Trans-esterification of fatty acids from microorganisms and human blood serum by trimethylsulfonium hydroxide (TMSH) for GC analysis

Summary Trimethylsulfonium hydroxide (TMSH) can convert fatty acids into the corresponding fatty acid methyl esters (FAMEs) in a single step. These fatty acids may also be bound in biomolecules such as phospholipids and/or glycerides. Complex mixtures of saturated and unsaturated FAMEs which may contain hydroxy and cylopropyl groups are obtained by trans-esterification; they can easily be separated in most cases by capillary GC. When FAMEs are generated from different microorganisms e.g. bacteria the patterns of the chromatograms are characteristic. Examples of characteristic patterns of bacteria with different cell wall structures are shown. The described method of transesterification can also be applied directly to blood serum without sophisticated sample pretreatment. The profiles of the chromatograms match well those described in the literature obtained by other methods of trans-esterification or sample preparation.     

Determination of lipids in infant formula powder by direct extraction methylation of lipids and fatty acid methyl esters (FAME) analysis by gas chromatography.

Fatty acid methyl esters (FAMEs) of pure triglyceride standards, oils, and fat from dry matrixes were formed by transesterification using sodium methoxide in methanol-hexane. FAMEs were produced by direct addition of sodium methoxide-hexane to samples and heating to simultaneously extract and transesterify acyl lipids. FAMEs were quantitated by capillary gas chromatography (GC) over a fatty acid concentration range of 0 to 1.7 mg/mL (r > or = 0.9997). Total fat was calculated as the sum of individual fatty acids expressed as triglyceride equivalents, in accordance with nutrition labeling guidelines. Saturated, polyunsaturated, and monounsaturated fats were calculated as sums of individual free fatty acids. Absolute recoveries determined from individual fatty acids in test samples ranged from 69.7 to 106%. Recoveries (relative to the C13:0 internal standard) for individual fatty acids in test samples ranged from 95 to 106%. Reproducibility was constant at each fatty acid level in the reaction mixture (n = 5, coefficient of variation [CV] < 2%). Absolute recovery determined from the sum of total fatty acids in standard reference material (SRM) 1846 (powdered infant formula) was 96.4%. Analysis of SRM 1846 gave results that agreed closely with the certified fat and fatty acid values. Analysis of commercial infant formula gave results that were comparable to those obtained with AOAC Method 996.01. The direct extraction methylation procedure is rapid, and the transesterification of acyl lipids to form FAMEs is complete within 15 min. Classical saponification and refluxing are not required. This method provides FAMEs free of interferences and easily quantitated by GC or confirmed by GC/mass spectrometry (MS). Unambiguous MS identification of individual FAMEs derived from pure standards, SRM 1846, and powdered infant formula product was obtained.     

Trimethylsulfonium hydroxide as derivatization reagent for the chemical investigation of drying oils in works of art by gas chromatography

A procedure for the determination of fatty acids (FA) and glycerol in oils has been developed. The method includes a derivatization step of the FAs into their methyl esters or a transesterification of the triacylglycerols with trimethylsulfonium hydroxide (TMSH), respectively. The analysis is carried out by gas chromatography with parallel flame ionization and mass spectrometric detection. The parameters involved in the transesterification reaction were optimized. Only the stoichiometric ratio of TMSH:total FA amount showed a significant influence on the reaction yield. Relative standard deviations for 10 replicates were below 3% for all FAs studied and their linearity range was 0.5-50 mmol/L, when using heptadecanoic acid as an internal standard. The final procedure was rapid and required little sample handling. It was then tested on fresh oil samples and presented satisfying results, in agreement with previous works.     

Production of biodiesel fuel by transesterification of rapeseed oil

Fatty acid methyl esters (FAMEs) show large potential applications as diesel substitutes, also known as biodiesel fuel. Biodiesel fuel as renewable energy is an alternative that can reduce energy dependence on petroleum as well as air pollution. Several processes for the production of biodiesel fuel have been developed. Transesterification processes under alkali catalysis with short-chain alcohols give high yields of methyl esters in short reaction times. We investigated transesterification of rapeseed oil to produce the FAMEs. Experimental reaction conditions were molar ratio of oil to alcohol, concentration of catalyst, type of catalyst, reaction time, and temperature. The conversion ratio of rapeseed oil was enhanced by the alcohol:oil mixing ratio and the reaction temperature.     

