological molecules is very important because it is a
simple and fairly direct way to investigate the properties of short-lived excited states
of these molecules.

Phosphorescence  generally takes much longer to complete (called metastable) than fluorescence
because of the transition from triplet state to ground state involves altering the
electron spin. If the emission is in visible light region, the light of excited
material fades gradually.
 Periods longer than 10-2 – 100 seconds and continue for minutes or even
hours after irradiation has ceased.
Absorption

Stimulated Emission

 Photon with right energy comes along and stimulates the transition of the electron from upper energy
to lower energy level. Two photons with the same phase, wavelength and direction are given out. It is
short lived.

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7
For Spontaneous emission:
The rate of decay for an electron (or atom or molecule) between the upper and lower levels is given by:
(rate) dn/dt = nuAul where nu is the density of electrons in the upper state and Aul is the Einstein
coefficient for spontaneous emission i.e. transition probability per unit time to go from upper level to
lower level by emission of a photon.

For Stimulated emission

The rate of stimulation emission is given by:-
(rate) dn/dt = nuBul I where Bul is the Einstein coefficient that gives the probability that the decay
will occur and I is the intensity of the radiation.

For absorption:-
The rate of electrons transitioning between the lower to the upper level due to absorption is given by:-
(rate) dn/dt = nlBlu I where Blu is the Einstein coefficient of absorption i.e. gives the probability
per unit time per unit intensity that a photon will be absorbed and nl is the density of electrons in the
lower level.

Absorption spectrum is a plot of absorbance versus  or  .

An atom, ion or molecule can absorb radiation if the energy matches separation between the energy states.
The study of frequency of absorbed radiation by the species provides a means for identification and
analysis of a sample.

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Typical excitation (absorbance) and fluorescence emission spectra for a globular protein

Electric Dipole and Magnetic dipole Moments

 Molecule subjected to external electric field  electrons drawn to +ve (anode) and nuclei
to –ve (cathode) electrodes.
 distortion of molecule  temporary

 Some molecules are permanently polarized.

 Permanent electric dipoles e.g. H2O, CH3COCH3, CHCl3, etc.
 dipolar molecules respond to an applied electric field.
 An electric field interacts with a molecular electric dipole moment.

 A spinning proton (electric charge) creates a local magnetic field or dipole and interacts
with externally applied magnetic field.

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Absorption

Instrumentation

Experimental measurements are usually made in terms of transmittance (T), which is defined
as:

T = I / Io

Where I is the light intensity after it passes through the sample and Io is the initial light
intensity. The relation between A and T is:

A = -log T = - log (I / Io).

The ratio of the intensity of the light entering the sample (Io) to that exiting the sample (It) at a
particular wavelength is defined as the transmittance (T). This is often expressed as the percent
transmittance (%T), which is simply the transmittance multiplied by 100. The absorbance (A) of
a sample is the negative logarithm of the transmittance.

% T = (Io / It ) x 100

A = - log (T)

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Beer – Lambert Law

 Gives relationship between concentration, absorbance and transmitted light intensity i.e.
absorbance (A) is directly proportional to the path length (l) and the concentration of the
absorbing molecule (c).

Units for  are l mol-1 cm-1 or 100 cm2 mol-1 or dm3 mol-1 cm-1 It is characteristic of the absorbing
molecules at the wavelength of the incident radiation  Can also depend on the environment e.g. solvent
polarity. It is a measure of the intensity of absorption (ranges 0 – 106  units of 100 cm2 mol-1).
 It is a constant for a particular compound at a given wavelength and mostly expressed as
max.
  max  104  high-intensity absorption
 max  103  low-intensity absorption

 Mostly used with UV-Vis absorption spectroscopy.

 Can be used with FT-IR

The Linearity of Beer- Lambert Law

 limited by chemical and instrumental factors

Causes of non-linearity include:

 deviations in absorptivity at high concentrations ( 0.01M) due to electrostatic

interactions between molecules in close proximity.
 Scattering of light due to particulates in the sample.
 Fluorescence or phosphorescence of the sample

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 Changes in refractive index at high analyte concentration.
 Shifts in chemical equilibria as a function of concentrations.
 Non – monochromatic radiation, deviations can be minimized by using a relatively flat
part of the absorption spectrum such as the maximum of an absorption band.
 Stray light.
 Works with relatively dilute samples.
 Does not work with turbid samples.
 Need to avoid scattering.
 Fixed single wavelength/fixed temperature.

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UV-Visible Spectroscopy (electronic)

UV: 200 – 400 nm  595 – 299 kjmol-1

Visible: 400 – 750 nm  299 – 149 kjmol-1

Region below 200 nm  vacuum UV purged with N2 since O2 absorbs strongly in this region.

Importance of UV – Vis Spectroscopy

 Ability to measure the extent of multiple bonds (conjugation) or aromatic conjugation

within a molecule.
 Nonbonding electrons on oxygen, nitrogen and sulphur can also extend the
conjugation of multiple bonds.

Relative energies of orbitals most involved in electronic spectroscopy

Non–conjugated chromophores

max (nm) max(10-2m2mol-1) chromophore Transition causing

absorption
 150 C-H or C–H δ-bonded δ → δ
electrons (alkanes)
~ 185 – 195 -X: (X = O, N, S) lone n → δ
pair of electrons

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 ~ 300 15 C=O: n → π (symmetry
forbidden, low
~ 190 5,000 Lone pair electrons intensity)
n → δ
~ 160 18,000
π → π (most intense
but needs high energy)
~ 190(~175) C=C (isolated) π-bond π → π
electrons

 Intensity of the major absorption (and of course the  value) increases as the
chromophore increases in length.
 Systems with a higher degree of conjugation have greater intensity absorption bonds with
larger  values.

