Page 56 - Modern Derivatization Methods for Separation Sciences

Document Página 1 de 2

Page 18

amino acids are called conjugated proteins. The representative conjugated proteins are glycoprotein and
lipoproteins. Peptides include oligopeptide (the number of amino acids is 2-10), and polypeptides (the
number of amino acids is 10-50). Peptides and proteins are either analysed by the high molecule itself
or by amino acids, the hydrolysates of peptides and proteins. Most HPLC methods may employ the
latter case because the amino acid composition of proteins is often studied.

Fig. 1.2.1.
Electropherogram of nine APTS-derivatized monosaccharides at 1.0 µM
each. conditions: untreated fused-silica capillary, 20 µm (i.d.) × 27 cm;
light source, 488 nm argon-ion laser; emission filter, 520 ± 20 nm (Oriel,
Startford, CT) and a notch filter at 488 nm (Barr Associates);
applied potential, 20 kV/19 µA; buffer, 100 mM borate, pH 10.2;
sample injection, 20 s, 3.5 kPa pressure; outlet, cathode. Peak identification:
(1) N-acetylgalactosamine; (2) N-acetylglucosamine; (3) rhamnose; (4)
mannose; (5) glucose; (6) fructose; (7) xylose; (8) fucose; and (9) galactose;
(x) impurity peak derived from APTS. (Reproduced from ref. 62: Anal.
Chem., (1995) 67, p. 2244, Fig. 5.).

Amino Acids

The analysis of amino acids has traditionally been based on ion-exchange chromatography followed by
post-column derivatization with ninhydrin. This approach is reliable and the resolution of the amino
acids is reasonable but the analysis time is rather long with limited detectability. Although the method
is still the main method in use, the following HPLC methods tend to supersede the classical method.
Due to the use of simpler instrumentation in pre-column derivatization and the lower cost of such
systems compared with post-column derivatization, the pre-column derivatization is generally
preferred. Typical reagents for pre-column derivatization are phenyl isothiocyanate (PITC) [66-68]. o-
phthalaldehyde (OPA) [69-71], 9-fluorenylmethyl chloroformate (FMOC) [72-75], 2,4-
dinitrofluorobenzene (DNFB) [76], dansyl chloride (DNS) [77-79], 6-aminoquinolyl-N-
hydroxysuccinimidyl carbamate (AQC) [80,81], PHI [82], N-(acridinyl) maleimide [83], and
fluorescamine [84,85]. Some of the features of these methods for amino acid analysis of protein
hydrolysates were summarized by Sawar et al. [86].

http://emedia.netlibrary.com/nlreader/nlreader.dll?bookid=17968&filename=Page_18.html 30/09/2003