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The determination of micromolar concentrations of ammonia with 1-fluoro, 2:4-dinitrobenzene Gadsby, Peter James 1966

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THE DETERMINATION OF MICROMOLAR CONCENTRATIONS OF AMMONIA WITH 1-FLUORO, 2:4-DINITROBENZENE  by  PETER JAMES GADSBY B.Sc,  Hons., S h e f f i e l d U n i v e r s i t y , 1963  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  i n t h e Department o f Chemistry and I n s t i t u t e o f Oceanography  We accept t h i s t h e s i s as conforming t o the required  standard  The  University of British November, 1966  Columbia  In presenting  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements  f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t t h e L i b r a r y s h a l l , make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e studyo  I f u r t h e r agree t h a t p e r m i s s i o n - f o r  and  extensive copying o f t h i s  t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e s .  I t Is u n d e r s t o o d t h a t  copying  or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n  permission.  Department o f The U n i v e r s i t y o f B r i t i s h Vancouver 8 , Canada Date  Columbia  ABSTRACT  The 1-fluoro,  conversion  o f ammonia t o 2 : 4 - d i n i t r o a n i l i n e by r e a c t i o n w i t h  2 : 4 - d i n i t r o b e n z e n e and  the subsequent c o n v e r s i o n  to a diazo-dye w i t h N - ( l - n a p h t h y l ) e t h y l e n e d i a m i n e as an a n a l y t i c a l method f o r determining level.  P a r t i c u l a r emphasis was  (pH g r e a t e r than 8) and  dinitrobenzene conversion, pH The  and  as a separate  which was  dinitroaniline  has been i n v e s t i g a t e d  ammonia at the micromolar  D i n i t r o a n i l i n e was  r e q u i r e d the presence o f the f l u o r o -  phase f o r i n i t i a t i o n o f the r e a c t i o n .  l i g h t s e n s i t i v e , was  y i e l d o f d i n i t r o a n i l i n e had  benzene content, concentrations  a c c e l e r a t e d by  i t then decreased w i t h h i g h e r  fluorodinitrobenzene.  increases i n  concentration  fluorodinitrobenzene  f u r t h e r r e a c t i o n between d i n i t r o a n i l i n e  In both d i s t i l l e d and  l e v e l was  found t o be  i n sea water o f s a l i n i t y  30.4  %»  55-58%. was  and  sea water, the maximum  The  o n l y 42%  micromolar  absorbance o f the  diazo-  o f t h a t observed i n d i s t i l l e d  A l t h o u g h s u f f i c i e n t l y s e n s i t i v e f o r a p p l i c a t i o n t o sea water  a n a l y s i s , the p r e c i s i o n o f the c o n v e r s i o n dye  yield.  increasing fluorodinitro-  y i e l d o f d i n i t r o a n i l i n e from s o l u t i o n s c o n t a i n i n g ammonia at the  water.  The  a marked dependence on the amount o f  i n i t i a l l y i n c r e a s i n g with  suggesting  this  formed under a l k a l i n e  temperature, but n e i t h e r o f these f a c t o r s improved the f i n a l  fluorodinitrobenzene;  dye  concentration  p l a c e d upon the p o s s i b i l i t y o f a p p l y i n g  method t o the a n a l y s i s o f sea water. conditions  of  i n sea water was  o f d i n i t r o a n i l i n e t o the  poor compared t o t h a t achieved  in distilled  diazo-  water.  iii  TABLE OF CONTENTS  Page INTRODUCTION  1  APPARATUS AND REAGENTS  4  EXPERIMENTAL  6  I.  D i a z o t i s a t i o n and C o u p l i n g o f D i n i t r o a n i l i n e i n D i s t i l l e d Water  9  a)  General  9  b)  S u l p h i t e Concentration  c)  Naphthylethylenediamine  d)  Time and C o n d i t i o n s o f D i a z o t i s a t i o n  11  e)  A c i d Concentration  13  f)  Summary  15  II.  Formation  Procedures  9 Concentration  of Dinitroaniline i n D i s t i l l e d  10  Water  17  a)  Amount o f FDNB  19  b)  pH  21  c)  Temperature  21  d)  R e l a t i o n s h i p between t h e Y i e l d o f D i n i t r o a n i l i n e and Ammonia C o n c e n t r a t i o n  21  III.  A p p l i c a t i o n t o Sea Water  25  DISCUSSION  29  CONCLUSION  34  REFERENCES  36  LIST OF TABLES  Table  I  II  Page  The r a t e o f f o r m a t i o n and s t a b i l i t y o f the diazonium s a l t o f 2 : 4 - d i n i t r o a n i l i n e  13  Comparison o f the s e n s i t i v i t y o f methods t o the a n a l y s i s o f ammonia i n s e a water.  29  applied  LIST OF FIGURES  The s p e c t r a o f t h e d i a z o - d y e formed from n a p h t h y l e t h y l e n e d i a m i n e and d i n i t r o a n i l i n e ( s o l i d l i n e ) and the compound produced by the r e a c t i o n o f n i t r i t e w i t h n a p h t h y l e t h y l e n e d i a m i n e (dashed l i n e )  The v a r i a t i o n i n t h e absorbance o f the d i a z o - d y e w i t h naphthylethylenediamine concentration. Dinitroaniline c o n c e n t r a t i o n , 9.11 micromoles/1.  The time dependence o f t h e absorbance o f t h e d i a z o - d y e formed i n s o l u t i o n s o f v a r y i n g s u l p h u r i c a c i d concentration. D i n i t r o a n i l i n e c o n c e n t r a t i o n , 9.11 micromoles/1.  The r e l a t i o n s h i p between the absorbance o f t h e d i a z o - d y e formed i n d i s t i l l e d water and the d i n i t r o a n i l i n e concentration.  The e f f e c t o f the amount o f FDNB on the pH and y i e l d o f d i n i t r o a n i l i n e a f t e r 24 hours. Ammonia c o n c e n t r a t i o n , 17.3 micromoles/1.  Dependence o f the y i e l d o f d i n i t r o a n i l i n e i n d i s t i l l e d water a f t e r 24 hours on the i n i t i a l pH. Ammonia c o n c e n t r a t i o n , 17.3 micromoles/1.  The r e l a t i o n s h i p between t h e absorbance o f the d i a z o - d y e formed i n d i s t i l l e d water and the ammonia c o n c e n t r a t i o n .  The time dependence o f the absorbance o f t h e d i a z o - d y e i n d i s t i l l e d water and s e a water ( s a l i n i t y , 3 0 . 4 % o ) . Dinitroa n i l i n e c o n c e n t r a t i o n , 9.11 micromoles/1.  The r e l a t i o n s h i p between the absorbance o f t h e d i a z o - d y e formed i n sea water ( s a l i n i t y , 30.4%«) and t h e d i n i t r o a n i l i n e concentration.  