- Email: [email protected]
Chapter 19 Amines, Nitrogen Bases and Other Related Nitrogen-Containing Compounds
343
Chapter 19 AMINES, NITROGEN BASES AND OTHER RELATED NITROGEN-CONTAINING COMPOUNDS L i q u i d column chromatography has been a p p l i e d t o t h e a n a l y s i s o f a l i p h a t i c mono-, d i - and polyamines, a r o m a t i c amines and n i t r o g e n - c o n t a i n i n g h e t e r o c y c l e s . O f t h e s e compounds, a l i p h a t i c d i - and polyamines and catecholamines a r e o f s p e c i a l b i o c h e m i c a l and p h y s i o l o g i c a l i n t e r e s t , w h i l e c e r t a i n a r o m a t i c amines and aza-arenes a r e i n t e r e s t i n g as i n d u s t r i a l p r o d u c t s and i n t e r m e d i a t e s , components o f raw m a t e r i a l s and e n v i r o n m e n t a l p o l l u t a n t s w i t h p o t e n t i a l c a r c i n o g e n i c p r o p e r t i e s . Bases o f n u c l e i c a c i d s a r e t r e a t e d i n t h e c h a p t e r on n u c l e i c a c i d c o n s t i t u e n t s and s u b s t i t u t e d ureas i n c h a p t e r on p e s t i c i d e s . 19.1.
CHROMATOGRAPHY OF ALIPHATIC AMINES AND POLYAMINES ON I O N EXCHANGERS
Columns packed w i t h s t r o n g sulphonated c a t i o n exchangers, such as Dowex 50W, A m b e r l i t e CG-120 and Aminex A-4 and A-5, o r w i t h weak c a r b o x y l i c c a t i o n e x changers, such as A m b e r l i t e IRP-64, A m b e r l i t e CG-50 and Zeo-Karb 226, were used m a i n l y i n e a r l i e r a p p l i c a t i o n s o f g r a d i e n t e l u t i o n l i q u i d chromatography t o t h e s e p a r a t i o n o f a l i p h a t i c amines. G r a d i e n t s o f i n c r e a s i n g pH o r s a l t c o n c e n t r a t i o n i n s o l u t i o n s o f phosphate, a c e t a t e , c i t r a t e , b o r a t e o r s a l i c y l a t e b u f f e r s were employed t o a c h i e v e t h e s e p a r a t i o n o f amines. I n t h e s e systems, t h e separat i o n s were l e n g t h y and t o o k s e v e r a l hours, even i f f i n e ion-exchange r e s i n s w i t h p a r t i c l e d i a m e t e r o f 10-20 urn were
F o r example, t h e s e p a r a t i o n o f a
m i x t u r e o f s i x t e e n amines (methylamine, e t h y l a m i n e , a l l y l a m i n e , n - p r o p y l a m i n e , i s o b u t y l a m i n e , n - b u t y l a m i n e , 1,2-propanediamine,
h i s t a m i n e , isoamylamine,
n-amylamine, t y r a m i n e , p u t r e s c i n e , p h e n y l e t h y l a m i n e , cadaverine, s e r o t o n i n and hexamethylenediamine) on an Aminex A-4 column ( 1 7 urn, 120 x 6 mm I.D.)
was
achieved i n 8 h u s i n g a combined s t e p w i s e - g r a d i e n t e l u t i o n w i t h c i t r a t e , b o r a t e and s a l i c y l a t e b u f f e r s (pH 5.28-12.50) 4
.
I n t h e s e t e c h n i q u e s , t h e amines i n t h e column e f f l u e n t s were q u a n t i t a t e d u s i n g post-column d e r i v a t i z a t i o n w i t h n i n h y d r i n r e a g e n t and c o n t i n u o u s m o n i t o r i n g o f t h e c o l o u r developed by t h i s r e a c t i o n a t 570 and 440 nm, as usual i n c l a s s i c a l automated amino a c i d a n a l y s i s systems. Because t h e same columns and s i m i l a r b u f f e r s c o u l d be used as i n amino a c i d a n a l y s e r s , these i n s t r u m e n t s c o u l d be employed f o r t h e chromatography o f a l i p h a t i c a m i n e ~ ~ -Using ~. a modification o f References on p . 350.
344 the b u f f e r system, basic amino acids and primary a l i p h a t i c amines could be simultaneously analysed i n a s i n g l e chromatographic run. The modified b u f f e r gradient system allowed the a n a l y s i s time f o r amine mixtures such as i n the above 5 example t o be shortened t o approximately 3 h
.
The i n f l u e n c e o f the p a r t i c l e s i z e o f the cation-exchange r e s i n (sulphonic type) and o f the c a r e f u l s e l e c t i o n o f b u f f e r s f o r gradient e l u t i o n may be i l l u s t r a t e d by a simpler chromatographic example, where o n l y t h e separation o f the polyamines putrescine, spermine and spermidine i s o f i n t e r e s t . This separat i o n took 3 h on a 90 x 5 nun I.D. column packed w i t h Dowex 50W-X4 (200-400 mesh) w i t h a gradient o f potassium i o n (from 0.5 t o 3.0 m o l / l ) i n 0.02 mol/l phosphate b u f f e r a t pH 88, whereas i t could be achieved i n 2 h on an Aminex A-5 (13-20 pm) column (150 x 4.6 mm I.D.)
using a convex pH gradient from 9.2 t o 11.8',
i n o n l y 22 min using a 45 x 4 mm
and
I.D. column packed w i t h Technicon C-2 c a t i o n -
exchange r e s i n (8% cross-linked, 8-12 pm p a r t i c l e diameter) and a gradient o f 7 sodium i o n from 2 t o 3.5 mol/l i n c i t r a t e b u f f e r o f pH 5.25
.
These t h r e e polyamines and t h e i r N-acetyl d e r i v a t i v e s (N-monoacetylputrescine and two isomeric N-monoacetylated spermidines) c o u l d be separated i n 40 min by chromatography on a cation-exchange column (250 x 3.5 mm I.D.), packed w i t h the chemically bonded c a t i o n exchanger P a r t i s i l PXS 10-25 SCX, using an acetate b u f f e r (pH 4.6) w i t h a gradient o f sodium s a l t (added as n i t r a t e ) from 0.01 t o 1.0 mol/l as the mobile phase 10
.
Free homologous a l i p h a t i c primary amines (C,-C7) were separated by chromatography on a column packed w i t h anion-ex'change r e s i n p i t a c h i 2632 (OH-), 550 x 9 mm I.D.]
18 pm, using e l u t i o n w i t h a l i n e a r gradient o f acetone i n water and
a continuous f l o w coulometric d e t e c t i o n i n the eluate. The separation i s based on a reversed-phase mechanism. The coul ometric d e t e c t o r employs measurement o f the c u r r e n t r e s u l t i n g from the e l e c t r o l y t i c o x i d a t i o n o f hydroquinone i n a basic 25
medium provided by the presence o f an amine i n t h e eluate
.
The importance o f the chromatographic analysis o f putrescine, spermidine, cadaverine and spermine increased a f t e r the discovery on increased l e v e l s o f these polyamines i n b i o l o g i c a l f l u i d s i n s i t u a t i o n s such as h e a r t hypertrophy and growth. This means t h a t t h e i r determination i n human u r i n e o r blood may be used f o r the e a r l y diagnosis o f malignant tumour growth i n humans and a r a p i d and s e n s i t i v e a n a l y t i c a l method was needed f o r t h i s purpose. This was the reason f o r a search f o r chromatographic methods other than ion-exchange techniques, which are generally lengthy ( w i t h a few exceptions, such as o f the techniques described i n r e f s . 7 and 10).
345 CHROMATOGRAPHY OF AMINE DERIVATIVES ON POLAR CHEMICALLY BONDED PHASES
19.2.
