Download NMR

UV, IR, NMR, CD
Isariya Techatanawat, PhD
Director of Bioequivalence Study Group,
Research and Development Institute,
The Government Pharmaceutical
Organization
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).
Spectroscopy
• When a beam of white light strikes a
triangular prism it is separated into its
various components. This is known as a
spectrum.
Spectroscopy
• There are many other forms of light which
are not visible to the human eye and
spectroscopy is extended to cover all these.
Ultraviolet &Visible
Spectroscopy
Ultraviolet and Visible Spectroscopy
• Ultraviolet (UV) &visible radiation comprise
only a small part of electromagnetic spectrum.
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)].
Some Natural Organic Pigments
• Colored compounds is a system of
extensively conjugated pi-electrons.
Energy Associated with
Electromagnetic Radiation
• E = hν
where E = energy (in joules),
h = Planck’s constant (6.62×10-34Js)
ν = frequency (in seconds).
Electronic Excitation
Chromophore
Chromophore
Example
C=C
Ethene
π
__>
π*
171
15,000 hexane
C≡C
1-Hexyne
π
__>
π*
180
10,000 hexane
n
π
n
π
__>
π*
__> π*
__> π*
__> π*
290
180
275
200
15
10,000
17
5,000
hexane
hexane
ethanol
ethanol
n
n
__>
σ*
__> σ*
205
255
200
360
hexane
hexane
C=O
Ethanal
Nitrometha
N=O
ne
Methyl
C-X X=Br bromide
X=I
Methyl
Iodide
Excitation λmax, nm
ε
Solvent
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.
UV Spectrometer
UV Spectrum
UV Spectrum
Infrared
Spectroscopy
(IR)
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.
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.
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’.
Mode of Vibration
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.
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.
Infrared Spectrometer
Identification of Structural Features
IR Spectrum
Nuclear Magnetic
Resonance (NMR)
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.
NMR
• Just as electrons with opposite spin pair
up with each other, a similar thing
happens with protons and neutrons in the
nucleus.
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.
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.
NMR
NMR
• NMR is particularly useful in the
identification of the positions of hydrogen
atoms (1H) in molecules.
1H
NMR
1H
NMR spectrum
Ethyl benzene,
C6H5CH2CH3
1H-NMR
spectrum of
hen egg white lysozyme
1D NMR Spectrum of Protein
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.
2D NMR
NOESY spectrum for 55 amino acid
domain from a protein
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.
Carbon Environment
Chemical Shift (ppm)
C=O (in ketones)
205 - 220
C=O (in aldehydes)
190 - 200
C=O (in acids and esters)
160 - 185
C in aromatic rings
125 - 150
C=C (in alkenes)
115 - 140
RCH2O-
50 - 90
RCH2Cl
30 - 60
RCH2NH2
30 - 65
R3CH
25 - 35
CH3CO-
20 - 50
R2CH2
16 - 25
RCH3
10 - 15
13C
NMR
13C
NMR
Circular Dichroism
(CD)
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.
Circular Dichroism (CD)
• Circular dichroism
= ΔA(λ)
= A(λ)LCPL ‐ A(λ)RCPL
• where λ is the wavelength
LCPL = Left-handed circularly polarised light
RCPL = Right-handed circularly polarised light
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.
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.
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.
Circular Dichroism (CD)
Circular Dichroism (CD)
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.
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.
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.
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 wildtype.
CD for biological molecules
• For analysis of biopharmaceutical
products to confirm that they are still in a
correctly folded active conformation.
References
• https://www2.chemistry.msu.edu/faculty/reusch/VirtT
xtJml/Spectrpy/UV-Vis/spectrum.htm
• An introduction to circular dichroism spectroscopy.
University of California.
http://www.chem.uci.edu/~dmitryf/manuals/Fundamental
s/CD%20spectroscopy.pdf
• D.A. Skoog, F.J. Holler and S.R. Crouch, Principles of
Instrumental Analysis, 6th Edition, Thomson Brooks/Cole
Publishers, 2007.