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of the 1990s, in practical use are derived from the findings of the 70s and 80s [6]. In the 1990's,
fluorescence derivatization has been a major detection technique when highly sensitive detection is
required in HPLC and capillary electrophoresis (CE) [7,8]

In this chapter, the derivatization method for HPLC and CE connecting with UV-VIS detection will be
described. The term derivatization in analysis means basically the reactions caused by chemical
reagents or physical methods to convert a poor detector-responding analyte into a highly detectable
product which has enhanced chromatographic or electrophoretic properties. The properties are: (1)
detector response; (2) linear response range; and (3) ability of separation. In other words, derivatization
aims to increase the detectability of the analyte and, sometimes to achieve the addition of a
chromatographic or electrophoretic handle onto the analyte molecule.

Derivatization procedures both in the pre and post-column or capillary cause problems for HPLC or CE.
On-line and off-line derivatization also has inherent problems which need to be considered [8].

We do not discuss derivatization with an enzyme, chelating reagent for the determination of metals and
photochemical derivatizations, rather we have tried to select those papers which help to construct a
view of UV-VIS derivatization of organic compounds involved in a creature's life.

2.2—
Label of Amines and Amino Acids (-NH , NH)
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The detection and determination of amines are concerned not only with biochemical but also industrial
analysis. Thus various derivatization reagents for the chromatographic determination of primary and
secondary amines as well as tertiary amines, are available. In many biologically important compounds,
an amino groups exists as amino acids and polyamines. When HPLC or CE analysis of these amino
compounds with direct UV-VIS detection is difficult because of the weak UV-VIS absorption of the
compounds, derivatization of amines with a reagent having a strong UV-VIS absorbing structure is one
of the considerable methods to choose from.

2.2.1—
Nitrobenzenes

2.2.1.1—
1-Fluoro-2,4-dinitrobenzene (FDNB, 2,4-Dinitro-1-fluorobenzene, Sanger Reagent)

Pre-column derivatization of aminoglycosides with FDNB prior to HPLC used an important analytical
technique for the determination of various antibiotics. Aminoglycosides, such as amikacin [9,10],
tobramycin [11,12], gentamicin [13], sisomicine [13], neamine, neomycin B, neomycin C [14] in
microliter quantities of biological fluids were measured by HPLC using FDNB derivatization. The 2,4-
dinitrophenyl (DNP) derivative produced is stable. A drawback of FDNB is its toxicity; it must be
handled with protective gloves. FDNB reacts with primary and secondary amino groups [14]. The
chemical structure of FDNB derivative of amikasin is shown in Fig. 2.1 [10]. Also the major FDNB
derivatives of tobramycin (Tb) were considered as a Tb derivative with all five amino groups 2,4-
dinitrophenylated TB(NDNB) and one hydroxyl group 2,4-dinitrophenylated TB(NDNB) (ODNB)
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[11].
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The amino acid N , N , N -trimethyllysine was also derivatized by FDNB. Trimethyllysine and N N ,
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N -triethyllysine (internal standard, IS) were isolated from urine specimens
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