P o l a r c h e m i c a l l y bonded phases have appeared u s e f u l f o r t h e r a p i d chromatography o f u r i n a r y a l i p h a t i c d i - and polyamines i n t h e f o r m o f UV-absorbing o r f l u o r e s c e n t d e r i v a t i v e s . A column (1000 x 2.1 mm I.D.)
packed w i t h an e t h e r e a l
p o r o u s - l a y e r bonded phase ( Z i p a x Permaphase ETH, 30-50 urn) was used t o s e p a r a t e t o s y l a t e d (p-toluenesulphonylated) d e r i v a t i v e s o f p u t r e s c i n e , spermine and s p e r m i d i n e i n 25 min u s i n g e l u t i o n w i t h an e x p o n e n t i a l g r a d i e n t o f a c e t o n i t r i l e i n w a t e r ( f r o m 25 t o 75%) a t 35OC. The s e p a r a t e d d e r i v a t i v e s were d e t e c t e d by
UV a b s o r p t i o n a t 254 nm”.
Dansyl (5-dimethylaminonaphthalene-l-sulphonyl )
d e r i v a t i v e s ( f l u o r e s c e n t ) o f p u t r e s c i n e , s p e r m i d i n e and spermine were s e p a r a t e d 1 i n ca. 30 min“ and d a n s y l d e r i v a t i v e s o f m o n o a c e t y l p u t r e s c i n e , N - a c e t y l 8 s p e r m i d i n e and N - a c e t y l s p e r m i d i n e were r e s o l v e d i n ca. 15 min13 on a bonded n i t r i l e phase ( M i c r o Pak CN-10, 250 x 2.5 mm I.D.)
u s i n g concave o r l i n e a r
t e r n a r y g r a d i e n t s o f d i c h l o r o m e t h a n e and i s o p r o p a n o l i n n-hexane w i t h f l u o r i m e t r i c ( o r UV) d e t e c t i o n . REVERSED-PHASE CHROMATOGRAPHY OF AMINE AND ISOCYANATE DERIVATIVES AND OF
19.3.
FREE ALIPHATIC AMINES Reversed-phase chromatography on m i c r o p a r t i c u l a r o c t a d e c y l - I4-l9 or octylcolumns (5-10 pm) has been a p p l i e d f o r r a p i d s e p a r a t i o n s o f f l u o r e s c e n t
silica“
dansyl 14-16y20y43
and f l ~ o r e s c a m i n e ~d e~ r-i v~a~t i v e s o f diamines and polyamines
formed by pre-column d e r i v a t i z a t i o n r e a c t i o n s , u s i n g f l u o r i m e t r i c d e t e c t i o n (Aex
J
360-390 nm; Xem
= 450-510 nm). L i n e a r 1 4 o r m u l t i - l i n e a r 2 O c o n c e n t r a t i o n
g r a d i e n t s o f methanol20 o r a c e t o n i t r i l e 1 4 y 4 3 i n w a t e r ( s e e s e p a r a t i o n i n F i g . 19.1)20 o r l i n e a r c o n c e n t r a t i o n g r a d i e n t s o f methanol i n aqueous b o r a t e 1 7 o r acetate”
b u f f e r s were employed f o r e l u t i o n . I o n - p a i r reversed-phase g r a d i e n t
e l u t i o n chromatography has been a p p l i e d s u c c e s s f u l l y f o r r a p i d s e p a r a t i o n s (ca. 3 0 m i n ) o f d a n ~ y l - ’ and ~ f l u o r e s c a m i n e I6,l8 d e r i v a t i v e s o f d i - and polyamines, u s i n g e l u t i o n w i t h a g r a d i e n t o f methanol I6’l8 o r a ~ e t o n i t r i l e ’i ~n m o b i l e phases c o n t a i n i n g 0.02 m o l / l h e p t a n e s u l p h o n i c a c i d 1 5 o r 0.035 m o l / l benzenes u l p h o n i c a c i d 1 6 y 1 8 as t h e i o n - p a i r i n g r e a g e n t . Reversed-phase chromatography o f t h e f l u o r e s c e n t d e r i v a t i v e s o f d i - and polyamines has been a p p l i e d s u c c e s s f u l l y t o a n a l y s e s o f u r i n e and b l o o d and samples o f o t h e r b i o l o g i c a l m a t e r i a l s . The d e t e c t i o n l i m i t s r e p o r t e d a r e gene r a l l y i n t h e picomole range. An a1 t e r n a t i v e t o t h e chromatography o f f l u o r e s c e n t d e r i v a t i v e s i s o f f e r e d by t h e use o f post-column d e r i v a t i z a t i o n r e a c t i o n s w i t h f l u o r e s c a m i n e r e a g e n t
References on p . 350.
346
I I
C I
I
b,'
0
10
20
t(min)
I
I
I
I
I
I
% CH,OH
I
-90 - 80
15
30
40
50
F i g . 19.1. S e p a r a t i o n o f a m i x t u r e o f dansyl amine d e r i v a t i v e s by reversed-phase g r a d i e n t e l u t i o n chromatography. Column: H i b a r pre-packed L i C h r o s o r b RP-8, 7 urn, 250 x 3 mm I . D . G r a d i e n t : m u l t i - l i n e a r , as i n d i c a t e d by t h e b r o k e n l i n e ; ( a ) 20 min 0.5% B/min; ( b ) 10 min 1.5% B/min; ( c ) 6 min 3% B/min. S o l v e n t s : A, w a t e r ; B, methanol. F l o w - r a t e : 1 ml/min. D e t e c t i o n : f l u o r i m e t r i c ; Xex = 360 nm; Xem = 510 nm. Peaks: 1 = 5-dimethylaminonaphthalene-I-sulphonic a c i d ( h y d r o l y s e d r e a g e n t ) ; 2-16 = dansyl d e r i v a t i v e s o f : ( 2 ) ammonia; ( 3 ) ethanolamine; ( 4 ) methyl amine; ( 5 ) 2 - o x o p y r r o l i d i n e ( r e a c t i o n p r o d u c t o f 4 - a m i n o b u t y r i c a c i d ) ; ( 6 ) d i methylamine; ( 7 ) b i s - d e r i v a t i v e o f agmatine; ( 8 ) 2-phenylethylamine; ( 9 ) b i s d e r i v a t i v e o f p u t r e s c i n e ; ( 1 0 ) b i s - d e r i v a t i v e o f cadaverine; ( 1 1 ) b i s - d e r i v a t i v e o f 1,6-diaminohexane; (12) b i s - d e r i v a t i v e o f histamine; (13) 0,N-bisderivative o f serotonin; (14) 0,N-bis-derivative o f p-tyramine; (15) t r i s - d e r i v a t i v e o f spermidine; ( 1 6 ) t e t r a k i s - d e r i v a t i v e o f spermine. (Reproduced f r o m r e f . 20 w i t h permission. 1
- 2-mercaptoethanol
o r o-phthalaldehyde
r e a g e n t and m o n i t o r i n g t h e f l u o r e s c e n c e
i n t h e e l u a t e . T h i s method has been a p p l i e d i n t h e cation-exchange chromatography o f polyamines8-10 and a l s o i n t h e s i n g l e - r u n reversed-phase chromatography o f amino a c i d s and d i - and polyamines i n b i o l o g i c a l samples on a WBondapak CI8 column (300 x 3.9 mm I.D.),
using e l u t i o n w i t h a l i n e a r g r a d i e n t o f a c e t o n i t r i l e 21
i n t h e m o b i l e phase c o n t a i n i n g aqueous sodium a c e t a t e
.
Reversed-phase chromatography on c h e m i c a l l y bonded a l k y l s i l i c a column i s s u i t a b l e f o r t h e s e p a r a t i o n o f f l u o r e s c e n t d e r i v a t i v e s o f homologous p r i m a r y a l k y l amines. Dansyl d e r i v a t i v e s a r e a p p r o p r i a t e f o r t h e purpose22, b u t t h e s e n s i t i v i t y can be improved by u s i n g pre-column d e r i v a t i z a t i o n w i t h 1,2-naphthoylenebenzi m i d a z o l e 6 - s u l p h o c h l o r i d e ( 1 ,2-NBI-6-S02C1). s e p a r a t i o n o f homologous d e r i v a t i v e s o f C1-Cl0
F i g . 19.2 shows t h e chromatographic p r i m a r y a l i p h a t i c amines on a n
o c t a d e c y l s i l i c a column w i t h a l i n e a r g r a d i e n t o f methanol i n w a t e r (70-100%) as 23 t h e m o b i l e phase
.
