Characterization of Palm Fatty Acid Distillate, Diacylglycerol Regioisomers, and Esterification Products Using High-Performance Size Exclusion Chromatography.

High-performance size exclusion chromatography (HPSEC) equipped with an evaporative light scattering detector (ELSD) was utilized for characterization of palm fatty acid distillate (PFAD) and its esterified products, with a particular focus on lipid profiles and diacylglycerol (DAG) regioisomers. The separation of triacylglycerol (TAG), DAG, monoacylglycerol (MAG), and free fatty acid (FFA) was achieved through a single 100-Å Phenogel column, coupled with a 2-cm C18 guard, utilizing toluene/acetic acid (100:0.25, v/v) as the mobile phase. This separation was based on size sieving principles and the interactions between the hydroxyl group(s) and the Phenogel matrix. The limit of detection (LOD) and limit of quantification (LOQ) for the esterified PFAD products analyzed by this method fell within the range of 4.8-5.5 µg/mL and 14.7-16.7 µg/mL, respectively. Additionally, the same column, paired with a 2-cm silica guard and a mobile phase comprised of toluene/isooctane/acetic acid (35:65:0.15, v/v/v), was used for the characterization of DAG regioisomers within the esterified PFAD. LODs and LOQs for sn-1,3-DAG and sn- 1,2-DAG were determined to be 39.2 and 118.7 µg/mL, and 32.8 and 99.5 µg/mL, respectively. Investigation of esterified PFAD products prepared using 4% H2SO4 at 120°C. After 2 h, the analysis revealed the highest MAG content at 31.85%, accompanied by 51.54% DAG, 2.35% TAG, and a residual 14.27% FFA. Notably, as the reaction time extended, the MAG content decreased, while both DAG and TAG levels exhibited an increasing trend. Further examination of DAG regioisomers during PFAD esterification, under varying catalyst concentrations (2-10%) and reaction temperatures (80-140°C), demonstrated a significant increase in the percentage of sn-1,3-DAG, inversely correlated with the reduction in FFA from 2% H 2 SO 4 and 80°C onwards. Remarkably, the percentage of sn-1,2-DAG remained relatively stable regardless of changes in catalyst concentrations or temperatures, confirming its susceptibility to isomerization into the thermodynamically more stable sn-1,3-DAG form. This study provides valuable insights into the composition and behavior of esterified PFAD products.

Simultaneous Determination of Vitamin E and γ-Oryzanol in Rice Bran Oil via HPSEC-PDA without Sample Pretreatment.

Vitamin E (tocopherols and tocotrienols) and γ-oryzanol are two minor constituents of rice bran oil (RBO) and are known to be potential bioactive compounds. The content of γ-oryzanol, a unique antioxidant found only in RBO, is a key factor in determining the retail price of the oil. Limitations of conventional HPLC columns for vitamin E and γ-oryzanol analysis are the alteration of these components and the time-consuming need for pretreatment of samples by saponification. High-performance size exclusion chromatography (HPSEC) equipped with a universal evaporative light scattering detector (ELSD) is a versatile tool for screening optimum mobile phase conditions because components of the sample can be separated and detected in the same run. In this work, the RBO components (triacylglycerol, tocopherols, tocotrienols, and γ-oryzanol) assessed on a single 100-Å Phenogel column using ethyl acetate/isooctane/acetic acid (30:70:0.1, v/v/v) as the mobile phase provided baseline separations (R s >1.5) with a total run time of 20 min. The HPSEC condition was then transferred to determine the content of tocopherols, tocotrienols, and γ-oryzanol in RBO products using a selective PDA detector. The limit of detection (LOD) and limit of quantification (LOQ) of α-tocopherol, α-tocotrienol, and γ-oryzanol were 0.34 and 1.03 µg/mL, 0.26 and 0.79 µg/mL and 2.04 and 6.17 µg/mL, respectively. This method was precise and accurate, with a percentage of relative standard deviation (%RSD) of the retention time of less than 0.21%. The intra-day and inter-day variations were 0.15-5.05% and 0.98-4.29% for vitamin E and γ-oryzanol, respectively. The recoveries of tocopherols, tocotrienols, and γ-oryzanol ranged between 90.75% and 107.98%. Thus, the developed HPSEC-ELSD-PDA method is a powerful analytical tool for determining the vitamin E and γ-oryzanol present in oil samples without requiring any sample pretreatment.

