**4. IR spectroscopy**

Infrared spectroscopy is based on molecular vibrations, characteristic to the specific chemical bonds or groups. The energy of most molecular vibrations (stretching, twisting and rotating) corresponds to that of the infrared region of the electromagnetic spectrum. There are many vibrational modes that do not represent a single type of bond oscillation but are strongly dependent on the neighbouring bonds and functional groups. One of the great advantages of this analytical technique for natural products, is due to the fact that spectra can be obtained form almost any environment (aqueous solution, organic solvents, etc.) and from relatively small quantities of sample.

There are a large number of IR spectroscopic studies regarding the structure of amino acids and peptides; some of the approached subjects are the following: infrared spectra of potassium ion tagged amino acids and peptides (Polfer et al., 2005[5]), IR spectra of deprotonated amino acids (Oomens et al., 2009[6]). Also, there have been reported studies regarding the IR spectra of some derivatives of the amino acids, namely the amides (Kasai et al., 1979[7]). The results show the appearance of the C=O group around 1675-1680 cm-1 for most of the studied compounds. An exception is represented by the L-tyrozine amide, which shows a vibration frequency of the C=O group at 1705 cm-1, fact that is probably due to the intermolecular hydrogen bonds between the N atom of the amidic group and the phenolic OH group (Kasai et al, 1979[7]). Linder et al. have presented the IR spectra of 5 natural amino acids, namely valine, proline, isoleucine, phenylalanine and leucine (Linder et al., 2005[8]). The five spectra are very similar as regards the position of C=O group, and the absorption frequencies of OH groups. Slight differences appear only in the case of the stretching vibrations of the C-H groups (Linder et al., 2005[8]).

The amino acid and peptide absorption bands in the 3400 cm-1 region is due to O–H and N– H, bond stretching. The broad absorption bands in the region 3030-3130 cm-1 are attributed to asymmetric valence vibrations of the ammonium (NH3+) group. The symmetric absorption vibrations in 2080 -2140 cm-1 or 2530-2760 cm-1, depend on amino acid chemical structures. The ammonium group deformation vibrations are located at 1500-1600 cm-1, together with 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) vibration.

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

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

138 Analytical Chemistry

various extracts of *Angelica* [4].

**3. UV-Vis spectroscopy** 

**4. IR spectroscopy** 

dyes or absorb at the detection wavelength.

from relatively small quantities of sample.

stretching vibrations of the C-H groups (Linder et al., 2005[8]).

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

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

Infrared spectroscopy is based on molecular vibrations, characteristic to the specific chemical bonds or groups. The energy of most molecular vibrations (stretching, twisting and rotating) corresponds to that of the infrared region of the electromagnetic spectrum. There are many vibrational modes that do not represent a single type of bond oscillation but are strongly dependent on the neighbouring bonds and functional groups. One of the great advantages of this analytical technique for natural products, is due to the fact that spectra can be obtained form almost any environment (aqueous solution, organic solvents, etc.) and

There are a large number of IR spectroscopic studies regarding the structure of amino acids and peptides; some of the approached subjects are the following: infrared spectra of potassium ion tagged amino acids and peptides (Polfer et al., 2005[5]), IR spectra of deprotonated amino acids (Oomens et al., 2009[6]). Also, there have been reported studies regarding the IR spectra of some derivatives of the amino acids, namely the amides (Kasai et al., 1979[7]). The results show the appearance of the C=O group around 1675-1680 cm-1 for most of the studied compounds. An exception is represented by the L-tyrozine amide, which shows a vibration frequency of the C=O group at 1705 cm-1, fact that is probably due to the intermolecular hydrogen bonds between the N atom of the amidic group and the phenolic OH group (Kasai et al, 1979[7]). Linder et al. have presented the IR spectra of 5 natural amino acids, namely valine, proline, isoleucine, phenylalanine and leucine (Linder et al., 2005[8]). The five spectra are very similar as regards the position of C=O group, and the absorption frequencies of OH groups. Slight differences appear only in the case of the