 Increased conjugation decreases the energy difference between the HOMO and the
LUMO of a particular system  less energy needed for transition.
 The longer the chromophore, the longer the wavelength of the absorption maximum.

e.g. But-3-en-2-one (methyl vinyl ketone, MVK): π → π; λmax = 219nm and max = 3600
n → π ; λmax = 324nm and max = 24
Propanone: π → π; λmax = 187nm and max = 900
n → π ; λmax = 270nm and max = 15

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NB: n → π is symmetry forbidden.
Intense band  K – band (π → π )
Less intense band  R – band ( n → π )
 Increase in the λmax towards the red end of the spectrum is call a red shift ( shift to longer
, lower energy) or a bathochromic shift

NB: Not all transitions are allowed.

Spin & Symmetry rules

 Allowed if: 1. Orientation of the spin does not change during the transition

2. Symmentry of the initial and final functions is different.

 Forbidden transitions have low intensity.

In general, the energy of transition increases in the following order.

n → *   → *   → *   → *

Definitions

Chromophore

 Part of a molecule containing the electrons involved in electronic transitions which give
rise to an absorption or covalently unsaturated group responsible for electronic
absorption or part of molecule responsible for absorption.
 Wavelength of the maximum of the broad absorptions is labeled max.
 Maximum value of the molar absorptivity is labeled max.
 Intensity of the major absorption (also max) increases as the chromophore increases in
length (conjugation increases).
 Conjugation decreases the energy difference between HOMO and LUMO level.

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 Auxochrome  a saturated group or atom with non-bonded electrons which when attached to a
chromophore alters both the wavelength and intensity of absorption e.g.-OH, -OR, -NH2,
-NR2, -Cl, etc.
Bathochronic Shift  Shift to longer wavelength (lower energy)  red shift.
Hypsochronic shift  Shift to shorter wavelength  blue shift
The shift could be due to substitution or solvent effect.
Hyperchromic effect  increase in absorption intensity.
Hypochromic effect  decrease in absorption intensity.

For unconjugated organic molecules:

 → *  below 150m
n → *,  → *  below 200 nm
n → *  200 – 400 nm

Absorption properties of a chromophore are sensitive to the environment and, particular to :-

 The interaction with solvent
 The interaction with other chromophores,
These generally modify position, intensity and shape of absorption bands  hence can be used
to monitor the structural changes of a macromolecule during a physical - chemical process.

For majority of proteins and nucleic acids, most biologically significant solvent is water, buffered at pH
close to 7.0, in the presence of salt ( 100 mMol) to mimic conditions in vivo.

 UV measurement performed at   170 nm.

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Approximate max and max for biological chromophores in the new UV/Visible region under
physiological conditions

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Others

Refinal (in rhodophsin) 500 42,000

Haem (in haemoglobin) 0xy - 414 131,000
Haem (in haemoglobin), deoxy- 432 138,000
NAD 260 18,000
NADH 340 6,200
Cholophyll 418 111,000

NB:
Absorption spectra are characterized by their shape, the peak wavelength (max) and the peak height or
molar extinction coefficient (max).

Effect of pH
e.g.

(Neutral or Phenoxide anion

Acidic conditions) (Basic conditions)
max (H2O) 210 (6,000) max (H2O) 235 (9,400)
270 (1,500) 287 (2,600)

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  Increased conjugation of the lone pairs on oxygen with
the -system of the aromatic ring.

 leads to decrease in the energy difference between the

HOMO and LUMO orbitals

 red (bathochromic) shift  shift to longer wavelengths

and increase in the intensity of absorption.

 Increasing pH shifts equilibrium to right.

 More non-bonding electrons in phenoxide ion
 Higher max
 Greater delocalization  Bathochromic shift

Phenolpthalein

π → π λmax 231 nm(max 25800) λmax 230 nm(max 25800)

n → π λmax 275 nm (max 4200) λmax 553 nm (max 3600)

NB:
Exposure of tyrosine by obtaining spectrum of a protein at higher pH. Tyrosyl residue deprotonated with
a pka ≅ 10  “red shift” and increased intensity.
 assumed overall conformation not changed by increase in pH.

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 OH-
H2C OH H2C O-

pH 6: 274 nm pH 13: 295 nm

NB: Protparam (http: //ca.expasy.org/tools/protparam-doc.html) allows one to calculate  based on

sequence → useful for concentration determination.
In general it is used to compute the physico-chemical properties that can be deduced from a
protein.

The calculated parameters

The parameters computed by ProtParam include the molecular weight, theoretical pI, amino acid
composition, atomic composition, extinction coefficient, estimated half-life, instability index,
aliphatic index and grand average of hydropathicity (GRAVY). Molecular weight and theoretical
pI are calculated as in Compute pI/Mw. The amino acid and atomic compositions are self-
explanatory. All the other parameters will be explained below.

Aniline Protonated aniline

(Neutral or basic conditions) (Acidic conditions)
 max (H2O) 235 (14,800)  max (H2O) 203 (7,500)
285 (2,800) 254 (180)

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  loss in overlap between the amine lone pair
and the aromatic  system
 blue (hypsochromic) shift
 shifts to shorter wavelength and a decrease in
intensity of absorption

Solvent effects

 Interaction with solvent has the effect of stabilizing or destabilizing the electronic
excited states relative to the ground state of the chromophore.
 The interactions depend on chromophore, solvent and electronic transition in
question.
e.g.
* Conjugated dienes and aromatic hydrocarbons  very little solvent effect.
* ,  - unsaturated carbonyl compounds  solvent polarity affects the  → *.

Polarity Effects of Solvent

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Solvent Role

The Franck–Condon Principle states that the electronic transitions involve the movement of
electrons, including those of the solvent, but not the movement of atoms.

