WRC RESEARCH REPORT 156
DEVELOPMENT O F METHODS FOR T H E D E T E C T I O N O F TRACE AMOUNTS
O F S E L E C T E D C A R C I N O G E N I C AND M U T A G E N I C A M I N E S I N WATER
Jay C. Means
I n s t i t u t e f o r Environmental Studies, U n i v e r s i t y of
I l l i n o i s a t Urbana-Champaign, and Chesapeake B i o l o g i c a l L a b o r a t o r y and Department of chemistry,
Uni v e r s i ty o f Mary1and
Edward G. P e r k i n s
Department o f Food Science, U n i v e r s i t y o f I 11 i n o i s
a t Urbana-Champaign
David D. E l l i s
I n s t i t u t e f o r Envi ronrr~entalStudies, U n i v e r s i t y of
I l l i n o i s a t Urbana-Champaign
Muhammad M. Chaudry
Department o f Food Science, U n i v e r s i t y of Illi n o i s
a t Urbana-Champai gn
FINAL REPORT
P r o j e c t B-107-ILL
UNIVERSITY OF ILLINOIS
WATER RESOURCES CENTER
2535 Hydrosystems Laboratory
208 N o r t h Romine S t r e e t
Urbana, I 1 1 in o i s 61801
February 1981
The work upon which t h i s p u b l i c a t i o n i s based was supported i n p a r t by
funds p r o v i d e d by t h e U.S. Department o f t h e I n t e r i o r as a u t h o r i z e d under
t h e Water Resources Research and Development A c t o f 1978, P.L. 88-379,
Agreement No. 14-34-0001-8081.
Contents o f t h i s p u b l i c a t i o n do n o t n e c e s s a r i l y
r e f l e c t t h e v i e w s and p o l i c i e s o f t h e O f f i c e o f
Water Research and T e c h n o l o g y , U.S. D e p a r t m e n t o f
t h e I n t e r i o r , n o r does m e n t i o n o f t r a d e names o r
c m e r c i a l p r o d u c t s c o n s t i t u t e t h e i r endorsement
o r r e c o m n e n d a t i o n f o r use by t h e U . S . Government.
ABSTRACT
A g r e a t deal of concern e x i s t s o v e r t h e presence o f p o t e n t i a l l y
c a r c i n o g e n i c and/or mutagenic substances i n d r i n k i n g water suppl i e s .
The
general a n a l y t i c a l scherr~es c u r r e n t l y a p p l i e d t o water a r e l e s s s u i t e d
than s p e c i f i c methods f o r t h e d e t e c t i o n o f c e r t a i n classes o f o r g a n i c
contaminants such as aromatic amines because o f t h e i r r e a c t i v i t y .
We
have concentrated o u r e f f o r t s a t developing a n a l y t i c a l schemes by which
we a r e a b l e t o r e l i a b l y d e t e c t , separate, and q u a n t i t a t e t r a c e l e v e l s of
a number o f aromatic and h e t e r o c y c l i c amines.
chromatographi c methods have been developed.
l i m i t a t i o n s o f t h e methods a r e discussed.
Both l i q u i d and gas
The r e 1 a t i ve s t r e n g t h s and
F i e l d e v a l u a t i o n s of t h e
f i n a l methods were c a r r i e d o u t and r e p o r t e d .
Means, Jay C. ; Edward G. Perkins; David D. E l l i s ; and Muhammad M. Chaudry
DEVELOPMENT OF METHODS FOR THE DETECTION OF TRACE AMOUNTS OF SELECTED
CARCINOGENIC AND MUTAGENIC AMIMES I N WATER
F i n a l r e p o r t t o t h e Water Resources Center, U n i v e r s i t y o f I 1 1 i n o i s
a t Urbana-Champaign, February 1981.
KEYWORDS:
aromatic aminesl c a r c i nogens1 e s t u a r i es/ gas chromatography/
1 i q u i d chromatography/ mutagens/ water p o l 1 u t i o n / w a t e r qua1 it y
ACKNOWLEDGMENTS
The a u t h o r s w i s h t o acknowledge t h e s u p p o r t and f a c i l i t i e s p r o v i d e d
by t h e Water Resources Center, t h e I n s t i t u t e f o r Environmental S t u d i e s ,
t h e Department o f Food Science, and t h e A g r i c u l t u r a l Experiment S t a t i o n s
a t t h e U n i v e r s i t y o f I l l i n o i s a t Urbana-Champaign.
The C e n t e r f o r E n v i r o n -
mental and E s t u a r i n e S t u d i e s a t t h e U n i v e r s i t y o f Maryland a l s o p r o v i d e d
s u p p o r t and f a c i l i t i e s ; t h i s r e p o r t i s f i l e d t h e r e as C o n t r i b u t i o n No. 1028.
The a u t h o r s a l s o w i s h t o acknowledge t h e c o n t r i b u t i o n s o f Ben B.
Ewing, d i r e c t o r o f t h e I n s t i t u t e f o r Environmental S t u d i e s , who h e l p e d
i n i t i a t e t h e s t u d y and was a p r i n c i p a l i n v e s t i g a t o r f o r t h e l a t t e r phase
o f the project.
S p e c i a l r e c o g n i t i o n i s g i v e n t o Anne Weeks, Water
Resources Center, who e d i t e d t h e r e p o r t and p r e p a r e d i t f o r p u b l i c a t i o n .
CONTENTS
Page
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Acknowl edglvents .
Figures . . . . .
Tables . . . . . .
Introduction . . .
Abstract
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iii
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2
Development o f L i q u i d Chromatographic Methods
. . . . . . . . . . .
B. Experimental M a t e r i a l s . . . . . . . . . .
C . E x p e r i m e n t a l Procedures . . . . . . . . .
D. Sample E x t r a c t i o n / C o n c e n t r a t i on S t u d i e s .
E. F i e l d E v a l ~ ~ a t i o n o f M e t h o d s. . . . . . .
F. R e s u l t s . . . . . . . . . . . . . . . . .
V I I . Development o f Gas Chromatographic Methods . .
A . Review o f L i t e r a t u r e . . . . . . . . . . .
B . E x p e r i m e n t a l M a t e r i a1 s . . . . . . . . . .
C . E x p e r i m e n t a l Procedures . . . . . . . . .
D. Sample E x t r a c t i o n / C o n c e n t r a t i o n S t u d i e s .
E. F i e l d Eva1 u a t i o n o f Methods . . . . . . .
F. R e s u l t s . . . . . . . . . . . . . . . . .
V I I I . Conclusions . . . . . . . . . . . . . . . . .
I X. Literature Cited . . . . . . . . . . . . . . . . . . . . . . .
A.
Review o f L i t e r a t u r e
iv
vii
ix
1
2
4
4
5
6
6
25
25
26
26
28
28
29
47
48
FIGURES
Page
1.
2.
S e p a r a t i o n o f 1-naphthylamine, 2 , 4 - d i c h l o r o a n i l i n e ,
N - m e t h y l a n i l i n e on a C o r a s i l I / C d column . . . . .
. . . . . . . . .
and
S e p a r a t i o n o f a t r a z i n e , Simazine, and Bladex on a
~ B o n d a p a kC18 column . . . . . . . . . . . . . . .
. . . . . . . .
3.
S e p a r a t i o n o f p - c h l o r o a n i 1 i n e , 2 , 4 - d i c h l o r o a n i 1 i n e , and
2,4,6- t r i c h l o r o a n i 1 i n e on a ~ B o n d a p a kC18 column . . . .
4.
S e p a r a t i o n of s e l e c t e d amines on a ~ B o n d a p a kC18 column
5.
L i n e a r response o f u l t r a v i 01 e t absorbance d e t e c t o r t o
a t r a z i n e and L i n u r o n . . . . . . . . . . . . . . . . .
6.
Sampling s i t e s on t h e Chesapeake Bay and i t s
7.
S e p a r a t i o n of s t a n d a r d amine m i x t u r e on 10% Apiezon L +
2% KOH on 80% Chromasorb W AW . . . . . . . . . . . . .
8.
S e p a r a t i o n o f s t a n d a r d amine m i x t u r e on 3% SP-2340 on
100/120 S u p e l c o p o r t . . . . . . . . . . . . . . . . .
9.
S e p a r a t i o n o f s t a n d a r d amine m i x t u r e on 3% SP-2250 DB on
1001120 Supel c o p o r t . . . . . . . . . . . . . . . . . .
S e p a r a t i o n o f ' s t a n d a r d amine m i x t u r e on Tenax GC 60180
11.
S e p a r a t i o n o f s t a n d a r d amine m i x t u r e on 3% OV 17 on
801100 SupeScoport . . . . . . . . . . . . . . . . .
13.
14.
15.
16.
.10
. . . . . . 12
. . . . . . 13
. . . . . . . 16
t r i b u t a r i e s . . . . . . 22
10.
12.
8
. . . . . . 30
. . . . . . . 31
. . . . . . 32
rnesh . . . . 33
. . . . . . .
P l o t o f area responses of 2 , 7 - d i m e t h y l q u i n o l i n e , a n i l i n e , and
a t r a z i ne versus amounts i n j e c t e d (ng ) as c a l c u l a t e d f r o m
a n a l y s i s by flame i o n i z a t i o n d e t e c t i o n . . . . . . . . . . . .
.34
. . . 37
P l o t o f area responses o f o - t o 1 u i d i n e , b e n z i d i n e , o - t o 1 i d i n e ,
and 2-naphthyl amine versus amounts i n j e c t e d ( n g ) as c a l c u l a t e d from a n a l y s i s by flame i o n i z a t i o n d e t e c t i o n . . . . .
. . . . 38
P l o t o f area responses of l e p i d i n e , o - t o l u i d i n e , and a n i l i n e
versus amounts i n j e c t e d ( n g ) as c a l c u l a t e d from a n a l y s i s by
n i trogen s p e c i f i c detectioti . . . . . . . . . . . . . . . .
. . . . 43
P l o t o f area responses o f 2-naphthylamine versus amounts
i n j e c t e d (ng) as c a l c u l a t e d from a n a l y s i s by n i t r o g e n
specific detection . . . . . . . . . . . . . . . . . . .
. . . . . . 44
P l o t o f area responses o f benzidine, a t r a z i n e , and o - t o l i d i n e
versus amounts i n j e c t e d ( n g ) as c a l c u l a t e d from a n a l y s i s by
n i t r o g e n speci f i c d e t e c t i o n . . . . . . . . . . . . . . . .
. . . . 45
TABLES
Page
Retention Indices of Bladex, Atrazine, and Simazine on a
~BondapakC I 8 Column . . . . . . . . . . . . . . . . . .
Retention Indices of Selected Amines on a VBondapak
C I 8 Column . . . . . . . . . . . . . . . . . . . . .
....
.ll
.......
Detection Limits of Several Aromatic and Heterocyclic
Amines Using Ultraviolet Absorbance (254 nm) . . . . .
