**5. Chromatographic methods**

These techniques insure the separation of closely related compounds in a mixture, by differences in the equilibrium or partition distributions of the components between two immiscible phases, the stationary and the mobile phases. These differences in the equilibrium distribution are a result of chemical structures and the degree of interactions of the components between these two phases. Under the influence of a mobile phase (one or a mixture of solvents), the target compounds percolate through the stationary phase, which is a porous medium (usually, silica or alumina). For successful amino acid and peptide isolations and purifications from natural products, have been developed different chromatographic methods (e.g., paper, thin layer, gas chromatography, column and high performance liquid chromatography, etc.). From the enormous variety of methods of separation and isolation useful for natural products, adsorption or partition chromatography represents one of the most useful techniques of general application.

Thin layer chromatographic (TLC) is the simplest technique used to separate and identify natural products of interest. This method readily provides qualitative information and possibly quantitative data. The stationary phase is usually silica gel on the TLC or HPTLC (high performance TLC) plate, which is made up of silica adhered to glass or aluminium or a plastic, for support. The eluent (solvent mixture) acts as the mobile phase. Practically, the compounds of interest need to be soluble to varying degrees. Separation again results from the partition equilibrium of the components in the mixture. The separation depends on several factors: 1) solubility in the mobile phase, 2) attractions or adsorption between the compound and silica, the more the compound interacts with silica, the less it moves upwards, 3) size or MW of the compound, for the larger the compound, the slower it moves up the plate.

Since amino acids are colourless compounds, ninhydrin is routinely used to detect them, with the result of a coloured product, due to the formation of Ruhemann'purple complex. The familiar violet color which is associated with the reaction of amino acids with ninhydrin is attributed to the anion of the reagent (derivatizing agent). Another technique uses anisaldehyde-H2SO4 reagent for detection of amino acids, followed by heating (120° C, 5 minutes).

Different organic solvents (e.g., alcohol, dioxane, methyl cellosolve, pyridine, and phenol) are used to accelerate the development of color, to varying degrees. Ultimately a phenolpyridine system was adopted as the most effective solvent. Exposure to 105°C for 3 to 5 minutes gives quantitative yields of color for all amino acids, except for tryptophan and lysine. The Rf (retardation factor) value for each compound can be calculated and compared 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 conditions.

140 Analytical Chemistry

up the plate.

minutes).

derivatization and HPLC) are usually performed.

**5. Chromatographic methods** 

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

These techniques insure the separation of closely related compounds in a mixture, by differences in the equilibrium or partition distributions of the components between two immiscible phases, the stationary and the mobile phases. These differences in the equilibrium distribution are a result of chemical structures and the degree of interactions of the components between these two phases. Under the influence of a mobile phase (one or a mixture of solvents), the target compounds percolate through the stationary phase, which is a porous medium (usually, silica or alumina). For successful amino acid and peptide isolations and purifications from natural products, have been developed different chromatographic methods (e.g., paper, thin layer, gas chromatography, column and high performance liquid chromatography, etc.). From the enormous variety of methods of separation and isolation useful for natural products, adsorption or partition

chromatography represents one of the most useful techniques of general application.

Thin layer chromatographic (TLC) is the simplest technique used to separate and identify natural products of interest. This method readily provides qualitative information and possibly quantitative data. The stationary phase is usually silica gel on the TLC or HPTLC (high performance TLC) plate, which is made up of silica adhered to glass or aluminium or a plastic, for support. The eluent (solvent mixture) acts as the mobile phase. Practically, the compounds of interest need to be soluble to varying degrees. Separation again results from the partition equilibrium of the components in the mixture. The separation depends on several factors: 1) solubility in the mobile phase, 2) attractions or adsorption between the compound and silica, the more the compound interacts with silica, the less it moves upwards, 3) size or MW of the compound, for the larger the compound, the slower it moves

Since amino acids are colourless compounds, ninhydrin is routinely used to detect them, with the result of a coloured product, due to the formation of Ruhemann'purple complex. The familiar violet color which is associated with the reaction of amino acids with ninhydrin is attributed to the anion of the reagent (derivatizing agent). Another technique uses anisaldehyde-H2SO4 reagent for detection of amino acids, followed by heating (120° C, 5

Different organic solvents (e.g., alcohol, dioxane, methyl cellosolve, pyridine, and phenol) are used to accelerate the development of color, to varying degrees. Ultimately a phenolpyridine system was adopted as the most effective solvent. Exposure to 105°C for 3 to 5 minutes gives quantitative yields of color for all amino acids, except for tryptophan and lysine. The Rf (retardation factor) value for each compound can be calculated and compared 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 presented in the Table 1.