When the solvent electrons can rearrange to stabilize the excited state of a molecule, the energy
different between the electronic levels of the molecule is lowered and the absorption moves to
higher wavelength.
NB:
1. Conjugated dienes and aromatic hydrocarbons  very little solvent shift.
2. , - unsaturated carbonyl compounds:-
(i) π → π band moves to longer wavelength (red shift)
(ii) n → π band moves to shorter wavelength (blue shift)

 due to solvation, stabilizes , * and n-orbitals especially with H–bonding solvents e.g. EtOH,
H2O etc.
 * are more stabilized by salvation then - orbitals  presumably because * orbitals are more
polar.
 Interaction of the lone pair with solvent lowers the energy of n–orbital  increases energy
required for n → *

 → * > s → *s  moves to longer wavelength

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  red shift
 less energy

n → * < ns → *s  moves to shorter wavelength

 blue shift
 more energy

NB:
 Main use of UV-Vis  determination of concentration.
 Environmental factors such as solvent pH and polarity may influence the
absorption  can be used to extract information indirectly about the
absorbing species.
 In the absence of prosthetic groups, an average protein has maximum
absorption in the near UV at approximately 280 nm.
 If max at 280 nm of the proteins is known, it is possible to derive the protein
concentration.
 If the amino acid composition of the protein is known, then can use the molar
extinction coefficients of the chromophoric side-chains to estimate the
extinction coefficient of the entire protein.  normally done at 280 nm where
the accepted 280 values are:

Trp = 5690
Tyr = 1280
Cys (half cysteines only) = 60
Phe does not absorb significantly at 280 nm.
e.g.: A globular protein of RMM 23,500 contains 6 Trp, 4 Tyr and 3 Phe residues.
(a) What is the 280, for this protein.
(b) What will be the absorbance at 280 nm for a 0.5 mg cm-3 solution in a 1 cm cuvette?

Answer
(a). 280 (protein) = nTrp x 280 (Trp) + nTyr x 280 (Tyr) = nPhe x 280 (Phe)

= (6 x 5690) + (4 x 1280) + (3 x 0)
= 39260 mol-1lcm-1 (mol-1dm3cm-1)
(b). molar concentration :
0.5mgcm-3  0.5gdm-3  0.5/23500 = 2.13 x 10-5 moldm-3
Hence A =  x c x l = 39260 x 2.13 x 10-5 x 1 = 0.836

Tryptopham: at  max 280; 1 mm path length; aqueous solution, 0.50 mmol l-1  54% of light passes
through. Calculate the value of .

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Answer
A = -log T = cl
 = -log 0.54/5.0 x 10-4 moll-1 x 1mm = 5.4 x 102 lmol-1mm-1 = 5.4 x 103 lmol-1cm-1

Absorbance for 5mm path length

A = 5.4 x 103 lmol-1mm-1 x 5 mm x 5.0 x 10-4 moll-1 = 1.35

Absorbance for 1mm path length

A = -log T = -Log 0.54 = 0.27

Nuclei Acids  DNA, RNA

 Absorbance between 250 – 300 nm  due to the  → * transitions in the

aromatic purine (A,G) and pyrimidine (U,C,T) rings of the nucleotide bases.
 The sugar phosphate backbone groups absorb only in the far–UV ( 250 nm)
 DNA and RNA spectra quite sensitive to conformation.
 Absorption properties strongly influenced by interactions with neighbouring
chromophores.
 Interaction of  electrons between the bases.
 Because of base–stacking interactions that perturb the spectroscopic properties of
the closely packed aromatic properties of the closely packed aromatic rings,
helical polynucleotides have a lower absorbance in the 260 nm region than would
be expected from the free nucleotide alone.
 used to examine macromolecular stability
 Denaturation of nucleic acids and proteins can be monitored by UV
spectroscopy.

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 AMP - isolated
 Increase in absorbance can amount to as much as 40%  useful for measuring
the thermal unfolding or “melting” of polynucleotides.

e.g. UV spectra of a solution of DNA at a low temperature (e.g. 400C) and a higher
temperature (e.g. 900C).

 The difference is due to the unstacking of the base pairs as the DNA double –
helix unfolds (or “melts”) at higher temperature.
 can be used to follow the unfolding process with temperature.

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  The melting temperature (Tm)  the temperature at which the denaturation is
half complete.
 The steepness of the melting curve is an indication of the co-operativity (“all-or-
none” character) of the denaturation.

Poly–L-lysine{ (Lys)n } can adopt 3 different conformations merely by varying the pH and
temperature.
 Random coil at pH 7.0
  - helix at pH 10.8
  - form at pH 11.1 after heating to 520C and re-cooling.

π → π of the peptide bond

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 If the protein has substantial helical content, denaturation will be accompanied by
a significant increase in absorption in the far UV since the random coil peptide
bond absorbs more strongly than the  - helical peptide bond.
 Peptide bond absorbance spectrum strongly influenced by secondary structure.
 an interaction between the transition dipoles of neighbouring chromophores
can cause an absorbance band to split into two components that will differ in
intensity.
 Absorbance at 260nm used to determine the concentration of nucleic acids.
 At concentration of 1mg ml-1 at path length 1 cm;
 double– stranded DNA has A260 = 20.
 RNA and single stranded DNA have A260 = 25.
(But this depends on base composition and secondary structure).
 A protein solution has A280 = 1.
Purity of DNA is given by the ratio of A260 and A280 .

If A260/A280 = 1.8  double–stranded DNA

If A260/A280 ≅ 2.0  pure RNA.

Protein; max = 280 nm has A260/A280 < 1 (0.5)

If DNA sample has A260/A280 > 1.8  It has RNA contamination

If < 1.8  It has protein contamination

Prosthetic groups and other chromophores

 In addition to the intrinsic chromophores of proteins and nucleic acids, there are a
number of other co-factors and prosthetic groups that have characteristic
absorbances in the UV and visible regions.

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 e.g. Green plants  primarily absorbance of red light by chlorophylls and other
photosynthetic pigments, mainly bound in specific protein structures.

 Mammalian haem proteins (haemoglobin, myoglobin)  red colour 

absorption at the blue end of the pectrum by the iron-porphyrin prosthetic group
(haem) also responsible for oxygen binding.

 Spectral properties of prosthetic groups are frequently modified by covalent and

non-covalent interactions with their host protein.

e.g. Major chromophore of the visual pigment protein rhodopsin is retinal

(Vitamin A aldehyde)  conjugated polyene.

 Free retinal absorbs at around 370 nm.