347
9
I
8
J
I
V(ml)
20
I
10
t I
0
F i g . 19.2. S e p a r a t i o n o f a homologous s e r i e s o f 1,2-naphthoylenebenzimidazole6 - s u l p h o n y l d e r i v a t i v e s o f s a t u r a t e d a l i p h a t i c n-amines by reversed-phase g r a d i e n t e l u t i o n l i q u i d chromatography. Column: S i l a s o r b C1 , 10 ym, 300 x 4.2 mm I.D. G r a d i e n t : l i n e a r , 70-100% B i n 18 min (1.67% B/miny. S o l v e n t s : A , w a t e r ; 6, methanol. F l o w - r a t e : 1 ml/min. D e t e c t i o n : f l u o r i m e t r i c ; Xer = 365 nm; Aem > 400 nm. Peaks: 1 = 1,2-naphthoylenebenzimidazol-6-sulphonic a c i d h y d r o l y s e d r e a g e n t ) ; 2 and 3 = amide and i m i d e d e r i v a t i v e o f ammonia; 4-13 = d e r i v a t i v e s o f amines: (4) m e t h y l ; ( 5 ) e t h y l ; ( 6 ) n - p r o p y l ; (7) n - b u t y l ; (8) n-amyl; (9) n - h e x y l ; (10) n - h e p t y l ; (11) n - o c t y l ; ( 1 2 ) n - n o n y l ; (13) n - d e c y l . P r i m a r y a l i p h a t i c amines have a l s o been s e p a r a t e d on an o c t a d e c y l s i l i c a column as N , N ' - d i s u b s t i t u t e d
ureas formed by pre-column d e r i v a t i z a t i o n w i t h phenyl i s o -
cyanate. E l u t i o n w i t h a l i n e a r g r a d i e n t o f a c e t o n i t r i l e i n w a t e r and UV d e t e c t i o n a t 240 nm were employed, w i t h a r e p o r t e d d e t e c t i o n l i m i t i n nanogram range
24 .
T h i s d e r i v a t i z a t i o n r e a c t i o n has a l s o been used f o r t h e chromatography o f a l i p h a t i c and a r o m a t i c d i i s o c y a n a t e s , which were c o n v e r t e d i n t o ureas by r e a c t i o n w i t h N-4-nitrobenzyl-N-n-propylamine i n heptane s o l u t i o n and t h e n s u b j e c t e d t o chromatography on a s i l i c a column ( P a r t i s i l 5, 50 x 4.5 mm I.D.) References on p . 350.
using e l u t i o n
w i t h a l i n e a r g r a d i e n t o f isopropanol i n dichloromethane ( I - 9 % ) w i t h UV d e t e c t i o n a t 254 nm. The method can be a p p l i e d t o t h e a n a l y s i s o f isocyanates used i n t h e urethane foam i n d u s t r y (these compounds cause s k i n and r e s p i r a t o r y t r a c t i r r i t a 26 tions)
.
19.4.
CHROMATOGRAPHY OF CATECHOLAMINES
Biogenic catecholamines have been separated u s i n g g r a d i e n t e l u t i o n chromatography on cation-exchange columns. E l u t i o n w i t h v a r i o u s g r a d i e n t s u s i n g borate, carbonate o r phosphate b u f f e r s i n t h e pH range 10-12 allowed t h e s e p a r a t i o n o f p h e n o l i c amines on columns packed w i t h f i n e - p a r t i c l e sulphonated p o l y s t y r e n e cation-exchange r e s i n s t o be achieved27. Concentration g r a d i e n t s o f phosphate b u f f e r s (0.03-0.5 m o l / l NaH2P04, a t pH 3-4.5) were used f o r t h e chromatography 28,29 o f catecholamines on s u p e r f i c i a l l y porous cation-exchange columns (Zipax S C X ) L i n e a r g r a d i e n t s o f methanol 30744 o r a c e t o n i t r i l e 3 ' i n potassium dihydrogen phosphate b u f f e r make i t p o s s i b l e t o accomplish r a p i d separations o f c a t e c h o l amines and t h e i r m e t a b o l i t e s on m i c r o p a r t i c u l a t e o c t a d e c y l s i l i c a columns. F o r example, t h e s e p a r a t i o n o f norepinephrine,
kynurenine, epinephrine, dopa,
dopamine, methyl dopa, deoxyepinephri ne , tyramine , isoproterenol tryptophan, s e r o t o n i n , tryptoplian,5-hydroxyindole-3-acetic
, 5-hydroxy-
acid, a n t h r a n i l i c
a c i d , indoleacetamide, i n d o l e - 3 - l a c t i c acid, i n d o l e - 3 - a c e t i c a c i d and i n d o l e - 3 p r o p i o n i c a c i d c o u l d be achieved i n about 30 min u s i n g t h i s technique. UV d e t e c t i o n a t 254 o r 280 nm2 8 y 3 0 y 3 1 o r f l u o r i m e t r i c d e t e c t i o n u t i l i z i n g UV a b s o r p t i o n o r t h e n a t i v e fluorescence 3 0 y 4 4 o f some o f these aromatic and h e t e r o c y c l i c compounds may be used f o r t h e d e t e c t i o n o f catecholamines and r e l a t e d compounds i n t h e e l u a t e ; t h e s e n s i t i v i t y o f t h e method can be improved and t h e d e t e c t i o n l i m i t s decreased t o ca. 20-30 pg o f t h e i n d i v i d u a l compounds u s i n g post-column d e r i v a t i z a t i o n by r e a c t i o n w i t h o-phthalaldehyde
-
2-mercaptothiazole
reagent o r by r e a c t i o n w i t h t r i h y d r o x y i n d o l e and continuous m o n i t o r i n g o f t h e fluorescence formed i n t h e r e a c t i o n w i t h a f l u o r i m e t r i c detector2'.
An e l e c t r o -
chemical ( v o l tammetric) d e t e c t i o n method u s i n g a t h r e e - e l e c t r o d e d e t e c t o r w i t h a glassy carbon working e l e c t r o d e has a l s o been a p p l i e d s u c c e s s f u l l y t o t h e 31 s e n s i t i v e d e t e c t i o n o f these o x i d i z a b l e compounds
.
These techniques have been a p p l i e d t o t h e d e t e r m i n a t i o n o f catecholamines 29-31 and t h e i r m e t a b o l i t e s i n u r i n e and v a r i o u s b i o l o g i c a l t i s s u e s
.
19.5. CHROMATOGRAPHY OF OTHER AROMATIC AMINES, AMIDES AND NITRO COMPOUNDS Various aromatic amino, amido and n i t r o compounds, i m p o r t a n t as t e c h n i c a l products and as p o t e n t i a l c a r c i n o g e n i c environmental p o l l u t a n t s , have been
.
349 analysed by gradient elution liquid column chromatography with UV detection. Aniline and six substituted halogeno anilines were separated in 15 min on a 150 nun long octadecylsilica column (Zorbax C18) using a linear concentration gradient from 20 to 60% of acetonitrile in 0.15 mol/l phosphate buffer32 The acaricide amitraz and its metabolites 2,4-dimethylaniline and 2,4-dimethylformanilide were chromatographed on a nitro-bonded phase (Nucleosil NO2, 5 pm, 200 x 4 mm I.D.) using a linear gradient of diethyl ether (from 10 to 100%) in n-hexane containing 0.3% triethylamine 33 . A silica gel column (Partisil, 10 urn, 250 x 4.6 nun I.D.) was employed for the separation of eight carcinogenic nitro-, amino- and acetylamino-anthracenes and 34 -anthraquinones using a linear gradient of isopropanol in n-hexane (1-10%) Substituted amino-, nitro-, dinitro-, acetoxy- and acetamidobiphenyls were chromatographed on silica gel or on bonded nitrile phase columns using a linear gradient from 0 to 100% of dichloromethane in i s ~ o c t a n eor~ ~a linear ternary gradient of isopropanol-dichloromethane (30 : 70) in n - h e ~ a n e ~or~ ,on octadecylsilica columns using linear gradients of methanol or acetonitrile in water, with addition of 0.1% acetic acid 36 . Using similar operating conditions, substituted amino-, amido- and nitrofluorenes could be separated on a silica gel column using elution with a gradient of propanol and dichloromethane (30 : 70) in n - h e ~ a n eor~ ~on chemically bonded octadecylsilica phases using linear gradients of methanol or acetonitrile in water containing 0.1% acetic acid 37,38
.
