10 – experimental aspects of nmr 1.the nmr tube 2.nmr solvents 3.the sample 4.how much time does...

10 – Experimental aspects of NMR

1. The NMR tube

2. NMR solvents

3. The sample

4.How much time does an NMR experiment require?

5.Line shape

6.NMR coils

7.Shimming an NMR Magnet (Pearson)

8.Signal-to-Noise

9.Signal processing

10-Experimental aspects of NMR (Dayrit) 2

• The quality of the NMR tube is very important.

• The NMR tube affects the resolution of the NMR spectrum and this becomes more important as one uses higher magnetic field instruments and carries out long accumulation experiments.

• Select appropriate NMR tube

• Clean and scratch-free.

1. The NMR tube

Experimental Aspects of NMR 3

A. Standard 5mm tube B&C. 10 mm with microcell


Typical specifications and approximate prices for 5 and 10 mm NMR tubes.

NMR Tube Specifications Approx. Cost (P)

5 mm, for routine NMR analysis o.d.: 4.97 + (0.00 - 0.13) i.d.: 4.20 + (0.00 - 0.13) concentricity: 51

700

5 mm, for high resolution, long acquisition and multidimensional experiments

o.d.: 4.97 + (0.00 - 0.13) i.d.: 4.20 + (0.00 - 0.13) concentricity: 13

850

10 mm, for high resolution o.d.: 10.00 + (0.00 - 0.13) i.d.: 8.16 + (0.00 - 0.13) concentricity: 38

2,100


2. NMR Solvents

The solvent is the most abundant substance inside the NMR tube!

The NMR solvent plays a very important role in determining the quality of the NMR spectrum. The NMR spectra of some compounds are solvent-dependent.

The most important functions of the NMR solvent are:

• to dissolve the sample adequately and appropriately

• to provide the deuterium nuclei for the lock signal.

mitsopoulos.com


Key considerations in NMR solvents

1. % Deuterium content

2. Residual moisture

3. Position of 13C

4. Solvent effects

5. Use of NMR solvent as reference

6. Melting point and boiling point.

7. Solvent viscosity

8. Solvent impurities

9. Sample recovery

10. Cost


1. % Deuterium. 2H is used as the lock nucleus. However, deuterated solvents normally contain residual 1H.

• Signal overlap?

• % Deuteration should be as high as possible. For example, a 99.5% D2O contains ~0.5% 1H (~ 0.6 mM 1H). This

seemingly small amount of 1H can be enough to dominate the spectrum of a dilute sample (eg, 5 mg of sample with MW~500).

• Use of residual 1H as internal 1H reference (instead of TMS).


2. Residual moisture. Dry solvents readily pick up moisture in the air and this gives rise to a water peak in the spectrum. Although this problem is most serious for hydroscopic solvents, even “hydrophobic” solvents pick up moisture.

• For solvents with exchangeable protons (in particular, water and methanol), moisture exchanges the -OD in the solvent for -OH.

• Moisture in most solvents give rise to a water or -OH peak at about 4.5 but in CDCl3 this appears at about 1.6.


3. Position of 13C . Standard NMR solvents contain 13C in natural abundance (1.1%).

• The 13C peak(s) from the NMR solvent can overlap with the 13C signals from the sample, and if the sample is dilute, the solvent peaks can overwhelm those from the sample.

• The 13C peak(s) from the NMR solvent can be used as a secondary reference (to replace TMS).

u-of-o-nmr-facility.blogspot.com


4. Solvent effects. The NMR spectrum is solvent-dependent (solubility and inter-molecular interactions). There may be differences in the spectra of the same compound taken in different solvents. Some specific examples:

a. Ring current effects in aromatic solvents: benzene, toluene, pyridine;

b. hydrogen bonding: water, methanol, acetone, pyridine, dioxane

c. Hydrogen exchange between the solvent and the sample: water, methanol.


5. Use of NMR solvent as reference. Although it is common practice to reference the NMR spectrum using a solvent peak, this is not an absolute reference.

• There may be slight shifts in the “standard” chemical shift values, specially if the solvent chemical shift is temperature-dependent.

•A number of references give different chemical shift values for the same solvent.


6. Melting point and boiling point. These factors are important when selecting a solvent for variable temperature work.

7. Solvent viscosity. Solvent viscosity affects T2.

• Solvents of low viscosity give sharper lock signals and better homogeneity. The net effect is better resolution.

• Deuterated solvents are generally more viscous than the normal solvent.

8. Solvent impurities. The presence of solvent impurities can sometimes interfere or cause confusion specially in samples of very low concentration.


