Direct methods based on (1)H NMR spectroscopic techniques have been developed for the determination of neutral lipids (triglycerides and free fatty acids) and polar lipids (glyceroglycolipids/phospholipids) in the solvent extracts of oleaginous microalgal biomasses cultivated on a laboratory scale with two species in different media. The chemical shift assignments observed in the (1)H and (13)C NMR spectra corresponding to unsaturated (C18:N, N = 1-3, C20:3, C20:5, C22:6, epoxy) and saturated (C14-C18) fatty acid ester components in a complex matrix involving overlapped resonances have been unambiguously confirmed by the application of 2D NMR spectroscopy (total correlation spectroscopy and heteronuclear single quantum coherence-total correlation spectroscopy). The study of the effect of a polar lipid matrix on the determination of neutral lipids by an internal reference blending process by a systematic designed experimental protocol has provided absolute quantification. The fatty acid composition of algal extracts was found to be similar to that of vegetable oils containing saturated (C16-C18:0) and unsaturated (C18:N, N = 1-3, C20:N, N = 3-4, C22:6) fatty acids as confirmed by NMR spectroscopy and gas chromatography-mass spectrometry analyses. The NMR methods developed offer great potential for rapid screening of algal strains for generation of algal biomass with the desired lipid content, quality, and potential for biodiesel and value-added polyunsaturated fatty acids in view of the cost economics of the overall cost of generation of the biomass.

In the present investigation, the application of NMR spectroscopic techniques was extensively used with an objective to explore the biodiesel potential of biomass cultivated on a lab scale using strains of Chlorella vulgaris and Scenedesmus ecornis. The effect of variation in the composition of culturing medium on the neutral and polar lipids productivity, and fatty acid profile of solvent extracts of microalgae biomass was studied. Determination of unsaturated fatty acid composition (C18:N = 1-3, ω3 C20:5, ω3 C22:6), polyunsaturated fatty esters (PUFEs), saturated fatty acids (SFAs), unsaturated fatty acids (UFAs), free fatty acids (FFAs), and iodine value were achieved from a single (1)H NMR spectral analysis. The results were validated by (13)C NMR and GC-MS analyses. It was demonstrated that newly developed methods based on (1)H and (13)C NMR techniques are direct, rapid, and convenient for monitoring the microalgae cultivation process for enhancement of lipid productivity and their quality aspects in the solvent extracts of microalgal biomasses without any sample treatment and prior separation compared to other methods. The fatty acid composition of algae extracts was found to be similar to vegetable and fish oils, mostly rich in C16:0, C18:N (N = 0 to 3), and n-3 omega polyunsaturated fatty acids (PUFAs). The lipid content, particularly neutral lipids, as well as most of the quality parameters were found to be medium specific by both the strains. The newly developed methods based on NMR and ultrasonic procedure developed for efficient extraction of neutral lipids are cost economic and can be an effective aid for rapid screening of algae strains for modulation of lipid productivity with desired biodiesel quality and value-added products including fatty acid profile.

Analytical strategies based on NMR ( 1 H and 13 C), IR (infrared), and GC (gas chromatography) techniques have been developed for the molecular level characterization of Soxhlet and ultrasonic solvent extracts of yeast biomass samples generated on a lab scale by different yeast, feed, and diverse culture conditions, with an objective to explore biodiesel potential. The extraction efficiency of each solvent (cyclohexane, chloroform, methanol) toward extraction of neutral lipids (total glycerides (TG), free fatty acids (FFA), and polar lipids have been determined and compared with regards to the nature of fatty acid components extracted in each solvent fractions. The fatty acid composition of yeast extracts has been found to be similar to vegetable oils, mostly rich in C16:0, 18:0, and C18:N (N = 1−3) fatty acids as indicated by the combined NMR, GC, and GC-MS analyses. The analytical protocol developed has established that 1 H NMR techniques can be used directly and rapidly without any sample treatment and prior separation to determine total neutral lipid content (TG, FFA), nature of fatty acids/ester, polyunsaturated fatty esters (PUFE), iodine value, etc. NMR results of nature of unsaturated fatty acids/esters (C18:N, N = 1−3) have been validated by GC and GC-MS analyses. The results have shown the presence of C18:1 and C18:2 as the predominant unsaturated fatty acid components besides common saturated fatty acids. The content and composition of biomass has been found to be specific to types of yeast and feed used for cultivation. The NMR methods offer great potential for rapid screening of yeast for generation of yeast biomass with desired lipid content, quality, and biodiesel potential and value added PUFE, keeping in view of the cost economics of overall generation cost of the biomass.

Dedicated to the memory of Detlef Schröder for his outstanding contributions to mass spectrometryKeywords: Acidity / Carboxylic acids / Mass spectrometry / Phenols / ProtomersTo address the title question, the relative intrinsic acidities of phenol and benzoic acid as well as the isomeric family of ortho-, meta-, and para-hydroxybenzoic acids were compared. Dissociation of the [PhCO 2 ···H···OPh] -proton-bound dimer showed slightly greater acidity for benzoic acid. Using traveling-wave ion mobility mass spectrometry (TWIM-MS) with CO 2 as the drift gas and post-TWIM collision-induced dissociation, the gaseous deprotonated molecules of the isomeric hydroxybenzoic acids were properly separated and characterized. For the para isomer, an intrinsic gas-phase acidity order inverse to that in solution was found, as before, that is, the phenol site of para-hydroxybenzoic acid was found to be considerably more acidic than its benzoic acid

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A direct, rapid, and convenient method for the determination of iodine value (IV) of biodiesels from vegetable oils has been developed by the application of nuclear magnetic resonance (500 and 600 MHz 1 H NMR) techniques. The method is based on the correlation of integrated intensities (I us ) of unsaturated hydrogen in the 1 H NMR spectra of biodiesels with (1) the predetermined iodine values by the standard iodometry based method (EN 14111), ( 2) iodine values determined from the fatty acid profile by GC-MS method, and (3) iodine values from the absolute number of double bonds (ADB) determined by 1 H NMR. The simple mathematical equation, IV = K(I us ) (K = 15.78), derived from the correlations, provides a direct method for the determination of iodine value in the range of 5−140 g of I 2 /(100 g). The constant "K" has been found to be independent of the nature and source of vegetable oils. It is equally applicable to blends of biodiesels, blends of biodiesel and oils in all proportions, and biodiesel samples containing glyceride impurities. The statistical comparison between NMR, EN 14111, and GC-MS methods indicates that NMR method provides equivalent results with acceptable precision. The quantitative NMR (q 1 H NMR) protocol for the optimization of acquisition and processing parameters have been established for achieving better precision on 500 and 600 MHz instruments.

PAPERMarcos N. Eberlin et al. Typifi cation and quality control of the Andiroba (Carapa guianensis) oil via mass spectrometry fi ngerprinting Typification and quality control of the Andiroba (Carapa guianensis) oil via mass spectrometry fingerprinting

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