.
.
19.6. CHROMATOGRAPHY OF NITROGEN-CONTAINING HETEROCYCLES
Various nitrogen-containing heterocycles (aza-arenes) with two to five rings could be rapidly separated and determined using UV detection, either on microparticulate silica gel columns using elution with linear gradients of isopropanol in chloroform39 or methanol in d i c h l o r ~ m e t h a n eor ~ ~ on an octadecylsilica bonded phase by elution with linear gradients of acetonitrile4’ or isopropan01~~ in water. The method has been employed for the analysis of air pollution by azaarenes on solid particles suspended in air39’41 and for the study of the metab40 olism of aza-arenes in biological tissues . 5-Chloro-8-hydroxy-7-isoquinoline conjugates were analysed by anion-exchange chromatography on Zipax SAX (500 x 2 mm I.D.) columns, using a linear gradient of sodium perchlorate (0.1-0.5 mol/l) in a borate buffer at pH 9.5, with UV detection at 254 nm42. Carcinogenic 4-nitroquinol ine-I-oxide and its 4-hydroxylamino and 4-amino metabolites in blood plasma were determined by reversed-phase chromatography on an octadecylsilica column using elution with an increasing concentration gradient of methanol in water45
.
References on p . 3 5 0 .
350 P l a n t hormones o f t h e i n d o l e t y p e i n b i o l o g i c a l samples were separated and determined by reversed-phase chromatography on a m i c r o p a r t i c u l a t e o c t a d e c y l s i l i c a 46 column u s i n g a l i n e a r g r a d i e n t o f a c e t i c a c i d i n e t h a n o l f o r e l u t i o n
.
19.7. REFERENCES 1 S. H o l d e r and H.J. Bremer, J . Chromatogr., 25 (1966) 48. 2 T.L. P e r r y and W.A. Schroeder, J . Chromatogr., (1963) 358. (1969) 3 M. Yoshioka, A. Ohara, H. Kondo and H. Kanazawa, Chem. Pharm. Bull., 1276. 4 H. Hatano, K. Sumizu, S. Rokushika and F. Murakami, Anal. Biochem., 35 (1970) 377. 5 S. Rokushika, S. Funakoshi, F. Murakami and H. Hatano, J . Chromatogr., 56 (1971) 137. 6 L.J. Marton and P.L.Y. Lee, C l i n . Chem., 21 (1975) 1721. 7 H. A d l e r , M. Margoshes, L.R. Snyder and C T S p i t z e r , J . Chromatogr., 143 (1977) 125. 8 B. Kneip, M. Raymondjean, D. Bogdanovsky, L. Bachner and G. Shapira, J . Chromatogr., 162 (1979) 547. 9 H. Veening, W x P i t t and G. Jones, J . Chromatogr., 90 (1974) 129. 10 P.K. Bondy and Z.N. C a n e l l a k i s , J . Chromatogr., 224 TT981) 371. 11 T. Hayashi, T. S u g i u r a , S. Kawai and T. Ohno, J . Ckromatogr., 145 (1978) 141. 12 N.E. Newton, K. Ohno and M.M. Abdel-Monem, J . Chromatogr., 1 2 4 1 7 9 7 6 ) 277. 13 M.M. Abdel-Monem and J.L. Merdink, J . Chromatogr., 222 (19875-363. 14 Y. Saeki, N. Uehara and S. Shirakawa, J . C h r o m a t o g r T 1 4 5 (1978) 221. 15 N.D. Brown, R.B. Sweet, J.A. K i n t z i o s , H.O. Cox and B . T D o c t o r , J . Chromatogr., 164 (1979) 35. 16 T H a r a g u c h i , M. K a i , K. Kohashi and Y. Ohkura, J . Chromatogr., 202 (1980) 107. 17 K. Samejima, J . Chromatogr., 96 (1974) 250. 18 M. K a i , T. Ogata, K. H a r a g u c h r a n d Y. Ohkura, J . Chromatogr., 163 (1979) 151. 19 A. L i c h t , R.L. Bowman and S. S t e i n , J . Liq. Chromatogr., 4 ( I s m 825. 20 N. S e i l e r , 6. Knodgen and F. E i s e n b e i s s , J . Chromatogr., T45 (1978) 29. 21 N. S e i l e r and 6. Knodgen, J . Chromatogr., 221 (1980) 227.22 P. Jandera, V. Riha and J. ChurdEek, C h e m . L i s t y , 74 (1980) 1209. 23 P. Jandera, H. Pechovd, J. Krblovskg, D. T o c k s t e i n G d and J . ChurdEek, Ozromatographia, 16 (1982) 275. 24 B. B j B r k q v i s t , J . Ckromatogr., 204 (1981) 109. 25 K. Tanaka, T. I s h i z u k a and H. S E h a r a , J . Chromatogr., 172 (1979) 484. (1977) 451. 26 C.R. H a s t i n g s Vogt, Chan Yan KO and T.R. Ryan, J . ChromaGr., 27 T.A. Wheaton and I . S t e w a r t , Anal. Biochem., 12 (1965) 585. 28 A. Kojima-Suda, Ind. Hlth. Jap., 13 (1975) 69, 29 K . I . Okamoto, Y. I s h i d a and K. As=, J . Chromatogr., 167 (1978) 205. 30 A.M. K r s t u l o v i c and A.M. Powell, J . Chromatogr. , 171 m 7 9 ) 345. 31 A.M. K r s t u l o v i c , M. Zakaria, K. Lohse and L. B e r t a n i - D z i e d z i c , J . Chromatogr., 186 (1979) 733. Lores, D.W. B r i s t o l and R.F. Moseman, J . Chromatogr. S c i . , E ( 1 9 7 8 ) 358. 32 33 F.E. R i c k e t t , J . Chromatogr., 142 (1977) 705. 34 6. Shaikh, M.R. Hallmark, R . K . m l m a r k , W.B. Manning, A. Pinnock and J.C. Kawalek, J . Chromatogr., (1980) 392. 35 Spectra-Physics Chromatogr. Rev. , 3 , NO. 1 (1977) 1 . 36 H.J. Johnson, S.F. Cernosek and R.M. Gutierez-Cernosek, J . Ckromatogr. , 161 (1978) 259. 37 H.J. Johnson, S.F. Cernosek and R.M. Gutierez-Cernosek, J . Chromatogr., (1979) 297. 38 S.S. T h o r g e i r s s o n and W.L. Nelson, Anal. Biochem., 75 (1976) 122.
12
134
m.
195
177
351 39 40 41 42
M.W. Dong, D.C. Locke and D. Hoffmann, E m i r o n . Sei. Technol., 1 1 (1977) 612. C. S t u b l e y , J.G.P. S t e l l and D.W. Mathieson, J. Chromatogr., 177-(1979) 313. M. Dong, D.C. Locke and D. Hoffmann, J . Chrornatogr. Sci., 15 m 7 ) 32. C.T. Chen, K. Hayakawa, T. Imanari and Z. Tamura, Chem. Phzrn. B u l l . , 23
(1975) 2173. 43 E. dohnson, A. Abu-Shumays and S.R. Abbott, J. Chrornatogr., 134 (1977) 107. 44 A.M. K r s t u l o v i c and C. Matzura, J. Chromatogr., 163 (1979) 7 2 . 45 H. Lund, D.J. Speelman, A. C u l l i n g , J.S. K i t t r e d g e a n d T. A l b r e c h t , J . Liq. Chrornatogr., 4 (1981) 299. 46 M. Wurst, Z. F p i k r y l and V. VanEura, J . Chrornatogr., 191 (1980) 129.
