Name: nmr spectroscopy as a robust tool for the rapid evaluation of the lipid profile of fish oil supplements
Uploaded: 2017-05-01T11:45:00
Description: Watch this Scientific Journal Video about NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements at JoVE.com

This work was supported by the Foods for Health Discovery Theme at The Ohio State University and the Department of Food Science and Technology at The Ohio State University. The authors would like to thank the NMR facility at The Ohio State University and the NMR facility at Penn State University.

1. NMR Sample Preparation
Note: Caution, please consult all relevant material safety data sheets (MSDS) before use. Deuterated chloroform (CDCl3) used in sample preparation is toxic. Please use all the appropriate safety practices when performing sample preparation including the use of a fume hood and personal protective equipment (safety glasses, gloves, lab coat, full length pants, closed-toe shoes).
<ol>
	<li>Preparation of 1H and 13C samples</stro...

<ol>
	<li>Simopoulos, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+the+ratio+of+omega-6/omega-3+essential+fatty+acids.">The importance of the ratio of omega-6/omega-3 essential fatty acids.</a> Biomed. Pharmacother. 56 (8), 365-379 (2002).</li><li>Goodnight, S. H., Harris, W. S., Connor, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+dietary+omega+3+fatty+acids+on+platelet+composition+and+function+in+man:+a+prospective,+controlled+study.">The effects of dietary omega 3 fatty acids on platelet composition and function in man: a prospective, controlled study.</a> Blood. 58 (5), 880-885 (1981).</li><li>Harper, C., Jacobsen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+omega-3+fatty+acids+and+the+prevention+of+coronary+heart+disease.">Usefulness of omega-3 fatty acids and the prevention of coronary heart disease.</a> Am. J. Cardiol. 96 (11), 1521-1529 (2005).</li><li>Kremer, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+high-dose+fish+oil+on+rheumatoid+arthritis+after+stopping+nonsteroidal+antiinflammatory+drugs.+Clinical+and+immune+correlates.">Effects of high-dose fish oil on rheumatoid arthritis after stopping nonsteroidal antiinflammatory drugs. Clinical and immune correlates.</a> Arthritis and Rheumatol. 38 (8), 1107-1114 (1995).</li><li>Malasanos, T., Stackpoole, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+effects+of+omega-3+fatty+acids+in+diabetes+mellitus.">Biological effects of omega-3 fatty acids in diabetes mellitus.</a> Diabetes Care. 14, 1160-1179 (1991).</li><li>Han, Y., Wen, Q., Chen, Z., Li, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+Methods+Used+for+Microalgal+Lipid-Content+Analysis.">Review of Methods Used for Microalgal Lipid-Content Analysis.</a> Energ. Procedia. 12, 944-950 (2011).</li><li>Guill&#233;n, M., Ruiz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1H+nuclear+magnetic+resonance+as+a+fast+tool+for+determining+the+composition+of+acyl+chains+in+acylglycerol+mixtures.">1H nuclear magnetic resonance as a fast tool for determining the composition of acyl chains in acylglycerol mixtures.</a> Eur. J. Lipid Sci. Technol. 105, 502-507 (2003).</li><li>Sacchi, R., Medina, I., Aubourg, S. P., Addeo, F., Paolillo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proton+nuclear+magnetic+resonance+rapid+and+structure+specific+determination+of+&#969;-3+polyunsaturated+fatty+acids+in+fish+lipids.">Proton nuclear magnetic resonance rapid and structure specific determination of &#969;-3 polyunsaturated fatty acids in fish lipids.