1. UV, IR, NMR, CD
Isariya Techatanawat, PhD Director of Bioequivalence Study Group, Research and Development Institute,
The Government Pharmaceutical Organization

2. Spectroscopy Study of interaction of electromagnetic radiation.
Interaction might give rise to electronic excitations, (e.g. UV), molecular vibrations (e.g. IR) or nuclear spin orientations (e.g. NMR).

3. Spectroscopy
When a beam of white light strikes a triangular prism it is separated into its various components. This is known as a spectrum.

4. Spectroscopy
There are many other forms of light which are not visible to the human eye and spectroscopy is extended to cover all these.

5. Ultraviolet &Visible Spectroscopy

6. Ultraviolet and Visible Spectroscopy
Ultraviolet (UV) &visible radiation comprise only a small part of electromagnetic spectrum.

7. Ultraviolet and Visible Spectroscopy
Wavelength: Distance between adjacent peaks (or troughs).
Frequency: Number of wave cycles that travel past a fixed point per unit of time [cycles per second, or hertz (Hz)].

8. Some Natural Organic Pigments
Colored compounds is a system of extensively conjugated pi-electrons.

9. Energy Associated with Electromagnetic Radiation
E = hν
where E = energy (in joules),
h = Planck’s constant (6.62×10-34Js)
ν = frequency (in seconds).

10. Electronic Excitation

11. Methyl bromide Methyl Iodide
Chromophore
Chromo-phore
Example
Excitation
λmax, nm ε
Solvent
C=C
Ethene
π  __>   π*
171
15,000
hexane
C≡C
1-Hexyne
180
10,000
C=O
Ethanal
n  __>  π* π  __>  π*
15 10,000
hexane hexane
N=O
Nitromethane
17 5,000
ethanol ethanol
C-X   X=Br       X=I
Methyl bromide Methyl Iodide
n  __>  σ* n  __>  σ*

12. UV/Vis Absorbance
Proteins absorb at 280 nm due to presence of amino acids with aromatic rings.
Proteins absorb at 200 nm due to peptide bonds.

13. UV Spectrometer

14. UV Spectrum

15. UV Spectrum

16. Infrared Spectroscopy (IR)

17. Infrared Spectroscopy (IR)
Absorption of infrared radiation brings about changes in molecular vibrations within molecules and 'measurements' of the ways in which bonds vibrate gives rise to infrared spectroscopy.

18. Infrared Spectroscopy (IR)
Atom size, bond length and bond strength vary in molecules and so the frequency at which a particular bond absorbs infrared radiation will be different over a range of bonds and modes of vibration.

19. The Different Types of Bonds
An organic molecule may contain quite a number of different bonds. All of these bonds will be vibrating, and clearly, different bonds will be vibrating at different frequencies.
There are two basic modes of vibration – ‘stretching’ and ‘bending’.

20. Mode of Vibration

21. Infrared Spectrometer
Infrared spectrometer analyses compound by passing infrared radiation, over a range of different frequencies, through a sample and measuring the absorptions made by each type of bond in the compound.
This produces a spectrum, normally a ‘plot’ of % transmittance against wavenumber.

22. Infrared Spectrometer
Since no 2 organic compounds have the same IR spectrum, a compound can be identified with certainty by comparing its spectrum with that of a known pure compound.
If they are identical, then they are one and the same.

23. Infrared Spectrometer

24. Identification of Structural Features

25. IR Spectrum

26. Nuclear Magnetic Resonance (NMR)

27. Nuclear Magnetic Resonance (NMR)
When some atoms are placed in a strong magnetic field, their nuclei behave like tiny bar magnets aligning themselves with the field.
Electrons behave like this too, and for this reason both electrons and nuclei are said to possess “spin”.
Any spinning electric charge has an associated magnetic field.

28. NMR
Just as electrons with opposite spin pair up with each other, a similar thing happens with protons and neutrons in the nucleus.

