**2. Sample preparation**

136 Analytical Chemistry

**Figure 1.** Amino acids chemical structures

nuclear magnetic resonance spectroscopies. On the basis of such information will be

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

proposed a likely and reasonable structure(s) for the studied natural product.

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

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 often involved with the use of enzymes.

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 analyzed in detail by various spectroscopic and chromatographic techniques.

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]

and genipin [3]. Also, it has also been reported on the use of IR spectroscopy for the study of various extracts of *Angelica* [4].

Peptide and Amino Acids Separation and Identification from Natural Products 139

the absorptions characteristic of the carboxylate ion. The asymmetrical deformation bands from 1610-1660 cm-1 is associated with a carboxylate (COO-) group, and it usually represents a weak absorption. The bands in the 1724-1754 cm-1 region correspond to the carbonyl (C=O)

In the following Figure 3, the IR spectrum of the *Chelidonium majus L.* extract is presented:

**Figure 3.** IR spectrum of the aqueous part of the *Chelidonium majus L.* extract (after successive

extractions with hexane, ethyl acetate, chloroform and n-butyl alcohol)

In the next figure (Figure 2), is presented the FT-IR spectra of L-leucine.

vibration.

**Figure 2.** FT-IR spectra of leucine

## **3. UV-Vis spectroscopy**

The UV-Vis spectra of natural compounds contain information about different properties (such as: chemical composition and structure). Such methods are simple, fast, inexpensive, and safe to perform; which accounts for their popularity. However, these methods have disadvantages, because the result's accuracy depends on many factors: e.g., variations in the length of the polypeptide chain, amount and types of amino acid residues, accessibility of dye reagents, presence of final buffers, stabilizers, and other excipients, which can react with dyes or absorb at the detection wavelength.
