Derivatives for HPLC Analysis

Derivatives for HPLC Analysis
Diploma Thesis
by
Mrs. Laurence Coppex
Faculty of Chemistry and Pharmacy
University of Genf
November 1999 - February 2000

TABLE OF CONTENTS
1 INTRODUCTION...............................................................................................................4
2 DETECTION METHODS.................................................................................................6
2.1 SPECTROPHOTOMETRIC DETECTION..............................................................................6
2.1.1 UV-VIS Detection................................................................................................6
2.1.1.1 Primary and secondary amines..................................................................6
2.1.1.1.1 Nitrobenzenes........................................................................................6
2.1.1.1.2 Ninhydrin.................................................................................................8
2.1.1.1.3 Disuccinimido Carbonate (DSC)........................................................9
2.1.1.1.4 4-N,N-dimethylaminoazobenzene-4’-sulfonyl Chloride (Dabsyl-Cl,
DABS-Cl) .............................................................................................10
2.1.1.1.5 Solid-phase reagents with UV or VIS light absorbing moiety ........11
2.1.1.2 Tertiary aliphatic amines............................................................................14
2.1.1.3 Polyamines..................................................................................................14
2.1.1.4 Aromatic amines ........................................................................................15
2.1.1.4.1 Dimethylaminobenzaldehyde.............................................................15
2.1.1.4.2 Phenylisocyanate (PHI) [1].................................................................15
2.1.2 UV-VIS and fluorimetric detection ...................................................................16
2.1.2.1 Primary amines...........................................................................................16
2.1.2.1.1 o-phtalaldehyde (OPA).......................................................................16
2.1.2.1.2 Fluorescamine.....................................................................................20
2.1.2.2 Primary and secondary amines................................................................22
2.1.2.2.1 Sulfonyl chlorides.................................................................................22
2.1.2.2.2 Carbonyl chlorides and fluorides .......................................................25
2.1.2.2.3 1,2-naphtoquinone-4-sulfonate (NQS)..............................................28
2.1.2.2.4 Isocyanates et Isothiocyanates..........................................................29
2.1.2.2.5 Succinimidyls.......................................................................................31
2.1.2.2.6 Benzofurazans .....................................................................................32
2.1.2.2.7 Oxazoles...............................................................................................34
2.1.2.2.8 Solid phase reagents..........................................................................35
2.1.2.2.9 Ions Al3+ ................................................................................................38
2.1.2.2.10 Potassium ferricyanide (K3Fe(CN)6).................................................38
2.1.2.3 Particular amines........................................................................................39
2.1.2.3.1 Catecholamines ..................................................................................39
2.1.2.3.2 Indolamines..........................................................................................40
2.1.2.3.3 5-hydroxyindoles (serotonine related compounds)..........................40
2.1.2.3.4 Guanidino compounds........................................................................42
2.2 ELECTROCHEMICAL DETECTION (ECD).......................................................................43
2.2.1 Introduction.........................................................................................................43
2.2.2 Primary and secondary amines.......................................................................44
2.2.2.1 O-phtalaldehyde (OPA)..............................................................................44
2.2.2.2 Naphtalene-2,3-dicarbaldehyde ...............................................................45
2.2.2.3 Ferrocene....................................................................................................45
2.2.2.4 Salicylic acid chloride ................................................................................47
2.2.2.5 2,4-dinitrofluorobenzene ............................................................................48
2.2.2.6 1,2-diphenylethylenediamine.....................................................................48
2.2.2.7 Bolton and Hunter reagents.......................................................................49
2.2.2.8 Other reagents............................................................................................51
3 DERIVATIZATION FOR THE RESOLUTION OF CHIRAL AMINES.........................52
3.1 DETECTION METHODS ..................................................................................................53
3.1.1 UV-VIS absorption detection ...........................................................................53
3.1.1.1 Isocyanates and isothiocyanates..............................................................54
3.1.1.2 Succinimidyls ..............................................................................................57
3.1.1.3 Acid chlorides .............................................................................................57
3.1.1.4 Tartric acids ................................................................................................58
3.1.2 Fluorescence detection ....................................................................................58
3.1.2.1 OPA .............................................................................................................60
3.1.2.2 Chloroformates...........................................................................................62
3.1.2.2.1 (+)-1-Ethyl (9-fluorenyle) chloroformate chloride (FLEC-Cl) ...........62
3.1.2.2.2 4-(6-Methoxy-2-naphtyl)-2-butyl Chloroformate (NAB-C)................63
3.1.2.2.3 Other chloroformates ..........................................................................63
3.1.2.3 Acid chlorides and ? -alkyl-? -arylacetic acids ........................................64
3.1.2.4 L-Proline derivatives ..................................................................................65
3.1.2.4.1 Dansyl-L-proline ...................................................................................65
3.1.2.4.2 L-Proline fluoroalkyl derivatives .........................................................65
3.1.2.5 (-)-? -Methoxy-? -(trifluoromethyl)phenylacetic (MTPA) and analogues 65
3.1.2.6 Other reagents............................................................................................66
3.1.3 Electrochemical detection................................................................................66
4 CONCLUSION................................................................................................................68


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