14
. . . . . . 17
Detection Limits of Several Aromatic and Heterocyclic
Amines Using Fluorescence . . . . . . . . . . . . . .
Recoveries of Selected
Recoveries of Selected
. . . . . . 17
Amines,by Solvent Extraction . . . . . . . 19
Amines on XAD-4 Resin . . . . . . . . . . . 21
Concentrations ( p p b ) of Amines Detected in Fourteen Field
Samples . . . . . . . . . . . . . . . . . . . . . . . . .
Recoveries of Several Amines from Estuarine Water
. . . .23
. . . . . . . . 24
Detector Response for Selected Standard Amines Using Flarne
IonizationDetector . . . . . . . . . . . . . . . . . . .
Percent Recoveries of Standard Carcinogenic Amines from
Distilled Water Using Flame Ionization Detector . . . .
Detector Response for Sel ected Standard Arni r~esUsi ng
Nitrogen Specific Detector . . . . . . . . . . . . .
Percent Recoveries of Standard Carcinogenic Ami nes
from Water Using Nitrogen Specific Detector . . .
. . .
.36
. . . . . 40
. . . . . . . 41
. . . . . . . . 46
INTRODUCTION
Aromatic amines have widespread use i n b o t h i n d u s t r y and a g r i c u l t u r e
and may occur as contaminants i n groundwater and s u r f a c e water as w e l l as
i n biological tissues.
These compounds e x e r t p h y s i o l o g i c a l e f f e c t s i n c l u d i n g
t o x i c i t y , m u t a g e n i c i t y , and c a r c i n o g e n i c i t y .
Several aromatic amines a r e
l i s t e d by t h e Occupational S a f e t y and H e a l t h A d m i n i s t r a t i o n as carcinogens
( ~ e d .Reg. 1973; Fed. Reg. 1974).
Although aromatic amines p r e s e n t
a p o t e n t i a l environmental problem, r e l a t i v e l y 1ittl e d a t a assessing t h e
presence o f t h e compourlds i n t h e environment have been r e p o r t e d .
Most o f
t h e a v a i l a b l e l i t e r a t u r e deals w i t h t h e s e p a r a t i o n and d e t e c t i o n of
p h y s i o l o g i c a l amines.
The concern f o r t h e presence o f amines i n n a t u r a l waters seems t o be
worldwide.
The presence o f a r o m a t i c arnir~es has been c o n f i r r r ~ e di n f i s h
taken from t h e B u f f a l o , Delaware, and o t h e r r i v e r s , i n t h e v i c i n i t y o f
t e x t i l e and d y e s t u f f manufacturers.
Diachenko (1979) d e t e c t e d i n f i s h
t h e presence o f 1-naphthylamine, N-ethyl-N-phenylbenzylamine,
and N-ethyl-N-
(m-t o l y l )-benzylamine by u s i n g gas chromatography, a f t e r d i g e s t i n g t h e
amines w i t h NaOH, p a r t i t i o n i n g w i t h benzene, and c l e a n i n g up w i t h g e l
permeation chromatography.
A s t u d y conducted i n t h e USSR (Vlasova 1974) has r e p o r t e d t h e screeni n g o f some l a k e s f o r amines and o t h e r o r g a n i c corr~pounds. Many such compounds have been found t o e x h i b i t t o x i c , mutagenic, and c a r c i n o g e n i c e f f e c t s
i n experimental animals. Weisburger e t a l . (1978) t e s t e d 21 environmental
aromatic ami nes o r d e r i v a t i v e s f o r long-term t o x i c i ty o r c a r c i n o g e n i c i t y i n
male r a t s and male and female mice.
They found t h a t o - t o l u i d i n e ,
toluene-
diamine, and t r i r n e t h y l a n i l i n e s l e d t o tumors i n a1 1 t h r e e o f t h e animal
model s.
Other ami nes s t u d i e d had v a r y i n g degrees o f a c t i v i ty i n one
animal model o r t h e o t h e r .
Most r e c e n t l y , aromatic amines have been
i d e n t i f i e d by gas chromatography/mass spectrometry (GC/MS) i n dyemanufacturing waste leachates. The amines were l i n k e d w i t h f i s h tumors
(Nelson and H i t e s 1980).
Aromatic amines have been d e t e c t e d o r determined by u s i n g v a r i o u s
a n a l y t i c a l techniques:
t i t r a t i o n w i t h NaN02 ( Z e l e n i n a and Krasnov 1974),
m i c r o d i f f u s i o n by bromocresol p u r p l e r e a c t i o n (~emenove t a1 . 1974),
.
and measurement o f t h e m e l t i n g p o i n t s of t h e i r r e a c t i o n products w i t h Nc h l o r o a c e t a n i 1 i d e ( L e g r a d i 1975),
Legradi (1975) i d e n t i f i e d t h e products
by n u c l e a r magneti c resonance spectroscopy.
More r e c e n t l y gas chromatography
and l i q u i d chromatography have been a p p l i e d t o t h e a n a l y s i s of aromatic
amines.
A r e v i e w and d i s c u s s i o n of a n a l y t i c a l techniques f o r t h e d e t e c t i o n
o f a r o m a t i c amines has been presented by Y l l o (1977).
DEVELOPMENT OF L I Q U I D CHROMATOGRAPHIC METHODS
REVIEW OF LITERATURE
Many of t h e high-performance l i q u i d chromatographic (HPLC)
s e p a r a t i o n s of aromatic ami nes which have been r e p o r t e d were accompl ished
using s i l i c a g e l m o d i f i e d w i t h metal i o n s .
Chow and Grushka (1977, 1978)
o b t a i n e d b e t t e r s e p a r a t i o n s o f s e v e r a l s e t s o f a r o m a t i c amine isomers
u s i n g copper(1 I ) - a 1 kylami ne-bonded s i l i c a g e l , copper(1 I ) - a 1 k y l d i t h i o carbamate-bonded s i 1ica g e l , and copper(1 I)-a1 k y l d i ketone-bonded s i 1 ica
g e l than u s i n g s i l i c a g e l .
Increased r e s o l u t i o n o f t h e t h r e e c h l o r o a n i l i n e
isomers was achieved by Vogt e t a l . (1977) u s i n g s i l i c a g e l h y d r o t h e r m a l l y
t r e a t e d w i t h CdC12 as compared w i t h s i l i c a g e l .
S i r n i l a r r e s u l t s were a l s o
r e p o r t e d f o r 1-naphthylamine and 2-naphthylamine (Vogt e t a l . 1977).
Kunzru and F r e i (1974) r e p o r t base1 i n e s e p a r a t i o n o f t h e t h r e e c h l o r o a n i l i n e
isomers and near-baseline s e p a r a t i o n o f t h e t h r e e t o l u i d i n e isomers u s i n g
cadmium-impregnated s i l i c a g e l which had been p r e t r e a t e d w i t h NaOH.
Silver
has a l s o been used t o m o d i f y s i l i c a g e l t o enhance s e p a r a t i o n s o f
a r o m a t i c amines.
e t a1
.
By hydrothermal l y t r e a t i n g s i l i c a g e l w i t h AgN03, Vogt
(1978) have achieved near-base1 i n e r e s o l u t i o n o f t h e t h r e e isomers
o f chloroaniline.
A i g n e r e t a l . (1976) and V i v i l e c c h i a e t a l . (1972) have
r e p o r t e d s e p a r a t i o n s o f aza-heterocycl i c s u s i n g s i 1ver-modi f i e d s i 1 i c a g e l .
S i l i c a g e l m o d i f i e d w i t h metal i o n s has a l s o been used i n t h e s e p a r a t i o n s
o f v a r i o u s u n s a t u r a t e d acetates, aldehydes, and hydrocarbons (Heath e t a l .
1975; Mike:
e t a1 . 1973).
Various o t h e r methods o f s e p a r a t i n g aromatic amines have been
reported.
Englehardt e t a1
.
(1974) separated N,N-diethylani 1 i n e , N ,&dimethyl
a n i l i n e , and N - m e t h y l a n i l i n e u s i n g s i l i c a g e l coated w i t h H20 (0.6g H20/
g s i l i c a gel).
Gel permeation chromatography has been used by P r o t i v o v i and
p o s p j S i 1 (1974) t o separate amine a n t i o x i d a n t s and antiozonants, i n c l u d i n g
-
a n i l i n e , 2-naphthylamine, b e n z i d i ne, and o - t o 1 i d i n e .
Reverse-phase separa-
t i o n s u s i n g m i x t u r e s o f CH3CN and 0.15M phosphate b u f f e r a t pH 2.1 were
r e p o r t e d by Lores e t a l . (1978).
A n i l i n e , 2-amino-5-chlorophenol,
p - c h l o r o a n i l i n e , p-brornoani 1 i n e , m - c h l o r o a n i l ine, o - c h l o r o a n i l i n e , and
3 , 4 - d i c h l o r o a n i l i n e were separated u s i n g an 8-min g r a d i e n t from 80% 0.15M
phosphate b u f f e r / 2 0 % CH3CN t o 40% 0.15M phosphate b u f f e r / 6 0 % CH3CN.
A
v a r i e t y o f HPLC c o n d i t i o n s w i t h c a p a c i t y f a c t o r s i s g i v e n f o r approximately
50 aromatic amines by Young and McNair (1976).
The use o f t h r e e common methods o f d e t e c t i o n - u l t r a v i o l e t absorbance,
fluorescence,
reported.
and e l e c t r o c k ~ e m i c a l p o t e n t i a l -wi t h aromatic amines' has been
C l a r k e t a1
.
(1977) and C l a r k and Wells (1978) have r e p o r t e d
enhancement o f t h e UV absorbance o f amines by f o r m i n g t h e corresponding
4-methoxybenzamides o r 4-ni trobenzamides.
Sei l e r (1977) has w r i t t e n a
r e v i e w o f t h e chrorr~atography o f b i o g e n i c amines which i n c l u d e s a good
s e c t i o n on t h e use 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 amines.
Included i n
t h i s r e v i e w a r e t h e f o l l o w i n g agents used t o f o r m f l u o r e s c e n t d e r i v a t i v e s
o f amines:
4-chloro-7-nitrobenzo-[cl-1,2,5-ozydiazole
c h l o r i d e s , i s o t h i o c y a n a t e s , f l uorescamine, p y r i d o x a l
phosphate, U-formyl -0-hydroxyacetophenone,
,
sulphonyl
pyridoxal-5-
and benzo-y-pyrene.
Samejima (1974) and Samejima e t a l . (1976) r e p o r t t h e s y n t h e s i s and
s e p a r a t i o n o f f l uorescamine d e r i v a t i v e s o f diamines and polyamines.
o-Phthalaldehyde was found t o form more f l u o r e s c e n t d e r i v a t i v e s o f
p r i m a r y amines than e i t h e r n i n h y d r i n o r f l uorescamine by Benson and
Hare (1975).