</a> J. Am Oil Chem Soc. 70, 225-228 (1993).</li><li>Igarashi, T., Aursand, M., Hirata, Y., Gribbestad, I. S., Wada, S., Nonaka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nondestructive+quantitative+acid+and+n-3+fatty+acids+in+fish+oils+by+high-resolution+1H+nuclear+magnetic+resonance+spectroscopy.">Nondestructive quantitative acid and n-3 fatty acids in fish oils by high-resolution 1H nuclear magnetic resonance spectroscopy.</a> J. Am. Oil Chem. Soc. 77, 737-748 (2000).</li><li>Plans, M., Wenstrup, M., Saona, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Infrared+Spectroscopy+for+Characterization+Dietary+Omega-3+Oil+Supplements.">Application of Infrared Spectroscopy for Characterization Dietary Omega-3 Oil Supplements.</a> J. Am. Oil Chem. Soc. 92, 957-966 (2015).</li><li>Jian-hua, C. I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+Spectrum+Detection+of+Fish+Oil+DHA+Content+Based+on+Empirical+Mode+Decomposition+and+Independent+Component+Analysis.">Near-infrared Spectrum Detection of Fish Oil DHA Content Based on Empirical Mode Decomposition and Independent Component Analysis.</a> J Food Nutr Res. 2 (2), 62-68 (2014).</li><li>Millen, A. E., Dodd, K. W., Subar, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+vitamin,+mineral,+nonvitamin,+and+nonmineral+supplements+in+the+United+States:+The+1987,+1992,+and+2000+National+Health+Interview+Survey+results.">Use of vitamin, mineral, nonvitamin, and nonmineral supplements in the United States: The 1987, 1992, and 2000 National Health Interview Survey results.</a> J. of Am. Diet Assoc. 104 (6), 942-950 (2004).</li><li>Dwyer, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+developing+analytical+and+label-based+dietary+supplement+databases+at+the+NIH+office+of+dietary+supplements.">Progress in developing analytical and label-based dietary supplement databases at the NIH office of dietary supplements.</a> J. Food Compos. Anal. 21, S83-S93 (2008).</li><li>Monakhova, Y. B., Ruge, I., Kuballa, T., Lerch, C., Lachenmeier, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+determination+of+coenzyme+Q10+in+food+supplements+using+1H+NMR+spectroscopy.">Rapid determination of coenzyme Q10 in food supplements using 1H NMR spectroscopy.</a> Int. J. Vitam. Nutr. Res. 83 (1), 67-72 (2013).</li><li>Monakhova, Y. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardless+1H+NMR+determination+of+pharmacologically+active+substances+in+dietary+supplements+and+medicines+that+have+been+illegally+traded+over+the+internet.">Standardless 1H NMR determination of pharmacologically active substances in dietary supplements and medicines that have been illegally traded over the internet.</a> Drug Test. Anal. 5 (6), 400-411 (2013).</li><li>Berger, S., Braun, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 200 and more NMR experiments: a practical course. , (2004).</li><li>Knothe, G., Kenar, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+fatty+acid+profile+by+1H-NMRspectroscopy.">Determination of the fatty acid profile by 1H-NMRspectroscopy.</a> Eur. J. Lipid Sci. Technol. 106, 88-96 (2004).