**4. Structural identification of phytochemicals**

The chemical structures of plant compounds must be identified or elucidated, which may provide the necessary basis for further study on the bioactivities, structure-activity relationships, metabolisms in vivo, structural modification, and synthesis of the active phytochemicals.

The quality of physiological active substances isolated from plants is often small, sometimes only a few milligrams, and the structural studies are often difficult to carry out with classical chemical methods, such as chemical degradation, derivative synthesis, etc. Therefore, spectral analysis is mainly used, that is, consuming sample as little as possible to obtain structural information as much as possible by measuring various spectra. Then comprehensive analysis is carried out with the assistance of literature data. If necessary, chemical means would be integrated into the former methods to determine the planar- and even the stereo-structures of the compounds.

#### **4.1 Determination of the purity of the compounds**

Before the structural investigation of an active compound, the purity must be determined, which is a prerequisite for the structural identification.

#### *4.1.1 Measurement of physical properties*

The crystals of each compound have certain shape, color, and melting point, which can be used as the basis for the preliminary determination of the purity. Generally, the crystal shape of a specific compound under the same solvent is consistent, the color is pure, and has a short melting range (generally at 1~2°C). But for compounds with double melting points or amorphous substances, the purity cannot be determined by this method.

#### *4.1.2 Thin layer chromatography (TLC)*

TLC, such as silica gel and paper chromatography, is the most commonly used method to determine the purity of compounds. Generally, a specific sample, showing an only spot (Rf value at 0.2~0.8) in three different developing agents, could be considered as a pure compound. In some cases, both normal and reverse phase chromatographic methods are needed.

## *4.1.3 Gas chromatography (GC) and high performance liquid chromatography (HPLC)*

GC and HPLC are important methods in the purity determination of phytochemicals. GC is widely used in the analysis of volatile compounds. Both volatile and nonvolatile substances could be analyzed with HPLC, which possesses various advantages of high speed, high efficiency, sensitivity, and accuracy.

#### **4.2 Major procedures of structural determination**

The general procedures of structural determination of phytochemicals are shown roughly in **Figure 2**.

The structural identification of phytochemicals can be greatly simplified according to the researchers' habits, experiences, and skill levels of different technologies. However, the literature search almost runs through the whole process of structural

**59**

*Analytical Methods of Isolation and Identification DOI: http://dx.doi.org/10.5772/intechopen.88122*

the compound was "known" or "unknown".

*The main procedures for studying the structures of phytochemicals.*

*4.3.1 Ultraviolet-visible spectra (UV-Vis)*

identification of phytochemicals are introduced briefly.

show strong absorption in UV spectra because of n → π\*

**4.3 Spectral technologies**

**Figure 2.**

spectra are recorded.

systems in the structures.

research, no matter for known or new compounds. A large number of facts have been proved that taxonomically related plants, that is to say, plants of same or similar genus often contain chemical constituents of similar or even same chemical structures. Therefore, it is necessary to investigate literatures of chemical studies of the study object and the plants of its same and similar genera. It is necessary to understand not only the components from different plants of similar genera, but also their extraction methods, physicochemical properties, spectral data, and biosynthesis pathways before the extraction and separation of one specific plant. The SciFinder Scholar database is used most widely to quickly determine whether

At present, spectrum analyses have become the main means to determine the chemical structures of plant chemicals. Particularly, with the developing of the superconducting nuclear magnetic resonance (NMR) and mass spectroscopic (MS) technologies, the speed of structural determination is greatly accelerated and the accuracy is improved. Here, the applications of infrared (IR), ultraviolet (UV), nuclear magnetic resonance (NMR), and mass (MS) spectra in the structural

UV-vis spectrum is a kind of electron transition spectrum, which is generated after the molecules absorbing the electromagnetic waves with wavelength at the range of 200–800 nm. The valence electrons in the molecules absorb light of certain wavelengths and jump to the excited state from the ground state, and then UV

Compounds containing conjugated double bonds, α,β-unsaturated carbonyl groups (aldehydes, ketones, acids, and esters), and aromatic compounds could

Therefore, UV spectrum is mainly used to identify the presence of conjugated

or π → π\*

transitions.

*Analytical Methods of Isolation and Identification DOI: http://dx.doi.org/10.5772/intechopen.88122*

#### **Figure 2.**

*Phytochemicals in Human Health*

compounds.

**4. Structural identification of phytochemicals**

**4.1 Determination of the purity of the compounds**

synthesis of the active phytochemicals.

*4.1.1 Measurement of physical properties*

be determined by this method.

*4.1.2 Thin layer chromatography (TLC)*

chromatographic methods are needed.

*(HPLC)*

shown roughly in **Figure 2**.