Chapter 19 AMINES, NITROGEN BASES AND OTHER RELATED NITROGEN-CONTAINING COMPOUNDS L i q u i d column chromatography has been a p p l i e d t o t h e a n a l y s i s o f a l i p h a t i c mono-, d i - and polyamines, a r o m a t i c amines and n i t r o g e n - c o n t a i n i n g h e t e r o c y c l e s . O f t h e s e compounds, a l i p h a t i c d i - and polyamines and catecholamines a r e o f s p e c i a l b i o c h e m i c a l and p h y s i o l o g i c a l i n t e r e s t , w h i l e c e r t a i n a r o m a t i c amines and aza-arenes a r e i n t e r e s t i n g as i n d u s t r i a l p r o d u c t s and i n t e r m e d i a t e s , components o f raw m a t e r i a l s and e n v i r o n m e n t a l p o l l u t a n t s w i t h p o t e n t i a l c a r c i n o g e n i c p r o p e r t i e s . Bases o f n u c l e i c a c i d s a r e t r e a t e d i n t h e c h a p t e r on n u c l e i c a c i d c o n s t i t u e n t s and s u b s t i t u t e d ureas i n c h a p t e r on p e s t i c i d e s . 19.1.
CHROMATOGRAPHY OF ALIPHATIC AMINES AND POLYAMINES ON I O N EXCHANGERS
Columns packed w i t h s t r o n g sulphonated c a t i o n exchangers, such as Dowex 50W, A m b e r l i t e CG-120 and Aminex A-4 and A-5, o r w i t h weak c a r b o x y l i c c a t i o n e x changers, such as A m b e r l i t e IRP-64, A m b e r l i t e CG-50 and Zeo-Karb 226, were used m a i n l y i n e a r l i e r a p p l i c a t i o n s o f g r a d i e n t e l u t i o n l i q u i d chromatography t o t h e s e p a r a t i o n o f a l i p h a t i c amines. G r a d i e n t s o f i n c r e a s i n g pH o r s a l t c o n c e n t r a t i o n i n s o l u t i o n s o f phosphate, a c e t a t e , c i t r a t e , b o r a t e o r s a l i c y l a t e b u f f e r s were employed t o a c h i e v e t h e s e p a r a t i o n o f amines. I n t h e s e systems, t h e separat i o n s were l e n g t h y and t o o k s e v e r a l hours, even i f f i n e ion-exchange r e s i n s w i t h p a r t i c l e d i a m e t e r o f 10-20 urn were
F o r example, t h e s e p a r a t i o n o f a
m i x t u r e o f s i x t e e n amines (methylamine, e t h y l a m i n e , a l l y l a m i n e , n - p r o p y l a m i n e , i s o b u t y l a m i n e , n - b u t y l a m i n e , 1,2-propanediamine,
h i s t a m i n e , isoamylamine,
n-amylamine, t y r a m i n e , p u t r e s c i n e , p h e n y l e t h y l a m i n e , cadaverine, s e r o t o n i n and hexamethylenediamine) on an Aminex A-4 column ( 1 7 urn, 120 x 6 mm I.D.)
was
achieved i n 8 h u s i n g a combined s t e p w i s e - g r a d i e n t e l u t i o n w i t h c i t r a t e , b o r a t e and s a l i c y l a t e b u f f e r s (pH 5.28-12.50) 4
.
I n t h e s e t e c h n i q u e s , t h e amines i n t h e column e f f l u e n t s were q u a n t i t a t e d u s i n g post-column d e r i v a t i z a t i o n w i t h n i n h y d r i n r e a g e n t and c o n t i n u o u s m o n i t o r i n g o f t h e c o l o u r developed by t h i s r e a c t i o n a t 570 and 440 nm, as usual i n c l a s s i c a l automated amino a c i d a n a l y s i s systems. Because t h e same columns and s i m i l a r b u f f e r s c o u l d be used as i n amino a c i d a n a l y s e r s , these i n s t r u m e n t s c o u l d be employed f o r t h e chromatography o f a l i p h a t i c a m i n e ~ ~ -Using ~. a modification o f References on p . 350.
344 the b u f f e r system, basic amino acids and primary a l i p h a t i c amines could be simultaneously analysed i n a s i n g l e chromatographic run. The modified b u f f e r gradient system allowed the a n a l y s i s time f o r amine mixtures such as i n the above 5 example t o be shortened t o approximately 3 h
.
The i n f l u e n c e o f the p a r t i c l e s i z e o f the cation-exchange r e s i n (sulphonic type) and o f the c a r e f u l s e l e c t i o n o f b u f f e r s f o r gradient e l u t i o n may be i l l u s t r a t e d by a simpler chromatographic example, where o n l y t h e separation o f the polyamines putrescine, spermine and spermidine i s o f i n t e r e s t . This separat i o n took 3 h on a 90 x 5 nun I.D. column packed w i t h Dowex 50W-X4 (200-400 mesh) w i t h a gradient o f potassium i o n (from 0.5 t o 3.0 m o l / l ) i n 0.02 mol/l phosphate b u f f e r a t pH 88, whereas i t could be achieved i n 2 h on an Aminex A-5 (13-20 pm) column (150 x 4.6 mm I.D.)
using a convex pH gradient from 9.2 t o 11.8',
i n o n l y 22 min using a 45 x 4 mm
and
I.D. column packed w i t h Technicon C-2 c a t i o n -
exchange r e s i n (8% cross-linked, 8-12 pm p a r t i c l e diameter) and a gradient o f 7 sodium i o n from 2 t o 3.5 mol/l i n c i t r a t e b u f f e r o f pH 5.25
.
These t h r e e polyamines and t h e i r N-acetyl d e r i v a t i v e s (N-monoacetylputrescine and two isomeric N-monoacetylated spermidines) c o u l d be separated i n 40 min by chromatography on a cation-exchange column (250 x 3.5 mm I.D.), packed w i t h the chemically bonded c a t i o n exchanger P a r t i s i l PXS 10-25 SCX, using an acetate b u f f e r (pH 4.6) w i t h a gradient o f sodium s a l t (added as n i t r a t e ) from 0.01 t o 1.0 mol/l as the mobile phase 10
.
Free homologous a l i p h a t i c primary amines (C,-C7) were separated by chromatography on a column packed w i t h anion-ex'change r e s i n p i t a c h i 2632 (OH-), 550 x 9 mm I.D.]
18 pm, using e l u t i o n w i t h a l i n e a r gradient o f acetone i n water and
a continuous f l o w coulometric d e t e c t i o n i n the eluate. The separation i s based on a reversed-phase mechanism. The coul ometric d e t e c t o r employs measurement o f the c u r r e n t r e s u l t i n g from the e l e c t r o l y t i c o x i d a t i o n o f hydroquinone i n a basic 25
medium provided by the presence o f an amine i n t h e eluate
.
The importance o f the chromatographic analysis o f putrescine, spermidine, cadaverine and spermine increased a f t e r the discovery on increased l e v e l s o f these polyamines i n b i o l o g i c a l f l u i d s i n s i t u a t i o n s such as h e a r t hypertrophy and growth. This means t h a t t h e i r determination i n human u r i n e o r blood may be used f o r the e a r l y diagnosis o f malignant tumour growth i n humans and a r a p i d and s e n s i t i v e a n a l y t i c a l method was needed f o r t h i s purpose. This was the reason f o r a search f o r chromatographic methods other than ion-exchange techniques, which are generally lengthy ( w i t h a few exceptions, such as o f the techniques described i n r e f s . 7 and 10).
345 CHROMATOGRAPHY OF AMINE DERIVATIVES ON POLAR CHEMICALLY BONDED PHASES
19.2.