 When bound to the protein it forms a covalent protonated imine (Schiff base)
linkage to specific lysine side-chain which, together with non-covalent
interactions with other amino acids in the binding site, shifts the absorbance max
to 400 – 600 nm visible region.

 The oxidized and reduced forms of important electron transfer co – factors such
as NAD (nicotinamide adenine dinucleotide) differ in their UV absorbing
properties.

 Useful in following the redox state of these prosthetic groups during

metabolic pathways.

 The UV absorbance changes also used in developing methods for measuring

the kinetics of enzyme – catalysed reactions involving such groups.

Other applications

 Identification  the absorption spectrum of a substance can offer insight into its
identity e.g. Cytochromes are distinguished on the basis of their absorption
spectra.

 The absorption spectrum can also be used to monitor purification progress.

 Monitor ligand – binding  requires only that the spectrum of either the
macromolecule or ligand undergo a measurable alteration (change in  or ) upon
binding.

 Iron–sulfur clusters in a native and a mutant protein.

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 S S S
Fe
Fe
S S S

Absorption band between 400 – 600 nm which is diagnostic of 2Fe – 2S cluster.

 Enzyme assays (quantitation of product formation or substrate utilization). E.g.

measuring production/utilization of NADH at 340 nm ( = 6.2 mMol-1cm-1).

 Monitoring growth of microorganisms by monitoring OD600 (light scattering)

OD600 =  c l where  is analogous to 

Disadvantages of UV – vis Spectroscopy

(i) Work within range which obeys Beer-Lambert’s Law.

 Threshold for deviations is strongly system – and instrument – dependant.
 Causes of deviations include: “stray light”, scattering, interactions.

(ii) Absorbing impurities can cause error (detergents; other proteins, DNA)
(iii) Single – wavelength measurements (at low absorbance values) can have errors.

Lipids and Carbohydrates

 Most lack the extensive electron delocalization or aromatic groups necessary for
the relatively low-energy electronic transitions required for near – UV or visible
absorbance.

 Absorb at short wavelengths (<200 nm).

 The insolubility of lipids in water and their tendency to form bilayer and other
aggregates gives rise to light scattering particularly at short wavelengths.

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  difficult to measure their true absorbance properties.

Working Curve

 From series of solutions of known concentration e.g. plot absorbance (A) at a

fixed wavelength against known concentration of standards.

Woodward–Friesser Rules

 Many classes of compounds show regularities in their absorption spectra 

enables calculations for max possible.
 Used in structure determinations by distinguishing between two or more isomers.
3 systems

 Acyclic dienes or dienes contained in non-fused ring systems

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  Dienes or polyenes in fused ring system
 Conjugated enones and dienones

Parent acyclic dienes (not homoannualr) - 217(215) nm

Parent homoannular dienes - 253 nm
Parent heteroannular dienes - 214 (215) nm
Parent acyclic trienes - 245 nm

NB:

1. Delocalization of electrons by conjugation decreases the energy difference between

HOMO and LUMO energy levels  leads to a red shift.

2. Alkyl substitution on a conjugated system leads to a smaller red shift.

 interaction between  - bonded electrons of the alkyl group with the  - bond system.

31
32
NB: Reduction in effective conjugation raises the energy requirement for →* transitions.

 max (calc) for strained dienes do not agree with the observed values.
Parent = 215 nm
2 exocyclic
double bonds = 10 nm
2 ring residues = 10 nm
Total = 235 nm

Observed = 220 nm

Observed = 248 nm

More strained.

33
  Woodward-Fieser Rules work well up to 4 double bonds.
  for more than 4 conjugated double bonds: Fieser-Kuhn Rules
 λmax = 114 + 5(# alkyl substituents) + n(48 - 1.7n) - 16.5(# endo) - 10(# exo)

OR
max = 114 + 5m + n (48 – 1.7 n) – 16.5 Rendo – 10 Rexo

λmax = (1.74 x 104)n

n = no. of conjugated double bonds

m = no. of alkyl or alkyl like substituents on the conjugated system

Rendo = no. of rings with endocyclic double bonds in the conjugated system

Rexo = no. of rings with exocyclic double bonds

For example, Lycopene  deep – red compound that is partially responsible for the red
colour of ripe fruits especially tomatoes.

Lycopene and beta-carotene

 λmax = 114 + 5(8) + 11(48 - 1.7•11) - 0 - 0 = 476 nm

 λmax (actual) = 474 nm

34
 λmax = 114 + 5(10) + 11(48 - 1.7•11) - 16.5(2) - 0 = 453.3 nm
 λmax (actual) = 452 nm

,  - Unsaturated carbonyl compounds (in EtOH)

λmax for π → π  K-band

π → π  πs → πs

Red shift

35
36
 Parent = 202 nm
O beta R = 12 nm
gamma R = 17 nm
delta R = 17 nm
extension
of conjugation = 30 nm
1 exocyclic
double bond = 5 nm
Total = 283 nm
HO2C
Observed = 281 nm

CO2Et

Parent = 197 nm
alpha R = 10 nm
beta OR = 30 nm
O Total = 237 nm
Observed = 236 nm

37
38
39
 Substituted benzene derivatives

Scott’s rule: e.g.

Parent = 246 nm
para Cl = 10 nm
Total = 256 nm

Observed = 254 nm

Cl

O
OH
Parent = 230 nm
meta Br = 2 nm
Total = 232 nm

Observed = 232 nm

Br

Stereochemical factors in electronic spectroscopy

 Angular strain or steric overcrowding

 distorts geometry of the chromophore
 reduced  - orbital overlap  reduced conjugation
e.g. Biphenyls:

Leads to shorter wavelength shift and diminished intensity.

