Chapter 13: NMR 1 Dr. Sivappa Rasapalli Chemistry and Biochemistry University of Massachusetts...

Chapter 13: NMR 1 Dr. Sivappa Rasapalli Chemistry and Biochemistry University of Massachusetts Dartmouth CARBON-13 ( 13 C) NMR SPECTROSCOPY

both give us information about the number of chemically nonequivalent nuclei (nonequivalent hydrogens or nonequivalent carbons) both give us information about the environment of the nuclei (hybridization state, attached atoms, etc.) Carbon-13 NMR Spectroscopy H 1 and C 13 NMR Spectroscopy

SPIN PROPERTIES OF ATOMIC NUCLEI What is spin? The Simple explanation Spin is a fundamental property of nature like electrical charge or mass. Spin is a measure of angular momentum (rotation about an axis) hence the term Spin comes in multiples of 1/2 (0, 1/2, 1, 3/2, 2, 5/2) and can be + or -. Protons, electrons, and neutrons possess spin. Individual unpaired electrons, protons, and neutrons each possesses a spin of 1/2 Atomic nuclei composed of neutrons and protons may also possess spin. The spin of an atomic nucleus is determined by the number of protons and neutrons in the nucleus. Atoms with an odd number of protons will have spin Atoms with an odd number of neutrons will have spin Atoms with an odd number of both protons and neutrons will have spin Atoms with an even number of both protons and neutrons will not have spin The value of nuclear spin is represented by the symbol I, the nuclear spin quantum number. (I = 0, 1/2, 1, 3/2, 2, 5/2.) A nucleus with spin of I can exist in (2I+1) spin states. The shell model for the nucleus tells us that nucleons (protons and neutrons), just like electrons, fill orbitals. When the number of protons or neutrons equals 2, 8, 20, 28, 50, 82, and 126, orbitals are filled. Because nucleons have spin, just like electrons do, their spin can pair up when the orbitals are being filled and cancel out. Odd numbers mean unfilled orbitals, that do not cancel out.

A moving perpendicular external magnetic field will induce an electric current in a closed loop An electric current in a closed loop will create a perpendicular magnetic field A Basic Concept in Electromagnetic Theory- Direct Application to NMR

Carbon-13 NMR Spectroscopy 5 Wheres Waldo?

13C NMR Spectroscopy 6 E= B o h 2 B o = external magnetic field strength = magnetogyric ratio 1 H= 26,752 13 C= 6.7 One carbon in 3 molecules of squalene is 13 C

Nucleus (10 6 rad/Tesla sec) Field strength B 0 (Tesla) Frequency (MHz) 1H1H267.531.0042.6 4.70200. 7.05300. 2H2H41.11.006.5 13 C67.281.0010.7 4.7050.0 7.0575.0 19 F251.71.0040.0 13C Transition Energy

BoBo = h / 4 In the absence of external field, each nuclei is energetically degenerate Add a strong external field (B o ) and the nuclear magnetic moment: aligns with (low energy) against (high-energy) Magnetic alignment RANDOM ORIENTATION

Nuclear Spin (cont.) to convert lower energy spin state into higher energy spin state, require external energy source.........irradiate with radiofrequency (rf) radiation! 1. At zero external magnetic field, spins are degenerate! 2. Apply an external magnetic field spins states will differ in energy depending upon relative orientation with respect to external field. -nuclei with I = will adopt two specific orientations with respect to an externally- applied magnetic field... external magnetic field BoBo -spin state +1/2 (lower energy) -spin state -1/2 (higher energy) radiofrequency energy source

Spins Orientation in a Magnetic Field (Energy Levels) to convert lower energy spin state into higher energy spin state, require external energy source.........irradiate with radiofrequency (rf) radiation! 1. At zero external magnetic field, spins are degenerate! 2. Apply an external magnetic field spins states will differ in energy depending upon relative orientation with respect to external field. -nuclei with I = will adopt two specific orientations with respect to an externally- applied magnetic field... radiofrequency required depends on E E depends on strength of B o h E apply magnetic field increasing B o -spin: +1/2 lower energy -spin: -1/2 higher energy no external magnetic field