ACKNOWLEDGMENTS  I wish t o thank my s u p e r v i s o r , Dr. E. V. G r i l l ,  for his  a d v i c e and c o - o p e r a t i o n .  I am i n d e b t e d t o t h e I n s t i t u t e o f Oceanography f o r f i n a n c i a l support  throughout t h e course o f my s t u d i e s .  INTRODUCTION  The purpose  o f t h i s r e s e a r c h was t o i n v e s t i g a t e t h e use o f  1 - f l u o r o , 2 : 4 - d i n i t r o b e n z e n e as an a n a l y t i c a l reagent f o r ammonia.  The  method s t u d i e d was t h e c o n v e r s i o n o f f l u o r o d i n i t r o b e n z e n e t o d i n i t r o a n i l i n e and the subsequent  c o n v e r s i o n o f t h i s substance to a d i a z o - d y e .  Of  p r i m a r y i n t e r e s t was the d e t e r m i n a t i o n o f ammonia a t t h e micromolar c o n c e n t r a t i o n l e v e l s found i n n a t u r a l waters, p a r t i c u l a r l y s e a water where the ammonia c o n c e n t r a t i o n o r d i n a r i l y is"between 0 and 3 m i c r o m o l e s / l i t r e . The N e s s l e r method commonly used f o r t h e e s t i m a t i o n o f ammonia i n water i s n o t , g e n e r a l l y , d i r e c t l y a p p l i c a b l e t o s e a water a n a l y s i s . A l t h o u g h t h e i n t e r f e r e n c e due t o t h e magnesium i o n can be a v o i d e d by p r e c i p i t a t i o n o r complexing,  t h e method l a c k s p r e c i s i o n because  c o l l o i d a l n a t u r e and i n s t a b i l i t y o f t h e c o l o u r e d p r o d u c t 1933).  o f the  (Wirth and Robinson,  The methods developed f o r t h e a n a l y s i s o f s e a water may be d i f f e r -  e n t i a t e d i n t o two c l a s s e s . sample by d i s t i l l a t i o n diffusion  In t h e f i r s t ,  ammonia i s ' s e p a r a t e d from the  (Krogh, 1934; R i l e y ,  ( R i l e y and S i n h a s e n i , 1957),  determined by a c o l o r i m e t r i c t e c h n i q u e .  1953; G i l l b r i c h t ,  1963) o r  c a p t u r e d * i n d i l u t e a c i d and f i n a l l y D i s t i l l a t i o n or d i f f u s i o n  circumvents  the i n t e r f e r e n c e s due t o o t h e r n o n - v o l a t i l e substances p r e s e n t i n s e a water and p r o v i d e s a method t h a t , because  o f the p o s s i b i l i t y o f concentrating  t h e ammonia, i s g e n e r a l l y more s e n s i t i v e than d i r e c t t e c h n i q u e s t h a t do not r e q u i r e p r e l i m i n a r y s e p a r a t i o n o f ammonia from t h e sample.  Unfortunately,  such procedures a r e time consuming and r e q u i r e equipment not W e l l t o r o u t i n e use on board  adapted  ship.  The second c a t e g o r y c o n s i s t s o f c o l o r i m e t r i c t e c h n i q u e s r u n d i r e c t l y on t h e raw s e a water samples.  The common i n d o p h e n o l - b l u e t e s t f o r ammonia  2.  ( R i l e y , 1953; R i l e y and S i n h a s e n i , 1957) i s n o t d i r e c t l y a p p l i c a b l e t o sea water a n a l y s i s due t o the. p r e c i p i t a t i o n o f magnesium and c a l c i u m i n the a l k a l i n e pH range  a t which the r e a c t i o n o c c u r s .  Newell  and D a l Pont  (1964)  a v o i d e d t h i s i n t e r f e r e n c e by c o n v e r t i n g t h e ammonia t o quinone c h l o r i m i d e , e x t r a c t i n g w i t h hexanol the o r g a n i c s o l v e n t .  and completing t h e f o r m a t i o n o f t h e indophenol i n  In an a l t e r n a t e method d e v i s e d by Roskam and de Langen  (1964), magnesium and c a l c i u m p r e c i p i t a t i o n was a v o i d e d by use o f a c h e l a t i n g agent.  By s u b s t i t u t i n g thymol  f o r p h e n o l , a more s t a b l e and more s e n s i t i v e  dye was formed than t h a t o b t a i n e d i n the i n d o p h e n o l - b l u e method o f R i l e y and S i n h a s e n i (1957).  The p y r i d i n e - p y r a z o l o n e method o f Kruse and M e l l o n  (1953) has been m o d i f i e d f o r s e a water a n a l y s i s by A t k i n s (1957) and S t r i c k l a n d and A u s t i n (1959). procedure  R i c h a r d s and K l e t s c h (1964) d e s c r i b e d a  i n which ammonia'is-oxidised*to  s u b s e q u e n t l y assayed as a d i a z o - d y e .  nitrite  and t h e n i t r i t e i s  Although r e p o r t e d t o have a p r e c i s i o n  as good as o r b e t t e r than d i s t i l l a t i o n p r o c e d u r e s , t h e method i s s u b j e c t to  i n t e r f e r e n c e s s i n c e t h e amino n i t r o g e n o f a number o f amino a c i d s was  shown t o a l s o undergo o x i d a t i o n t o n i t r i t e . 1-fluoro, 2:4-dinitrobenzehe Sanger (1945) f o r t h e purpose Its  (FDNB) was f i r s t  employed by  o f i d e n t i f y i n g t h e t e r m i n a l groups o f p r o t e i n s .  use has s i n c e been extended  t o the a n a l y s i s o f various nitrogenous  compounds such as amino a c i d s (Levy, 1954; Rapp, 1963). and amines ( L o c k h a r t , 1956).  Palmork  (1962) used FDNB t o i s o l a t e and i d e n t i f y microgram  q u a n t i t i e s o f amino a c i d s from s e a water. Levy  (1954) and Lockhart  ammonia a t about pH 9.  (1956) r e p o r t e d t h a t FDNB r e a c t s w i t h  By d e t e r m i n i n g t h e d i n i t r o a n i l i n e formed by a b s o r p t i o n  3.  spectrophotometry f o l l o w i n g Bradbury  i t s i s o l a t i o n by paper chromatography,  (1960) s u c c e s s f u l l y employed t h i s r e a c t i o n t o e s t i m a t e  amounts o f ammonia.  small  Such a p r o c e d u r e , however, would appear t o be t o o  cumbersome f o r r o u t i n e  a n a l y t i c a l work.  d i n i t r o a n i l i n e t o an i n t e n s e l y c o l o u r e d  The p o s s i b i l i t y o f c o n v e r t i n g d i a z o - d y e suggested an a l t e r n a t i v e  procedure; one by which the r e a c t i o n between FDNB and ammonia might be made t h e b a s i s  o f a s e n s i t i v e and i n t e r f e r e n c e  f r e e t e s t f o r ammonia.. I t  was hoped t h a t such a t e s t would prove a p p l i c a b l e t o s e a water a n a l y s i s .  4.  APPARATUS AND  Absorbance  REAGENTS  measurements were made w i t h a Beckman model DU  spectrophotometer, u s i n g a s l i t width o f 0.01 c e l l s o f e i t h e r 1 cm.  o r 5 cm.  