Separation Of Phenolic Compounds Research Articles

Development of a Comprehensive NPxRP System for the Characterization of Stevia rebaudiana

A comprehensive NPxRP system in combination with ultraviolet detection has been developed for the analysis of active components in Stevia rebaudiana extracts. Stevia rebaudiana is a perennial shrub of the Asteraceae (Compositae) family native to certain regions of South America (Paraguay and Brazil). Stevia plant material and extracts are used as sweeteners. The most common approach for the separation of Stevia compounds is normal phase chromatography. However, reversed phase chromatography might also be useful for the separation of phenolic compounds and flavonoids occurring in these matrices.

Concentration of Rutin Model Solutions from Their Mixtures with Glucose Using Ultrafiltration

Separation of polyphenolic phytochemical compounds from their mixtures with sugars is necessary to produce an added-value sugar-reduced extract with high biological activity from fruit juice processing industry waste streams. The separation characteristics of a binary mixture of rutin and glucose using a Pellicon-2 regenerated cellulose ultrafiltration membrane with an area of 0.1 m2 having nominal MWCO of 1,000 Da were investigated, to demonstrate the separation of phenolic compounds from sugars. The effects of the operating variables–transmembrane pressure, feed solution temperature and pH, initial feed concentration and feed flow rate–on the permeate flux and enrichment of rutin, were determined. The permeate flux increased with the increase in transmembrane pressure up to a certain limit and after that the flux remained more or less constant. The optimum transmembrane pressure was within 4–5 bar. The flux increased with the increase in feed solution temperature because of reduced feed viscosity, and better solubility. The concentration of rutin was optimum at lower temperature (30°C), with an enrichment factor of 1.3. The effect of pH on permeate flux was less obvious. Lowering the feed solution pH increased the retention of rutin and the optimum separation was obtained within pH 3–4. The permeate flux decreased with the increase in feed concentration of rutin (concentration range 0.1–0.5 g/L). The enrichment of rutin was significant in the glucose concentration range 0.35–0.5 g/L. The feed flow rate had a significant effect on the flux and separation characteristics. Higher cross-flow through the membrane reduced the fouling by providing a shear force to sweep away deposited materials from the membrane surface. At high feed flow rate, more rutin was retained by the membrane with less sugar permeating through. The optimum feed flow rate was 1.5 L/min. For the separation of rutin (in the retentate) and glucose (in the permeate), the best results were obtained at rutin enrichment of 2.9 and recovery 72.5%, respectively. The performance of this system was further improved by operating it in a diafiltration mode, in which only approx. 11% of glucose remained in the retentate.

1-Hexadecyl-3-methylimidazolium Ionic Liquid as a New Cationic Surfactant for Separation of Phenolic Compounds by MEKC

The ionic liquid 1-hexadecyl-3-methylimidazolium bromide ([C16MIm]Br) has been used as a novel cationic surfactant for separation of phenolic compounds, including quinol, phloroglucinol, resorcinol, phenol, p-cresol, and m-nitrophenol, by micellar electrokinetic capillary chromatography (MEKC). The effects of buffer concentration and pH, concentration of [C16MIm]Br, and applied potential were studied. Use of the optimized buffer (25 mmol L−1 NaH2PO4), 10 mmol L−1 [C16MIm]Br, and an applied potential of −15 kV enables optimum separation with regard to resolution and migration time. The phenolic compounds were detected at 214 nm. The micelle of this long-alkyl-chain imidazolium ionic liquid acts as a pseudo-stationary phase in this MEKC separation.

Comparison of separation power of ultra performance liquid chromatography and comprehensive two‐dimensional liquid chromatography in the separation of phenolic compounds in beverages

In this study, 1-D and 2-D liquid chromatographic systems, namely, conventional HPLC, UPLC, HPLC x HPLC and HPLC x UPLC systems were developed and evaluated for the separation of phenolic acids in wine and juices. In the LC x LC studies, the first dimension separation was based on RPLC and the second dimension was performed with ion-pair chromatography. Three different columns, namely two short columns packed with either 2.5 or 1.7 microm particles and a monolithic column, were tested for the fast second dimension separation. The best results were obtained when the monolithic column was applied for the second dimension separation. The peak capacities for comprehensive 2-D systems varied from 330 to 616.

