Phenolic Acid Extraction Procedures

Simple phenolic acids are colorless solids when pure, but usually oxidize and become dark on exposure to air (Robinson 1967). Water solubility increases as the number of hydroxyl side groups, solution temperature and/or solution pH values 

A range of extractants and extraction procedures has been used to extract phenolic acids from soil (Dalton 1999). Many of these extractants and extraction procedures, however, recover phenolic acids that are not directly involved in plant-plant allelopathic interactions (e.g., phenolic acids sorbed in the recalcitrant organic matter). Thus considerable efforts were made to identify extraction procedures that would provide reasonable estimates of available phenolic acids ( free phenolic acids in soil solutions and reversibly sorbed phenolic acids on soil particles) in soils (Dalton et al. 1983, 1987, 1989a, b Blum et al. 1994 Blum 1997 Dalton 1999). 

For our soils water, EDTA, citrate, NaOH, etc. extractions were carried out on individual samples or subsamples of soils with or without amendment phenolic acid(s). Thus the recovery for each extractant comprised the sum of the free , reversibly sorbed, and/or fixed (not immediately available to roots or microbes) phenolic acids that were recoverable by a given extractant. Differences between water extractions (primarily free phenolic acids) and EDTA or citrate ( free and reversibly sorbed phenolic acids) extractions were utilized to estimate reversibly sorbed phenolic acids. Differences between EDTA or citrate extractions and NaOH extractions were utilized to estimate the fraction of fixed phenolic acids that could be recovered by NaOH. For additional details see Section 2.4.3. This approach has been criticized because free phenolic acids were not removed from soil samples before reversibly sorbed phenolic acids were extracted (Ohno and First 1998). However, in a preliminary study extracting our soils by the traditional method (removing free  

2 Plant-Plant Allelopathic Interactions. Phase I The Laboratory 

To extract phenolic acids in plant tissues/residues ground plant tissues/residues were extracted with water, EDTA, citrate plus or minus imidazole, KCl, or dibasic sodium phosphate and the water-autoclave procedure (Blum et al. 1992 Blum 1997). For procedures used for C-labeled phenolic acids see Section 2.2.10 

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RNA isolation and cDNA synthesis. Total RNA was isolated from cultured skin fibroblasts using the acid guanidinium thiocyanate-phenol-chloroform extraction procedure and used to prepare cDNA. ... 

Dissociation extraction is the process of using chemical reac tion to force a solute to transfer from one liquid phase to another. One example is the use of a sodium hydroxide solution to extract phenolics, acids, or mercaptans from a hydrocarbon stream. The opposite transfer can be forced by adding an acid to a sodium phenate stream to spring the phenolic back to a free phenol that can be extrac ted into an organic solvent. Similarly, primary, secondary, and tertiary amines can be protonated with a strong acid to transfer the amine into a water solution, for example, as an amine hydrochloride salt. Conversely, a strong base can be added to convert the amine salt back to free base, which can be extracted into a solvent. This procedure is quite common in pharmaceutical production. 

Procedure. Prepare four test solutions of phenol by placing 200 mL of boiled and cooled distilled water in each of four stoppered, 500 mL bottles, and adding to each 5g of sodium chloride this assists the extraction procedure by salting out the phenol. Add respectively 5.0, 10.0, 15.0 and 20.0 mL of the standard phenol solution to the four bottles, then adjust the pH of each solution to about 5 by the careful addition of 5M hydrochloric acid (use a test-paper). Add distilled... 

Following the same procedures described in the above-mentioned study, additional extractive data were obtained for the epoxy phenolic enamel that was irradiated at 4.7-7.1 Mrad at 25 and — 30 °C in the presence of distilled water, 3% acetic acid, and n-heptane. The changes in the amount of extractives resulting from the irradiation treatment are shown in Table IX. In the case of the water and acetic acid extractives, there was no change in either the chloroform-soluble fractions or the chloroform-insoluble fractions. In the case of the n-heptane extractives, the amount of extractives decreased when the irradiation temperature was reduced from +25 to — 30°C. Infrared spectra of the chloroform-soluble residues from the water and acetic acid extractives of the unirradiated and irradiated enamel were identical to the chloroform-soluble residues from the solvent blanks. In other words, the epoxy phenolic... 

Preparation of seedlings for treatments with extract-amended nutrient solution was similar to that described for testing the effects of phenolic acids, except 40 plants were used per treatment and no replacement of the nutrient solution was made during the treatment period. Data collection procedures were modified in that only ab-axial leaf resistance was obtained and water potential was determined from four plants each day. Prior work established that abaxial resistance provided an adequate indicator of stomatal effects. The data were analyzed as described in experiments with pCA and FA. 

Michalkiewicz A, Biesaga M and Pyrzynska K. 2008. Solid-phase extraction procedure for determination of phenolic acids and some flavonols in honey. J Chromatogr A 1187( 1-2) 18-24. 

Fiamegos, Y.C. et al.. Analytical procedure for the in-vial derivatization-extraction of phenolic acids and flavonoids in methanolic and aqueous plant extracts followed by gas chromatography with mass-selective detection, J. Chromatogr. A, 1041, 11, 2004. 

Reversed-phase HPLC is used for the analysis of the different groups of phenols, phenolic acids, hydroxycinnamic acids, flavonoids, and procyanidins in grapes and wines (22,46,47,77-80). However, due to the presence of a large quantities of various compounds, wine analysis is difficult. Thus, different sample preparation procedures, including fractionation and extraction, are often applied when various groups of phenolic compounds are studied together. 