The most s e n s i t i v e method o f d e t e c t i o n o f aromatic amines i s
electrochemical detection.
Using e l e c t r o c h e m i c a l d e t e c t i o n , subnanogram
and picorrlole q u a n t i t i e s o f aromatic amines have been d e t e c t e d (Lores e t a l .
1978; M e f f o r d e t a1
.
1977).
Using s o l v e n t e x t r a c t i o n o f aqueous s o l u t i o n s ,
d e t e c t i o n 1 i m i t s can be lowered.
R i g g i n and Howard (1979) have compared
t h e d e t e c t i o n 1 i m i t s o f benzidine, 3 , 3 ' - d i c h l o r o b e n z i d i n e ,
and 1,2-diphenyl-
h y d r a z i n e u s i n g d i r e c t i n j e c t i o n , s o l v e n t e x t r a c t i o n , and r e s i n a d s o r p t i o n .
Less t h a n 1 ~ g / lcould be d e t e c t e d by d i r e c t i n j e c t i o n .
These 1 i m i t s were
improved t o 50 n g / l by s o l v e n t e x t r a c t i o n and 100 n g / l by r e s i n a d s o r p t i o n .
EXPERIMENTAL MATERIALS
Equi pment
1.
Dual Waters 6000A S o l v e n t D e l i v e r y System w i t h Model 660
Gradi e n t Programmer
2.
I S C O Model UA-5 F i x e d Wave1 ength U l t r a v i o l e t D e t e c t o r
3.
S c h o e f f e l Vlodel 970 V a r i a b l e Wavelength Fluorescence D e t e c t o r
4.
Waters Model 440 F i x e d Wavelength U l t r a v i o l e t D e t e c t o r
5.
I S C O Model 1440 I s o c r a t i c L i q u i d Chromatograph
Reagents and Suppl i e s
1.
Amine standards were prepared f r o m reagent grade (99+% pure)
m a t e r i a l s o b t a i n e d from A l d r i c h Chemical Company.
2.
A1 1 s o l v e n t s used were of d i s t i l l e d - i n - g l a s s qua1 it y ( B u r d i c k and
A1 1 o t h e r chemicals
Jackson L a b o r a t o r i e s , I n c . , Muskegon, MI).
were Baker analyzed r e a g e n t grade.
3.
The f o l l o w i n g chromatographic columns were used i n these s t u d i e s :
a.
b.
c.
d.
e.
f.
g.
4.
Waters
Waters
Waters
Waters
Waters
Dupont
Waters
~Bondapak Cl
~Bondapak CN
~BondapakPhenyl
Corasi 1 I impregnated w i t h cadmi um
Poragel PS
ODs
Porasil A
A m b e r l i t e XAD-4 and XAD-8 r e s i n s were purchased from Rohm and Haas.
EXPERIMENTAL PROCEDURES
High-performance 1 i q u i d chromatographic (HPLC) analyses were r u n on an
I S C O model 1440 l i q u i d chromatograph and a Waters Associates g r a d i e n t e l u t i o n
pumping system i n c l u d i n g two model 6000A chromatography pumps and a model 660
solvent programer.
The amines were d e t e c t e d u s i n g b o t h a f i x e d wavelength
I S C O model UA-5 u l t r a v i o l e t - v i s i b l e d e t e c t o r and a S c h o e f f e l 970 v a r i a b l e
wavelength f l u o r e s c e n c e d e t e c t o r . Separations were attempted u s i n g each o f
t h e f o l l o w i n g columns:
Poragel PS (Waters A s s o c i a t e s ) , a v i n y l p y r i d i n e ,
s t y r e n e , v i n y l benzene
t e r p o l y m e r i c a d s o r p t i o n m a t e r i a l ; ODs ( ~ u p o n t ,) a
reverse-phase p a r t i t i o n m a t e r i a l ; Corasi 1 I (Waters A s s o c i a t e s ) , a p e l 1 i c u l a r
s i l i c a a d s o r p t i o n m a t e r i a l ; Corasi 1 I impregnated w i t h c a d m i u n . ~ ( I I;) Porasi 1 A
(Waters A s s o c i a t e s ) , a porous s i 1 i c a a d s o r p t i o n m a t e r i a l , impregnated w i t h
cadmium; ~BondapakC 1 8 (Waters A s s o c i a t e s ) , a reverse-phase p a r t i t i o n m a t e r i a l ;
and ~ B o n d a p a kCN (Waters A s s o c i a t e s ) , a reverse-phase c y a n o - d e r i v a t i z e d
a d s o r p t i o n l p a r t i ti on m a t e r i a l .
Normal -Phase Separations ( A d s o r p t i o n )
Standard amines were d i s s o l v e d s i n g l y and i n m i x t u r e s i n methanol
and d i 1 u t e d t o known c o n c e n t r a t i o n s i n t h e ppm (pg/ml) range.
Twentym i c r o l i t e r a l i q u o t s o f these s o l h t i o n s were i n j e c t e d a t a t i m e onto t h e
columns which were b e i n g e v a l u a t e d and e l u t e d w i t h t h e a p p r o p r i a t e s o l v e n t
system.
The areas under t h e peaks observed were measured by an e l e c t r o r ~ i c
i n t e g r a t o r o r by p l a n i m e t r y .
some cases.
prepared.
Peak h e i g h t measurements were a l s o used i n
R e t e n t i o n i n d i c e s ( k ' ) and 1 i n e a r response curves were
Flow r a t e s o f 0.5 t o 2.0 ml p e r rni nute were used.
Reverse-Phase Separation ( A b s o r p t i o n )
The same s e r i e s o f standard amines and m i x t u r e s of amines were used
t o e v a l u a t e t h e u t i l i t y o f f o u r reverse-phase columns. T w e n t y - m i c r o l i t e r
s eluted using
samples o f each s o l u t i o n were i n j e c t e d o n t o t h e c o l u ~ i i r ~and
s o l v e n t g r a d i e n t s o f 0-100% methanol i n water o r 0-100% a c e t o n i t r i l e i n
water.
Flow r a t e s o f 0.5 t o 2.0 m l p e r minute were used.
Retention
i n d i c e s ( k ' ) values and l i n e a r response curves were prepared as above.
SAMPLE EXTRACTION/CONCENTRATION STUDIES
One-1 i t e r sarnples o f pure w a t e r o r n a t u r a l water were s p i k e d w i t h
standard amines a t t h e 1-100 ppb l e v e l . Samples were prepared i n t r i p l i c a t e
and t r e a t e d i n one o f two ways.
One s e t o f samples was a d j u s t e d t o pH 12 w i t h 50% NaOH and e x t r a c t e d
w i t h methylene c h l o r i d e .
Three e x t r a c t i o n s were performed u s i n g a t o t a l o f
200 m l (100 + 50 + 50) of s o l v e n t .
The combined e x t r a c t s were d r i e d over
anhydrous sodium s u l f a t e and evaporated i n a Kuderna-Danish evaporator t o a
f i n a l known v o l ume o f one m i 1 1i1it e r .
A second s e t o f samples was concentrated u s i n g XAD-4 o r XAD-8 r e s i n .
The l i t e r o f water, a d j u s t e d t o pH 10, passed through a 209 bed o f pree x t r a c t e d XAD-4 r e s i n a t a r a t e o f 3-5 m l p e r minute. The r e s i n was precleaned
by soaking i n 5% ammonium carbonate and s e q u e n t i a l r e f l u x e x t r a c t i o n w i t h
d i s t i 1 l e d water, acetone, methanol, methyl ene c h l o r i d e , methanol
, and
water.
The bed was washed w i t h 50 m l of d e i o n i z e d water t o remove excess base, and
then t h e r e s i n was e x t r a c t e d w i t h 100 ml 10% methanol i n methylene c h l o r i d e .
The s o l v e n t was d r i e d o v e r anhydrous sodium s u l f a t e and evaporated i n a
Kuderna-Danish e v a p o r a t o r t o a known volume o f one m i l l i l i t e r .
FIELD EVALUATION OF METHODS
A s e r i e s of f o u r t e e n f i e l d samples was c o l l e c t e d i n f r e s h and e s t u a r i n e
r e g i o n s , c o n c e n t r a t e d u s i n g t h e XAD-4 r e s i n , and analyzed u s i n g reverse-phase
g r a d i e n t s e p a r a t i o n on a ~BondapakC1,
column.
Q u a n t i t a t i o n was achieved u s i n g
u l t r a v i o l e t absorbance a t 254 nm and peak area i n t e g r a t i o n w i t h an i n t e r n a l
s t a n d a r d ( d i p h e n y l amine). Two of t h e samples were analyzed i n d u p l i c a t e ,
and two were analyzed w i t h a known s p i k e o f 10 ppb o f s e v e r a l amines.
The
c o n c e n t r a t i o n s of i d e n t i f i e d amines as w e l l as r e c o v e r i e s o f t h e s p i k e d compounds
were c a l c u l a t e d .
RESULTS
E v a l u a t i o n o f Normal-Phase Column Separations
The cadmium-impregnated C o r a s i l I column was prepared u s i n g a m o d i f i c a t i o n
of t h e procedure r e p o r t e d by Kunzru and F r e i (1974).
n i g h t a t %120°c.
C o r a s i l I was baked over-
A f t e r cool i n g i n a vacuum d e s i c c a t o r , t h e C o r a s i l I was
s t i r r e d w i t h a 15% s o l u t i o n o f NaOH i n CH30H f o r one hour. T h i s suspension
was vacuum f i l t e r e d and washed t h r e e times w i t h CH30H. The C o r a s i l I was then
t h e n s t i r r e d w i t h a 50% s o l u t i o n o f Cd12 i n CH30H f o r 90 minutes.
vacuum f i l t e r e d and d r i e d a t %120°c.
T h i s s l u r r y was
The cadmium-impregnated C o r a s i l I was
packed i n a s t a i n l e s s s t e e l colhmn 57.6 cm i n l e n g t h by 2.5 m1.n I D u s i n g t h e
" t a p - f i l l " method (Snyder and K i r k l a n d 1976, p. 189).
P r i o r t o packing, t h e
empty column was cleaned by t h e method d e s c r i b e d by Karger e t a1
.
(1970).
The cadmium c o n t e n t of t h e C o r a s i l I was analyzed u s i n g a method s i m i l a r
t o t h a t d e s c r i b e d by Kunzru and F r e i (1974).
A known amount (%0.2 g ) o f t h e
cadmium-impregnated C o r a s i l I was s t i r r e d w i t h 10 m l o f %5N HN03 f o r two hours.
The suspension was c e n t r i f u g e d , and one ml o f t h e supernatant was d i l u t e d t o
50 m l w i t h H20.
The r e s u l t i n g s o l u t i o n , analyzed w i t h atomic a b s o r p t i o r ~
spectrophotometry, c o n t a i n e d 0.7 mg/l cadmium.
