Quantification of soybean oil ethanolysis with <Superscript>1</Superscript>H NMR

In this study, proton NMR spectroscopy (200 MHz) was used for quantifying the content of ethyl esters in known mixtures of soybean oil and ethyl soyate (biodiesel). For this purpose, the peak areas of ester ethoxy and glycerol methylenic peaks in the region of 4.05–4.40 ppm were measured and a calibration plot of the respective peak areas vs. the known composition of the oil/ethyl ester mixtures was used. The transesterification values determined in this way were compared with viscosity and total glycerol determinations and a good correlation was obtained. Therefore, for routine analysis, the conversion (in %) of oil to ethyl esters was determined. The methodology presented in this work proved to be quicker and simpler than others reported in the literature, such as GC and/or HPLC.     

The use of <Superscript>1</Superscript>H NMR for yield determination in the regioselective epoxidation of squalene

¹H NMR can be used to determine the epoxide yield rapidly in the oxidation of squalene. Moreover, unequivocal distinction can be made between internal and terminal epoxide bonds. To underline the power of this technique, different stoichiometric and catalytic epoxidation procedures were carried out using squalene as substrate. They were characterized in terms of substrate conversion and regioselectivity of the epoxide fraction.     

<Superscript>1</Superscript>H NMR characterization of milk lipids: A comparison between cow and buffalo milk

Characterization of the lipid fraction of raw cow and buffalo milk samples, collected in different breeding areas in Apulia, a region of southern Italy, were performed by means of ¹H NMR. The aim of this work was to establish whether FA composition data obtained by ¹H NMR can be used in the differentiation of buffalo and cow milk samples according to species. A complete assignment of the signals present in the spectrum was attempted by COSY, heteronuclear coherence spectra. Quantification of FA was carried out by inserting the integrals of particular peaks in suitable calculations. Multivariate statistical analysis, conducted on the results of the quantification, permitted buffalo and cow milks to be distinguished.     

Role of the functional group position in producing viscoelasticity in micellar solutions: A <Superscript>1</Superscript>H NMR study

¹H NMR spectra of cetyltrimethylammonium bromide (CTAB) micellar solutions in the absence and presence of sodium anthranilate (NaAn) have been recorded. Studies were performed at a fixed CTAB concentration in the presence of different NaAn concentrations. The specificity of the functional group attached to the salt in producing changes (i.e., viscoelasticity) in CTAB micellar solutions was explored. It was concluded that the nature of the salt counterion together with its intercalation/adsorption in the head group region is the principal cause of viscoelasticity.     

Acyl Migration Kinetics of Vegetable Oil 1,2-Diacylglycerols

The acyl migration kinetics of long-chain 1,2-diacylglycerol (1,2-DAG) to form 1,3-diacylglycerol (1,3-DAG) over the temperature range of 25–80 °C were examined using ¹H-NMR spectroscopy. Lipase-catalyzed ethanolysis of high-oleic sunflower oil, followed by a series of solvent extraction steps, generated high purity 1,2-DAG (0.93 mol fraction of the DAG content). The 1,2-DAG mole fraction of 0.32 at equilibrium was found to be insensitive to temperature, indicating that long-chain acyl group migration is neither endothermic nor exothermic. Determination of the temperature-dependent, first-order reaction kinetic parameters revealed a 1,2-DAG half life (t _1/2) of 3,425 h and 15.8 h at 25 and 80 °C, respectively. A comparison of 1,2-DAG with 2-monoacylglycerol indicated that there is no difference between the two in the potential energy state (ΔG ^‡) of their respective transitions states or cyclic intermediates. Product names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may be suitable.     