Spins Orientation in a Magnetic Field (Energy Levels) radiofrequency required depends on E E depends on strength of B o h E apply magnetic field increasing H 0 -spin: +1/2 lower energy -spin: -1/2 higher energy no external magnetic field Difference in energy between the two states is given by: E = h B o / 2 E = h where: B o = external magnetic field h = Plancks constant; gyromagnetic ratio B o =0 B o >0

= Bo/2 The value, , is the magnetogyric ratio

Frequency of absorption: = B o / 2 Transition from the low energy to high energy spin state occurs through an absorption of a photon of radio-frequency (RF) energy RF Spins Orientation in a Magnetic Field (Energy Levels)

Nuclear Spin (cont.) Difference in energy between the two nuclear spin states: depends on strength of external magnetic field (MHz) For nucleus of H atom (proton), spin energy differences: 0 4.736.358.4611.75 -1/2 +1/2 100 200300360500 2.34 H 0 (Tesla) Thus, at H 0 = 4.7 T (Tesla), use rf radiation of 200 MHz for 1 H Nuclei ENERGY OF A PHOTON E = h SPIN STATE ENRGY DIFFERENCE E = hB 0 /2 WHEN E = E, SPIN FLIP OCCURS h hB 0 /2 THE NECESSARY FREQUENCY IS: B 0 /2

Nuclear Spin (cont.) Difference in energy between the two nuclear spin states: depends on strength of external magnetic field (MHz) For nucleus of H atom (proton), spin energy differences: 0 4.736.358.4611.75 -1/2 +1/2 25 507590125 2.34 H 0 (Tesla) Thus, at B 0 = 4.7 T (Tesla), use rf radiation of 50 MHz etc for 13 C nuclei. ENERGY OF A PHOTON E = h SPIN STATE ENRGY DIFFERENCE E = hB 0 /2 WHEN E = E, SPIN FLIP OCCURS h hB 0 /2 THE NECESSARY FREQUENCY IS: B 0 /2

Fourier Transform NMR 13 C-spectra of CH 3 CH 2 CH 2 CH 2 CH 2 OH average of 200 scans after one scan

Features of 13 C NMR Spectra Each unique C in a structure gives a single peak in the spectrum; there is rarely any overlap. The C NMR spectrum spans over 200 ppm; chemical shifts only 0.001 ppm apart can be distinguished; this allows for over 2x10 5 possible chemical shifts for carbon. The intensity (size) of each peak is NOT directly related to the number of that type of carbon. Other factors contribute to the size of a peak: Peaks from carbon atoms that have attached hydrogen atoms are bigger than those that dont have hydrogens attached. Carbon chemical shifts are usually reported as downfield from the carbon signal of tetramethylsilane (TMS).

H 1 and C 13 NMR Spectroscopy Number of peaks Chemical shifts Integration Spin-Spin Splitting Number of peaks Chemical shifts Integration Spin-Spin Splitting

13 C NMR 19

13 C NMR 20

Predicting 13 C Spectra

Predicting 13 C NMR

Each unique carbon in a molecule gives rise to a 13 C NMR signal. Therefore, if there are fewer signals in the spectrum than carbon atoms in the compound, the molecule must possess symmetry. Examples: Symmetry in C-13 NMR

How many signals would you expect?

* * * * * Enantiotopic vs Diastereotopic carbonss

Determine the number of signals in the proton-decoupled C-13 NMR spectrum of each of the following compounds: Predicting 13 C NMR

1H and 13C NMR compared: 13 C signals are spread over a much wider range than 1 H signals making it easier to identify and count individual nuclei The following slides show the 1 H NMR and the 13 C spectrum of 1-chloropentane. It is much easier to identify the compound as 1-chloropentane by its 13 C spectrum than by its 1 H spectrum.

