Spectroscopy Applications In The Food Industry Biology Essay

Nuclear Magnetic Resonance ( NMR ) is a robust non-selective analytical tool that allows you to determine molecular construction inclusive of comparative constellation, comparative every bit good as absolute concentrations, and even intermolecular interactions without the decay of the analyte. NMR spectrometry is utilized to analyze chemical make-up doing usage of basic unidimensional methods. Planar techniques are being used to infer the construction of more complex molecules. These sort of techniques are steadily replacing x-ray crystallography for the finding of protein construction. Time sphere NMR spectroscopic methods are being utilized to examine molecular kineticss in solutions. Solid province NMR spectrometry is being used to infer the molecular fundamental law of solids. Scientists have besides developed NMR methods for quantifying diffusion coefficients.

The flexibleness of NMR causes it to be invasive in the scientific disciplines. Research workers every bit good as pupils are happening out that acquaintance with the scientific discipline and engineering of NMR is necessary for using, in add-on to developing, advanced applications for it. NMR isA without a doubtA an imperativeA toolA for theA contemporaryA scientist. NMR techniquesA which were one time challengingA and specialized have becomeA everyday. Chemists, withA minimum understanding ofA NMR, A at this recent timeA are able toA obtainA 2-A and evenA three-dimensional spectra with aA few clicksA ofA theA button. CareA should beA taken, however, when usingA such ‘black box ‘ methods.A AlthoughA theA customaryA parametersA utilised inA the set-upA macros for the survey may good beA passable forA oneA sample, they may beA incorrect for another.A One individual falsely set parametric quantity can intend the difference between obtaining a dependable, accurate & A ; practical spectrum and acquiring a meaningless spectrum.

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2.0 HistoryC: UsersUserDesktopMcGill Logo.jpg

Isidor Rabi and co. were the first to detect the phenomenon of Nuclear Magnetic Resonance ( NMR ) utilizing molecular beams in the twelvemonth 1937. Isidor Rabi received the Nobel Prize in Physics in 1944 for this work.A Edward Mills Purcell, Pound and Torrey detected proton magnetic resonance in paraffin wax. Felix Bloch and Packard independently observed the phenomenon of NMR in H2O. Felix Bloch and Edward Mills Purcell were jointly awarded the Nobel Prize in Physics for their finds. After these finds, NMR emerged as a tool for dependable and precise measuring of magnetic minutes of the karyon and Earth ‘s magnetic field. During the following two decennaries following the find of NMR in majority stuffs, the basic constructs and theories underlying NMR parametric quantities, such as atomic quadrupole interactions, chemical displacements, relaxation rates, atomic spin-spin yokes, dipolar interactions, Nuclear Overhauser Nffect ( NOE ) and consequence of chemical exchange on line-shapes were established. The power of NMR in chemical research improved significantly during this period due to the assorted proficient & A ; technological developments. Some technological developments: usage of signal averaging to better signal to resound ( S/N ) ratio ; better investigation designs ; usage of field/frequency lock to stabilise magnetic Fieldss ; electronic shims to better field homogeneousness ; usage of dual resonance techniques & A ; development of magnets with higher and homogenous magnetic Fieldss. Spin-Echo ( SE ) by Hahn was one of the major finds during this period. It plays a important function both in Magnetic Resonance Imaging ( MRI ) & A ; Magnetic Resonance Spectroscopy ( MRS ) . The power of NMR spectrometry was extremely enhanced with the promotions in electronics and computing machine engineering ensuing NMR going an indispensable tool in chemical and physical scientific disciplines. Ernst and Anderson, in 1966 developed the technique of Fourier Transform ( FT ) spectrometry which was considered as a major discovery at that clip. They used short and powerful radiofrequency ( RF ) pulsation for excitement, attainment of the signal in time-domain, followed by Fourier Transformation of the acquired signal, which improved the sensitiveness of Nuclear Magnetic Resonance experiment several fold. This technique ( FT ) shortly displaced the antecedently employed slow transition uninterrupted moving ridge ( CW ) method which relies on altering the magnetic field to make resonance status. Ernst was awarded to the Nobel award in 1991 for his work in FT spectrometry. Promotions in computing machine package, hardware and magnet engineerings coupled with improved design of the Radio Frequency ( RF ) investigations opened new boundaries for NM R applications in biological scientific disciplines. Damadian in 1971 discovered that the 1H relaxation rates of H2O in normal and malignant cells are dissimilar. This determination uncovered the possibility of exerting this belongings for medical diagnosing. Paul Lauterbur utilised magnetic field gradients to turn up NMR signal information in infinite. His work showed that it can be used to bring forth images of objects. This work formed the footing for the Magnetic Resonance Imaging ( MR1 ) . Peter Mansfield engineered the technique of echo-planar imagination. Lauterbur and Mansfield received the Nobel Prize for their work in 2003.