Influence of Solid Supports on Acyl Migration in 2-Monoacylglycerols: Purification of 2-MAG via Flash Chromatography

Soybean oil 2-monoacylglycerol (2-MAG) was synthesized via the Novozym 435-catalyzed ethanolysis of triacylglycerols and purified by conventional liquid–liquid extraction. The 2-MAG was subjected to incubation at 20 and 40 °C in the presence of five solid commercial support materials, Lewatit, Silica Gel 60, Alumina–Neutral Brockman Activity 1, Amberlyst-15, and SBA-15, to determine their effects on acyl migration rates. Lewatit and SBA-15 did not catalyze acyl migration rates after 144 h, while silica gel slightly increased migration rates. The more polar alumina and the cationic Amberlyst 15 significantly increased migration rates. Flash chromatography purification of 2-MAG using silica gel as the immobile phase developed with an acetone/hexane binary gradient proved to be a comparable purification method to liquid–liquid extraction, resulting in 60 % 2-MAG yield, no residual triacylglycerol, diacylglycerol, or glycerol co-products, and a 95 mol% 2-MAG purity vs. 1-MAG, demonstrating that flash chromatography did not catalyze acyl migration.     

Synthesis,Purification, and Acyl Migration Kinetics of 2-Monoricinoleoylglycerol

2‐Monoricinoleoylglycerol (2‐MRG) was synthesized by the Novozym 435 catalyzed alcoholysis of castor oil in excess ethanol (1:70 mol:mol) at ambient temperature. Due to the fatty acid C12‐OH group, conventional liquid–liquid extraction methods developed for less polar, non‐hydroxylated 2‐monoacylglycerols (2‐MAG) proved inadequate for 2‐MRG purification. Alternatively, 2‐MRG was purified by normal‐phase flash chromatography (FC) on silica gel using a binary acetone‐hexane gradient mobile phase. Gram quantities of 2‐MRG were isolated in 63 % yield and contained no residual diacylglycerol (DAG), which fail to separate using liquid–liquid extraction methods. The 2‐MRG was obtained at ~90 mol% relative to 1‐MRG, proving that the FC method did not appreciably catalyze acyl migration. ¹H‐NMR spectroscopy was used to monitor the spontaneous acyl migration of isolated 2‐MRG from 20 to 80 °C. The relative energy of activation calculated from the Arrhenius relationship of the 2‐MRG acyl migration rate constants was 82.9 kJ/mol. This was ~two‐fold higher than the theoretical ΔG_298.15 calculated from molecular modeling using density functional calculations (B3LYP/6?31 + G*) of 2‐MRG, the ketal ring transition state, and 1‐monoricinoleoylglycerol (1‐MRG). The synthesis and isolation methods reported herein provide a convenient means to access useful intermediates for functionalized structured lipids.     

Epoxide yield determination of oils and fatty acid methyl esters using <Superscript>1</Superscript>H NMR

Product mixtures of epoxidized fatty compounds can be analyzed by using ¹H NMR. Conversion of double bonds and selectivities to different products can easily be calculated. Moreover, if diunsaturated substrates are used in epoxidation reactions, yields to mono- and diepoxidized products can be determined. The effectiveness of this method is proven by comparing some NMR results with those found by GC analysis.     

Characterization of sacha inchi (<Emphasis Type="Italic">Plukenetia volubilis</Emphasis> L.) oil by FTIR spectroscopy and <Superscript>1</Superscript>H NMR. Comparison with linseed oil

Three oil samples obtained from sacha inchi (Plukenetia volubilis L.) seeds were studied by means of FTIR and ¹H NMR. Frequency data of the most significant bands of the IR spectrum of this oil are given. These data show that sacha inchi oil has a high degree of unsaturation. The same fact is deduced from the ratio between the absorbance of the bands due to the stretching vibrations of the cis olefinic CH double bonds at 3010.5 cm⁻¹ and to the methylene symmetrical stretching vibrations at 2855.1 cm⁻¹. The proportions of monounsaturated, polyunsaturated, and saturated acyl groups were predicted from the frequency of some IR bands, and these were in satisfactory agreement with the values obtained through FAME generation and their quantification by GC. Likewise, simple observation of the ¹H NMR spectra provided a great deal of information about sacha inchi oil, with regard not only to the relative proportions of the different acyl groups but also to their nature. Thus, the presence of γ-linolenic acyl groups was discounted. Furthermore, the area of some ¹H NMR signals was used to determine the proportion of saturated and mono-, di-, and triunsaturated acyl groups, which also were in satisfactory agreement with the values obtained by classical methods. IR and ¹H NMR determinations take very little time in comparison with classical methods and do not require chemical manipulation or transformation of the sample. A comparison was also made between the compositions of sacha inchi and linseed oil. Both oils are important sources of the healthful n−3 linolenic acyl groups, and sacha inchi also contains high proportions of the n−6 linoleic acyl groups.     

