C13 & 2D NMR

CONTENTS
• INTRODUCTION
• COMPARE .
13
𝐶 & .
1
𝐻 NMR
• WHY .
13 𝐶 NMR IS REQUIRED?
• .
13
𝐶 NMR SPIN-SPIN COUPLING
• SOLVENTS
• CHEMICAL SHIFT
• FACTORS AFFECTING CHEMICAL
SHIFT
• PROBLEMS
• PROBLEMS CAN BE OVERCOME
BY…
• ADVANTAGES OF .
13
𝐶 NMR OVER .
1
𝐻
NMR
• 2D NMR
• APPLICATIONS
• REFERENCE
27-12-2019V.K. VIKRAM VARMA 2

INTRODUCTION
•FIRST NMR OBSERVATION REGARDING .
13 𝐶 NUCLEI WERE
REPORTED IN 1957.
•GIVES INFORMATION ABOUT THE CARBON SKELETON
• .
13 𝐶 HAS ONLY ABOUT 1.1% NATURAL ABUNDANCE
• .
13 𝐶 NUCLEUS IS ABOUT 400 TIMES LESS SENSITIVE THAN
H NUCLEUS.

CONTD.
•CHEMICAL SHIFT RANGE: 0-220𝑝𝑝𝑚
.
𝟏𝟐
𝑪 .
𝟏𝟑
𝑪
Spin number zero Spin number 1
2
Even mass & charge Odd mass & charge
NMR inactive NMR active
e.g. .
12 𝐶, .
16 𝑂 e.g. .
1 𝐻, .
13 𝐶, .
19 𝐹

COMPARE .
1
𝐻& .
13
𝐶 NMR
•BOTH GIVE INFORMATION ABOUT THE NUMBER OF CHEMICALLY
NON EQUIVALENT NUCLEI & ABOUT THE ENVIRONMENT OF
NUCLEI.
.
1
𝐻 .
13
𝐶
ABUNDANCE 99% 1%
CHEMICAL SHIFT 0-15𝑝𝑝𝑚 0-220𝑝𝑝𝑚
REFERENCE TMS TMS
COUPLING YES NO

WHY .
13
𝐶 NMR IS REQUIRED?
•CARBON NMR CAN BE USED TO DETERMINE THE
NUMBER OF NON-EQUIVALENT CARBON ATOMS WHICH
MAY PRESENT IN COMPOUND.
• .
13
𝐶 SIGNALS SPREAD OVER A MUCH WIDER RANGE THAN
.
1 𝐻 SIGNALS MAKING IT EASIER TO IDENTIFY & COUNT
INDIVIDUAL NUCLEI.

.
13
𝐶 NMR SPIN-SPIN COUPLING
• .
1 𝐻: SPLITTING REVEALS NUMBER OF HYDROGEN NEIGHBOURS.
• .
13
𝐶: LIMITED TO NUCLEI SEPERATED BY JUST ONE SIGMA BOND;
NO 𝜋 BOND.
•HOMONUCLEAR SPIN-SPIN COUPLING BETWEEN .
13
𝐶- .
13
𝐶 ATOMS
NOT POSSIBLE DUE TO LOW ABUNDANCE.
•HETERONUCLEAR SPIN-SPIN COUPLING BETWEEN .
13 𝐶- .
12 𝐶 ATOMS
NOT POSSIBLE DUE TO SPIN QUANTUM NUMBER OF .
12 𝐶 IS ZERO.

SOLVENTS
•SHOULD NOT CONTAIN PROTON, INEXPENSIVE, LOW
BOILING POINT & NON POLAR IN NATURE.
• 𝐶𝐷𝐶𝑙3 GENERALLY USED
•IF SAMPLE IS SOLUBLE IN POLAR SOLVENT THEN
𝐷2 𝑂, 𝐶𝐶𝑙4, 𝐶𝐹3, 𝐶𝑂𝑂𝐻 ARE USED AS SOLVENT.

CHEMICA
L SHIFT

CONT
D.

