Chem-806Identification of organic and inorganic

compounds by advance NMR techniques

Tool box

2D-NMR: Homonuclear

2D-NMR: Heteronuclear

3D-NMR

Parameter Consideration• Receptivity

– Spin Quantum Number– Resonance Frequency– Sensitivity– Natural abundance

• Frequency Shift– Absolute Frequency– Relative Chemical Shift Scale (Referencing)

• Relaxation – T1 and T2

– Definition– Mechanisms– Measurement– NOE

Multinuclear NMR

Z Nuclei Spin I Frequency % natural abundance

1 1H ½ 100.00 99.98

6 13C ½ 25.15 1.108

7 14N 1 7.23 99.935

15N ½ 10.136 0. 365

9 19F ½ 94.103 100

15 31P ½ 40.43 100

At Bo=2.35 Tesla

When sampling a nuclei, following parameters shoud be considered:

• Sensitivity• Natural abundance• Relaxation time : T1 recycle time, T2 acquisition Time• Influence of H-decoupler: {NOE and J}

n0 =gB0

2p

Sensitivity and Receptivity

The sensitivity of a nuclei depends on:

1. Magnetic Field ( g)2. Population excess ( g)3. Magnetic field induced in receiver coil ( g)

Sensitivity = k * gx3 * Ix(Ix +1)

e.g. g(13C)/g(1H) = 1/4 13C less sensitive than proton (64 less)

Receptivity Rx = ax * Sensitivity

Where ax = natural abundance

e.g. Relative receptivity of 1H and 13C

R =CH aC * gC

3

aH * gH3 = 0.01 * (25)3

1 * (100)3 = 1.6 * 10-4 R = 0.83FH

T1 considerationsSpin lattice relaxation time T1 “lifetime” of First Order Rate process

z

yx

90x

z

yx

Mo

My

z

yx

z

yx

z

yx

Mo5 T1t ..t

Magnitude of T1 is highly dependant on : 1. the type of nuclei2. State of the sample

T1 governs the efficiency of the NMR experiment : recycle time

For 1H in solution T1 can be 0.01 to 100 sec.For low g nuclei – spin ½ - relaxation can be much longer!

Recovery of the magnetization along the Z axis

Relaxation (T1)

Dinitrobenzene: T1

NO2

NO2

HH

H

H

H2H4/H6 H5

180 90

t

Inversion recovery : 13C

180 90

tD1= 5T1

t = 0.03 s

t = 1.5 s

t = 3 s

t = 6 s

t = 50 s

3

2

4

1

5

6

7CH39

CH38

OH

CH310

T1 = tnull / ln2 = 1.443 * tnull

e.g. C2 => T1 = 4.3 s (tnull=3 )

Helping relaxationOne approach of reducing relaxation time is by the addition of paramagnetic relaxation reagent (Chromium III acetylacetonate => Cr(acac)3)

1s delay, 30o pulseWithout Cr(acac)3

With Cr(acac)3

Intensity vs Pulse Interval

Mz

t

M(t) = Mo(1-e-t/T1)

PW=90o

D1 AQ

NSz

yx

90x

z

yx

Mo

My

t = pulse interval = D1 + AQ

t1 * T1

M(t)0.63 M0

2 * T1 0.86 M0

3 * T1 0.95 M0

4 * T1 0.98 M0

5 * T1 0.99 M0

10 * T1 0.99995 M0

Optimum recycle delay (pulse interval) with 90` pulse

Total experiment time fixed

During the experiment, t (D1+AQ) is repeated for NS

PW=90o

D1 AQ

NS

t = pulse interval = D1 + AQ

t Sensitivity

.1 T1 .3

.2 T1 .41

.5 T1 .56

.75 T1 .61

1 T1 .63

1.26 T1 .64

1.5 T1 .63

2 T1 .55

Optimum delay

Optimum angle with D1 < T1

PW<90o

D1 AQ

NS

D1 = 0

t = pulse interval = AQ

z

yx

PWx

Mo M

z

yx

Optimum angle Ernst angle

a = cos-1 et/T1

t aE T1 (t=1)

100 T1 90o .01

10 T1 90o .1

2.5 T1 86.3o .4

1.5 T1 77.1o .67

1. T1 68.4o 1

0.5 T1 52.7o 2

0.25 T1 38.8o 4

0.1 T1 25.2o 10

0.01 T1 8.1o 100

Short T1

Long T1

Sensitivity curves for different pulse and different delays and relaxation time

Steady State

T2 Consideration

T2

Refocusing of field inhomogeneity

Carr-Purcell-Meiboom-Gill

CPMG used to get rid of broad signals

Polystyrene (50,000) + camphor

90 180

t

t = 1.5 ms

t

SW and Memory sizeO1

SW

The spectral window (SW) and the carrier offset (O1) are chosen to match entire spectra (to avoid Fold-over and aliasing)

For a given SW, the time (Dwell time) between 2 data point is defined by the Nyquist theorem (1/2SW).

