TECHNIQUES FOR THE PURIFICATION AND CHARACTERIZATION OF ACTIVE COMPOUNDS

8.7.1 PURIFICATION TECNIQUES

The bioactive compounds of medicinal plants are isolated by separation and characterization teclmiques according to the chemical structures of the compounds of interest.

The compounds regularly purified by different separation teclmiques, including liquid-liquid partitioning, gravitational liquid chromatography, vacuum liquid chromatography, liquid flash chromatography, TLC, GC, ion-exchange chromatography, medium-pressure liquid chromatography (MPLC), HPLC, and ultrahigh-performance liquid chromatography (UPLC). The selection and implementation of the teclmique are based on the chemical structure of each compound as well as the affinity it has for both the mobile phase and the stationary phase (Cela et al., 2002).

In some types of chromatographic teclmiques, it is necessary to establish adequately the critical conditions for the separation process to be efficient and reproducible. Therefore, in the separation of compounds by gravitational liquid chromatography, vacuum liquid chromatography, and liquid flash chromatography, it is necessary to define the diameter of the separation column according to the amount of extract (Hernandez-Ocura et al., 2017;

Harris, 2001). In these chromatographic techniques, the separation columns with a porous glass filter with a diameter according to the amount of the extract were used. Extracts of less than 100 mg use diameters of 0.5-1 cm, extracts of 0.5-1 g use 2.5-4 cm, and extracts of 1-10 g use diameters greater than 5 cm.

The conventional height of the column used for vacuum liquid chromatography is 4-5 cm, while the height of the column to carry out gravitational liquid chromatography and liquid flash chromatography goes from 10-20 cm. If it is necessary to scale the separation process, the diameter should be modified but not the height of the column (Harris, 2001).

On the other hand, one of the critical factors to achieve a better separation in TLC is the selection of the eluent. Therefore, in most cases, separation profiles are made to choose the mixture of solvents that separate the extract. The eluent that separates the components of the extract with ratio of fronts (RF) of 0.1-0.2 will be the optimum for the separation. The RF is the ratio between the distances travelled by the extract and by the eluent. This value is measured from the area of application of the sample. RF = distance travelled by the extract/distance traveled by the eluent. Another factor to take into account is that the thickness and the measurements of the chromatographic support used in the separation are always the same (Hemandez-Ocura et al., 2017; Wagner and Bladt, 2001). In ion-exchange chromatography and HPLC-UPLC, one of the most important factors is the selection of the chromatographic support. Therefore, the characteristics of the chromatographic support used in the separation should be consulted. The selection of the chromatographic support is based mainly on the particle size and in some cases on the ionogenic groups interlaced to the chromatographic support that is regularly Sephadex (Cela et al., 2002).

Currently, there are prefabricated columns filled with the chromatographic support. However, it is sometimes necessary to prepare the column manually. Therefore, it is necessary to condition the Sephadex particles in MeOH for 24 h at rest. Because of this, it calculates the amount of support according to the area of the column and the degree of expansion of the chromatographic support. This data is regularly in the labeling of the material. Once the column is packed, the extract completely dissolved is eluted (Cela et al., 2002; Hams, 2001).

Purification techniques that employ pressures, such as vacuum liquid chromatography, liquid flash chromatography, ion-exchange chromatography, HPLC, and UPLC, establish parameters that allow reproducible elution, such as the amount of sample, flow, injection, detector, among others.

A liquid-liquid partition is usually carried out, followed by a separation process in column chromatography to obtain partially purified fractions; then, the automated methods such as HPLC are used to achieve the purification of some compounds.

Currently, the development of analytical separation and purification techniques, such as HPLC, TLC, and GC, and identification teclmiques, such as MS, NMR, GC-MS, and liquid chromatography coupled to mass spectrometry (LC-MS), has allowed the identification of a large number of secondary metabolites: flavonoids, teipenes, phenolic compounds and their esters, sugars, hydrocarbons, and mineral elements present in various plants (Huang et al., 2014).

The HPLC has displaced the GC-MS technique for the quantification of compounds since it has the disadvantage of derivatization of components. The most used detection systems have been UV detectors, diode arrays, and mass spectrometers (Gomez Caravaca et al., 2006). Colorimetric methods have also been used (Chang, 2002); spectroscopic teclmiques (Popovaa et al., 2007), capillary electrophoresis in the zone capillary electrophoresis (CZE) zone, micellar electrokinetic chromatography (MECK) micellar, and IR spectroscopy (Zhang et al., 2013), having a large number of analytical tools, allow characterizing various secondary metabolites.

8.7.2 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

It is a technique that allows the distribution of the analyte of interest between two phases, a mobile and a stationary one (Gilbert, 1987). This analytical teclmique is the most used for the characterization of compounds; the chromatographic conditions of HPLC methods include a reverse-phase C₁₈ column, a UV-vis or diode array detector, and a binary solvent system containing acidified water and a polar organic solvent. HPLC-MS MS is the most used technique for the separation and determination of metabolites; however, it has some disadvantages in the limit of detection and sensitivity, especially for plant extracts of complex matrixes and environmental samples. Therefore, the initial preconcentration and purification of the compounds are important before instrumental analysis (Pellati et al., 2011; Falcao et al., 2013). The most common detectors in HPLC are absorbance, fluorescence, electrochemical, refractive index, conductivity, Fourier transfonn infrared (FT-IR) spectrometer, and light scattering and MS detectors (Gilbert, 1987).