01.02.03.04.05.06.07.08.09.010.0 Chemical shift ( , ppm) ClCH 2 Proton Spectrum CH3CH3CH3CH3 ClCH 2 CH 2 CH 2 CH 2 CH 3 1H1H1H1H

Chemical shift ( , ppm) Carbon Spectrum ClCH 2 CH 2 CH 2 CH 2 CH 3 020406080100120140160180200 13 C CDCl 3 a separate, distinct peak appears for each of the 5 carbons

30 200.3137.1 132.8 128.5 128.0 17.8 40.5 13.9 TMS CDCl 3 137.1 132.8 128.5 128.0 Intensity of 13 C NMR signals

13C-NMR: Integration 1 H-NMR: Integration reveals relative number of hydrogens per signal 13 C-NMR: Integration reveals relative number of carbons per signal Rarely useful due to slow relaxation time for 13 C time for nucleus to relax from excited spin state to ground state

13 C Chemical shifts are most affected by: electronegativity of groups attached to carbon hybridization state of carbon

13 C NMR Chemical Shifts Several functionalities appear directly on 13 C NMR which are not visible in 1 H NMR: - Quaternary carbons - ipso carbons - Carbonyl carbons downfield (ppm) upfield deshielded shielded higher E lower E 220200180160140120100806040200.0 carbonyl carbons aromatic carbons alkene carbons alkyne carbons sp 3- EWG sp 3 carbon

13 C NMR

Chemical Shift - 13 C-NMR Trends RCH 3 < R 2 CH 2 < R 3 CH Electronegative atoms cause downfield shift Pi bonds cause downfield shift C=O 160-210 ppm

36 Chemical Shift Range of 13 C Note the carbonyl range Chemical Shift - 13 C-NMR

Carbonyl Carbon Chemical Shifts 220210200190180170160150140130120110 ketones conj. Ketones aldehydes carboxylic acids anhydridesnitriles esters acid chlorides amides

Alkane: 2-methylpentane

Alcohol: 2-hexanol

Alkyl Halide: 3-bromopentane

Alkene: 1-hexene

Aromatic Ring: eugenol

Carboxylic Acid: pentanoic acid

Ester: ethyl valerate

Amide: pentanamide

Ketone: 3-methyl-2-pentanone

Aldehyde: 2-methylpentanal

ppm Carbon # 139.07 1 131.62 2 124.07 3 123.71 4 59.16 5 39.64 6 26.51 7 25.66 8 17.66 9 16.24 10 8 9 Carbon-13 NMR Spectrum of Geraniol

Spin-Spin Coupling in 13 C NMR Homonuclear coupling of 13 C- 13 C is possible in theory. However, due to the low natural abundance of 13 C, it is rare to find two 13 Cs in the same molecule, let alone adjacent to one another. No need to consider 13 C- 13 C coupling except for enrichment studies! Heteronuclear coupling between 13 C and the 1 H atoms attached to them is observed ( 1 H abundance ~99%). Because the 1 H atoms are directly attached, the coupling constants ( 1 J)are large, typically 100-250 Hz. When such spectra are observed, they are referred to as proton coupled spectra (or non-decoupled spectra).

Carbon-13 NMR Spectroscopy 50 Wheres Waldo?

Spin-Spin Coupling in 13 C NMR Homonuclear coupling of 13 C- 13 C is possible in theory. However, due to the low natural abundance of 13 C, it is rare to find two 13 Cs in the same molecule, let alone adjacent to one another. No need to consider 13 C- 13 C coupling except for enrichment studies! Heteronuclear coupling between 13 C and the 1 H atoms attached to them is observed ( 1 H abundance ~99%). Because the 1 H atoms are directly attached, the coupling constants ( 1 J)are large, typically 100-250 Hz. When such spectra are observed, they are referred to as proton coupled spectra (or non-decoupled spectra).

1 H NMR (Proton with Carbon-13 coupling)

13 C NMR Spectrum Carbon 13 and Proton-Coupled Proton-Coupled

1 H 13 C Splitting The splitting follows the simple N+1 rule: The multiplet analysis gives useful information, but there are two major limitations: 1) If the 13 C signal is weak (common) the outer peaks of the multiplet may be lost in the noise of the spectrum. 2) Due to the large J-constants, the multiplets quickly begin to overlap and become congested. quaternary singlet methine doublet methylene triplet quaternary quartet

Effect of Coupling Coupling can cause 13 C NMR spectra to become very complicated (convoluted) quite easily. 1 H Coupled Three equal intensity lines at 77 ppm CDCl 3 solvent 13 C- 2 D coupling

