*4.2.2. The use of HPLC-DAD/UV-VIS and HPLC-MS*

The high-performance liquid chromatography (HPLC), referred in the past as high-pressure liquid chromatography, like other chromatography methods is a technique used to separate, identify and quantify phytochemicals from plant mixtures and relies on pumps to pass a pressurized solvent containing the plant samples, foods or other matrices through a column filled with a solid adsorbent material (e.g., silica) [137]. The HPLC methods, however, differ from other liquid methods, particularly from "low pressures," because it uses high pressures (ranging from 50 to 350 bar), while the others normally use the force of gravity to pass the mobile phase through the column [137, 138]. Each component of the samples interacts differently with the adsorbent material of the column, causing a different flow rate for the different components in the mixture, thus leading to the separation of the components as they flow out the column. The columns used in the HPLC methods are made with smaller adsorbent particles size ranging from 2 to 50 µm [137, 138].

Although there is many variations in the HPLC equipment available in the market, the basic HPLC equipment includes a sampler (to carry the sample mixture into the mobile phase), pumps (to deliver mobile phase through the column, with a specific flow) and a detector (such as UV/Vis or photodiode array (PDA), which generates signal proportional to the amount of compound present in the sample mixture [138]. The signal detected allows the identification and quantification of sample components. Each compound detected has a specific retention time; however, due to the interaction strength of interactions between the analytes and stationary phases, the retention time can vary. Nowadays, modern HPLC equipment has a digital processor, which uses a software interface to control the instruments and provides data analysis. Other modern models are equipped with several pumps, which allow different combinations of various solvents at different ratios changing in time, creating a gradient in the mobile phase.

values; (xi) the identification and quantification of components in the mixtures are done by comparison with pure commercial standards of known commercial concentration, through

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

(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,

(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

(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

(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

The high-performance liquid chromatography (HPLC), referred in the past as high-pressure liquid chromatography, like other chromatography methods is a technique used to separate, identify and quantify phytochemicals from plant mixtures and relies on pumps to pass a pressurized solvent containing the plant samples, foods or other matrices through a column filled with a solid adsorbent material (e.g., silica) [137]. The HPLC methods, however, differ from other liquid methods, particularly from "low pressures," because it uses high pressures (ranging from 50 to 350 bar), while the others normally use the force of gravity to pass the mobile phase through the column [137, 138]. Each component of the samples interacts differently with the adsorbent material of the column, causing a different flow rate for the different components in the mixture, thus leading to the separation of the components as they flow out the column. The columns used in the HPLC methods are made with smaller adsorbent

analytical curves.

is classified as:

materials.

of purification.

and (3) capillary gas chromatography.

74 Phenolic Compounds - Natural Sources, Importance and Applications

chromatography, along with supercritical fluid chromatography.

*4.2.2. The use of HPLC-DAD/UV-VIS and HPLC-MS*

particles size ranging from 2 to 50 µm [137, 138].

Nowadays, the classic HPLC evolved to HPLC coupled with a mass spectrometry detector (MS), called as LC-MS or HPLC-MS [138]. This new technique allows a more accurate identification which is based on the specific fragmentation of each separated molecule. This enhances the sensitivity and is oriented for the separation of chemicals with specific masses in a complex mixture. The separation of molecules or fragments occurs according to their mass-to-charge ratio in an analyzer by electromagnetic fields. The ions are detected by a qualitative and quantitative analysis, and the signal is processed into mass spectra. The HPLC-MS equipment is in general composed of three modules: (1) an ion source, which converts gas phase sample molecules into ions; (2) a mass analyzer, which sorts the ions by their masses by applying electromagnetic fields; and (3) a detector, which measures the value of the signal detected and provides data for the quantification of each ion present. In the last years, this new method has been strongly


**Table 4.** Some recent examples of the usage of HPLC-MS system for separation and quantification of polyphenols in different matrices.

implemented in academies in basic research, pharmaceutical and agro-chemical industries to study physical, chemical and biological properties of a great diversity of compounds, as well, and quality control of drugs, foods and natural products. In **Table 4** are presented some recent works [139–145] in which HPLC-MS was effectively used for polyphenol characterization of plants and food with very accurate results.