A Simple and Efficient Method for Synthesis and Extraction of Ethyl Ferulate from γ-Oryzanol.

Ethyl ferulate (EF) is a ferulic acid (FA) derivative with high commercial value. It is not found naturally and is mostly synthesized from FA via esterification with ethanol. The present work aimed to synthesize the EF from γ-oryzanol, a natural antioxidant from rice bran oil via acid-catalyzed transethylation at refluxing temperature of ethanol. The reaction was optimized by central composite design (CCD) under response surface methodology. Based on the CCD, the optimum condition for the synthesis of EF from 0.50 g of γ-oryzanol was as follows: γ-oryzanol to ethanol ratio of 0.50:2 (g/mL), 12.30% (v/v) H2SO4, and a reaction time of 9.37 h; these conditions correspond to a maximum EF yield of 87.11%. Moreover, the optimized transethylation condition was further validated using 12.50 g of γ-oryzanol. At the end of the reaction time, distilled water was added as antisolvent to selectively crystallize the co-products, phytosterol and unreacted γ-oryzanol, by adjusting the ethanol concentration to 49.95% (v/v). The recovery yield of 83.60% with a purity of 98% of EF was achieved. In addition, the DPPH and ABTS assays showed similar antioxidant activities between the prepared and commercial EF.

Simulation of peak position and response profiles in comprehensive two-dimensional gas chromatography.

Approaches to simulate peak time and intensity profiles of compounds in comprehensive two dimensional gas chromatography (GC × GC) were developed, and which are demonstrated for separation of a mixture of saturated and unsaturated fatty acid methyl esters (FAME) using a range of column sets. The simulation of first and second dimension time (1tR and 2tR) of FAME relies on use of a Gibbs energy additivity approach to correlate with the structures of FAME. First and second dimension peak standard deviations (1σ and 2σ) of the compounds were further calculated from the 1tR and 2tR data according to the plate height concept which provided good agreement between the predicted and experimental peak widths at half height in one dimension GC (1DGC) with an overall R2 of 0.9628. The effect of 1σ distortion caused by the modulation process was also taken into account in the peak width simulation where the simulated 1σ data were rounded up to multiples of the scale of modulation period (PM). Two dimension Gaussian equations were then used to generate GC × GC results (2D contour plots) from simulated 1tR, 2tR, 1σ and 2σ data for FAME separation on different column sets employing ionic liquid and conventional columns. Good overall correlations between experimental and simulated 1tR and 2tR were obtained with R2 of 0.9951 and 0.9802, respectively, and the simulated 2D contour plots were an acceptable match with the experimental results.

Recovery of γ-Oryzanol from Rice Bran Acid Oil by an Acid-base Extraction Method with the Assistance of Response Surface Methodology.

A rapid and low energy consumption method for the recovery of γ-oryzanol from rice bran acid oil (RBAO), a byproduct of rice bran oil (RBO) refining, is presented. The RBAO was converted to the fatty acid ethyl ester (FAEE) and was used as the starting material. The dissolved γ-oryzanol was separated from the FAEE using an acid-base extraction method with alkaline aqueous ethanol and hexane as extraction media. A systematic investigation of the extraction yield was carried out by applying response surface methodology (RSM) based on central composite design (CCD) and Derringer's desirability function. The concentration of NaOH, the percentage of ethanol in water, the hexane content and their interactions showed significant effects on the yield of γ-oryzanol and FAEE. The optimal extraction conditions were as follows: extraction time of 1 min at room temperature (28-32°C); extraction medium: 1.855 M NaOH; 75.91% ethanol in water and 20.59% hexane in the total volume of the extractant; and FAEE to extractant ratio of 1:10 corresponding to a maximum γ-oryzanol yield of 75.82±3.44% and the desired FAEE yield of 54.42±7.80%. The γ-oryzanol-rich fraction was further purified by washing with a 2% Na2CO3 solution, obtaining 69.94% recovery yield with 89.90% purity of γ-oryzanol. The purified γ-oryzanol showed good scavenging activity on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and the ABTS radical and was comparable to the commercial product, clearly suggesting that the presented process was efficient and feasible.

Structure and rheology of oleogels made from rice bran wax and rice bran oil.

