Tutorial on Nuclear magnetic resonance Spectroscopy (NMR). Prepared by Lawrence Kok http://lawrencekok.blogspot.com

Analytical Techniques Spectroscopy analysis Study on Identification, Structural Determination, Quantification and Separation Classical method Qualitative analysis Quatitative analysis Separation analysis Flame test Chemical test Melting/boiling point Gravimetric Titration Distillation Precipitation Chemical test Involve Qualitative and Quantitative analysis Quantitative – Amt present in sample/mix Qualitative – Identity species present in impure sample Structural – Determination of structure of molecule Separation of mix – Chromatographic Techniques Identification of functional gps Purity of substances Qualitative analysis Flame test Classical method Gravimetric Quatitative analysis Titration Analytical Techniques InfraRed /UV Spectroscopy Mass Spectroscopy Spectroscopy analysis Nuclear Magnetic Resonance Spectroscopy Instrumental method Atomic Absorption/Emission Spectroscopy High Performance Liquid Chromatography Separation analysis Gas Liquid Chromatography Paper/Thin Layer/Column Chromatography

E = hf Spectroscopy Gamma/X ray UV or visible InfraRed Microwave Spectroscopy measures interaction of molecules with electromagnetic radiation Particles (molecule, ion, atom) can interact/absorb a quantum of light Electromagnetic Radiation High Energy Radiation Low Energy Radiation Velocity of light (c ) = frequency (f) x wavelength (λ) - c = f λ All electromagnetic waves travel at speed of light (3.00 x 108ms-1) Radiation with high ↑ frequency – short ↓ wavelength Electromagnetic radiation/photon carry a quantum of energy given by h = plank constant = 6.626 x 10-34 Js f = frequency λ = wavelength E = hf Click here notes spectroscopy Gamma/X ray UV or visible InfraRed Microwave Radiowaves Transition of inner electrons Transition of outer most valence electrons Molecular vibration Molecular rotation Nuclear spin Ultra Violet Spectroscopy Atomic Absorption Spectroscopy Infra Red Spectroscopy Nuclear Magnetic Resonance Spectroscopy

Electromagnetic Radiation and Spectroscopy Electromagnetic Radiation Interact with Matter (Atoms, Molecules) = Spectroscopy Electromagnetic Radiation Radiowaves Infrared UV or visible Nuclear spin Molecular vibration Transition of outer valence electron Nuclear Magnetic Resonance Spectroscopy Infrared Spectroscopy UV Spectroscopy Atomic A Spectroscopy Organic structure determination MRI and body scanning Organic structure determination Functional gp determination Measure bond strength Measure degree unsaturation in fat Measure level of alcohol in breath Quantification of metal ions Detection of metal in various samples

Nuclear Magnetic Resonance Spectroscopy (NMR) Involve nucleus (proton + neutron) NOT electron Proton + neutrons = Nucleons Nucleons like electrons have spin and magnetic moment (acts like tiny magnet) Nuclei with even number of nucleon (12C and 16O) Even number of proton and neutron – NO net spin Nucleon spin cancel out each other –Nucleus have NO overall magnetic moment – NOT absorb radiowave Nuclei with odd number of nucleon (1H, 13C, 19F, 31P) Nucleon have net spin – Nucleus have NET magnetic moment – Absorb radiowave Nuclei with net spin – magnetic moment will interact with radiowaves Nuclei have a “spin” associated with them (i.e., they act as if they were spinning about an axis) due to the spin associated with their protons and neutrons. Nuclei are positively charged, their spin induces a magnetic field NMR spectroscopy does not work for nuclei with even number of protons and neutrons - nuclei have no net spin. Spin cancel each other Net spin

NMR spectrum CH3CH2Br Chemical Shift Nuclear Magnetic Resonance Spectroscopy (NMR) Main features of HNMR Spectra 1. Number of diff absorption peaks – Number of diff proton/chemical environment 2. Area under peaks - Number of hydrogen in a particular proton/chemical environment (Integration trace) - Ratio of number of hydrogen in each environment 3. Chemical shift - Chemical environment where proton is in - Spinning electrons create own magnetic field, creating a shielding effect - Proton which are shielded appear upfield. (Lower frequency for resonance to occur) - Proton which are deshielded appear downfield. (Higher frequency for resonance to occur) - Measured in ppm (δ) 4. Splitting pattern - Due to spin-spin coupling - Number of peak split is equal to number of hydrogen on neighbouring carbon +1 (n+1) peak Click here khan NMR videos. NMR spectrum CH3CH2Br Chemical Shift Number of peaks Splitting pattern Area under peaks Chemical shift

Nuclear Magnetic Resonance Spectroscopy (NMR) Absence of External magnetic Field (EMF) TWO nuclear spin have same energy level. Presence of External Magnetic Field (EMF) TWO nuclear spin split to TWO diff energy level Presence of External Magnetic Field (EMF) External MF applied to atomic nuclei, MF of nuclei align themselves either with or against MF Nuclei have a slight preference for parallel alignment with the applied field as it has a slightly lower energy Nuclei can absorb energy to move/flip to higher energy level by absorbing energy in radio freq region High spin nuclei align against magnetic field Presence of EMF Two spins in diff energy level Lower spin nuclei absorb radio freq equivalent to ∆E Move to higher energy level ∆E Absence of EMF Two spins in same energy level Lower spin nuclei align with magnetic field

