Fluorescence and Nuclear Magnetic Resonance (NMR) Spectroscopy
Murphy, B. (2017). Fluorescence and Nuclear Magnetic Resonance Spectroscopy: Lecture 3.
Lecture presented at PHAR 423 Lecture in UIC College of Pharmacy, Chicago.
FLUORESCENCE SPECTROSCOPY
• Electron is excited by absorption and then emits fluorescence upon relaxation
• Stokes shift = difference between excited and emitted wavelengths
• Fluorophore = molecules or functional groups that have the capacity to exhibit
fluorescence
o Require extended conjugation of pi bonds
o More conjugated → less energy required for excitement → longer wavelength
can be used for excitation
• Fluorescent probes used to identify biological processes
o Green fluorescent protein (GFP) – fluoresces green light when exposed to light
in the blue to UV range
▪ Can make its own color using oxygen only
▪ Slight modifications can allow for different colors to be emitted. Gives
researcher a toolbox of probes for in vivo imaging studies
o Can study specific proteins or cellular movements → disease states
▪ Must be careful → too much modification of the protein can impact its
natural functioning
• Protein tagging
o Can add the fluorescent probe to the C- or N-terminus. Glycine allows for more
flexibility
• Cellular tagging
o Can visualize the G1 phase and the S/G2/M phase
• Weakness- hemoglobin and melanin can also absorb fluorescent light
o Optimal viewing window is near IR region, not visible light region
o Near IR probes – increase tissue penetration and resolution of image
▪ Can use small organic molecules or inorganic nanoparticles
▪ Just need a certain degree of conjugation
• Forster resonance energy transfer (FRET) – studying energy transfer between
fluorophore molecules → allows study of protein interactions in the cell
o The excited energy fluorophore passes its energy to the lower energy
fluorophore via a dipole-dipole interaction
• Photosensitizers – dyes that can generate reactive oxygen species (ROS) light
o Photodynamic therapy – using a photosensitizer in tumor cells to kill them with
targeted therapy
NUCLEAR MAGNETIC RESONANCE (NMR)
• Involves analyzing nuclear spin of the atom (in the molecule) being studied
o Nuclei absorb electromagnetic radiation
o Only certain nuclear can exhibit this nuclear spin: 1H, 13C, 14N, 17O, 19F
o Have to use a deuterated solvent
▪ Deuterium = 2H or D
▪ Otherwise the solvent would interfere with the results if we used normal 1H
• Electrons shield each nucleus from the magnetic field
o For example, oxygen is fairly electronegative so it can pull electrons away from
carbon and “deshield” it. This would give a signal on the spectrum that is more
“downfield”
o Signals that appear “upfield” (to the right) are from nuclei that are more
shielded (next to an electron donating group)
o Chemical shift – electronic environment around a nuclei giving a certain
resonance signal on the NMR spectrum
• Integration – area under the peak correlates with how many nuclei there are
o Can distinguish between CH3, CH2, CH, etc
• Will need to use 13C NMR for molecules that don’t have a lot of hydrogens
DIFFERENT NMR EXPERIMENTS
• COSY – determines connectivity of 1H spin systems
• NOESY – will distinguish between stereoisomers (cis vs trans)
o Spatial configurations; doesn’t say anything about bond connectivity
• HSQC – will determine 13C-1H connectivity
• HMBC – will determine 1H connectivity to multiple carbons
MRI (MAGNETIC RESONANCE IMAGING)
• The magnetic field at the feet is slightly different than the magnetic field at the head
o Can fine tune to focus on different areas of the body