Nuclear Magnetic Resonance

1Nuclear Magnetic ResonancePrinciple

Nuclear Spin2Nuclear Magnetic ResonanceEffect of External Magnetic Field

PrinciplePrincipleNuclear Magnetic Resonance

Zeeman EffectNuclear Magnetic ResonancePrincipleAbsorption of RadiationResonant Frequency

is the gyromagnetic ratio, characteristic of each nuclei:B0 is the magnitude of the external magnetic field.

Resonant frequency for all nuclei of same element in a molecule need not be the same.

Depends on the electronic environment of the nucleus considered.

Electrons have magnetic moment of their own, which acts opposite to the external field and reduces the energy gap between the spin states of the nucleus.

Reduced energy gap gives rise to lower resonant (NMR) frequency.

This change in NMR frequency is called chemical shift.

Chemical Shift

Nuclear Magnetic ResonanceContinuous Wave NMR Spectroscopy (CWNMR)NMR SpectrometerWorkingFourier Transform NMR Spectroscopy (FTNMR)Nuclear Magnetic ResonanceWorkingAn NMR Spectrometer consists of three main parts:

NMR Powerful Magnet NMR Spectrometer ConsoleRadio frequency detectorRadio frequency generatorSignal Amplifier Computer Workstation

Nuclear Magnetic ResonanceWorking

Nuclear Magnetic ResonanceWorkingThe process:1. The magnetic field is held constant.2. The sample is irradiated with a short pulse (approximately 10-5s) of radio-frequency energy.3. The radio-frequency energy flips the spins of all susceptible nuclei simultaneously.4.When the pulse is discontinued, the excited nuclei begin to lose their excitation energy and return to their original spin state, or relax. Since the molecule contains many different nuclei, many different frequencies of EM radiation are emitted simultaneously. This emission is calledfree-induction decayorFIDsignal as shown below:

5. The intensity of the FID decays with time and falls to zero as nuclei return to their equilibrium state.6. A computer records the intensity-versus-time information (time domain) and then uses aFourier Transformto convert the information to intensity-versus-frequency information (frequency domain).

Nuclear Magnetic ResonanceWorking5. The intensity of the FID decays with time and falls to zero as nuclei return to their equilibrium state.

6. A computer records the intensity-versus-time information (time domain) and then uses aFourier Transformto convert the information to intensity-versus-frequency information (frequency domain).Nuclear Magnetic ResonanceWorking

Why do we see peaks?Why are the peaks at different position?Doublet Triplet and n+1 rule

Nuclear Magnetic ResonanceApplicationsProtein StructureDeterminationsMagnetic Resonance Imaging (MRI)PharmaceuticalsFood SciencesNuclear Magnetic ResonanceProteins Structure Determination

Kurth Wthrich Nobel Prize 2002

Nuclear Magnetic ResonanceApplicationsProteins Structure DeterminationNuclear Magnetic ResonanceApplicationsProteins Structure DeterminationProtein SolutionNMR SpectroscopySequential resonance AssignmentCollection of Conformational ConstraintsCalculation of 3D structureSTEPSNuclear Magnetic ResonanceApplicationsProteins Structure DeterminationSample PreparationsProteinspHIonic StrengthMolecular WeightPhysiological ConsiderationsPurityTemperatureConcentration19Nuclear Magnetic ResonanceApplicationsProteins Structure DeterminationSpectroscopy

The chemical shift is very sensitive to the micro environment of a particular nucleus.Nuclear Magnetic ResonanceApplicationsProteins Structure DeterminationCan the amino acid residues be connected by NMR?If so, one can get the primary structureNuclear Magnetic ResonanceApplicationsProteins Structure DeterminationResonance Assignment

Use to connect nuclei of amino acid residues which are neighbors in the sequence

Nuclear Magnetic ResonanceApplicationsProteins Structure DeterminationDetermining the arrangement of amino acids by NMR