Analysis of the Triglycerides and the Free and Derivatized Fatty Acids in Fish Oil by Capillary Electrochromatography

Capillary ElectroChromatography (CEC) on a fused silica capillary column (40 cm L × 100 μm i.d.) packed with 3 μm octadecylsilica (ODS) was evaluated for the analysis of the triglycerides and their fatty acids in fish oils, and more especially in the oil of Moroccan Sardinia pilchardus. The very high complexity of the lipids in fish oil is well illustrated by CEC with a nonaqueous mobile phase consisting of acetonitrile/isopropanol/n-hexane in the ratio 57/38/5 to which 50 mM ammonium acetate has been added. In order to unravel the complexity, the oil was hydrolyzed and the free fatty acids (FFAs), the methyl esters (FAMEs), and the phenacyl esters (FAPEs) were analyzed by CEC on the same column used for the analysis of the triglycerides. Isocratic elution was achieved with the mobile phase acetonitrile/50 mM MES pH 6 in ratio 9/1. The migration characteristics of FFAs, FAMEs, and FAPEs are compared. The analysis of FAPE derivatives has the advantage that quantification applying UV detection is possible and moreover that the number of double bonds in the fatty acid chains can be elucidated by measuring the UV abundance ratio 240/210 nm.     

Accurate determination of the degree of substitution of long chain cellulose esters

The determination of the degree of substitution (DS) of fatty acid cellulose esters with alkyl chain lengths from C8 to C18 was performed by direct transesterification with trimethylsulphonium hydroxide (TMSH) using tert-butyl methyl ether (MTBE) as a solvent. Transesterification was demonstrated to be quantitative at 75 °C in 60 min. The quantification of the formed fatty acid methyl esters was performed by gas chromatography (GC). After the optimization of the method, long chain cellulose esters (LCCE) could be analyzed in a wide range of DS. The obtained values were compared to those given by other existing protocols. LCCE with DS-values in a range of 5 × 10⁻⁵ to 3 were analyzed with high accuracy. Reproducibility is weakened for high DS values if the sample has a compact aspect limiting the accessibility of TMSH to the ester functions. This method can also be suitable for the analysis of mixed cellulose esters.     

Accurate and reliable quantification of total microalgal fuel potential as fatty acid methyl esters by <Emphasis Type="Italic">in situ</Emphasis> transesterification

In the context of algal biofuels, lipids, or better aliphatic chains of the fatty acids, are perhaps the most important constituents of algal biomass. Accurate quantification of lipids and their respective fuel yield is crucial for comparison of algal strains and growth conditions and for process monitoring. As an alternative to traditional solvent-based lipid extraction procedures, we have developed a robust whole-biomass in situ transesterification procedure for quantification of algal lipids (as fatty acid methyl esters, FAMEs) that (a) can be carried out on a small scale (using 4–7 mg of biomass), (b) is applicable to a range of different species, (c) consists of a single-step reaction, (d) is robust over a range of different temperature and time combinations, and (e) tolerant to at least 50% water in the biomass. Unlike gravimetric lipid quantification, which can over- or underestimate the lipid content, whole biomass transesterification reflects the true potential fuel yield of algal biomass. We report here on the comparison of the yield of FAMEs by using different catalysts and catalyst combinations, with the acid catalyst HCl providing a consistently high level of conversion of fatty acids with a precision of 1.9% relative standard deviation. We investigate the influence of reaction time, temperature, and biomass water content on the measured FAME content and profile for 4 different samples of algae (replete and deplete Chlorella vulgaris, replete Phaeodactylum tricornutum, and replete Nannochloropsis sp.). We conclude by demonstrating a full mass balance closure of all fatty acids around a traditional lipid extraction process.     

AgOTf‐catalyzed transesterification of β‐keto esters

AgOTf proved to be an effective catalyst for the transesterification of β‐keto esters with primary, secondary and tertiary alcohols. The products were obtained in high yield within a reasonable reaction time period. The kinetics of the transesterification reaction were also studied and the reaction was found to follow second‐order kinetics. Copyright © 2012 John Wiley & Sons, Ltd.     

Phase and chemical equilibria in the transesterification reaction of vegetable oils with supercritical lower alcohols

Calculations of thermodynamic data are performed for fatty acid triglycerides, free fatty acids, and fatty acid methyl esters, participants of the transesterification reaction of vegetable oils that occurs in methanol. Using the obtained thermodynamic parameters, the phase diagrams for the reaction mixture are constructed, and the chemical equilibria of the esterification reaction of free fatty acids and the transesterification reaction of fatty acid triglycerides attained upon treatment with supercritical methanol are determined. Relying on our analysis of the obtained equilibria for the esterification reaction of fatty acids and the transesterification reaction of triglycerides attained upon treatment with lower alcohols, we select the optimum conditions for performing the reaction in practice.     