Study of Ozonated Sunflower Oil Using 1H NMR and Microbiological Analysis

Prior studies have proven that ozonated vegetable oils present a high germicidal power. Ozonation of sunflower oil at different applied ozone dosage was carried out and peroxide and aldehydes indices along with antimicrobial activity were determined. The reaction products were identified using Proton Nuclear Magnetic Resonance Spectroscopy (¹H NMR). The principal signals intensity values were used for following the reaction course between ozone and sunflower oil. The reaction was following up to peroxide index values of 1202 mmol-equi/kg. The intensities of olefinic proton signals decreased with the gradual increase in ozone concentration but without disappearing completely. The Criegee ozonides obtained at 107.1 mg/g of ozone doses were approximately 3.9-fold higher than that at beginning of the reaction. The aldehyde protons were observed as a weak intensity signal in all the spectra. The signals belonging to olefinic protons from hydroperoxides appeared weak and increased with the increase in ozone doses. Signals from other oxygenated groups were assigned. The highest action spectrum of antimicrobial activity was obtained with the higher peroxide index. It was concluded that at higher applied ozone doses, the higher the antimicrobial activity potential of ozonized sunflower oil     

Visualization of Bile Homeostasis Using <Superscript>1</Superscript>H-NMR Spectroscopy as a Route for Assessing Liver Cancer

Changes in bile synthesis by the liver or alterations in the enterohepatic circulation due to a variety of etiological conditions may represent a novel source of liver disease-specific biomarkers. Bile from patients with liver diseases exhibited significant changes in the levels of glycine- and taurine-conjugated bile acids, phospholipids, cholesterol and urea relative to non-liver disease controls. Cholangiocarcinoma and non-malignant liver diseases (NMLD) showed the most significant alterations. Further, hepatocellular carcinoma (HCC) could be differentiated from NMLD (p = 0.02), as well as non-liver disease controls (p = 0.02) based on the amounts of bile acids, phospholipids and/or cholesterol. HCC also differed with cholangiocarcinoma although not significantly. Urea increases somewhat in non-malignant liver disease relative to non-liver disease controls, while the bile acids, phospholipids and cholesterol all decrease significantly. The ratio between some major bile metabolites also distinguished NMLD (p = 0.004–0.01) from non-liver disease controls. This snapshot view of bile homeostasis, is obtainable from a simple nuclear magnetic resonance (NMR) approach and demonstrates the enormous opportunity to assess liver status, explore biomarkers for high risk diseases such as cancers and improve the understanding of normal and abnormal cellular functions.     

Roles of adsorbed OH and adsorbed H in the oxidation of hydrogen and the reduction of UO<Stack><Subscript>2</Subscript><Superscript>2+</Superscript></Stack> ions at Pt electrodes under non-conventional conditions

The roles of adsorbed hydroxyl radicals, OH, at a high temperature and adsorbed hydrogen atoms, H, in an acidic solution were investigated in the electrochemical reactions on Pt electrode by using potentiodynamic polarisation experiment, cyclic voltammetry and constant-potential electrolysis combined with UV/VIS analysis. From the analysis of the polarisation curves obtained from Pt electrode in a 0.185 M H₃BO₃ solution at 473 K, it was found that the reducing capability of dissolved hydrogen is significantly enhanced due to the increases of the mass transfer and the electron transfer rates. Especially, it is suggested that the stable Pt-OH_ad plays a significant role in the passivation reaction in the potential range from 0.60 to 0.75 V_SHE. From the analyses of the experimental results for the electrochemical reduction of UO₂²⁺ ions on Pt surface in a 1.0 M HClO₄ solution, it is recognised that the reduction reaction of UO₂²⁺ to U⁴⁺ ions is strongly dependent on the hydrogen atoms adsorbed on Pt electrode (indirect reduction of UO₂²⁺) as well as on the electrons transferred from Pt electrode (direct reduction of UO₂²⁺). In addition, the reduction mechanism of UO₂²⁺ ions involved in Pt-H_ad is also proposed.     

