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found to be important to many areas of science. Because of its extremely high sensitivity, laserinduced
fluorescence (LIF) detection has been successfully introduced to HPLC and CE analyses, and the LIF
instrument equipped with heliumcadmium (He-Cd) and argon (Ar) ion lasers is now available
commercially. The successful design of derivatization reagents for He-Cd and Ar ion LIF detections,
relies on the development of excellent fluorophore with highly intense fluorescence and an excitation
maximum nearing the 325 nm and 488 nm output wavelengths of the He-Cd and Ar ion lasers,
respectively. Numerous reagents for LIF detection, using these lasers, are proposed. Recently, (visible)
diode LIF has been focused as an ultrasensitive and selective on-line detection method in both HPLC
and CE, because of the reduction of background fluorescence and Raman scattering light. The diode
lasers have several advantages; they are stable, compact, easy to perform, highly efficient and
inexpensive. Most naturally occurring substances do not show fluorescence in the far-red and near-
infrared wavelength regions in which the diode lasers emit. Therefore, derivatization reagents are
required for the diode LIF detection of the substances. In order to utilize the potential diode LIF
detection, it is essential to develop new labeling reagents with special fluorophores that absorb, and
fluorescence in the far-red and near-infrared regions.

In this section, analytical features of fluorescence derivatization of biologically and environmentally
important compounds and their applications to HPLC and CE with fluorescence detection are surveyed.

3.2—
Reagents for Amines and Amino Acids

Amines and amino acids are present in most biological and environmental samples. Hence, numerous
fluorescence derivatization reagents have been proposed for the determination of the amino compounds.
The amino moieties of the compounds are so reactive that the fluorescence derivatization generally
proceeds under mild conditions.

3.2.1—
General Amino Compounds

3.2.1.1—
Primary Amines and Amino Acids

OPA has been widely used for the derivatization of primary amines and amino acids. OPA reacts only
with primary amino groups in alkaline pH in the presence of an alkylthiol such as 2-mercaptoethanol to
give highly fluorescent isoindole derivatives (Fig. 3.1 A) [1]. The derivatization is complete within
about 2 min in a mixture of borate buffer (pH 6-8 for amines, pH 9.7-10.0 for amino acids) and
methanol, even at room temperature. The OPA reaction can be applied to post-column derivatization
because OPA itself is non-fluorescent [2]. As OPA derivatives, however, are not sufficiently stable to
afford reproducible results, the post-column OPA reaction is introduced for an automatic amino acid
analyzer including cation-exchange chromatography. An automated system, based on the OPA pre-
column derivatization is also developed. Using the system, the highly sensitive and reproducible
analysis for amino compounds are attained [3-6]. 2-Ethanethiol [7], 3-mercaptopropionic acid [8] and
N-acetyl-L-cysteine [9,10] afford more stable fluorescent derivatives than 2-mercaptoethanol. Sample
preparation and OPA reagent stability are still improving. The addition of nitriloacetic acid to reaction
mixture results in a four-fold improvement in stability [11]. The detection limits [signalto-noise ratio
(S/N) = 3] of amino acids in precolumn derivatization HPLC are around 1 pmol on-column.

General Derivatization Procedure of Primary Amines with OPA

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