9. Sample recovery. • Consider boiling point of NMR solvent.• Avoid leaving sample in NMR tube for a long time.

10. Cost. The prices of NMR solvents vary widely depending on the solvent itself and the % deuterium enrichment.

• For standard high magnetic field experiments, solvents with enrichments of > 99.5% should be used. .

• A standard 5 mm NMR tube requires at least 0.5 mL of solvent.


Properties of common NMR solvents. The cost quoted is the average for deuteration of >99%. Values enclosed in (x) are not suitable as reference. Alternative values from other literature sources are given. The chemical shifts of solvents may vary with experimental conditions.

NMR Solvent mp ~ bp (C)

Residual 1H ,

13C, Cost (P/0.5mL)

Acetic Acid-d4 17 ~ 118 2.03 (11.53)

20.0 (178.4)

Acetone-d6 -94 ~ 57 2.05 30.3 / 29.8 (206.0)

65

Acetonitrile-d3 -45 ~ 82 1.95 1.3; 118.2 120

Benzene-d6 5 ~ 80 7.2 / 7.16 128.0 80

Chloroform-d -64 ~ 62 7.24 / 7.26 / 7.27

77.0 9

Deuterium oxide, D2O 3.8 ~ 101 (4.7) - 40

DMSO-d6 18 ~ 189 2.50 39.5 7


Properties of common NMR solvents. The cost quoted is the average for deuteration of >99%. Values enclosed in (x) are not suitable as reference. Alternative values from other literature sources are given. The chemical shifts of solvents may vary with experimental conditions.

NMR Solvent mp ~ bp (C)

Residual 1H ,

13C, Cost (P/0.5mL)

Methanol-d4 -98 ~ 65 3.31 49.8 / 49.0 230

Pyridine-d5 -42 ~ 114 8.71 (); 7.55 (); 7.19 ()

149.9 (); 135.5 (); 123.5 ()

220

Toluene-d8 -93 ~ 110 7.09 (o); 7.00 (m); 6.98 (p);

2.09

137.1 (C1); 128.9 (o); 128.0 (m);

125.2 (p); 20.4

190


3. The Sample

The main concerns with regards the sample are: purity, quantity and selectivity.

•Purity: For structural elucidation, purity should be 98%.

•Quantity: NMR is a insensitive technique. For structural elucidation of unknown with MW ~ 400, recommended minimum amount is 4-5 mg.

• Selectivity: One can select particular nucleus and not observe other interfering nuclei. For example, if one carries out a 31P NMR in a biological sample, only P-containing compounds will be detected.


4. How much time does an NMR experiment require?

The time required for a NMR experimental depends on the type of pulse sequence and the amount of sample.

• In general, a sample of ca. 10 mg of a compound of MW 250, will require 1 scan for a standard 1H NMR and 64 - 128 scans for a standard 13C NMR.

•As the amount of sample decreases, the number of scans required increases by a factor of (1/x)2 where x is the relative amount of sample. For example, a 5 mg sample of the same MW may require 16 scans for a 1H spectrum and over 1000 scans for a 13C spectrum.


Typical accumulation times for a number of standard NMR experiments. Pulse sequence Number of

Scans Accumulation time

(hr : min) Conditions

non 1 8 32

14 s 1 min 4 min

cosy 4 8 64

0 : 18 0 : 35 4 : 40

CLPNT = 256

bcm 64 128

1000 10,000

4 min 7 min 0 : 50 8 : 20

dept 64 128 512

3 min 6 min 0 : 24

chshf 16 64

1 : 26 5 : 41

CLPNT = 256

hmqc 16 64

1 : 15 5 : 00

CLPNT = 256

hmbc 16 64

1 : 32 6 : 10

CLPNT = 256


5. Line shape

• Resolution refers to the extent of the separation of peaks, e.g., 0.1 Hz resolution. An alternative to measuring peak separation is the measurement of a related property-- line shape.

• A well-defined singlet in the spectrum is selected and its width at half-height (½, in Hz) is measured.

This yields information on the relaxation time, T2, which is an

indication of homogeneity.


• The ideal NMR line shape is Lorentzian because it arises from the Fourier transformation of an FID which decays exponentially. The exponential decay is typical of samples in the liquid state and is characteristic of damped oscillatory motion. The alternative line shape is Gaussian and is characteristic of rapidly decaying signals. Lorentzian lines are narrower around mid-height and are taller than Gaussian lines.

Lorentzian Gaussian


(from: JEOL)

SCM coils immersed in liq. He (4 K).