29. NMR
If a nucleus has an even number of protons and neutrons (e.g. 12C), their magnetic fields cancel each other out and there is no overall magnetic field.
If the number of protons and neutrons is odd (e.g.13C and 1H ), the nucleus has a magnetic field.

30. NMR
If the substance is placed in external magnetic field, nuclear magnet lines up with the field, in the same way as a compass needle lines up with a magnetic field.

31. NMR

32. NMR
NMR is particularly useful in the identification of the positions of hydrogen atoms (1H) in molecules.

33. 1H NMR

34. 1H NMR spectrum
Ethyl benzene, C6H5CH2CH3

35. 1H-NMR spectrum of hen egg white lysozyme

36. 1D NMR Spectrum of Protein

37. 2D NMR
1D protein spectra are too complex for interpretation as most of the signals overlap heavily.
By introduction of additional spectral dimensions, these spectra are simplified and some extra information is obtained.

38. 2D NMR

39. NOESY spectrum for 55 amino acid domain from a protein

40. 13C NMR 13C has only about 1.1% natural abundance
12C does not exhibit NMR behavior.
Magnetic moment of 13C nucleus is much weaker than that of a proton. NMR signals from 13C nuclei are much weaker than proton signals.
Chemical shift range is normally 0 to 220 ppm.
Chemical shifts are measured with respect to tetramethylsilane (TMS), (CH3)4Si.

41. C=O (in acids and esters)
Carbon Environment
Chemical Shift (ppm)
C=O (in ketones)
C=O (in aldehydes)
C=O (in acids and esters)
C in aromatic rings
C=C (in alkenes)
RCH2O-
RCH2Cl
RCH2NH2
R3CH
CH3CO-
R2CH2
RCH3

42. 13C NMR

43. 13C NMR

44. Circular Dichroism (CD)

45. Circular Dichroism (CD)
Difference in absorption of left-handed circularly polarised light and right-handed circularly polarised light
Occurs when molecule contains one or more chiral chromophores.

46. Circular Dichroism (CD)
= A(λ)LCPL ‐ A(λ)RCPL
where λ is the wavelength
LCPL = Left-handed circularly polarised light
RCPL = Right-handed circularly polarised light

47. Circular Dichroism (CD)
CD of molecules is measured over a range of wavelengths.
Use to study chiral molecules.
Analyse the secondary structure or conformation of macromolecules, particularly proteins.

48. Circular Dichroism (CD)
Observe how secondary structure changes with environmental conditions or on interaction with other molecules.
Measurements carried out in the visible and ultra-violet region.

49. Circular Dichroism (CD)
Molecule contains chiral chromophores then one CPL state will be absorbed to a greater extent than the other.
CD signal over the corresponding wavelengths will be non-zero.

50. Circular Dichroism (CD)

51. Circular Dichroism (CD)

52. CD for biological molecules
Majority of biological molecules are chiral.
To understand the higher order structures of chiral macromolecules such as proteins and DNA.
Each structure has a specific circular dichroism signature.

53. CD for biological molecules
To identify structural elements and to follow changes in the structure of chiral macromolecules.
To study secondary structural elements of proteins such as the α-helix and the β sheet.

54. The secondary structure conformation and the CD spectra of protein structural elements.
Right : a peptide in an α-helix
Left: a peptide in a β-sheet.
Centre: CD spectra for these different conformations.

55. CD for biological molecules
To compare 2 macromolecules, or the same molecule under different conditions and determine if they have a similar structure.
To ascertain if a newly purified protein is correctly folded.
To determine if a mutant protein has folded correctly in comparison to the wild-type.

56. CD for biological molecules
For analysis of biopharmaceutical products to confirm that they are still in a correctly folded active conformation.

57. References
An introduction to circular dichroism spectroscopy. University of California.
D.A. Skoog, F.J. Holler and S.R. Crouch, Principles of Instrumental Analysis, 6th Edition, Thomson Brooks/Cole Publishers, 2007.