1 H Decoupling To simplify the 13 C spectrum, and to increase the intensity of the observed signals, a decoupler is used to remove the spin effects of the 1 H nucleus. A second RF generator irradiates at the 1 H resonance frequency causing the saturation effectively averaging all their spin states to zero 1 H channel- 13 C channel 13 C pulse 13 C FID

Proton-decoupled mode, a sample is irradiated with two different radiofrequencies. One to excite all 13 C nuclei, a second to cause all protons in the molecule to undergo rapid transitions between their nuclear spin states. On the time scale of a 13 C-NMR spectrum, each proton is in an average or effectively constant nuclear spin state, with the result that 1 H- 13 C spin-spin interactions are not observed and they are decoupled. Decoupling

13 C Proton Decoupled Spectrum 13 C{ 1 H}

13C Off-resonance & Broadband decoupled spectra Broadband Off-resonance

Effect of Decoupling 1 H Coupled 1 H Decoupled

H1 Decoupling Techniques J values for C-H are typically 110-300 Hz (C-C-H and C-C-C-H are 0-60Hz). Thus a CH3 group would appear as a quartet, CH2-triplet CH-doublet etc. The H1 nuclei are irradiated with a broadband Rf to remove coupling to Carbon.

CH 3 OH Attached Protons Affect T1 and Signal Intensity 7 carbons give 7 signals, but intensities are not equal Chemical shift ( , ppm) 020406080100120140160180200

Example: Ethanol 1 H- 13 C spin-spin coupling: spin-spin coupling tells how many protons are attached to the 13 C nuclei. (i.e., primary, secondary tertiary, or quaternary carbon) 13 C spectra are usually collected with the 1 H- 13 C coupling turned off (broad band decoupled). In this mode all 13 C resonances appear as singlets.

Other isomers of C 5 H 12 pentane CH 3 CH 2 CH 2 CH 2 CH 3 3 peaks 2,3-dimethylpropane(CH 3 ) 4 C2 peaks There are four chemically different carbon atoms in the molecule so there are four peaks in the C-13 nmr spectrum. NO SPLITTING WITH C-13 ONLY ONE PEAK FOR EACH CARBON NO SPLITTING WITH C-13 ONLY ONE PEAK FOR EACH CARBON chemically equivalent carbon atoms H C C C C H H H CH3CH3 H H H H H 2-methylbutane (CH 3 ) 2 CHCH 2 CH 3

Example: cyclohexane

Example: cyclohexene

Example: 1,3-cyclohexadiene

Example: 1,4-cyclohexadiene

Example: m-nitrotoluene 1 2 3 4 6 7 5 3 1 2 6 5 7 4

C 6 H 12 O 2

Isomers of C 3 H 7 Br 3 peaks 2 peaks all three carbons are different the two outer carbons are similar H C C C Br H H H H H H HCCCHHCCCH H H Br H H H

Can we tell the isomers apart?

Edited C 13 NMR Edited C 13 NMRs APT= Attached Proton Test APT DEPT= Distortionless Enhancement by Polarization Transfer

Multiplicity detection DEPT : CH, CH3 CH 2 APT : CH, CH3 C, CH 2 Normal C13

Attached Proton Test APT Acquiring a 13 C after 1/J seconds: methine and methyl Cs produce negative peaks (odd number of attached Hs) methylene and quaternary Cs produce positive peaks (even number of attached Hs) Remember 1 J CH is essentially the same for all tetrahedral carbons Thus acquiring the C signal after a pre-determined time can give positive peaks, negative peaks, or even no peaks at all, depending on how many Hs are attached. APT has been superseded by DEPT Distortionless Enhancement by Polarization Transfer Complex pulse sequence allowing selective reception of signals from different C types: -C, -CH, -CH 2, -CH 3 DEPT spectra (Distortionless Enhancement by Polarization Transfer) a modern 13 C NMR spectra that allows you to determine the number of attached hydrogens.

Measuring a 13C NMR spectrum involves In DEPT, a second transmitter irradiates 1 H during the sequence, which affects the appearance of the 13 C spectrum. some 13 C signals stay the same some 13 C signals disappear some 13 C signals are inverted

13 C NMR - DEPT Distortionless enhancement by polarization transfer (DEPT) spectra permit identification of CH 3, CH 2, and CH carbon atoms. DEPT 45 shows 1 o, 2 o,and 3 o carbons. DEPT 90 shows only 3 o carbons. DEPT 135 shows 1 o and 3 o carbons as positive peaks and 2 o carbons as negative peaks.