Outer insulated chamber immersed in liq. N2 (77 K).

Superconducting coils

Room temp. shim coils

Bore

Insert probe here

6. NMR coils


NMR tube

Spinner

Probe

Shim coils

Probe (without cover)

(web.mit.edu)

The NMR tube is placed in the center of 2 RF

coils (HF and LF channels). The inner

and outer coils transmit RF to the sample and detect the RF signal

from the sample.

(from: Jacobsen, 2007)

Helmholtz (“saddle”) RF coil design

u-of-o-nmr-facility.blogspot.com


• The variation of magnetic field with position is a GRADIENT. Because the frequency of the NMR signal is directly proportional to the strength of the magnetic field, the NMR signal will broaden if there are variations in the strength of the magnetic field.

• To obtain a sharp proton peak of 0.4 Hz at 400 MHz, the field felt by the sample must vary by less than (0.4 Hz / 400,000,000 Hz) or one part in 109. This is affected by the diamagnetic susceptibility of the solvent. The homogeneity of the field is affected by the quality of the NMR tube, temperature gradients, suspended particles, paramagnetic compounds, etc.

• To obtain a homogeneous magnetic field over the sample, SHIM COILS are placed in the vicinity of the sample and SHIM CURRENTS are adjusted to create gradients of the desired strength so as to correct inhomogeneous gradients in the NMR sample.

7.


(from: Jacobsen, 2007)

Shimming


8. Signal-to-Noise• “Noise” is defined as random (and unwanted) electrical or

electromagnetic energy that interferes with the observation of the signals and data from the sample.

• S/N (number of scans)½

• To double S/N, number of scans must be increased by 22 =4 times.

• In NMR, noise is added to the FID from a number of sources: • Stationary 'receiver noise' which originates both in the sample and

in the receiver system and determines the achievable signal-to-noise ratio.

• Random fluctuations of the main magnetic field (called “field noise”)

• Random fluctuations of magnetic field inhomogeneity across the sample (field-inhomogeneity noise or field-gradient noise).


Typical sources of magnetic-field instabilities (1)

• Intrinsic (type A)• Electronic noise in field stabilizers (shim coils) and lock systems.

• Environmental (type A)• Stray alternating fields from mains power wiring, both external

and internal to the instrument. • Stray alternating fields from magnetic devices such as AC power

transformers (including those located within the instrument's own power supplies).

• Motion induced (type B)• Sample spinning; sample movement induced by environmental

vibrations (pumps, acoustic waves, floor tremble, gas flows, etc.)

(from: Sykora, F”ield noise effects on NMR signals: FID's and 1D spectra” http://www.ebyte.it/library/docs/nmr06a/NMR_FieldNoise_Fid.html)


Typical sources of magnetic-field instabilities (2)

• It is impossible to completely suppress receiver noise and field noise. In high-resolution NMR spectroscopy (HRNMR):the resulting artifacts include:

• Rotational sidebands: Sidebands at integral multiples of the mains frequency.

• Broadening of spectral peaks during repeated-scans averaging.• Reduced efficiency of noise suppression by repeated-scans averaging.• t1-noise in 2D spectra

(from: Sykora, F”ield noise effects on NMR signals: FID's and 1D spectra” http://www.ebyte.it/library/docs/nmr06a/NMR_FieldNoise_Fid.html)


8. Signal Processing

noisesignal + noise

time, s


(use matched LB = digital resolution)

LB = line broadening


Zero filling


Window functions


Starting with an FID (raw time-domain data), we need to carry out the following operations:(save raw data; create copy; zero-fill copy)(a) Multiply the FID by a multiplier or window function.(b) Fourier transform the time domain data to obtain a frequency domain spectrum.(c) Correct for phase errors by adjusting the phase.(d) Find a reference standard peak and set its chemical shift to the reference value inparts per million.(e) Expand the desired region of the full spectral window to be plotted.(f) Plot the spectrum.

(From: Jacobsen, 2007)


In addition, there are several optional operations we might want to perform:(g) Add zeroes to the end of the FID to increase digital resolution (“zero fill”).(h) Flatten the spectrum baseline (average of noise regions where there are no peaks).(i) Measure the area under individual peaks by integration.(j) Plot the chemical shift values of peaks on the spectrum, or print a separate list.(k) List the acquisition and processing parameters on the spectrum or in a printout.(l) Expand and plot smaller regions of the spectral window.

(From: Jacobsen, 2007)

10 – experimental aspects of nmr 1.the nmr tube 2.nmr solvents 3.the sample 4.how much time does...

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