Structural and rheological properties of oleogels consisting of 0.5-25 wt% rice bran wax (RBX) in rice bran oil (RBO) were explored. RBX was an efficient, thermoreversible oleogelator capable of structuring RBO at concentrations as low as 0.5 wt% RBX. A qualitative temperature-composition phase diagram showed that oleogels containing higher concentrations of RBX were expectedly the most resistant to melting. In oleogels at higher RBX concentrations, polarized light microscopy revealed the presence of a network of interlinked, long aspect ratio wax crystal needles up to 50 µm long. Upon heating, RBX crystals did not undergo any structural transition, based on the constant short spacings at ~ 4.16 and ~ 3.73 Å, indicative of an orthorhombic subcell, and d001 long spacing at 74-76 Šthat persisted until RBX fusion. This long spacing was ascribed to the presence of wax esters consisting of long-chain saturated fatty acids (C24 and C22) esterified to C28 - C34 saturated fatty alcohols. During cooling from 90 to 20 °C, the increase in oleogel viscosity resulting from the RBX liquid-solid phase transition was corroborated by DSC-based crystallization onset and enthalpy data. Similarly, elastic moduli and hardness both rose with increasing RBX concentration. This study, which demonstrated that RBX can structure RBO with distinct concentration-dependent properties, serves as the foundation for the development of oleogel-based approaches to saturated and trans fats replacement in processed foods.

Ionic liquid phases with comprehensive two-dimensional gas chromatography of fatty acid methyl esters.

New generation inert ionic liquid (iIL) GC columns IL60i, IL76i and IL111i, comprising phosphonium or imidazolium cationic species, were investigated for separation of fatty acid methyl esters (FAME). In general, the iIL phases provide comparable retention times to their corresponding conventional columns, with only minor selectivity differences. The average tailing factors and peak widths were noticeably improved (reduced) for IL60i and IL76i, while they were slightly improved for IL111i. Inert IL phase columns were coupled with conventional IL columns in comprehensive two-dimensional GC (GC × GC) with a solid-state modulator which offers variable modulation temperature (TM), programmable TM during analysis and trapping stationary phase material during the trap/release (modulation) process, independent of oven T and column sets. Although IL phases are classified as polar, relative polarity of the two phases comprising individual GC × GC column sets permits combination of less-polar IL/polar IL and polar IL/less-polar IL column sets; it was observed that a polar/less-polar column set provided better separation of FAME. A higher first dimension (1D) phase polarity combined with a lower 2D phase polarity, for instance 1D IL111i with 2D IL59 gave the best result; the greater difference in 1D/2D phase polarity results in increasing occupancy of peak area in the 2D space. The IL111i/IL59 column set was selected for analysis of fatty acids in fat and oil products (butter, margarine, fish oil and canola oil). Compared with the conventional IL111, IL111i showed reduced column bleed which makes this more suited to GC × GC analysis of FAME. The proposed method offers a fast profiling approach with good repeatability of analysis of FAME.

Gibbs energy additivity approaches to QSRR in generating gas chromatographic retention time for identification of fatty acid methyl ester.

The Gibbs energy additivity method was used to correlate the retention time (t R) of common fatty acid methyl esters (FAMEs) to their chemical structures. The t R of 20 standard FAMEs eluted from three capillary columns of different polarities (ZB-WAXplus, BPX70, and SLB-IL111) under both isothermal gas chromatography and temperature-programmed gas chromatography (TPGC) conditions were accurately predicted. Also, the predicted t R of FAMEs prepared from flowering pak choi seed oil obtained by multistep TPGC with the BPX70 column were within 1.0% of the experimental t R. The predicted t R or mathematical t R (t R(math)) values could possibly be used as references in identification of common FAMEs. Hence, FAMEs prepared from horse mussel and fish oil capsules were chromatographed on the BPX70 and ZB-WAXplus columns in single-step and multistep TPGC. Identification was done by comparison of t R with the t R of standard FAMEs and with t R(math). Both showed correct identifications. The proposed model has six numeric constants. Five of six could be directly transferred to other columns of the same stationary phase. The first numeric constant (a), which contained the column phase ratio, could also be transferred with the adjustment of the column phase ratio to the actual phase ratio of the transferred column. Additionally, the numeric constants could be transferred across laboratories, with similar correction of the first numeric constant. The TPGC t R predicted with the transferred column constants were in good agreement with the reported experimental t R of FAMEs. Moreover, hexane was used in place of the conventional t M marker in the calculation. Hence, the experimental methods were much simplified and practically feasible. The proposed method for using t R(math) as the references would provide an alternative to the uses of real FAMEs as the references. It is simple and rapid and with good accuracy compared with the use of experimental t R as references.