</li><li>Sacchi, R., Medina, J. I., Aubourg, S. P., Paolillo, I. G. L., Addeo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+High-Resolution+13C+NMR+Analysis+of+Lipids+Extracted+from+the+White+Muscle+of+Atlantic+Tuna+(Thunnus+alalunga).">Quantitative High-Resolution 13C NMR Analysis of Lipids Extracted from the White Muscle of Atlantic Tuna (Thunnus alalunga).</a> J. Agric. Food Chem. 41 (8), 1247-1253 (1993).</li><li>Dais, P., Misiak, M., Hatzakis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+marine+dietary+supplements+using+NMR+spectroscopy.">Analysis of marine dietary supplements using NMR spectroscopy.</a> Anal. Methods. 7 (12), 5226-5238 (2015).</li><li>Pickova, J., Dutta, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+Oxidation+in+Some+Processed+Fish+Products.">Cholesterol Oxidation in Some Processed Fish Products.</a> J. Anal. Oil Chem. Soc. 80 (10), 993-996 (2003).</li><li>Siddiqui, N., Sim, J., Silwood, C. J. L., Toms, H., Iles, R. A., Grootveld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicomponent+analysis+of+encapsulated+marine+oil+supplements+using+high-resolution+1H+and+13C+NMR+techniques.">Multicomponent analysis of encapsulated marine oil supplements using high-resolution 1H and 13C NMR techniques.</a> J. of Lipid Rsrch. 44 (12), 2406-2427 (2003).</li><li>Sua&#180;rez, E. R., Mugford, P. F., Rolle, A. J., Burton, I. W., Walter, J. A., Kralovec, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13C-NMR+Regioisomeric+Analysis+of+EPA+and+DHA+in+Fish+Oil+Derived+Triacylglycerol+Concentrates.">13C-NMR Regioisomeric Analysis of EPA and DHA in Fish Oil Derived Triacylglycerol Concentrates.</a> J. Am. Oil Chem. Soc. 87, 1425-1433 (2010).</li><li>Youlin, X. A., Moran, S., Nikonowiczband, E. P., Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Z-restored+spin-echo+13C+1D+spectrum+of+straight+baseline+free+of+hump,+dip+and+roll.">Z-restored spin-echo 13C 1D spectrum of straight baseline free of hump, dip and roll.</a> Magn. Reson. Chem. 46, 432-435 (2008).</li><li>Tengku-Rozaina, T. M., Birch, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positional+distribution+of+fatty+acids+on+hoki+and+tuna+oil+triglycerides+by+pancreatic+lipase+and+13C+NMR+analysis.">Positional distribution of fatty acids on hoki and tuna oil triglycerides by pancreatic lipase and 13C NMR analysis.</a> Eur. J. Lipid Sci. Technol. 116 (3), 272-281 (2014).</li><li>Berry, S. E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triacylglycerol+structure+and+interesterification+of+palmitic+and+stearic+acid-rich+fats:An+overview+and+implications+for+cardiovascular+disease.">Triacylglycerol structure and interesterification of palmitic and stearic acid-rich fats:An overview and implications for cardiovascular disease.</a> Nutr. Res. Rev. 22 (1), 3-17 (2009).</li><li>Hunter, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+effects+of+dietary+fatty+acids+as+related+to+their+position+on+triglycerides.">Studies on effects of dietary fatty acids as related to their position on triglycerides.</a> Lipids. 36, 655-668 (2001).</li><li>Vlahov, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regiospecific+analysis+of+natural+mixtures+of+triglycerides+using+quantitative+13C+nuclear+magnetic+resonance+of+acyl+chain+carbonyl+carbons.">Regiospecific analysis of natural mixtures of triglycerides using quantitative 13C nuclear magnetic resonance of acyl chain carbonyl carbons.</a> Magnetic Res. in Chem. 36, 359-362 (1998).</li></ol>