Recovery of Natural Antioxidants from Olive Mill Wastewater Using Genapol‐X080

AbstractThe possibility of applying cloud point extraction (CPE) using Genapol X‐080 as surfactant for the separation of phenolic compounds from olive mill wastewater was examined. The ability of the surfactant to recover individual and mixtures of polyphenols and tocopherols in various concentrations from aqueous solutions was tested before its application for the recovery of phenols from olive mill wastewater. Many of the examined individual polyphenols were recovered at high percentage. Especially, in the case of Luteolin, low surfactant concentrations were sufficient for quantitative removal. The recovery appeared proportional to the surfactant concentration. The complete recovery of tocopherols was also possible. Total phenol recovery by simple and successive CPE of olive mill wastewater with various concentrations (2, 5 and 20%, v/v) of Genapol X‐080 was up to 89.5%.

Analytical applications of glassy carbon electrodes modified with multi-wall carbon nanotubes dispersed in polyethylenimine as detectors in flow systems

This work reports the advantages of using glassy carbon electrodes (GCEs) modified with multi-wall carbon nanotubes (CNT) dispersed in polyethylenimine (PEI) as detectors in flow injection and capillary electrophoresis. The presence of the dispersion of CNT in PEI at the electrode surface allows the highly sensitive and reproducible determination of hydrogen peroxide, different neurotransmitters (dopamine (D) and its metabolite dopac, epinephrine (E), norepinephrine (NE)), phenolic compounds (phenol (P), 3-chlorophenol (3-CP) and 2,3-dichlorophenol (2,3CP)) and herbicides (amitrol). Sensitivities enhancements of 150 and 140 folds compared to GCE were observed for hydrogen peroxide and amitrol, respectively. One of the most remarkable properties of the resulting electrode is the antifouling effect of the CNT/PEI layer. No passivation was observed either for successive additions (30) or continuous flow (for 30 min) of the compounds under investigation, even dopac or phenol. A critical comparison of the amperometric and voltammetric signal of these different analytes at bare- and PEI-modified glassy carbon electrodes and pyrolytic graphite electrodes is also included, demonstrating that the superior performance of CNT is mainly due to their unique electrochemical properties. Glassy carbon electrodes modified with CNT–PEI dispersion also show an excellent performance as amperometric detector in the electrophoretic separation of phenolic compounds and neurotransmitters making possible highly sensitive and reproducible determinations.

High-performance liquid chromatographic method for the quantification of phenolics in ‘Chyavanprash’ a potent Ayurvedic drug

Quantification of bioactive principles through modern analytical tools is essential for establishing the authenticity, creditability, prescription and usage of Ayurvedic medicines/herbal formulations. ‘Chyavanprash’ is one of the oldest and most popular Ayurvedic preparations, used widely as a health promotive and disease preventive ‘Rasayana’ drug in India and elsewhere. The rejuvenating and tonic properties of ‘Chavanprash’ are considered majorly due to their antioxidant principles, which in turn is due to the presence of phenolic compounds. A simple high-performance liquid chromatography (HPLC) method for the separation and quantitative determination of the major antioxidant compounds from ‘Chyavanprash’ has been developed. The use of Waters Symmetry ® column and an acidic mobile phase enabled the efficient separation of phenolic compounds (catechin, quercetin-3- O-rutinoside, syringic acid and gallic acid) within a 35 min analysis. Validation of the method was done with a view to demonstrate its selectivity, linearity, precision, accuracy and robustness. In addition optimization of the complete extraction of phenolic compounds were also studied.

Separation of phenolic compounds by NP and RP two-dimensional thin layer chromatography on connected plates

Separation selectivity has been optimized in two-dimensional thin-layer chromatography (2D TLC) by connecting diol or silica plates (on which NP chromatograms were developed) to RP-18W plates (on which reversed-phase chromatograms were developed). Retention of test substances was investigated to select optimum chromatographic systems for separation of selected phenolic compounds by 2D TLC. Selection of optimal mobile phases was performed on the basis of plots of retention against mobile phase composition. The next step of the optimization was calculation of statistical data for correlation of R M values for pairs of chromatographic systems — NP diol-RP C 18 or silica-RP C 18 . Orthogonal chromatographic systems were selected on the basis of these correlations and used to separate phenolic compounds present in plant extracts. For example, extracts from Polygonum sp. and Verbascum sp. were separated by use of optimum 2D TLC systems.