The chemical structures of plant compounds must be identified or elucidated,

The quality of physiological active substances isolated from plants is often small, sometimes only a few milligrams, and the structural studies are often difficult to carry out with classical chemical methods, such as chemical degradation, derivative synthesis, etc. Therefore, spectral analysis is mainly used, that is, consuming sample as little as possible to obtain structural information as much as possible by measuring various spectra. Then comprehensive analysis is carried out with the assistance of literature data. If necessary, chemical means would be integrated into the former methods to determine the planar- and even the stereo-structures of the

Before the structural investigation of an active compound, the purity must be

The crystals of each compound have certain shape, color, and melting point, which can be used as the basis for the preliminary determination of the purity. Generally, the crystal shape of a specific compound under the same solvent is consistent, the color is pure, and has a short melting range (generally at 1~2°C). But for compounds with double melting points or amorphous substances, the purity cannot

TLC, such as silica gel and paper chromatography, is the most commonly used method to determine the purity of compounds. Generally, a specific sample, showing an only spot (Rf value at 0.2~0.8) in three different developing agents, could be considered as a pure compound. In some cases, both normal and reverse phase

*4.1.3 Gas chromatography (GC) and high performance liquid chromatography* 

advantages of high speed, high efficiency, sensitivity, and accuracy.

**4.2 Major procedures of structural determination**

GC and HPLC are important methods in the purity determination of phytochemicals. GC is widely used in the analysis of volatile compounds. Both volatile and nonvolatile substances could be analyzed with HPLC, which possesses various

The general procedures of structural determination of phytochemicals are

The structural identification of phytochemicals can be greatly simplified according to the researchers' habits, experiences, and skill levels of different technologies. However, the literature search almost runs through the whole process of structural

determined, which is a prerequisite for the structural identification.

which may provide the necessary basis for further study on the bioactivities, structure-activity relationships, metabolisms in vivo, structural modification, and

**58**

*The main procedures for studying the structures of phytochemicals.*

research, no matter for known or new compounds. A large number of facts have been proved that taxonomically related plants, that is to say, plants of same or similar genus often contain chemical constituents of similar or even same chemical structures. Therefore, it is necessary to investigate literatures of chemical studies of the study object and the plants of its same and similar genera. It is necessary to understand not only the components from different plants of similar genera, but also their extraction methods, physicochemical properties, spectral data, and biosynthesis pathways before the extraction and separation of one specific plant. The SciFinder Scholar database is used most widely to quickly determine whether the compound was "known" or "unknown".

#### **4.3 Spectral technologies**

At present, spectrum analyses have become the main means to determine the chemical structures of plant chemicals. Particularly, with the developing of the superconducting nuclear magnetic resonance (NMR) and mass spectroscopic (MS) technologies, the speed of structural determination is greatly accelerated and the accuracy is improved. Here, the applications of infrared (IR), ultraviolet (UV), nuclear magnetic resonance (NMR), and mass (MS) spectra in the structural identification of phytochemicals are introduced briefly.

#### *4.3.1 Ultraviolet-visible spectra (UV-Vis)*

UV-vis spectrum is a kind of electron transition spectrum, which is generated after the molecules absorbing the electromagnetic waves with wavelength at the range of 200–800 nm. The valence electrons in the molecules absorb light of certain wavelengths and jump to the excited state from the ground state, and then UV spectra are recorded.

Compounds containing conjugated double bonds, α,β-unsaturated carbonyl groups (aldehydes, ketones, acids, and esters), and aromatic compounds could show strong absorption in UV spectra because of n → π\* or π → π\* transitions. Therefore, UV spectrum is mainly used to identify the presence of conjugated systems in the structures.

UV spectra could provide the following information: (1) the compounds show no UV absorption at 220–800 nm, indicating the compounds were aliphatic hydrocarbons, aliphatic cyclic hydrocarbons, or their simple derivatives. (2) The compounds show strong absorption at 220–250 nm, indicating that the compounds possess conjugated diene, α,β-unsaturated aldehyde, or ketone substructures. (3) The absorption at 250–290 nm is moderately strong, indicating that the compounds possess benzene rings or aromatic heterocycles. (4) Weak absorption at 250–350 nm indicates the presence of carbonyl or conjugated carbonyl groups. (5) Strong absorptions at above 300 nm indicate that the structures possess long conjugated chains.

Generally, UV spectrum can only provide part of the structural information, rather than the whole structural information of a compound, so it can only be used as an auxiliary method to identify the structures. It possesses practical value to determine the structures of phytochemicals with conjugated substructures.