Calculations of phase equilibria for mixtures of triglycerides,fatty acids,and their esters in lower alcohols

The objects of study were mixtures containing triglycerides and lower alcohols and also the products of the transesterification of triglycerides, glycerol and fatty acid esters. The Redlich-Kwong-Soave equation of state was used as a thermodynamic model for the phase state of the selected mixtures over wide temperature, pressure, and composition ranges. Group methods were applied to determine the critical parameters of pure substances and their acentric factors. The parameters obtained were used to calculate the phase diagrams and critical parameters of mixtures containing triglycerides and lower alcohols and the products of the transesterification of triglycerides, glycerol and fatty acid esters, at various alcohol/oil ratios. The conditions of triglyceride transesterification in various lower alcohols providing the supercritical state of reaction mixtures were selected.     

Unnatural Amino Acids. 2. Simple Method of Obtaining Esters of Aziridine-2-carboxylic Acids by a Transesterification Reaction

A series of N-unsubstituted esters of aziridine-2-carboxylic acid has been obtained by transesterification in basic medium using primary, secondary, and tertiary alcohols. Methods of transesterification using various bases (K₂CO₃, ROLi, t-BuOK) have been compared. Transesterification with lithium alcoholates also affords the possibility of obtaining esters of N-substituted aziridine-2-carboxylic acids. Transesterification of chiral esters proceeds with retention of the configuration of the chiral center.     

毛细管气相色谱法鉴定分析杆菌

本文利用ＯＶ－１０１熔融石英毛细管柱，对１５种标准分枝杆菌的色谱特征进行了研究，得到了特异性指纹图。并对４２株临床菌株进行了鉴别。     

Candida antarctica B Lipase-Loaded Microreactor for the Automated Derivatization of Lipids

A key bottleneck in the profiling of lipids is the multistep derivatization required prior to gas chromatography (GC) analysis. A single in-vial lipid derivatization and analysis may significantly minimize sample loss and improve analytical sensitivity. A cotton fiber-supported poly(glycidylmethacrylate-co-ethylene glycol dimethacrylate) polymer microbrush microreactor loaded with Candida antarctica lipase B was developed for the facile conversion of triacylglycerols into fatty acid ethyl ester derivatives for gas chromatograph–mass spectrometry (GC–MS) analysis. The polymer microbrush microreactor was fabricated in effort to provide efficient, simplified, cost effective, and high-throughput GC–MS determination of triacylglycerols. The polymer microbrush microreactor was used as an in-vial triacylglycerol transesterification platform, with economical sample consumption of less than or equal to 100?µL and significant reduction of reagents. To evaluate the polymer microbrush microreactor performance for lipids, a triolein standard and camelina oil triacylglycerols were quantitatively transformed into ethyl oleate and fatty acid ethyl esters, respectively, following a 3?h reaction time. The lipase-loaded cotton fiber-supported poly(glycidylmethacrylate-co-ethylene glycol dimethacrylate) polymer microbrush microreactors were reusable for up to five times for quantitative transesterification with minimal loss of lipase activity.     

Fast GC for the analysis of fats and oils

Fast and conventional GC techniques were both applied to ten different lipidic matrices and the results then compared. The fats and oils were of fish, animal, and vegetable origin and were all simultaneously transesterified with acidic methanol before performing batch analysis of the fatty acid methyl esters (FAMEs) obtained. All FAMEs samples were consecutively analyzed three times by each method. The fast method significantly reduced the time required for analysis by a factor of 5 while maintaining a similar resolution. Furthermore, the reproducibility of relative quantitative data was measured on going from one method to the other. Peak identification was achieved through conventional GC‐MS in combination with linear retention index values contained in a home library and information derived from comprehensive 2D GC group patterns.     

Reaction efficiency of organic alkalis with various classes of lipids during thermally assisted hydrolysis and methylation

Reaction efficiencies of two organic alkalis, tetramethylammonium hydroxide (TMAH) and trimethylsulfonium hydroxide (TMSH), with lipids during thermally assisted hydrolysis and methylation (THM) were examined focusing on (1) the types of lipids and (2) degree of unsaturation of fatty acid moieties. Different types of lipids such as triglycerides, phospholipids, free fatty acids and cholesteryl esters containing saturated, monounsaturated or polyunsaturated fatty acid (PUFA) residues were subjected to THM-gas chromatography (GC) in the presence of TMAH or TMSH. The obtained results revealed that the THM reaction using TMAH allowed almost quantitative methylation of saturated and monounsaturated fatty acid components independently of the classes of lipids. However, strong alkalinity of TMAH brought about isomerization and/or degradation of PUFA components. In contrast, the use of TMSH was effective to highly sensitive detection of PUFA as well as saturated and monounsaturated fatty acid components contained in triglycerides, phospholipids (phosphatidylcholines) and free fatty acids. On the other hand, TMSH was proved to react hardly with any kind of fatty acid residues in cholesteryl esters due to their steric hindrance.     