The total number of data point (TD) acquired is related to the Acquisition time AQ (DW*TD)

The digital resolution depends on the window and on the number of points placed in that window

Digital resolution = 2 * SW/ TD = 1/AQ

TD = 2 * SW * AQ

Sharp lines have long FID (long T2*),

broad peaks have short FID (short T2*),

AQ ~ 3 * T2

Nyquist Theorem

DIGITALLIZATION

• Accuracy• Speed

Digitallization: Convert FID (Volt/Time) in Digital formDigitallization process is limited by:

Carrier Offset or Transmitter Offset or “O1” is the frequency of the irradiating field. It is also the “Reference” or “Rotating Frame” frequency

The “Window” or “Spectral Width” also called “SW” define the range of frequencies that can be measured

r.f.

O1SW

The Sampling Rate => 2 Points/Cycle Dwell Time = DW= _1__2*SW

If Maximum Frequency to be sampled is fmax = SW

We must sample at a rate no less than 2 * SWsec.

Digital Resolution

The amount of memory limit the accuracy of the signal to be recorded

For a given # of memory (# Points -> TD (time domain)), one obtain:

NP (real) and NP (Imaginary) 2 2

Digital Resolution = D.R. = Df (Separation between 2 points)

D.R. = 2 * SW NP

Digitallization : resolution and Acquisition time

Example

At 200 MHz If: SW=2000 Hz (10 ppm)

TD = 16,000 points (16K)

What is the Digital Resolution:

D.R. = 2*SW/TD = 4000 / 16,000 = 1 / 4 = 0.25 Hz

What is the Acquisition Time AQ:

AQ = TD * DW = TD / (2 * SW) = 4 seconds

D.R. = 1 / AQ = 2 * SW / TD

C13-NMR : proton decoupling

C

O

OH CH2

CHCH3

OH

Heteronuclear nOe

For nuclei having positive g : (e.g. 13C)Decoupling proton can produce higher signals due to nOe.Enhancement is dependant on motion and distance between interacting nuclei

4a

8a

5

8

6

7

3

2

4

O O

CH39

AcO

AcO

As a consequence, quaternary carbons are much smaller than protonated carbons

Heteronuclear nOe

• Can yield higher positive signal for nuclei with positive g (e.g. 13C)

• For nuclei with negative g (e.g. 29Si, 15N) can yield larger (negative) signals or for partial T1DD can null the signal!!!

e.g. 15N {1H}

Without NOES = A0 =1

NOEmax gH

2 gN

~ -5

S =A0+NOE= -4

100% NOE50% NOE

NOE=-2.5

S =A0+NOE= -1.5

20% NOE

S =A0+NOE= 0

NOE=-1

Refocusing : echo formation

Delay as a sequence building block

JAX

dA

J2

J2

-

AX Spin system A=1H, X=13C, JAX

For each isochromats the distance they run in the xy frame during a delay t is:

Distance = Frequency (cycles/sec) * delay (sec)

Distance = 2p * (+/-) J/2 * t

x x x x

J= 10 Hz t (1/2J) = 0.05 s

J=100 Hz t (1/2J) = 0.005 s

J=140 Hz t (1/2J) = 0.00357 s

DelayDist.

t=00

t=1/4Jp/4 (45o)

t=1/2Jp/2 (90o)

t=1/Jp (180o)

APT experiment

APT Pulse Sequence

Multiplet Modulation

APT : 8 msec delay

APT : 6 msec delay

INEPT experiment

INEPT

Comparing NOE Enhancement and

INEPT Enhancement

INEPT sequence

Multiplet distortion in INEPT: 29Si

Multiplet distortion in coupled INEPT: 13C

Multiplet distortion in coupled INEPT: 13C

29Si – INEPT coupled

Refocused INEPT

Multiplicity Modulation : HX

Multiplicity Modulation : XH2

Multiplicity Modulation : XH3

Decoupled INEPT: Menthol

Optimum delay in decoupled INEPT

Decoupled INEPT: compare normal and INEPT 29Si

DEPT

Intensity vs

pulse angle

Normal 13C

DEPT-45

DEPT-90

DEPT-90

DEPT-135Menthol in Acetone-d6

DEPT Ipsenol

DEPT Editing

ADEPT Editing

The BIRD pulse

Refocusing : echo formation

Field Gradient in NMR

Gradient Field: A Way to Speed up 2D & 3D NMR =>

replace phase Cycling

If gradient gz is applied during tg

Each isochromats accumulate phase:

F = 2p * Df * tg

Coherences precess at n * Df Where n is the coherence order

GE-COSY: Gradient Enhance COSY

Homonuclear 2DJ-NMR

Different format in 2D-NMR

2DJ stack plot

2DJ- contour

2DJ- expansion

2DJ-ROTATE

2DJ-rotate

NMR processing: apodization Window

• Line Broadning (LB) : Exponential Multiplication improves the signal to noise ratio (at the expense of resolution)

Processing : line broadening

Processing : line broadening

NMR processing: apodization Window

• Line broadning : Exponential multiplication improves the signal to noise ratio (at the expense of resolution)

• Resolution Enhancement reverse exponential + Gaussian function also traf function, also sine function (for 2D – magnitude mode)

Processing : resolution enhancement

Processing : sine bell (phased mode)

Processing : sine bell (magnitude mode)

Processing : sine bell (Power mode)

Processing : Qsine (phased mode)

Processing : Qsine (magnitude mode)