INTRODUCTION:- 13C is a natural, stable isotope of carbon. 13C NMR is analogous to proton NMR. NMR spectroscopy is based on the measurement of absorption of EMR in radiofrequency region of roughly 4 to 900 MHz with applied magnetic field. Nuclei of atoms are involved. NMR technique can be classified as PMR:- 1H NMR Isotopic NMR:- 12C NMR, 19F NMR, 31P NMR. 3
The 13C-NMR spectra are recorded by the pulsed –FT-NMRmethod, with the sensitivity enhanced by several spectra. The spin quantum number of 12C =zero, therefore it is non-magnetic and hence does not give NMR signal. Both 13C and 1H have spin quantum number i.e. I = ½, so wecan expect to see coupling in the spectrum between: a) 13C – 13C b) 13C – 1H 4
The probability of the two 13C atoms being together in thesame molecule is so low that 13C – 13C coupling are notobserved. Only 1.1% of carbon atoms in 13C are magnetic and thesenuclei split the protons in 13C-H groups into doublet and hencethe 13C-H coupling is seen in the spectra. However these couplings make the 13C spectra extremelycomplex and can be eliminated by decoupling. The spectroscopy that is done using this nucleus 13C NMRgives the information about the carbon chains in the compound. 5
This information is complimentary to thatobtained from 1H NMR spectroscopy:- The number of signals tells us how many different carbonsor different sets of equivalent carbons are present in amolecule. The splitting of a signal tells us how much hydrogen isattached to each carbon. The chemical shift tells us the hybridization (sp3,sp2, & sp)of each carbon. The chemical shift tells about the electronic environment ofeach carbon with respect to other, nearby carbon or functionalgroups. 6
Importance of 13C-NMR / why should 13C-NMR berecorded when PNMR is present: 13C NMR is a non-destructive and non-invasivemethod.13C NMR can be used in biological systems andeasy assessment of the metabolism of carbon and itspathway. Chemical shift for 13C NMR ranges from (δ = 0-240)when compared to proton NMR (δ =0-14). Sincechemical shift gives information regarding the physico-chemical environment of compound, i.e. whenchemically closely related metabolites are under NMRscan, they are often well separated and resolved to 7obtain clearly identifiable spectra.
As 13C nuclei have low abundance, thus tagging the specificcarbon position by selective 13C enrichment, thus 13C labelingincreases the signal intensities and often helps to trace thecellular metabolism Labeling with 13C helps to know the fate of specific carbonthroughout the metabolism with out need for tedious isolationand purification. The danger involved in using radioactive isotopes in tracing isavoided as 13C nuclei are stable carbon isotope. Labeling at multiple carbon sites in the same molecule andhomonucleus 13C-13C spin coupling provides novel biochemicalinformation. 8
Difficulties of recording 13C spectra than 1H spectra arebecause of the following reasons: 1. Natural low abundance of 13C . 2. Magnetic movement and Gyro magnetic ratio. 3. Chemical shift. 4. Decoupling phenomenon pronounced C-13 and H-1 spin-spin interactions. 9
1. Natural abundance:- The natural abundance of 13C is only 1.1% that of 12C, which is not detectable by NMR, that renders CMR less sensitive that PMR.2. Magnetic movement and Gyro magnetic ratio: - The 13C nucleus has only a weak magnetic movement and consequently a small gyromagnetic ratio. Sensitivity of CMR is much reduced due to the presence of only 1.1% magnetic isotope (13C) in the sample. 13C sensitivity is 1/4th that of C(Overall sensitivity of 13C compared with 1H is about 1/5700). The gyromagnetic ratio of 13C is 1.4043 as compared to 10 5.5854 of a proton 1H
These factors show that 13C CMR is much less sensitive thanPMR. The weak signals observed in 13C-CMR are thereforescanned i.e., recorded routinely by Pulse-irradiation, signal-summation and Fourier transforms. The low sensitivity of CMR is overcome by the use of largesamples, upon 2ml in 15mm tubes and by enhancement anddecoupling techniques in conjunction with highly stablespectrometers operating at high fields 12
The problems arises during recording CMR can be readilyeliminated by adopting following methods:- a) Fourier transform technique b) NOE C) DECOUPLING 13
FT NMR to record the spectra:It is a Fourier Transform NMR Spectroscopy.TYPES OF FT NMR:Multi-Dimensional:The use of pulses of different shapes, frequencies and durationsin specifically designed patterns or pulse sequences allows thespectroscopist to extract different types of information about themolecule.Multi-dimensional nuclear magnetic resonance spectroscopy isa kind of FT-NMR in which there are at least two pulses and, asthe experiment is repeated, the pulse sequence is varied.In multidimensional nuclear magnetic resonance there will be asequence of pulses and, at least, one variable time period. 14
In three dimensions, two time sequences will be varied. In fourdimensions three will be varied.2-dimensional and multidimensional FT-NMR into a powerfultechnique for studying biochemistry,in particular for thedetermination of the structure of biopolymers such as proteins oreven small nucleic acids. Pulsed radiofrequency-fourier transforms NMRspectroscopy:The NMR spectrometer operates by exciting the nuclei of theisotope under observation only one type at a time. In the case of 1H nuclei each distinct type of proton(phenyl, methyl, and vinyl) is excited individually and itsresonance peak is observed and recorded independently of allthe others. Scanning is done individually until all types have come 15into resonance.
