Nuclear Magnetic Resonance Spectroscopy

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Table of Contents

Introducing
Project goals
Not all nuclei possess the property called spin
Abstract
Resonance Instruments MARAN Ultra spectrometer
Example
Conclusion
Bibiography

Introducing

One of the most useful components in NMR spectroscopy the application of the peak area for the quantitative analysis. Although the use of proton NMR has been applied for the determination of trace components such as the residual hydrogen in D2

O, the mixtures of dinitrotoluene, and the mixtures of drugs, its great merits have been ignored in the field of quantitative analysis because NMR method is inherently less sensitive than many other spectroscopic methods. The quantitative analysis using NMR spectroscopy, however, has been increased significantly during the last few years because of the development of new techniques including high-field NMR. Its applications are generally limited to the use of hydrogen nucleus.

In our previous study, we reported that the accuracy and precision of the P-NMR methods are either comparable or superior to that of the conventional method (ASTM D 515).

In a continuing study of heteronuclear NMR application in quantitative analysis, we attempted the analysis of sodium (23Na) in solution. The sodium is one of the most abundant and the most frequently analyzed elements and its analysis methods are well established. In modern laboratories, the concentration of sodium in the solution is generally analyzed by AAS or ICP methods using an external standard.

These classical methods, however, could have factors, which disturb correct analysis, such as spectral, physical, chemical, and ionization interference. Thus, these matrix effects, which could repress or enhance the radiation of the element to be determined to derive considerable error, are well documented.

Therefore, if the composition of sample is known, the error can be compensated by using calibrating solution through the time-consuming procedures. On the other hand, the unknown matrix sample could not be calibrated by this procedure and could produce a result containing significant error. In this study we report a new preferred alternative NMR method for the quantitative analysis of sodium in solution. Because of the natural abundance (100%) and quadrupole relaxation (I=3/2), sodium is a very amenable nucleus for NMR study.

Two case studies, which are the most extreme cases containing cobalt(Co) and silver(Ag) ion in the sample, are employed to show that this NMR method is comparable or superior to that of the conventional flame or ICP method in the analysis of sodium. The representative 23 Na-NMR spectra obtained on a Bruker DRX-300 spectrometer at 79.3 MHz equipped with 10 mm broadband probe are shown in the Figure 1. A single peak (δ 3.2 ppm) shown with same intensity in all spectrum (a-g) is the peak of the 50 µg/mL sodium which is contained with 7.7 mM of Dy(TTHA) 3−at a 5 mm NMR tube and placed co-axially in a 10 mm NMR tube containing various concentrations of sodium. The complex of dysprosium used here was a shift reagent and was made by the literature procedure.

This complex is widely used as a reagent to differentiate the content of intracellular and extracellular sodium in in vivo study.

The spectra were collected with 0.01 s relaxation delay, 0.9 s acquisition time, and 4 k data points over a 1,500 Hz spectral width using a 90 o pulse. Because of the fast pulsing due to the quadrupole relaxation of sodium, the total acquisition time of each experiment was only about 10-60 min to get reasonable signal-to-noise ratio depending on the sample concentration. The Na integration ratio of individual samples to the 50, 150, and 250 µg/mL sodium with shift reagent as an external standard showed the excellent linear regression coefficient (R2 = 0.9998) in the range of 0.1 to 500 µg/mL sodium concentration (Figure 2).

The different slope of calibration line for sodium concentration by the integration ratio of sample and reference solution clearly indicates that Figure 1. A stack plot of 23 Na-NMR spectra at various concentrations with 50 µg/mL Na and 7.7 mM Dy(TTHA) as reference. The concentrations (a-g) are 0.2, 1.0, 10, 20, 50, 200, and 500 µg/mL sodium.

Project goals

Ultraviolet/Visible

Nuclear Magnetic Resonance – Proton

Identify the number of different kinds of protons in an unknown compound from its proton NMR spectrum and assign those different kinds of protons to likely chemical environments.
Identify the relative numbers of each of the different kind of protons in an unknown, using integration curves (or shrewd guesswork) and/or the formula of the unknown.
Identify the presence (and number) or absence of neighboring protons for each different kind of proton (coupling).
Distinguish spectral information from solvent and standard reference NMR signals.
Predict the appearance of an NMR spectrum from its structure (number, size and multiplicity of absorptions).
Use the proton NMR spectrum to assist in determining the structure of an unknown compound.
Use and identify the following terms: coupling, chemical shift, integration, intensity, TMS, doublet, triplet, quartet, multiplet, noise.
Explain the reason for doublet, triplet and quartet coupling patterns, both multiplicity and intensity.

