Derivatización por Dansyl o Dabsyl

8/13/2019 Derivatizacin por Dansyl o Dabsyl

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Ibolya Molnftr-Perl (Editor)Quantitation of mino cids and mines by ChromatographyJournal of ChromatographyLibrary, Vol. 709 2005 Elsevier B.V. All rights reserved

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H P L C o f A m i n o A c i d s as D a n s y l a n d D a b s y l D e r i v a ti v e s

Toyohide Takeuchi

o n t e n t s

Summary

1. Introduction

1.1. Dansyl derivatives

1.2. Dabsyl derivatives

2. Derivatization conditions

2.1. D ansyl derivatization

2.2. D absyl derivatization

3. Chromatographic conditions for dansyl and dabsyl amino acids

3.1. Dansy l amino acids

3.1.1. Detection

3.1.2. Separation

3.2. Dabsy l amino acids

3.2.1. D etection

3.2.2. Se paration

4. Applications

4.1. Dansyl amino acids

Am ino acids / enantiomeric sep aration / on-colum n fluorimetric detection

4.2. Dab syl amino acids

Summary

Pre-column derivatizat ion of amino acids w ith 5-dimethylam inonaphthalene-l-sulfonyl chlo-

ride (dansyl chloride) or 4-dimethylaminoazobenzene-4'-sulfonyl chloride (dabsyl chloride) is

described. These pre-column derivatization methods enable a sensitive HPLC analysis of

amino acids.

Da nsyl de rivatization has originally been a pplied to the sequential analysis o f peptides

and proteins. It finds another use in biochem istry for the fluorogenic labeling o f proteins and

enzymes. Dansyl chloride is the most widely used for the derivatization of amino acids. Dan-

syl chloride readily reacts with primary and seconda ry amino groups o f amino acids. The re-


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230 Toyohide Takeuchi

action med ium is usually an aqueou s-organic mixture e.g., 1:1 acetone-water) adjusted to a

pH of 9.5-10. Dansylation reaction is usually carried out at elevated temperatures. Various

dansy lation reaction conditions are reported, involving 60 ~ for 60 min or at 38 ~ for 90 -120 min. Dansyl amino acids absorb light in the UV region. For example, absorption maxima

are observed at 214, 246 and 325 nm for dansyl glycine, and the absorption at 214 nm is the

strongest. Dansyl group is also fluorescent and dansyl amino acids can therefore be detected

by a fluorimetric detector. The excitation and emission wavelengths for dansyl glycine are

324 and 559 nm, respectively. Dansyl amino acids are mostly separated in the reversed-phase

mo de on a C8 or C 18 column with a linear gradient.

Dab syl chloride has a num ber of advantages over other derivatization methods, includ-ing a simple derivatization procedure, very good stability, good reproducibili ty and a good

limit of detection for the method, complete HPLC separation of all the amino acids, and spe-

cific detection at a wavelength in the visible region. Dabsyl chloride also reacts with primary

and secondary amino groups of amino acids as does dansyl chloride. Dabsylation reagent so-

lution can be prepared by dissolving dabsy l chloride in acetone or in acetonitrile, followed by

mixing w ith buffer e.g., carbonate, pH 8.5-9.5). Derivatization of amino acid with dabsyl

chloride is also carded out at elevated temperatures as for dansylation. For example, sampleswere incubated at 70 ~ for 15-30 min.

There have been a number of l i teratures dealing with application of dansylation to the

determ ination o f amino acids contained in various sam ple sources, involving biological fluids,

tissues, foods, peptide or protein hydrolyzates, etc. Dabsylation also covers nearly the same

application areas as for dansylation. Enantiomeric se paration of dansyl and dabsyl am ino ac-

ids can be ca rded out by l igand-exchange chromatography. The use o f native, and derivat ized

13- and ~,-cyclodextrin station ary ph ases as w ell as the u se o f 13-cyclodextrin mo bile p has e ad-ditive are other op tions for the chiral separation of these derivatized a mino acids.

1 In t r o d u c t i o n

1 1 Da nsyl derivatives

Pre-colunm derivat ization with 5-dimethylaminonaphthalene-l-sulfonyl chloride dansyl

chloride) is known to enable a sensitive HPLC analysis of amino acids, as do derivatization

with o-phthalaldehyde, phenylisothiocyanate, fluorescamine or 2,4-dinitrofluorobenzene.Dansyl derivatization has originally been developed by Gray and co-workers [1-3] and ap-

plied to the sequential analysis o f peptides and proteins. It finds another use in biochem istry

for the fluoroge nic labeling o f proteins and enzym es.