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

widely-used plant in the Chinese traditional medicine; DBCEC (2-[2-(dibenzocarbazole) ethoxy] ethyl chloro-formate) was used as derivatization reagent, and the modified amino acids were detected by means of liquid chromatography with fluorescence detection. Li et al. [16] have proposed a new method for the detection of amino acids from the asparagus tin. After performing the derivatization of the samples with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), solid phase extractions on C18 cartridges have been performed. The purified amino acid derivatives were then subjected to the HPLC analysis. Zhang et al.[17] have proposed an improved chromatographic method (by the optimization of mobile phases and gradients) for the simultaneous detection of 21 free amino acids in tea leaves.

Peptide and Amino Acids Separation and Identification from Natural Products 143

OPA is another reagent used both for post-column or pre-column derivatization. Orthophthaldehyde (OPA) reacts at an amino group, generally in the presence of a thiol (mercaptoethanol), resulting in a fluorescent derivative, UV active at 340 nm. Other reagents for the precolumn derivatization of free amino groups from amino acids, are: PITC (phenylisothiocyanate), DABS-Cl (dimethylamino-azobenzenesulfonyl chloride), Fmoc-Cl (9-fluorenylmethyl-chloroformate), NBD-F (7-fluoro-4-nitro-2-aza-1,3-diazole), and others. The reaction time depends on the type of derivatization reagent and the reacting, functional group involved. For instance, from practically nearly instantaneous derivative formation in the case of the reagent, fluorene chloroformate, OPA is 1 minute and PITC is about 20

Ion pair, reverse phase liquid chromatography coupled with mass spectroscopy, IPRPLC-MS/MS, is a technique which allows for the analysis of amino acids without derivatization, thus reducing the possible errors introduced by reagent, interferences and derivative instability, side reactions, etc. Using volatile reagents, the IP separation is based on two different mechanisms: a) the IP-reagent is adsorbed at the interface between stationary and mobile phases; and b) the formation of a diffuse layer and the electrostatic surface potential depends on superficial (surface) concentration of IP reagent. There are other, possible

Gas chromatography (GC) can be used for the separation and analysis of compounds that can be vaporized without decomposition. The derivatization procedure most commonly employed is silylation, a method through which acidic hydrogens are replaced by an alkylsilyl group. Typically, silylation reagents are: BSTFA (*N*,*O*-bis-(tri-methyl-silyl) trifluoroacetamide and MSTFA (*N*-methyl-silyl-trifluoro-acetamide). A possible disadvantage of this approach, is due to the reagent and derivative being sensitive to moisture and possible, derivative instability. Some amino acids are unstable (e.g., arginine and glutamic acid). Arginine descomposes to ornithine, and glutamic acid undergo a rearrangement to pyro-glutamic acid. Another GC-derivatization method includes acylation or esterification, now using an aldehyde and alcohol (pentafluorpropyl or trifluoracetic aldehyde and isopropanol) or alkyl chloroformate and alcohol. Silylation takes place through the direct conversion of carboxylic groups to esters and amino groups to carbamates. Such reactions are catalyzed by a base (pyridine or picoline). Alkyl esters are

**GC-MS** represents an analysis method with excellent reproducibility of retention times, and the method can be easily automated. The major disadvantage is due to the possible temperature instabiity of some compounds and/or their derivatives, which then cannot be

**Mass spectrometry** represents one of most efficient techniques for natural product, structure elucidation. It functions by a separation of the ions formed in the ionisation source of the mass spectrometer, according to their mass-to-charge (m/z) ratios. The technique allows for accurate MW measurements, sample confirmation, demonstration of the purity of a sample, verification of amino acid substitutions, and amino acid sequencing. This procedure is

mechanisms suggested in the literature for how IPRPLC operates.

extremely stable and can be stored for long periods of time.

easily analyzed under most GC conditions.

minutes.