TALOS+. Predicts protein backbone torsion angles from chemical shift data. Frequently used to generate further restraints applied to a structure model during refinement.Nuclear Magnetic ResonanceApplicationsProteins Structure DeterminationDetecting the secondary structure of protein by NMR

Nuclear Magnetic ResonanceApplications

GeNMRGEnerate NMR structurePROSESSProtein Structure Evaluation Suite & Server

Assessment of protein structural models by NMR chemical shifts as well as NOEs, geometrical, and knowledge-based parameters.Proteins Structure DeterminationNuclear Magnetic ResonanceApplicationsProteins Structure Determination

NMR can also be used to determine the protein ligand interaction by observing the changes in the peaks of chemical shift before and after ligand binding.26

OverviewMagnetic Resonance Imaging (MRI)Nuclear Magnetic ResonanceApplicationsHistory

Nuclear Magnetic ResonanceApplicationsMagnetic Resonance Imaging (MRI)Comparison with CTAdvantagesDisadvantagesCost of ProcessingTime for ProcessingSafetyContrastNuclear Magnetic ResonanceApplicationsMagnetic Resonance Imaging (MRI)ApplicationsNuclear Magnetic ResonancePharmaceuticalsDrug DiscoveryImpurity Profile AnalysisConformation of DrugComposition of Drug TargetClinical DiagnosticsDissolution of TabletInvestigating the metabolites of the drug in the bodyNuclear Magnetic ResonanceApplicationsPharmaceuticalsDrug And Purity DetectionCase Study : Heparin

Nuclear Magnetic ResonanceApplicationsPharmaceuticalsLow Molecular Weight Heparins (LMWHs v/s Unfractioned Heparin (UFH)Limitation of UFHUnpredictable Anticoagulant EffectPlatelets ActivationRisk Of HitAdvantage of LMWHMore Predictable Anti Coagulant responseReduced Protein BindingLess Risk Of HitNuclear Magnetic ResonanceApplicationsPharmaceuticalsOverall characterization can be done by 1H & 13C NMR.

All LMW heparin varies in the Disaccharide and Sulphur content.

Molecular weight of LMW Heparin & its structural information can be determined by 13C NMR (provide high resolution).

1H NMR is used to determine the ratio of Iduronic acid and Glucouronic acid and the position of sulfation in the chain.

Due to presence of different reduced end anomeric carbon and all other anomeric carbon impurities can be easily determined. NMR concept involvedNuclear Magnetic ResonanceApplicationsFood ScienceNuclear Magnetic Resonance (NMR) spectroscopic techniques, based on selective nucleus such as 1H, 13C, 31P and 19F have been utilized for the study of the food analysis because they found in abundance in food and show high sensitivity for NMR spectroscopy.

ParametersFood analysis for solids and multiphase systems, parameters are isotope ratio measurements and magnetic resonance imaging.

Nuclear Magnetic ResonanceApplicationsFood ScienceMilk Analysis by NMR31P NMR work in food science has been covered as part of general NMR reviews for milk, meat, and lipids or olive oil analysis.

The application of 31P NMR spectroscopy in the study of milk by identifying the signals of inorganic phosphates Pi, phosphoserine (SerP) residues in casein in spectra of milk and skim milk in bulk.

Distribution of phosphorus compounds in milk from various species, concluding that the technique may be of value in milk authentication, and also showed that 31P NMR can be used to measure changes during lactation.Nuclear Magnetic ResonanceApplicationsFood ScienceAdvantage over ChromatographyIn general, NMR spectroscopic techniques present several advantages compared to the chromatographic ones such as they are very fast while chromatographic techniques are time consuming they require less solvents thus are cheaper, since one has bought the equipment they do not destroy the sample.

Nuclear Magnetic ResonanceApplicationsFood ScienceDisadvantage of using NMR The big disadvantage of NMR is its low sensitivity.

But this is partially compensated by the high resolution of the NMR spectra. In the aspect of the development of novel fast NMR methods of enhanced sensitivity, scientist has synthesized new fluorine derivatives as probes for the food analysis.THANK YOU