This indicates t h a t the
cadmium-impregnated C o r a s i l I i s 0.2% cadmium.
T h i s v a l u e i s much l o w e r than
t h a t r e p o r t e d by Kunzru and F r e i (1974).
An a t t e m p t was made .to i n c r e a s e t h e
cadmium c o n c e n t r a t i o n by t r e a t i n g t h e C o r a s i l I w i t h 50% CdI,
f o r 150 minutes i n s t e a d of 90 minutes.
cadmium c o n c e n t r a t i o n .
CH30H a l s o f a i l e d .
i n CH30H
T h i s procedure f a i l e d t o i n c r e a s e t h e
Treatment w i t h a s a t u r a t e d s o l u t i o n o f CdI,
in
T h i s f a i l u r e can be e x p l a i n e d by t h e s t r u c t u r a l
differences of t h e s i l i c a packing m a t e r i a l s used.
Corasil I i s a t h i n
l a y e r of s i l i c a g e l fused t o a g l a s s bead, whereas LiChrosorb S I 60, used
by Kunzru and F r e i ( 1 9 7 4 ) ~ i s a c o m p l e t e l y porous s i 1 i c a g e l packing
material.
LiChrosorb S I 60, b e i n g porous, has a g r e a t e r c a p a c i t y f o r
i m p r e g n a t i o n by cadmium t h a n C o r a s i l I.
S e p a r a t i o n o f amine m i x t u r e s on the cadmium-impregnated C o r a s i l I
column was i n i t i a l l y attempted u s i n g 2% CH30H i n hexane as t h e s o l v e n t
system, as r e p o r t e d by Kunzru and F r e i (1974).
W i t h t h i s s o l v e n t system,
t h e r e was no r e s o l u t i o n o f N - m e t h y l a n i l i n e , a n i l i n e , and 2 , 4 , 6 - t r i c h l o r o aniline.
T h i s i s e x p l a i n e d by t h e l o w e r cadmium c o n t e n t o f t h e cadmium-
impregnated C o r a s i l I column as compared t o t h e cadmium-impregnated
LiChrosorb S I 60 column used by Kunzru and F r e i .
Using t h e cadmium-
impregnated C o r a s i l I collrrnn, t h e b e s t s e p a r a t i o n s were achieved u s i n g
m i x t u r e s of e t h y l a c e t a t e and hexane.
With a 100-to-1 r a t i o of hexane
t o e t h y l a c e t a t e , near-baseline s e p a r a t i o n o f 1-naphthylamine, 2,4d i c h l o r o a n i 1i n e , and N-methylanil i n e was achieved ( F i g . 1 ) .
However,
,
line,
u s i n g t h e same s o l v e n t , a m i x t u r e o f N - m e t h y l a r ~ i l i r ~ e2,4-dichloroani
1-naphthylamine, p - c h l o r o a n i l i n e , and 2-naphthylamine c o u l d n o t be separated.
Complete r e s o l u t i o n o f t h e above f i v e amines was a l s o n o t achieved u s i n g a
400-to-1 r a t i o o f hexane t o e t h y l a c e t a t e .
On t h e assumption t h a t the f a i l u r e o f t h i s c o l ulnrl t o r e s o l v e t h e m i x t u r e
i s due t o t h e s t r u c t u r e as d e s c r i b e d above, a new column was prepared u s i n g
P o r a s i l A, a c o m p l e t e l y porous s i l i c a a d s o r p t i o n m a t e r i a l .
T h i s column was
prepared u s i n g t h e method described above w i t h t h e e x c e p t i o n t h a t a 25%CdI,
s o l l r t i o n was s u b s t i t u t e d f o r t h e 50%-CdI? s o l u t i o n .
N-methylaniline,
2,4-dichloroaniline,
p - c h l o r o a n i l i n e was attempted.
1-naphthylamine,
Separation of
2-naphthylamine, and
Using hexane, t h e amines were n o t separated.
Using e i t h e r e t h y l a c e t a t e o r a one-to-one r a t i o o f e t h y l a c e t a t e t o hexane,
N-methylani 1i n e , 2,4-di c h l o r o a n i 1 ine, and 1-naphthylamine were i n c o m p l e t e l y
separated, and p - c h l o r o a n i 1 i n e and 2-naphthylamine were e x c e s s i v e l y r e t a i n e d .
The Poragel PS column gave the b e s t s e p a r a t i o n s u s i n g m i x t u r e s of
CH30H and H20.
g i t h a CH30H/H20 (10/1) m i x t u r e , b a s e l i n e s e p a r a t i o n o f
TIME ( M I N I
F i g u r e 1.
S e p a r a t i o n o f l-naphthylamine, 2 , 4 - d i c h l o r o a n i l ine, and
N-methylani 1 ine.
Col umn: Corasi 1 I/Cd. Sol vent: e t h y l acetate/hexane (1/100).
Flow r a t e : 1 ml/min. UV absorbance range: 0.05 AUFS (absorbance u n i t s f u l l s c a l e ) .
a n i 1 i n e and N-methylani 1 i n e was accompl ished, b u t 2 , 4 , 6 - t r i c h l o r o a n i
1 i n e was
excessively retained.
When t h e r a t i o o f CH30H t o H20 was changed t o 20 t o
1, t h e peaks from a n i l i n e and N - m e t h y l a n i l i n e became merged, b u t 2,4,6t r i c h l o r o a n i 1 i n e was no l o n g e r e x c e s s i v e l y r e t a i n e d .
These r e s u l t s suggested
t h e use o f a g r a d i e n t system t o r e s o l v e t h e amines; however, t h e peaks were
t o o broad t o be r e s o l v e d by t h i s method.
Eva1 u a t i o n o f Reverse-Phase Col umns
The Dupont ODs column gave t h e b e s t s e p a r a t i o n s u s i n g CH30H and H20
mixtures.
A l l o f t h e i s o c r a t i c systems attempted f a i l e d t o r e s o l v e a m i x t u r e
of N , N - d i m e t h y l a n i l i n e ,
l-naphthylamine, and 2 , 4 - d i c h l o r o a n i l i n e .
The
i s o c r a t i c systems used were CH3CN, CH30H, a l - t o - 1 r a t i o of CH3CN t o H20,
and m i x t u r e s of CH30H and H20 i n t h e f o l l o w i n g r a t i o s :
and 1 t o 3.
However, near-base1 i n e s e p a r a t i o n
10 t o 1, 1 t o 1,
o f l-naphthylamine, 2,4-
d i c h l o r o a n i 1i n e , and N-methylanil i n e was achieved u s i n g a 10-rnin convex
g r a d i e n t from 100% H20 t o 85% H20/15% CH30H.
The b e s t o v e r a l l s e p a r a t i o n s were o b t a i n e d u s i n g t h e ~BondapakCI8
column.
These s e p a r a t i o n s were achieved u s i n g m i x t u r e s o f CH30H and H20
as we1 1 as m i x t u r e s o f CH30H and an ~ 0 . 0 8 Maqueous s o l u t i o n of t r i e t h y l ammonium phosphate.
The ~ 0 . 0 8 M t r i e t h y l a m o n i u m phosphate was prepared by
d i l u t i n g 11.3 m l o f c o n c e n t r a t e d phosphoric a c i d t o 2 l i t e r s w i t h H20 and i n c r e a s i n g t h e pH t o ~ 7 w i t h t r i e t h y l a m i ne.
Using a 10-min concave g r a d i e n t
from 50% H20/50% CH30H t o 40% H20/60% CH30H, b a s e l i n e s e p a r a t i o n o f Bladex,
a t r a z i n e , and Simazine was achieved ( F i g . 2 and Table 1 ) . B a s e l i n e s e p a r a t i o n
o f p - c h l o r o a n i l i n e , 2,4-dichloroani 1 ine, and 2 , 4 , 6 - t r i c h l o r o a n i
1 i n e was achieved
u s i n g 75% CH30H/25% 0.08M triethylammonium phosphate as t h e s o l v e n t ( F i g . 3 ) .
Using a 20-mi n concave g r a d i e n t from 40% CH30H/60% 0.08M t r i e t h y l ammoni um
phosphate t o 70% CH30H/30% 0.08M triethylammonium phosphate, b e n z i d i n e ,
o-tolidine,
o-toluidine,
N-rnethylaniline, p - c h l o r o a n i l i n e , l-naphthylamine,
N,N-dimethylaniline,
2,4-dichloroaniline,
were p a r t i a l l y r e s o l v e d ( F i g . 4 and Table 2 ) .
2-naphthylamine,
and 2 , 4 , 6 - t r i c h l o r o a n i l i n e
Baseline separation o f benzidine,
o - t o l i d i n e , N - m e t h y l a n i l i n e , N,N-dimethylaniline, 2 , 4 - d i c h l o r o a n i l i n e , and
2 , 4 , 6 - t r i c h l o r o a n i l i n e was achieved. N - m e t h y l a n i l i n e and p - c h l o r o a n i l i n e
were p a r t i a l l y merged, and l-naphthylamine, 2-naphthylamine,
were co-eluted.
and o - t o l u i d i n e
0
2
4
6
8
10
'
12
TIME (MINI
F i g u r e 2.
S e p a r a t i o n o f a t r a z i n e , Simazine, and Bladex.
S o l v e n t : 10-min concave g r a d i e n t f r o m 50% H20/50% CH30H t o
Column: ~ B o n d a p a k
40% H20/60% CH30'H. Flow r a t e : 2.0 ml/min. UV absorbance range: 0.5 AUFS (absorbance
units f u l l scale).
C 1 8 e
14
.uwnLo3 8T3 yedepuoqfl e uo
a u k z e w s pue ' a u k z e ~ ~' yx a p ~ ~40
g s a ~ k p uuokquaqad
~
0
2
4
6
TIME ( M I N 1
F i g u r e 3.
S e p a r a t i o n o f p - c h l o r o a n i 1 i n e , 2 , 4 - d i c h l o r o a n i 1 ine, and
2,4,6-trichloroaniline.
Col umn: ~ B o n d a p a kC18.
Sol vent: 75% CH30H/25% 0.08M t r i e t h y l ammoni um phosphate. Flow r a t e : 2.0 ml/min. UV absorbance
range: 0.5 AUFS (absorbance u n i t s f u l l s c a l e ) .
DETECTOR RESPONSE
0 D J - c
3 w
3 3 0
- 0 3
\ 3 P
3 2.w
2.C-a
.
3 3 m
0
3
0
-h
wl
I D ,
U
50
COW
ID
0
ewca
rD
-0DJ 5
UDJ
m d v ,
P
d
DJ
3
o m 5
2.
'j
3
urt<
DJOID
3
3
sc
2
N - Methylaniline
ID
wl
.
s?
lv
DJ 0 0
'j
3
I
I
row 3
rD 02.
-.
