View source for Physics:Isotopic analysis by nuclear magnetic resonance

'''Isotopic analysis by nuclear magnetic resonance''' allows the user to quantify with great precision the differences of isotopic contents on each site of a [[Physics:Molecule|molecule]] and thus to measure the specific natural [[Physics:Isotope fractionation|isotope fractionation]] for each site of this molecule. The '''SNIF-NMR''' analytical method was developed to detect the (over) sugaring of wine and enrichment of grape musts, and is mainly used to check the authenticity of foodstuffs (such as wines, [[Chemistry:Liquor|spirits]], fruit juice, [[Chemistry:Honey|honey]], [[Chemistry:Sugar|sugar]] and [[Chemistry:Vinegar|vinegar]]) and to control the naturality of some aromatic molecules (such as [[Chemistry:Vanillin|vanillin]], [[Chemistry:Benzaldehyde|benzaldehyde]], [[Chemistry:Raspberry ketone|raspberry ketone]] and [[Chemistry:Anethole|anethole]]). The SNIF-NMR method has been adopted by the [[Organization:International Organisation of Vine and Wine|International Organisation of Vine and Wine]] (OIV) and the [[Social:European Union|European Union]] as an official method for wine analysis. It is also an official method adopted by the Association Of Analytical Chemists (AOAC) for analysis of fruit juices, maple syrup, vanillin, and by the European Committee for Standardization (CEN) for vinegar.

== Background ==

* 1981: Invention of the SNIF-NMR method by Professor Gerard Martin, Maryvonne Martin and their team at the University of Nantes / CNRS <ref name="Eurofins Scientific">Eurofins Scientific – Worldwide laboratory testing services, “Eurofins Scientific - 1987 - 1997 - The Start-up Phase”, (consulted the 2nd of January 2014). http://www.eurofins.com/en/about-us/our-history/start-up-phase.aspx {{Webarchive|url=https://web.archive.org/web/20150210234100/http://www.eurofins.com/en/about-us/our-history/start-up-phase.aspx |date=2015-02-10 }}</ref>
* 1987: Creation of Eurofins Nantes Laboratories - specializing in wine analysis, and purchase of operating the CNRS patent rights<ref name="Eurofins Scientific"/> (this patent is now public and the name “SNIF-NMR” is now a registered [[Social:Trademark|trademark]]<ref name="INPI">INPI : Institut National de la Propriété Industrielle, "Bases de Données Marques", (consulted the 6th of January 2014). http://bases-marques.inpi.fr/</ref>
The OIV adopts it as an official method
* 1987-1990: Eurofins Laboratories apply the SNIF-NMR method to the analysis of fruit juices and certain natural [[Chemistry:Flavoring|flavors]]
* 1990: The SNIF-NMR method is recognized by the European Union as an official method for the analysis of wines<ref name="wine">Commission Regulation of the European communities, 1990. (EEC) n° 000/90: “determining community methods for the analysis of wine”. Brussels, Official Journal of the European communities, p.64-73</ref>

[[File:Implantation SNIF-NMR dans le monde.jpg|thumb|SNIF-NMR in the world]]
→ Implementation of the SNIF-NMR method for official laboratories in Europe
* 1990-1992: the method is tested on aromatic molecules
* 1996: The SNIF- NMR method is recognized in the United States by the AOAC for fruit juices<ref name="fruit juice">AOAC official Method 995.17, Beet Sugar in Fruit Juices, SNIF-NMR, AOAC International 1996</ref>

→ Implementation of the SNIF-NMR< method for official laboratories in US
* 2001: The SNIF-NMR< method is recognized by the AOAC for vanillin
* 2013: The SNIF-NMR method is recognized by the CEN for [[Chemistry:Acetic acid|acetic acid]]
→ Implementation of the SNIF-NMR method for official laboratories in Asia

== Principle ==

=== Isotopic distribution ===

[[File:Natural existence of hydrogen, carbon and oxygen.jpg|thumb|Figure 2]]
[[File:Figure 3 - Isotopic Fractionation Sources.JPG|thumb|Figure 3 - Isotopic Fractionation Sources]]

The atoms [[Chemistry:Hydrogen|hydrogen]], [[Chemistry:Oxygen|oxygen]], and [[Chemistry:Carbon|carbon]] co-exist naturally in specific proportions with their stable isotopes, 2H (or D), 18O and 13C respectively, in different proportions as shown in the figure 2 below.