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HP LC of Amino Acids as D ansyl and Dabsyl Derivatives 231

Dansyl chloride is the most widely used for the derivatization of amino acids. Dansyl

chloride readily reacts with primary and secondary amino groups of amino acids. It can also

react with phenols, thiols and imidazoles less rapidly, and very slowly with alcohols. There-fore, some amino acids can form a few different dansylated derivatives depending on the de-

rivatization conditions. For example, tyrosine forms N-dansylated, O-dansylated and didansy-

lated derivatives [4 ]. Da nsyl am ino acids have usually been separated in the reversed-phase

mode , fol lowed by f luorimetric or UV spectrophotometric detection. The y have m ost ly been

separated by g radient elution, but isocratic separations have also been reported.

1 2 Dab syl derivat ivesAfter introduc tion of 4-dim ethylam inoazob enzen e-4 -sulfonyl chloride (dabsyl chloride) as a

prec olum n derivatization agent for amino acids [5], num erous applications have been pub-

lished, and most of them related to the analysis of amino acids in biological fluids or protein

hydrolyzates by HPLC [6, 7]. Dabsyl chloride has a number of advantages over other deri-

vatization methods, including a simple derivatization procedure, very good stability, good

reprodu cibili ty and a goo d limit of detection for the me thod, comp lete HP LC separation of all

the amino acids, and specific detection at a wavelength in the visible region [5-8]. Dabsylchloride also reacts with primary and secondary amino groups of amino acids as does dansyl

chloride, and it forms mono-dabsyl derivatives as well as bis-dabsyl derivatives with amino

acids such as lysine, tyrosine and histidine [9].

2 D e r i v a t i z a t i o n c o n d i t i o n s

2 1 Da nsyl der ivat izat ion

The rea ction med ium is usually an aqueou s-organic mixture (e.g., 1:1 acetone-w ater) adjustedto a pH o f 9.5-10 . Figure 1 shows the reaction of a primary or sec ondary am ino acid

(HNR1R2) with dansyl chloride. Dansylation reaction is usually carried out at elevated tem-

peratures. Various dan sylation reaction conditions are reported, involving 60 ~ for 60 rain

[10] or at 38 ~ for 90-1 20 rain [11].

The dansylation reaction provides high yield derivatization while the yield appears to

be inde pende nt o f the ratio of dansyl chloride to amino acid over 1000-fold range [12]. In

addition, dan syl chloride stock solution is kept in a freezer to avoid degradation.


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232 Toyohide Takeuchi

H 3 C ~ N ~ C H 3

R I ~ N I R 2

H

-t-

H 3 C ~ N I C H 3

SO2 SO2

C R , ,,,N ~ R 2

Figure 1 Dansyl derivatization of amino acids

2 2 Dab syl derivatization

Dabsylation reagent solution can be prepared by dissolving dabsyl chloride in acetone or in

acetonitrile, followed by mixing with buffer (e.g., carbonate, pH 8.5-9.5). Derivatization of

amino acid with dabsyl chloride is also carried out at elevated temperatures as for dansylation.

For example, samples w ere incubated at 70 ~ for 15-30 min [ 13, 14]. It should be noted that

dabsyl chloride stock solution is also kept in a freezer.

H a C ~ N / C H 3 H 3 C ~ N I C H 3

R n ~ N R 2N ~ N - - I "~ N ~ N

H

SO SO

Ci RI,, , ,N~R2

Figure 2 Dabsyl derivatization of amino acids


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HP LC of Amino Acids as Dansyl and Dabsyl Derivatives 233

3 Ch rom atographic conditions for dansyl and d absyi am ino acids

3 1 Dan sy l amino ac ids

3 1 1 De tec t ionDansyl amino acids absorb l ight in the UV region. Figure 3 shows UV absorpt ion spectra of

0.01 and 0.1 mM dansyl glycine aqueous solut ion. I t can be seen that absorpt ion maxima are

observed at 214, 246 and 325 nm, and the absorpt ion at 214 nm is the s t rongest . The molar

extinc tion coefficie nts o f dans yl glycine at 214, 246 and 325 nm are 3.5x104, 1.3x104 and

0.39x 104, resp ective ly see Table 1).

The d ansyl group is fluorescent and dansyl am ino acids can therefore be d etected by a

f luorimetr ic detector [15] . The exci tat ion and emission wavelengths for dansyl glycine are324 and 559 nm, respe ct ively [16] . The exci tat ion wav elength is c lose to the wave length of a

He/C d laser, which leads to highly sensi t ive detect ion of dansyl amino acid [17]. Fluorimetr ic

detect ion with a packe d f low cel l was appl ied to dansyl am ino acids in LC using convent ional

s ize colum ns [18, 19] and micro colum ns [20] . Both peak area and peak height of analytes

increased w ith increasing retent ion ow ing to focusing and environm ental effects of the s ta-

t ionary phase [18-20] . Chro ma togram s obtained in the presence o f the s tat ionary phase are

dem onstrated in Figure 4 [18].

0 5

0 4l

= 0 3

0 2

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Derivatización por Dansyl o Dabsyl