The amino acid and peptide absorption bands in the 3400 cm-1 region is due to O–H and N– H, bond stretching. The broad absorption bands in the region 3030-3130 cm-1 are attributed to asymmetric valence vibrations of the ammonium (NH3+) group. The symmetric absorption vibrations in 2080 -2140 cm-1 or 2530-2760 cm-1, depend on amino acid chemical structures. The ammonium group deformation vibrations are located at 1500-1600 cm-1, together with 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)

The wavenumbers that appear in the IR spectra can be attributed to: OH (3405.67 cm-1), CH2 and CH3 (2975.62 cm-1), C=C (1644.02 cm-1), and C-O (1382.71 cm-1). Also, the UV-Vis spectra of the aqueous part of *Chelidonium majus L.*, showed the existence of three absorption bands: 734 nm, 268 nm and 198 nm, respectively. For a complete study, further analysis (including derivatization and HPLC) are usually performed.

Peptide and Amino Acids Separation and Identification from Natural Products 141

with their reference values, in order to identify specific amino acids. The Rf value for each known compound should remain the same, provided the development of the plate is done with the same solvents, type of TLC plates, method of spotting and under exactly the same

High performance liquid chromatography (HPLC) allows for the most efficient and appropiate separations of consitutents from natural product, complex mixtures. It has been shown that HPLC is the premier separation method that can be used for amino acid analysis (AAA), from natural products, allowing for the separation and detection by UV absorbance or fluorescence. However, most common amino acids do not contain a chromophoric group, and thus some form of derivatization is usually required before HPLC or post-column.

Amino acids are highly polar molecules, and therefore, conventional chromatographic methods of analysis, such as, reversed-phase high performance liquid chromatography (RP-HPLC) or gas-chromatography (GC) cannot be used without derivatization. The derivatization procedure has several goals, such as: to increase the volatily, to reduce the reactivity, or to improve the chromatographic behaviour and performance of compounds of interest. In the case of amino acids, derivatization replaces active hydrogens on hydroxyl, amino and SH polar functional groups, with a nonpolar moiety. The great majority of derivatization procedures involve reaction with amino groups: usually primary amines, but also secondary amines (proline and hydroxyproline), or the derivatization of a carboxyl function of the amino acids. Some of the most common derivatization reagents are

As it was mentioned before, prior derivatization of the amino acids is necessary due to the lack of UV absorbance in the 220-254 range. The paper of Moore and Stein [9] is actual even nowadays. Their method, that used a modified nynhidrin reagent for the photometric determination of the amino acids, represents the basis for various derivatization methods. There is a continuous increasing number of amino acids derivation reagents. There will be mentioned, as follows, some of the them: Melucci et al. [10] presents a method for the quantization of free amino acids that implies a pre-column derivatization with 9 fluorenylmethylchloroformate, followed by separation by reversed-phase high-performance liquid chromatography. Kochhar et al. [11] use the reverse-phase high-performance liquid chromatography for quantitative amino acids analysis and, as derivatization agent, 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide, known as Marfey's reagent. The method was successfully applied for the quantization of 19 L-amino acids and it is based on the stoichiometric reaction between the reagent and the amino group of the amino acids [11]. Ngo Bum et al. [12] have been used the cation exchange chromatography and post-column derivatization with ninhydrin for the detection of the free amino acids from the plant extracts. Culea et al. [13] have used the derivatization of amino acids with trifluoroacetic anhydride, followed by the extraction with ion exchangers and GC/MS analysis. Warren proposed a version of CE, CE-LIF, for quantifying the amino acids from soil extracts. The advantage of the method is represented by the low detection limits that are similar to the ones corresponding to the chromatographic techniques. In 2010, Sun et al. [15] have presented another method for the detection of amino acids from Stellera chamaejasme L., a

conditions.

presented in the Table 1.