**Table 1.** Reagents for Derivatization

There have been already developed, several liquid chromatography methods for amino acid quantification. General approaches are ion-exchange chromatography (IEC) and reversedphase HPLC (RP-HPLC). Both approaches require either a post-column or pre-column derivatization step. Even this technique offers satisfactory resolutions and sensitivity, but the necessary derivatization step provides an increased complexity, cost, and analysis times.

Ion-exchange chromatography with postcolumn ninhydrin detection is one of the most commonly used methods employed for quantitative amino acid analysis. Separation of the amino acids on an ion-exchange column is accomplished through a combination of changes in pH and ionic (cation) strength. A temperature gradient is often employed to enhance separation.

But, perhaps the most effective method is cation exchange chromatography (CEC) in the presence of a buffer system (usually a lithium buffer system), and a post-column derivatization step with ninhydrin. Detection is performed with UV absorbance. In this way one achieves the desired amino acid separation, according to the colour (structure) of the derivatived compound formed. Amino acids which contain primary amines, except an imino acid, give a purple color, and show the maximum absorption at 570 nm. The imino acids such as proline give a yellow color, and show the maximum absorption at 440 nm. The postcolumn reaction between ninhydrin and an amino acid eluted from the column is monitored at 440 and 570 nm.

OPA is another reagent used both for post-column or pre-column derivatization. Orthophthaldehyde (OPA) reacts at an amino group, generally in the presence of a thiol (mercaptoethanol), resulting in a fluorescent derivative, UV active at 340 nm. Other reagents for the precolumn derivatization of free amino groups from amino acids, are: PITC (phenylisothiocyanate), DABS-Cl (dimethylamino-azobenzenesulfonyl chloride), Fmoc-Cl (9-fluorenylmethyl-chloroformate), NBD-F (7-fluoro-4-nitro-2-aza-1,3-diazole), and others. The reaction time depends on the type of derivatization reagent and the reacting, functional group involved. For instance, from practically nearly instantaneous derivative formation in the case of the reagent, fluorene chloroformate, OPA is 1 minute and PITC is about 20 minutes.

142 Analytical Chemistry

widely-used plant in the Chinese traditional medicine; DBCEC (2-[2-(dibenzocarbazole) ethoxy] ethyl chloro-formate) was used as derivatization reagent, and the modified amino acids were detected by means of liquid chromatography with fluorescence detection. Li et al. [16] have proposed a new method for the detection of amino acids from the asparagus tin. After performing the derivatization of the samples with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF), solid phase extractions on C18 cartridges have been performed. The purified amino acid derivatives were then subjected to the HPLC analysis. Zhang et al.[17] have proposed an improved chromatographic method (by the optimization of mobile phases and gradients) for the simultaneous detection of 21 free amino acids in tea leaves.

**DERIVATIZATION REAGENT Reference** 

9-fluorenylmethylchloroformate 10 trifluoroacetic anhydride 13

4-chloro-3,5-dinitrobenzotrifluoride 16

1-fluoro-2,4-dinitrophenyl-5-L-alanine amide 11 2-[2-(dibenzocarbazole)-ethoxy] ethyl chloroformate 15

There have been already developed, several liquid chromatography methods for amino acid quantification. General approaches are ion-exchange chromatography (IEC) and reversedphase HPLC (RP-HPLC). Both approaches require either a post-column or pre-column derivatization step. Even this technique offers satisfactory resolutions and sensitivity, but the necessary derivatization step provides an increased complexity, cost, and analysis times. Ion-exchange chromatography with postcolumn ninhydrin detection is one of the most commonly used methods employed for quantitative amino acid analysis. Separation of the amino acids on an ion-exchange column is accomplished through a combination of changes in pH and ionic (cation) strength. A temperature gradient is often employed to enhance

But, perhaps the most effective method is cation exchange chromatography (CEC) in the presence of a buffer system (usually a lithium buffer system), and a post-column derivatization step with ninhydrin. Detection is performed with UV absorbance. In this way one achieves the desired amino acid separation, according to the colour (structure) of the derivatived compound formed. Amino acids which contain primary amines, except an imino acid, give a purple color, and show the maximum absorption at 570 nm. The imino acids such as proline give a yellow color, and show the maximum absorption at 440 nm. The postcolumn reaction between ninhydrin and an amino acid eluted from the column is

ortho-phthaldehyde (OPA) phenylisothiocyanate dimethylamino-azobenzenesulfonyl chloride 7-fluoro-4-nitro-2-aza-1,3-diazole

**Table 1.** Reagents for Derivatization

monitored at 440 and 570 nm.

separation.