I -Naphthylamine
2-Naphthylamine
I 3
\
O W 0
0 0
was3
0
D O D
C.
<
7 O I D
0 0 3
3-
N , N- Dimethylaniline
2,4-Dichloroaniline
aqeydsoyd wnkuowwelLy3ak~3
W80'0 %OE/HoEH3 %OL 03 a2eydsoyd
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R e s o l u t i o n o f benzidine, O - t o l u i d i n e , o - t o l i d i n e , 1-naphthylamine,
2-naphthylamine, P - c h l o r o a n i l i n e , 2 , 4 - d i c h l o r o a n i l i n e ,
2,4,6-trichloroanil
ine
14-methylani 1i n e , and N,N-dimethylani 1i n e was l e s s complete using e i t h e r t h e
~BondapakCN o r ~BondapakPhenyl column than i t was u s i n g the uBondapak
column.
C18
The b e s t separations using these columns were achieved using e i t h e r
a 20-min g r a d i e n t from 100% H20 t o 50% H20/50% CH,OH
o r a 20-min concave
g r a d i e n t from 100% 0.08M triethylammoni um phosphate t o 70% 0.08M t r i e t h y l ammonium phosphate/30% CH30H.
Separations using t h i s column were n o t
reproducible.
L i n e a r i t y of U l t r a v i o l e t and Fluorescent Response o f Selected Amines
Standard s e r i a l d i l u t i o n s o f t h e amines used i n t h i s s t u d y were
i n j e c t e d s i n g l y and i n m i x t u r e t o determine i f l i n e a r responses could be
obtained u s i n g e i t h e r u l t r a v i o l e t absorbance a t 254 nm o r i n t r i n s i c
fluorescence.
I n a l l cases, the u l t r a v i o l e t absorbance versus c o n c e n t r a t i o n
p l o t s obtained f o r t h e amines were l i n e a r over a t l e a s t a two-order-ofmagnitude range i n concentration.
F i g u r e 5 shows t h e 1inear response obtained
f o r two amines, a t r a z i n e and Linuron.
r e p o r t e d i n Table 3.
D e t e c t i o n l i m i t s f o r several amines a r e
The d e t e c t i o n l i m i t was d e f i n e d i n t h i s case t o be t h e
mass o f m a t e r i a l r e q u i r e d t o produce a s i g n a l t w i c e t h a t o f the n o i s e l e v e l a t
0.02 absorbance u n i t s f u l l s c a l e (AUFS).
A l l o f these d e t e c t i o n l i m i t s
correspond t o approximate concentrations i n t h e a n a l y t e s o l u t i o n i n t h e range of
0.1 t o 0.2 pprn.
Depending upon t h e volumle e x t r a c t e d and t h e concentratiorl
f a c t o r employed, these d e t e c t i o n l i m i t s corrrespond t o concentrations a t t h e
0.1 ppb l e v e l i n n a t u r a l waters.
A s i m i l a r study was performed t o determine the d e t e c t i o n l i m i t s o f
several amines u s i n g i n t r i n s i c fluorescence.
L i n e a r response curves were
obtained f o r a1 1 compounds t e s t e d over a t 1e a s t a two-order-of-magni tude
range i n c o n c e n t r a t i o n .
The d e t e c t i o n l i m i t was again d e f i n e d as t h e amount
of a compound which gave a s i g n a l t w i c e t h a t of t h e n o i s e l e v e l .
was measured w i t h an e x c i t a t i o n wavelength o f 254 nm.
The fluorescence
These d e t e c t i o n l i m i t
s t u d i e s were done u s i n g t h e ~BondapakCI8 column and CH30H as t h e s o l v e n t .
The r e s u l t s o f t h i s study a r e presented i n Table 4. The l a r g e d i f f e r e n c e s
between t h e d e t e c t i o n 1 i m i t s o f t h e s - t r i a z i n e s ( a t r a z i n e , Bladex, and
Simazine) and those of t h e o t h e r aromatic amines t e s t e d are probably due
t o t h e d i f f e r e n c e i n t h e molecular s t r u c t u r e o f t h e aromatic r i n g s .
Atrozine Slope = 0 . 8 0 5 /ppm
Linuron Slope = 0 . 7 3 8 / p p m
r 2 = 1.00
ppm ATRAZ l NE / Ll NURON
Figure 5.
L i n e a r response o f u l t r a v i o l e t absorbance d e t e c t o r t o a t r a z i n e
and Linuron.
AREAAT:
area o f compound response.
AREAIS:
area o f i n t e r n a l standard response.
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Benzidi ne, 2-naphthyl amine, o - t o 1 u i d i ne, and o - t o 1 i d i n e c o n t a i n t r u e
c a r b o c y c l i c a r o m a t i c r i n g s , w h i l e a t r a z i n e , Bladex, and Simazine c o n t a i n
n i t r o g e n o u s h e t e r o c y c l i c pseudoaromatic r i n g s .
The d e t e c t i o n l i m i t s f o r
t h e a r o m a t i c amines correspond t o a n a l y t e c o n c e n t r a t i o n i n t h e range o f 1
t o 100 ppb.
Depending upon t h e volumes e x t r a c t e d and t h e c o n c e n t r a t i o n
f a c t o r employed, t h e d e t e c t i o n 1i m i t s correspond t o c o r ~ c e n t r a t i o n s a t t h e
1 t o 100 p a r t - p e r - t r i l l i o n l e v e l i n n a t u r a l w a t e r s .
I t i s c l e a r t h a t , f o r compounds which e x h i b i t good i n t r i n s i c
f l u o r e s c e n c e , u l t r a t r a c e a n a l y s i s may be accomplished.
However, w i t h
b o t h fluorescence and u l t r a v i o l e t techniques, i n t e r f e r e n c e s from c o - e l u t i n g
substances a r e p o s s i b l e .
f a c t o r s a r e employed.
T h i s i s e s p e c i a l l y t r u e when l a r g e c o n c e n t r a t i o n
A good understanding o f t h e m a t r i x p r e s e n t i n any
n a t u r a l w a t e r sample i s e s s e n t i a l i f a c c u r a t e q u a n t i t a t i o n i s t o be achieved.
Mass s p e c t r a l c o n f i r m a t i o n should p r o b a b l y be a p p l i e d as a r o u t i n e measure
t o back up t h e q u a n t i t a t i v e data.
Sample E x t r a c t i o n / C o n c e n t r a t i o n S t u d i e s
A s e r i e s o f amines were e x t r a c t e d and c o n c e n t r a t e d u s i n g methylene
c h l o r i d e as d e s c r i b e d i n t h e s e c t i o n on sample e x t r a c t i o n and c o n c e n t r a t i o n
on page 5.
The amines were s p i k e d a t l e v e l s o f 1.0 ppb, 10 ppb and 100
ppb i n t o l a b o r a t o r y w a t e r and analyzed by l i q u i d chromatography u s i n g t h e
u l t r a v i o l e t absorbance method.
The r e c o v e r i e s observed a t t h e t h r e e
c o n c e n t r a t i o n l e v e l s a r e r e p o r t e d i n T a b l e 5.
I t appears t h a t a l l of t h e
ami nes t e s t e d can be r e 1 i a b l y and q u a n t i t a t i v e l y recovered by s o l v e n t
extraction.
However, c a u t i o n must be taken t h a t samples a r e n o t overheated
d u r i n g t h e c o n c e n t r a t i o n step.
S t u d i e s were performed t o determine t h e u t i l i t y o f XAD-4 and XAD-8
p o l y m e r i c a d s o r p t i o n r e s i n s i n t h e c o l l e c t i o n and c o n c e n t r a t i o n of a r o m a t i c
amines.
There was a t w o - f o l d purpose f o r t h i s s t u d y :
f i r s t , t o t e s t the
r e t e n t i o n c a p a c i t y of t h e columns, and second, t o t e s t t h e r e c o v e r y o f
t h e amines a f t e r a d s o r p t i o n t o t h e column.
F i r s t , 100 ml o f a 993 n g / l
aqueous s o l u t i o n o f p - c h l o r o a n i l i n e was passed through each o f t h r e e columns
f i l l e d w i t h XAD-8 r e s i n .
The e f f l u e n t from each column was analyzed
,
by HPLC u s i n g t h e uBor~dapak CN columr~ and 10% CH,OH/90% HO
as t h e s o l v e n t .
No v - c h l o r o a n i l i n e was d e t e c t e d by f l u o r e s c e n c e i n a 2 0 - ~ 1 i n j e c t i o n o f
t h i s effluent,
i n d i c a t i n g t h a t t h e p - c h l o r o a n i 1 i n e was f u l l y r e t a i n e d .
each column was r i n s e d w i t h 100 ml o f CH30H.
Then,
The f i r s t 10 m l t h a t were
e l u t e d c o n t a i n e d no d e t e c t a b l e p - c h l o r o a n i l i n e and were d i s c a r d e d .
The
remainder o f t h e e f f l u e n t was d i l u t e d t o 100 m l w i t h CH30H and analyzed
by HPLC u s i n g t h e above c o n d i t i o n s .
The average r e c o v e r y was 100.4% w i t h
a standard d e v i a t i o n o f 0.64%.
A d d i t i o n a l work was a l s o performed t o determine i f t h e XAD-4 r e s i n
c o u l d be used t o r e l i a b l y c o n c e n t r a t e s e l e c t e d amines a t environmental
levels.
The amines were s p i k e d i n t o l a b o r a t o r y w a t e r a t l e v e l s o f 1.0 ppb,
10 ppb, and 100 ppb, c o n c e n t r a t e d on t h e XAD-4 r e s i n , recovered, and
analyzed by l i q u i d chromatography u s i n g t h e u l t r a v i o l e t absorbance method.
The r e c o v e r i e s observed a t t h e t h r e e c o n c e n t r a t i o n l e v e l s a r e r e p o r t e d
i n Table 6.
The r e c o v e r i e s o f t h e amines a t a l l l e v e l s t e s t e d were w e l l
w i t h i n t h e acceptabl e range (90-100%).
F i e l d Eva1 u a t i o n o f Methods
The f o u r t e e n f i e l d samples were c o l l e c t e d a t a number o f s i t e s on
t h e Chesapeake Bay, t h e Patuxeht R i v e r , and t h e Susquehanna R i v e r a t two
times d u r i n g t h e l a s t y e a r (October 10-12,
1979 and A p r i l 24 and 26, 1980).
F i g u r e 6 shows t h e l o c a t i o n s where t h e samples were c o l l e c t e d . The samples
were f i l t e r e d t h r o u g h 0.45 pm m i l l i p o r e f i l t e r pads a t t h e t i m e o f c o l l e c t i o n ,
c o n c e n t r a t e d i n XAD-4 r e s i n , and c o n c e n t r a t e d and analyzed u s i n g reversephase chromatography and u l t r a v i o l e t absorbance d e t e c t i o n .