Ammonia f r e e water  and matched q u a r t z  o p t i c a l path length.  (afterwards r e f e r r e d t o as d i s t i l l e d water)  o b t a i n e d by p a s s i n g d i s t i l l e d water'through column and f i n a l l y  mm  a mixed bed i o n exchange r e s i n  through Dowex 50 immediately b e f o r e use.  Standard d i n i t r o a n i l i n e s o l u t i o n was of 2:4-dinitroaniline  p r e p a r e d by d i s s o l v i n g 0.020 gm  i n d i s t i l l e d water c o n t a i n i n g 100 ml o f c o n c e n t r a t e d  sulphuric  a c i d and a d j u s t i n g the volume t o 1 l i t r e .  a working  s o l u t i o n c o n t a i n i n g 9.11  micromoles  Naphthylethylenediamine s o l u t i o n s a s t o c k s o l u t i o n c o n t a i n i n g 0.30  dinitroaniline /  weeks (Bendschneider and Robinson,  The s t o c k s o l u t i o n was over p e r i o d s o f  stored  several  1952).  Standard ammonia s o l u t i o n was  p r e p a r e d by d i s s o l v i n g 0.114  o f ammonium s u l p h a t e i n 1 l i t r e o f d i s t i l l e d water.  each two  litre.  were p r e p a r e d by d i l u t i n g  i n an amber g l a s s b o t t l e i n which i t i s s t a b l e  micromoles  T e n - f o l d d i l u t i o n gave  gm o f N - ( l - n a p h t h y l ) e t h y l e n e d i a m i n e  d i h y d r o c h l o r i d e i n 100 ml d i s t i l l e d water.  c o n t a i n e d 1.73  was  ammonia/ml.  The r e s u l t a n t  T h i s s o l u t i o n was  made up  gm solution  freshly  days. S a t u r a t e d sodium b o r a t e s o l u t i o n was  and d i s t i l l e d water and was might have been p r e s e n t .  p r e p a r e d from sodium b o r a t e  b o i l e d p r i o r t o use t o e x p e l any ammonia t h a t  5.  Sodium n i t r i t e s o l u t i o n was sodium n i t r i t e i n 100 ml d i s t i l l e d  p r e p a r e d by d i s s o l v i n g 1 gm  water.  1 - f l u o r o , 2 : 4 - d i n i t r o b e n z e n e was liquid  (m.p.  25-27°C).  of  added t o the samples as the pure  EXPERIMENTAL  The method o f a n a l y s i s r e l i e s on two s e p a r a t e the c o n v e r s i o n  steps;  firstly,  o f ammonia t o 2 : 4 - d i n i t r o a n i l i n e and, s e c o n d l y , t h e  q u a n t i t a t i v e e s t i m a t i o n o f d i n i t r o a n i l i n e by t h e development o f a d i a z o dye. to  Before  i n v e s t i g a t i o n o f the f i r s t  step c o u l d p r o c e e d , i t was  necessary  develop adequate methods f o r c a r r y i n g out t h e second s t e p . The  c o u p l i n g agent s e l e c t e d was  N-(1-naphthyl)ethylenediamine.  T h i s reagent  had p r e v i o u s l y been employed as a c o u p l i n g agent by Bendschneider  and Robinson  (1952) and R i c h a r d s  and K l e t s c h  (1964) and was  considered  s u i t a b l e f o r t h i s work s i n c e i t forms i n t e n s e l y c o l o u r e d dyes and couples under t h e s t r o n g l y a c i d i c c o n d i t i o n s r e q u i r e d t o d i a z o t i s e d i n i t r o a n a l i n e . P h e n o l i c agents, which couple under m i l d l y a l k a l i n e c o n d i t i o n s , were found to  be l e s s s a t i s f a c t o r y because o f t h e d i f f i c u l t y o f n e u t r a l i z i n g t h e  initially dye  s t r o n g l y a c i d i c d i a z o t i s a t i o n mixture t o a r e p r o d u c i b l e pH.  formed from d i n i t r o a n i l i n e and n a p h t h y l e t h y l e n e d i a m i n e  The  i s rose-red,  is  s t a b l e under s t r o n g l y a c i d i c c o n d i t i o n s and has an a b s o r p t i o n  at  525 my which i s s u f f i c i e n t l y i n t e n s e t o r e a d i l y d i f f e r e n t i a t e between  micromolar c o n c e n t r a t i o n s  of dinitroaniline  To o b t a i n q u a n t i t a t i v e c o n v e r s i o n diazonium s a l t  maximum  (Fig. 1). of dinitroaniline to i t s  and ensure t h a t t h e y i e l d i s independent o f t h e n i t r i t e  c o n c e n t r a t i o n , d i a z o t i s a t i o n must be c a r r i e d out i n the p r e s e n c e o f of n i t r i t e .  N i t r i t e , however, i n t e r f e r e s d u r i n g t h e subsequent  r e a c t i o n since naphthylethylenediamine but  also with n i t r i t e  (Benschneider  an excess  coupling  not o n l y r e a c t s w i t h diazonium  and Robinson, 1952) forming,  salts,  i n the  l a t t e r case, a mauve c o l o u r e d m a t e r i a l w i t h an a b s o r p t i o n peak a t 565 my ( F i g . I t was found t h a t sodium s u l p h i t e reduced t h i s mauve product  t o a near-  c o l o u r l e s s m a t e r i a l w i t h no a p p r e c i a b l e absorbance a t 525 my and t h a t t h e  7.  0.400 _  0  1  400  500  600  WAVELENGTH, Millimicrons Fig. 1. The spectra of the diazo-dye formed from naphthylethylenediamine and d i n i t r o a n i l i n e (solid line) and the compound produced by the reaction o f n i t r i t e with naphthylethylenediamine (dashed l i n e ) .  8.  s u l p h i t e c o u l d be added i n s o l u t i o n a l o n g w i t h without i n t e r f e r i n g i n the f o r m a t i o n o f the  naphthylethylenediamine  dizao-dye.  Before attempting t o a p p l y t h i s procedure  t o sea water, the  optimum c o n d i t i o n s f o r c a r r y i n g out the r e a c t i o n s i n d i s t i l l e d water were studied.  9.  I.  D i a z o t i s a t i o n and (a)  Coupling of D i n i t r o a n i l i n e i n D i s t i l l e d  G e n e r a l Procedures The  optimum c o n d i t i o n s  determined u s i n g Sulphuric  f o r d i a z o t i s a t i o n and  a standard s o l u t i o n containing  to 10 ml  d i s t i l l e d water.  The  e f f e c t e d by by the  the  the  the  The the by  a d d i t i o n o f 1 ml  acid.  and  o f d i s t i l l e d water and  and  d e t h y l e n e d i a m i n e and  by  1 ml  Diazotisation and  containing  sodium s u l p h i t e .  the  preventing interference  coupling  adding 5 ml  agent was  varying  0.01%  amounts o f sodium s u l p h i t e . pipet-te.  