The amount and distribution of the different isotopes in a molecule is influenced by: 
* Environmental (climatic and geographical) conditions - for natural products 
* Chemical or biochemical processes such as the photosynthetic metabolism in plants

This phenomenon is known as natural [[Physics:Isotope fractionation|isotopic fractionation]] (see Figure 3). The resulting isotopic fingerprint can provide information on the origin - botanical, synthetic, geographical - of the molecule or product.

=== General principle ===

The Principle of the SNIF-NMR is built on: “The Natural Isotopic Fractionation”. Routinely for food authenticity, two nuclei are used:
* The Hydrogen nuclei: <sup>2</sup>H-SNIF-NMR method was the initial application of SNIF-NMR, it measures the ratio of [[Physics:Deuterium|deuterium]]/hydrogen on each site of a sample molecule
* The Carbon nuclei: <sup>13</sup>C-SNIF-NMR method has opened new possibilities of analysis by SNIF-NMR (new molecules and new applications). This method will use the ratios of <sup>13</sup>C over <sup>12</sup>C on each site of a molecule

=== Steps of the method ===
[[File:Figure 5 -Steps of SNIF-NMR of ethanol - Official method.jpg|thumb|Figure 5 -Steps of SNIF-NMR of ethanol - Official method]]
The SNIF-NMR is applied on pure (or purified) molecules. Therefore, some preparation steps may be required in the lab before analysis.
For example, for the SNIF-NMR of ethanol, according to official methods:
* [[Biology:Fermentation|Fermentation]] (for fruit juice)
* Quantitative extraction of [[Chemistry:Ethanol|ethanol]] by [[Distillation|distillation]] 
* Standardized preparation of NMR samples
* NMR acquisition
* Interpretation of results and report on the authenticity

At each step of the SNIF-NMR analysis, efforts should be made to avoid parasite isotopic fractionation. Control measures such as determining the alcoholic strength of the intermediate products of the analysis (fermented juice or distillate) are performed on each sample.

=== Advantages of the method ===
[[File:Figure 6 - Principle of the IRMS.jpg|thumb|Figure 6 - Principle of the IRMS]]
The isotopic ratios of a molecule can also be determined by isotope ratio mass spectrometry (IRMS), sample quantity for IRMS is much lower than for NMR, and there is the possibility of coupling the mass spectrometer to a chromatographic system to enable on-line purification or analyses of several components of a complex mixture. However the sample is burnt after a physical transformation such as [[Physics:Combustion|combustion]] or [[Chemistry:Pyrolysis|pyrolysis]]. Therefore, it gives a mean value of the concentration of the isotope studied between all sites of the molecule.  IRMS is the official AOAC technique used for the average ratio <sup>13</sup>C/<sup>12</sup>C (or δ<sup>13</sup>C) of sugars or ethanol, and the official CEN and OIV method for the 18O/16O in [[Chemistry:Water|water]].

The SNIF-NMR method (Site-Specific Natural Isotope Fractionation studied by Nuclear Magnetic Resonance) is able to determine, to a high level of accuracy, the isotopic ratios for each of the sites of the molecule, which enables a better discrimination. For example, for ethanol (CH<sub>3</sub>CH<sub>2</sub>OH), the three ratios ((D/H)CH<sub>3</sub>, (D/H)CH<sub>2</sub> and (D/H)<sub>OH</sub>) can be obtained.

== Examples of applications of <sup>2</sup>H-SNIF-NMR ==
[[File:Figure 7 - Official Isotope method recognition for food application – 2013.jpg|thumb|Figure 7 - Official Isotope method recognition for food application – 2013]]

=== Application for fruit juice and maple syrup ===

AOAC Official Method for detecting the addition of sugar in a fruit juice<ref name="fruit juice"/> or in maple syrup. It is the only reliable method to detect addition of C3 sugar (ex: beet sugar).

=== NMR Spectrum (example of the <sup>2</sup>H-SNIF-NMR) ===
[[File:Figure 8 - 2H-NMR spectrum of ethanol.jpg|thumb|Figure 8 - 2H-NMR spectrum of ethanol]]
Ethanol molecules obtained after complete fermentation of the sugar coexists with 3 naturally monodeuterated isotopomers (CH<sub>2</sub>D-CH<sub>2</sub>-OH, CH<sub>3</sub>-CHDOH and-CH<sub>3</sub>-CH<sub>2</sub>OD). Their presence can then be quantified with relative precision.<ref name="plant analysis">G. Martin, M.L. Martin. Modern Methods of Plant Analysis: “The Site-Specific Natural Isotope Fractionation-NMR Method Applied to the Study of Wines”, edition: HF Linskens and JF Jackson Springer Verlag, Berlin, 1988, p. 258-275</ref>

On the following <sup>2</sup>H-NMR spectrum (Figure 8), a peak corresponds to one of the three observed isotopomers of ethanol. 
In the AOAC official method, the ratios of (D/H)CH<sub>3</sub> and (D/H)CH<sub>2</sub> are calculated by comparison with an Internal standard, [[Chemistry:Tetramethylurea|tetramethylurea]] (TMU), with a certified (D/H) value.