FT NMR is an alternate approach to use powerful but short ofenergy called pulse that excites all of the magnetic nuclei in themolecule simultaneously for example, all of the 1H nuclei areinduced undergo resonance at the same time. An instrument with T magnetic fields uses a short burst of 90MHz energy to accomplish this. The source is turned on and off very quickly and generates apulse. Similarly FT NMR operates in case of carbon also. 12Cnucleus is not magnetically active because spin number I=0 butthe 13C nucleus like the 1H nucleus has a spin number of ½,however since the natural abundance of 13C is only about 1.1%that of 12C and its sensitivity is only about 1.6% that of 1H, theoverall sensitivity of 13C compared with 1H is about 1/5700. 16Pulsed FT NMR permits simultaneously irradiation of all 13Cnuclei and hence 13C spectra.
A pulse is a powerful but short burst of energy. According tothe variation of the Heisenberg Uncertainly principle, eventhough the frequency of the oscillator generating this pulse isset to 90 MHz, if the duration of the pulse is very short, thefrequency content of the pulse is uncertain because theoscillator was on long enough to establish a solid fundamentalfrequency. Therefore, the pulse actually contains a range of frequenciescentered about the fundamental. This range of frequencies isgreat enough to excite all of the distinct types of the carbons inthe molecule at once with this single burst of energy. 17
When pulse is discontinued, the excited nuclei begin tolose their excitation energy and return to their original spinstate or relax.As each excited nucleus relaxes, it emits theelectromagnetic radiation. Since the molecule contains manydifferent nuclei, many different frequencies of theelectromagnetic radiation are emitted simultaneously, thisemission is called “free induction decay” signal.The intensity of FID decays with time as all of the nucleieventually lose their excitation. This FID is complex and it is asuperimposed combination of all the frequencies emitted. Theindividual frequencies due to different nuclei can be extractedby using a computer and by Fourier transform analysis. 18
An Advantages of Fourier Transforms: -FT NMR spectroscopy is one of the principal techniques usedto obtain physical, chemical, electronic and structural informationabout a molecule. It is the only technique that can providedetailed information on the exact three-dimensional structure ofbiological molecules in solution. Also, FT nuclear magneticresonance is one of the techniques that have been used to buildelementary quantum computers. Fourier transform is more sensitive. It takes few seconds to measure FID. With computer and fast measurement, it is possible to repeatand average the measurement of the FID signal. This is a real advantage when the sample is small in which19case the FID is weak in intensity and has a great amount ofnoise associated with
NOISE:-Noise is random electronic signals that are usually visible asfluctuations of the baseline in the signal. Since noise israndom; it normally cancels out of the spectrum after manyinteractions of the spectrum are added together. Using thisprocedure one can show that signal to noise ratio improves asa function of the square root of the number of the scans, n. S/N = f√ nTherefore pulsed FT-NMR is especially suitable forexamination of the nuclei that are not very abundant in nature. Nuclei that is not strongly magnetic. Or very dilute sample 20
INTERPRETATION OF C-13 NMR SPECTRA: -Chemical shifts in C-13 NMR Spectra: -The range of shifts generally encountered in routine C-13studies is about 240 ppm. Therefore C-13 chemical shiftsrepresent the spread of chemical shifts of about 12 times thatof the proton The peak assignment or chemical shifts in CMR are madeon the basis of reference compounds. 21
13C NMR spectrum of 2-Amino-5-(4-methylphenyl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazole (1b) H 3C S N N S NH2Molecular Formula: C11H11N3S2 CH3 24
13C NMR spectrum of 2-(Alanyl)-Amino-5-(4-methylphenyl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazole (3c) S H 3C N S N NH 3c O CH3 H 2N Molecular Formula: C14H16N4OS2 CH3 C=O CH 25
13C NMR spectrum of 2-(Alanyl)-Amino-5-(4-chlorophenyl)-1,3,4-thiadiazole (2a) N N Cl S NH O 2a H 2N Molecular Formula: C11H11ClN4OS CH3 CH C=O 26
13C NMR spectrum of 5-(4-methylphenyl)-N-[(1E)-phenylmethylene][1,3]thiazolo[4,3- b][1,3,4]thiadiazol-2-amine (3d) S H 3C N S N 3d NM olecular F orm ula = C1 8H 1 5N 3S 2 CH3 CH 27
13C NMR spectrum of 3-chloro-1-[5-(4-methylphenyl)[1,3]thiazolo[4,3-b][1,3,4]thiadiazol-2-yl]-4- phenylazetidin-2-one (4d) S S N O N N Cl H 3C 4d CH3 M olecular F orm ula = C 20 H 16 C lN 3 O S 2 CH-Cl C=O CH 28
Factors Influencing Chemical Shifts :- Shifts are mainly related to hybridization and substituentelectronegativity. Solvent effects are also very important as inproton 1H spectra. Chemical shifts for 13C are affected by substituents as farremoved as the δ position. Pronounced shifts for 13C arecaused by substituents at the ortho, Meta, and para positions inthe benzene ring. Steric compression causes 13C chemical shifts to move upfield significantly. Up field shifts my also occur on dilution. Hydrogen bonding effects may cause downfield especiallywith polar solvents. 29
Calculation of chemical shifts using the correlation data: - ALKANES: -e.g: shifts for c-atoms of 3-methyl pentane: CH3CH3 CH2 CH CH2 CH3δ -calculations are made using the formula: δ= -5.2+ ∑nA.Where,δ= predicted shift for a C atom.A= additive shift parameter 31n= number of C-atoms for each shift parameter.-5.2= the shift of C-13 of methane.