The  from  determined  of concentrated  of a s o l u t i o n containing  added by means o f a s y r i n g e  a d d i t i o n by  with  was  sulphuric  A f t e r c o o l i n g t o about 10-15°C, 2 drops o f 1% sodium n i t r i t e  were added f o l l o w e d  was  was  Concentration  optimum s u l p h i t e c o n c e n t r a t i o n  15 ml  acid  solution  o f an aqueous s o l u t i o n  s i d e r e a c t i o n between n i t r i t e taking  brought t o 20 ml  samples under running tap water.  adding 2 drops o f a 1% sodium n i t r i t e  Sulphite  dinitroaniline.  w i t h d i s t i l l e d water-  a d d i t i o n o f the  N-(l-naphthyl)ethylenediamine dihydrochloride  (b)  were  micromoles/1  f i n a l volume was  heat g e n e r a t e d by  d i s s i p a t e d by h o l d i n g  coupling  o f the s t a n d a r d d i n i t r o a n i l i n e s o l u t i o n i n a g l a s s -  s t o p p e r e d graduated c y l i n d e r and  coupling  9.11  a c i d - e i t h e r c o n c e n t r a t e d or d i l u t e d 1:1  added by p i p e t t e  was  Water  solution  of naphthylThis  second s o l u t i o n  samples were mixed a f t e r each  i n v e r t i n g the g l a s s s t o p p e r e d c y l i n d e r s i n which they were  contained.  0.1%  The  a f f e c t o f sodium s u l p h i t e c o n c e n t r a t i o n s r a n g i n g between  and  5% was  observed.  I t was  found t h a t  a 2% sodium s u l p h i t e  gave a near c o l o u r l e s s b l a n k which faded i n a few  solution  seconds t o y i e l d a  pale  10.  green  s o l u t i o n w i t h no a p p r e c i a b l e absorbance a t 525 my.  s u l p h i t e c o n c e n t r a t i o n s t h e mauve c o l o u r e d product  With  faded l e s s  In a l l cases, t h e mauve m a t e r i a l c o u l d be r e g e n e r a t e d  through  o x i d a t i o n when t h e samples were a g i t a t e d , but t h e remaining bleached  lower rapidly. aerial  s u l p h i t e again  out t h i s c o l o u r when t h e samples were a l l o w e d t o s t a n d .  a 2% s u l p h i t e s o l u t i o n , i t was found t h a t f i l l i n g t h e  spectrophotometer  c e l l s a t l e a s t 10 minutes b e f o r e r e a d i n g s were t o be made ensured removal o f t h e i n t e r f e r e n c e .  Using  complete  H i g h e r s u l p h i t e c o n c e n t r a t i o n s o f f e r e d no  improvement and were, i n any case, u n d e s i r a b l e s i n c e they might endanger the success  o f t h e method by r e d u c t i o n o f t h e diazonium  salt.  The c o u p l i n g  s o l u t i o n was made up f r e s h l y each day from s o l i d sodium s u l p h i t e and a stock s o l u t i o n o f N-(l-naphthyl)ethylenediamine.  c)  Naphthylethylenediamine  The n a p h t h y l e t h y l e n e  Concentration  diamine c o n c e n t r a t i o n n e c e s s a r y  maximum colour, development was determined  t o produce  by t a k i n g 10 ml a l i q u o t s o f  the s t a n d a r d d i n i t r o a n i l i n e s o l u t i o n and making these up t o 20 ml w i t h s u l p h u r i c a c i d d i l u t e d 1:1 w i t h d i s t i l l e d water.  The s o l u t i o n s were c o o l e d  t o about 10-15°C and 2 drops o f a 1% sodium n i t r i t e  s o l u t i o n were added.  A f t e r s t a n d i n g f o r 10 minutes, 1 ml o f a s o l u t i o n c o n t a i n i n g 2% sodium s u l p h i t e and n a p h t h y l e t h y l e n e d i a m i n e  i n c o n c e n t r a t i o n s r a n g i n g from 0.005%  t o 0.08% was admitted by means o f a s y r i n g e p i p e t t e .  Time s e r i e s  observations  e s t a b l i s h e d t h a t t h e dye had o b t a i n e d maximum development a f t e r about 15 minutes and was s t a b l e f o r p e r i o d s o f a t l e a s t an hour. a f f e c t o f v a r y i n g the naphthylethylenediamine  To compare t h e  c o n c e n t r a t i o n s on t h e f i n a l  c o l o u r i n t e n s i t y , r e a d i n g s were taken a g a i n s t a d i s t i l l e d water o p t i c a l  b l a n k a t 525 mu i n a 1 cm c e l l develop f o r 30 minutes. prepared  a f t e r the c o l o u r had been allowed t o  These r e a d i n g s  were compared w i t h reagent  by s u b s t i t u t i n g d i s t i l l e d water f o r t h e d i n i t r o a n i l i n e .  blanks  The  r e s u l t s , d e p i c t e d i n F i g . 2, i n d i c a t e d t h a t maximum c o l o u r i n t e n s i t y be a c h i e v e d when t h e c o u p l i n g s o l u t i o n c o n t a i n e d ethylenediamine  dihydrochloride.  could  0.03% N-(1-naphthyl)  This concentration, i n conjunction  with  a 2% sodium s u l p h i t e c o n c e n t r a t i o n , was employed i n subsequent s t u d i e s .  (d) The  Time and c o n d i t i o n s o f d i a z o t i s a t i o n a f f e c t o f t h e time o f d i a z o t i s a t i o n upon t h e f i n a l  colour  i n t e n s i t y o f t h e diazo-dye was s t u d i e d f o r s o l u t i o n s p r e p a r e d  by t h e  a d d i t i o n o f 10 ml o f 1:1 s u l p h u r i c a c i d t o 10 ml o f t h e 9.11 micromolar dinitroaniline solution.  The samples were c o o l e d t o about 10-15°C and  2 drops o f a 1% sodium n i t r i t e  s o l u t i o n were added.  A f t e r i n t e r v a l s between  2 and 25 minutes, 1 ml o f t h e c o u p l i n g s o l u t i o n was added and t h e absorbance o f t h e dye was measured a t 525 mu i n c e l l s o f 1 cm o p t i c a l p a t h a f t e r t h e c o l o u r had developed f o r 30 minutes.  length  The r e s u l t s , shown i n t h e  t a b l e below, i n d i c a t e d t h a t t h e diazonium s a l t was formed q u i c k l y and was s t a b l e under t h e experimental  c o n d i t i o n s f o r p e r i o d s up t o 20 minutes.  To ensure maximum c o l o u r development, i t was d e c i d e d t o add t h e c o u p l i n g s o l u t i o n 10 minutes a f t e r t h e a d d i t i o n o f the n i t r i t e s o l u t i o n .  I t was  observed t h a t t h e absorbance d i d not i n c r e a s e s i g n i f i c a n t l y i f t h e d i a z o t i s a t i o n and c o u p l i n g were c a r r i e d out a t 0°C.  12.  0.250.  0.02  Fig-  2.-  0.04  0.06  0.08  % NAPHTHYLETHYLENEDIAMINE  The variation i n the absorbance of the diazo-dye with naphthylethylenediamine concentration. Dinitroaniline concentration, 9.11 micromoles/1.  13.  Table I.  The r a t e o f f o r m a t i o n and s t a b i l i t y of  (e)  o f t h e diazonium  2:4-dinitroaniline.  