Chemical Shift (Shielding Effect) Without SHIELDING EFFECT Proton in nucleus – have spin – generate its magnetic field (MF) Electron around nucleus – have spin- also generate its MF Proton shielded by MF produced by electron - appear UPFIELD Proton deshielded by electron withdrawing gp - appear DOWNFIELD Downfield Upfield Presence of EMF Two spins in diff energy level Lower spin nuclei absorb radio freq equivalent to ∆E Move to higher energy level S H ׀ H – C – H Without SHIELDING EFFECT Energy of ∆E absorb by H to move to higher energy level ∆E MF H nucleus proton N Absence of EMF Two spins in same energy level Presence of EMF Two spins at diff energy level S SHIELDING EFFECT Electron around H produce MF and shield the H H in CH3 will experience less EMF (SHIELDED) Absorb at lower radiofreq to move to higher level ∆E absorb by H to move to higher energy level is less Appear upfield. H ׀ H – C MF ∆E is smaller - H proton H nucleus shield by electron MF N Absence of EMF Two spins in same energy level Presence of EMF Two spins at diff energy level

Chemical Shift (Deshielding Effect) Without SHIELDING EFFECT Proton in nucleus – have spin – generate its magnetic field (MF) Electron around nucleus – have spin- also generate its MF Proton shielded by MF produced by electron - appear UPFIELD Proton deshielded by electron withdrawing gp - appear DOWNFIELD Downfield Upfield Presence of EMF Two spins in diff energy level Lower spin nuclei absorb radio freq equivalent to ∆E Move to higher energy level S H ׀ H – C – H ∆E Without SHIELDING EFFECT Energy of ∆E absorb by H to move to higher energy level MF proton H nucleus N Absence of EMF Two spins in same energy level Presence of EMF Two spins at diff energy level S DESHIELDING EFFECT Electron withdrawn by C=O gp Carbonyl gp has electron withdrawing effect Less electron around H in CH3 H in CH3 deshielded, experience greater EMF ∆E absorb by H, to move to high energy is higher Absorb at higher radiofreq, to move to high level Appear downfield MF ∆E is higher H nucleus deshield by elec MF proton N Absence of EMF Two spins in same energy level Presence of EMF Two spins at diff energy level

Chemical Shift (Shielding and Deshielding Effect) Absence of EMF Two spins in same energy level Presence of EMF Two spins at diff energy level Deshielding Effect S DESHIELDING EFFECT Electron withdrawn by C=O gp Carbonyl gp has electron withdrawing effect Less electron around H in CH3 H in CH3 deshielded, experience greater EMF ∆E absorb by H, to move to high energy is higher Absorb at higher radiofreq, to move to high level Appear downfield MF ∆E is higher H nucleus deshield by elec MF proton N No shielding S H ׀ H – C – H MF Without any SHIELDING EFFECT Energy of ∆E absorb by H to move to higher energy level ∆E H nucleus proton N Shielding Effect S SHIELDING EFFECT Electron around H produce MF and shield H H in CH3 experience less EMF (SHIELDED) ∆E absorb by H to move to higher energy level is less Appear upfield. MF ∆E is smaller proton N H nucleus shield by elec MF

Downfield Upfield Chemical Shift (Shielding and Deshielding Effect) Shielding/Deshielding: Electron circulate nucleus, create MF opposing external MF. Each nucleus experience a slightly diff magnetic field (Sum external field and field from electron cloud). Energy a nucleus achieve resonance depend on its surrounding. Freq absorption depend on electron density around nucleus Chemical shift of various electron withdrawing gp Downfield Upfield 12 9.7 - Electron withdrawn from CH3 by C=O Deshield H in CH3 Absorb at slightly higher freq Upfield ≈ 2.1 Electron withdrawn from CH2 by COO Stronger electron withdrawing effect Higher ↑ Deshielding effect on H in CH2 Absorb at Higher ↑ freq Slightly Downfield ≈ 4.1 Electron withdrawn from H by CHO Very strong electron withdrawing effect Higher ↑ Deshielding effect on H in CHO Absorb at Very High ↑ freq Very Very Downfield ≈ 9.7 Electron withdrawn by benzene Stronger electron withdrawing effect Higher ↑ deshielding effect on H Absorb at Very high ↑ freq Very Downfield ≈ 7.3 - 8 Electron withdrawn by COOH Very strong electron withdrawing effect Highest deshielding effect on H Absorb at Very High ↑ freq Very Very Very Downfield ≈ 12

