Presented by:

Naveen Kadian

K.L.E.S’s College of Pharmacy, BELAGAVI.

CONTENTS:

INTRODUCTION

PRINCIPLE OF 13C NMR SPECTROSCOPY

IMPORTANCE

DIFFICULTIES ENCOUNTERED IN 13C NMR

13C CHEMICAL SHIFT

APPLICATIONS

REFERENCES

INTRODUCTION: The first NMR observation regarding 13C nuclei

were reported in 1957. The experiment concluded that the direct

observation of carbon nuclei had greater utility over the equivalent protons studies.

The study of carbon nuclei through magnetic resonance spectroscopy is important technique for determining the structure of organic compounds,using it with proton NMR and IR spectroscopy organic chemist can often determine the complete structure of unknown compounds.

Thus, carbon NMR provides direct information about the carbon skeleton of the molecule.

INTRODUCTION: contd….

13C NMR ( CMR) Proton NMR ( PMR)It is study of spin changes of carbon nuclei.

It is study of spin changes of proton nuclei.

Chemical shift range is 0-240 ppm. Chemical shift range is 0-14 ppm.

Fourier transform Technique is used. Continuous wave method is used

Very fast process.

Gyromagnetic ratio is 1.4043

slow process.

Gyromagnetic ratio is 5.5854

Coupling constant range is 125-250Hz. Coupling constant range is 0-15Hz.

Solvent peak is observed. Solvent peak is not observed.

Area under the peak is not considered. Area under the peak is considered

TMS peak is quartet. TMS peak is singlet.

Effect of substitute on adjacent carbon atom cannot varies chemical shift.

Effect of substituent on adjacent carbon atom can varies chemical shift.

Any nucleus with odd mass number spins on its own axis By the application of an external magnetic field (Ho), the nucleus spins on its own axis and a magnetic moment is created,.In this ground state the magnetic field caused by a spin of nuclei is aligned with external magnetic field.When the energy in the form of radio frequency is applied and when applied frequency is equal to processional frequency, absorption of energy occurs and NMR signal is recorded.Because of absorption of energy, the nucleus moves from ground state to excited state, which results in spin reversal or anti-parallel orientation in which magnetic field caused by the spin of nucleus opposes the external applied magnetic field.

Nuclear SpinNuclear Spin

A spinning charge, such as the nucleus of 1H or 13C, generates a magnetic field. The magnetic field generated by a nucleus of spin +1/2 is opposite in direction from that generated by a nucleus of spin –1/2.

+ +

++

+

+

+

The distribution of nuclear spins is random in the absence of an external magnetic field.

++

+

+

+An external magnetic field causes nuclear magnetic moments to align parallel and antiparallel to applied field.

HH00

Energy Differences Between Nuclear Spin States

Energy Differences Between Nuclear Spin States

no difference in absence of magnetic field

proportional to strength of external magnetic field

+

+

EE E E ''

increasing field strengthincreasing field strength

Radio WaveTransceiverRadio WaveTransceiver

A Modern NMR Instrument

IMPORTANCE OF 13C NMR CMR is nondestructive and noninvasive method. CMR of biological materials allows for the

assessment of the metabolism of carbon. CMR, chemical shift range is wider than PMR. The low natural abundance of 13C nuclei (1.1%)

can be made use of tagging the specific carbon position by selective 13C enrichment.

13C nucleus is a stable isotope, hence not subjected to dangers related to radiotracers.

Homo nuclear coupling of 13C provides novel biochemical information.

DIFFICULTIES ENCOUNTERED IN 13C NMR

The 13C nucleus is magnetically active

and which is similar to 1H nucleus.

Recording of CMR nucleus is difficult

due to the following reasons:

1. Natural abundance.

2. Gyro magnetic ratio.

3. Coupling phenomenon.

1. Natural abundance:

The most abundant isotope of carbon 12C is not

detected by NMR, as it is magnetically inactive (I=0).

The low natural abundant isotope 13C is magnetically

active (I=1/2).

As a result of the natural abundance of 13C is 1.1% , the

sensitivity of 13C nuclei is only 1.6% that of 1H nuclei.

The availability of FT instrumentation enhances the

sensitivity of 13C nucleus.

2. Gyro magnetic ratio:

The gyro magnetic ratio of 13C is 1.4043 as

compared to 5.5854 of a proton.13C nucleus resonance frequency is only 1/4th of

PMR at a given magnetic field.

Thus, CMR is less sensitive than PMR

Sensitivity of CMR can be increased by adopting

FT technique.

3. Coupling phenomenon: Both 13C and 1H have I =0, so that we expect coupling in the spectrum between 13C - 13C and 13C - 1H.

The probability of two 13C nuclei adjacent to each other in the same molecule is extremely rare due to low natural abundance of 13C.

So that 13C- 13C coupling will not usually exist. However the 13C - 1H coupling have observed in CMR spectrum.

As a result of coupling makes the 13C spectrum extremely complex , consequently there is an overlap of multiplets.

These 13C - 1H coupling can be eliminated by adopting following techniques.

a) FT technique

b) Decoupling technique

c) Nuclear overhauser phenomenon for enrichment of the carbon signal.

FT technique: Earlier, the continuous wave method is used to record

13C spectra but it is slow procedure, require large sample and for assessing takes long time .FT technique increases activity of 13C nuclei

FT technique permits simultaneous irradiation of all 13C nuclei .

In this method sample is irradiated with a strong pulse of radio frequency radiation in desired range at once in a fixed magnetic field .

Advantages 1)The scanning takes place rapidly

compared to continuous wave NMR. 2)The sensitivity problems are eliminated in NMR,

therefore which helps in a) Analyses the sample at low conc. b) NMR studies on nuclei with low

natural abundance and with low gyro magnetic ratio.