A reversed-phase HPLC procedure was proposed for the determination of seven phenolic acids in green coffee samples (144). The sample preparation technique involved extraction, alkaline hydrolysis, and liquid/liquid extraction. The chromatographic separation was achieved us-... 

Sequential Methylation Technique. A sequential methylation-extraction technique has been devised primarily to separate phenolics from aromatic and aliphatic acids. The procedure, outlined below, allows quantitative conversion of phenolic hydroxyls to methyl ethers in one step and esterification of carboxyl groups in another. 

The vaccine formulation comprises a mixture of the outer-membrane lipopolysaccharides from bacteria of the seven different serotypes. The lipopolysaccharides may be obtained from cultures of each of the seven types by the standard procedure of Westphal (11), involving phenol—water extraction of the cells. However, for large-scale purposes, the older procedure of Boivin, (12) involving extraction of the cells with trichloroacetic acid, was... 

The lipid material precipitated upon mild acid treatment of the Boivin-extracted lipopolysaccharide is here termed a lipoidal precipitate. The fatty-acid profile (Table IV) of hydrolyzates of this material shows little variation between the seven lmmunotype strains. If the original lipopolysaccharides are first treated by phenol—water extraction, and the resultant materials then subjected to hydrolysis to release the lipid, the composition of the latter is significantly different it corresponds closely to the classic composition expected for lipid A. It is noteworthy that material extracted by the phenol—water (Westphal) method is rich in the Csaturated acid and in the hydroxy fatty acids having ten and twelve carbon atoms, whereas the and C g saturated acids present in the lipoidal precipitate, as prepared by the Boivin procedure, are absent or present at much lower levels in the lipid prepared by the Westphal procedure -9). It... 

Extraction by chemically active solvents. Not infrequently the crude organic product from a reaction may contain a mixture of acidic (phenols and carboxylic acids), basic and neutral components in various combinations. Some of these components may of course be impurities, but none the less, whether as a preliminary purification stage or as a means of separating the mixture, a carefully planned solvent extraction procedure may be adopted using acidic and basic reagents which react chemically with the basic and acidic components of the mixture respectively. The following full account of a typical procedure may be abbreviated in practice according to the complexity of the mixture to be handled. 

Tanaka et al. [ 16] have described a spectrophotometric method for the determination of nitrate in vegetable products. This procedure is based on the quantitative reaction of nitrate and 2-sec-butylphenol in sulfuric acid (5 + 7), and the subsequent extraction and measurement of the yellow complex formed in alkaline medium. The column reaction is sensitive and stable and absorbances measured at 418 nm obey Beer s law for concentrations of nitrate-nitrogen between 0.13 and 2.5 xg/ml. In this procedure, the vegetable matter is digested at 80 °C with a sodium hydroxide silver sulfate solution, concentrated sulfuric acid and 2-sec-butylphenol are added, and after 15 minutes of standing time the nitrated phenol is extracted with toluene. Finally, the toluene layer is back-extracted with aqueous sodium hydroxide and evaluated spectrophotometrically at 418 nm. The standard deviation of the whole procedure was 1.4%, and analytical recoveries ranged between 91 and 98%. 

Experimentally, C14-aminoacyl sRNA was incubated with rat liver microsomes or ribosomes, GTP, various fractions obtained from the nonparticulate portion of rat liver homogenates, and buffered salt-sucrose medium in a total volume of approximately 2 ml. (6-10). The C14-aminoacyl sRNA was prepared by the phenol-extraction procedure from the pH 5 amino acid-activating enzymes, fraction of rat liver after incubation with C14-L-amino acids (9, 13). C14-leucyl sRNA (approximately 1000 c.p.m.), having a specific radioactivity of approximately 55,000 c.p.m. per mg. of RNA, and containing a complement of endogenous, unlabeled, bound amino acids, was used in most of these studies. The microsomes were sedimented from the post-mitochondrial supernatant at 104,000 x g (10) and the ribosomes were prepared from them by extraction with deoxycholate (16). 

An HPLC-DAD method was developed for the separation and the determination of flavonoid and phenolic antioxidants in commercial and freshly prepared cranberry juice.Two sample preparation procedures were used with and without hydrolysis of the glycoside forms of flavonoids carried out by the addition of HCl in the step prior to solid-phase extraction (SPE). The flavonoid and phenolic compounds were then fractionated into neutral and acidic groups via a solid-phase extraction method (Sep-Pak Cig), followed by a RP HPLC separation with gradient elution with water-methanol-acetic acid and a detection at 280 and 360 nm. A comparison of the chromatograms obtained for extracts prepared with and without hydrolysis showed that flavonoids and phenolic acids exist predominantly in combined forms such as glycosides and esters. In a freshly squeezed cranberry juice, for instance, 400 mg of total flavonoids and phenolics per liter of sample was found, 56% of which were flavonoids. Quercetin was the main flavonoid in the hydrolyzed products, where it accounted for about 75% of the total flavonoids, while it was absent in the unhydrolyzed products. 

Sample handling is a very important part of the method development for HPLC determination of phenolic acids in natural plants. Because of the great variability of phenolic acids (different polarity, acidity, number of hydroxyl groups, and aromatic rings), the various concentration levels of individual analytes, and the very complex natural matrix with many interfering components, the choice of the technique for their isolation and quantification differs from one described HPLC assay to the next. In some cases, only a one-step extraction and simple clean-up procedure are sufficient before the HPLC analysis, but the most often described HPLC assays include two or more steps of sample preparation, especially in the case of fruits and vegetable samples. It is obvious that each step contributes, on one hand, to the higher sensitivity and selectivity, but, on the other hand, it could increase the number of errors and decrease the recovery of the method. 

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