Page 54 - Modern Derivatization Methods for Separation Sciences

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To analyse glucosides, immobilized β-glucosidase was used for the hydrolysis of β-D-glucose,
followed by luminol chemiluminescence detection of hydrogen peroxide produced by GOD reactor [46]
and immobilized GAM was used to transform maltooligosaccharide to glucose followed by PAD
detection [47]. The detection limit of these HPLC methods with immobilized enzyme reactors for
glucose is 2-10 ng, and the sensitivity is 1000 times higher than that of RI detector (10 µg). However,
exchange of reactors is required because the lifetime of these immobilized enzyme reactors is reported
to be three months for GOD [42,43], one month for GAM [43] and eight days for INV [45].

The use of the pre-column derivatization method is reported for the reaction with p-nitorobenzoyl
chloride (PNB-C1) followed by UV detection at 260 nm [48,49], with phenyl isocyanate (PHI) by UV
detection at 240 nm [50,51], and with isatoic anhydride to form fluorescent anthraniloyl derivatives by
fluorescent detection (λex360 nm, λem420 nm) [52]. PNB-C1 was used for the analysis of biological
samples with excellent sensitivity, but the washing procedure after the reaction is complicated. On the
other hand, PHI reacts highly with the free hydroxyl groups of carbohydrates and sugar alcohols. The
resulting derivatives are very stable and show excellent sensitivity. UV monitoring at 240 nm permits
detection down to the nanogram level. However, PHI derivatives of reducing sugars gave a peak for
each enantiomer while non-reducing sugar alcohols gave a single peak. PNB-C1 derivatives were
analysed with normal-phase HPLC, PHI derivatives with reversed-phase HPLC and anthraniloyl
derivatives with reversed-phase or normal-phase HPLC.

Analysis by Capillary Electrophoresis

Capillary electrophoresis (CE) for the analysis of saccharides commonly employs UV detection, and the
drawback is sensitivity. Introduction of boron in the alkaline mobile phase allows 2-20 fold higher
sensitivity [53], which may be explained by anion electrification of saccharides which results in the
reaction of the hydroxy group of saccharides with boron ion. To improve sensitivity, the addition of
sorbic acid which acts as both an electrolyte and a chromophore with detection at 265 nm was
developed (detection limit, 2 pmol) [54].

A derivatization method with 1-phenyl-3-methyl-5-pyrazolone [55], 2-aminopyridine (AP) [56], N-2-
pyridylglycamine [57] and ethyl-p-aminobenzoate [58] with detection at UV 240 nm-305 nm was
reported. The detection limit of these CE ranges from 10 fmol to 10 pmol [54,57,58]. Pre-column
fluorophore derivatization with AP [59], 5-aminonaphthalene-2-sulfonate (ANA) [60], 8-
aminonaphthalene-1,3,6-trisulfonic acid(ANTS) [61] and 9-aminopyrene-1,4,6-trisulfonate (APTS)
[62], as reductive amination, is reported (Fig. 1.2.1). The sugars were derivatized through the Shiff base
formation between the aromatic amine of a reagent and the aldehyde form of a sugar, followed by
reduction of the Shiff base to a stable product. Other fluorescence derivatization methods use aminated
reduced sugar and aminosaccharides with 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA)
[63-65]. These fluorescent derivatives are excited by laser ray, AP, ANA and ANTS derivatives by
heliumcadmium laser (λex325 nm, λem375 nm for AP, 475 nm for ANA, 514 nm for ANTS), and
APTS by argon-ion laser (λex457 nm, λem550 nm for CBQCA; λex488 nm, λex512 nm for APTS).
The limit of laser-induced fluorescence detection for APTS derivatives of sugars is 2 pmol, for CBQCA
is at atto-mole level.
The CE analysis of saccharides in this section is limited to standards of sugars and saccharides and
hydrolysis products. The application of CE for food materials which contain a complex matrix would
require such preliminary separation as clean-up.

1.2.2.2—

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