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3.0 NMR Principles

3.1 Nuclear spin and the splitting of energy degrees in a magnetic field

Electrons, protons and neutrons can be conceived as whirling on their axes. In certain atoms, these spins are paired against each other ensuing in the absence of overall spin of the karyon of the atom. While in some atoms ( such asA 1H andA 13C ) the karyon does consist an overall spin. The regulations which determine the net spin of a karyon are as follows:

The karyon has no spin, if the figure of neutrons and the figure of protons is uneven.

The karyon has a half-integer spin ( ex. 1/2, 3/2, 5/2 ) , if the figure of neutronsA plusA the figure of protons is uneven.

The karyon has an whole number spin ( ex. 1, 2, 3 ) , if the figure of neutronsA andA the figure of protons are both uneven.

The overall spin designated as ‘I ‘ , is of import. Harmonizing to quantum mechanics a karyon of spinA IA will hold 2IA + 1 possible orientations. For illustration, a karyon with spin 1/2 will hold 2 possible orientations. These orientations are of equal energy in the absence of an external magnetic field. The energy degrees split in presence of a magnetic field. Each degree is assigned aA magnetic quantum number.http: //teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/nmrlev1.gif

In a karyon exposed to a magnetic field, thermodynamics decide the initial populations of the energy degrees, as explained by the Boltzmann distribution. This is important, and it signifies that the lower energy degree will incorporate somewhat more nuclei than the higher degree. These karyons can be excited to the upper degrees with electromagnetic radiation. The frequence of radiation required is determined by the difference in energy between the energy levels.C: UsersUserDesktopMcGill Logo.jpg

3.2 Calculating passage energy

The karyon has a positive charge and is whirling. This generates a little magnetic field. The nucleus therefore possesses a magnetic minute, A Aµ , which is relative to its spin, I.

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I“ is theA magnetogyric ratio and is a cardinal atomic changeless particular to a karyon. It varies organize one karyon to the other. hA is the Plancks invariable.

The energy of a peculiar energy degree is given by:

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Where, A BA is the strength of the magnetic fieldA at the karyon.

The passage energy which is the difference in energy between degrees can be found from:

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From the above look we can infer that, if the magnetic field, A B, is increased, so isA a?†E. It besides means that if a karyon has a comparatively big magnetogyric ratio, thenA a?†EA is correspondingly big.

i?·oA =A i?§BoA … ( 1 ) ( the Larmor frequence, in Hz ) A

3.3 The soaking up of radiation by a karyon in a magnetic fieldC: UsersUserDesktopMcGill Logo.jpghttp: //teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/precess.gif

In quantum mechanical footings, the atomic magnetic minute of a karyon can aline with an externally applied magnetic field of strengthA BA in merely 2I+1 ways, either re-inforcing or opposingA B. A karyon ( say, of spin 1/2 ) that is revolving on its axis in the magnetic field & A ; present in the decreased energy degree ( i.e. its magnetic minute wo n’t oppose the applied field ) , can aline with the field in TWO agencies. In the being of a magnetic field, this axis of rotary motion will precess round the magnetic field.

The frequence of precession is known as the Larmor frequence that is similar to the passage frequence. The possible energy of this precessing karyon is given by:

E =A -A mA B cosA Eµ

Where, A EµA is the angle between the way of the applied field and the axis of atomic rotary motion.

If energy is absorbed by the karyon, so the angle of precession, A Q, will alter. For a karyon of spin 1/2, soaking up of radiation “ somersaults ” the magnetic minute so that itA opposesA the applied field ( the higher energy province ) .