ALKENES: -The alkenes Cs give signals in the range of δ 80-145. The base valuefor –CH2=CH2 is δ 123. in case of alkenes the influence of nearestsubstituent (α,β,γ) differ from the influence of the most distantsubstituent (α1,β1,γ1) as shown below.Chemical shifts δ=123+Σ (increments for carbon atoms) C – C – C – C =C – C – C - C γ--β--α γ--β—γ Increments -2 7 10 -8 -2 2 δ = 123 32
E.g.: predict the 13C-chemical shift values for the alkenes in 2-penteneCH2-CH=CH-CH2-CH3 base value : δ = 123C2 1α,1α’,1β’ C3 1α,1β, 1α’δ =123+10-8-2=123. δ 123+10+7-8=132.ALKYNES: -E.g.: - HC ≡ C - O - CH2 - CH3; H3C - C ≡ C - O - CH3 23.2 89.4 28.0 88.4 Base value to HC ≡ CH is δ =72. 33
Influence of Functional Group substituents on Alkene &Aromatic Chemical Shifts:-Alkene δ values will be affected by substitution at pointsfurther along the carbon chain (as in allyl alcohol CH2 = CH-CH2-OH). But, systematic correlations have not beencompiled. However the major influence on the alkene Cs willbe the direct substituent (in case of allyl alcohol, it is the –CH2 group). So we can say, if a substituent is identifiable as–CH2X, it should simply be treated as –CH3.The same principles hold good in predicting the shifts in thearomatic δ values. The deviation from predicted values isoften due to or associated with H- bonding and steric effects. 34
E.g.: - compounds related to salicylic acid show suchdeviation because of the strong intramolecular H-bondingbetween the –OH group and the ortho-carbonyl group. 35
Using the correlation data: BASE ISOPROPYL(R) NITRO TOTALC1 128 +21 +1 150C2 128 0 -5 123C3 128 0 +20 148C4 128 -2 -5 121C5 128 0 +1 129C6 128 0 +6 134To predict the δ values of isopropyl group carbon atoms the benzenering is considered to be the substituent isopropyl as alkane. 36
CARBONYL GROUP CHEMICAL SHIFTS:Major strength of C-13 NMR is the ability to observe the NMRcharacteristics of carbonyl C directly. The carbonyl resonanceis at very high frequency and also, different classes appearwithin narrow ranges (advantage). So that quite finedistinctions can be made, in the knowledge that, the influencesof unaccounted factors will be minimal.Introduction of alkyl group on the Cs directly attached to- COusually shifts the –CO signal by 2-3 ppm. conjugation with –CO group causes –CO resonance’s shift upfield (lowerfrequency). The anions of carboxylic acids are not muchshifted in range from the free acids, inspite of the fact that theC-O bonding in carboxylate anions is weaker than the trueC=O bond in acid. This theory fails to offer a convincingexplanation. 37
READING THE C-13 SPECTRUM:The first steps in deducing the structure of an organiccompound, using the C-13 NMR spectrum are; - Count the number of signals in the spectrum; tis is thenumber of non-equivalent C environments in the molecule.(Identify and discount the signals from solvent). Use figure (δ values table) to assign signals approximatelythe regions δ 0-80, δ 80-150 and δ 160-220(carbonyl carbons). Note the intensities of the peaks: non-proton bearing Csgive lower intensity signals, and groups of two or moreequivalent Cs give higher intensity signals. Take account of any multiplicity into (q, t, d or s). Use the correlation tables to predict the chemical shifts of all 38Cs inn each putative structure.