Fractionation of Soybean Globulins Using Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript>: A Comparative Analysis

The substitution of CaCl₂ by MgCl₂ was undertaken in Deak’s two-step process of separating the soybean 11S and 7S globulins, aiming at higher purities and lower phytic acid (PA) contents of recovered protein fractions. The effects of pH and the addition of NaCl were also evaluated. Compared with CaCl₂, MgCl₂ reduced the PA content of the 11S-rich fraction by 63–71% but increased that of the 7S-rich fraction by 14–28%, depending on pH. Correspondingly, more Ca²⁺ was recovered in the 11S-rich fraction, while more Mg²⁺ co-precipitated with the 7S-rich fraction. NaCl increased the purity of the 11S-rich fraction and reduced its PA content, but the purity of the 7S-rich fraction was reduced by using 50–100 mM NaCl. Lowering pHs from 6.4 and 4.8 to 5.6 and 4.0 in the two precipitation steps increased the yield of both fractions. The optimized fractionating procedure was as follows: the 11S-rich fraction was precipitated at pH 5.8 by using 5 mM MgCl_2, 10 mM NaHSO₃ and 20 mM NaCl, followed by the precipitation of the 7S-rich fraction at pH 4.5. The new method provided both fractions with satisfactory protein yields (22% for 11S and 16% for 7S), purities (88% for 11S and 80% for 7S) and PA contents (0.356% for 11S and 0.882% for 7S).     

Linseed oil and mixture with maleic anhydride: <Superscript>1</Superscript>H and <Superscript>13</Superscript>C NMR

It was shown that ¹H NMR allowed a rapid determination of the ratio of the linolenic residues over all the others (linoleic + oleic + saturated) and ¹³C NMR allowed a rapid determination of the ratio of linolenic over (linoleic + oleic) residues as well as the linoleic/oleic ratio in a few minutes on less than 20 mg of crude LO. After thermal treatment (220°C for 2 h, followed by cooling to room temperature), a 1∶1 mixture of linseed oil (LO) and maleic anhydride (MA), which was a suspension, became limpid and remained limpid. Moreover, the viscosity of the mixture was higher than that of pure LO treated in the same way. It was shown by ¹³C NMR and quantitative recovery of the constituents (LO and MA) through solvent separation that no reaction occurred between LO and MA during this thermal treatment. This result is in accord with DSC analysis of such a 1∶1 LO/MA mixture that exhibited an exothermic effect too small (about 34 kcal/mol) to correspond to formation of a C−C bond.     

1H NMR Study of Methyl Linoleate Ozonation.

Unsaturated fatty acids are essential components of vegetable oils and cellular membranes and the involved aspect of unsaturated fatty acids ozonation have been widely studied by different authors. In this paper, in vitro ozonolysis of unsaturated fatty acids with addition of water or ethanol has been studied by Proton Nuclear Magnetic Resonance (¹H NMR) at 250?MHz in order to explore the possibility of this technique for the detection of Criegee ozonides in characterizing ozone reaction with these substrates. The ozonolysis of methyl linoleate showed that signal intensities from formed ozonides were increased with ozone concentration increments. However, the signal intensities with addition of water were higher than those in ethanol addition. Signal intensities from olefinic double bonds were found to decrease with the increment in ozonide signals. Thus, a correspondence of the behavior of these signals is observed with a proportional rate reaction between the number of double bonds in the substrate molecule. Signals from aldehyde formation were poorly detected at lower ozone concentration. It was concluded that the evaluation of ozonide and olefinic double bond signals from 250?MHz ¹H NMR can be a useful tool in assessing ozone reaction with biomolecules. The reaction mechanism for the ozone reaction with unsaturated fatty acids in the presence of water or ethanol is analyzed.     