Cis- and trans- isomers

40
Alkenes:  trans- isomer exhibits longer wavelength absorption and higher intensity than cis –
isomer.

Angular distortion:  steric inhibition of conjugation.

ring strain cross-conjugation

Woodward rules not applicable

E.g.
For A
Parent = 215 nm
beta substituent = 12 nm
A Total = 227 nm

For B
O Parent = 215 nm
B 2 beta substituents = 24 nm
1 exocyclic double bond = 5 nm
Total = 244 nm
Observed = 244 nm

Infra Red (IR) Spectroscopy (1 – 10 mg)

 Vibrational excitations
 bond stretching
 bond bending (deformation)

 For Biochemists, main groups include:

O-H, S-H, N-H, C=O, -CO2H, -COO, aromatic rings etc.
Hooke’s Law

41
  X – H  highest frequencies
 Bond strength order C  C > C = C > C  C
C  O>C O

 Shows presence or absence of functional groups.

 Diagnosis of finer structural detail such as conformation and intramolecular
hydrogen bonding.
 Identification of unknown by finger printing.
 Studies of macromolecular dynamics by hydrogen – deuterium exchange.
 Wavenumber ( ῡ )  number of waves per cm cm -1
 Vibrational IR covers the region: 4000 – 400 cm-1
 Vibrational modes of – CH2 -, - NH2 (AX2)

Stretching and bending vibrations of bonds in organic molecules e.g. -CH2- , -NH2

42
  Pass a bean of infrared radiation of constantly varying frequency through a sample of the
compounds.
 Detector generates a plot of % transmission of radiation versus wave number or
wavelength of the transmitted radiation.
 Each spike (absorption band) represents absorption of energy.

Sample

1. Gas  gas cells

2. Liquid  thin film on NaCl cells
 solutions
3. Solids  KBr pellets (disc)
0.1 – 2% by weight with KBr
 Mulls → pastes → using liquids paraffin (Nujol) but interference because of
C–H stretching and C-H bending.
 use hexachlorobutadiene (HCB)
 C=C interference
 use chlorofluorocarbon oils

Factors influencing vibrational frequencies

1. Vibrational coupling

43
  Takes place between two bonds vibrating with similar frequency, provided that
the bonds are reasonably close in the molecule.
 For isolated C – H bond  one stretching vibration frequency.
 C – H in – CH2 -  2 coupled vibrations with difference frequencies, e.g. sym,
asym.
 Coupling of stretching and bending vibrations e.g. secondary acyclic amides
show strong absorption in the 1650 – 1515 cm -1 region.

Amide bands
 Coupling of stretching and bending vibrations e.g. secondary acyclic amides (exists
predominantly in the trans–conformation) show strong absorption in the 1650–1515 cm-1
region  involves coupling of N–H bending and C=O stretching vibrations.

The most important for proteins are:

I  near 1650 cm-1  80% C = O stretching

(1600 – 1690) <20% in – plane
N – H bending

II  near 1550 cm-1  60% N – H bending

(1480 – 1425) 40% C – N stretching

III  near 1300 cm-1  40% C-N stretching

30% N-H bending (in – plane)

 Coupling of the two N – Hstr bands in the 3497 – 3077 cm-1 region in primary
amines and primary amide spectra  gives 2 bands.
 Also for the two C=O str bands in the 1818–1720 cm-1 region in carboxylic
acid anhydrides (with separation of 40 - 80 cm-1).

 Coupling between the two carboxyl groups via oxygen  coplanar system.
 higher frequency band  sym C=O strecting.

44
Fermi Resonance  Enrico Fermi
 fundamental vibrations may couple with the overtone of some other vibration  Fermi
resonance.

e.g. “doublet” appearance of C=O str. of cyclopentanone.

1st overtone of C–H bend and C=O stretching.

45
 alters force constant of both groups

X–H str.  moves to lower frequencies usually with increased intensity and broadening.

Free O–H str.: 3650 cm-1 (sharp)

Hydrogen–bonded O–H str.: 3350 cm-1 (broad)

46
  Intramolecular H-bonding not affected by dilution.

Enols

e.g. 2, 4 – pentanedione

3. Electronic effects
 Changes in the absorption frequencies for a particular group take place when the
substituents in the neighbourhood of that particular group are changed.

It includes:
 Inductive effects
 Mesomeric effects (resonance)
 Field effects (through space influence)

47
48
Phenyl esters
Alkyl esters
O O O O
O

O R O O
O

Weakens C=O bond

1740 cm-1 1760 cm-1 1735 cm-1
Lowers C=O str. freq.

Strengthens C=O bond

Increases C=O str freq.

Field effects  electrostatic and/ or steric in nature.

E.g.

C=O str not shifted

C=O str shifted O

OCH3

CH3
O
O

Cl
Cl

4. Bond angles

Strained bond-angle  higher C=O str freq.

If bond  120o  opposite effects.
E.g.

49
 about 1697 cm-1

greater than 120o

Correlation Tables

Alkanes: C – Hstr 3000 – 2840 cm-1; C – Cstr 1300 – 800 cm-1

Alkenes: C – H str 3080 – 3020 cm-1; C = Cstr 1667 – 1640 cm-1

Conjugated C = C str 1640 – 1600 cm-1 (increased intensity)

Aromatic (mononuclear): C – Hstr 3100 – 3000 cm-1 (weak)

C – Hdef ≅ 710 – 675 cm-1 (out–of–plane)
C – CStr (skeletal) 1600 – 1450 cm-1 (medium strength)

Amine: - NH2  N – HStr 3500 – 3300 cm-1

Primary (10)  2 bands
Secondary (20)  1 band.

Amino acids (Zwitter ions): N – HStr 3130 – 3030 cm-1

Sometimes accompanined by broad bands near 2500 and 2000 cm-1.

Amides: N – HStr 3460 – 3400 and 3100 – 3070 (2 bands)

C = OStr 1700 – 1500 cm-1
 The N – Hstr bands lowered by hydrogen bonding and solid state samples.

R – SH (thiols): S – HStr 2600 – 2550 cm-1

 weaker than O – HStr and less affected by hydrogen bonding.

Aldehydes: C = OStr 1740 – 1720 cm-1 (strong)

 shifted to 1655 – 1625 cm-1 due to intramolecular hydrogen bonding.
C – HStr 2830 – 2695 cm-1 (2 moderately intense bands)  Fermi resonance:
 C – HStr coupling with 1st C – H def (≅ 1390 cm-1) overtone.