Nuclear Magnetic Resonance – Carbon-13

Identify the number of different kinds of carbons in an unknown compound from its proton noise-decoupled 13C NMR spectrum and assign those different carbons to likely chemical environments.
Distinguish solvent and reference NMR signals from that of the sample.
Use the proton noise-decoupled 13C NMR spectrum to assist in the determination of the structure of an unknown compound.
Predict the appearance of the proton noise-decoupled 13C NMR spectrum from the structure of a compound.
Use the relative intensity of the signal to provide structural information about an unknown compound (why do NMR spectra seldom have peaks of equal intensity for equal numbers of carbons?)
Use the proton-coupled or off-resonance decoupled spectrum to assist in the determination of the structure of an unknown.
Explain some advantages of (pulsed) Fourier transform spectroscopy has over scanning spectroscopy (also applies to IR and proton NMR). Explain why 13C spectra take longer to obtain than 1H spectra.

Mass Spectrometry

Using a low resolution mass spectrum, identify the peak most likely to be the molecular ion.
Explain and use the difference between the mass of the molecular ion and the molecular weight calculated from the periodic table.
Recognize the presence of Cl and Br in a compound from its mass spectrum.
Use several characteristic daughter ions, e.g. 77, 91, to determine features of the structure of a compound from its mass spectrum.
Use the mass spectrum of an unknown compound to assist in determining its structure.
Use and identify the following terms: molecular ion, daughter ion, base peak, fragmentation; describe the fate of a molecule analyzed by mass spectrometry.

NMR

Nuclear magnetic resonance, or NMR as it is abbreviated by scientists, is a phenomenon that occurs when the nuclei of certain atoms are immersed in a static magnetic field and exposed to a second oscillating magnetic field. Some nuclei experience this phenomenon, and others do not, dependent upon whether they possess a property called spin.

Most of the matter you can examine with NMR is composed of molecules. Molecules are composed of atoms. Here are a few water molecules. Each water molecule has one oxygen and two hydrogen atoms. If we zoom into one of the hydrogens past the electron cloud we see a nucleus composed of a single proton. The proton possesses a property called spin which:

can be thought of as a small magnetic field, and
will cause the nucleus to produce an NMR signal.

Not all nuclei possess the property called spin

The versatility of NMR makes it pervasive in the sciences. Scientists and students are discovering that knowledge of the science and technology of NMR is essential for applying, as well as developing, new applications for it. Unfortunately many of the dynamic concepts of NMR spectroscopy are difficult for the novice to understand when static diagrams in hard copy texts are used. The chapters in this hypertext book on NMR are designed in such a way to incorporate both static and dynamic figures with hypertext.

This book presents a comprehensive picture of the basic principles necessary to begin using NMR spectroscopy, and it will provide you with an understanding of the principles of NMR from the microscopic, macroscopic, and system perspectives. (Joseph P. Hornak, Ph.D. Department of Chemistry, Rochester Institute of Technology, Rochester, NY 14623-5603).

Spectroscopy

Spectroscopy is the study of the interaction of electromagnetic radiation with matter. Nuclear magnetic resonance spectroscopy is the use of the NMR phenomenon to study physical, chemical, and biological properties of matter. As a consequence, NMR spectroscopy finds applications in several areas of science. NMR spectroscopy is routinely used by chemists to study chemical structure using simple one-dimensional techniques. Two-dimensional techniques are used to determine the structure of more complicated molecules. These techniques are replacing x-ray crystallography for the determination of protein structure. Time-domain NMR spectroscopic techniques are used to probe molecular dynamics in solutions. Solid-state NMR spectroscopy is used to determine the molecular structure of solids. Other scientists have developed NMR methods of measuring diffusion coefficients.

(Joseph P. Hornak, Ph.D. Department of Chemistry, Rochester Institute of Technology, Rochester, NY 14623-5603.)