Modifications and Strategies for Troubleshooting
Spectral quality. The linewidth of the NMR signal and thus the resolution of the NMR spectrum is highly dependent on shimming, which is a process for the optimization of the homogeneity of the magnetic field. For routine analysis, 1D shimming is adequate and a 3D shimming is not required, given that it is performed by NMR personnel on a regular basis. If this is not the case, a 3D shimming must be performed prior to analysis using ...

The consumption of n-3 fatty acids in the diet has proven to be beneficial against several conditions such as heart disorders1,2,3, inflammatory diseases4 and diabetes5. The Western diet is considered poor in n-3 fatty acids and thus the consumption of fish oil supplements is recommended to improve the n-6/n-3 balance in consumer&#39;s nutrition1. Despite the recent increase in fish oil supplement consumption, questions remain about the safety, authenticity, and quality of some of these products. The rapid and accurate compositional analysis of fish oil supplements is essential to properly evaluate the quality of these commercial products and ensure consumer safety.
The most common methodologies for the assessment of fish oil supplements are gas chromatography (GC) and Infrared Spectroscopy (IR). While these are highly sensitive methods, they suffer from several drawbacks6. GC analysis is time consuming (4-8 h) because separation and derivatization of individual compounds is required7 and lipid oxidation may occur during the analysis8,9. While IR spectroscopy can be quantitative, a prediction model is required to be constructed using partial least squares regression (PLSR), although there are exceptions in which IR bands can be attributed to a single compound10. PLSR requires the analysis of a large number of samples, which increases the time of the analysis11. For this reason, there is an increasing interest in the development of new analytical methodologies that allow accurate and fast analysis of a large number of fish oil samples. Organizations such as the Office of Dietary Supplements (ODS) at the National Institutes of Health (NIH) and the Food and Drug Administration (FDA) have collaborated with the Association of Official Analytical Chemists (AOAC) to develop these new methods12,13.
One of the most promising analytical methods for the screening and the evaluation of multi-component matrices, such as dietary supplements, is Nuclear Magnetic Resonance (NMR) spectroscopy14,15. NMR spectroscopy has several advantages: it is a non-destructive and quantitative technique, it requires minimal to no sample preparation, and it is characterized by excellent accuracy and reproducibility. In addition, NMR spectroscopy is an environmentally friendly methodology because it utilizes only small amounts of solvents. The main drawback of NMR spectroscopy is its relatively low sensitivity compared to other analytical methods, however, recent technological advances in instrumentation such as stronger magnetic fields, cryogenic probes of various diameters, advanced data processing, and versatile pulse sequences and techniques have increased the sensitivity up to the nM range. While NMR instrumentation is high cost, the long-life of NMR spectrometers and the many applications of NMR lower the cost of the analysis in the long run. This detailed video protocol is intended to help new practitioners in the field avoid pitfalls associated with 1H and 13C NMR spectroscopic analysis of fish oil supplements.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Avance III 850 NMR instrument</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Avance III 500 NMR instrument</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TCI 5mm probe</td>
			<td>Bruker</td>
			<td></td>
			<td>Helium cooled inverse (proton deetected) NMR probe featuring three independent channels (1H, 13C, 15N)</td>
		</tr>
		<tr>
			<td>BBO prodigy 5mm probe</td>
			<td>Bruker</td>
			<td></td>
			<td>Nitrogen cooled observe (X-nuclei detected) probe, featuring two channels; one for 1H and 19F detectionand one for X-nuclei (covering from 15N to 31P)</td>
		</tr>
		<tr>
			<td>Spinner turbin</td>
			<td>Bruker</td>
			<td></td>
			<td>NMR spinners are made by polymer materials and they have a rubber o-ring to hold the NMR tube securely in place</td>
		</tr>
		<tr>
			<td>Topspin 3.5</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>deuterated chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;865-49-6</td>
			<td>99.8 atom % D, contains 0.03 TMS</td>
		</tr>
		<tr>
			<td>2,6-Di-tert-butyl-4-methylphenol,</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;128-37-0</td>
			<td>purity &#62;99%</td>
		</tr>
		<tr>
			<td>Fish oil samples</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NMR tubes</td>
			<td>New Era</td>
			<td>NE-RG5-7</td>
			<td>5mm OD Routine &#8220;R&#8221; Series NMR Sample Tube</td>
		</tr>
		<tr>
			<td>BSMS</td>
			<td>Bruker</td>
			<td></td>
			<td>Bruker Systems Management System; control system device</td>
		</tr>
	</tbody>
</table>

nmr spectroscopy as a robust tool for the rapid evaluation of the lipid profile of fish oil supplements

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential nutrients. The objective of this work is to demonstrate the qualitative and quantitative analysis of encapsulated fish oil supplements using high-resolution 1H and 13C NMR spectroscopy utilizing two different NMR instruments; a 500 MHz and an 850 MHz instrument. Both proton (1H) and carbon (13C) NMR spectra can be used for the quantitative determination of the major constituents of fish oil supplements. Quantification of the lipids in fish oil supplements is achieved through integration of the appropriate NMR signals in the relevant 1D spectra. Results obtained by 1H and 13C NMR are in good agreement with each other, despite the difference in resolution and sensitivity between the two nuclei and the two instruments. 1H NMR offers a more rapid analysis compared to 13C NMR, as the spectrum can be recorded in less than 1 min, in contrast to 13C NMR analysis, which lasts from 10 min to one hour. The 13C NMR spectrum, however, is much more informative. It can provide quantitative data for a greater number of individual fatty acids and can be used for determining the positional distribution of fatty acids on the glycerol backbone. Both nuclei can provide quantitative information in just one experiment without the need of purification or separation steps. The strength of the magnetic field mostly affects the 1H NMR spectra due to its lower resolution with respect to 13C NMR, however, even lower cost NMR instruments can be efficiently applied as a standard method by the food industry and quality control laboratories.