Ketones: C = OStr 1725 – 1705 cm-1

 strong and lowered by conjugation and hydrogen bonding.

Carboxylic acids: C = OStr 1725 – 1680 (strong)

50
 O – HStr 3600 – 2500
 lowered by hydrogen bonding

Carboxylate ion
C = OStr 1610 – 1550 (strong)

Ester: C = OStr 1750 – 1735 (strong)

C – OStr 1050 – 1300 cm-1

Phosphate: P = OStr 1240 – 1180 (strong)

O - HStr 2700 – 2560 (hydrogen bonded)

Alcohols: O – HStr 3600 – 3200 cm-1 (broad)

R – OH C – OStr 1025 – 1200 cm-1

Ethers: C – OStr 1070 – 1150 cm-1

R–O–R

Application of IR to Biomolecules

 IR spectrum of a protein provides a lot of information on structure and

environment of the protein backbone and of the amino acid side chains.

Hence useful tool for the investigation of:

(i) Protein structure

(ii) Molecular mechanism of protein reactions
(iii) Protein folding, unfolding and misfolding

e.g. N-HStr Amide I Amide II

(C=OStr) (N-Hdef)

 - helix: 3290 – 3300 1648 – 1660 1540 – 1550

 - sheet: 3280 – 3300 1625 – 1640 (strong) 1520 – 1530

1690 (weak)

 Information about structure and environment of amino acid chains can be

deduced from the following spectral parameters:

51
 (i) Band position  protonation state, coordination of cations and hydrogen
bonding.

(ii) Band width  a measure of conformational freedom with flexible

structures giving broader bands e.g. used to characterize the environment
of the phosphorylated Asp residue of the sarcoplasmic reticulum Ca2+ -
ATPase.

(iii) Absorption coefficient  increases with the change of dipole moment

during the vibration  often correlated with the polarity of the vibrating
bonds.

FT – IR

 Proteins in different environment can be characterized.

Biological systems  lipids, proteins, peptides, bio-membranes, nucleic acids,
animal tissues, microbial cells, plants and clinical samples have all been
successfully studies by using IR spectroscopy.

Environments: Protein  in single crystals, in aqueous solution, organic solvents, detergent

micelles, lipid membranes, etc.

Nuclear Magnetic Resonance (NMR)

 Nuclei isotopes possessing an odd number of protons or odd number of neutrons

or both exhibit mechanical spin associated with spin angular momentum
 characterized by a nuclear spin quantum number I  O.

e.g.

Nuclei Natural Abundance Spin Quantum No. (I)

1H 99.99% ½
13C 1.11% ½
11B 100% 3/2
14N 99.64% 1
15N 0.37% ½
2H 0.015% 1
17O 0.037% 5/2

52
 19F 100% ½
31P 100% ½

12 16 32
Non–magnetic nuclei: C, O, S.

 I = O

 Important nuclei for biochemical studies in NMR  1H, 13C, 31P, 15N, 17O.
1
H NMR

 Shows the number of the difference types of H atoms  group of signals.

 What type of H atoms  chemical shift.
 Number of H atoms of each type  integration.
 Connectivity  neighbours  coupling patterns.
 Like an electron, a proton has two spin states with quantum numbers of +½ and
-½.  given by 2I + 1, when I = ½.

* Spinning proton behaves like a tiny bar magnetic and has a magnetic moment
associated with it.

 Placed in external applied magnetic field (Bo), a proton’s own magnetic field
caused by the nuclear spin can align with or against the external field.

53
 when placed in external magnetic field (Bo), a proton’s own magnetic field caused by the
nuclear spin can align either with or against the external field.

NB: The nuclei can adopt (2I+1) orientations in the field with m1 (magnetic quantum
number) values of -I, -I + 1, … , I.

Transitions with m1 = ± 1 are allowed  selection rules in NMR.

54
55
 Frequency of electromagnetic  Energy difference  Magnetic
radiation (Hz or s-1) between nuclear states field (T)
-1
(kjmol )
 If sample is irradiated with radiation in the radio-wave region (MHz), the proton can
absorb the necessary energy and be promoted to the higher less–favourable state (spin
-½)  this absorption is called “resonance” since the frequencies of the applied radiation
and the precession coincide or resonate.

56
 Irradiate with short, intense burst of radiofrequency radiation (the pulse) which excites all
the protons in the molecule at the same time.

 Free-Induction Decay (FID)

57
58
  FID stored in computer and converted into a spectrum by a mathematical process known
as a fourier transform.

Radiationless relaxations

1. Spin-lattice relaxation  energy transferred to the molecule framework (“Lattice”) or

solvent molecule (surrounding environment) and is lost as translational or vibrational
energy (heat).

2. Spin-Spin relaxation  transfer of energy from one nucleus to the neighbouring nuclei,
provided that particular value of E is common to both nuclei.

The frequency at which any nucleus will resonate in the NMR spectrum  chemical shift.

59
OR Shift in frequency of nuclei depending on their chemical environments.

OR The difference in the absorption of a particular proton from the absorption

position of a reference proton.

60
1
H Chemical Shifts

Shielding or

Diamagnetic shift

Deshielding or

Praramagnetic shift

 The term deshielding often used to describe the decreased shielding of one proton
relative to another.

61
1. Electronegativity of neighbouring groups or atoms

e.g. CH3-CH3 CH3-Cl CH3–OCH3

 0.26  3.06  3.24

(shielding effects) (deshielding effects)

62
 2. Hybridization

In general: Methine H > Methylene H > Methyl H

SP2 > SP3, e.g. C-C-H appears at δ 0.5 – 1.5 ppm

3. Magnetic anisotropic effects

-electrons create non uniform fields in the vicinity of protons  for some protons the
effect of these fields will be shielding while for others deshielding.