Time o f d i a z o t i s a t i o n (minutes)  Absorbance  2  0.170  5  0.205  8  0.201  10  0.208  12  0.210  15  0.205  20  0.207  25  0.202  Acid The  Concentration stability  o f t h e diazo-dye was found t o be dependent upon  the f i n a l a c i d c o n c e n t r a t i o n o f t h e s o l u t i o n .  T e s t s were conducted by  adding v a r y i n g amounts o f c o n c e n t r a t e d s u l p h u r i c s t a n d a r d d i n i t r o a n i l i n e s o l u t i o n and b r i n g i n g o f 20 ml w i t h d i s t i l l e d water. addition  salt  a c i d t o 10 ml o f t h e  t h e samples t o a f i n a l volume  D i a z o t i s a t i o n was a f f e c t e d by t h e  o f 2 drops o f 1% sodium n i t r i t e s o l u t i o n and t h e c o u p l i n g s o l u t i o n  was added a f t e r 10 minutes.  The development o f t h e diazo-dye was f o l l o w e d  w i t h time by measuring i t s absorbance a t 525 my i n a 1 cm c e l l a d i s t i l l e d water o p t i c a l b l a n k .  versus  The r e s u l t s are shown i n F i g . 3.  l e s s than 3 ml o f c o n c e n t r a t e d s u l p h u r i c  When  a c i d were p r e s e n t , t h e absorbance  o f t h e s o l u t i o n r a p i d l y a c h i e v e d a maximum and then faded.  As t h e a c i d  TIME, Minutes Fig.  3.  The time dependence, o f the absorbance o f the d i a z o - d y e formed i n s o l u t i o n s o f varying sulphuric acid concentration. D i n i t r o a n i l i n e c o n c e n t r a t i o n , 9.11 micromoles/1  15.  c o n c e n t r a t i o n i n c r e a s e d , t h e c o l o u r was formed l e s s r a p i d l y , b u t remained more s t a b l e .  In t h e case where 5 ml. o f a c i d were p r e s e n t , maximum c o l o u r  i n t e n s i t y was o b t a i n e d i n l e s s than 20 minutes whereas w i t h 10 ml o f a c i d maximum development was a c h i e v e d o n l y a f t e r 160 minutes. for a l l  acid concentrations that:  I t was v e r i f i e d  a) reagent b l a n k s i n which d i s t i l l e d  water  was s u b s t i t u t e d f o r t h e d i n i t r o a n i l i n e s o l u t i o n had a n e g l i g i b l e absorbance; b) t h a t d i a z o t i s a t i o n was accomplished w i t h i n a few minutes a t 10-15°C and t h a t diazonium s a l t was s t a b l e f o r p e r i o d s o f up t o 20 minutes.  These r e s u l t s  i n d i c a t e d t h a t a c i d c o n c e n t r a t i o n s a p p r o x i m a t e l y 5 ml o f c o n c e n t r a t e d  sulphuric  a c i d i n a t o t a l volume o f 20 ml would be s u i t a b l e f o r f u r t h e r work p r o v i d e d t h a t t h e absorbance was r e c o r d e d 30 minutes a f t e r t h e a d d i t i o n o f t h e c o u p l i n g agent.  f)  Summary C o n c e n t r a t i o n s o f d i n i t r o a n i l i n e i n t h e range 0-10 micromoles/1  can be e s t i m a t e d by t h e f o l l o w i n g p r o c e d u r e . To 10 ml o f t h e d i n i t r o a n i l i n e s o l u t i o n t o be a n a l y z e d add 10 ml o f 1:1 s u l p h u r i c a c i d and c o o l t o 10-15°C.  Add 2 drops o f a 1% sodium  nitrite  s o l u t i o n and, a f t e r 10 minutes, 1 ml o f a s o l u t i o n c o n t a i n i n g 0.03% N - ( 1 - n a p h t h y l ) e t h y l e n e d i a m i n e d i h y d r o c h l o r i d e and 2% sodium s u l p h i t e .  Measure  the absorbance a t 525 mu i n a 5 cm c e l l a f t e r c o l o u r development has proceeded f o r 30 minutes and compare w i t h a reagent b l a n k . c e l l s s h o u l d be f i l l e d  The spectrophotometer  a t l e a s t 10 minutes b e f o r e r e a d i n g s a r e t o be made.  The s e l e c t i o n o f a 10 minute, i n t e r v a l between t h e a d d i t i o n o f the n i t r i t e and c o u p l i n g s o l u t i o n , r e s p e c t i v e l y , was j u s t i f i e d by t h e  16.  consistency o f the r e s u l t s obtained.  The r e l a t i o n s h i p between the i n t e n s i t y  o f the dye produced and the amount o f d i n i t r o a n i l i n e p r e s e n t was  studied  u s i n g the method o f a n a l y s i s d e s c r i b e d above ( r e c o r d i n g the absorbance i n 5 cm c e l l s ) and was found t o be l i n e a r  (Fig. 4).  F i g . 4. The r e l a t i o n s h i p between the absorbance o f the d i a z o - d y e formed i n d i s t i l l e d water and the d i n i t r o a n i l i n e c o n c e n t r a t i o n .  18.  II.  The Formation o f D i n i t r o a n i l i n e i n D i s t i l l e d Water P r e v i o u s work (Levy, 1954; Palmork,  1962) i n d i c a t e d t h a t FDNB  r e a c t s w i t h n i t r o g e n o u s compounds under m i l d l y a l k a l i n e c o n d i t i o n s  (pH 8-10).  In  t h e f o l l o w i n g experiments t e s t samples were made up by d i l u t i n g  1 ml  of  s t a n d a r d ammonia s o l u t i o n t o 100 ml w i t h d i s t i l l e d water g i v i n g a  s o l u t i o n 17.3 micromolar i n ammonia, and adding 2 ml o f s a t u r a t e d  sodium  b o r a t e s o l u t i o n t o b u f f e r t h e s o l u t i o n t o pH 9.3. FDNB has g e n e r a l l y been employed by the a d d i t i o n o f e i t h e r an acetone o r e t h a n o l i c s o l u t i o n  (Sanger, 1945; Bradbury, 1960).  p r a c t i c e was f o l l o w e d i n the i n i t i a l  This  experiments, adding 1 ml o f a 2%  s o l u t i o n o f FDNB i n acetone o r e t h a n b l t o 100 ml o f t e s t sample in  t i g h t l y stoppered c l e a r o f l i g h t - t i g h t glass b o t t l e s .  contained  The samples were  a g i t a t e d a t room temperature e i t h e r by a magnetic s t i r r e r o r a B u r r e l w r i s t a c t i o n shaker and 10 ml a l i q u o t s were withdrawn dinitroaniline analysis.  a t v a r i o u s times f o r  Using the p r e v i o u s l y described procedure,  no d i n i t r o a n i l i n e c o u l d be d e t e c t e d , even a f t e r t h e experiments had been a l l o w e d t o p r o c e e d f o r as long as 48 hours. i n i t i a l pH o f t h e sample of  F u r t h e r experiments i n which t h e  c o n t a i n i n g b o r a t e was a d j u s t e d over t h e range  7.5 t o 12 by t h e a d d i t i o n o f sodium h y d r o x i d e o r h y d r o c h l o r i c  acid,  r e s p e c t i v e l y , i n d i c a t e d t h a t no d i n i t r o a n i l i n e was formed when t h e FDNB was added i n s o l u t i o n . When, however, pure l i q u i d FDNB was added so t h a t a two phase system was produced, slow c o n v e r s i o n o f ammonia t o d i n i t r o a n i l i n e was obtained.  