ETHYL ETHANOTE
E
X
A
M
P
L
E
S

ACETOPHENONE
E
X
A
M
P
L
E
S

FACTORS AFFECTING CHEMICAL
SHIFT
•ELECTRONEGATIVITY
•HYBRIDISATION
•HYDROGEN BONDING
•ANISOTROPIC

PROBLEMS
•RECORRDING OF CARBON NMR (CMR) NUCLEI IS
DIFFICULT DUE TO FOLLOWING REASONS:
•NATURAL ABUNDANCE
•GYROSCOPIC RATIO
•COUPLING PHENOMENA

 GYROSCOPIC RATIO
•VERY LOW 1%
 NATURAL ABUNDANCE
• .
13 𝐶 NUCLEUS GYROMAGNETIC RATIO IS MUCH LESSER
THAN PROTON NUCLEUS

CONTD.
.
13 𝐶- 1.404 (100𝑀𝐻𝑧, 75𝑀𝐻𝑧, 125𝑀𝐻𝑧)
.
1 𝐻-5.585 (400𝑀𝐻𝑧, 300𝑀𝐻𝑧)
•CMR IS LESS SENSITIVE THAN PMR
•SENSITIVITY OF CMR CAN BE INCREASED BY FT
TECHNIQUE.
• .
13
𝐶 RESONANCE FREQUENCY IS ONLY 1
4 OF PMR AT A
GIVEN MAGNETIC FIELD.

 COUPLING PHENOMENA
• .
13 𝐶 & .
1 𝐻 HAVE SPIN QUANTUM NUMBER (I)=1
2 SO THAT COPLING BETWEEN THEM
MAY OCCUR.
• .
13 𝐶- .
13 𝐶 NOT POSSIBLE, HOWEVER .
13 𝐶- .
1 𝐻 IS POSSIBLE & CAN SEE IN CMR.
• AS A RESULT COUPLING MAKES .
13 𝐶 SPECTRUM EXTREMELY COMPLEX→
MULTIPLETS.
• .
13 𝐶- .
1 𝐻 CAN BE ELIMINATED BY ADOPTING
FT TECHNIQUE
PROTON DECOUPLING TECHNIQUE
NOE(NUCLEAR OVER HAUSER ENHANCEMENT)

PROBLEMS CAN BE OVERCOME
BY...
•FOURIER TRANSFORM TECHNIQUE (FT-NMR)
•DECOUPLING TECHNIQUE
a)BROADBAND DECOUPLING
b)OFF RESONANCE DECOUPLING
c)DEPT (DISORTIONLESS ENHANCEMENT BY POLARISATION OR
PULSE DECOUPLING)
•NUCLEAR OVER HAUSE PHENOMENA

 SIGNAL AVERGING & FT-
NMR
•LOW ABUNDANCE OF .
13 𝐶 IS OVERCOME BY THIS
•SIGNAL AVERGING(INCREASE INSTRUMENT SENSITIVITY)
RANDOM FREQUENCY ARE ADDED TOGETHER TO
CANCEL THE NOISE TO ZERO & NMR SIGNAL
ENHANCES.

CONTD.
•FT-NMR (INCREASE INSTRUMENT SPEED)
SAMPLE IRRADIATED WITH ENTIRE RANGE OF USEFUL
FREQUENCIES.
 .
13
𝐶 NUCLEI IN THE SAMPLE RESONANTE AT ONCE GIVING
COMPLEX, COMPOSITE SIGNAL THAT IS MATHEMATICALLY
MANIPULATED BY FT TO SEGGREGATE INDIVIDUAL SIGNALS &
CONVERT THEM TO FREEQUENCIES.
ADVANTAGES: MORE SENSITIVE & VERY FAST