Abstract

Magnetic resonance measurements on the electronic (free radicals) and nuclear (protons) moments in diphenylpicrylhydrazil were made using a superconducting tuned circuit as the sensing element. Two operating frequencies, 30 MHz and 0.95 MHz, were used in these experiments. Signal-to-noise improvements between 5,000 and 16,000 were obtained over the non-superconducting mode.

The quality factor of the superconducting resonant circuits ranged from a low of 10,000 to a high of 2,800,000. A major limitation to high-quality factor was the low-temperature loss properties of the dielectrics used in the construction of the tuned circuit. Three types of detection schemes were used:

Rollins type,
Rollins type with MOS Fet amplifier held at 4.2K., and
Robinson-type marginal oscillator.

Design criteria are presented to circumvent line broadening due to magnetic flux expulsion of the Meissner effect from the superconducting radiofrequency coil. (Paul J. Gemperline and David Littlejohn1965)

The method of quantitatively analyzing a mixture of two or more compounds through use of a pulsed FT NMR spectrometer having an adjustable variable gain, comprising the steps of:

forming a solution by dissolving said mixture in a solvent having a relaxation time substantially longer than the longest relaxation of any one of said compounds;

placing said solution in said spectrometer and pulsing said solution with a 180°-τ-90° pulse sequence where τ is equal to the time at which said mixture is at equilibrium while said solvent is only partially relaxed so as to minimize any free induction decay signal from said solvent following said 90° pulse, to generate a spectrum wherein any spectral peak of said mixture is unobstructed by any spectral peak of said solvent, said pulse sequence being applied at a given gain GA of said spectrometer;

dividing said spectrum into a number N of regions which number is at least as great as the number of compounds in said mixture and measuring the area Ai under each region i of said spectrum where i varies from 1 to N;

and solving a series of simultaneous equations of the form ##EQU5## where aij is the area of spectral region i produced by a known concentration of compound j due to a gain Gc on said spectrometer, and fj is the concentration of compound j to be found by solving said equations.

determining aij by operating said spectrometer at gain Gc to form spectra from solutions of each pure compound j dissolved in a solvent which does not contribute to the spectra, dividing each said spectra into a number of regions corresponding to those of said first mentioned spectrum, measuring the areas in each region of said spectra, and dividing such areas by the weight concentration of pure compounds j to obtain aij.

Gains Gc and GA are each maximized to produce a spectrum using the full dynamic range of said spectrometer.

The method of quantitatively analyzing the concentrations of compounds in a mixture through use of a pulsed FT NMR spectrometer having an adjustable gain, comprising the steps of:

preparing a series of separate solutions each with a known concentration of a pure compound and using a solvent that will produce a spectral peak free from interfering with any peak due to the compounds dissolved thereby;
operating said spectrometer to generate a series of spectra, one for each pure compound, at a gain Gc ;
dividing each spectra into the same number of regions which number is as great as the number of compounds in said mixture;
determining in each region the area per unit concentration of each compound;
preparing a solution of said mixture in a solvent having a relaxation time at least 5 times longer than the longest relaxation time of any one of said compounds;
operating said spectrometer at a gain GA by using an inversion recovery pulse sequence so that said solution of said mixture is pulsed at a point in time where said solute is at equilibrium while said solvent is only partially recovered from being inverted, to produce a mixture spectrum;
dividing said spectrum into regions corresponding to said regions of said spectra and measuring the areas of said mixture spectrum in each region;
and determining the respective concentrations of each compound of said mixture by determining how much each compound contributed to the respective areas in each mixture region using said area per unit concentration and equating the different gains GA and Gc.

The last-mentioned steps comprise solving a series of simultaneous equations of the form ##EQU6## where aij equals the area per unit concentration

Ai is the area under the mixture spectrum in region i

N is the numer of compounds in the mixture and

Fj is the concentration of compound j to be determined. (Colin A. McGill 1980)

Quantitative analysis of process NMR signals in the time domain

Process NMR

Low-resolution NMR has been used to determine moisture and fat content
However, chemical shift information is required to obtain chemical composition
High-field NMR spectrometer not suited to the process environment
Use low-field NMR spectrometer employing a permanent magnet which provides medium resolution

NMR spectrometer

Resonance Instruments MARAN Ultra
1H, 19F, 31P
Permanent magnet (29 MHz for 1H)
Small, robust instrument (53 x 50 x 30 cm3)
Suitable for on-line and at-line measurements
Additional features:
- shim coils.
- lock channel.