Use of Correlation Tables: -Two principle predictable influences that we can quantify indetermining the chemical shift positions of a C atom: 1. The number of other carbon atoms attached to it (andwhether these are CH3, CH2, CH, and C groups). 2. Natural of all other substituents attached (or nearby along achain of other C atoms). But it is important to compute 1 before 2.LIMITATIONS OF 13C NMR STUDIES: - Sensitivity of C-NMR compared to PMR, chromatography,spectrophotometry, radiochemical studies, etc. is poor. Limitation factors of C-NMR like intrinsically low sensitivity ofmagnetic resonance techniques, low gyromagnetic ratio of 13Cand low natural abundance of 13C. 39
NUCLEAR OVER HAUSER EFFECT (NOE):In NMR spectroscopy, changes brought about in the energypopulations of one nucleus by the decoupling of a neighboringnucleus are named the Nuclear Overhauser Effect of (NOE). Two conditions that always apply to NOE are It arises only during the double irradiation of one nucleus, andaffects another nucleus which must be close but not necessarilycoupled with the irradiated nucleus. It is associated with dipolar relaxation mechanisms.MaximumNOE operates on CH3, CH2 and CH carbons, whereas noenhancement arises for 4 carbons (includes carbons on aromaticrings with substituents attached.) 40
THEORY OF NOE Consider a hypothetical molecule in which 2 protons Ha Hb are in close proximity . In such compound, if we double irradiate Hb,then this protonC C gets stimulated and the stimulation is transferred through space to the relaxation mechanism of Ha. 41
Thus, due to increase in spin lattice relaxation of Ha, itssignal will appear more intense by 15 to 50%. So, if theintensity of absorption of Ha signal is increased by doubleirradiating Hb, then protons Ha and Hb must be in closeproximity in a molecule 42
The Nuclear Overhauser Effect (NOE)The carbon-13 spectrum from CH3I. The NMR spectrum from the carbon-13 nucleus will yield one absorption peak in the spectrum.In reality, we see a single line with a relative intensity of 24. Adding the nuclear spin from one hydrogen will split the carbon-13 peak into two peaks. Adding one more hydrogen will split each of the two carbon-13 peaks into two, giving a 1:2:1 ratio. The final hydrogen will split each of the previous peaks, giving a 1:3:3:1 ratio.If the hydrogen spin system is saturated, the four lines collapse into asingle line having an intensity which is eight times greater than the outer 43peak in the 1:3:3:1 quartet since 1+3+3+1=8 .
The Nuclear Overhauser Effect (NOE)If the hydrogen spin system is saturated, the four lines collapse into asingle line having an intensity which is eight times greater than the outerpeak in the 1:3:3:1 quartet since 1+3+3+1=8 .In reality, we see a single line with a relative intensity of 24.This is because of the Nuclear Overhauser Effect (NOE).The NOE is one of the ways that spin system can release energy.Magnetization transfer between spins is mediated bydipolar coupling. 44
The Nuclear Overhauser Effect (NOE)To describe the NOESY experiment, consider a pair of spin I andS, which are in close spatial proximity so as to have the dipolarinteraction. The first 900 pulse brings the magnetization of spin I down to the x-y plane. After the evolving period t1, the second pulse flips the magnetization of I back to the z-axis. 45
The Nuclear Overhauser Effect (NOE) During the delay tM, cross relaxation between spin I and S occurs and some of the spin I magnetization is transferred to S. In the detection period t2, magnetization of spin S is detected but the signal (at the frequency of spin S) is amplitude-modulated at the frequency of spin I.The result is the cross peak in the NOESY spectrum. By adjusting themixing time tM, the maximum distance between spins for which cross peaks 46will be seen can be adjusted.
The Nuclear Overhauser Effect (NOE) Another description of the NOE using energy level diagrams: Here is a 2 spin system. In the diagram, W represents the transition probability (the rate at which certain transitions can occur). At equilibrium, single quantum transitions are allowed (i.e. W1I and W1S).Double quantum transitions (W01S and W21S) are forbidden.The W1I and W1S transitions are related to spin-lattice relaxation.Relaxation due to dipolar coupling takes place when the spins give offenergy close to the Larmor frequency. 47
In pulse acquire experiment the x and y components of the freeinduction signalcould be computed by thinking about the evolution of themagnetizationduring the acquisition time. we assumed that the magnetizationstarted out along the −y axis as this is where it would be rotated tobya 90◦ pulse. For the purpose we are going to assume that themagnetization starts out along x; we will see later that this choice ofstartingposition is essentially arbitrary. 48
From fig we can easily see that the x and ycomponents of the magnetization are:.The signal that we detect is proportional to these magnetizations. Theconstantof proportion depends on all sorts of instrumental factors which need notconcern us here; we will simply write the detected x and y signals, Sx (t ) andwhere S0 gives is the overall size of the signal and we have remindedourselvesthat the signal is a function of time by writing it as Sx (t ) etc.It is convenient to think of this signal as arising from a vector of lengthS0rotating at frequency ; the x and y components of the vector giveSx and Sy,as is illustrated in Fig. 4.3. 49
NMR Pulse SequencesThe 90o-FID SequenceIn the 90-FID pulse sequence, netmagnetization is rotated down into theXY plane with a 90o pulse.The net magnetization vector begins toprecess about the +Z axis.The magnitude of the vector alsodecays with time. 51
NMR Pulse Sequences The 90o-FID SequenceA timing diagram is a multiple axis plot of some aspect of a pulsesequence versus time. A timing diagram for a 90-FID pulse sequencehas a plot of RF energy versus time and another for signal versustime. When this sequence is repeated, for example when signal-to-noise improvement is needed, the amplitude of the signal (S) will depend on T1 and the time between repetitions, called the repetition time (TR), of the sequence.In the signal equation below, k is a proportionality constant and is thedensity of spins in the sample. S=k ( 1 - e-TR/T1 ) 52
NMR Pulse SequencesThe Spin-Echo SequenceIn the spin-echo pulse sequence, a 90opulse is first applied to the spinsystem.The 90o degree pulse rotates themagnetization down into the XY plane.The transverse magnetization begins todephase.At some point in time after the 90o pulse,a 180o pulse is applied. This pulse rotatesthe magnetization by 180o about the Xaxis.The 180o pulse causes the magnetization 53to at least partially rephase and to producea signal called an echo.