Using DEPT to Count Hydrogens Attached to 13C

Chemical shift ( , ppm) 020406080100120140160180200 Proton Decoupled SpectrumOC C CH CHCH CH 2 CH 3 CCH 2 CH 2 CH 2 CH 3 O

Chemical shift ( , ppm) 020406080100120140160180200 DEPT 135 SpectrumCH CHCH CH 2 CH 3 CCH 2 CH 2 CH 2 CH 3 O CH and CH 3 unaffected C and C=O nulled CH 2 inverted

94 CH 3 CH 2 Broad-band decoupled DEPT 6 5 24 1 7 8 3 CH CH 2 s give negative resonances CHs and CH 3 s give positive resonances Quaternary carbon (no attached Hs) are not observed DEPT 135 Spectrum

DEPT DEPT Distortionless Enhancement by Polarization Transfer Allows us to observe the number of hydrogens attached to a particular carbon. DEPT Pulse Sequence methylmethylenemethinequaternary DEPT-45Positive peak Not observed DEPT-90No obs. peak Positive peakNot observed DEPT-135Positive peakNegative Peak Positive PeakNot observed

13C Citronellol

DEPT-135 CH 3 positive CH 2 negative CH positive C not observed DEPT-135 Ipsenol

DEPT - Ipsenol CH 3 (+), CH 2 (-), CH (+), C (none) DEPT-135 DEPT-90 CH 3, CH 2 (none), CH (+), C (none) 7 42 9,1 35108 6

DEPT Spectra of Codeine

DEPT Spectra of 1-phenyl-1-butanone DEPT 135 DEPT 90 DEPT 45 CH 3, CH 2, CH (+), C (none) CH 3, CH 2 (none), CH (+), C (none) CH 3 (+), CH 2 (-), CH (+), C (none)

DEPT Spectra of CH 3, CH 2 (none), CH (+), C (none) CH 3 (+), CH 2 (-), CH (+), C (none)

Summary of Edited 13 C NMR

Summary Number of signals indicates the number of types of carbon in the sample. (Is symmetry present?) Chemical shifts show what types of carbons are in the sample. Quaternary/ipso carbons will be smaller than carbons with protons attached. DEPT differentiates between primary, secondary, and tertiary carbons.

How many peaks would you expect there to be in the carbon-13 spectrum of butaneCH 3 CH 2 CH 2 CH 3 2-methylpropaneCH 3 CH(CH 3 )CH 3 butanalCH 3 CH 2 CH 2 CHO butanoneCH 3 COCH 2 CH 3 pentan-2-oneCH 3 COCH 2 CH 2 CH 3 pentan-3-oneCH 3 CH 2 COCH 2 CH 3 cyclohexaneC 6 H 12

How many peaks would you expect there to be in the carbon-13 spectrum of butaneCH 3 CH 2 CH 2 CH 3 2 2-methylpropaneCH 3 CH(CH 3 )CH 3 2 butanalCH 3 CH 2 CH 2 CHO4 butanoneCH 3 COCH 2 CH 3 4 pentan-2-oneCH 3 COCH 2 CH 2 CH 3 5 pentan-3-oneCH 3 CH 2 COCH 2 CH 3 3 cyclohexaneC 6 H 12 1 19

Identify the isomers of C 4 H 8 O

A butanal B butanone C 2-methylpropanal

The 13 C nucleus is present in only 1.08% natural abundance. Therefore, acquisition of a spectrum usually takes much longer than in 1 H NMR. The magnetogyric ratio of the 13 C nucleus is about 1/4 that of the 1 H nucleus. Therefore, the resonance frequency in 13 C NMR is much lower than in 1 H NMR. (75 MHz for 13 C as opposed to 300 MHz for 1 H in a 7.04 Tesla field). At these lower frequencies, the excess population of nuclei in the lower spin state is reduced, which, in turn, reduces the sensitivity of NMR detection. Unlike 1 H NMR, the area of a peak is not proportional to the number of carbons giving rise to the signal. Therefore, integrations are usually not done. Each unique carbon in a molecule gives rise to a 13 C NMR signal. Therefore, if there are fewer signals in the spectrum than carbon atoms in the compound, the molecule must possess symmetry. When running a spectrum, the protons are usually decoupled from their respective carbons to give a singlet for each carbon atom. This is called a proton-decoupled spectrum. C 13 NMR-Important points