CONTD.
E
X
A
M
P
L
E
S

 DECOUPLING TECHNIQUES
•BROADBAND DECOUPLING:
A SAMPLE IRRADIATED WITH 2 DIFFERENT FREQUENCIES
1 − 𝑡𝑜 𝑒𝑥𝑐𝑖𝑡𝑒 𝑎𝑙𝑙 𝐶 𝑛𝑢𝑐𝑙𝑒𝑖
2 − 𝑡𝑜 𝑐𝑎𝑢𝑠𝑒 𝑎𝑙𝑙 𝑝𝑟𝑜𝑡𝑜𝑛𝑠 𝑟𝑎𝑝𝑖𝑑 𝑡𝑟𝑎𝑛𝑠𝑖𝑡𝑖𝑜𝑛
RAPID TRANSITION
DECOUPLED ANY SPIN-SPIN INTERACTION BETWEEN .
13 𝐶 & .
1 𝐻
NUCLEI → DUE TO RAPID CHANGE ALL SPIN INTERACTIONS ARE
AVERAGE TO ZERO → APPEAR ONLY .
13 𝐶 SPECTRUM.
DISADVANTAGE: INFORMATION OF ATTACHED HYDROGEN IS
LOST

CONTD.
•IT AVOID SPIN-SPIN SPLITTING OF .
13 𝐶 LINES BY .
1 𝐻
NUCLEI.E
X
A
M
P
L
E
S

CONTD.
E
X
A
M
P
L
E
S

CONTD.
•OFF RESONANCE TECHNIQUE:
1000-2000𝐻𝑧 ABOVE THE SPECTRAL RANGE
COUPLNG BETWEEN EACH CARBON ATOM & EACH
HYDROGEN ATTACHED DIRECTLY TO IT, 𝑛 + 1 RULE.
IT SIMPLIFIES THE SPECTRUM BY ALLOWING SOME
OF THE SPLITTING INFORMATION TO BE RETAINED.

CONTD.
E
X
A
M
P
L
E
S

CONTD.
•PULSE/DEPT (DISTORTIONLESS ENHANCEMENT BY POLARISATION)
DECOUPLING:
3 STAGES:
ORIDINARY BROADBAND- LOCATE CHEMICAL SHIFTS OF ALL
CARBONS.
DEPT 90- ONLY SIGNALS DUE TO CH CARBON APPEARS
DEPT 135- 𝐶𝐻3 & CH RESONANCE APPEAR POSITIVE & 𝐶𝐻2
SIGNAL APPEAR NEGATIVE (BELOW BASELINE)
USED TO DETERMINE NUMBER OF HYDROGENS ATTACHED TO
EACH CARBON.

CONTD.
E
X
A
M
P
L
E
S

ADVANTAGES OF .
13
𝐶NMR OVER .
1
𝐻
NMR
• .
13 𝐶 GIVES INFORMATION ABOUT THE BACKBONE OF
MOLECULES.
• .
13
𝐶 IS EASIER TO ANALYSE THAN .
1
𝐻 SPECTRA
BECAUSE THE SIGNALS WILL NOT SPLIT.

2D NMR
•SET OF NMR METHODS WHICH GIVE DATA PLOTTED IN A SPACE
DEFINED BY 2 FREQUENCY AXES.
•TYPES OF 2D NMR:
COSY(CORRELATION SPECTROSCOPY)
EXSY(EXCHANGE SPECTROSCOPY)
NOESY (NUCLEAR OVER HAUSER EFFECT SPECTROSCOPY)
J-SPECTROSCOPY

CONTD.
•2D NMR PROVIDE MORE INFORMATION ABOUT A
MOLECULE THAN 1D-NMR SPECTRA.
•USEFUL IN DETERMINING THE STRUCTURE OF A
MOLECULE, PARTICULARLY FOR MOLECULES THAT
ARE TOO COMPLICATED TO WORK WITH USING 1D-
NMR.