1H and 13C NMR spectra were collected for commercially available fish oil supplements using two NMR instruments; an 850 MHz and a 500 MHz spectrometer. These spectra can be used for the quantitative determination of components of fish oil, such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), as well other compounds such as n-1 acyl chains and nutritionally important index such as the n-6/n-3 ratio. The quantification can be p...

Watch this Scientific Journal Video about NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements at JoVE.com

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

The overall goal of this method is to determine the lipid profile of fish oil supplements using Nuclear Magnetic Resonance spectroscopy. This method can help answer key questions for lipid analysis such as the lipid contents of fish oil supplements and the positional distribution of various fatty acids on the glycerol skeleton. The main advantage of this technique is that it makes the rapid and robust analysis of several different molecules feasible in one snapshot without using separation and purification steps.To prepare the samples for proton and carbon NMR analysis, first extract 120 microliters, which is around 110 milligrams of fish oil, from a dietary capsule using a syringe and place it in a 4-mL glass vial. Record the weight of the fish oil. Use 500 microliters of a BHT stock solution to dissolve 100 milligrams plus or minus 10 milligrams of fish oil.After dissolving the oil, transfer all of the solution directly into a high quality, five millimeter NMR tube and attach a cap. Analyze the samples within 24 hours of preparing the samples. Insert the NMR tube into a spinner turbine.Place the spinner and the tube on the top of a graded depth gauge and gently push the top of the tube until its bottom part touches the bottom of the gauge. Then place the NMR tube in an open spot of the sample case. Note the slot number the sample is placed in.To load the sample in the NMR instrument, return to the control computer and type SX-number, where number is the slot in the sample case holding the sample. Wait for the deuterium signal of deuterated chloroform to appear on the screen. As soon as the deuterium signal is visible, type lock"on the command line and select deuterated chloroform from the solvents list to lock the sample using its deuterium resonance.Type bsmsdisp"in the command line to ensure spinning is not active. If the spin button is green, click it to deactivate spinning. After typing the command new"to create a new data set, enter a name for the data set in the name tab and the experiment number in the experiment number tab titled EXPNO.Use number one in the process number tab titled PROCNO. Write the title of the experiment in the TITLE tab. In the Experiment tab, hit Select and choose the C13IG parameter file.Then hit Set selected item in editor"and click OK.Type getprosol"in the command line to obtain the standard parameters for the current NMR probe and solvent. Then type the command atma"to perform automatic tuning and to matching of the probe for both carbon and proton nuclei. Next, perform one-dimensional gradient shimming to achieve a highly homogeneous magnetic field and thus optimum line shape for the NMR signals.To do so, use the standard automatic procedure for 1D shimming simply by sequently executing the commands qu topshim 1dfast ss;qu topshim tuneb ss;and qu topshim report"in the command line. After parameter optimization as described in the text protocol, acquire one dimensional carbon NMR spectra. To do so, go to the carbon data set and use an inverse gated decoupled pulse sequence by typing pulprog zgig"in the command line.Type commands on the command line to set up the spectral with nppm, the center of the RF transmitter, the number of scans, the number of dummy scans, the number of data points, and the pulse duration for a ninety degree pulse angle. Type digmod baseopt"in the command line to acquire a spectrum with an improved base line. Then set a relaxation delay of 60 seconds for the 850 megahertz instrument by typing di 60s"in the command line.Set the receiver gain to an appropriate value using the command rga"for automatic calculation of receiver gain. Finally, start the acquisition by typing the pulse acquire command zg"in the command line. To process the data, first type si 64K"in the command line to apply zero filling and set the size of the rail spectrum to 64K.Then set the line broadening parameter to 1.0 hertz by typing lb 1.0"in the command line to apply a waiting function with a line broadening factor of 1.0 hertz prior to 4ea transform. Execute 4ea transformation by typing efp"in the command line. Next perform automatic phase correction by typing the command apk"in the command line.If additional phase adjustments are required to further improve the spectrum, click on the Process tab. Then click on the Adjust Phase icon and the Phase Correction icons for zero order and first order phase correction. While clicking on the zero order and first order phase correction icons, drag the mouse until all of the signals are in positive absorption mode.Apply and save the phase correction values by clicking the Return and Save button to exit the Phase Correction mode. Apply a polynomial forth order function for baseline correction upon integration by typing the command abs n"in the command line. To report chemical shifts in ppm from TMS, zoom in on the TMS signal, then click on the Calibration icon and place the cursor with the red line on top of the NMR signal to be referenced, TMS in this case.To adjust the threshold intensity, use the middle mouse button if needed. Left click and type in 0. When using BHT as an internal standard, integrate the peak at delta 151.45 extending five hertz from each side of the peak.Then set the integral equal to the micromoles of BHT per 0.5 milliliters of the stock solution. Integrate the peaks of interest extending fiver hertz from each side of the peak. If there is a need to focus on a region, click on the highlight icon to deactivate it.Then left click and drag to zoom in on the region. Click on the highlight icon again to make the integration function active and move to the next peak. Left click and drag the cursor through the integral.Click the Return Save Regions button. Shown here is a representative spectrum of the Carbon NMR analysis in the carbonyl carbon region. The NMR signals of EPA and DHA on the sn-1, 3 and sn-2 positions are shown.These signals can be used for the quantitative determination of EPA and DHA. Shown here is a representative spectrum of the proton NMR analysis. The NMR signals of EPA and DHA that can be used for their determination are shown.The proton NMR spectrum is characterized by a narrower spectral width as compared to the carbon NMR spectrum and thus a lower spectral resolution. Once mastered, this technique can be done in less than 30 minutes if it is performed properly. After its development, this technique paved the way for researchers in the field of lipid analysis to rapidly analyze various lipid components that appear in multi-component matrices, such as dietary supplements, vegetable oil, and biofuels.After watching this video, you should have a good understanding of how to apply proton and carbon, NMR spectroscopy for the analysis of the lipid profile of fish oil supplements. Don't forget that working with strong magnetic fields produced by NMR spectrometers can be extremely hazardous to pacemakers and surgical protheses as well as electronic items such as credit cards and watches.