It is critical to understand that simply a bantam ratio of “ mark ” nuclei tend to be within the lower energy province ( which enables them to absorb radiation ) . There is likeliness that through exciting these karyons, the dwellers belonging to the greater and lesser energy degrees will stop up going indistinguishable. In the event that this takes topographic point, so there wo n’t be any extra soaking up of radiation. The spin system is saturated. The likeliness of impregnation implies that we need to be knowing of the relaxation processes which return karyons to the reduced energy states.http: //teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/nmrflip.gif

3.4 Relaxation processesC: UsersUserDesktopMcGill Logo.jpg

The karyon in the higher energy province de-excite to the lower energy province through either emanation of radiation or non-radiative relaxation procedures which are based on thermodynamic rules. Emission of radiation is decidedly invisible sing that the chance of re-emission of photon alterations with the regular hexahedron of frequence. At wireless frequences, re-emission is negligible. In non-radiative relaxation processes the NMR spectroscopists presumptively wish the relaxation rate is speedy, but non excessively rapid. In the event the relaxation rate is rapid, so impregnation is decreased. If the relaxation rate is excessively rapid, libe-broadening in the attendant NMR spectrum is noticed.

The two main relaxation procedures are:

Spin – lattice ( longitudinal ) relaxation

Spin – spin ( transverse ) relaxation

3.4.1 Spin – lattice relaxation.

In an NMR experiment karyon are in a sample. This sample is held in what is termed as the lattice. The nuclei present inside the lattice are in vibrational and rotational gesture, that generates an luxuriant magnetic field. This peculiar magnetic field attributable to motion of karyon within the lattice is referred to as lattice field. This specific lattice field pocesses several constituents. A few of these constituents will be indistinguishable in frequence every bit good as stage to the Larmor frequence of your karyon of involvement. Such constituents of the lattice field can interact with karyon in the upper energy province, and besides do them to lose energy ensuing in deexcitation to a lower province. The energy that the nucleus loses raises the figure of quiver and rotary motion inside the lattice which consequences in a small addition in the temperature of the sample ) .

The relaxation clip, T1 which is the typical life-time of a karyon in the upper energy province is reliant on the magnetogyric ratio of the karyon every bit good as the mobility of the lattice. As mobility improves, the vibrational and rotational frequences addition, doing it more likely for any component of the lattice field to hold the ability to interact with aroused karyon. However, at inordinate mobility rates, the chance of a constituent of the lattice field being able to interact with aroused nuclei reduces.

3.4.2 Spin – spin relaxation.

Spin – spin relaxatiion explains the interaction among neighboring karyons and similar precessional frequences yet typical magnetic quantum provinces. With this scenario, the karyon can trade quantum provinces ; a karyon from the lower energy degree will be excited, while the aroused karyon relaxes to the lesser energy province. There is n’t a attendant alteration in the populations of the energy provinces, nevertheless, the mean life-time of the karyon in an aroused province will cut down. This perchance can ensue in line-broadening.C: UsersUserDesktopMcGill Logo.jpg

3.5 Chemical shift/ Nuclear Shielding

The magnetic field at the karyon will non be equal to the administrated magnetic field ; negatrons around this peculiar karyon shield the karyon from the applied field. The difference between all of the applied magnetic field and the field at the karyon is termed the atomic shielding.

S-orbital negatrons have spherical symmetricalness and revolve inside the administrated field, which aproduces a magnetic field that will opposes the applied field. In other words the administrated field strength ought to be elevated for the karyon to absorb at its passage frequence. This sort of upfield displacement is normally termed diamagnetic displacement.

Electrons in p-orbitals do non hold any spherical symmetricalness. They generate resonably important magnetic Fieldss at the karyon, which give a lessened field displacement. This “ deshielding ” is termed paramagnetic shift.In proton ( 1H ) NMR, p-orbitals are absent, ensuing in simply a little scope of chemical displacement ( 10 ppm ) . the consequence of s-electrons on the chemical displacement can be determined by sing substituted methanes, CH3X. As Ten becomes more and more negatively charged and so the negatron denseness environing the protons reduces, and they resonate at reduced field strengths ( increasing dH values ) .http: //teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/espin.gif

Nuclear shielding is a map of the karyon and its peculiar environment. Its determined relation to a mention compound. For 1H NMR, the mention is normally tetramethylsilane, Si ( CH3 ) 4.

