COMPARISION OF PROTON NMR WITH 13C NMR

Presented by: Sumit Kaushik M.PHARM (Part-I) Dept. of PHARMACEUTICS KLES College of Pharmacy, BELGAUM.

CONTENTS:
INTRODUCTION PRINCIPLE OF NMR SPECTROSCOPY IMPORTANCE
DIFFICULTIES ENCOUNTERED IN 13C NMR
13C

CHEMICAL SHIFT

APPLICATIONS REFERENCES

INTRODUCTION
13C

NMR ( CMR)

Proton NMR ( PMR)

It is study of spin changes of proton nuclei. Chemical shift range is 0-14 ppm. Continuous wave method is used slow process. Gyromagnetic ratio is 5.5854 Coupling constant range is 0-15Hz.

It is study of spin changes of carbon nuclei. Chemical shift range is 0-240 ppm. Fourier transform Technique is used. Very fast process. Gyromagnetic ratio is 1.4043 Coupling constant range is 125-250Hz.

Solvent peak is observed.

Area under the peak is not considered. TMS peak is quartet. Effect of substitute on adjacent carbon atom cannot varies chemical shift.

Solvent peak is not observed.

Area under the peak is considered TMS peak is singlet. Effect of substituent on adjacent carbon atom can varies chemical shift.

Principle
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 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 parallel and antiparallel to applied field.

+
H0

+ +

Energy Differences Between Nuclear Spin States

DE
+

DE '

increasing field strength

no difference in absence of magnetic field proportional to strength of external magnetic field

Basic Features of NMR Spectrometer

A Modern NMR Instrument

Radio Wave Radio Wave Transceiver Transceiver

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 .

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.

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.

THE CHEMICAL SHIFT

The 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:

parts per million

chemical shift

shift in Hz = spectrometer frequency in MHz

= ppm

This division gives a number independent of the instrument used. A particular proton in a given molecule will always come at the same chemical shift (constant value).

Downfield Decreased shielding

Upfield Increased shielding

(CH3)4Si (TMS)

10.0

9.0

8.0

7.0

6.0

5.0

4.0

3.0

2.0

1.0

Chemical shift (d, ppm) measured relative to TMS

Factors affecting chemical shift

Electronegativity Hybridization Hydrogen bonding Anisotropic

Applications of 13C NMR

1. 2. 3. 4. Metabolic studies Metabolic studies on human Brain function Glucose metabolism in liver Glucose metabolism in muscle 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.