Phenolic compounds sample preparation

From fractions 6 and 7 (CHC1,), after further separations by preparative TLC, a mixture of 8-sitosterol (4) and stigmasterol (3) was obtained. The identity was established by comparison of Hi NMR and MS data with those of authentic samples. Another TLC band provided minor amounts of a phenolic compound which on the basis of... 

SEC in combination with multidimensional liquid chromatography (LC-LC) may be used to carry out polymer/additive analysis. In this approach, the sample is dissolved before injection into the SEC system for prefractionation of the polymer fractions. High-MW components are separated from the additives. The additive fraction is collected, concentrated by evaporation, and injected to a multidimensional RPLC system consisting of two columns of different selectivity. The first column is used for sample prefractionation and cleanup, after which the additive fraction is transferred to the analytical column for the final separation. The total method (SEC, LC-LC) has been used for the analysis of the main phenolic compounds in complex pyrolysis oils with minimal sample preparation [974]. The identification is reliable because three analytical steps (SEC, RPLC and RPLC) with different selectivities are employed. The complexity of pyrolysis oils makes their analysis a demanding task, and careful sample preparation is typically required. 

Revilla E and Ryan JM. 2000. Analysis of several phenolic compounds with potential antioxidant properties in grape extracts and wines by high-performance liquid chromatography—photodiode array detection without sample preparation. J Chromatogr 881(1-2) 461 169. 

Antolovich, M., Prenzler, P., Robards, K., and Ryan, D., Sample preparation in the determination of phenolic compounds in fruit. Analyst, 125, 989, 2000. 

The Falin-Ciocalteu reagent (FCR) is a complex formed in a reaction between sodium tungstate and sodium molybdenate in hydrochloric add and phosphoric acid, which turns yellow after lithium sulphate is added. The reagent reads in an alkaline environment with reducing compounds. Such a reaction gives a blue chromophore which is observed by colorimetry. The Folin-Ciocalteu method is highly sensitive - both to phenolic and non-phenolic compounds, e.g. proteins, vitamin C, vitamin Bj, folic acid, Cu(I). The method is applied most frequently to determine the total content of phenolic compounds [34,35]. If that is the case, a sample for determination should be prepared in a proper manner to minimise the effect of non-phenolic... 

A reversed-phase liquid chromatographic method was developed for simultaneous determination of carboxylic acids, phenolic compounds, and SA in white wines (84). The diluted samples are injected into a Spherisorb ODS-2 column with a gradient of sulphuric acid (pH 2.5)/methanol as mobile phase. A diode array detector is used, set at 210 nm for carboxylic acids and altered to 278 nm, during the run, for phenolics and SA. The identification of compounds is based on retention time and UV spectra. Some cleanup methods (Sep-Pak C18 and an ion-exchange column) were tested and did not improve the results. The analysis was considered simple, with no sample preparation. Application of this method was illustrated by analyses of Brazilian Welchriesling wines (84). 

Many different sample preparation procedures have been employed, ranging from simple filtration of juice products to solvent extraction, and extraction by SPE using C, 8, Sephadex LH-20 (49,50,52), and Amberlite XAD-2 (51,54,57). The Amberlite XAD-2 cleaning step has been used for many phenolic extracts, especially for fruit purees, to remove the sugars and other polar compounds. However, due to the low recovery rate with Amberlite XAD-2 for certain phenol glycosides, a modified sample preparation technique is needed, especially for quantification of ar-butin in pear juice and blends (54). Figure 6 describes the fractionation procedure for phenolics using a Sephadex LH-20 column (58). 

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. 

However, due to the artifacts resulting from oxidation, hydrolysis of esters or ethers, or isomerization of phenolics during pretreatment of wines, as well as due to the low recovery rates of some phenolics, analysis of wine phenolics via direct injection of the filtered wine into the chromatographic column is often selected (80,82-84). For the red wine and musts (80), which were injected directly into the HPLC without sample preparation, a ternary-gradient system was often employed for phenolic compounds. Twenty-two phenolic compounds, including 10 anthocyanins, were analyzed from red wine. The separation of cinnamic acid derivatives (313 nm),... 

For the red wines (82-84), which were injected directly into the HPLC without sample preparation, a ternary-gradient system using aqueous acetic acid (1% and 5% or 6%), and acidified acetonitrile (acetonitrile-acetic acid-water, 30 5 6) was used for cinnamic acid derivatives, catechins, flavonols, flavonol glycosides, and proanthocyanidins. Due to the large number of peaks, the gradient was extended to 150 min for the resolution of many peaks of important phenolics. This direct injection method was able to separate phenolic acids and esters, catechins, proanthocyanidins, flavonols, flavonol glycosides, and other compounds (such as tyrosol, and rrans-resveratrol) in wine in a single analysis. However, use of acetic acid did not permit the detector (PDA) to be used to record the UV spectra of phenolics below 240 nm (84). 

Castillo, M., D. Puig, and D. Barcelo. 1997. Determination of priority phenolic compounds in water and industrial effluents by polymeric liquid-solid extraction cartridges using automated sample preparation with extraction columns and liquid chromatography Use of liquid-sohd extraction cartridges for stabilization of phenols. J. Chromatogr. A 778 301-311. 

Marinez-Ortega, M.V. Garcia-Parilla, M.C. Troncoso, A.M. 2004. Comparison of different sample preparation treatments for the analysis of wine phenolic compounds in human plasma by reversed phase high-performance liquid ehromatog-raphy. Anal. Chim. Acta. 502 49-55. 

Analytical determination of peptides in wine requires sample preparation, involving their isolation from the remaining components, mainly high molecular weight nitrogen compounds, free amino acids and phenols. Table 6B.1 summarizes the procedures used in the literature for the extraction of wine peptides before their analysis by different analytical techniques and with different detection systems. 

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. 

Most papers dealing with phenolic acid HPLC analysis in herbs describe only simple liquid extraction without the hydrolysis step. Acetone, methanol, or alcoholic-water or acetone-water mixtures are applied. Very rarely, pure water is used as the extraction solvent. " It was found that the extraction recoveries for water extracts are often lower in comparison to alcoholic-water mixtures, especially when the simultaneous separation of polar and less polar phenolic acids has been performed. Sometimes, the control of pH can improve the recovery. If necessary, n-hexane, chloroform, diethyl ether, benzene-acetone, petroleum ether, or other less polar solvents are recommended for removing interfering compounds. The extraction is usually performed by refluxing the samples for a specific time in a Soxhlet apparatus, with simple mechanical or magnetic stirring of the sample with the extraction solvent, or by plant sample maceration. The application of an ultrasonic bath for the liquid extraction has also become popular in recent years. The hydrolysis steps have also been recommended for medicinal species preparation, especially when other phenolic compounds are also analyzed simultaneously with phenolic acids in herbs. 

---