NMR SPECTROSCOPY

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A.Solairajan 1 st year M.pharm (analysis) 1 H NMR SPECTROSCOPY

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Introduction -Spin-spin Coupling Fundamental principles and theory -Coupling constant Instrumentation -2D-NMR Solvents -NOE Chemical shift -NOESY Factors affecting chemical shift -COSY Interpretation of proton -Applications NMR spectra Proton chemical shifts Presentation outline

Introduction:-:

Introduction:- Nuclear Magnetic Resonance (NMR) is a spectroscopy technique which is based on the absorption of electromagnetic radiation in the radio frequency region 4 to 900 MHz by nuclei of the atoms. Proton Nuclear magnetic resonance spectroscopy is one of the most powerful tools for elucidating the number of hydrogen or proton in the compound. It is used to study a wide variety of nuclei: 1 H 15 N 19 F 19 F 13 C 31 P

Theory of NMR:-:

Theory of NMR:- Spin quantum number (I) is related to the atomic and mass number of the nucleus. Elements with either odd mass or odd atomic number have the property of nuclear “spin”.

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If an external magnetic field is applied, the number of possible orientations calculated by (2I+1). Hydrogen has spin quantum number I=1/2 and possible orientation is (2*1/2+1=2) two +1/2 and -1/2.

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The theory behind NMR comes from the spin of a nucleus and it generates a magnetic field. Without an external applied magnetic field, the nuclear spins are random in directions. But when an external magnetic field(B o ), is present the nuclei align themselves either with or against the field of the external magnet. Principles of NMR

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If an external magnetic field is applied, an energy transfer (∆E) is possible between ground state to excited state. when the spin returns to its ground state level, the absorbed radiofrequency energy is emitted at the same frequency level. The emitted radiofrequency signal that give the NMR spectrum of the concerned nucleus. .

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The emitted radio frequency is directly proportional to the strength of the applied field. γ B o 2 П B o = External magnetic field experienced by proton γ = Magnetogyric ratio ( The ratio between the nuclear magnetic moment and angular moment) v =

NMR spectrum:

NMR spectrum The NMR spectrum is a plot of intensity of NMR signals VS magnetic field (frequency) in reference to TMS

NMR instrumentation:

NMR instrumentation 1. Sample holder 4. Sweep generator 2. Permanent magnet 5. Radio frequency transmitter 3. Magnetic coils 6. Radio frequency receiver 7.Read out systems

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1.Sample holder :- Glass tube with 8.5 cm long,0.3 cm in diameter 2.Permanent magnet :- It provides homogeneous magnetic field at 60-100 MHZ 3.Magnetic coils :- These coils induce magnetic field when current flows through them. 4.Sweep generator :- To produce the equal amount of magnetic field pass through the sample

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5.Radio frequency :- A radio transmitter coil transmitter that produces a short powerful pulse of radio waves 6.Radiofrequency :- A radio receiver coil that detects Receiver radio frequencies emitted as nuclei relax to a lower energy level 7.Readout system :- A computer that analyses and record the data

Solvents used in NMR:

Solvents used in NMR The following solvents are normally used in which hydrogen replaced by deuterium. CCl4 - carbon tetrachloride CS2 - carbon disulfide CDCl3 - Deuteriochloroform C6D6 - Hexa deuteriobenzene D 2 O - Deuterium oxide

Chemical shift:

Chemical shift A chemical shift is defined as the difference in parts per million (ppm) between the resonance frequency of the observed proton and tetramethylsilane (TMS) hydrogens . TMS is the most common reference compound in NMR, it is set at δ=0 ppm

:

Shielding of protons:- High electron density around a nucleus shields the nucleus from the external magnetic field and the signals are upfield in the NMR spectrum Deshielding of protons:- Lower electron density around a nucleus deshields the nucleus from the external magnetic field and the signals are downfield in the NMR spectrum

Factors affecting chemical shift:-:

Factors affecting chemical shift:- Electronegative groups Magnetic anisotropy of π- systems Hydrogen bonding Electronegative groups:- Electronegative groups attached to the C-H system decrease the electron density around the protons, and there is less shielding ( i.e. deshielding) and chemical shift increases Compound Chemical shift CH 3 I 2.16 CH 3 Br 2.65 CH 3 Cl 3.10 CH 3 F 4.26

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Magnetic anisotropy of π- systems:- The word "anisotropic" means "non-uniform". So magnetic anisotropy means that there is a "non-uniform magnetic field". Electrons in π systems (e.g. aromatics, alkenes, alkynes, carbonyls etc .) interact with the applied field which induces a magnetic field that causes the anisotropy. It causes both shielding and deshielding of protons. Example:-Benzene Hydrogen bonding:- Protons that are involved in hydrogen bonding are typically change the chemical shift values. The more hydrogen bonding, the more proton is deshielded and chemical shift value is higher.