Effect of bioactive compounds from Sainfoin ( Onobrychis viciifolia Scop.) on the in vitro larval migration of Haemonchus contortus : role of tannins and flavonol glycosides

Anthelmintic bioactivity against gastrointestinal nematodes has been associated with leguminous forages supporting the hypothesis of a role of condensed tannins. However, the possibility that other compounds might also been involved has received less consideration. Using bio-guided fractionation, the current study aimed at characterizing the biochemical nature of the active compounds present in sainfoin (Onobrychis viciifolia ), previously identified as an anthelmintic leguminous forage. The effects of sainfoin extracts were evaluated on 3rd-stage larvae (L3) of Haemonchus contortus by using a larval migration inhibition (LMI) assay. Comparison of extracts obtained with several solvent systems showed that the bioactivity was associated with the 70ratio30 acetone/water extract. Further fractionation of the later allowed the separation of phenolic compounds. By use of a dialysis method, compounds were separated with a molecular weight cut-off of 2000 Da. The in vitro anthelmintic effect of the fraction with condensed tannins was confirmed. In the fraction containing molecules of MW <2000 Da, 3 flavonol glycosides were identified as rutin, nicotiflorin and narcissin. At 1200 mug/ml, each inhibited significantly the migration of larvae. Addition of polyvinyl pyrrolidone (PVPP) to both fractions before incubation restored larval migration. These results confirmed the role of both tannins and flavonol glycosides in the anthelmintic properties of sainfoin.

Comparison of microemulsion electrokinetic chromatography and micellar electrokinetic chromatography methods for the analysis of phenolic compounds

In this study, microemulsion electrokinetic chromatography (MEEKC) and micellar electrokinetic chromatography (MEKC) were compared for their abilities to separate and detect thirteen phenolic compounds (syringic acid, p-coumaric acid, vanillic acid, caffeic acid, gallic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, (+)-catechin, (-)-epigallocatechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate, (-)-epicatechin, and (-)-gallocatechin), and two other ingredients (caffeine and theophylline) in teas and grapes. Separation of phenolic compounds was improved by changing the SDS concentration for MEEKC, but the SDS concentration rarely affected the resolution for MEKC. Organic modifier (acetonitrile or methanol) was found to markedly influence the resolution and selectivity for both MEEKC and MEKC systems. In addition, a higher voltage and a higher column temperature improved the separation efficiency without any noticeable reduction in resolution for MEEKC whereas they caused a poor resolution for the MEKC system. Although separations with baseline resolution were achieved by the optimized MEEKC and MEKC methods, the separation selectivity resulting from the proposed MEEKC method was completely different from that of MEKC.

Analysis of phenolic components in Croatian red wines by thin-layer chromatography

A thin-layer chromatographic (TLC) method has been used for determination of phenolic compounds in two samples of wine commercially available in Croatia. Sample preparation was by liquid-liquid extraction with diethyl ether at pH 2.0. Extracts and standard solutions were applied to 20 cm × 20 cm silica gel 60 F 254 TLC plates. After treatment of the developed plates with ammonia vapor, phenolic compounds were detected on chromatograms by their colors or by fluorescence in UV light at λ = 244 nm and at λ = 366 nm before and after spraying with 1% ethanolic AlCl 3 . The efficiency of eleven mobile phases was tested by three mathematical techniques — calculation of the information content ( I ) derived from Shannon’s equation, determination of discriminating power ( DP ), and formation of clusters and dendrogram. It was shown that the best mobile phase for separation of phenolic compounds in the wine extracts was benzene-ethyl acetate-formic acid, 30 + 15 + 5 ( v / v ). Six phenolic compounds were identified...

Chromatographic Separation of Phenolic Compounds from Rapeseed by a Sephadex LH‐20 Column with Ethanol as the Mobile Phase

Phenolic compounds from rapeseed were chromatographed using an open column filled with Sephadex LH‐20 as the stationary phase and ethanol as the mobile phase. Typically, methanol has been used as the eluent of choice in previous studies, but it was hypothesized that ethanol's lesser polarity would provide better separation of the rapeseed phenolics. In this work, three fractions (I–III) containing phenolic compounds were collected; their characteristics were evaluated qualitatively and quantitatively by UV spectrophotometry, a colorimetric assay, and capillary electrophoresis (CE). Although fraction I possessed the greatest relative proportion of the crude extract (62.2%), fraction III contained the richest source of rapeseed phenolics (176 mg trans‐sinapic acid equivalents g−1). Capillary electrophoresis identified sinapine as the dominant phenolic compound of fraction I and indicated that a number of phenolic compounds were present in fractions II and III. Although free and esterified derivatives of phenolic acids were constituents of fraction II, the electropherogram revealed that sinapic acid was the chief phenolic compound present, based on retention time mapping. Application of ethanol as the eluent, rather than methanol, gave a better baseline separation of the phenolic constituents from rapeseed.