2D CONCEPT
• IT CONSISTS OF A SEQUENCE OF RADIOFREQUENCY PULSES WITH DELAY
PERIODS IN BETWEEN THEM.
• 2D NMR HAVE 4 STAGES/ PHASES:
PREPERTION PERIOD- MAGNETIASTION COHERENCE IS CREATED
THROUGH A SET OF RF PULSES.
EVOULTION PERIOD- DETERMINED LENGTH OF TIME DURING WHICH NO
PULSES ARE DELIVERED & NUCLEAR SPINS ARE ALLOWED TO FREELY
ROTATE.
MIXING PERIOD- THE COHERENCE MANIPULATED BY ANOTHER SERIES OF
PULSES TO GIVE AN OBSERVABLE SIGNAL.

CONTD.
DETECTION PERIOD- FREE INDUCTION DECAY SIGNAL FROM THE SAMPLE
IS OBSERVED
• THE 2DS OF A 2D-NMR ARE 2 FREQUENCY AXES REPRESENTING A CHEMICAL
SHIFT
EACH OF THE 2 FREQUENCY IS ASSOCIATED WITH
THE EVOLUTION TIME
THE DETECTION TIME
THEY ARE EACH CONVERTED FROM A TIME SERIES TO FREQUENCY SERIES
USING A FOURIER TRANSFORM.

CONTD.
• IN TWO DIMENSIONAL EXPERIMENTS, BOTH THE X & THE Y AXES HAVE CHEMICAL
SHIFT SCALES & THE 2D SPECTRA ARE PLOTTED AS A GRID LIKE A MAP.
• INFORMATION IS OBTAINED FROM THE SPECTRA BY LOOKING AT THE PEAKS IN THE
GRID & MATCHING THEM TO THE X AND Y AXES.
• COSY - CORRELATION SPECTROSCOPY
• BOTH AXES CORRESPOND TO THE PROTON NMR SPECTRA.
• THE COSY SPECTRA INDICATES WHICH H ATOMS ARE COUPLING WITH EACH
OTHER.
• AN EXAMPLE OF A COSY IS PROVIDED BELOW.
• HETCOR - HETERONUCLEAR CORRELATION SPECTROSCOPY
• PROTON NMR SPECTRA ON ONE AXIS AND THE 13C NMR SPECTRA ON THE OTHER.
• THE HETCOR SPECTRA MATCHES THE H TO THE APPROPRIATE C.

COSY SPECTRA
• THE INFORMATION ON THE H THAT ARE COUPLING WITH EACH OTHER IS
OBTAINED BY LOOKING AT THE PEAKS INSIDE THE GRID. THESE PEAKS ARE
USUALLY SHOWN IN A CONTOUR TYPE FORMAT, LIKE HEIGHT INTERVALS ON A
MAP.
• IN ORDER TO SEE WHERE THIS INFORMATION COMES FROM, LET'S CONSIDER
AN EXAMPLE SHOWN BELOW, THE COSY OF ETHYL 2-BUTENOATE

CONTD.

CONTD.
• FIRST LOOK AT THE PEAK MARKED A IN THE TOP LEFT CORNER. THIS
PEAK INDICATES A COUPLING INTERACTION BETWEEN THE H AT 6.9 PPM
AND THE H AT 1.8 PPM. THIS CORRESPONDS TO THE COUPLING OF THE
CH3 GROUP & THE ADJACENT H ON THE ALKENE.
• SIMILARLY, THE PEAK MARKED B INDICATES A COUPLING INTERACTION
BETWEEN THE H AT 4.15 PPM & THE H AT 1.25 PPM. THIS CORRESPONDS
TO THE COUPLING OF THE CH2 & THE CH3 IN THE ETHYL GROUP.
• NOTICE THAT THERE ARE A SECOND SET OF EQUIVALENT PEAKS, ALSO
MARKED A & BON THE OTHER SIDE OF THE DIAGONAL.

HECTOR SPECTRA
•THE INFORMATION ON HOW THE H ARE C ARE MATCHED
IS OBTAINED BY LOOKING AT THE PEAKS INSIDE THE
GRID. AGAIN, THESE PEAKS ARE USUALLY SHOWN IN A
CONTOUR TYPE FORMAT, LIKE HEIGHT INTERVALS ON A
MAP.
•IN ORDER TO SEE WHERE THIS INFORMATION COMES
FROM, LET'S CONSIDER AN EXAMPLE SHOWN BELOW, THE
HETCOR OF ETHYL 2-BUTENOATE.