The c o n c e n t r a t i o n s
o f t h e amines found i n t h e f o u r t e e n samples a r e r e p o r t e d i n Table 7.
Low l e v e l s o f s e v e r a l amines a s s o c i a t e d w i t h a g r i c u l t u r e were
d e t e c t e d a t t h e sub-ppb l e v e l .
amines were observed.
No r e s i d u e s of d y e - i n d u s t r y - a s s o c i a t e d
These r e s u l t s a r e c o n s i s t e n t w i t h t h e f a c t s t h a t no
dye m a n u f a c t u r i n g i s t a k i n g p l a c e i n t h i s geographical r e g i o n and t h a t
a g r i c u l t u r e ( i n c l u d i n g corn, soybean, and tobacco f a r m i n g ) i s widespread
on lands s u r r o u n d i n g t h e Chesapeake and i t s t r i b u t a r i e s .
R e p l i c a t e analyses of t h e CHB-2 samples i n d i c a t e t h a t t h e method
used can y i e l d r e l i a b l e and r e p r o d u c i b l e d a t a a t t h e ppb l e v e l .
Table 8
shows t h e r e c o v e r i e s of several arni r ~ e ss p i ked i n t o PAX-1 water a t a 1eve1
o f 10 ppb a f t e r t h e w a t e r had been f i l t e r e d .
A l l o f t h e r e c o v e r i e s were
observed t o be i n t h e acceptable range (93-100%).
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DEVELOPMENT OF GAS CHROMATOGRAPHIC METHODS
REVIEW OF LITERATURE
Due t o i t s ease of o p e r a t i o n and v e r s a t i l i t y , gas chromatography
i s w i d e l y used f o r t h e d e t e c t i o n , i d e n t i f i c a t i o n , d e t e r m i n a t i o n , and
q u a n t i t a t i o n o f amines.
Various c o n d i t i o n s , column packings , and d e t e c t i o n
systems have been ernployed by d i f f e r e n t workers.
Flame i o n i z a t i o n d e t e c t i o n
was used by Markacheva and Kogan (1976) and Golovnya e t a l . (1978); e l e c t r o n
c a p t u r e d e t e c t i o n by Bowen (1976), Pashkevich e t a l . (1977), and Nony and
Bowman (1978) ; thermal c o n d u c t i v i t y d e t e c t i o n by Pasechni k and Rogovi k (1973) ;
and n i t r o g e n s p e c i f i c d e t e c t i o n by Bowen (1976) and Diachenko (1979).
The f o l l o w i n g phases have been err~ployed t o separate aromatic amines by
gas chromatography:
Apiezon M (Andersons e t a l . 1973a and 1973b); Apiezon L
(Golovnya e t a l . 1978); Tenax GC (Daemen e t a l . 1975; Bowen 1976) ; T r i t o n
X-305 (Golovnya e t a l . 1978); S i l i c o n OV 101 (Grimmer e t a l . 1978) ; p o l y e t h y l e n e g l y c o l 1000 (Golovnya e t a1
.
1978) ; p o l y e t h y l e n e g l y c o l 2000
(Andersons e t a l . 1973a and 1973b); Chromaton N c o n t a i n i n g 20% p o l y p h e n y l e t h e r
(Markacheva and Kogar~ 1976) ; 20% Reoplex-400 (Czerwiec and Markowski 1975) ;
25% polymethyl phenyl s i loxane-4 (Pasechni k and Rogovi k 1973) ; and 5% SE 60/
Chromaton N (Pashkevich e t a l . 1977).
Aromatic amines have a l s o been
determined by s e v e r a l o t h e r workers u s i n g v a r i o u s c o n d i t i o n s (Smi kun
e t a l . 1974; Farroha and Emeishi 1975; Nony and Bowman 1978; and Tsuda
e t a l . 1977).
Trace analyses o f c a r c i n o g e n i c amines and t h e i r analogs have been
c a r r i e d o u t by Nony and Bowman (1978) i n wastewater and human u r i n e , by
Diachenko (1979) i n f i s h , by H a e f e l f i n g e r (1975) i n b l o o d and plasma, and
by Neurath e t a l . (1977) i n t h e hurlan environment and foods i n general.
E x t r a c t i o n and c o n c e n t r a t i o n s t u d i e s f o r amines were performed f o r
m o n i t o r i n g t h e safe d i s p o s a l of wastewater (Nony and Bowman 1978).
Salient
elements of t h e procedure d e s c r i b e d by t h e above workers a r e :
extraction
o f p h e n o l i c and n e u t r a l r e s i d u e s from t h e a c i d i f i e d sample, 1 i q u i d - 1 i q u i d
p a r t i t i o n i n g cleanup and s e p a r a t i o n of n e u t r a l from phenol ic r e s i d u e s a t
pH 14 and 10.2,
a c i d h y d r o l y s i s o f t h e n e u t r a l component, subsequent
a1 k a l i r ~ i z a t i o no f t h e sarr~pleand e x t r a c t i o n o f t h e b a s i c r e s i d u e s of t h e
f r e e amines, c o n v e r s i o n o f a l l r e s i d u e s t o t h e corresponding p e n t a f l u o r o p r o p i o r ~ y l (PFP) d e r i v a t i v e s , and q u a n t i f i c a t i o n by e l e c t r o n c a p t u r e gas
chromatography.
The a u t h o r s were a b l e t o d e t e c t t h e r e s i d u e s i n wastewater
and u r i n e a t 0 . 1 and 1.0 ppb l e v e l s , r e s p e c t i v e l y .
EXPERICIENTAL MATERIALS
Equi pment
1.
Hewlett-Packard 5700A gas chromatograph equipped w i t h a flame
i o n i z a t i o n d e t e c t o r and a Hew1e t t - P a c k a r d 3385A autornati on
system f o r data processing
2.
Hewlett-Packard 5840A gas chromatograph equipped w i t h a n i t r o g e n phosphorus s p e c i f i c d e t e c t o r a r ~ da GC t e r m i n a l f o r d a t a p r o c e s s i n g
3.
Kuderna-Dani sh c o n c e n t r a t o r (Kontes Glass Company, V i n e l and, NJ)
4.
R o t a r y Evaporator ( F l a s h-Evaporator, B u c h l e r Instruments, F o r t Lee, NJ)
Reagents and S u p p l i e s
1.
Carcinogenic amine standards were a c q u i r e d as 20 rng/rnl s o l u t i o n s
i n methanol, from Supelco, I n c . , Supel co Park, Be1 l e f o n t e , PA.
2.
The f o l l o w i n g column packings were purchased from Supelco, I n c :
a.
b.
c.
d.
e.
f.
3.
4.
10% Apiezon L + 2% KOH on 80/100 Chromasorb W AW
3% OV 17 on 80/100 S u p e l c o p o r t
Tenax GC, 60180 mesh
1.5% SP-2250/1.95% SP-2401 on 100/120 S u p e l c o p o r t
10% SP-2340 on 100/120 S u p e l c o p o r t
32 SP-2100 DB on 100/120 Supelcoport
3% SP-2250 DB on 100/120 S u p e l c o p o r t
g.
A1 1 t h e s o l vents used were o f g l a s s - d i s t i 1 l e d qua1 it y (Burdick-Jackson
L a b o r a t o r i e s , Inc., Muskegon, M I ) , and r e a g e n t grade chemicals were
used, i f needed.
Glass columns and gas chromatographic s u p p l i e s were purchased from
Hewlett-Packard, 5201 T o l l v i e w D r i v e , R o l l i n g Meadows, I L .
EXPERIMENTAL PROCEDURES
The d e t e c t i o n of c a r c i n o g e n i c amines was i n v e s t i g a t e d w i t h f l a m e
i o n i z a t i o n , e l e c t r o n capture, and n i t r o g e n s p e c i f i c d e t e c t i o n gas chromatography.
S e l e c t e d amines were s t u d i e d t o determine t h e i r response t o each
t y p e o f d e t e c t i o n a t s e v e r a l d i l u t i o n s o f t h e standards.
Flame Ionization Detection
Standard amines dissolved in methanol were diluted t o known concent r a t i o n s (nanograrns/~l)of each. 1- t o 5 - ~ 1injections representing 10-500
nanograms of sample were introduced into the gas chromatograph. The area
units reproduced by the integrator were processed to calculate the
response factors and 1 i neari ty of detection f o r various amines .
Electron Capture Detection
The selected carcinogenic amines were studied t o determine t h e i r
response to electron capture detection a t picogram amounts. The amines
were derivatized by adding 0.5 ml benzene and 0.1 ml 0.05M trimethylamine
in benzene
to the samples in a 5-ml centrifuge tube, followed by the
addition of 10 ~1 of heptafluorobutyric anhydride.
The reaction mixture
was heated in capped tubes for 15 minutes a t 50°c, then cooled; excess
anhydride was hydrolyzed by adding 1.0 ml water.
Heptafl uorobutyric acid
was removed by addition of one ml of 5% aqueous ammonia solution to the
sample, followed by vigorous shaking f o r 5 minutes and then by
centrifugation. 1- t o 2 - ~ 1 injections were made from the benzene phase.
Nitrogen Specific Detection
The standard amines were evaluated t o determine t h e i r response a t
low concentrations. The amines dissolved in methanol were diluted t o
,
contain from one to several nanograms of each standard amine per ~ 1 and
1 t o 5 ~1 of each amine were analyzed by nitrogen s p e c i f i c detection. A
6-foot-long x 2-mm-ID glass column packed with 3% OV 17 on 80/100
Supelcoport was employed f o r the analyses. Helium was used as c a r r i e r gas,
flow-controlled a t 20.0 + 0.1 ml per minute. Flow rates f o r hydrogen and
a i r were 30 ml per minute and 50 ml per minute respectively. The voltage
f o r the detector was adjusted before every s e t of injections t o give a
recorder pen deflection of 100 mm a t an attenuation of 8. The area units
printed out by the integrator were processed to calculate the responses Per
nanogram amount and t o establish the l i n e a r i t y of detection f o r various
ami nes .
SAMPLE EXTRACTION/CONCENTRATION STUDIES
A l i t e r o f d i s t i l l e d water was s p i k e d w i t h s t a n d a r d amines a t two
c o n c e n t r a t i o n l e v e l s f o r flame i o n i z a t i o n d e t e c t i o n .
Level A c o n t a i n e d 500
ppb each of a n i l i n e , o - t o l u i d i n e , 2-naphthylamine, benzidine, o - t o l i d i n e ,
2,7-dirriethylquinoline,
and 1.0 ppm of a t r a z i n e .
Level B c o n t a i n e d 100 ppb
each of t h e above s t a n d a r d arnines except a t r a z i n e , which was s p i k e d a t 200
ppb. Samples were prepared i n t r i p l i c a t e and t h e pH a d j u s t e d t o 11.5. The
samples were e x t r a c t e d w i t h chloroform. Three e x t r a c t i o n s were performed
u s i n g a t o t a l o f 200 ml (100 + 50 + 50) c h l o r o f o r m .
