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Dansyl derivatives of 24 amino acids can be separated by EMKC with UV absorption at 214 nm [40].

Recent interest in the field of amino acid analysis is placed on chiral separation of D and L-amino acids
by HPLC and CE in which high sensitivity and high resolution are essential. A maximum number of six
pairs of DL-dansylamino acids was separated by cyclodextrin-modified MEKC in a single run [66].

DNS-Cl Application: Optimized Derivatization Procedure for PAPC [64]

Dansylation was carried out by adding 2.0 ml of TEA buffer (at pH 11), 2.0 ml PAPC solution and 3.0
ml DNS-Cl solution to a 10-ml volumetric. The sample was then diluted to volume with CH CN and
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water to give a final ratio of 60:40 organic to aqueous. The final concentrations in the derivatization
solutions were 14 nmol/ml PAPC, 17 µmol/ml TEA and 1.3 µmol/ml dansyl chloride. The sample
solutions were allowed to react at room temperature in the dark for 4, 8 and 24 h. The reaction was
stopped by adding a 2.0 ml aliquot of the reaction solution to 2 ml of glycine (2 mg/ml) in a 10 ml
volumetric. After 10 min the neutralized solutions were diluted to volume with 0.04 M pH 5 acetic
acid-CH3CN (30:70). HPLC was performed under the above conditions, detection limits for DNS-
PAPC were found by injecting 0.7 pmol in column at an S/N ratio of 2.1.

2.2.5.3—
4-N,N-Dimethylaminoazobenzene-4sulfonyl Chloride (DABS-Cl, Dabsyl-Cl) [53]

The sulfonyl group of DABS-Cl reacts with primary and secondary amino groups, thiol, imidazole,
phenols and aliphatic hydroxyl groups. In an original study by Chang et al., the intense chromophoric
dabsyl amino acid permits the detection of amino acids as colored spots in the range 0.1-0.01 nmol
visible directly on a silica gel TLC plate [67] and on a polyamide layer sheets [68]. The dabsyl
derivatives of various amino acids show absorption maxima ranging from 448 to 468 nm. Actually
absorbance detection wavelength at 436 nm [69,70], 456 nm [69] and 425 nm [71] was also utilized in
HPLC. The stability of the dabsyl amino acid derivaties is as good as that of dansyl amino acid.

Dabsyl-Cl Application: Derivatization of Amino Acids [69,70]

The derivatization reagent consisted of a solution of 4 nmol/1 DABS-Cl in CH CN. This solution was
3
prepared daily. CH CN (40 µ1) and DABS-Cl reagent (40 µ1) were added to 20 µ1 of the standard
3
solution. For each sample, 100 µ1 of sample were added to 100 µ1 of DABS-Cl reagent. The mixture
was heated for 10 min at 70 °C in an oven (the mixture will become completely soluble after heating).
A 900 µ1 volume of a solution of sodium hydrogen phosphate (0.1 mol/1, pH 7.0)-CH3CNMeOH
(70:20:10, v/v/v) was added to the reaction mixture. A 20 µ1 volume of this mixture was injected on to
a C18 HPLC column.

2.2.6—
9-Fluorenylmethyl Chloroformate (9-Fluorenylmethoxycarbonyl Chloride, FMOC-CL, Fluorescence
and UV Detection)

An automatic pre-column derivatization procedure is developed for the determination of amino acids.
Carbonyl chlorides react with primary, secondary and also tertiary amines and alcohols. The reactivity
of the chloride is thought to be higher than that of sulfonyl chlorides. Carratú et al. [72] reported that
the results of determination for amino acids obtained with FMOC-Cl by UV detection (265 nm) were
similar to those obtained by fluorescence detection (excitation at 265 nm and emission at 340 nm)
except for tryptophan, which was not detectable by fluorimetry because the fluorescence of the

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