2D NMR: COSY AND HETCOR

2D NMR Terminology 1D NMR = 1 frequency axis 2D NMR = 2 frequency axes COSY = Correlated Spectroscopy 1 H- 1 H COSY provides connectivity information by allowing one to identify spin-coupled protons. x,y-coordinates of cross peaks are spin-coupled protons

1H-1H COSY CH 3 CCH 2 CH 2 CH 2 CH 3 O 1H1H 1H1H

HETCOR 1 H and 13 C spectra plotted separately on two frequency axes Coordinates of cross peak connect signal of carbon to protons that are bonded to it.

1H-13C HETCOR CH 3 CCH 2 CH 2 CH 2 CH 3 O 13 C 1H1H

Solving Combined Spectra Problems: 116 Mass Spectra: Molecular Formula Nitrogen Rule # of nitrogen atoms in the molecule M+1 peak # of carbons Degrees of Unsaturation: # of rings and/or -bonds Infrared Spectra: Functional Groups C=OO-H C=CN-H C CCO-OH C N 1 H NMR: Chemical Shift ( ) chemical environment of the H's Integration # of H's giving rise to the resonance Spin-Spin Coupling (multiplicity) # of non-equivalent H's on the adjacent carbons (vicinal coupling). 13 C NMR: # of resonances symmetry of carbon framework Type of Carbonyl Each piece of evidence gives a fragment (puzzle piece) of the structure. Piece the puzzle together to give a proposed structure. The proposed structure should be consistent with all the evidence.

119 13 C NMR: 212.1 35.5 7.9 C 5 H 10 O

120 = 7.4-7.1 (m, 5H) = 2.61 (sextet, J=7, 1H) = 2.61 (pentet, J=7, 2H) = 2.61 (d, 3H) = 2.61 (t, 3H) C 10 H 14 147.6 128.2 127.0 125.7 41.7 31.2 21.8 12.3

121 Infrared (IR): Characteristic OH stretching absorption at 3300 to 3600 cm 1 Sharp absorption near 3600 cm -1 except if H-bonded: then broad absorption 3300 to 3400 cm 1 range Strong CO stretching absorption near 1050 cm 1 O-H C-O cm -1 % T

122 = 1.5, q, 2H = 0.9, d, 3H = 3.65, t, 2H = 1.7, m, 1H = 2.25, br s, 1H CDCl 3 41.7 61.2 24.7 22.6 1 H NMR: protons attached to the carbon bearing the hydroxyl group are deshielded by the electron-withdrawing nature of the oxygen, 3.3 to 4.7 O-HC-O

123 Usually no spin-spin coupling between the OH proton and neighboring protons on carbon due to exchange reaction The chemical shift of the -OH proton occurs over a large range (2.0 - 5.5 ppm). It chemical shift is dependent upon the sample concentration and temperature. This proton is often observed as a broad singlet (br s). Exchangable protons are often not to be observed at all.

124 13 C NMR: The oxygen of an alcohol will deshield the carbon it is attached to. The chemical shift range is 50-80 ppm DMSO-d 6 (solvent) CH 3 CH 2 CH 2 CH 2 OH 62 35 19 14

125 13 C: 145.2 128.8 127.8 126.5 76.3 32.3 10.6 13 C: 138.6 129.4 128.4 126.3 68.8 45.8 22.7

Magnetic Resonance Imaging (MRI) 126 MRI uses the principles of nuclear magnetic resonance to image tissue MRI normally uses the magnetic resonance of protons on water and very sophisticated computer methods to obtain images. Other nuclei within the tissue can also be used ( 31 P) or a imaging (contrast) agent can be administered

127 Normal 25 years old Normal 86 years old Alzheimers Disease 78 years old fMRI: MRI images

Chapter 13: NMR 1 Dr. Sivappa Rasapalli Chemistry and Biochemistry University of Massachusetts...

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