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Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic ...

HKUST-1 as a Heterogeneous Catalyst for the Synthesis of Vanillin

Vanillin (4-hydoxy-3-methoxybenzaldehyde) is the main component of the extract of vanilla bean. The natural vanilla scent is a mixture of approximately ...

Spin Saturation Transfer Difference NMR (SSTD NMR): A New Tool to Obtain Kinetic Parameters of Chemical Exchange Processes

Spin Saturation Transfer Difference NMR SSTD NMR: A New Tool to Obtain Kinetic Parameters of Chemical Exchange Processes

This detailed protocol describes the new Spin Saturation Transfer Difference Nuclear Magnetic Resonance protocol (SSTD NMR), recently developed in our ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Coulomb Explosion Imaging as a Tool to Distinguish Between Stereoisomers

This article shows how the COLTRIMS (Cold Target Recoil Ion Momentum Spectroscopy) or the "reaction microscope" technique can be used to distinguish ...

Electrochemical Impedance Spectroscopy as a Tool for Electrochemical Rate Constant Estimation

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). ...

Color Spot Test As a Presumptive Tool for the Rapid Detection of Synthetic Cathinones

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and ...

A New Straightforward Method for Lipophilicity (logP) Measurement using 19F NMR Spectroscopy

A New Straightforward Method for Lipophilicity logP Measurement using 19F NMR Spectroscopy

Fluorination has become an effective tool to optimize physicochemical properties of bioactive compounds. One of the applications of fluorine introduction ...