63
Carbonyl group

64
Integrals in 1H NMR

 Area under each peak signal is proportional to the number of hydrogen atoms in that
environment  relative quantities of the resonance nuclei with different chemical
environment.

Spin–Spin splitting (coupling)

 A magnetic “through bond” interaction between the two nuclei which are coupled 
brought about by the interactions between local magnetic fields and bonding electrons.
 Related to the number of possible spin orientations that these neighbours can adopt.

1st order splitting  n + 1 rule where n = number of neighbouring protons.

The splitting is due different magnetic environments.

Examples

The number of split peaks in the signal is called multiplicity.

65
66
NB: (i) Peak splitting (coupling) generally not observed among chemically and
magnetically equivalent protons.
Two protons are defined as being magnetically equivalent if each couples
equally to a third neighbouring proton (coupling equivalent).

(ii) Two protons are defined as being chemically equivalent if by virtue of

symmetry within the molecule, their electronic environments are
indistinguishable  posses same value of chemical shift (chemical shift
equivalent)

67
 2 protons which are chemically non-equivalent must also be magnetically non-

68
 equivalent but 2 protons can be magnetically non-equivalent but chemically
equivalent.
 Normally coupling upto 3 bonds (3J)

Successive splitting  use of scale diagram

 Interacting nuclei represented by the letters of the alphabet  gap between the letters
indicating the separation between the nuclei in chemical shift.

Pascal’s triangle or Binomial Coefficients

69
AX and AB spin system

70
AX3 Spin system

E.g. CH3-CH

A2X2 Spin system

E.g.
Y-CH2-CH2-Z

71
72
A2X3 e.g. CH3CH2Cl

AMX

E.g.

73
 doublet of doublets

7.0 - 5.5 ppm

HX 7.5 - 7.0 ppm
HM
doublet of doublets

HA CHO
O
9.5 ppm
8.0 - 7.5 ppm

doublet of doublets

JMX = 0.7 Hz; JAX = 1.7 Hz; JAM = 0.7 Hz

Non – first order AB splitting pattern

(a) First order AX (b) AB quartet

74
The AB Spin System and Higher Order Spin
Systems
AB spin systems show coupling between two nuclei of similar chemical shift and
show a variable intensity pattern that depends on the relative magnitude of the shift
difference (in Hz) a - b and the coupling constant J(A,B).

75
a) J / a - b) = 1: 3;
b) J / a - b) = 1: 1;
c) J / a - b) = 5: 3;
d) J / a - b) = 5: 1

Spectra a) to d) which show deviations from the binomial intensity pattern are called
higher order spectra.

Coupling Constants

 The difference between the peaks arising from a coupling interaction between
neighbouring protons with different chemical shifts.
 Expressed in Hertz (Hz)
 Independent on the magnitude of the applied field.
 JH-H
 Characteristic of the type of protons causing the splitting
 High field NMR spectrometers are very useful since stronger fields do not affect J but
influence .
 Spectra run in high fields are more likely to be first order, because the ratio ∆⁄𝐽 is larger.
 1st order if δA – δB (Hz) ≥ 10JAB (Hz)

76
77
 The effects of spectrometer field strength on the ability to resolve NMR coupling information is
illustrated in this set of spectra of ethylbenzene, plotted at a constant Hz scale. The aromatic
signals go from nearly a singlet at 60 MHz to a reasonably resolved set of peaks at 600 MHz
(spectra courtesy of Kris Kolonko). This increase in information content and greater ease of
interpretation of NMR spectra at higher magentic field strength is the main justification for the
additional expense of more powerful magnets.

78
Factors affecting J values

1. Number of bonds between the coupling nuclei

E.g 1
JCH  120 – 250 Hz
2
JHH  10 – 20 Hz (aliphatic germinal)
3
JHH  5 – 8 Hz

79
80
81
 H H H

trans: J = 12 - 18 Hz cis: J = 6 - 12 Hz

82
83
RCOOH becomes ROOD; ROH becomes ROD and RCONH2 becomes
RCOND2 etc.

84
Decoupled, NOE (Double Resonance) and COSY Spectra

 If it is not obvious which protons in a molecule are coupled to which  can determine
coupled signals through the use of decoupling experiments.
 Irradiate one nucleus while detecting all the other nuclei in the molecule i.e. “double
resonance” using two pulses of radiation  signal due to any nucleus coupled to that
being irradiated increases in intensity.

COSY  Correlated SpectroscopY

1
H1H COSY  helps to correlate the 1H shifts of all the coupling partners in the molecule.
 “normal” 1H spectrum plotted on both the X– and Y–axes and a projection along the
diagonal (with the peak heights represented by contours).
 Off the diagonal peaks (or cross peaks) are the key signals.
 Cross peaks appear on either side of the diagonal axis and are symmetrical about it.
 A particularly popular 2D NMR experiment is the COSY experiment.
 COSY experiments are often 1H-1H experiments but there are many other correlations in
the literature.

 Makes it possible to assign protons to specific amino acids.

 The (H,H) COSY experiment establishes the connectivity of a molecule by giving cross
peaks (these are the off diagnonal peaks) for pairs of protons that are in close proximity.
For the example of Glutamic acid below, we obtain cross peaks for the proton pairs (2,3)
and (3,4). We do not observe a cross peak for the pair (2,4)  because these protons are
not directly adjacent.

85
500 MHz (H,H) COSY Spectrum of Glutamic acid. 1-D spectra left and top. 10 mg of compound
in 0.5 mL of D2O, 5 mm sample tube, 256 spectra, digital resolution of 2.639 Hz/data point.
Total measurement time ca. 3h.
A relayed COSY experiment goes one step beyond a COSY experiment by showing cross peaks
not just for pairs of adjacent protons, but for triples as well. As a result, we observe additional
cross peaks like the one for the pair 2,4 in Glutamic acid below. Relayed COSY experiments can
give cross peaks for protons that are too distant to show coupling in the 1D NMR spectrum

86
  TOCSY  Total correlation spectroscopy.

NOE Spectra
NOE  Nuclear Overhauser Effect
2D-NOE Techniques
Irradiate one nucleus with a low – level radiofrequency signal (double resonance) and monitor
what is happening in the rest of the spectrum  enhancement of the signals for nearby nuclei is
observed.

 through–space interactions

87
  Helps to get which protons are close in space to each other in a molecule
 Used to study conformation e.g. proteins.
 2D version which gives all the NOE effects in the molecule in a single spectrum 
NOESY.