=== Interpretation of SNIF-NMR isotopic values ===
[[File:Figure 9 - Adulteration triangle - repartition of isotopic ratios on ethanol molecules.jpg|thumb|Figure 9 - Adulteration triangle: repartition of isotopic ratios on ethanol molecules]]
The following Figure 9 summarizes the principle of interpretation of:

* Results measured by IRMS (isotopic deviation of δ <sup>13</sup>C), which enables discrimination of plants according to their CO<sub>2</sub> photosynthetic metabolism (C4 like can or maize versus C3 like beet, orange or grape)
* With results measured by SNIF-NMR ((D / H)I ) that can differentiate the botanical origin of sugars within the same metabolic group (beet versus orange or grape)

Values obtained on a test sample are then compared with the values of authentic samples (Database).

=== Application for authenticity of wines ===
[[File:Figure 10 - Applications of SNIF-NMR and IRMS to wine authenticity.JPG|thumb|Figure 10 - Applications of SNIF-NMR and IRMS to wine authenticity]]
SNIF-NMR is the official method of the OIV to determine the authentication of wine origin. It is the only method to detect C3 sugar addition (like beet sugar).

The isotopic parameters of both water and ethanol are related to the [[Physics:Humidity|humidity]] and [[Physics:Temperature|temperature]] of the growing region of the [[Biology:Plant|plant]]. Therefore, considerations of meteorological data of the region and of the year help to make a diagnosis. In the case of wine and fruits, the isotopic parameters of ethanol have been shown to respond even to subtle environmental variations and they efficiently characterize the region of production,.<ref name="plant analysis" /><ref name="food chem">Martin GJ, Guillou C, Martin ML, Cabanis MT, Tep Y, Aerny J. J. Agric. Food Chem. 1988;36:316–22</ref>

Since 1991, an isotopic data bank is built in the Joint Research Centre of the European Commission (EC-JRC) concerning wines of all European members. The database contains several thousand entries for European wines <ref name="official journal">Official Journal of the European Communities. Off. J. Eur. Commun. 1991;L214:39–43</ref> and is maintained and updated every year. This database is accessible for all official public laboratories. Private companies involved in food and beverage controls have also collected authentic samples and built up specific data banks.<ref name="OIV">Guillou C, Jamin E, Martin GJ, Reniero F, Wittkowski R, Wood R. Bulletin OIV. 2001;74:26–36</ref>

Thus, by comparing the specific natural isotope fractionation corresponding to each site of a molecule of ethanol of wine with that of a molecule known and referenced in a database.  The geographical origin, botany and method of production of the ethanol molecule and thus the authenticity of wine can be checked.<ref name="natural factors">G. Martin, C. Guillou, Y.L. Martin, Natural Factors of Isotope Fractionation and the characterization of Wines”, Journal of agricultural and food chemistry, n°36, 1988, p. 316-322</ref>

=== Application for vinegar and acetic acid ===
[[File:Figure 11 - Application for vinegar.jpg|thumb|Figure 11 - Application for vinegar]]
The origins of vinegars obtained by bacterial or chemical oxidation of ethanol resulting from the fermentation of various sugars can be identified by the <sup>2</sup>H-SNIF-NMR. It allows to control the quality of vinegar and to determine if it comes from sugar cane, wine, malt, cider, and alcohol or from a chemical synthesis.<ref name="acetic acid">Site specific isotope fractionation of Hydrogen in the oxidation of ethanol into acetic acid. Application to vinegars. (C. Vallet, M. Arendt, G. Martin), Biotechnology Techniques, vol. 2 N° 2, 1988</ref>

=== Application for vanillin ===

<sup>2</sup>H-SNIF-NMR is the official AOAC method for determining the natural vanillin.

The abundance of five monodeuterated isotopomers for vanillin can be measured by <sup>2</sup>H-SNIF-NMR. The vanillin molecule is represented in figure 11, all observable sites for which the site specific deuterium concentrations can be measured are referenced with a number.