CONTD.

CONTD.
• FIRST LOOK AT THE PEAK MARKED A NEAR THE MIDDLE OF THE GRID. THIS
PEAK INDICATES THAT THE H AT 4.1 PPM IS ATTACHED TO THE C AT 60
PPM. THIS CORRESPONDS TO THE -OCH2- GROUP.
• SIMILARLY, THE PEAK MARKED B TOWARDS THE TOP RIGHT IN THE GRID
INDICATES THAT THE H AT 1.85 PPM IS ATTACHED TO THE C AT17 PPM. SINCE
THE H IS A SINGLET, WE KNOW THAT THIS CORRESPONDS TO THE CH3- GROUP
ATTACHED TO THE CARBONYL IN THE ACID PART OF THE ESTER AND NOT THE
CH3- GROUP ATTACHED TO THE -CH2- IN THE ALCOHOL PART OF THE ESTER.
• NOTICE THAT THE CARBONYL GROUP FROM THE ESTER HAS NO "MATCH"
SINCE IT HAS NO H ATTACHED IN THIS EXAMPLE.

NUCLEAR OVER HAUSER
ENHANCEMENT
•NEARBY ATOMS UNDERGO CROSS RELATION.
•NOE CROSS RELAXATION BETWEEN NUCLEAR SPINS
DURING MIXING PERIOD IS USED TO ESTABLISH THE
CORRELATIONS.
•SPECTRUM OBTAINED IS SIMILAR TO COSY WITH
DIAGONAL PEAKS & CROSS PEAKS.

CONTD.
•ALSO CONTAIN EXTRA AXIAL PEAKS WHICH DON’T PROVIDE EXTRA
INFORMATION & CAN BE ELIMINATED THEM BY REVERSING THE
PHASE OF THE FIRST PULSE.
•USED IN STUDY OF LARGE BIOMOLECULES SUCH AS IN PROTEIN
NMR.
•IMPORTANT TOOL TO IDENTIFY STEREOCHEMISTRY OF A
MOLECULE IN SOLVENT.
•USEFUL IN DETERMINING WHICH SIGNALS ARISE FROM PROTONS
THAT ARE CLOSE TO EACH OTHER IN SPACE EVEN IF THEY ARE
NOT BONDED.

APPLICATIONS OF .
13
𝐶 NMR
•METABOLIC STUDIES
•INDUSTRIAL APPLICATIONS IN
SOLIDS
•METABOLIC STUDIES ON
HUMAN
BRAIN FUCTION
GLUCOSE METABOLISM IN
LIVER
GLUCOSE METABOLISM IN
MUSCLE
IN DISEASED STATE
CHARACTERSTIC OF BODY
FLUID & ISOLATED TISSUES
DETERMINATION OF DEGREE
OF UNSATURATION OF FATTY
ACIDS IN ADIPOSE TISSUE

REFERENCE
• INTRODUCTION TO SPECTROSCOPY BY PAVIA.
• A TEXTBOOK OF ORGANIC CHEMISTRY BY BAHL ARUN & BAHL B.S.
• HTTP://WWW.CHEM.UCALGARY.CA/COURSES/350/CAREY5TH/CH13/CH13-NMR-2B.H
• HTTPS://TEACHING.SHU.AC.UK/HWB/CHEMISTRY/TUTORIALS/MOLSPEC/NMR1.HTM
• HTTPS://ORGSPECTROSCOPYINT.BLOGSPOT.COM/P/BASIC-1D-NMR-SPECTRA-SMALL-
AND-SIMPLE.HTML
• HTTPS://EN.WIKIPEDIA.ORG/WIKI/NUCLEAR_MAGNETIC_RESONANCE
• WWW.YOUTUBE.COM
• WWW.SLIDESHARE.COM
• WWW.GOOGLE.COM

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C13 & 2D NMR