To e l i m i n a t e t r a c e s of
moisture, t h e combined chloroforrn e x t r a c t s were d r i e d by passing through a
c o l ulnn o f s o d i u~ns u l f a t e .
The e x t r a c t s were c o n c e n t r a t e d t o approximately
one ml i n a Kuderna-Danish c o n c e n t r a t o r and evaporated t o dryness w i t h a strearn
Each sample was r e d i s s o l v e d i n one m l o f methanol and analyzed
o f nitrogen.
on a flame i o n i z a t i o n gas chrorrtatograph.
To e v a l u a t e t h e e f f i c i e n c y o f t h e e x t r a c t i o n procedure a t l o w e r
c o n c e n t r a t i o n s , s e v e r a l standard amines were s p i k e d i n t o one l i t e r o f d i s t i l l e d
w a t e r a t 50 ppb c o n c e n t r a t i o n l e v e l s .
The samples were e x t r a c t e d as d e s c r i b e d
above u s i n g 200 ml o f chloroforrn and/or methylene c h l o r i d e .
evaporated f r o m t h e e x t r a c t s w i t h a f l a s h evaporator.
The s o l vents were
The recovered standard
ami r ~ e swere d i s s o l v e d i n one m l of methanol and analyzed w i t h t h e n i t r o g e n
s p e c i f i c gas chromatograph.
FIELD EVALUATION OF METHODS
A sample o f w a t e r a c q u i r e d from t h e Fox R i v e r was e x t r a c t e d w i t h
chloroform,
as p r e v i o u s l y described, t o screen f o r t h e p o s s i b l e presence o f
small amounts of arornatic amines.
The e x t r a c t from one l i t e r o f w a t e r was
r e d i s s o l v e d i n t o one m l o f methanol ; 1-5 v1 were subsequently i n j e c t e d i n t o a
gas chromatograph
equipped w i t h a n i t r o g e n s p e c i f i c d e t e c t o r .
Another l i t e r o f water from t h e Fox R i v e r was s p i k e d w i t h s e v e r a l
s t a n d a r d amines a t 50 ppb c o n c e n t r a t i o n l e v e l s , e x t r a c t e d , and concentrated
as d e s c r i b e d above.
1- t o 5-p1 i n j e c t i o n s were rnade i n t o a gas chromatograph
with the nitrogen s p e c i f i c detector.
Percent r e c o v e r i e s were c a l c u l a t e d from
t h e p r i n t o u t s o f area responses f o r recovered amines compared t o area responses
f o r standard amines. The r e c o v e r i e s o f amines from d i s t i l l e d w a t e r and Fox
R i v e r water, b o t h of which had been spiked a t 50 ppb c o n c e n t r a t i o n l e v e l s ,
were compared.
RESULTS
Eva1 u a t i o n o f S t a t i o n a r y Phases
The e f f e c t s o f v a r i o u s s t a t i o n a r y phases o f column packings on t h e
e l u t i o n p a t t e r n s of s e l e c t e d c a r c i n o g e n i c amines were determined by flame
Using i s o t h e r m a l c o n d i t i o n s , t h e e l u t i o n and s e p a r a t i o n
o f a l l t h e amines under study were found t o be impossible. Various prograrr~rned
i o n i z a t i o n detection.
temperature modes were then i n v e s t i g a t e d f o r each packing and m i x t u r e of
standard arr~ines.
Figure
7 shows e l u t i o n o f a n i l i n e , O - t o l u i d i n e , l e p i d i n e , and
2-naphthylamine from a 10% Apiezon L + 2% KOH column a t t h e programmed
temperature o f 1 0 0 ' ~ t o 200'~.
Under these c o n d i t i o n s , o t h e r amines present
The use o f temperatures higher.
i n t h e m i x t u r e were r e t a i n e d by t h e packing.
A
than 2 0 0 ' ~ would have d e t e r i o r a t e d t h e s t a t i o n a r y phase on t h e packing.
column packed w i t h 3% SP-2340 was then used t o separate a standard m i x t u r e
(Fig. 8).
The f i g u r e shows t h a t , by temperature programming t h e column from
1 2 5 ' ~ t o 2 7 0 ' ~ e~ x c e l l e n t separations were achieved f o r most of t h e amines.
B u t a t t h e maximu~ntemperature b e n z i d i n e and o - t o l i d i n e t o o k a much l o n g e r
t i m e t o e l u t e , and t h e s e p a r a t i o n between these two amines was n o t
compl e t e .
E v a l u a t i o n o f 3% SP-2250 DB, a b a s e - t r e a t e d packing, was performed by
e l u t i n g t h e standard amines from t h e column a t a programmed temperature o f
9 0 ' ~ t o 2 7 5 ' ~ a t a r a t e o f 8 ' ~p e r minute.
The r e p r e s e n t a t i o n ( F i g . 9) shows
good s e p a r a t i o n s , b u t column b l e e d was excessive above a column temperature
of 250'~; however, i t t o o k a much l o n g e r t i m e t o e l u t e o - t o l i d i n e from t h e
column a t a temperature o f 2 5 0 ' ~ than a t 275'.
The base1 i n e d r i f t was excessive
a t l o w e r a t t e n u a t i o n s which were necessary t o d e t e c t small amounts o f standard
amines.
Another packing commonly used f o r p e s t i c i d e a n a l y s i s , 1.5% SP-2250 +
1.95% SP-2401 on 100/200 Supel coport, e x h i b i t e d s i m i l a r s e p a r a t i o n s , b u t peak
t a i l i n g and b a s e l i n e d r i f t were excessive.
F i n a l l y , t h e nonpolar column packings c o n t a i n i n g OV 17 and Tenax GC were
i n v e s t i g a t e d i n an e f f o r t t o f i n d t h e most s u i t a b l e packing t o r e s o l v e a m i x t u r e
o f arr~ines w i t h c o n s i d e r a b l y d i f f e r e n t v o l a t i 1iz a t i o n c h a r a c t e r i s t i c s .
Most of
t h e amines under s t u d y were r e s o l v e d on Tenax GC ( F i g . 10) and 3% OV 17
( F i g . 11);
3% OV 17 a l s o r e s o l v e d t h e hydrocarbons.
Both o f these packings
uo k ~ e kuo
z L awe 14 :wa7 sAs uo k ~ ~ a ~ a a
- u k w / ~ w02 : S P ~J ~ L J J P D se U ~ ~ O J J L N '30002 :z a ~ n ~ e ~ a d w a l
- u p 1 / 3 ~ 8:aJed .upu 2 :I auk1 -3,001 :I aJnJeJadwa1
'MY M qJosewoJV3 %08 uo
HO] %2 + 1 uozatdv %01 uo aJnJxpu aukwe p ~ e p u e ~40s u o k g e ~ e d a ~' L a ~ n 6 ~ j
Figure 8.
Separation of standard amine mixture on 3% SP-2340 on 100/120
Supelcoport.
Temperature 1: 125OC. Time 1: 4 min. Rate: 16OC/min.
Temperature 2: 270°C. Nitrogen a s c a r r i e r gas: 20 ml/min.
Detection system: flame i o n i z a t i o n .
Inject
TIME ( M I N I
Figure 11.
Separation of standard amine mixture on 3% OV 17 on 80/100
Supelcoport.
Temperature 1: 100°C. Time 1: 2 min. Rate: 8"C/min.
Temperature 2: 300°C. Nitrogen a s c a r r i e r gas: 20 ml/min.
Detection system: flame i o n i z a t i o n .
were more s t a b l e a t h i g h e r temperatures than were t h e packings p r e v i o u s l y
evaluated.
Tenax GC r e q u i r e d h i g h e r column temperatures t o accomplish
e l u t i o n s comparable t o 3% OV 17; a t r a z i n e and 2-naphthylamine appeared as
an u n r e s o l v e d d o u b l e t , and phenyl h y d r a z i ne e x h i b i t e d two s e p a r a t e peaks.*
Because of t h e above reasons, Tenax GC was dropped i n f a v o r of 3% OV 17,
which was employed t o c a l c u l a t e d e t e c t o r responses o f v a r i o u s s t a n d a r d amines
and t o determine t h e s e n s i t i v i t y o r l i m i t s o f d e t e c t i o n .
D e t e c t o r Response of Standard Amines on a Flame I o n i z a t i o n D e t e c t o r
I t was observed t h a t Simazine, one o f t h e s e l e c t e d standard amines, was
n o t a p p r e c i a b l y s o l u b l e i n methanol i n d e s i r e d c o n c e n t r a t i o n s ; i t was t h e r e f o r e
dropped f r o m t h e study.
I t was a l s o found t h a t phenylhydrazine, a n o t h e r
s e l e c t e d s t a n d a r d amine, was u n s t a b l e a t lower c o n c e n t r a t i o n s i n methanol.
I n f r e s h l y prepared s o l u t i o n s , phenyl h y d r a z i ne was r e s o l v e d as a sharp and
d i s t i n c t peak upon a n a l y s i s by gas chromatography; b u t , w i t h subsequent i n j e c t i o n s from t h e same v i a l , t h e peak area decreased and u l t i m a t e l y disappeared.
The d e t e c t o r responses o f each s t a n d a r d amine were c a l c u l a t e d compared t o an
i n t e r n a l s t a n d a r d ( 2 , 7 - d i m e t h y l q u i n o l i n e ) b y t h e use o f flame i o n i z a t i o n detect i o n (Table 9).
I t was found t h a t a n i l i n e , o - t o l u i d i n e , and 2-naphthylarnir~e
have r e l a t i v e responses c l o s e t o t h e i n t e r n a l standard.
Atrazine exhibited
t h e l o w e s t d e t e c t o r response o f a l l t h e standard amines t e s t e d .
L i n e a r i t y o f D e t e c t o r Response Over a Range o f Sample Sizes
on a Flame I o n i z a t i o n D e t e c t o r
S e n s i t i v i t y o f t h e method was determi ned by i n j e c t i n g 10-20 nanograrrls
o f s t a n d a r d arnines i n t o t h e columrr.
A1 though t h e standards were d e t e c t e d a t
these low l e v e l s by t h e flame i o n i z a t i o n d e t e c t o r , t h e responses were n o t
found t o be 1 i n e a r below 50 ng f o r a n i l i n e , a t r a z i n e , and 2,7-dimethylq u i n o l i n e and below 100 ng f o r o- t o 1 u i d i n e , 2-naphthylamine,
and o - t o l i d i n e .
benzidine,
A l l t h e standard amines e v a l u a t e d gave a l i n e a r response
up t o 500 ng as shown i n Fig. 12 and F i g . 13.