Thermal decomposition and stability of fatty acid methyl esters in supercritical methanol

In recent years, non-catalytic supercritical processes for biodiesel production have been proposed as alternative environmentally friendly technologies. However, conditions of high temperature and pressure that occur while biodiesel is in supercritical fluid can cause fuel degradation, resulting in low yield. In this study, we performed the thermal decomposition of fatty acid methyl esters (FAMEs) in supercritical methanol at temperatures ranging from 325 °C to 420 °C and pressure of 23 MPa to investigate the degradation characteristics and thermal stability of biodiesel. The primary reactions we observed were isomerization, hydrogenation, and pyrolysis of FAMEs. The main pathway of degradation was deduced by analyzing the contents of degradation products. We found that if FAME has shorter chain length or is more saturated, it has higher thermal stability in supercritical methanol. All FAMEs remained stable at 325 °C or below. Based on these results, we recommend that transesterification reactions in supercritical methanol should be carried out below 325 °C (at 23 MPa) and 20 min, the temperature at which thermal decomposition of FAMEs begins to occur, to optimize high-yield biodiesel production.     

Analysis of biodiesel/petroleum diesel blends with comprehensive two-dimensional gas chromatography

Comprehensive two-dimensional gas chromatography (GCxGC) is used to analyze petroleum diesel, biodiesel, and biodiesel/petroleum diesel blends. The GCxGC instrument is assembled from a conventional gas chromatograph fitted with a simple, in-line fluidic modulator. A 5% phenyl polydimethylsiloxane primary column is coupled to a polyethylene glycol secondary column. This column combination generates chromatograms where the fatty acid methyl esters (FAMEs) found in biodiesel occupy a region that is also populated by numerous cyclic alkanes and monoaromatics found in petroleum. Fortunately, the intensities of the petroleum hydrocarbon peaks are far lower than the intensities of the FAME peaks, even for blends with low biodiesel content. This allows the FAMEs to be accurately quantitated by direct integration. The method is calibrated by analyzing standard mixtures of soybean biodiesel in petroleum diesel with concentrations ranging from 1 to 20% v/v. The resulting calibration curve displays excellent linearity. This curve is used to determine the concentration of a B20 biodiesel/petroleum diesel blend obtained from a local retailer. Excellent precision and accuracy are obtained.     

Biodiesel Production from Vegetable Oils and Animal Fat over Solid Acid Double-Metal Cyanide Catalysts

Biodiesel comprises of fatty acid alkyl esters prepared from vegetable oils or animal fat by esterification/transesterification with short-chain alcohols (methanol or ethanol, for example). It is a biodegradable renewable fuel. Its production is growing exponentially due to greater concerns about environmental protection and depletion of fossil fuel resources. Further, its production from non-edible oils and animal fat is more desirable than from edible oils due to lower cost of non-edible feedstocks and elimination of food verses fuel issues. Solid acid catalysts are ideal for conversion of such low-grade oils to biodiesel. Biodiesel from non-edible oils can be produced by two methods: (1) simultaneous esterification of fatty acids and transesterification of fatty acid glycerides and (2) hydrolysis of glycerides followed by esterification. This account reports the catalytic performance of solid, Fe–Zn double-metal cyanide (DMC) complexes and other acid catalysts in these transformations for biodiesel production. The factors influencing the catalytic performance of the solid acid catalysts in biodiesel production are discussed.     

An alternative approach for predicting peak width in gas chromatography

Summary The linear relationship between natural logarithm of width factor (lnp′)and natural logarithm of retention factor (lnk) is demonstrated. This relationship is then used to establish the relationship between (lnp′), absolute temperature (T), and carbon number (z), as follows: Inp′=A+bz+c/T+dz/T where A, b, c and d are thermodynamically related constants. The above equation is used to predict the unadjusted widths (w _R ) ofn-alkanes, fatty alcohols and fatty acid methyl esters (FAMEs) at various temperatures, predicted values are in good agreement with experimental values. The above equation can be used to predict the width of FAMEs from rice bran oil. The largest difference between the experimental and predicted values is 0.66 s or 6.32%.     

Preparation of methyl esters of fatty acids with trimethylsulphonium hydroxide—an appraisal

The rates of reaction of the new basic transesterification catalyst, trimethylsulphonium hydroxide (TMSH), have been compared with an established sodium methoxide-catalysed procedure. Although the latter is more rapid, the difference is only of practical importance when cholesterol esters are transesterified. Some losses of polyunsaturated components from fish oils were observed when methods were employed in which TMSH was injected directly into the heated injection port of the gas chromatograph, as had been recommended.