Proton NMR:

Proton NMR The most common for of NMR is based on the hydrogen-1 ( 1 H), nucleus or proton. It can give information about the structure of any molecule containing hydrogen atoms .

1H NMR chemical shift:

1 H NMR chemical shift

Proton NMR spectra of Ethanol:-:

Proton NMR spectra of Ethanol:- 1 H spectrum of Ethanol:- 3 types of proton CH 3, CH 2, OH

1H NMR spectra of Benzene:

1 H NMR spectra of Benzene Aromatic Hydrogen shows peak in the chemical shift scale 6.5-8.0 ppm . From the above spectrum Benzene has same type of protons and it shows single peak at 7.26

Interpretation of 1HNMR spectra:-:

Interpretation of 1 HNMR spectra:- Number of signals - Indicates how many "different kinds" of protons are present. Position of signals - Indicates something about ( chemicalshift ) magnetic (electronic) environment of protons Relative intensity of - Proportional to number of protons present signals Splitting of signals - Indicates the number of nearby nuclei (spin spin coupling) usually protons

n+1 rule:-:

n+1 rule:- The multiplicity of signal is calculated by using n+1 rule. This is one of the rule to predict the splitting of proton signals. This is considered by the nearby hydrogen nuclei. Therefore, n= Number of protons in nearby nuclei Zero H atom as neighbour n+1=0+1=1(singlet) One H atom as neighbour n+1=1+1 = 2(doublet) Two H atom as neighbour n+1=2+1 =3(triplet)

Spin-spin coupling (splitting):

Spin-spin coupling (splitting) The interaction between the spins of neighbouring nuclei in a molecule may cause the splitting of NMR spectrum.This is known as spin-spin coupling or splitting. The splitting pattern is related to the number of equivalent H-atom at the nearby nuclei. Ethyl acetate

Rules for spin-spin coupling:-:

Rules for spin-spin coupling:- * Chemically equivalent protons do not show spin-spin coupling. * Only nonequivalent protons couple. * Protons on adjacent carbons normally will couple. * Protons separated by four or more bonds will not couple .

Coupling Constant:

Coupling Constant The distance between the peaks in a given multiplet is a measure of the splitting effect known as coupling constant . It is denoted by symbol J, expressed in Hz. Coupling constants are a measure of the effectiveness of spin-spin coupling and very useful in 1 H NMR of complex structures.

2D NMR:

2D NMR Basics of 2D NMR Experiment:- Basis: Interaction of nuclear spins ( 1 H with 1 H, 1 H with 13 C, etc.) plotted in two dimensions Normal spectra( 1D NMR) are plots of intensity Vs frequency. In 2D NMR intensity is plotted as function of two frequencies called f 1 & f 2 • In general, 2D’s can be divided into two types, Homonuclear Heteronuclear • Each type can provide either through-bond (COSY-type) or through space (NOESY-type) coupling information

2D-Correlation spectra:

2D-Correlation spectra

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2-D NMR –Signal is recorded as a function of two time variables, t1 and t2. •Rf pulses are generally applied during the preparation and mixing periods.

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Applications of 2D NMR:- Simplifies analysis of more complex or ambiguous cases such as proteins. Obtain structural information not accessible by one- dimensional NMR methods.