Heteroconjugation-based capillary electrophoretic separation of phenolic compounds in acetonitrile and propylene carbonate.

A mixture of methyl- and hydroxy-substituted phenols was separated by capillary electrophoresis in pure acetonitrile and propylene carbonate. Interactions between undissociated phenolic compounds and the background electrolytes were investigated. In the present work, benzyltriethylammonium chloride, tetrabutylammonium acetate, and two room temperature-molten salts, 1-butyl-3-methyl imidazolium trifluoroacetate and 1-butyl-3-methyl imidazolium heptafluorobutanoate, were used as background electrolytes. The formation of a negative complex between background electrolyte anion and neutral phenolic compound was observed and the formation constant calculated. The formation constants for anion-analyte complexes were approximately the same in propylene carbonate and in acetonitrile. In both solvents the formation constants were the highest for acetate and the lowest for trifluoroacetate. The separation of analytes was slightly influenced by the nature of the solvent: in acetonitrile the resolution between peaks was higher for 1,3-dihydroxyphenol and 1,3,5-trihydroxyphenol, in propylene carbonate 3-methylphenol and phenol were better separated. It was demonstrated that traces of water influence the mobilities of anion-phenol complexes in propylene carbonate.

Limits for Alkaline Detoxification of Dilute-Acid Lignocellulose Hydrolysates

In addition to fermentable sugars, dilute-acid hydrolysates of lignocellulose contain compounds that inhibit fermenting microorganisms, such as Saccharomyces cerevisiae. Previous results show that phenolic compounds and furan aldehydes, and to some extent aliphatic acids, act as inhibitors during fermentation of dilute-acid hydrolysates of spruce. Treatment of lignocellulose hydrolysates with alkali, usually in the form of overliming to pH 10.0, has been frequently employed as a detoxification method to improve fermentability. A spruce dilute-acid hydrolysate was treated with NaOH in a factorial design experiment, in which the pH was varied between 9.0 and 12.0, the temperature between 5 and 80 degrees C, and the time between 1 and 7 h. Already at pH 9.0, >25% of the glucose was lost when the hydrolysate was treated at 80 degrees C for 1 h. Among the monosaccharides, xylose was degraded faster under alkaline conditions than the hexoses (glucose, mannose, and galactose), which, in turn, were degraded faster than arabinose. The results suggest that alkali treatment of hydrolysates can be performed at temperatures below 30 degrees C at any pH between 9.0 and 12.0 without problems with sugar degradation or formation of inhibiting aliphatic acids. Treatment with Ca(OH)2 instead of NaOH resulted in more substantial degradation of sugars. Under the harsher conditions of the factorial design experiment, the concentrations of furfural and 5-hydroxymethylfurfural decreased while the total phenolic content increased. The latter phenomenon was tentatively attributed to fragmentation of soluble aromatic oligomers in the hydrolysate. Separate phenolic compounds were affected in different ways by the alkaline conditions with some compounds showing an increase in concentration while others decreased. In conclusion, the conditions used for detoxification with alkali should be carefully controlled to optimize the positive effects and minimize the degradation of fermentable sugars.

Preparative isolation of polyphenolic compounds from Vitis vinifera by centrifugal partition chromatography

This study deals with a centrifugal partition chromatography developed for the separation of phenolic compounds from Vitis vinifera. EtOAc grape seed extracts were separated using the solvent system hexane–ethyl acetate–ethanol–water (1:8:2:7; v/v) in two fractions: one containing about 75% of flavanol monomers (catechin and epicatechin) corresponding to 18% of crude extract and another fraction B-type dimers (22% of crude extract). From the stalk extracts, we could separate stilbenoid compounds (resveratrol and its oligomers; 12% of crude extract) which were eluted in less than 30 min from flavanols (which required a few hours of additional elution). Using the same solvent system but in different ratios (4:5:3:3; v/v), we isolated the trans-resveratrol (7‰; 90% purity).

