Page 160 - Modern Derivatization Methods for Separation Sciences

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other volatile products were then removed by evaporation under vacuum. A derivatized sample could be
stored dry at 4 °C for 24 h or frozen at -20 °C for a week or more. The derivatives were dissolved either
in 50 µ1 (cerebrospinal fluid, blood spots) or 100 µ1 (plasma, amniotic fluid, urine) of a diluent
composed of 5 mM Na phosphate buffer (pH 7.4) containing 50 ml/1 CH CN. Usually 20 µ1 of sample
3
solution were injected onto the column provided by the Waters Pico-Tag amino acid analysis system.

2.2.3.3—
Butylisothiocyanate (BITC) [44]

BITC reacts with amino acids including hydroxyproline and proline and produces butylthiocarbamyl
(BTC) derivatives. All derivatives of 22 protein amino acid were quantitatively resolved in 27 min by
C18 RP-HPLC, except asparagine and serine, and detected at UV 250 nm (absorption maximum
wavelength is ca. 234 nm, but the wavelength is the most efficient wavelength to avoid the absorption
spectra of the impurities and the electrolyte, ammonium acetate, in the solvent). With the PTC
derivatives, the derivatization reaction was completed in 10-20 min at room temperature, but with the
BTC derivatives 120 min were required. The calibration graphs showed good linearity in the range 0.5-
2.5 nmol. The BTC derivatives were stable at room temperature for ca. 8 h.

2.2.3.4—
4-N,N-Dimethylaminoazobenzene-4isothiocyanate (DABITC) [45-50]

This modified Edman reagent can be used to derivatize peptides (Fig. 2.9) after a tryptic or
chymotryptic treatment and offers distinct advantages as described in detail by Chang [49]. The
introduction of the DABITC reagent to the derivatization of peptides and to the microsequencing
analysis of proteins and peptides with the DABITC/PITC double coupling method [51,52] is thought to
be a very efficient tool for microsequencing studies at picomole levels [53]. The thiohydantoin amino
acid derivatives (DABTHs) of DABITC are red and molar

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