NMR Pulse Sequences The Spin-Echo SequenceA timing diagram shows the relative positions of the two radiofrequency pulses and the signal.The signal equation for a repeated spin echo sequence as afunction of the repetition time, TR, and the echo time (TE) definedas the time between the 90o pulse 54
NMR Pulse SequencesThe Inversion Recovery SequenceIn this sequence, a 180o pulse is firstapplied. This rotates the netmagnetization down to the -Z axis.The magnetization undergoesspin-lattice relaxation and returnstoward its equilibrium position alongthe +Z axis.Before it reaches equilibrium, a 90opulse is applied which rotates thelongitudinal magnetization into theXY plane. In this example, the 90opulse is applied shortly after the 55180o pulse.
NMR Pulse Sequences The Inversion Recovery SequenceOnce magnetization is present in the XY plane it rotates aboutthe Z axis and dephases giving a FID.The timing diagram shows the relative positions of the tworadio frequency pulses and the signal. 56
Decoupling phenomenon/spin-decoupling methods: Non decoupled (proton coupled) 13C spectra usually showcomplex overlapping multiplets that are very difficult tointerpret, but some spectra are simple and can be interpretedeasily. 57
Various decoupling methods are as follows: -a) Multiplicity & Proton (1H) Decoupling- Noise Decoupling.b) Coherent & Broadband Decoupling.c) Off-Resonance Decoupling.d) Selective Proton Decoupling. 59
a) Multiplicity & Proton (1H) Decoupling- Noise Decoupling: -Both 13C & 1H have I=1/2, so that we would expect to seecoupling in the spectrum between a) 13C-13C b) 13C-1H However the probability of 2 C13 atoms being together in thesame molecule is so low that 13C-13C couplings are not usuallyobserved.The complicating effects of proton coupling in 13C spectra i.e.,in 13C-1H coupling can be eliminated by decoupling the 1H nucleiby double irradiation at their resonant frequencies. this is anexample of Heterounuclear De-coupling. 60
Here specific protons are not decoupled but allprotons are simultaneously decoupled by doubleirradiation while recording the 13 C spectrum. Adecoupling signal is used that has all the 1Hfrequencies spread around 80-100 Hz & is therefore aform of radio frequency noise. Spectra derived thus are1H decoupled or nose decoupled.The proton-decoupled spectrum is recorded byirradiating the sample at 2 frequencies. The First radio frequency signal is used to effectcarbon magnetic resonance (CMR), while simultaneousexposure to the second signal causes all the protons inresonance at the same time and flip their α & β spinsvery fast. 61
In the noise decoupled spectrum of diethyl phthalate:- 62
b) Coherent and Broadband Decoupling: - The most widely used spin-decoupling technique involves simplybroadband decoupling of all proton resonances to reduce the 13Cspectrum (of most organic compounds) to a set of sharp peakseach directly reflecting a 13C chemical shift.The requirements for broadband decoupling are: - 1. A Sufficiently strong decoupling field strength. 2. Method of modulation that will “spread” the decoupling fieldover the range of proton chemical shifts. Satisfying the requirement of sufficiently strong decouplingmethod strength requires use of an radiofrequency power amplifierthat is capable of supplying several watts of radiofrequency powerof the decoupler coil in the probe. However the limitation here is theability remove heat from the problem and the sample with a 64reasonable airflow.