Application of normal- and reversed-phase 2D TLC on a cyanopropyl-bonded polar stationary phase for separation of phenolic compounds from the flowers ofSambucus nigraL.

Two-dimensional thin-layer chromatography on a cyanopropyl-bonded polar stationary phases has been used to separate phenolic compounds occurring in Flos Sambuci L. The separation was realized by development of the chromatographic plate in two directions with different mobile phases in each direction. Optimum systems were selected by analysis of retention data obtained for a variety of concentrations of polar solvents in a non-polar diluent ( n -hexane) for NP TLC and polar solvents in water for RP TLC, both on thin layers of cyanopropyl-bonded polar adsorbents. The first development was performed by with a non-aqueous mobile phase and the second with an aqueous mobile phase. Eight flavonoids and three phenolic acids were separated by use of the method described.

Rapid HPLC Analysis of Phenolic Compounds in Red Wines

A new direct and rapid (18 min) reversed-phase high-performance liquid chromatography method for the separation of phenolic compounds (benzoic acids, flavan-3-ols, cinnamic acids, flavonols, and anthocyanins) in red wines is described. A column that allows for low pH conditions and high flow was used, with a gradient of two solvents: water and acetonitrile, both with 0.2% trifluoroacetic acid. To improve selectivity, each compound was monitored at its absorbance maximum. Precision, linearity, and sensitivity (limit of detection and limit of quantitation) were established. While this rapid method cannot resolve all wine constituents, it is appropriate for measuring major components and quantifying total amounts of particular classes of phenolic compounds. The method was applied to a set of new and aged red wines.

Liquid−Liquid Equilibria for Phenolic Compounds, Neutral Oils, and Nitrogen Bases at 313.15 K

Liquid−liquid extraction with triethylene glycol as solvent, water as cosolvent, and hexane as countersolvent is being investigated as a process for the separation of phenolic compounds from neutral oils and nitrogen bases. To this end, liquid−liquid equilibria at 313.15 K and atmospheric pressure have been determined for the systems triethylene glycol + hexane + water + (phenol + aniline + benzonitrile + 5-ethyl-2-methyl pyridine + mesitylene) or (m-cresol + o-tolunitrile + o-toluidine + indene + pseudocumene + undecane) or (2,4-xylenol + 3,4-xylenol + 3,5-xylenol + indane + naphthalene + dodecane). From the experimental results it is concluded that triethylene glycol, hexane, and water are effective solvents for the separation of phenolic compounds from neutral oils and nitrogen bases by means of liquid−liquid extraction. LLE binary parameters for the NRTL equation were obtained for each of the three systems by regression of the equilibrium data. The NRTL equation fit the experimental data with a root-mean-square-deviation (RMSD) of 0.0037, 0.0100, and 0.0058 for the systems containing phenol, m-cresol, and the xylenol isomers, respectively.

Separation of phenolic compounds by high-performance liquid chromatography with absorbance and fluorimetric detection

Phenolic compounds including phenolic aldehydes, acids and flavonoids are separated by high-performance liquid chromatography (HPLC) with analysis time shorter than described in the literature. The use of a fluorescence detector in series with absorbance detector allowed increasing selectivity and sensitivity for the determination of catechin, vanillic acid, syringic acid, epicatechin and trans-resveratrol in wine samples. An optimised sample preparation method using liquid–liquid extraction with diethyl ether at pH 2.0 was used. The optimised method was applied to analyse wine samples with good results.

Computer assisted scale up from analytical HPLC to preparative MPLC for the separation of phenolic compounds

A computer assisted method for the scale up of a column liquid chromatographic separation from the analytical to preparative scale is successfully proposed to make the up-scaling process easier and faster. The test sample consisting of six phenolic compounds was chromatographed on an analytical HPLC column using two different gradient runs. Based on the chromatographic data achieved from these initial analyses, elution behaviour of compounds could be simulated for alternative conditions using the DryLab program. A simulative replacement of the analytical column with a preparative scale medium pressure LC (MPLC) column allowed the determination of gradient profile to allow sufficient separation in the preparative scale. A test run carried out in suggested simulated conditions matched well with expected elution times. Furthermore, this upscaling procedure was successfully applied to a plant extract.