HO-CH2-CH3 1 2 3 12 TMS Nuclear Magnetic Resonance Spectroscopy (HNMR) OH chemical shift ≈ 4.8 integration = 1 H No split (Singlet) CH2 chemical shift ≈ 3.8 integration = 2 H split into 4 CH3 chemical shift ≈ 1 integration = 3 H split into 3 1 2 3 12 Downfield Upfield 3 diff proton environment Ratio of 3:2:1 TMS CH3 ׀ H3C – Si – CH3 Tetramethyl Silane (TMS) as STD Strong peak upfield (shielded) Silicon has lower EN value < carbon Electron shift to carbon H in CH3 more shielded Experience lower EMF, absorb ↓ freq UPFIELD ≈ 0 Advantages using TMS Volatile and can be removed from sample All 12 hydrogen in same proton environment Single strong peak, upfield, wont interfere with other peak All chemical shift, in ppm (δ) are relative to this STD, ( zero) Click here Spectra database (Ohio State) Click here Spectra database (NIST) Click here for more complicated proton chemical shift

O HO-C-CH2-CH3 12 O ‖ CH3-C-O-CH2-CH3 B A C 2 3 3 ‖ A B C 1 2 3 1H NMR Spectrum O ‖ CH3-C-O-CH2-CH3 B A C 2 3 3 3 diff proton environment, Ratio H - 3:2:3 Peak A – split to 3 (2H on neighbour C) Peak B - No split Peak C – split to 4 (3H on neighbour C) O ‖ HO-C-CH2-CH3 A B C 1 2 3 12 3 diff proton environment, Ratio H - 3:2:1 Peak A – split to 3 (2H on neighbour C) Peak B – split to 4 (3H on neighbour C) Peak C – No split

HO-CH2-CH3 A B C 1 2 3 O ‖ CH3-C-CH2-CH2-CH3 A C D B 2 3 2 3 1H NMR Spectrum HO-CH2-CH3 A B C 1 2 3 3 diff proton environment, Ratio H - 3:2:1 Peak A – split to 3 (2H on neighbour C) Peak B – split to 4 (3H on neighbour C) Peak C – No split O ‖ CH3-C-CH2-CH2-CH3 A C D B 2 3 2 3 4 diff proton environment, Ratio H - 3:2:2:3 Peak A – split to 3 (2H on neighbour C) Peak B – split to 6 (5H on neighbour C) Peak C – No split Peak D – split to 3 (2H on neighbour C)

2 diff proton environment, Ratio H - 3:1 1H NMR Spectrum O ‖ CH3-C-O-CH2-CH2-CH3 A D C B 2 3 2 3 4 diff proton environment, Ratio H – 3:2:2:3 Peak A – split to 3 (2H on neighbour C) Peak B – split to 6 (5H on neighbour C) Peak C – No split Peak D – split to 3 (2H on neighbour C) O ‖ H-C-CH3 A B 3 1 9.8 2 diff proton environment, Ratio H - 3:1 Peak A – split to 2 (1H on neighbour C) Peak B – split to 4 (3H on neighbour C)

Molecule with plane of symmetry Molecule with plane of symmetry 1H NMR Spectrum CH3 ׀ H-C-OH Molecule with plane of symmetry C A B 1 1 6 3 diff proton environment, Ratio H - 6:1:1 Peak A – split to 2 (1H on neighbour C) Peak B – No split Peak C – split to 7 (6H on neighbour C) O CH3 ‖ ׀ CH3-C-O-C-H ׀ CH3 Molecule with plane of symmetry A B C 3 6 1 3 diff proton environment, Ratio H - 6:3:1 Peak A – split to 2 (1H on neighbour C) Peak B – No split Peak C – split to 7 (6H on neighbour C)

Molecule with plane of symmetry Molecule with plane of symmetry 1H NMR Spectrum O ‖ CH3-CH2-C-CH2-CH3 Molecule with plane of symmetry A B 4 6 2 diff proton environment, Ratio H – 6:4 Peak A – split to 3 (2H on neighbour C) Peak B – split to 4 (3H on neighbour C) O CH3 ‖ ׀ H-C-C-CH3 ׀ CH3 Molecule with plane of symmetry A B 1 9 2 diff proton environment, Ratio H – 9:1 Peak A – No split Peak B – No split

Molecule with plane of symmetry Molecule with plane of symmetry 1H NMR Spectrum CH3 ׀ HO-CH2-CH Molecule with plane of symmetry A B D C 2 1 1 6 4 diff proton environment, Ratio H- 6:1:1:2 Peak A – split to 2 (1H on neighbour C) Peak B – split to 7 (6H on neighbour C) Peak C – No split Peak D – split to 2 (1H on neighbour C) Molecule with plane of symmetry CH3-CH-CH3 ׀ CI A B 1 6 2 diff proton environment, Ratio H – 6:1 Peak A – split to 2 (1H on neighbour C) Peak B – split to 7 (6H on neighbour C)

Acknowledgements Thanks to source of pictures and video used in this presentation Thanks to Creative Commons for excellent contribution on licenses http://creativecommons.org/licenses/ Prepared by Lawrence Kok Check out more video tutorials from my site and hope you enjoy this tutorial http://lawrencekok.blogspot.com