#### *4.3.2 Infrared spectra (IR)*

IR is caused by the vibration-rotational energy level transition of the molecule, ranging from 4000 to 625 cm<sup>−</sup><sup>1</sup> . The region above 1250 cm<sup>−</sup><sup>1</sup> is functional group region, and the absorption of characteristic functional groups such as hydroxyl, amino, carbonyl, and aromatic rings occurs in this region. The region of 1250 to 625 cm<sup>−</sup><sup>1</sup> is fingerprint region, and the peaks appear mainly due to the stretching vibrations of C-X (X = C, O, N) single bonds, and various bending vibrations. IR is mainly used for the determination of functional groups and the types of aromatic ring substitution. In some cases, IR can also be used to determine the configuration of plant chemical constituents. For example, there is a significant difference between 960 and 900 cm<sup>−</sup><sup>1</sup> for 25R and 25S spirostanol saponins.

#### *4.3.3 Mass spectrometry (MS)*

In a mass spectrometer, mass and strength information of molecular and fragment ions is recorded after the molecules are ionized and enter into the collector under the action of electric and magnetic fields. The abscissa represents the mass-to-charge ratio (m/z) and the ordinate represents the relative intensity in a MS spectrum. Unlike IR, UV, and NMR spectra, MS is mass spectrum, which characterizes fragment ions, not an absorption spectrum. Its role is to determine weights, formulas, and fragment structures of molecules.

With the rapid development of modern techniques, new ion sources have emerged in recent years, which make MS play more important role in determining the molecular weights, elemental composition, detecting functional groups by cleavage fragments, identifying compound types, and determining carbon skeletons [25]. In the structural analysis, the information of molecular weights could be obtained on the basis of molecular ion peaks, and the molecular formula could be obtained by high-resolution mass spectrometry (HR-MS). Fragment ion peaks, combined with molecular ion peak, could be applied to conjecture chemical structures. Tandem mass spectrometry even can isolate and analyze the mixed ions again. According to the types of ion sources, common mass spectrometry could classified as electron impact mass spectrometry (EI-MS), chemical ionization mass spectrometry (CI-MS), field desorption mass spectrometry (FD-MS), fast atom bombardment mass spectrometry (FAB-MS), matrix-assisted laser desorption mass spectrometry (MALDI-MS), electrospray ionization mass spectrometry (ESI-MS), tandem mass spectrometry (MS–MS), and so on.

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**Table 1.**

*Analytical Methods of Isolation and Identification DOI: http://dx.doi.org/10.5772/intechopen.88122*

*4.3.4 Nuclear magnetic resonance (NMR)*

*4.3.4.1 Commonly used deuterated reagents*

clide research such as 1

shown in **Table 1**.

cause the absorption).

With the birth of Fourier transform spectrometer, the great progress of radionu-

Samples used to measure NMR spectra include solids, liquids, and gases. Liquid high-resolution NMR is most widely used. The solvent used in the measurement of NMR must be deuterated. The commonly used deuterated reagents to dissolve samples and their chemical shifts of their residual proton and carbon signals are

Resonance absorption peaks are generated after hydrogen protons absorb elec-

trum can provide structural information of chemical shifts (*δ)*, coupling constants (*J*) that indicate the coupling relationships between different hydrogen nucleus, and the number of protons (the peak area is proportional to the number of protons that

different magnetic cloud densities and magnetic shielding effects caused by the rotation

different regions. Tetramethylsilane (TMS) is usually used as a reference compound. Compared with the general compounds, the shielding effect of protons and carbons on the methyl groups is stronger in TMS. Therefore, regardless of the hydrogen spectrum

**Solvent δ<sup>C</sup> δ <sup>H</sup>** CDCl3 77.0 7.24 CD2Cl2 53.8 5.32 CD3OD 49.0 3.3 Acetone-*d6* 29.8, 206.0 2.04 D2O — 4.7 DMSO-*d6* 39.5 2.49 C6D6 128.0 7.16 C5D5N 123.6135.6149.9 7.2, 7.6, 8.7

*Chemical shifts of common deuterated solvents (TMS is an internal standard).*

H-NMR

H-NMR spec-

H nuclei possess

H nuclear resonance signals appear in

tromagnetic waves of different frequencies in an external magnetic field. 1

possesses high sensitivity, easy measurement, and wide application. <sup>1</sup>

Because of the different surrounding chemical environment, the 1

around the nucleus, and then different types of 1

*4.3.4.2 Proton nuclear magnetic resonance spectroscopy (1H-NMR)*

and three-dimensional nuclear magnetic technology, NMR has become the most important spectroscopic method to determine chemical structures. Particularly, hydrogen spectrum and carbon spectrum are most widely used. During the operation of nuclear magnetic resonance spectrometer, compound molecules are irradiated by electromagnetic waves in a magnetic field, energy level transitions occur after the atomic nuclei with magnetic distance absorb a certain amount of energy, and then NMR spectrum is obtained by mapping the absorption strength with the frequencies of the absorption peaks. It can provide structural information about the type and number of hydrogen and carbon atoms in the molecule, the modes they are connected, the surrounding chemical environment, configuration, and conformation [26].

H, 13C, 15N, 19F, 31P, and the advancement of two-dimensional
