**1. Introduction**

134 Analytical Chemistry

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The natural extracts of plants are an important source for the identification of new biologically active compounds with possible applications in the pharmaceutical field. Phytotherapy embraces especially the isolation from herbs, of compounds with unique chemical structures, which are considered to be pharmacologically active. Recent statistics show that, on an annual basis, there are identified over 1500 new compounds from different species of plants, and about one quarter of prescription drugs contain substances of plant origin.

Although globally there are many studies on natural products, research in this area continues to be of great latent potential, in part due to the existing and still unexploited, potential and perspective development of new opportunities to recovery in major industrial areas and because of their socio-economic impact. A large number of plant extracts that have been used in the traditional medicine have also applications nowadays, both in the pharmaceutical and food industries (Busquet et al., 2005 [1]).

It is known that native Carpathian flora represents about 30% of plant species on the entire European continent. In Romanian, traditional medicine, there are used a number of plants with powerful therapeutic effects, and, as a result, an extensive investigation of their content is necessary. There are many classes of compounds that can be found in an alcoholic, natural extracts: amino acids, peptides, small proteins, phenols, polyphenols, saponins, flavonoids and sugars. Compounds of great interest are the free amino acids and the peptides from these extracts, which show important antitumor activities.

The process of structure elucidation of a natural product involves the determination of many physical-chemical properties: melting point, optical rotation, solubility, absorption, optical rotatory dispersion, circular dichroism, infrared spectroscopy, as well as mass and

© 2012 Neda et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Neda et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

nuclear magnetic resonance spectroscopies. On the basis of such information will be proposed a likely and reasonable structure(s) for the studied natural product.

Peptide and Amino Acids Separation and Identification from Natural Products 137

specific rotation, yet now more modern techniques, especially various spectroscopies, of analysis and characterization are extremely useful tools for a complete chemical screening of

The chemistry of natural products includes three main areas: isolation, structure elucidation, and synthetic methods. The isolation step is considered to be a part of structure elucidation, and therefore analysis and characterization methods, such as the UV-Vis and infrared spectroscopy, mass spectrometry and various chromatographic techniques, are all important tools for a proper identification of the components of an extract. Figure 1 illustrates the

A preliminary step, required for the proper separation of amino acids and peptides, consists in finding a suitable, partitioning scheme of the extract between various solvents, in order to remove the unwanted compounds, such as: polysaccharides, lipids, phenols and others.

**Capillary electrophoresis (CE)** allows the separation of amino acids without prior derivatization. A derivatization step is often necessary in order to improve the detectability using optical detection. A wide variety of labeling reagents have been reported, such as:

Typically, in amino acid analysis, peptide bonds must first be broken, into the individual amino acid constituents. It is known, that the sequence and nature of amino acids in a protein or peptide determines the properties of the molecule. There are different hydrolyzing methods commonly utilized before amino acid analysis, but the most common is acid hydrolysis. However, some of the amino acids can be destroyed using such an approach. Thus, methionine and cystine were either partially destroyed or oxidised to methionine sulphone and cysteic acid. Usually, it is often best to use a hot hydrochloric acid solution and 0.1% to 1.0% of phenol, which is added to prevent halogenation of tyrosine.

Alkaline hydrolysis method has limited applications due the destruction of arginine, serine, threonine, cysteine and cystine. Enzymatic hydrolysis represents perhaps the best method for the complete hydrolysis of peptide bonds, because it does not affect tryptophan, glutamine and asparagines. However, their applications are restricted, due to the difficulties

Separation and elucidation of the chemical composition of a natural product, from a medicinal plant, involves a very laborious procedure. For instance, in the case of *Chelidonium majus L,* a well –known herb, it was necessary to perform successive extractions with hexane, ethyl acetate, chloroform, and n-butyl alcohol. Every fraction obtained was

Recent scientific research has reported a number of increased and improved techniques for the identification of free amino acids, such as, spectroscopic identification by means of colorimetric methods. These have often used reagents such as 2,4-dinitrofluorobenzene [2]

analyzed in detail by various spectroscopic and chromatographic techniques.

chemical structures for most, widely found, naturally occurring amino acids.

FMOC, NDA, OPA or FITC (fluorescein isothiocyanate).

often involved with the use of enzymes.

the natural extracts.

**2. Sample preparation** 

If early methods for organic molecule characterisation used only a few physico-chemical parameters, such as: melting point, solubility, elemental analysis, molecular weight, and/or specific rotation, yet now more modern techniques, especially various spectroscopies, of analysis and characterization are extremely useful tools for a complete chemical screening of the natural extracts.

The chemistry of natural products includes three main areas: isolation, structure elucidation, and synthetic methods. The isolation step is considered to be a part of structure elucidation, and therefore analysis and characterization methods, such as the UV-Vis and infrared spectroscopy, mass spectrometry and various chromatographic techniques, are all important tools for a proper identification of the components of an extract. Figure 1 illustrates the chemical structures for most, widely found, naturally occurring amino acids.