P o l a r c h e m i c a l l y bonded phases have appeared u s e f u l f o r t h e r a p i d chromatography o f u r i n a r y a l i p h a t i c d i - and polyamines i n t h e f o r m o f UV-absorbing o r f l u o r e s c e n t d e r i v a t i v e s . A column (1000 x 2.1 mm I.D.)
packed w i t h an e t h e r e a l
p o r o u s - l a y e r bonded phase ( Z i p a x Permaphase ETH, 30-50 urn) was used t o s e p a r a t e t o s y l a t e d (p-toluenesulphonylated) d e r i v a t i v e s o f p u t r e s c i n e , spermine and s p e r m i d i n e i n 25 min u s i n g e l u t i o n w i t h an e x p o n e n t i a l g r a d i e n t o f a c e t o n i t r i l e i n w a t e r ( f r o m 25 t o 75%) a t 35OC. The s e p a r a t e d d e r i v a t i v e s were d e t e c t e d by
UV a b s o r p t i o n a t 254 nm”.
Dansyl (5-dimethylaminonaphthalene-l-sulphonyl )
d e r i v a t i v e s ( f l u o r e s c e n t ) o f p u t r e s c i n e , s p e r m i d i n e and spermine were s e p a r a t e d 1 i n ca. 30 min“ and d a n s y l d e r i v a t i v e s o f m o n o a c e t y l p u t r e s c i n e , N - a c e t y l 8 s p e r m i d i n e and N - a c e t y l s p e r m i d i n e were r e s o l v e d i n ca. 15 min13 on a bonded n i t r i l e phase ( M i c r o Pak CN-10, 250 x 2.5 mm I.D.)
u s i n g concave o r l i n e a r
t e r n a r y g r a d i e n t s o f d i c h l o r o m e t h a n e and i s o p r o p a n o l i n n-hexane w i t h f l u o r i m e t r i c ( o r UV) d e t e c t i o n . REVERSED-PHASE CHROMATOGRAPHY OF AMINE AND ISOCYANATE DERIVATIVES AND OF
19.3.
FREE ALIPHATIC AMINES Reversed-phase chromatography on m i c r o p a r t i c u l a r o c t a d e c y l - I4-l9 or octylcolumns (5-10 pm) has been a p p l i e d f o r r a p i d s e p a r a t i o n s o f f l u o r e s c e n t
silica“
dansyl 14-16y20y43
and f l ~ o r e s c a m i n e ~d e~ r-i v~a~t i v e s o f diamines and polyamines
formed by pre-column d e r i v a t i z a t i o n r e a c t i o n s , u s i n g f l u o r i m e t r i c d e t e c t i o n (Aex
J
360-390 nm; Xem
= 450-510 nm). L i n e a r 1 4 o r m u l t i - l i n e a r 2 O c o n c e n t r a t i o n
g r a d i e n t s o f methanol20 o r a c e t o n i t r i l e 1 4 y 4 3 i n w a t e r ( s e e s e p a r a t i o n i n F i g . 19.1)20 o r l i n e a r c o n c e n t r a t i o n g r a d i e n t s o f methanol i n aqueous b o r a t e 1 7 o r acetate”
b u f f e r s were employed f o r e l u t i o n . I o n - p a i r reversed-phase g r a d i e n t
e l u t i o n chromatography has been a p p l i e d s u c c e s s f u l l y f o r r a p i d s e p a r a t i o n s (ca. 3 0 m i n ) o f d a n ~ y l - ’ and ~ f l u o r e s c a m i n e I6,l8 d e r i v a t i v e s o f d i - and polyamines, u s i n g e l u t i o n w i t h a g r a d i e n t o f methanol I6’l8 o r a ~ e t o n i t r i l e ’i ~n m o b i l e phases c o n t a i n i n g 0.02 m o l / l h e p t a n e s u l p h o n i c a c i d 1 5 o r 0.035 m o l / l benzenes u l p h o n i c a c i d 1 6 y 1 8 as t h e i o n - p a i r i n g r e a g e n t . Reversed-phase chromatography o f t h e f l u o r e s c e n t d e r i v a t i v e s o f d i - and polyamines has been a p p l i e d s u c c e s s f u l l y t o a n a l y s e s o f u r i n e and b l o o d and samples o f o t h e r b i o l o g i c a l m a t e r i a l s . The d e t e c t i o n l i m i t s r e p o r t e d a r e gene r a l l y i n t h e picomole range. An a1 t e r n a t i v e t o t h e chromatography o f f l u o r e s c e n t d e r i v a t i v e s i s o f f e r e d by t h e use o f post-column d e r i v a t i z a t i o n r e a c t i o n s w i t h f l u o r e s c a m i n e r e a g e n t
References on p . 350.
346
I I
C I
I
b,'
0
10
20
t(min)
I
I
I
I
I
I
% CH,OH
I
-90 - 80
15
30
40
50
F i g . 19.1. S e p a r a t i o n o f a m i x t u r e o f dansyl amine d e r i v a t i v e s by reversed-phase g r a d i e n t e l u t i o n chromatography. Column: H i b a r pre-packed L i C h r o s o r b RP-8, 7 urn, 250 x 3 mm I . D . G r a d i e n t : m u l t i - l i n e a r , as i n d i c a t e d by t h e b r o k e n l i n e ; ( a ) 20 min 0.5% B/min; ( b ) 10 min 1.5% B/min; ( c ) 6 min 3% B/min. S o l v e n t s : A, w a t e r ; B, methanol. F l o w - r a t e : 1 ml/min. D e t e c t i o n : f l u o r i m e t r i c ; Xex = 360 nm; Xem = 510 nm. Peaks: 1 = 5-dimethylaminonaphthalene-I-sulphonic a c i d ( h y d r o l y s e d r e a g e n t ) ; 2-16 = dansyl d e r i v a t i v e s o f : ( 2 ) ammonia; ( 3 ) ethanolamine; ( 4 ) methyl amine; ( 5 ) 2 - o x o p y r r o l i d i n e ( r e a c t i o n p r o d u c t o f 4 - a m i n o b u t y r i c a c i d ) ; ( 6 ) d i methylamine; ( 7 ) b i s - d e r i v a t i v e o f agmatine; ( 8 ) 2-phenylethylamine; ( 9 ) b i s d e r i v a t i v e o f p u t r e s c i n e ; ( 1 0 ) b i s - d e r i v a t i v e o f cadaverine; ( 1 1 ) b i s - d e r i v a t i v e o f 1,6-diaminohexane; (12) b i s - d e r i v a t i v e o f histamine; (13) 0,N-bisderivative o f serotonin; (14) 0,N-bis-derivative o f p-tyramine; (15) t r i s - d e r i v a t i v e o f spermidine; ( 1 6 ) t e t r a k i s - d e r i v a t i v e o f spermine. (Reproduced f r o m r e f . 20 w i t h permission. 1
- 2-mercaptoethanol
o r o-phthalaldehyde
r e a g e n t and m o n i t o r i n g t h e f l u o r e s c e n c e
i n t h e e l u a t e . T h i s method has been a p p l i e d i n t h e cation-exchange chromatography o f polyamines8-10 and a l s o i n t h e s i n g l e - r u n reversed-phase chromatography o f amino a c i d s and d i - and polyamines i n b i o l o g i c a l samples on a WBondapak CI8 column (300 x 3.9 mm I.D.),
using e l u t i o n w i t h a l i n e a r g r a d i e n t o f a c e t o n i t r i l e 21
i n t h e m o b i l e phase c o n t a i n i n g aqueous sodium a c e t a t e
.
Reversed-phase chromatography on c h e m i c a l l y bonded a l k y l s i l i c a column i s s u i t a b l e f o r t h e s e p a r a t i o n o f f l u o r e s c e n t d e r i v a t i v e s o f homologous p r i m a r y a l k y l amines. Dansyl d e r i v a t i v e s a r e a p p r o p r i a t e f o r t h e purpose22, b u t t h e s e n s i t i v i t y can be improved by u s i n g pre-column d e r i v a t i z a t i o n w i t h 1,2-naphthoylenebenzi m i d a z o l e 6 - s u l p h o c h l o r i d e ( 1 ,2-NBI-6-S02C1). s e p a r a t i o n o f homologous d e r i v a t i v e s o f C1-Cl0
F i g . 19.2 shows t h e chromatographic p r i m a r y a l i p h a t i c amines on a n
o c t a d e c y l s i l i c a column w i t h a l i n e a r g r a d i e n t o f methanol i n w a t e r (70-100%) as 23 t h e m o b i l e phase
.