*The f i r s t peak was more prominent when a f r e s h s o l u t i o n was i n j e c t e d ; f o r
subsequent i n j e c t i o n s w i t h t h e same s o l u t i o n , t h e second peak became more
prominent-then b o t h peaks decreased i n h e i g h t and f i n a l l y disappeared.
The f i r s t peak i s p r o b a b l y t h e t r u e phenylhydrazine peak, and t h e second
one p r o b a b l y r e p r e s e n t s a decomposition p r o d u c t .
TABLE 9
D e t e c t o r Response f o r S e l e c t e d Standard Amines
U s i n g Flame I o n i z a t i o n D e t e c t o r
Standard
Ami ne
Arnoun t
I n j e c t e d (ng)
Response/ng
(area u n i t s )
2
Anil ine
500
7.00 x 10
o-To1 u i d i n e
500
7.46 x 10
2-Naphthyl amine
500
7.62 x 10
A t r a z i ne
1000
9.70 x 10
Benzi d i ne
500
4.56 x 10
o-To1 i d i n e
500
5.74 x 10
2,7-Dimethyl q u i n o l i n e
500
7.92 x 10
2
2
1
Relative
Response
0.934
0.942
0.962
0.245
2
2
0.576
0.725
2
1.000
A 2 ,7-Dimethylquinoline
Aniline
0 Atrazine
Figure 12.
,
Plot of area responses of 2,7-dimethylquinoline, aniline, and atrazine
versus amounts injected (ng) as calculated from analysis by flame
ionization detection.
-
Io- Toluidine
0 Benzidine
I
100
200
300
I
I
400
500
AMOUNT INJECTED (ng )
Figure 13. Plot of area responses of o-toluidine, benzidine, o-tolidine, and
2-naphthylamine versus amounts injected (ng) as calculated from
analysis by flame ionization detection.
E x t r a c t i o n of Standard Amines from D i s t i l l e d Water
The e x t r a c t i o n s t u d i e s were c a r r i e d o u t by s p i k i n g t h e w a t e r a t two
l e v e l s , 500 ppb and 100 ppb, f o r most of t h e standard amines.
The standard
amines were analyzed by a flame i o n i z a t i o n d e t e c t o r , and t h e r e c o v e r i e s were
c a l c u l a t e d as percentages
a able
10).
Recovered standard ami nes ranged from
27.86% f o r a n i l i n e t o 90.74% f o r 0 - t o l i d i n e , a t t h e h i g h e r l e v e l of s p i k e .
A t t h e lower l e v e l o f spike, 100 ppb, t h e r e c o v e r i e s f o r rnost o f t h e amines
were l o w e r except f o r a n i l i n e .
Recoveries ranged from 28.06% f o r a n i l i n e
t o 85.06% f o r o - t o l i d i n e .
D e t e c t o r Response o f Standard Amines t o E l e c t r o n Capture D e t e c t i o n
The h e p t a f l u o r o b u t y r i c d e r i v a t i v e s o f aromatic amines were e v a l u a t e d
i n an a t t e m p t t o o b t a i n h i g h e r s e n s i t i v i t y w i t h e l e c t r o n c a p t u r e gas
chromatography.
A t 100-pg l e v e l s , a n i 1i n e and 0-to1 u i d i n e were separated
as sharp peaks; b u t b a s e l i n e d r i f t s , even a t a low r a t e of temperature
programrr~ing, were excessive.
temperature i n c r e a s e .
The pen d e f l e c t e d t o f u l l s c a l e a f t e r a 15-20'
Because o f t h e above r e s u l t s , c a l c u l a t i o n s of d e t e c t o r
responses by e l e c t r o n c a p t u r e were dropped i n favor o f t h e n i t r o g e n s p e c i f i c
detector.
D e t e c t o r Response o f Standard Amines t o t h e N i t r o g e n S p e c i f i c D e t e c t o r
The usefulness and a p p l i c a b i l i t y o f n i t r o g e n s p e c i f i c d e t e c t i o n were
i n v e s t i g a t e d f o r t h e standard c a r c i n o g e n i c amines.
The d e t e c t o r responses
c a l c u l a t e d f o r v a r i o u s standard amines a r e presented i n Table 11.
Table
11 shows t h a t v a r i o u s amines e x h i b i t e d responses r a n g i n g from 0.532 t o
3.803,
r e l a t i v e t o 1.000 f o r t h e i n t e r n a l standard, l e p i d i n e .
From t h e
comparisons of area responses of standard amines on a n i t r o g e n s p e c i f i c d e t e c t o r
as compared t o a flame i o n i z a t i o n d e t e c t o r , i t seems t h a t t h e n i t r o g e n
s p e c i f i c d e t e c t o r g i v e s a t l e a s t a 9 - t i r n e s - b e t t e r response f o r a n i l i n e and
o-toluidine,
atrazine.
whereas t h e response was more than 400 times b e t t e r f o r
Moreover, e l i m i n a t i o n of i n t e r f e r i n g s o l v e n t peaks and peaks
due t o compounds n o t c o n t a i n i n g n i t r o g e n and phosphorus improves t h e
q u a n t i t a t i on and r e p r o d u c i b i 1-1t y of t h e analyses.
TABLE 10
P e r c e n t Recoveries of Standard Carcinogeni c Arni r ~ e s
f r o m D i s t i l l e d Water Using Flame I o n i z a t i o n D e t e c t o r
Standard
Arni ne
Percent
Recovered
(level A)l
Percent
Recovered
( l e v e l B)2
Aniline
27.86
28.06
o-To1 u i d i ne
51.82
40.00
2,7-Dimethylquinol i n e
90.06
79.22
A t r a z i ne
87.50
83.12
Benzi d i ne
90.03
84.44
l ~ t a n d a r damines s p i k e d a t 0.5 ppm l e v e l , e x c e p t a t r a z i n e a t 1 ppm l e v e l
2Standard ami nes s p i ked a t 100 ppb 1eve1 s , e x c e p t
a t r a z i n e a t 200 ppb l e v e l
TABLE 11
D e t e c t o r Response f o r S e l e c t e d Standard Amines
Usi ng N i t r o g e n S p e c i f i c D e t e c t o r
Standard
Anii ne
Aniline
A t r a z i ne
Benzi d i ne
Lepidine
Arnoun t
I n j e c t e d (ng)
35
Responselng
(Area U n i t s )
Re1a t i ve
Response
6.30 x 10'
0.566
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Jay30 40 s a k ~ a ~ o 3a ay ~l 'aldules s!.yq UOJJ p a ~ a ~ o 3 a 301.1
e
LaAal qdd-0s ay3 3e paykds saukue
aukLkuy ' ( 1 1 a l q e l ) Mol A ~ 6 u k y s ~ u o 3 saJaM
ay3 40 s a p a ~ o 3 aay3
~ 'saukue p ~ e p u e 3 saykds 03 x k ~ 3 e ue se pasn SPM JaAkd
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UOJJ
Ja3eM ay3 uayM
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a
saukue ay3 40 s3unoue 3ue3kjku6ks
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JOJ
ON
-saukue 3kua6ouk3~e340 a z ~ u a s a ~ d
p a u a a ~ 3 sSPM ~ a h k dx o j ay3 u o ~ 4Ja3eM 40 aldues y
sPoy3aW A J o 3 ~ J o q e l 40
. p a ~ a k y 3aq
~ 3ou plno3 uok33a3ap 40 s l a m 1
pue uok3e3k3uenb ' s ~ o 3 3 e & Jay30 pue sau;ue
s a 3 u a ~ a j j ~ay3
p 03 ana
M O ~asay3
LPkJl
PLakj
3e A ~ k ~ ~ q k 3 n p o ~ d a ~
p ~ e p u e 3 ssnokJeA 40 s a s u o d s a ~u l
'uok33a3ap 3 k j p a d s u a 6 0 ~ 3 ~Aq
u pa33a3ap aJaM ' p a 3 3 a f u ~
AMOUNT INJECTED (ng 1
F i g u r e 14.
P l o t o f area responses o f l e p i d i n e , o - t o l u i d i n e , and a n i l i n e
versus amounts i n j e c t e d (ng) as c a l c u l a t e d from a n a l y s i s by
nitrogen s p e c i f i c detection.
20
40
60
80
100
120
140
AMOUNT INJECTED ( n g )
Figure 15.
Plot of area responses of 2-naphthylamine versus amounts injected ( n g )
as calculated from analysis by nitrogen s p e c i f i c detection.
160
TABLE 12
Percent Recoveries o f Standard Carcinogenic Amines
from Water Using N i trogen S p e c i f i c D e t e c t o r
Standard
Ami ne l
% Recovery from
D i s t i l l e d Water
-
Aniline
o-To1 u i d i n e
Lepi d i ne
2-Naphthyl amine
Atrazine
Bl adex
Benzi d i n e
o -To1 id i ne
lSpiked a t a l e v e l o f 50 ppb each
% Recovery from
Fox R i v e r Water
CONCLUS I O N S
1.
Gas chromatography w i t h flame i o n i z a t i o n d e t e c t i o n and n i t r o g e n
s p e c i f i c d e t e c t i o n can s u c c e s s f u l l y be used f o r t h e a n a l y s i s o f
m i x t u r e s o f amines w i t h a wide range of v o l a t i l i z a t i o n constants,
u s i n g such f a i r l y nonpolar column packings as OV 17 and Tenax GC.
2.
Sorne o f t h e arrlines w i t h h i g h v o l a t i l i t i e s appear t o be l o s t d u r i n g
e x t r a c t i o n and c o n c e n t r a t i o n ; t t ~ e r e f o r e , XAD r e s i r ~ srnay be b e t t e r
s u i t e d f o r c o n c e n t r a t i o n o f t h e amines.
Resin columns a l s o e l i m i n a t e
t h e t e d i o u s steps of e x t r a c t i o n which a f f e c t t h e r e c o v e r y of amines.
3.
I t i s e v i d e n t from t h e use o f Fox R i v e r water as a m a t r i x t h a t some
o f t h e amines c o u l d be bound t o t h e b i o l o g i c a l and/or i n o r g a n i c
p a r t i c u l a t e m a t t e r present.
Procedures s t ~ o u l dbe developed t o account
f o r such bound residues.
4.
L i q u i d chromatography u s i n g e i t h e r u l t r a v i o l e t absorbance o r f l u o r e s c e n c e
d e t e c t o r s can s u c c e s s f u l l y be used f o r t h e q u a n t i t a t i v e a n a l y s i s of
complex m i x t u r e s o f aromatic and h e t e r o c y c l i c arnines.
Separation,
u s i n g g r a d i e n t s o f a c e t o n i t r i l e i n water and m i c r o p a r t i c u l a t e reversephase columns such as ~BondapakC 1 8 y
yields the highest resolution.
L i q u i d chromatography may o f f e r some advantages over gas chromatography
f o r t h e a n a l y s i s of thermal l y l a b i 1e compounds o r p o l a r metabol it e s
o f ami nes.
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