The two phase system e x i s t e d f o r o n l y about one q u a r t e r o f t h e  time n e c e s s a r y t o o b t a i n maximum development  of dinitroaniline.  By s u b s t i t u t i n g  1 ml o f a 2% aqueous d i n i t r o p h e n o l s o l u t i o n f o r l i q u i d FDNB and c o n d u c t i n g the  experiment under i d e n t i c a l c o n d i t i o n s , no d i n i t r o a n i l i n e was observed  19.  even a f t e r  72 hours i n d i c a t i n g  t o proceed.  As observed i n o t h e r work w i t h t h i s compound, t h e r e a c t i o n  found t o be s e n s i t i v e When c a r r i e d  t h a t FDNB was n e c e s s a r y f o r t h e r e a c t i o n  to light  (Peraino and Harper, 1961; P a t a k i ,  out i n amber g l a s s b o t t l e s ,  glass At  per  1964).  conversion to d i n i t r o a n i l i n e  o c c u r r e d ; no d i n i t r o a n i l i n e was formed when t h e r e a c t i o n clear  was  was conducted i n  bottles. room temperature, w i t h a sample c o n t a i n i n g 0.1 ml o f FDNB  100 ml o f t e s t s o l u t i o n  and pH a d j u s t e d t o 9.3 by t h e a d d i t i o n o f  2 ml s a t u r a t e d sodium b o r a t e , maximum c o n v e r s i o n was a c h i e v e d a f t e r a p e r i o d o f 32 hours.  Based on t h e absorbance o f s o l u t i o n s  o f known  d i n i t r o a n i l i n e content, 55% o f t h e ammonia o r i g i n a l l y p r e s e n t had been converted to d i n i t r o a n i l i n e .  I t was c o n s i s t e n t l y  found t h a t 45% c o n v e r s i o n  was o b t a i n e d a f t e r 24 hours.  a)  Amount o f FDNB  The  affect  o f v a r y i n g t h e FDNB content on t h e y i e l d o f d i n i t r o a n i l i n e  a f t e r 24 hours a t pH 9.3 was s t u d i e d .  As shown by F i g . 5, t h e amount o f  d i n i t r o a n i l i n e formed i n c r e a s e d i n a p p r o x i m a t e l y l i n e a r p r o p o r t i o n t o t h e FDNB up t o a maximum o f 0.1 ml FDNB/100 ml sample. the  amount o f FDNB r e s u l t e d  addition 45%  Further increases i n  i n a decreased y i e l d o f d i n i t r o a n i l i n e .  o f 0.1 ml FDNB t o 100 ml o f t h e t e s t s o l u t i o n  conversion a f t e r  f o r subsequent t e s t s . test solution  consistently  The gave  24 hours, and t h u s , t h i s volume o f t h e reagent was used Further observations indicated  decreased as t h e r e a c t i o n  h y d r o l y s i s o f FDNB t o d i n i t r o p h e n o l .  t h a t t h e pH o f the  proceeded, presumably due t o t h e  T e s t s were c a r r i e d out t o determine t h e  a f f e c t o f adding more sodium b o r a t e i n an attempt t o m a i n t a i n t h e pH o f t h e test not  solution. affected  I t was observed t h a t t h e amount o f d i n i t r o a n i l i n e formed was  by i n c r e a s i n g  t h e amount o f b o r a t e .  20. co  cc o  ml FDNB/100 ml sample Fig- 5. The e f f e c t o f the amount of FDNB on the pH and y i e l d o f d i n i t r o a n i l i n e after 24 hours. Ammonia concentration, 17.3 micromoles/1.  21.  b)  pH  T e s t s were conducted t o observe t h e a f f e c t o f pH on t h e r e a c t i o n . 2 ml o f s a t u r a t e d sodium b o r a t e s o l u t i o n were added t o 100 ml o f d i s t i l l e d water and t h e pH o f t h e s o l u t i o n was a d j u s t e d by t h e a d d i t i o n o f h y d r o c h l o r i c a c i d o r sodium h y d r o x i d e , r e s p e c t i v e l y .  0.1 ml o f FDNB and 1 ml o f t h e  s t a n d a r d ammonia s o l u t i o n were added and t h e r e a c t i o n was a l l o w e d t o p r o c e e d for  24 hours w i t h s h a k i n g .  A f t e r t h i s p e r i o d 10 ml samples  and a n a l y z e d f o r d i n i t r o a n i l i n e .  were withdrawn  I n each case a reagent b l a n k was r u n .  As shown i n F i g . 6, below pH 8, t h e amount o f c o n v e r s i o n was i n s i g n i f i c a n t ; above pH 8, t h e y i e l d i n c r e a s e d w i t h pH u n t i l pH 9.5. the  a maximum was reached a t about  F u r t h e r i n c r e a s e i n pH d i d n o t improve  the y i e l d .  By a l l o w i n g  r e a c t i o n t o go t o c o m p l e t i o n , i t was observed t h a t 55% c o n v e r s i o n o f  ammonia t o d i n i t r o a n i l i n e was a c h i e v e d f o r a l l samples pH g r e a t e r than 8.0.  w i t h an i n i t i a l  A t pH 10 maximum c o n v e r s i o n was o b t a i n e d a f t e r 28 hours,  4 hours q u i c k e r than a t pH 9.3. c)  Temperature  I n c r e a s i n g t h e temperature produced a marked a c c e l e r a t i o n i n t h e rate o f formation o f d i n i t r o a n i l i n e .  T e s t s were conducted by s h a k i n g t h e  r e a c t i o n v e s s e l s i n a t h e r m o s t a t i c a l l y c o n t r o l l e d water b a t h .  At higher  temperatures t h e same maximum percentage c o n v e r s i o n , 55%, as a t room temperatures was c o n s i s t e n t l y o b t a i n e d . reached c o m p l e t i o n a f t e r about  A t 50°C and pH 9.3 t h e r e a c t i o n  8 hours; a t pH 10 t h e p e r i o d n e c e s s a r y t o  a c h i e v e maximum c o n v e r s i o n was reduced t o l e s s than 3 h o u r s . d)  R e l a t i o n s h i p between t h e Y i e l d o f D i n i t r o a n i l i n e and t h e Ammonia C o n c e n t r a t i o n .  A s e r i e s o f s t a n d a r d ammonia s o l u t i o n s was p r e p a r e d i n d i s t i l l e d  22.  60  50  40  2  O  h-l C/J OS  to o u  30  20  10  INITIAL pH F i g . 6. The dependence o f the y i e l d o f d i n i t r o a n i l i n e i n d i s t i l l e d a f t e r 24 hours on the i n i t i a l pH. Ammonia c o n c e n t r a t i o n , 17.3 micromoles/1.  water  23.  water and  100 ml  sodium b o r a t e  and  samples were t r e a t e d at 50°C w i t h 0.1  ml FDNB.  A f t e r 10 hours, 10 ml a l i q u o t s were  withdrawn f o r d i n i t r o a n i l i n e a n a l y s i s . A b s o r b a n c i e s were measured i n 5 cm  The  r e s u l t s are d e p i c t e d i n F i g . 