Phenolic compounds sample preparation methods

Solid-phase microextraction (SPME), first described by Pawliszyn and coworkers [157-161], is a recent upcoming sample preparation method for phenolic and other organic compounds from water and air samples. It is a novel solvent-free sample preparation technique. SPME has advantage of simplicity, low cost, and rapid extraction. It has been successfully coupled with various techniques such as GC, HPLC, CE, and MS. 

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. 

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). 

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). 

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. 

RP HPLC has proved to be the method of choice for the separation of a variety of flavonoids in different samples. The phenolic nature of these compounds requires the use of acidic mobile phases for satisfactory separation and peak shapes, whereas the detection is usually carried out with photodiode array detectors which are also very helpful for their identification of the characteristic absorption spectra of the flavonoids. In the last decade, mass spectrometers connected to HPLC systems introduced a greater selectivity and sensitivity in flavonoid analysis. Improving the characteristics of the stationary phases and developing more sophisticated instruments as well as devices for more efficient and faster sample preparation are the challenges for all modem analysts. Discovering... 

Other plants (cereals, seeds, rye, nuts, barley, malt) can also be extracted with organic solvents or their mixtures with water, but, in many cases, less polar solvents are also used for the elimination of pigments, oils, non-polar and macromolecular compounds. Many of these assays have also included acidic and/or alkaline hydrolysis steps, column liquid clean-up procedures, or SPE. The simultaneous separation of phenolic acids and other phenolic compounds in these samples also complicates the preparation step. Very often, multi-step liquid extraction and sample clean-up assays are recommended. It is necessary to select extraction solvents according to the polarities of the analytes and the form of their bonding to the sample matrix. Very often, both free and bound phenolic acids are separated in plant samples, and hydrolyzed and non-hydrolyzed materials are analyzed separately. In some cases, two or more hydrolysis methods are applied for the analyses of cereals and nuts. ... 

For analytical studies, sample preparation is necessary to determine the composition of phenolic compounds in these matrices. The most widely used extraction system is UAE, which is an inexpensive method since it involves the use of low organic solvent and requires short extraction times. Multi-analyte methods with minimal sample manipulation are demanded. However, vegetables and fruits are very complex matrices and hence sample extraction and clean-up treatments must be carefully developed to reduce manipulation and total analysis time. Therefore, QuEChERS could be a useful tool for this purpose in future applications. 

The number of unsaturated bonds can be determined by a titration with a standard solution of bromine dissolved in glacial acetic acid. The sample is also dissolved in glacial acetic acid to which sodium and ammonium acetate are added, so as to give a final concentration of 0-5 and OT M respectively. The cathodic current of bromine is measured with a short circuit system using a rotating platinum indicator, and a calomel or chloranil reference electrode at the potential of the reference electrode. Several unsaturated open chain compounds and terpenes have been titrated in this way.( ) Other modifications of this technique have been used to determine the amount of unsaturation in fats in methanolic solutions. The standard solution of bromine is prepared in methanol, saturated with sodium bromide, and the methylester of stearic acid is added to suppress the maxima. If the methanolic solution is acidified, OT M potassium bromate with OT M sodium bromide can be used instead of bromine. The bromine generated in homogeneous solution can be used to analyse xylenol and cresol fractions of coal-tars. Other phenolic compounds may be similarly determined. The method was used for the determination of small concentrations of styrene... 

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