Preparative Extraction and Separation of Phenolic Compounds

Phenols are an important group of phytochemicals with significant health beneficial effects. Extraction of phenols firom the biological sources is a growing field of interest and is an integrated part of analytical methods. Some of the common methods of extraction of phenolic compounds are solvent extraction, accelerated solvent extraction, supercritical fluid extraction, ultrasonic extraction, and microwave extractimi. Separation is the next important step of analytical methods, which is done to separate the required phenolic components from the unwanted part of the extract. In case of phenols, methods such as 

Bioresource Engineering Department, McGill University, Ste-Anne-de-Bellevue, QC, Canada e-mail valerie.orsat mcgill.ca 

Bioavailability, that is, the availability of the bioactive compounds at the cellular level to an organism when consumed, is one of the major factors which govern the effectiveness of different bioactive compounds. Bioavailability is affected by various factors which have been discussed in different reviews [8, 9]. It has been observed that sometimes either the pure form of a polyphenol is more helpful or combinations of some particular polyphenols are more effective. Hence, extraction of the bioactive compounds with efficient processes, proper separation with least losses, and encapsulation with appropriate methods are required. For all the studies related to bioactive compounds such as polyphenols, including characterization, quantification, or biomedical in vivo or in vitro studies, extraction is the fundamental method which can be divided into the various steps, mainly preparation, extraction, separation, and purification [10]. This chapter concentrates on the phenols and properties of phenol which affect the extraction efficiency. Other factors affecting the extraction efficiency have also been discussed along with the different methods of extraction. Different methods of separatirni applied for the polyphenols have also been discussed. 

There are several ways in which phenols have been categorized. Harbome and Simmonds categorized polyphenols based on the number of carbon atoms, which includes simple phenols (Ce) phenolic acids and related compounds (Ce—Ci) acetophenones and phenyl acetic acids (Cg—C2) cinnamic acids, cinamyl aldehydes, and alcohols (Cg—C3) coumarins, isocoumarins, and chromones (Cg—C3) flavonoids (C15) biflavonyls (C30) stilbenes (Cg—C2—Cg) benzophenones and xanthones (Cg—C2—Cg) quinones (Cg, Cio, Cm) betacyanins (Cjg) and lignans, lignins, tannins, and phlobaphenes (which are dimmers, oligomers, or polymers) [19]. Polyphenols have also been categorized by some researchers based on their 

Structural differences between different polyphenols decide their chemical properties and thus affect their extractability. Antioxidant effect, which is one of the most commonly smdied effects of polyphenols, has been found to generally increase with the amount of polyphenols in the extracts [23-25]. However, the factors such as solvents and the reactivity and solubility of different polyphenols in different solvents also affect the extraction efficiency and antioxidant activity of the different polyphenols. Hence, the extraction processes and the solvents to be chosen are very much dependent on the type of polyphenols to be extracted, and the processes are chosen to obtain the highest amotmt of polyphenols possible. Study of the mechanisms of different extraction methods, their basic principles, and factors affecting the extraction processes can be helpful in deciding the best method for the extraction of different polyphenols from different sources. 

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Mono-nitro Phenols.—In the first reaction the product is a mixture of ortho-nitro phenol and para-nitro phenol. The two may be easily separated as the ortho compound is volatile with steam, crystallizing in beautiful yellow crystals, while the para compound is not volatile, being left behind when the mixture is distilled with steam. It is then extracted from the residue by boiling with hydrochloric acid, recrystallized from the same solvent and obtained as fine white needles. The preparation and separation of these two compounds is a very satisfactory laboratory exercise. The meta-nitro phenol can not be pre-... 

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. ... 

Since the nature of these chemicals in water varies from polar compounds like phenol to very nonpolar compounds such as pentachlorophenol (PCP), it is a challenge to analytical chemists to determine them collectively. Conventional analytical methods for these compounds are often extensive as they require numerous analytical steps to obtain significant results. The first and also one of the most important requirements is to find a suitable sample preparation technique that allows the separation of the substances of interest from the sample matrix. The analysis of phenols in water is normed by EPA Method 625 [9]. A main disadvantage of this time-consuming and cost-intensive method is the large sample volume required for the extraction and use of large volumes of toxic organic solvents. Therefore, current developments in the field of sample preparation aim for fast and low-cost treatment of enviromnental samples. 

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. 

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