Resonance Instruments MARAN Ultra spectrometer

1H NMR spectrum of a sample from a benzene production process

Quantitative analysis

Overlapping signals
multivariate, e.g. PLS, analysis of spectra
Construction of calibration model
reference technique
simulate samples
Validation and testing of model
Model maintenance and update

Processing of NMR signals

Data analysis – Analysis of FIDs.

Analysis of FIDs

Eliminate data processing steps that are difficult to automate, e.g. phasing
Potential for model-free analysis
Methods investigated:
- continuous wavelet transform (CWT)
- modification of generalised rank annihilation method (FID-GRAM)
- modification of direct exponential curve resolution algorithm (FID-DECRA)

Fid-Decra

Construction of Hankel matrix, H, from FID
Create 2 sub matrices, H1 and H2, from H
Obtain individual components from solution to generalized eigenproblem
Calculate amplitude, area and T2 for resolved signals

Example

Applications of Fid-Decra

Quality control
determination of ethoxy chain length in nonyl phenol ethoxylates (1H NMR)
Reaction monitoring
Dehydroxylation of tetrafluorohydroquinone (19F NMR)

Determination of ethoxy chain length in nonyl phenol ethoxylates Magnitude spectrum of FID-DECRA resolved components FID-DECRA results.

Conclusions – nonyl phenol ethoxylates

Results obtained using FID-DECRA comparable to those obtained from univariate analysis of spectral data
However, with FID-DECRA the FID is analyzed directly and no phase correction is required Þ could be automated

Dehydroxylation of tetrafluorohydroquinone 19F NMR spectrum of mixture (with 10 Hz line broadening) Magnitude spectrum of FID-DECRA resolved components TFHQ concentration v FID-DECRA area FID-DECRA v PLS Conclusions – fluorocarbons.

Possible to analyze quantitatively 19F NMR FIDs using FID-DECRA with a single calibration sample
Accuracy and precision of FID analysis using FID-DECRA (1 calibration sample) comparable to that of spectral analysis using PLS (10 calibration samples)

Overall conclusions

Quantitative results can be obtained from a single FID using FID-DECRA
No phase correction needed
Insensitive to solvent effects
FID-DECRA analysis could be automated Þ useful in process NMR spectrometry

Acknowledgements

Resonance Instruments.
ICI.

(Alison Nordon,. Colin A. McGill,. Paul J. Gemperline and David Littlejohn 1975).

Conclusion

One of the objects of the invention is to provide a method for making an accurate quantitative analysis of a solution in which the solvent produces a strong NMR spectral peak which may dominate, hide or be close to a solute peak, which method involves the use of an NMR spectrometer having a variable gain feature and the capability of conducting experiments with variable pulse sequences.

Another object is to provide an NMR quantitative analytical technique using a pulse modulated Fourier transform spectrometer to perform an inversion recovery experiment so as to produce a spectrum having a minimized solvent peak.

Another object is to provide an NMR analytical technique in which the areas under any solute peaks can be accurately measured by producing a spectrum at a relatively high gain setting of an NMR spectrometer.

Still another object is to provide an NMR analytical technique in which an inversion recovery Fourier transform NMR experiment can be used to produce a quantitative analysis of the components of a solution.

Briefly, the manner in which these and other objects of the invention are achieved is to first produce a frequency domain spectrum of the solution in which spectrum the solvent peak produces a maximum intensity at the given gain setting which will not saturate the detection system of the spectrometer and wherein the solvent peak may hide or be spaced relatively close to one or more solute peaks and wherein the relative intensities of the solute peaks are small compared to that of the solvent peaks.

Next, a second spectrum is obtained by exciting the sample with a 180° pulse followed after a given period of time by a 90° pulse, the period of time being chosen so that the solvent has not completely relaxed while the solute components have relaxed so that the resultant spectrum does not include any significant peaks due to the solvent. Such measurement can be made at increased gain settings on the spectrometer to increase the areas and hence increase the accuracy of making measurements of the relative areas. Next, the relative areas under the different component peaks are measured or determined.