TLC and <Superscript>31</Superscript>P-NMR Analysis of Low Polarity Phospholipids

High-performance TLC and ³¹P-NMR were assessed as methods of observing the presence of numerous low polarity phospholipids: bis-phosphatidic acid (BPA), semi-lyso bis-phosphatidic acid (SLBPA), N-acyl phosphatidylethanolamine (NAPE), N-(1,1-dimethyl-3-oxo-butyl)-phosphatidylethanolamine (diacetone adduct of PE, DOBPE), N-acetyl PE, phosphatidylmethanol (PM), phosphatidylethanol (PEt), phosphatidyl-n-propanol (PP), phosphatidyl-n-butanol (PB). Both techniques are non-discriminative and do not require the prior isolation of individual lipids. It appears that 2D TLC is superior to ³¹P NMR in the analysis of low polarity phospholipids. All phosphatidylalcohols were well separated by 2D TLC. However, some compounds which can present difficulty in separation by 2D-TLC (e.g., SLBPA and NAPE; or DOBPE and N-acetyl PE) were easily distinguished using ³¹P NMR so the methods are complimentary. A disadvantage of 2D TLC is that Rf values can vary with different brands and batches of TLC plates. The chemical shifts of ³¹P NMR were less variable, and so a library of standards may not be necessary for peak identification. Another advantage of ³¹P NMR is the ease of quantification of phospholipids. The applicability of the methods was tested on natural extracts of fish brain and cabbage stem.     

Investigation of the Structure-Directing Effect of Surfactants on the Distribution of Silicate Species Using <Superscript>29</Superscript>Si-NMR Spectroscopy

Silicate anions were studied using ²⁹Si NMR to investigate the structure-directing effect of a surfactant (sodium dodecylsulfate-SDS) on the distribution of silicate species present in alkaline silicate solutions. The distribution of the anions in the presence of polyelectrolytes was also investigated by ²⁹Si NMR through the use of different bases including tetramethyl ammonium hydroxide (TMAOH) and sodium hydroxide (NaOH). Both SDS and the polyelectrolytic solutions were shown to stabilize low molecular weight silicate species and thus inhibit polymerization.

Determination of Glycerol in Wines Using <Superscript>31</Superscript>P-NMR Spectroscopy

³¹P-NMR spectroscopy was employed to detect and quantify glycerol in red wines from various regions of Greece. This novel analytical method was based on the derivatization of the hydroxyl groups of glycerol with 2-chloro-4,4,5,5-tetramethyl dioxaphospholane, and identification of the phosphitylated compound on the basis of ³¹P chemical shifts. Quantification of glycerol in wines was accomplished by integration of appropriate signals in the ³¹P-NMR spectrum and the use of the phosphitylated cyclohexanol as the internal standard. The method was reproducible (CV (%) = 2.35) and accurate (CV (%) = 1.34). Its applicability to glycerol quantification in wines was tested against a weighted amount of a glycerol-model compound by linear regression analysis (R = 0.999; intercept = 0.074 ± 0.078; slope = 0.998 ± 0.003; p = 0.000). Furthermore, the NMR method was compared to the AOAC official method (HPLC) using the Bland and Altman statistical analysis. The distribution of the data points in the bias plot showed that 100% of the measurements of glycerol in 16 wine samples from various regions of Greece were within the limits of agreement of the two methods.     

Discrimination of Cod Liver Oil According to Wild/Farmed and Geographical Origins by GC and <Superscript>13</Superscript>C NMR

The objective of this study was to test the possibility of using lipid profiles obtained by gas chromatography (GC) and ¹³C nuclear magnetic resonance (NMR) in authentication of cod liver oils according to wild/farmed and geographical origin. GC and ¹³C-NMR data of cod liver oil from wild and farmed fish from different locations in Norway and Scotland were obtained, and analyzed by principal component analysis (PCA) and linear discriminant analysis (LDA) to test if it was possible to differentiate oil from wild and cultured cod (Gadus morhua L.), and to further elucidate differences between fish from the different farms/catch area. Cod liver oils of wild and farmed origin were clearly separated in the PCA score plot both from GC and NMR data. From NMR data it was also possible to observe groupings based on geographical origin (farm/catch area) of the different samples. Using LDA with cross validation the wild/farmed classification rates were 97% for GC data and 100% for NMR data. In the classification of cod liver oils according to geographical origin (38 samples from six different farms/catch area), the correct classification rate was 63% for GC data and 95% for NMR data.