7.  c e l l s at 525 my.  r e l a t i o n s h i p between c o n c e n t r a t i o n and y i e l d o f d i n i t r o a n i l i n e was  2 ml o f s a t u r a t e d  The  absorbance i n d i c a t e d t h a t  the  independent o f ammonia c o n c e n t r a t i o n f o r s o l u t i o n s  up t o at l e a s t 12 micromolar i n ammonia c o n c e n t r a t i o n and, q u a n t i t i e s o f ammonia can be d e t e c t e d and Moreover, the method appeared t o be t o sea water.  linear  thus, t h a t  small  d i f f e r e n t i a t e d by t h i s p r o c e d u r e .  sufficiently sensitive for application  24. 1.000  F i g . 7. The r e l a t i o n s h i p between the absorbance o f the d i a z o - d y e i n d i s t i l l e d water and the ammonia c o n c e n t r a t i o n .  25.  III.  A p p l i c a t i o n t o Sea Water T e s t s were conducted t o determine t h e a p p l i c a b i l i t y o f the  procedures developed t o s e a water systems.  In o r d e r t o i n v e s t i g a t e t h e  e f f e c t o f s e a water upon t h e d i a z o t i s a t i o n and c o u p l i n g s t e p s , s t o c k  dinitro-  a n i l i n e s o l u t i o n was d i l u t e d t e n - f o l d w i t h P a c i f i c Ocean water o f s a l i n i t y 33.6%o  which had been passed through a 0.45 y M i l l i p o r e f i l t e r .  The r e s u l t a n t  s o l u t i o n had a d i n i t r o a n i l i n e c o n c e n t r a t i o n o f 9.11 micromole/1 and a s a l i n i t y of 30.4%» .  Blanks were p r e p a r e d  dinitroaniline solution. o f these  solutions.  by s u b s t i t u t i n g d i s t i l l e d water f o r t h e  10 ml o f 1:1 s u l p h u r i c a c i d were added t o 10 ml  Tests with the blank i n d i c a t e d that the colour r e s u l t i n g  from t h e r e a c t i o n o f t h e n a p h t h y l e t h y l e n e d i a m i n e  w i t h n i t r i t e was d i s s i p a t e d  much more s l o w l y i n s e a water than i n d i s t i l l e d water.  T h i s c o l o u r was  d i s p e r s e d w i t h i n 35 minutes u s i n g a 2% s u l p h i t e s o l u t i o n and w i t h i n 20 minutes when t h e s u l p h i t e c o n c e n t r a t i o n was i n c r e a s e d t o 6%.  The absorbance  o f t h e b l a n k a t 525 my was s m a l l and c o n s i s t e n t when t h e c o u p l i n g s o l u t i o n contained  6% s u l p h i t e and t h e spectrophotometer c e l l s were f i l l e d  before reading.  25 minutes  D i z a o t i s a t i o n o f t h e d i n i t r o a n i l i n e o c c u r r e d q u i c k l y i n water  o f t h i s s a l i n i t y and t h e diazonium s a l t was found t o be s t a b l e over a p e r i o d s i m i l a r to-•.that i n d i s t i l l e d water.  S a t i s f a c t o r y r e s u l t s were o b t a i n e d by  adding 1 ml o f a s o l u t i o n c o n t a i n i n g 0.03% n a p h t h y l e t h y l e n e d i a m i n e  and 6%  sodium s u l p h i t e 10 minutes a f t e r t h e a d d i t i o n o f 2 drops o f 1% sodium n i t r i t e t o samples c o o l e d t o about 10-15°C.  The absorbance o f t h e dye was  r e a d a t 525 my a f t e r t h e c o l o u r had developed f o r 30 minutes.  Parallel  t e s t s conducted i n d i s t i l l e d water i n d i c a t e d t h a t c o u p l i n g was n o t a f f e c t e d by the i n c r e a s e i n s u l p h i t e c o n c e n t r a t i o n , b u t t h e p r e s e n c e o f s e a water s a l t s c o n s i d e r a b l y reduced t h e c o l o u r i n t e n s i t y o f t h e diazo-dye  (Fig. 8).  26.  0.250-  5 c  -O—H3  0.'200.  O — O  G  O  O — - O  O  CD—  O  •-' D i s t i l l e d  Water  Q  - Sea Water (30.4 %o s a l i n i t y )  ex. rt o  0.150_  o  LO CN LO  0.100J -©  -Q  &-  w CJ  < cc: o  c<a  CO  0.050.  —r 10  T"  20  30  40  50  60  TIME, M i n u t e s F i g - 8. The t i m e d e p e n d e n c e o f t h e a b s o r b a n c e o f t h e d i a z o - d y e i n d i s t i l l e d w a t e r and s e a w a t e r ( s a l i n i t y , 3 0 . 4 % ) . D i n i t r o a n i l i n e c o n c e n t r a t i o n , 9.11 m i c r o m o l e s / 1 . o  i  The absorbance o f t h e sample was  not i n c r e a s e d e i t h e r by i n c r e a s i n g t h e  p r o p o r t i o n o f s u l p h u r i c a c i d i n the sample o r by t h e amount o f n a p h t h y l e t h y l e n e d i a m i n e i n the c o u p l i n g  solution.  A l t h o u g h the c o l o u r i n t e n s i t y o f the dye i n sea water o f s a l i n i t y 30.4%o  was  reduced t o o n l y about 42% o f t h a t i n d i s t i l l e d water,  sensible  r e s u l t s c o u l d s t i l l be o b t a i n e d i n sea water systems by u s i n g o p t i c a l o f 10 cm p a t h l e n g t h .  A s e r i e s o f d i n i t r o a n i l i n e s o l u t i o n s i n the  c o n c e n t r a t i o n , range 0-rl7 micromoles/1 was have a f i n a l  cells  s a l i n i t y o f 30.4%o .  p r e p a r e d i n sea water so as t o  When a n a l y z e d as d e s c r i b e d above,  s c a t t e r o c c u r r e d about the b e s t s t r a i g h t l i n e through the p o i n t s  considerable  ( F i g . 9)  s u g g e s t i n g t h a t , under t h e c o n d i t i o n s used, t h e procedure would not be r e l i a b l e i n s e a water.  Reductions i n the amount o f n i t r i t e i n the d i a z o t i s a t i o n  s t e p and t h e a d d i t i o n o f s u l p h i t e a f t e r t h e c o u p l i n g agent d i d not r e s o l v e these  difficulties. T e n t a t i v e r e s u l t s were o b t a i n e d c o n c e r n i n g t h e c o n v e r s i o n o f  FDNB t o d i n i t r o a n i l i n e i n sea water o f s a l i n i t y 3 0 . 4 % t o which 17.3 micromoles/1 ammonia had been added was FDNB.  The s o l u t i o n was  24 hours.  . 100 ml o f s e a water t r e a t e d w i t h 0.1 ml o f  b u f f e r e d by the a d d i t i o n o f 2 ml o f a s a t u r a t e d  sodium b o r a t e s o l u t i o n and sodium h y d r o x i d e . was more r a p i d a t pH  0  At room temperature c o n v e r s i o n  10 than a t pH 9 and appeared t o r e a c h c o m p l e t i o n a f t e r  By comparing the absorbance c o r r e c t e d f o r a sea water b l a n k w i t h  t h a t o f a s o l u t i o n 17.3 micromolar i n d i n i t r o a n i l i n e i n water o f the same salinity,  i t was  found t h a t t h e r e was  ammonia t o d i n i t r o a n i l i n e .  