Here the decoupling frequency is phrase modulated with a50% duty cycle, 100Hz square wave. Residual broadening ofdecoupled off-resonance 13C peaks is significantly reducedusing this method in comparison to the former method. Thismethod is now being widely used in broadband (1H)decoupling. 65
C) Off Resonance Decoupling: - The off-resonance coupling not only simplifies the spectrum but alsoretains the residual 13C-H coupling information.This is a deliberately inefficient double irradiation of the protonfrequencies. The decoupler is offset by 1000-2000Hz upfield or about 2000-3000Hzdownfield from the frequency of TMS without using the nose generator. In off-resonance decoupling, while recording the CMR spectrum, thesample is irradiated at a frequency close (but not identical) to theresonance frequency of protons. Consequently, the multiples become narrow and not removedaltogether as in fully decoupled spectra i.e., the weak C-H coupling aredecoupled and strong couplings remain though somewhat distorted. 66
The residuals coupling constant Jr is < true J. Jr =2 J /γB2 = difference between decoupled frequencies andResonance frequencies of 1H of interest.J = true coupling constant.B2= strength of rotating magnetic field generated by thedecoupler frequencies. = gyro-magnetic ratio. 67
d) Selective Proton Decoupling: -•When a specific proton is irradiated at its exact frequency at a lowerpower level than is used for off-resonance decoupling, the absorbance ofthe directly bonded 13C becomes a singlet, while the other 13Cabsorptions show residual coupling. DEUTERIUM COUPLING: -The number of orientations, which any magnetic nucleus can adopt inmagnetic field, is (2I+1). I = spin quantum number. Thus for 1H & 13C where, I = ½,2 orientations arise either +I or –I. But fordeuterium whose I = 1, 3 orientations arise: a) Aligned with the magnetic field most stable will augment Bo b)Across the field on a plane Deuterium nucleus is précessing on a plane cutting across Bo(magnetic field) & will not change field strength (1H frequenciesunchanged). 69
c) Antiparallel/non-aligned with the Bo least stable will diminish Boproton frequency reduced (a) (b) (c) 70
Protons coupled with one deuterium nucleus come to resonance atthree different frequencies i.e., the 1H signal appears as a triplet; theline separation correspond to JH-D . If a group of protons signal is coupled to more than oneDeuterium then the Multiplicity of the proton signal is found from thegeneral formula (2nI=1). Thus two (equal) deuterium couplings give rise to quintets, &three deuterium gives septets & so on. Deuteriated solvents (deuteriochloroform CDCL3, deuteriobenzeneC6D6, deuterioacetone CD3COCD3 , or dexadeuteriodimethylsulphoxide CD3SOCD3 ) give rise to 13C signals, which are split bycoupling to deuterium. Thus in molecules with one deuteron attached to each carbon (asin CDCL3 & C6D6) the C-13 signal form the solvent are a 1:1:1triplet. For CD3 groups (CD3COCB, CD3SOCD3 ), the solvent gives 71rise to a septet with line intensities 1:3:6:7:6:3:1.
Relaxation Phenomenon: - What happens when protons absorb energy? Nuclei in the lower energy state undergo, transitions tothe higher energy state; the populations of the tow states mayapproach equality, and if this arises, no further net absorptionof energy can occur and the observed resonance signal willfade out saturation of the signal.However, during a normal NMR run, the populations in the 2spin states do not become equal, because higher E nuclei areconstantly returning to the lower energy spin state 73
How do the nuclear lose energy and undergotransition from the high to the low-energy state? The energy difference E can be re-emitted asradio frequency E that is monitored by a radio frequencydetector as evidence of resonance condition having beenreached.However 2 important radiation-less processed exist,which enable high-energy nuclei to lose energy. Spin-Lattice Relaxation Spin-Spin Relaxation 75
1) Spin-Lattice Relaxation The high energy nuclear can undergo energy loss (orrelaxation) by transferring E to some electromagnetic vectorpresent in the surrounding environment e.g.: a nearby solventmolecule undergoing continuous vibration and rotationalchanges, will have associated electrical and magneticchanges, which might just be properly oriented and of thecorrect dimension to absorb E. since the nuclear may besurrounded by a whole array of neighboring atoms either inthe same molecule or in solvent molecules, etc., thisrelaxation process is termed spin-lattice relaxation. 76
2) Spin-Spin Relaxation: - A 2nd relaxation process involves transferring E to c neighboringnucleus, provided that the particular value of E is common to both nucleithis mutual exchange of spin energy is termed spin-spin relaxation. Whileone nucleus loses energy, the other nucleus gains energy, so that no netchange in the population of the 2 spin states is involved.Relaxation phenomenon in terms of magnetization and vectors:- Aligned with the fieldOne nucleus is an eitherapplied either field orprecesses Opposed to the field 77
When the system of nuclear spins relaxes, two differentprocesses are identified:(a) the reduced z-axis component eventually increasesback to Mo(b) the y-axis component reduces to zero. 78
APPLICATIONS 13C-NMR is mainly used to study the metabolism in humans1. Brain function.2. Glucose metabolism and Glycogen quantification.3. Glucose metabolism in the muscle.4. Mechanism of hepatic glycogen repletion.5. Disease status.6. Characteristics of body fluids and isolated tissues. 79
2D NMRAll 2D experiments are a simple series of 1D experimentscollected with different timing.2D NMR differ from the conventional NMR in that responseintensity would be function of two frequency rather than a singlefrequency. 1D one time variable one intensity variable 2D two time variables two intensity variables 75 80
1-D NMR - ONE OR TWO- DIMENSIONS? 1-D NMR COMPRISES TWODIMENSIONS (ONE FREQUENCY AND ONE INTENSITY AXES) 81
2-D NMR • 2-D NMR CONSISTS OF TWOFREQUENCIES AND ONE INTENSITY AXES - INTENSITY NOT COUNTED 82
The two dimension of NMR based on dimension of time.One of the dimension is time domain with which we can collectthe free induction decay (FID) output from thespectrophotometer and which contain frequency &intensityinformation .The second dimension is refer to the time pass away /lapsing between application of some distribution to the systemand the onset of collection of data in the first time domain.The second time period is varied in regular way and series ofFID response collected corresponding to each period chosen . 83