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Techniques include: Correlation Spectroscopy (COSY) Heteronuclear Correlation Spectroscopy (HETCOR) Heteronuclear Multiple-Quantum Coherence (HMQC) Nuclear Overhauser Effect Spectroscopy (NOESY) Incredible Natural Abundance Double Quantum Transfer Experiment (INADEQUATE) Many others

Routine 2-D NMR Experiments:- :

Routine 2-D NMR Experiments:- COrrelationSpectroscopY (COSY) –Scalar Coupling »Identifies all coupled spins systems. Nuclear OverhauserEffect SpectroscopY (NOESY) –Dipolar Coupling »Identifies neighboring spin systems (≤5 Å) »Identifies chemical exchange. Heteronuclear Multiple/Single Quantum Correlation (HMQC/HSQC) –Scalar Coupling »Identifies coupling between heteronuclei (C-H, N-H)

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COSY : Homonuclear correlated spectroscopy. Correlation between protons that are coupled to each other TOCSY : Total Correlation Spectroscopy- Uses a spin-lock for coherence transfer. During the spin-lock all protons of a coupled system become "strongly coupled," leading to cross peaks between all resonances of a coupled system. HETCOR : Heteronuclear correlation, usually between 1 H and 13 C resonances mediated by J C-H .

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NOESY, ROESY : Proton-proton correlation mediated by dipolar coupling (NOE effect). Correlation between protons that are close in space. This is the single most powerful NMR technique for determining the 3- dimensional structure of molecules from conformations of small molecules to the 3-dimensional structure of small proteins . HOESY : Heteronuclear Overhauser Effect Spectroscopy. Correlation between protons and heteronuclei that are close in space.

COSY experiment:

COSY experiment Correlation Spectroscopy Commonly used for regiochemical assignment Cross-peaks appear if spin coupling is present Protons that are separated by 2 or 3 bonds are usually detected

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Nuclear Overhauser Effect NOE Caused by dipolar coupling between nuclei. The local field at one nucleus is affected by the presence of another nucleus. The result is a mutual modulation of resonance frequencies. N S N S

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Nuclear Overhauser Effect The intensity of the interaction is a function of the distance between the nuclei according to the following equation. I = A (1/r 6 ) I - intensity A - scaling constant r - internuclear distance 1 H 1 H r 1,2 1 2 1 H 3 r 1,3 r 2,3 Arrows denote cross relaxation pathways r 1,2 - distance between protons 1 and 2 r 2,3 - distance between protons 2 and 3 The NOE provides a link between an experimentally measurable quantity, I, and internuclear distance. NOE is only observed up to 5 Å.

GENERAL APPLICATIONS OF NMR SPECTROSCOPY :

GENERAL APPLICATIONS OF NMR SPECTROSCOPY NMR is used in biology to study the Biofluids , Cells, Per fused organs and biomacromolecules such as Nucleic acids(DNA, RNA), carbohydrates Proteins and peptides. And also Labeling studies in biochemistry. NMR is used in physics and physical chemistry to study High pressure Diffusion ,Liquid crystals ,liquid Crystal solutions , Membranes, Rigid solids. NMR is used in food science.

NMR SPECTROSCOPY ECTROSCOPY SCOPY :

NMR SPECTROSCOPY ECTROSCOPY SCOPY NMR is used in pharmaceutical science to study Pharmaceuticals and Drug metabolism. NMR is used in chemistry to ; Determine the Enantiomeric purity. Elucidate Chemical structure of organic and inorganic compounds. Macromolecules –ligand interaction. Contd…

1H-NMR SPECTROSCOPY applications :

1 H-NMR SPECTROSCOPY applications 1 H widely used for structure elucidation. Inorganic solids - In organic compounds are investigated by solid state 1H-NMR.eg CaSO4⋅ H2O . Organic solids - Solid-state 1 H NMR constitutes a powerful approach to investigate the hydrogen-bonding and ionization states of small organic compounds. Direct correlation with hydrogen-bonding lengths could be demonstrated, e.g. for amino acid carboxyl groups.

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Polymers and rubbers - Examine hydrogen bonding and acidity. Peptides and proteins Clinical and scientific research In vivo NMR studies - concerned with 1 H NMR spectroscopy of human brain. Many studies are concerned with altered levels of metabolites in various brain diseases. To determine the spatial distribution of any given metabolite detected spectroscopically IS (image selected in vivo spectroscopy).

Application of NMR in medicine:

Application of NMR in medicine MRI is specialist application of multi dimensional Fourier transformation NMR Anatomical imaging. Measuring physiological functions Flow measurements and angiography. Tissue perfusion studies. Tumors

References:-:

References:- Organic spectroscopy by William Kemp Instrumental methods of chemical analysis by Chatwal Instrumental methods of analysis by Willard Wikipedia.org

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