Lastly, the percentage weight composition is calculated by the simultaneous solution of equations in which the relative weight percentages are proportional to the component areas of the curves and to the gain settings of the spectrometer. (Alison Nordon 1970)

Wrote in recognition of the following writers:

Alison Nordon, Paul J. Gemperline.
Colin A. McGill.
Paul J. Gemperline.
Joseph P. Hornak, Ph.D.
David Littlejohn.

Bibiography

Publications – Original Research

Pomerat, C.M., Rounds, D.E., Raiborn, C.W. and Pollard, T.D. (1964) Observations on newborn ratdorsal root ganglia in vitro following gamma irradiation. In: “Response of the nervous system to ionizingirradiation” (Haley and Snider, eds.), Little, Brown and Company, pp. 175-200.
Ito, S., Shihman Chang, R. and Pollard, T.D. (1969) Cytoplasmic distribution of DNA in a strain of Hartmannellid amoeba. J. Protozool. 16:638-645.
Pollard, T.D., Shelton, E., Weihing, R.R. and Korn, E.D. (1970) Ultrastructural characterization of F-actin isolated from Acanthamoeba castellanii and identification of cytoplasmic filaments as F-actin by reaction with rabbit muscle heavy meromyosin. J. Mol. Biol. 51:91-97.
Pollard, T.D. and Ito, S. (1970) Cytoplasmic filaments of Amoeba proteus. I. The role of filaments inconsistency changes and movements. J. Cell Biol. 46:267-289.
Pollard, T.D. and Weiss, I.W. (1970) Acute tubular necrosis in a patient with march hemoglobinuria. New Eng. J. Med. 283:803.
Pollard, T.D. and Korn, E.D. (1971) Filaments of Amoeba proteus. II. Binding of heavy meromyosin by thin filaments in motile cytoplasmic extracts. J. Cell Biol. 48:216-219.
Stossel, T.P., Pollard, T.D., Mason, R.J. and Vaughan, M. (1971) Isolation and properties of phagocytic vesicles from polymorphonuclear leukocytes. J. Clin. Invest. 50:1745-1757.
Adelstein, R.S., Pollard, T.D. and Kuehl, W.M. (1971) Isolation and characterization of myosin and two myosin fragments from human blood platelets. Proc. Natl. Acad. Sci. USA 68:2703-2707.
Stossel, T.P., Mason, R.J., Pollard, T.D. and Vaughan, M. (1972) Isolation and properties of phagocytic vesicles. II. Alveolar macrophages. J. Clin. Invest. 50:605-614.
Adelstein, R.S., Conti, M.A., Johnson, G.S., Pastan, I. and Pollard, T.D. (1972) Isolation and characterization of myosin from cloned mouse fibroblasts. Proc. Natl. Acad. Sci. USA 69:3693-3697.
Pollard, T.D. and Korn, E.D. (1973) The contractile proteins of Acanthamoeba castellanii. Cold Spring Harbor Symposium on Quantitative Biology 37:573-583.
Pollard, T.D. and Korn, E.D. (1973) Electron microscopic identification of actin-associated with isolated amoeba membranes. J. Biol. Chem. 248:448-450.
Pollard, T.D., Eisenberg, E., Korn, E.D. and Kielley, W.W. (1973) Inhibition of Mg++‑ATPase of actin-activated Acanthamoeba myosin by muscle troponin tropomyosin: implications for the mechanism of control of amoeba motility and muscle contraction. Biochem. Biophys. Res. Comm. 51:693-698.
Pollard, T.D. and Korn, E.D. (1973) Acanthamoeba myosin. I. Isolation from Acanthamoeba castellanii of an enzyme similar to muscle myosin. J. Biol. Chem. 248:4682-4690.
Pollard, T.D. and Korn, E.D. (1973) Acanthamoeba myosin. II. Interaction with actin and with a new cofactor protein required for actin activation of Mg++ATPase activity. J. Biol. Chem. 248:4691-4697.

Last Updated: January 24, 2007

Internet Resources

Pollard, T.D. (2004) John Heuser’s contributions to the visualization of the actin cytoskeleton by electron microscopy. Eur. J. Cell Biol. 83:243-255. Web.

M D Harpen, J Powell Williams and J P Williams III 1987 Phys. Med. Biol. 32 421-430. Web.

Michael Shapiro. Novartis Institute for Biomedical Research, 556 Morris Avenue, Summit NJ 07901, USA. Web.

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