WHAT…?Stack of several 1D spectraEach 1D is different from thenext by a Small Change in theevolution time t1Parameters for eachsuccessive experiment in theseries are constant except thephase of the pulsesFT of the two time 84domains provides a mapof spin-spin correlations
WHY 2D-NMR…?The various 2D-NMR techniques are useful when 1D-NMRis insufficient, as the signals start overlapping because oftheir resonant frequencies are very similar.2D-NMR techniques can save time especially wheninterested in connectivity between different types of nuclei(e. g., proton and carbon).This method is useful when the multiplets overlap or whenextensive second order coupling complicates in the 1Dspectrum. 85
THEORYThe basic 2D NMR experiment consists of a pulse sequencethat excites the nuclei with two pulses or groups of pulses.The groups of pulses may be purely radiofrequency (rf) orinclude magnetic gradient pulses. The acquisition is carried outmany times, incrementing the delay (evolution time - t1) betweenthe two pulse groups.The first aim of the system (pulse) will be the preparation of thespin system.The variable Td is renamed as evolution time, T1. 89
Secondly mixing event, in which information from one partof the spin system is relayed to other parts.Finally, an acquisition period (T2) as with all 1Dexperiments. Schematically, it is presented as following:T1 is the variable delay time, and T2 is the normalacquisition time. This can be envisioned having f1 and f2, for both 90frequencies.
BASIC SEQUENCES OF 2D-NMR PREPARATION PERIOD:During this period, magnetization is prepared by applicationof a pulse or a series of pulses (generally 900 pulse and 1800refocusing pulse) to the spin system for evolution process.The nuclei is allowed to relax to their equilibrium state.For this reason, the actual time is usually set to about fivetimes the average relaxation time of the nuclei(about 2seconds) 91
EVOLUTION PERIOD:The preparation period is followed by evolution phase duringwhich the spin system evolves, sometimes under the influenceof chosen experimental conditions.The evolution period is critical as its duration T1 will affect theFID acquired during the detection time T2.The time interval serves as a variable whose value changesthe phase and amplitude of the peaks.The components of magnetization on the Y-axis depends onthe length of time allowed for the evolution of magnetizationbefore detection. 92
MIXING:Evolution phase is followed by mixing phase in which oneor more radio frequency pulse are applied and to generateobservable transverse magnetization.The mixing period may be of zero or finite duration andduring detection period it do not fixed the FID is acquired.ACQUISITION TIME:The essence of 2D experiments is that the time period T1is used to modulate the FID. 93
Fourier transformation of the FID acquired during the fixedtime T2 yields a series of spectra, each corresponding to adifferent value; a second transformation is then carried outover the period T1 which gives the two dimensional spectrum.Finally there is a detection phase in which the correlatedNMR signal is recorded. 94
J-RESOLVED SPECTROSCOPY (ROSY)In this technique, the scalar coupling are spread out along oneaxis of the plot whereas the other axis represents chemical shift.This is thus, a useful method for separating crowded spectrawith overlapping multiplets.In spectra, the chemical shift on one axis is plotted against themultiplicity on the other axis but the graph obtained indicates thatthe mid points of the multiplets lie on the middle row of the stackplot.It is represented using stacked plots which representing signal 97intensity perpendicular to plan of pages.
ADVANTAGE:J-Resolved 2D-NMR spectra allow identification of- 1.chemical shift position 2.Multiplicity 3.coupling constant-JDISADVANTAGE:It do not necessarily establish proton coupled with proton orcarbons 98
HOMONUCLEAR ROSYThe separate presentation of chemical shift and couplinginformation is the basic of homonuclear ROSY. 100
E.g. Ethyl acetate1. The normal ROSY spectra for ethyl acetate is at (a) and its simplicity does not require 2D treatment although it is a representative model.2. At (b), the chemical shift is plotted at one axis and the multiplicity on other.3. The additional information with its presentation reveals the projection spectrum at (c).ADVANTAGES: It helps in separation of overlapping multiplets. The decoupled projection spectrum can be much more facilitated by ROSY. 101
HETERONUCLEAR ROSY•In this spectrum, the multiplicity information for the carbon-proton coupling is plotted against the carbon is chemical shift.•E.g. Decalin•The projection spectrum in the case of trans Decalin would bethe broad band C-H NMR spectrum which is in any event easilyrecorded by simpler means. 102
CORRELATED 2D NMR (COSY)•Here, correlation is plotted in second dimension with theclassical chemical shift in the other dimension. It isrepresented by using counter plot which represents peakintensity•COSY help to establish - proton couple with proton - proton couple with carbon•While determine molecular structure from a high resolutionNMR spectrum . It is important to establish signal which iscomes from nuclei couple via the scalar interaction . 103
COSYWhile determine molecular structure from a high resolutionNMR spectrum . It is important to establish signal which iscomes from nuclei couple via the scaler interaction . Thescaler interaction allows to inter the location of nucei inmolecule because the coupling constant j- depend on - the no of chemical bond are separating from thosenuclei - whether the bonds are single or double - the angle they form with other bands 104
APPLICATION OF NMRQUANTITATIVE ANALYSISThe concentration of species can be determined directly bymaking use of signal area per proton and the area of thatidentifiable peak of one of the constituent for e.g. if the solventpresent in known amount were benzene, cyclohexane orwater, the area of single proton peak for these compound couldbe used in order to set the required information.ANALYSIS OF MULTICOMPONENT MIXTUREHollis has described a method for the determination ofaspirin, phenacetin and caffeine in commercial analgesicpreparation.