347
9
I
8
J
I
V(ml)
20
I
10
t I
0
F i g . 19.2. S e p a r a t i o n o f a homologous s e r i e s o f 1,2-naphthoylenebenzimidazole6 - s u l p h o n y l d e r i v a t i v e s o f s a t u r a t e d a l i p h a t i c n-amines by reversed-phase g r a d i e n t e l u t i o n l i q u i d chromatography. Column: S i l a s o r b C1 , 10 ym, 300 x 4.2 mm I.D. G r a d i e n t : l i n e a r , 70-100% B i n 18 min (1.67% B/miny. S o l v e n t s : A , w a t e r ; 6, methanol. F l o w - r a t e : 1 ml/min. D e t e c t i o n : f l u o r i m e t r i c ; Xer = 365 nm; Aem > 400 nm. Peaks: 1 = 1,2-naphthoylenebenzimidazol-6-sulphonic a c i d h y d r o l y s e d r e a g e n t ) ; 2 and 3 = amide and i m i d e d e r i v a t i v e o f ammonia; 4-13 = d e r i v a t i v e s o f amines: (4) m e t h y l ; ( 5 ) e t h y l ; ( 6 ) n - p r o p y l ; (7) n - b u t y l ; (8) n-amyl; (9) n - h e x y l ; (10) n - h e p t y l ; (11) n - o c t y l ; ( 1 2 ) n - n o n y l ; (13) n - d e c y l . P r i m a r y a l i p h a t i c amines have a l s o been s e p a r a t e d on an o c t a d e c y l s i l i c a column as N , N ' - d i s u b s t i t u t e d
ureas formed by pre-column d e r i v a t i z a t i o n w i t h phenyl i s o -
cyanate. E l u t i o n w i t h a l i n e a r g r a d i e n t o f a c e t o n i t r i l e i n w a t e r and UV d e t e c t i o n a t 240 nm were employed, w i t h a r e p o r t e d d e t e c t i o n l i m i t i n nanogram range
24 .
T h i s d e r i v a t i z a t i o n r e a c t i o n has a l s o been used f o r t h e chromatography o f a l i p h a t i c and a r o m a t i c d i i s o c y a n a t e s , which were c o n v e r t e d i n t o ureas by r e a c t i o n w i t h N-4-nitrobenzyl-N-n-propylamine i n heptane s o l u t i o n and t h e n s u b j e c t e d t o chromatography on a s i l i c a column ( P a r t i s i l 5, 50 x 4.5 mm I.D.) References on p . 350.
using e l u t i o n
w i t h a l i n e a r g r a d i e n t o f isopropanol i n dichloromethane ( I - 9 % ) w i t h UV d e t e c t i o n a t 254 nm. The method can be a p p l i e d t o t h e a n a l y s i s o f isocyanates used i n t h e urethane foam i n d u s t r y (these compounds cause s k i n and r e s p i r a t o r y t r a c t i r r i t a 26 tions)
.
19.4.
CHROMATOGRAPHY OF CATECHOLAMINES
Biogenic catecholamines have been separated u s i n g g r a d i e n t e l u t i o n chromatography on cation-exchange columns. E l u t i o n w i t h v a r i o u s g r a d i e n t s u s i n g borate, carbonate o r phosphate b u f f e r s i n t h e pH range 10-12 allowed t h e s e p a r a t i o n o f p h e n o l i c amines on columns packed w i t h f i n e - p a r t i c l e sulphonated p o l y s t y r e n e cation-exchange r e s i n s t o be achieved27. Concentration g r a d i e n t s o f phosphate b u f f e r s (0.03-0.5 m o l / l NaH2P04, a t pH 3-4.5) were used f o r t h e chromatography 28,29 o f catecholamines on s u p e r f i c i a l l y porous cation-exchange columns (Zipax S C X ) L i n e a r g r a d i e n t s o f methanol 30744 o r a c e t o n i t r i l e 3 ' i n potassium dihydrogen phosphate b u f f e r make i t p o s s i b l e t o accomplish r a p i d separations o f c a t e c h o l amines and t h e i r m e t a b o l i t e s on m i c r o p a r t i c u l a t e o c t a d e c y l s i l i c a columns. F o r example, t h e s e p a r a t i o n o f norepinephrine,
kynurenine, epinephrine, dopa,
dopamine, methyl dopa, deoxyepinephri ne , tyramine , isoproterenol tryptophan, s e r o t o n i n , tryptoplian,5-hydroxyindole-3-acetic
, 5-hydroxy-
acid, a n t h r a n i l i c
a c i d , indoleacetamide, i n d o l e - 3 - l a c t i c acid, i n d o l e - 3 - a c e t i c a c i d and i n d o l e - 3 p r o p i o n i c a c i d c o u l d be achieved i n about 30 min u s i n g t h i s technique. UV d e t e c t i o n a t 254 o r 280 nm2 8 y 3 0 y 3 1 o r f l u o r i m e t r i c d e t e c t i o n u t i l i z i n g UV a b s o r p t i o n o r t h e n a t i v e fluorescence 3 0 y 4 4 o f some o f these aromatic and h e t e r o c y c l i c compounds may be used f o r t h e d e t e c t i o n o f catecholamines and r e l a t e d compounds i n t h e e l u a t e ; t h e s e n s i t i v i t y o f t h e method can be improved and t h e d e t e c t i o n l i m i t s decreased t o ca. 20-30 pg o f t h e i n d i v i d u a l compounds u s i n g post-column d e r i v a t i z a t i o n by r e a c t i o n w i t h o-phthalaldehyde
-
2-mercaptothiazole
reagent o r by r e a c t i o n w i t h t r i h y d r o x y i n d o l e and continuous m o n i t o r i n g o f t h e fluorescence formed i n t h e r e a c t i o n w i t h a f l u o r i m e t r i c detector2'.
An e l e c t r o -
chemical ( v o l tammetric) d e t e c t i o n method u s i n g a t h r e e - e l e c t r o d e d e t e c t o r w i t h a glassy carbon working e l e c t r o d e has a l s o been a p p l i e d s u c c e s s f u l l y t o t h e 31 s e n s i t i v e d e t e c t i o n o f these o x i d i z a b l e compounds
.
These techniques have been a p p l i e d t o t h e d e t e r m i n a t i o n o f catecholamines 29-31 and t h e i r m e t a b o l i t e s i n u r i n e and v a r i o u s b i o l o g i c a l t i s s u e s
.
19.5. CHROMATOGRAPHY OF OTHER AROMATIC AMINES, AMIDES AND NITRO COMPOUNDS Various aromatic amino, amido and n i t r o compounds, i m p o r t a n t as t e c h n i c a l products and as p o t e n t i a l c a r c i n o g e n i c environmental p o l l u t a n t s , have been
.
349 analysed by gradient elution liquid column chromatography with UV detection. Aniline and six substituted halogeno anilines were separated in 15 min on a 150 nun long octadecylsilica column (Zorbax C18) using a linear concentration gradient from 20 to 60% of acetonitrile in 0.15 mol/l phosphate buffer32 The acaricide amitraz and its metabolites 2,4-dimethylaniline and 2,4-dimethylformanilide were chromatographed on a nitro-bonded phase (Nucleosil NO2, 5 pm, 200 x 4 mm I.D.) using a linear gradient of diethyl ether (from 10 to 100%) in n-hexane containing 0.3% triethylamine 33 . A silica gel column (Partisil, 10 urn, 250 x 4.6 nun I.D.) was employed for the separation of eight carcinogenic nitro-, amino- and acetylamino-anthracenes and 34 -anthraquinones using a linear gradient of isopropanol in n-hexane (1-10%) Substituted amino-, nitro-, dinitro-, acetoxy- and acetamidobiphenyls were chromatographed on silica gel or on bonded nitrile phase columns using a linear gradient from 0 to 100% of dichloromethane in i s ~ o c t a n eor~ ~a linear ternary gradient of isopropanol-dichloromethane (30 : 70) in n - h e ~ a n e ~or~ ,on octadecylsilica columns using linear gradients of methanol or acetonitrile in water, with addition of 0.1% acetic acid 36 . Using similar operating conditions, substituted amino-, amido- and nitrofluorenes could be separated on a silica gel column using elution with a gradient of propanol and dichloromethane (30 : 70) in n - h e ~ a n eor~ ~on chemically bonded octadecylsilica phases using linear gradients of methanol or acetonitrile in water containing 0.1% acetic acid 37,38
.