3.6 Spin – spin couplingC: UsersUserDesktopMcGill Logo.jpg

Protons have a magnetic field associated with them since they have a atomic spin. When placed in a magnetic field about half of the protons become aligned with the field and half become aligned against the field. It is the passage between these two provinces can be obderved in NMR.A .

In the simplest of instances a individual extremum for each type of proton is expected in a molecule. If a prton ( HA ) near another nonequivalent proton ( HB ) is considered, in half of the molecules the HAA proton will be next to an HBA aligned with the field and in the other half the HAA proton will be next to an HBA aligned against the field. Therefore, half the HA ‘s in the sample will experience a somewhat larger magnetic field than they would in the absence of HBA and half will experience a somewhat smaller magnetic field. Therefore, two soaking ups for the HAA proton are observed and frailty versa. This splitting of the HAA resonance into two extremums is termed “ spin-spin yoke ” or “ spin-spin splitting ” and the distance between the two extremums ( in Hz ) is called the “ yoke changeless ” ( represented as J ) . The spin-spin yoke is transmitted through the negatrons in the bonds and so depends on the bonding relationship between the two Hs.

A

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3.7Spectroscopy

The survey of the interaction of electromagnetic radiation with affair is termed as spectrometry. NMR spectrometry is the usage of the phenomenon, Nuclear Magnetic Resonance to understand physical, chemical, every bit good as biological belongingss associated with affair. In truth, NMR spectrometry finds intents in several countries of scientific discipline. NMR spectrometry is clip and once more utilised by chemists in order to analyze chemical make-up doing usage of basic unidimensional methods. Planar techniques are being used to infer the construction of more complex molecules. These sort of techniques are steadily replacing x-ray crystallography for the finding of protein construction.

4.0 NMR Spectroscopy: InstrumentationC: UsersUserDesktopMcGill Logo.jpg

The criterion set up of an NMR spectrometer is displayed in the figure below. The sample is placed in the magnetic field and aroused via pulsings in the wireless frequence input circuit. The realigned magnetic Fieldss generate a wireless signal in the end product circuit which can be utilised to bring forth the end product signal. Fourier analysis of the sophisticated end product provides the specific spectrum. The pulsation is replicated legion times to enable the signals to be recognized within the background noise.Description: Basic agreement of an NMR spectrometer

Two varities of NMR spectrometers are widely used, pulsed or Fourier-Transform ( FT-NMR ) spectrophotometers & A ; continuous-wave ( cw-NMR ) . Cw-NMR spectrometers have largely been replaced with pulsed FT-NMR instruments. Even so, as a consequence of lesser operating and up maintaining costs of cw

instruments, they are still being utilized for the 1H NMR spectrometry. Water can be used for chilling intents for electromagnets in low-resolution cw devices, whereas in FT-NMR spectrometers magnets are to be cooled utilizing liquid helium.Description: Contineous wave NMR

4.1 NMR – Continuous-wave Nuclear Magnetic Resonance

A continuous-wave NMR instrument comprises of the listed units: a magnet which to divide all the atomic spin energy provinces ; a lower limit of 2 radiofrequency channels, one of which furnishes the RF irradiation energy & A ; the other for field/frequency stabilisation and a sample investigation incorporating spirals which couple the sample with the RF field ; a sensor which processes the NMR signals ; a expanse generator which sweeps either the magnetic or RF field via the resonance frequences of the sample ; and a recording equipment which displays the spectrum.

The spectrum is normally scanned utilizing the existent field-sweep attack or the frequency-sweep method. While in the frequency-sweep process, the existent magnetic field is maintained changeless, which in bend maintains the atomic spin energy sums consistent, following of which the RF signal is swept in order to work out the frequences where energy is absorbed.While in field sweep process, the RF signal is unbroken consistent, so the magnetic field is swept, which frequently alters the energy degrees, to happen out the magnetic field strengths which will bring forth resonance at a fixed resonance frequency.C: UsersUserDesktopMcGill Logo.jpg

Description: Fourier transform NMR

4.2 NMR – Fourier-Transform Nuclear Magnetic Resonance

Fourier-Transform NMR spectrometers make usage of a pulsation of radiofrequency radiation to trip nucleiinside a magnetic field to assist toss straight into the greater-energy alliance. RF pulsation of length 1-10 Aµs is normally wide plenty to concurrently excite nucle in all of the local environments. The period of clip amongst pulsations T is by and large one to quite a few mere seconds. In the class of T, a time-domain RF signal referred to as the free initiation decay ( FID ) signal is released as karyon go back to their primary province. Description: Degree centigrade: UsersUserDesktop1.jpg

