**4.2. Chromatography**

**4. The identification and measurements of phenolics**

72 Phenolic Compounds - Natural Sources, Importance and Applications

are limited.

**4.1. The classical colorimetric methods**

of total amount of polyphenols is enough.

ods, and the AlCl<sup>3</sup>

absorbance at 510 nm.

There is a great diversity of studies about the development of new methods for polyphenol quantification. The high-performance liquid chromatography (HPLC) with or without mass spectrometry (MS) is one of the most commonly applied method to identify and quantify polyphenols. However, the classical spectrophotometric assay is still used, even if their results

The classical spectrophotometry UV/Vis method [124], even with modifications, is still widely used to measure total phenolic content in plant materials. This method is based on the chemical reduction of polyphenols in an alkaline medium to form a blue chromophore complex (phosphomolybdic/phosphotungstic acid) that can be quantified by visible-light spectrophotometry (at 760 765 nm). Many studies have discussed the advantages and disadvantage of using routinely this method to quantify the level of polyphenols, and most of them seems to agree that although they are easy to perform, low cost, rapid and applicable routinely in the most laboratories, they are not accurate. In addition, the reagents used in the method do not react specifically with only polyphenols, and they react with any reducing substance like ascorbic acid, pigments, aromatic amines and sugars [125], and thus, these methods measure the total reducing capacity and not just the polyphenols compounds. Also, their reagents react with some nitrogen-containing compounds such as hydroxylamine and guanidine [126], thiols, many vitamins and some inorganic ions [127] Therefore, many researchers have chosen to use this method only as an indicative tool of total reduction capacity and not for a specific quantification of polyphenol compounds. However, these methods are still considered useful for a quick and prior screening of numerous samples, and for many applications, a simple measure

Similar to total polyphenols, total flavonoids can be measured by spectrophotometry meth-

total flavonoid content. Vanillin and 4-(dimethylamino)-cinnamaldehyde (DMCA) assays are often used to determine the level of proanthocyanidins, in which the flavonoid catechin is used as standard [130, 131]. Like in total polyphenols, the vanillin or DMCA method can overestimate the amount of total flavonoids present in samples. The proanthocyanidins can also be determined by butanol-HCl [132] and bovine serum albumin (BSA) [133] methods. The butanol-HCl method is based on the cleavage of the flavonoid bonds by hot acid, followed by an auto-oxidation reaction which converts flavan-3-ols into anthocyanidins. The red extract formed has a maximum absorbance at around 550 nm. In the BSA method, the flavonoids complex is dissolved in an alkaline solution (sodium decyl sulphate-triethanolamine) followed by a reaction with ferric chloride solution to form a violet complex with a maximum

Another spectrophotometric method widely used in the quantification of polyphenols is the UV/Vis spectrophotometry method to determine the anthocyanin content. The anthocyanins constitute one of the main class of polyphenols largely present in plant samples, particularly

method [128, 129] is the most vulgarized method used to determine the

In the course of the last four decades, several chromatography methods were developed to overcome the main constraints of the classical spectrophotometry methods. The development of new technologies and software led to the appearance of improved methods capable of separation, identification and quantification of phytochemicals individually. These methods are generally based on the principle that a sample is composed of a mixture of components which are separated when the mixture passes through two phases: a mobile (liquid or gaseous) and a stationary (solid, liquid or gel). It is used for the qualitative and quantitative analysis, and the components are separated and analyzed according the properties of a given solution. The great diversity of combinations between the two phases makes possible an existence of several differentiated techniques.

### *4.2.1. Principles*

The basic principles of chromatography are universal [135] and thus widely accepted by all researchers. The main principles are more or less the following ones: (i) chromatography is a physical and chemical method of separating, identification and quantification of different components of samples; (ii) the separation always dependent of the interaction between the components of the mixture with the mobile and stationary phases, and, thus a large combinations of the three are possible; (iii) the interaction of the matrices components with the mix of both phases is influenced by different intermolecular forces including ionic, dipole, nonpolar and effects of specific affinity or solubility; (iv) the mobile phase is generally named as eluent, and the absorbent material, named as stationary phase; (v) the analyte is the compound to be separated; (vi) a chromatogram is the visual output of the chromatograph; (vii) the instrument used for qualitative and quantitative detection of analytes after separation is named as detector; (viii) the separation of components present in the mixture occurs according to the different chemical affinity for the stationary phase, and it happens as the eluent advances on the stationary phase; (ix) the separation of compounds is slower when compounds have strong interaction with the stationary phase and faster when the components have weaker interaction with the stationary phase, and by this, the compounds will be separated from each other as they move over the support material; (x) the component to be analyzed must have solubility with the mobile phase, and different compounds have different retention time values; (xi) the identification and quantification of components in the mixtures are done by comparison with pure commercial standards of known commercial concentration, through analytical curves.

The chromatography can be classified according to several criteria [135, 136], but in general, the chromatography applied in separation, identification and quantification of phytochemicals is classified as:

(i) Gas chromatography (GC), when gas chromatography makes use of a pressurized gas cylinder and a carrier gas (e.g., helium), to carry the solute through the column. The most common detectors used in this type of chromatography are of thermal conductivity and flame ionization detectors. There are three types of GC as follows: (1) gas adsorption, (2) gas-liquid, and (3) capillary gas chromatography.

(ii) Liquid chromatography (LC), when a liquid adsorbent is used. This method is used in large-scale applications since adsorbents are relatively inexpensive. There is a liquid-liquid chromatography which is analogous to gas-liquid chromatography. The three types of modern LC are as follows: (1) reverse phase, (2) high performance and (3) size exclusion liquid chromatography, along with supercritical fluid chromatography.

(iii) Ion exchange chromatography (IEC), when charged molecules mobile phase passes through the column, until a binding site in the stationary phase appears and retains the molecules. There are two types of ion exchange chromatography: (1) cation exchange in which the stationary phase carries a negative charge, and (2) anion exchange in which the stationary phase carries a positive charge. The method is mainly used in the purification of biological materials.

(iv) Affinity chromatography (AC), which is a technique that involves the chemical modification of a given compound by attaching another compound with a specific affinity for the desired molecules. This method requires that the compounds to be analyzed must be inert and easily to modify, and otherwise, it can be very difficult to perform, and a large number of impurities can appear. Therefore, this type of technique is only used in advanced processes of purification.