Decoupling technique:

Generally the probability occurrence of 13C- 13C coupling is rare , but the 13C - 1H coupling can usually observed . The problem of 13C - 1H coupling can be eliminated by decoupling the proton from carbon .

Types of decoupling in CMR

1) Proton decoupling or noise decoupling .

2) Coherent and broadband decoupling .

3) Off resonance decoupling .

1) Proton decoupling or noise decoupling: The proton decoupled CMR spectrum can be

recorded by irradiating the sample at two frequencies. The first radio frequency signal is used to affect

carbon magnetic resonance, while simultaneous exposure to second signal causes all the protons to be resonance at the same time they spin or flip very fast.

As they flip so fast, there is no coupling and each carbon appears as a single unsplit peak at corresponding chemical shift range.

Ex: Proton decoupled spectra of sec-butyl bromide.

2) Broadband decoupling:

In this technique, all the proton resonance can be reduced and to get sharp CMR spectral peaks, each directly reflecting a 13C chemical shift.

The NMR spectrum of nucleus A is split by nucleus B, because A can see B in different magnetic orientation.

Off resonance decoupling

1000-2000 Hz above the spectral region

In this primary carbon nuclei (bearing three

protons) yield a quartet of peaks, secondary

carbons give three peaks, tertiary carbon nuclei

appear as doublets, and quaternary carbons

exhibit a single peak.

This division gives a number independent of the instrument used.

parts permillion

THE CHEMICAL SHIFTTHE CHEMICAL SHIFTThe shifts from TMS in Hz are bigger in higher field instruments (300 MHz, 500 MHz) than they are in the lower field instruments (100 MHz, 60 MHz).

We can adjust the shift to a field-independent value,the “chemical shift” in the following way:

A particular proton in a given molecule will always come at the same chemical shift (constant value).

chemical shift

= = shift in Hz

spectrometer frequency in MHz= ppm

01.02.03.04.05.06.07.08.09.010.0

Chemical shift (Chemical shift (, ppm), ppm)

measured relative to TMSmeasured relative to TMS

UpfieldUpfieldIncreased shieldingIncreased shielding

DownfieldDownfieldDecreased shieldingDecreased shielding

(CH(CH33))44Si (TMS)Si (TMS)

Factors affecting chemical shift

Electronegativity Hybridization Hydrogen bonding Anisotropic

Applications of 13C NMR Metabolic studies Metabolic studies on human

1. Brain function

2. Glucose metabolism in liver

3. Glucose metabolism in muscle

4. Determination of degree of unsaturation of fatty acids in adipose tissue

5. Characteristic of body fluids and isolated tissues

6. In diseased state Industrial applications in solids

REFERENCES Morrison RT, Boyd RN. Organic chemistry. 6th

edition.2001; P.no 604-629. Sanders FRS, Jeremy KM, Hunters BK. Modern

NMR Spectroscopy. 2nd edition. 1993; P.no 46. Skoog, Holler, Nieman. Principles of

Instrumental analysis. 5th edition 1991; P.no 480-484.

Kemp W. Organic Spectroscopy 3rd edition 1991; P.no 110-130.

Silverstein RM, Webstar FX. Spectrometric Identification of Organic Compounds 6th edition. 1998; P.no 222.

A.The differences in the applied magnetic field strength (Angular Frequency of Precession) at which the various proton configurations in a molecule Resonate is extremely small.

A.The differences amount to only a few parts per million in the Magnetic field strength.

A.It is difficult to measure the precise field strength to less than a part per million.

A.Measuring the difference between absorption positions is much easier using the difference between the Resonance of the sample and the Resonance of a standard reference sample.

The Chemical Shift

The Chemical Shift (Con’t)

E. The actual procedure measures the difference between nuclei resonance energies relative to the universally accepted standard - Tetramethylsilane (CH3)4Si (TMS).

All protons in TMS have chemically and electronically similar environments.

They are highly shielded - nothing about them diminishes the electron density.

They resonate at same field strength, i.e., a single reference signal is produced.

The proton resonances in Tetramethylsilane (TMS) appear at a higher magnetic field strength than proton resonances in most all other molecules.

Tetramethylsilane (TMS)

Chemical Shift () = 0 ppm by Definition All protons in TMS are in chemically equivalent environments The protons are in regions of high electron density Silicon is less electronegative than Carbon Proton resonances appear at a higher field strength than proton

resonances in most all other molecules One signal is produced Small amount produces large signal

Reasons to use TMS as internal Standard

Si

H

C H

H

H

H C

HH C H

H

H

H C H

Observed Shift from TMS (Hz) Hz Chemical Shift () = = = PPM 60 MHz MHz

The Chemical Shift on (Con’t)

F. Thus, the protons in compounds of interest to the organic chemist resonate at frequencies greater than the protons in TMS, i.e., at lower magnetic field strengths.

G. A parameter called the “Chemical Shift ()” has been defined to give the position of the absorption of a proton a quantitative value.

H. The Chemical Shift values are reported in units of “Parts Per Million” (ppm).

The Chemical Shift (Con’t)

A. A NMR spectrometer increases the Magnetic field strength as the pen moves from left to right on the chart.

B. The TMS absorption is higher than will be obtained for just about all organic compounds.

C. Thus, the absorption signal for TMS appears at the far right side of the chart.

D. The Chemical Shift for TMS is arbitrarily set at “0” PPM.

E. By convention, the Chemical Shift values increase from right to left, with a range of 0 (TMS) to about 13 on the far left of the chart.

F. In other words: Chemical Shift values decrease with increasing Magnetic field strength!

OH does not have carbon

no 13C-NMR OH signal

Example: HOCH2CH2CH2CH3