.
.
19.6. CHROMATOGRAPHY OF NITROGEN-CONTAINING HETEROCYCLES
Various nitrogen-containing heterocycles (aza-arenes) with two to five rings could be rapidly separated and determined using UV detection, either on microparticulate silica gel columns using elution with linear gradients of isopropanol in chloroform39 or methanol in d i c h l o r ~ m e t h a n eor ~ ~ on an octadecylsilica bonded phase by elution with linear gradients of acetonitrile4’ or isopropan01~~ in water. The method has been employed for the analysis of air pollution by azaarenes on solid particles suspended in air39’41 and for the study of the metab40 olism of aza-arenes in biological tissues . 5-Chloro-8-hydroxy-7-isoquinoline conjugates were analysed by anion-exchange chromatography on Zipax SAX (500 x 2 mm I.D.) columns, using a linear gradient of sodium perchlorate (0.1-0.5 mol/l) in a borate buffer at pH 9.5, with UV detection at 254 nm42. Carcinogenic 4-nitroquinol ine-I-oxide and its 4-hydroxylamino and 4-amino metabolites in blood plasma were determined by reversed-phase chromatography on an octadecylsilica column using elution with an increasing concentration gradient of methanol in water45
.
References on p . 3 5 0 .
350 P l a n t hormones o f t h e i n d o l e t y p e i n b i o l o g i c a l samples were separated and determined by reversed-phase chromatography on a m i c r o p a r t i c u l a t e o c t a d e c y l s i l i c a 46 column u s i n g a l i n e a r g r a d i e n t o f a c e t i c a c i d i n e t h a n o l f o r e l u t i o n
.
19.7. REFERENCES 1 S. H o l d e r and H.J. Bremer, J . Chromatogr., 25 (1966) 48. 2 T.L. P e r r y and W.A. Schroeder, J . Chromatogr., (1963) 358. (1969) 3 M. Yoshioka, A. Ohara, H. Kondo and H. Kanazawa, Chem. Pharm. Bull., 1276. 4 H. Hatano, K. Sumizu, S. Rokushika and F. Murakami, Anal. Biochem., 35 (1970) 377. 5 S. Rokushika, S. Funakoshi, F. Murakami and H. Hatano, J . Chromatogr., 56 (1971) 137. 6 L.J. Marton and P.L.Y. Lee, C l i n . Chem., 21 (1975) 1721. 7 H. A d l e r , M. Margoshes, L.R. Snyder and C T S p i t z e r , J . Chromatogr., 143 (1977) 125. 8 B. Kneip, M. Raymondjean, D. Bogdanovsky, L. Bachner and G. Shapira, J . Chromatogr., 162 (1979) 547. 9 H. Veening, W x P i t t and G. Jones, J . Chromatogr., 90 (1974) 129. 10 P.K. Bondy and Z.N. C a n e l l a k i s , J . Chromatogr., 224 TT981) 371. 11 T. Hayashi, T. S u g i u r a , S. Kawai and T. Ohno, J . Ckromatogr., 145 (1978) 141. 12 N.E. Newton, K. Ohno and M.M. Abdel-Monem, J . Chromatogr., 1 2 4 1 7 9 7 6 ) 277. 13 M.M. Abdel-Monem and J.L. Merdink, J . Chromatogr., 222 (19875-363. 14 Y. Saeki, N. Uehara and S. Shirakawa, J . C h r o m a t o g r T 1 4 5 (1978) 221. 15 N.D. Brown, R.B. Sweet, J.A. K i n t z i o s , H.O. Cox and B . T D o c t o r , J . Chromatogr., 164 (1979) 35. 16 T H a r a g u c h i , M. K a i , K. Kohashi and Y. Ohkura, J . Chromatogr., 202 (1980) 107. 17 K. Samejima, J . Chromatogr., 96 (1974) 250. 18 M. K a i , T. Ogata, K. H a r a g u c h r a n d Y. Ohkura, J . Chromatogr., 163 (1979) 151. 19 A. L i c h t , R.L. Bowman and S. S t e i n , J . Liq. Chromatogr., 4 ( I s m 825. 20 N. S e i l e r , 6. Knodgen and F. E i s e n b e i s s , J . Chromatogr., T45 (1978) 29. 21 N. S e i l e r and 6. Knodgen, J . Chromatogr., 221 (1980) 227.22 P. Jandera, V. Riha and J. ChurdEek, C h e m . L i s t y , 74 (1980) 1209. 23 P. Jandera, H. Pechovd, J. Krblovskg, D. T o c k s t e i n G d and J . ChurdEek, Ozromatographia, 16 (1982) 275. 24 B. B j B r k q v i s t , J . Ckromatogr., 204 (1981) 109. 25 K. Tanaka, T. I s h i z u k a and H. S E h a r a , J . Chromatogr., 172 (1979) 484. (1977) 451. 26 C.R. H a s t i n g s Vogt, Chan Yan KO and T.R. Ryan, J . ChromaGr., 27 T.A. Wheaton and I . S t e w a r t , Anal. Biochem., 12 (1965) 585. 28 A. Kojima-Suda, Ind. Hlth. Jap., 13 (1975) 69, 29 K . I . Okamoto, Y. I s h i d a and K. As=, J . Chromatogr., 167 (1978) 205. 30 A.M. K r s t u l o v i c and A.M. Powell, J . Chromatogr. , 171 m 7 9 ) 345. 31 A.M. K r s t u l o v i c , M. Zakaria, K. Lohse and L. B e r t a n i - D z i e d z i c , J . Chromatogr., 186 (1979) 733. Lores, D.W. B r i s t o l and R.F. Moseman, J . Chromatogr. S c i . , E ( 1 9 7 8 ) 358. 32 33 F.E. R i c k e t t , J . Chromatogr., 142 (1977) 705. 34 6. Shaikh, M.R. Hallmark, R . K . m l m a r k , W.B. Manning, A. Pinnock and J.C. Kawalek, J . Chromatogr., (1980) 392. 35 Spectra-Physics Chromatogr. Rev. , 3 , NO. 1 (1977) 1 . 36 H.J. Johnson, S.F. Cernosek and R.M. Gutierez-Cernosek, J . Ckromatogr. , 161 (1978) 259. 37 H.J. Johnson, S.F. Cernosek and R.M. Gutierez-Cernosek, J . Chromatogr., (1979) 297. 38 S.S. T h o r g e i r s s o n and W.L. Nelson, Anal. Biochem., 75 (1976) 122.
12
134
m.
195
177
351 39 40 41 42
M.W. Dong, D.C. Locke and D. Hoffmann, E m i r o n . Sei. Technol., 1 1 (1977) 612. C. S t u b l e y , J.G.P. S t e l l and D.W. Mathieson, J. Chromatogr., 177-(1979) 313. M. Dong, D.C. Locke and D. Hoffmann, J . Chrornatogr. Sci., 15 m 7 ) 32. C.T. Chen, K. Hayakawa, T. Imanari and Z. Tamura, Chem. Phzrn. B u l l . , 23
(1975) 2173. 43 E. dohnson, A. Abu-Shumays and S.R. Abbott, J. Chrornatogr., 134 (1977) 107. 44 A.M. K r s t u l o v i c and C. Matzura, J. Chromatogr., 163 (1979) 7 2 . 45 H. Lund, D.J. Speelman, A. C u l l i n g , J.S. K i t t r e d g e a n d T. A l b r e c h t , J . Liq. Chrornatogr., 4 (1981) 299. 46 M. Wurst, Z. F p i k r y l and V. VanEura, J . Chrornatogr., 191 (1980) 129.