Separation of Vitamin E on a 100-Å Phenogel Column.

The effect of π-electrons and hydroxyl group on the separations of vitamin E on a swelling-controlled polystyrene-divinylbenzene (Phenogel) column using toluene/isooctane as the mobile phase was investigated. The effect of the π-electrons was demonstrated in the baseline separation of α-tocopherol and α-tocotrienol on a 100-Å Phenogel column. In addition, baseline separation of α-, (ß- + Î³-)- and δ-tocopherol could be achieved on this column. The separation mechanism of these isomers are due to the difference in the interactions between the hydroxyl group on the chromanol ring of each tocopherol and the gel matrix caused by the steric hindrance of methyl group(s). It was concluded that solutes of the same molecular size but different in the polar groups could be separated on a high performance size-exclusion chromatography by controlling the swelling of the gel matrix via modification of the mobile phase.

Prediction of gas chromatographic retention times of esters of long chain alcohols and fatty acids.

The linear free energy of solution (DeltaG) relationship (DeltaG=DeltaGo+zdeltaG) for compounds of different carbon atoms (z) in the same homologous series is expanded and modified to cover compounds with two different hydrocarbon side chains. The expanded equation is successfully used to predict the retention times (tR) of standard esters of long chain alcohols and fatty acids of different chain lengths in both isothermal and temperature-programmed gas chromatography (TPGC). Approximately 90% of the 125 predicted tR values have a difference of less than 1.00% from the actual tR and the highest difference is 1.26%. Two different temperature gradients in TPGC are tested. The expanded equation can be used to forecast the tR of TPGC with good accuracy. The highest difference is +/-1.40% and +/-1.00% for the temperature gradients of 2 degrees C and 4 degrees C/min, respectively. However, the increments in free energy per carbon atom (zdeltaG) of the alcohol and acid are approximately equal but have slightly different temperature sensitivities. Therefore, it is very difficult to separate esters of different acid and alcohol chain length but with the same total carbon numbers. Furthermore, the difference in temperature sensitivities for the acid and alcohol side chains renders them to be inversely eluted at different temperatures.

An alternative approach for the estimation of equivalent temperature in gas chromatography.

The temperature at which the isothermal retention time (tRiso) is equal to the temperature-programmed retention time (tRTPGC) or the isothermal retention index (Iiso) is equal to the temperature-programmed retention index (ITPGC) is defined as the equivalent temperature (Teq). The Teq of one-, two-, three-, and four-step temperature-programmed gas chromatography (TPGC) of unsaturated fatty acid methyl esters (FAMEs) from Chinese mustard seed oil is calculated. All of the tR values obtained at the Teq (tRTeq) are very close to the tRTPGC. The highest difference for each chromatogram is less than 0.5%. The slight deviation may partly arrive from the difficulty in setting the desired carrier gas flow rate. Also, good agreement among equivalent chain length values determined by different methods is observed, including the graphical method at the Teq. Therefore, it is speculated that the proposed method may facilitate the GC identification of FAMEs as well as other organic compounds in TPGC by using the available isothermal retention index database.

Molecular interconversion behaviour in comprehensive two-dimensional gas chromatography.

Comprehensive two-dimensional gas chromatography (GC x GC) is shown to provide information on dynamic molecular behaviour (interconversion), with the interconversion process occurring on both columns in the coupled-column experiment. The experiment requires suitable adjustment of both experimental conditions and relative dimensions of each of the columns. In this case, a longer column than normally employed in GC x GC allows sufficient retention duration on the second column, which permits the typical plateau-shape recognised for the interconversion process to be observed. The extent of interconversion depends on prevailing temperature, retention time, and the phase type. Polyethylene glycol-based phases were found to result in high interconversion kinetics, although terephthalic acid-terminated polyethylene glycol had a lesser extent of interconversion. Much less interconversion was seen for phenyl-methylpolysiloxane and cyclodextrin phases. This suggests that for the oximes, interconversion largely occurs in the stationary phase. Examples of different extents of interconversion in both dimensions are shown, including peak coalescence on the first column with little interconversion on the second column.