Page 154 - Modern Derivatization Methods for Separation Sciences

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Na CO was added and incubated for a further 5 min. After cooling the mixture, NTP-polyamines were
2 3
extracted twice with 2 ml of 2-methylbutane. The organic phase was evaporated to dryness. The residue
was reconstitute with 50 µ1 of MeOH and 10-µ1 aliquots were subjected to PR-HPLC with the
detection at 242 nm. The detection limits were 5 pmol for putrescine, 10 pmol for spermidine and 25
pmol for spermine.

2.2.1.3—
2,4,6-Trinitrobenzene-1-sulfonic Acid (TNBS) [18]

TNBS reacts with primary amino groups of amino acids and peptides in aqueous solution at pH 8 and at
room temperature without any undesirable side reactions. The N-trinitrophenyl derivatives produced
(Fig. 2.3) have a high molar absorptivity at 340 nm. The optimum pH for the reaction of TNBS with
amikacin is between 9.5 and 10.0. At temperatures below 70 °C and reaction times shorter than 30 min,
multiple derivatives of amikacin formed, due to the incomplete reaction.

2.2.1.4—
Na-(2,4-Dinitro-5-fluorophenyl)L-alanine amide (FDAA, 1-Fluoro-2,4-ddinitrophenyl-5-L-alanine
amide, Marfey's Reagent)

An HPLC-UV procedure for the separation and quantification of enantiomeric amino acid mixtures [19]
and D- and L-phosphoserine [20] were employed using pre-column derivatization with a chiral reagent,
FDAA (Fig. 2.4). The absolute sensitivity of the detector to the phosphoserine was found to be linear to
2 ng (11 pmol) on column.

Fig. 2.3.
The reaction of TNBS with primary amines.

Fig. 2.4.
Na-(2,4-Dinitro-5-fluorophenyl)
-L-alanineamide (FDAA).

2.2.2—
Acetic Anhydride [21]

Tertiary aliphatic amines can be separated on a HPLC column packed with Nucleosil 5N(CH ) resin,
3 2
and were detected by a post-column derivatization based on a reaction with a color reagent consisting of
an acetic anhydride solution of citric acid. The reaction temperature was 120 °C and the wavelength of
detection of wine red color was 550 nm. Primary and secondary amines do not react or interfere. The
lower limit of determination is 0.01 % for aliphatic mono, di-and trialkylamines.

2.2.3—

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