FID could be discovered utilizing a radio-receiver spiral which is perpendicular towards the inactive magnetic field. The FID transmittals can be digitized and saved in the computing machine system for informations analysis. Time-domain decay signals coming from many consecutive pulsations can be summed plus averaged to heighten the signal-to-noise per centum. This result is so altered to a frequency-domain signal by the manner of Fourier transmutation. The attendant frequency-domain end product is comparable to the spectrum generated by a scanning continuous-wave experiment.

5.0 NMR Spectroscopy: Applications in Food IndustryC: UsersUserDesktopMcGill Logo.jpg

High declaration NMR spectrometry is applied for the analysis of nutrient samples, biological tissues, every bit good as biofluids because it supplies complete and comprehensive information on all of the broad scope of constituents found in nutrient matrix in a individual experiment. It presents added advantages of being non-destructive, simplifying the sample readying and speed of analysis. On top of that, really small clip ( few proceedingss ) is required to obtain the NMR spectra, which in concert with mechanization enables analysis of legion samples with minimum operator input.

The application of NMR to the nutrient industry for analysis is fundamentally two types: ( 1 ) designation of distinguishable resonances and, hence, specific compounds, and ( 2 ) usage of chemometric profile analysis, in which the whole spectral profile is used without delegating peculiar resonances.

5.1 Oils & A ; Fats

5.1.1 Fatty Acid Profile

Fatty acid profiles influence the physical and chemical belongingss of oils, fats, and their derived functions. Gas chromatography ( CC ) is commonly employed for finding the fatty acid profile. Although GC provides dependable information about complete fatty acid profile, it does n’t supply any informations on fatty acerb distribution on the glycerin ground tackles, which is critical to find the functionality of the ingredient in processing. For illustration, for quality pie crusts or crescent rolls, the right type of fat is a necessity. The common unsaturated fatty acids, such as oleic, linoleic, and Iinolenic acids in an oil or fat sample, can be quantified utilizing 1H-NMR, by integrating of choice signals in the spectra. 13C-NMR analysis can be used to obtain the fatty acerb distribution on the glycerin ground tackles. There are two groups of resonances in the carbonyl part of the spectrum ; the first resonance is due to fatty acids in places 1,3, and the 2nd is from fatty acids in place 2 of the glycerin mediety.

5.1.2 Confirmation of Vegetable Oil Identity C: UsersUserDesktopMcGill Logo.jpg

The debasement of oils which are of high value with those of lesser value is an issue of commercial and economic importance. This is more prevailing with olive oil as it is expensive and has a high nutritionary value. Many surveies from Greece, Italy, and Spain which are the major olive oil-producing Mediterranean states, trade with acknowledging lower-value oils, such as hazelnut oil, used for loading olive oil. This job is complicated by the fact that the lower-value oils normally have fatty acid profiles similar to olive oil. ‘3C-NMR and ‘I-I-NMR spectroscopy are the methods used for analyzing potentially adulterated olive oil. For illustration, NMR is used in concurrence with multivariate statistical analyses of specific resonances in NMR spectra of olive oil diluted with hazelnut or sunflower oil. This method can be used to place the geographical beginning and assortment of the oil.

5.1.3 Monitoring of Oxidation

A important quality job in the nutrient industry is oxidization of vegetable oils and it frequently leads to farther impairment of the oil. The fatty acids, with bis-allylic methylene groups which are extremely unsaturated, are really susceptible to oxidization. Hydroperoxides and aldehydes which are the primary and secondary oxidization merchandises can be easy detected by ‘H-NMR analyses. 1H-NMR is utile for such analyses because the samples do non necessitate any extra interventions, such as derivatization, that could do debasement.

5.1.4 Solid Fat Content C: UsersUserDesktopMcGill Logo.jpg

A low-resolution pulsed NMR spectrometer can be used to observe the solid fat content ( SFC ) of a given sample. For illustration, the sum of triacylglycerols in an oil/fat at assorted temperatures can be determined utilizing NMR methodological analysis. This method utilizes the difference in relaxation times between solids and liquids. The NMR signal of the liquid fat is measured after a hold. The solid content is so estimated. Using Solid Fat Content ( SFC ) measurings, crystallisation mechanisms of fat blends besides can be studied.