NOE techniques complement techniques that involve scalar coupling because they establish the
proximity of non-bonded atoms. Most published data are on 1H, 1H experiments. Common pulse
sequences:

 NOESY (3-dimensional structure of molecules, exchange processes)

 zz-NOESY (solvent peak suppression, differentiation between chemical exchange and
NOE).

NOESY & HOESY

The cross peaks in NOESY are due to pairs of nuclei that are close enough (<2.5 Å) to have
strong dipolar interactions. Moreover, the size of 1H-1H NOESY cross peaks is proportional to
their distance. This is particularly valuable for the structure elucidation of proteins.

88
Sequence of Proteins

1. Find patterns of coupled interconnected spins (spin systems) belonging to amino acid residues
using COSY and TOCSY.  amino acid identification.

2. Connect neighbouring spin systems (in the amino acid sequence) using sequential NOEs.

3. Match stretches of connected spin systems with the (known) amino acid sequence for unique fits.

13C NMR Spectroscopy

C  abundances 98.9%  but nuclear spin (I) = 0.

12

C  1.1%  nuclear spin (I) = ½

13

 Carbon skeleton
 Generally less signal overlapping (simpler)
 Spread over a wider range O – 220 ppm; compare with 1H NMR  0 - < 20 ppm
 Low abundance of 13C gives low probability of 2 adjacent carbon atoms having 13C  no
13
C – 13C coupling but have 13C – 1H coupling  follows n + 1 rule where n = no. of H
atoms.
 Chemically and magnetically equivalent C atoms appear together on the spectrum.
 Area under each peak not always proportional to the number 13C nuclei giving rise to it
 NO integration of peaks.
 Data harder to obtain then 1H  use more scans and more sample.

89
90
Conventional 13C spectrum referred to as being proton decoupled and consists of a plot of
chemical shift against intensity.
 Irradiate the H over the entire proton frequency range while obtaining the carbon
spectrum (broad-band decoupled spectrum).
 Irradiation enhances the signal-to-noise ratio by as much as 300% (NOE)
 Coupling of 13C – 1H gives a complex spectrum  thus partial decoupling of 13C – 1H
signals produces off–resonance spectrum  only 1JCH is seen.

91
Alkanes:  δ 10 – 30 ppm

C H C CH3
C
H
most shielded
lowest delta value
most deshielded
highest delta value

92
93
Assignment techniques which can be run to aid interpretation and assignment of different 13C NMR
spectra are:

1. Distortionless Enhancement by Polarization Transfer (DEPT)

 Helps to differentiate between the above different types of carbon atoms.

94
2. Heteronuclear Multiple Quantum Correlation (HMQC)
 2D spectrum that shows correlation between a carbon atom and the attached proton (s)
i.e. shows 1JCH coupling  detect directly bond 13C 1H atoms.

95
  Also HETCOR  heteronuclear correlation.

3. Heteronuclear Multiple Bond Correlation (HMBC)

 Detect longer range 1H – 13C coupling and converts it into a correlation map.
 Shows the connectivities of carbon atoms through their 2JCH and 3JCH coupling.

 Spectra showing 13C – 13C shift correlations are called 2D 13C INADEQUATE.
 Identifies directly bonded carbon atoms.

96
  More recently 3D- and 4D-NMR spectroscopy have been developed and can be used to
determine more complex macromolecular biological systems.

 Also 15N NMR spectroscopy. Low abundance (< 0.4%). Needs 24 hrs scan time and is
more expensive.

 Isotope labeling of samples enriched with 13C, 15N or 2H (RNA)

 31P NMR spectroscopy is a routine NMR method (abundance of 100%)

X-Ray Spectroscopy

 Not limited to size of the protein and usually gives high resolution details of the atomic structure
of the protein in a crystal.
 Determination of complete 3-dimensional molecular structure and stereochemistry.
Structural information that can be studied by x-ray:
 Electronic structure (focused on valence and core electrons, which control the
chemical and physical properties among others).

97
 Geometric structure (which gives information about the locations of all or set of
atoms in a molecule at an atomic resolution). This gives bond lengths, angles and
distances between non-bonded atoms.

98
X-Ray Absorption Spectroscopy (XAS)
Used to probe empty states and shapes of molecules or local structures.

X-Ray Emission Spectroscopy (XES) or also known as X-Ray Flourescence Spectroscopy (XFS)

99
Very sensitive probe for examining the local electronic structure and chemical bonding of emitting atoms,
e.g. excitation of –O-H, -O- etc.

X-Ray Photoelectron Spectroscopy (XPS)

Investigate occupied electronic states.
Determines bonding energies of C, O N atoms.

100
X-Ray Auger Spectroscopy

101
 X-Ray spectroscopy is a powerful and flexible tool and an excellent complement to many
structural analysis techniques such as UV-Vis, IR, NMR, etc.
 Requires high quality single crystals which are not available for many proteins, e.g. most of the
membrane proteins.
 Structure of a protein in a crystal may not relate to its structure in solution.
 Globular proteins are comparatively easy to crystallize.
 If single crystals of sufficient size cannot be obtained, various other X-ray methods can be
applied to obtain less detailed information; e.g. fiber diffraction and powder diffraction.
 Solving the 3-dimensional structure of a protein by X-ray crystallography requires well-ordered
crystals that diffracts x-rays.
 A beam of X-rays (monochromatic or polychromatic) is directed at a protein crystal such that the
repeating pattering of protein molecules (atoms) in the crystal results in a characteristic pattern.
The repeating units are called unit cells and may contain one or more protein molecules.
 This requires a pure homogeneous protein sample of high purity (ideally > 97% purity).

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