Chamber lain and kolthoff have described a method for therapid analysis of benzene, ethylene glycol and water inmixture.ELEMENTAL ANALYSISThe total concentration of a given kind of magnetic nucleusin sample can also be determine by NMR for e.g. theintegrated NMR intensities ofProton peak for a large no. of organic compound havesuccessfully determined by Jungnikel and forbes. 108
IDENTIFICATION OF COMPOUND The structure of unknown compound from its NMR can beeasily decided by certain principles, some of them areThe no. of main NMR signal should be equal to the no ofequivalent protons in interested compound.The type of methylene hydrogen atom, methyl grouphydrogen, ether hydrogen etc. is indicated by chemical shift.The possible arrangement of group in the molecule isindicated by spin-spin splitting.The area under NMR is directly proportional to the no. ofnuclei present in each group. 109
HYDROGEN BONDINGHydrogen bonding causes a decreasing the electronshielding on the proton. Breaking of intermolecular hydrogenbond is indicated by an up field shift of the signal.The downfield shift depends upon the strength of hydrogenbonding.KETOENOL TAUTOMERISMThe keto-enol tautomerism has also been studied by NMRspectroscopy. 110
STRUCTURAL DETERMINATION NMR spectroscopy is very helpful in studying andestablishing the structure of complexes, organic andinorganic compounds.For e.g. A) structure of SOF4 - only one resolution field signal isobtained while 19F spectrum of SOF4 is recordedindicating that all the four fluorine in the molecule of SOF4are equivalent.B) Structure of HF2 if 19F magnetic resonance spectrumof HF2 is recorded, only one signal is recorded showingthat HF2 has linear structure. 111
INTERMOLECULAR CONVERSIONEXCHANGE EFFECTSThe physical state of the sample and the type of nucleus aretwo important factors upon which the width of absorptionband in NMR depends.The width is small (2-3Hz) for most of the liquids: althoughbroad bands have also been observed in the NMR spectraof liquids and this fact may be accounted for in terms ofexchange effects. 112
QUESTION 20 MARK 1(A).Explain the techniques used for decoupling its interpretation between 13C NMR & 1H NMR interaction in carbon-13 NMR. 1.(B)Describe the concept of NMR Spectroscopy. What are the factor affecting Chemical Shifts. (April 2008, Sept 2007) 2.What are Decoupling methods? What is significance in 13C NMR Spectroscopy? 10 MARK 1.What is Decoupling? What is its significance in 13C NMR Spectroscopy?(May 2010) 2.Discuss 13C NMR Spectroscopy & its application (May 113 2012)
5 MARK 1.Give Principles of 13C NMR Spectroscopy?(OCT 2010). 2.Explain Chemical Shifts in NMR.(2004) 3.Explain Brief account on 2-D NMR(May 2011) 4.Explain brief account on Nuclear overhouser effect.(2006,2008,April 2009) Give detail on NMR pulse sequense.(1996,2003,2006) 114
REFERENCES1. James Keeper. In: Understanding of NMR spectroscopy; Wiley VCH, NY.20022. Joseph B. Lambert, Eugene P. Mazzola. In: NMR spectroscopy; Pearson Education Inc. NJ.3. Jag Mohan. In: Organic spectroscopy; Narosa publication house.4. Skoog, Holler, Nieman. In: Principles of instrumental analysis; Harcourt asia pte ltd.5. G. Ganglitz, T. Vo-Dinh. In: Handbook of spectroscopy; Wiley VCH, NY.2003.6. Sharma BK. Instrumental methods of chemical analysis; GOEL publishing House, Meerut 1157. Some internet sources
References : -8.Organic spectroscopy by William Kemp.9.Spectroscopy of organic compounds by P.S.Kalsi.10.Spectrometer identification of organic compounds by Silverstein.11.Elementary organic spectroscopy by Y.R.Sharma. 116