5.2 Water

Glass transitionA is the reversible alteration inA amorphousA stuffs from a difficult and comparatively brickle province into a liquefied orA rubber-like province. It is a noteworthy belongings of nutrients, and the glass passage temperature ( Tg ) , which relies chiefly on H2O content, effects both the processing and the storage of nutrient merchandises. Tg can be obtained with an NMR province diagram. It is a curve which relates NMR relaxation clip to glaze passage temperature at assorted different wet contents. This information is important since processing and storage temperatures above Tg at any phase during production, distribution and ingestion of the merchandise is associated with fleet impairment. Spin-spin relaxation clip ( TZ ) is often employed as an indicant of proton mobility. The value of TZ differs above and below the Tg of a given merchandise. Though Differential Scanning Calorimeter ( DSC ) can be used for simple Tg analyses, NMR, with its ability to bring forth NMR province diagrams increases its value of for many applications.

5.3 Ingredient Assaies

The debasement of fruit juice is widespread and is non easy to observe by colour or gustatory sensation. For illustration, Citrus paradisi juice which is comparatively cheap can be blended with orange juice, but the presence of Citrus paradisi juice in a commercially sold orange juice merchandise presents some potentially unsafe wellness hazards for consumers with medical conditions. Juice signifier grape fruit has many coumarin-like flavonoids and other CYP450 inhibitors which are powerful and can negatively impact the metamorphosis of legion prescribed drugs. Hence, the sensing and bar of this sort of debasement is really important. Chemometric attacks based on NMR along with Independent Component Analysis, are now applied for the sensing intents. Specific selective parts of the 1H NMR spectra, which are recognized to consist characteristic flavonoid glycoside signals, are exactly analyzed in a comparatively short clip.

Distinguishing between newly squeezed juices and those manufactured from mush washes and subsequently added to fresh-squeezed orange juice to cut down industry costs is another issue of concern with respects to juice readying. ‘H NMR, in concurrence with Principal Component Analyses, can merely and exactly know apart the fresh-squeezed and pulp-wash orange juice.C: UsersUserDesktopMcGill Logo.jpg

Large transnational breweries use NMR methods to supervise batch-to-batch quality and production site differences in beer as they prepare their beers at assorted different geographic locations and require methods for quality control at an detailed molecular degree. NMR in combination with chief component analysis can be used to distinguish beer from different production sites based on lactic acid, dextran, adenosine, pyruvic acid, inosine, tyrosine, and uridine, 2-phenylethanol content. Manufacturers can place the production sites with greater variableness ( i.e. poorer quality control ) by quantifying these compounds.

Other manufacturers use NMR methods to better quality control in juice production, soft drink production, and vegetable oil fabrication. NMR methods besides are employed to supervise the quality of functional nutrients and neutraceuticals that are harvested from diverse geographic locations.

* Instumental Methods of Food Analysis 6th EditionC: UsersUserDesktopMcGill Logo.jpg

6.0 ReferencesC: UsersUserDesktopMcGill Logo.jpg

Books:

C. P. Slichter, “ Principles of Magnetic Resonance ” ( 3rd edn ) , Springer-Verlag, Berlin ( 1990 ) .

J. R. J. Pare , A J. M. R. Belanger, “ Instrumental methods in nutrient analysis ” ( 4th edn ) , Elsevier ( 1997 ) .

A N.S. Suzanne, “ Food Analysis ” ( 4th erectile dysfunction ) , Illus. , ( 2010 )

Ray Freeman, “ A short history of NMR ” ( Vol. 31 ) , No. 9, Khimiya Geterotsiklicheskikh Soedinenii ( 1995 )

R. K. Harris, “ Nuclear Magnetic Resonance Spectroscopy chemical View ” , Longman, London ( 1986 ) .

Webliography:

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hypertext transfer protocol: //www.chem.ucalgary.ca/courses/351/Carey/Ch13/ch13-nmr-1.html

hypertext transfer protocol: //www2.ups.edu/faculty/hanson/Spectroscopy/NMR/coupling.htm