Nynhidrin 9, 12

Ion pair, reverse phase liquid chromatography coupled with mass spectroscopy, IPRPLC-MS/MS, is a technique which allows for the analysis of amino acids without derivatization, thus reducing the possible errors introduced by reagent, interferences and derivative instability, side reactions, etc. Using volatile reagents, the IP separation is based on two different mechanisms: a) the IP-reagent is adsorbed at the interface between stationary and mobile phases; and b) the formation of a diffuse layer and the electrostatic surface potential depends on superficial (surface) concentration of IP reagent. There are other, possible mechanisms suggested in the literature for how IPRPLC operates.

Gas chromatography (GC) can be used for the separation and analysis of compounds that can be vaporized without decomposition. The derivatization procedure most commonly employed is silylation, a method through which acidic hydrogens are replaced by an alkylsilyl group. Typically, silylation reagents are: BSTFA (*N*,*O*-bis-(tri-methyl-silyl) trifluoroacetamide and MSTFA (*N*-methyl-silyl-trifluoro-acetamide). A possible disadvantage of this approach, is due to the reagent and derivative being sensitive to moisture and possible, derivative instability. Some amino acids are unstable (e.g., arginine and glutamic acid). Arginine descomposes to ornithine, and glutamic acid undergo a rearrangement to pyro-glutamic acid. Another GC-derivatization method includes acylation or esterification, now using an aldehyde and alcohol (pentafluorpropyl or trifluoracetic aldehyde and isopropanol) or alkyl chloroformate and alcohol. Silylation takes place through the direct conversion of carboxylic groups to esters and amino groups to carbamates. Such reactions are catalyzed by a base (pyridine or picoline). Alkyl esters are extremely stable and can be stored for long periods of time.

**GC-MS** represents an analysis method with excellent reproducibility of retention times, and the method can be easily automated. The major disadvantage is due to the possible temperature instabiity of some compounds and/or their derivatives, which then cannot be easily analyzed under most GC conditions.

**Mass spectrometry** represents one of most efficient techniques for natural product, structure elucidation. It functions by a separation of the ions formed in the ionisation source of the mass spectrometer, according to their mass-to-charge (m/z) ratios. The technique allows for accurate MW measurements, sample confirmation, demonstration of the purity of a sample, verification of amino acid substitutions, and amino acid sequencing. This procedure is useful for the structural elucidation of organic compounds and for peptide or oligonucleotide sequencing. The major advantadge in using MS is due to the need for very small amounts of sample (ng to pg). A disadvantage of conventional ionization methods (e.g., electron impact, API) is that they are limited to compounds with sufficient volatility, polarity and MW. Volatility can be increased by chemical modifications (derivatizations, such as: methylation, trimethylsilylation or trifluoro-acetylation). For peptides, there has been developed certain new, very efficient techniques, such as: electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI).

Peptide and Amino Acids Separation and Identification from Natural Products 145

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[3] S. W. Lee, J. M. Lin, S. H. Bhoo, Y. S. Paik, T. R. Hahn, Colorimetric determination of amino acids using genipin from *Gardenia jasminoides*, Anal. Chim. Acta 2003, 480, 267-

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[8] R. Linder, M. Nispel, T. Haber, K. Kleinermanns, "Gas-phase FT-IR spectra of natural

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[10] D. Melucci, M. Xie, P. Reschiglian, G. Torsi, "FMOC-Cl as derivatizing agent for the analysis of amino acids and dipeptides by the absolute analysis method",

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In the next Figure 4, is presented the mass spectrum of pure valine, recorded on a Bruker Daltonics High Capacity Ion Trap Ultra (HCT Ultra, PTM discovery) instrument.

**Figure 4.** Valine MS-specta

NMR spectroscopy offers the most useful and valuable information about the structure of perhaps any natural product. The method has the advantage of excellent reproducibility. Even though it is considered to be one of the more expensive techniques, NMR is relatively cheap, fast sensitive and easily used as a routine application for amino acid analysis.
