Analysis of amino acids

Due to zwitterionic character the analysis of amino acids has traditionally been based on ionexchange chromatography followed by post-column derivatization with ninhydrin or detection
with amperometric detector. Disadvantages of the method are rather long analysis time and
requirement of special instrumentation. Amino acids can be analyzed employing reversed-phase
HPLC or GC methods after appropriate derivatization. Typical reagents for pre-column
derivatization used in HPLC analysis of amino acids are phenyl isothiocyanate (PITC), ophthalaldehyde (OPA), 9-fluorenylmethyl chloroformate (FMOC), dansyl chloride (DNS) and
some other.
FMOC is a protective reagent for amino group used during peptide synthesis. FMOC reacts
with both, primary and secondary amino groups and forms stabile derivatives that can be
detected using UV (=263 nm) or highly sensitive fluorescent detection (excitation =270 nm,
detection =316 nm).

The problem with FMOC reagent is that it fluoresces itself, derivatization of aspartic and
glutamic acids proceed slower than with other amino acids and disubstituted FMOC
derivatization products of tyrosine and hystidine may be formed. Nonetheless, FMOC has been
widely used derivatizing reagent for the determination of amino acids.

Application: HPLC-UV-FL analysis of amino acids

Sample
Chromatography

Mixture of 24 amino acids, 20 g/ml

Derivatization: OPA & FMOC
HPLC;
Column: Zorbax Eclipse-AAA, (Agilent), 150 mm, 3.5
m
Gradient: 40 mM Na2HPO4 pH 7.8 / ACN:MeOH:W
(45:45:10)

Detection

UV =338 nm for OPA derivatives

UV =262 nm for FMOC derivatives
FLD for concentrations < 1 g/ml

For the GC or GC-MS analysis, amino acids have to be converted to volatile derivatives, i.e.
both, carboxyl and amino groups should be protected. The GC analysis, when coupled to a mass
spectrometer, has an important advantage over other methods because analytes are characterized
not only by their retention time but this method also provides molecular weight information.
Silylation, acylation, alkylation and conversion to alkoxycarbonyl esters are widely employed
methods for derivatization of amino acids.
The conversion of amino acids to their N(O,S)-alkoxycarbonyl derivatives is of considerable
interest, as it can be performed directly in aqueous media and the resulting derivatives are
extracted by organic solvents. In this one-step derivatization procedure amino acids are protected
using a mixture of alkylchloroformate, alcohol and pyridine as a catalyst.

Derivatization of amino acids by other methods must be performed in dry media some of them
may involve number of steps and are time consuming. Samples of amino acids often contain
difficult matrices and reducing interference of other components is one of derivatization goals.
Only the amino and carboxyl groups are protected using the above method while alcohols (for
example, sugars) remain in aqueous media. The method however has disadvantages
since alkoxycarbonyl derivatives are not stable therefore samples should be analyzed soon after
the derivatization; moreover, derivatization and GC analysis of some amino acids (threonine,
serine, arginine, citrulline, glutamine) may be problematic or not possible at all.
Sample
Derivatization
Chromatography
Ionization
Detection

Mixture of amino acids

Ethylchloroformate/ ethanol/ pyridine
GC; carrier gas helium. GC column: Thermo TR-5MS
EI
Polaris Q ion trap (Thermo Scientific)

The GC-MS chromatogram of N-ethoxycarbonyl ethyl esters of some amino acids