8.7.3 GAS CHROMATOGRAPHY

The determination of components by GC allows a qualitative as well as a quantitative analysis to be carried out. However, sample preparation is complex due to the need for removing impurities from the extract, which may affect the reproducibility of the results. GC-MS is the most used method since MS detection allows the acquisition of molecular mass data and structural information that facilitates the identification of compounds (Sawaya et al., 1983). It is a technique that has a great separation capacity and offers high sensitivity and selectivity when combined with MS (Pellati et ah, 2011). The common detectors in these systems are a flame ionization detector, nitrogen phosphorus detector, flame photometric detector, thermal conductivity detector, and electronic capture detector (Gilbert, 1987).

8.7.4 FOURIER TRANSFORM INFRARED SPECTROSCOPY

IR spectroscopy is an analytical teclmique that measures the wavelength and absoiption intensity of IR light in a sample; it is possible only because molecular absoiption generates changes in the state of vibrational and/or rotational energy. The resulting spectrum contains information specific to the aspects of molecular vibration of the sample, its physical properties, and its unique interaction with the instrument. The absorption data obtained in the spectrum give structural information that represents the molecular details of the sample that is measured. Each functional group of a molecule has a unique vibrational frequency that can be used to determine which functional groups are present in a sample. When the effects of all the different functional groups are together, the result is a unique “molecular fingerprint” that can be used to characterize the identity of a sample. Depending on the type of link, there are several types of molecular vibrations that absorb at unique wavelengths or near-IR (NIR) energy frequencies. Some NIR spectrometers are capable of measuring transmittance (direct and diffuse), reflectance (specular and diffuse), etc. The IR region is not especially useful, by itself, as a quantitative measurement technique. However, it is a quick, easy to perform, and nondestructive method that can record spectra of solid and liquid samples without any previous treatment. One of its main advantages is that it implies a low-cost operation and provides spectrum quickly, being able to predict physical and chemical parameters from a single spectrum.

The analytical information contained in the typically large and widely overlapping bands of NIR spectra is not selective and is influenced by a series of physical, chemical, and structural features. In addition, the differences between the samples can cause very slight spectral differences that are difficult to distinguish with a simple view (Blanco and Villaroya, 2002).

8.7.5 UV-VIS SPECTROPHOTOMETRY

At present, sufficient information can be obtained for rapid or “screening” evaluation of the total polyphenol content, total flavonoids, total catechin, total coumarins, etc. and used as a criterion of the nutritional value of food products derived from plants. These analyses can be easily performed from spectrophotometric measurements, using specific analytical reagents that have adequate selectivity to avoid obtaining false-positive values that affect the quality of the estimate. The detection of various compounds can be done through a UV-vis spectrophotometer since they efficiently absorb UV light. The advances of the “green chemistry” and the need for simultaneously carrying out a high number of samples have led to the development of innovations of the classical methods, drastically decreasing the contaminating residues of the laboratory and making the management of resources more efficient. In this way, the volumes of reagents have been adjusted for this new format, which allows the analysis of a high number of samples simultaneously in a 96-well microplate. This is how in recent years many modified methods have been developed for small volumes, resulting in being as efficient as the results obtained by classical methods (Herald et al., 2012).

8.7.6 NUCLEAR MAGNETIC RESONANCE

NMR is widely used for the elucidation of structures, mainly of an organic nature, since in principle it focuses on the study of proton NMR, subsequently expanding to other magnetically active nuclei. Therefore, this technique was almost exclusively a tool for organic chemists; however, with the development of magnets and superconducting materials, its field of application has been extended. Currently, the fields of application are biochemistry and biology, pharmacology and toxicology, medicine, environment, and food. It is noteworthy the great boom that has experienced in recent years in the field of analytical quantification. It is a spectroscopic method for nondestructive analysis, which is based on the absorption of electromagnetic radiation energy by atomic nuclei that have a magnetic moment. The nuclear spin electrons, neutrons, and protons, constituents of the nucleus of an atom, possess an intrinsic property that is given by the so-called spin term. The spin is the fourth quantum number that takes place when solving, in an approximate way, the wave equation. The spin represents a general property of the particles that can easily be explained by similarity to the properties of the electrons. The electrons of the atom circulating around its nucleus generate a magnetic field associated with an angular momentum described with the quantum number of spin +1/2 and -1/2. For NMR, the proton and neutron spins of the nucleus are very interesting. In the nucleus, the protons and neutrons are paired combining the spins +1/2 and -1/2. Following are some conditions for the purification and characterization of active principles of medicinal plants (Claridge, 2016).