As for the wine or the fruit, the interpretation of results in terms of origin is done by comparison of the isotopic parameters of the sample analyzed with those from a group of referenced molecules of known origin. 
It appears that all the origins of vanillin are well discriminated using <sup>2</sup>H-NMR data. Particularly, vanillin ex-bean can well be distinguished from the other sources, as we can see in figure 12 below.

Additionally, this method is the only one to discriminate between natural and biosynthetic sources of vanillin.<ref name="vanillin">AOAC Official Method 2006.05, Site-Specific Deuterium/Hydrogen (D/H) Ratios in Vanillin, AOAC International 2007</ref>

=== Application for other aromas ===

The naturality of different aroma can also be checked using SNIF-NMR: for example for anethole, abundance of only six monodeuterated isotopomers can be measured by <sup>2</sup>H-SNIF-NMR that allows differentiating the botanical origins fennel, star anise or pine.<ref name="pine">La Résonnance Magnétique Nucléaire du Deutérium en Abondance Naturelle, une nouvelle méthode d’identification de l’origine de produits alimentaires appliquée à la reconnaissance des Anétholes et des Estragoles. (G. Martin, M. Martin, F. Mabon, J. Bricout), Sciences des Aliments, 1983</ref>

''Other applications:''
The SNIF-NMR applied to benzaldehyde can detect adulterated [[Biology:Bitter almond|bitter almond]] and [[Biology:Cinnamon|cinnamon]] oils. It is demonstrated that the site specific deuterium contents of [[Chemistry:Benzaldehyde|benzaldehyde]] allow the determination of the origin of the molecule: synthetic (ex-[[Chemistry:Toluene|toluene]] and ex-[[Chemistry:Benzal chloride|benzal chloride]]), natural (ex-kernels from apricots, peaches, cherries and ex-bitter almond) and semisynthetic (ex-[[Chemistry:Cinnamaldehyde|cinnamaldehyde]] extracted from cinnamon).<ref name="cinnamon">G. Remaud, A. Debon, Y. Martin, G. Martin. Authentication of Bitter Almond Oil and Cinnamon Oil: Application of the SNIF-NMR Method to Benzaldehyde, Journal of agricultural and food chemistry, n° 45, 1997</ref>
Other applications have also been published: raspberry ketone),<ref name="ketone">Journal of High Resolution Chromatography, vol.18, May 1995,279-285</ref> heliotropine, ...

== <sup>13</sup>C-SNIF-NMR ==
[[File:Figure 14 - With this method C3, C4 and CAM plant metabolism are well separated.jpg|thumb|Figure 12 - With this method C3, C4 and CAM plant metabolism are well separated]]
As described in the work of E. Tenailleau and S. Akoka, an optimization of the technique parameters have enabled to reach a better accuracy for the <sup>13</sup>C NMR measurements).<ref name="routine">E. Tenailleau, S. Akoka . Adiabatic 1H decoupling scheme for very accurate intensity measurements in 13C NMR, Journal of Magnetic Resonance, n°185, 2007, p50-58</ref>

The <sup>13</sup>C-SNIF-NMR method is called method “new frontier” because it is the first analytical method that can differentiate sugars coming from C4-metabolism plants (cane, maize, etc.) and some crassulacean acid metabolism plants (CAM-metabolism) like [[Biology:Pineapple|pineapple]] or [[Biology:Agave|agave]].<ref name="tequila">F. Thomas, C. Randet, A. Gilbert, V. Silvestre, E. Jamin and al.  Improved Characterization of the Botanical Origin of Sugar by Carbon-13 SNIF-NMR Applied to Ethanol, Journal of agricultural and food chemistry, n° 58, 2010, p11580-11585</ref>

This method can also be applied to [[Biology:Tequila|tequila]] products, where it can differentiate authentic 100% agave tequila, misto tequila (made from at least 51% agave), and products made from a larger proportion of [[Biology:Sugarcane|cane]] or [[Biology:Maize|maize]] sugar and therefore not complying with the legal definition of tequila.<ref name="tequila" />

This method will certainly have further applications in future, in the field of food and beverage analysis authenticity.

== References ==
<references/>

==See also==
* [[Physics:Nuclear magnetic resonance|Nuclear magnetic resonance]]
* [[Chemistry:Food chemistry|Food chemistry]]
* [[Earth:Food security|Food security]]


[[Category:Nuclear magnetic resonance]]
[[Category:Isotopes]]
[[Category:Analytical chemistry]]

{{Sourceattribution|Isotopic analysis by nuclear magnetic resonance}}