a p p r o x i m a t e l y 58% c o n v e r s i o n o f  28.  0.700  0.600  0.500 _  0.400  0.300  0.200 -  O  r e p r e s e n t the r e s u l t s  0 • from t h r e e + • d i f f e r e n t runs  0.100  i  4  10  MICROMOLES DINITROANILINE/LITRE F i g . 9. The r e l a t i o n s h i p between the absorbance o f the diazo-dye formed i n s e a water ( s a l i n i t y , 30.4% ) and t h e d i n i t r o a n i l i n e c o n c e n t r a t i o  29.  DISCUSSION The s e n s i t i v i t y o f a s p e c t r o p h o t o m e t r y method o f a n a l y s i s i s d e f i n e d by S a n d e l l  (1950) as:  " . . . t h e number o f micrograms o f t h e element, c o n v e r t e d t o t h e c o l o u r e d p r o d u c t , which i n a column o f s o l u t i o n h a v i n g a c r o s s s e c t i o n o f one square c e n t i m e t r e shows an e x t i n c t i o n o f 0.001." The s e n s i t i v i t y o f t h e method under i n v e s t i g a t i o n was  compared  with other techniques devised f o r the estimation o f small q u a n t i t i e s o f ammonia i n aqueous s o l u t i o n s i n t h e t a b l e below.  Table I I .  Comparison o f t h e s e n s i t i v i t y o f methods a p p l i e d to  t h e a n a l y s i s o f ammonia i n s e a water.  Method  S e n s i t i v i t y x 10  FDNB  -  d i s t i l l e d water  1.04  FDNB  -  s e a water  2.48  R i c h a r d s and K l e t s c h  (1964)  0.504  Roskam and de Langen  (1964)  1.3  Newell and D a l Pont  (1964)  -  d i s t i l l e d water  0.321  Newell and Dal. Pont  (1964)  -  s e a water  0.512  R i l e y and S i n h a s e n i  (1957)  2.91  A l t h o u g h n o t as s e n s i t i v e as the methods o f R i c h a r d s and K l e t s c h (1964), Newell and D a l Pont (1964) and Roskam and de Langen  (1964), t h e FDNB  t e c h n i q u e , because o f i t s s i m p l i c i t y , would have a p p l i c a t i o n t o s e a water a n a l y s i s i f the p r e c i s i o n o f the d i n i t r o a n i l i n e d e t e r m i n a t i o n c o u l d be improved.  The p r o c e d u r e o f Newell and D a l Pont (1964), a l t h o u g h v e r y  30.  s e n s i t i v e , appears t o be too e l a b o r a t e  f o r routine shipboard  use.  The  methods d e v i s e d by Roskam and de Langen (1964) and R i c h a r d s and K l e t s c h require strongly basic conditions ammonia.  R i c h a r d s and K l e t s c h  (1964)  - pH g r e a t e r than 11- t o o x i d i s e the  (1964), by running comparisons o f t h i s  method on n a t u r a l sea water samples a g a i n s t the d i s t i l l a t i o n d e v i s e d by R i l e y (1953), showed t h a t under these c o n d i t i o n s  method there  was  a s i g n i f i c a n t e r r o r i n the ammonia e s t i m a t e d due, presumably, t o the d e g r a d a t i o n o f amino a c i d s and amines.  Further,  i n the Roskam and  de Langen (1964) method c a r e f u l p r e p a r a t i o n  o f the sample i s r e q u i r e d  i n order  ions present  t h a t a l l t h e magnesium  chelated with cyclo-hexyl  and c a l c i u m  t r a n s 1:2 d i a m i n e t e t r a a c e t i c  should  acid.  s l i g h t p r e c i p i t a t i o n i s a l l o w e d t o o c c u r , low r e s u l t s are The p o s s i b i l i t y o f i n t e r f e r e n c e from n i t r o g e n  be  I f even  obtained.  containing  compounds  such as amino a c i d s and amines on the FDNB procedure f o r ammonia  determination  was not s p e c i f i c a l l y i n v e s t i g a t e d .  these  I t i s u n l i k e l y , however, t h a t  would s i g n i f i c a n t l y c o n t r i b u t e t o the amount o f d i n i t r o a n i l i n e Levy (1954) and Lockhart y i e l d s were o b t a i n e d suggesting  appreciable and  quantitative  w i t h p r i m a r y and secondary amines and amino  that l i t t l e  t h i s source.  (1956) i n d i c a t e d t h a t v i r t u a l l y  recovered.  acids  c o n t r i b u t i o n o f d i n i t r o a n i l i n e would be l i k e l y from  Sanger (1945) and Bradbury (1960) found t h a t t h e r e was no breakdown o f the d i n i t r o p h e n y l d e r i v a t i v e s o f amino  acids  amines even when r e f l u x e d w i t h 20% h y d r o c h l o r i c a c i d f o r 8 hours;  w i t h h i s t d i n e , however, t h e i m i d a z o l e  group r e a c t e d  s l o w l y w i t h FDNB  so t h a t , under the c o n d i t i o n s  of hydrolysis described  d i n i t r o a n i l i n e was o b t a i n e d .  Under t h e e x p e r i m e n t a l c o n d i t i o n s  t h i s study t h i s i s an u n l i k e l y source o f e r r o r .  above, some 2:4employed i n  The  a d d i t i o n o f sodium s u l p h i t e t o the  diamine used f o r c o u p l i n g from the  appeared t o s u c c e s s f u l l y remove the  s i d e r e a c t i o n between n i t r i t e and  consistent water and  the  coupling  sea water and,  from t h i s s i d e r e a c t i o n .  thus, the  l a c k o f p r e c i s i o n i n the  Low  appear to be  and  distilled  determination  caused by  interference  Furthermore, the r e s u l t s shown i n F i g . 8 i n d i c a t e  The  reason f o r the poor p r e c i s i o n o f the d i n i t r o a n i l i n e  d e t e r m i n a t i o n i n sea water, t h e r e f o r e ,  i s obscure.  sea s a l t s on the r e a c t i o n c o u l d p o s s i b l y be  i n t o a c i d i f i e d d i s t i l l e d water and under c o n d i t i o n s  apparent a f f e c t o f  solvent,  extracting  re-extracting  c o n d u c t i n g the d i a z o t i s a t i o n and  the r e a c t i o n between FDNB and  ammonia, but  found t o i n f l u e n c e the u l t i m a t e  significantly  accelerated  n e i t h e r o f these f a c t o r s  y i e l d o f d i n i t r o a n i l i n e . The  ammonia s o l u t i o n , i s c o n s i d e r e d .  40°C aqueous s o l u t i o n s o f amines gave 100% derivatives within  80 minutes, but  o b t a i n e d 50%  conversion  could only obtain  ammonia t o d i n i t r o a n i l i n e even by h e a t i n g (1956) has  conversion  compares extreme  (1954) found t h a t t o the 25%  conversion  t o d i n i t r o a n i l i n e by Bradbury  at  dinitrophenyl  f o r 7 hours at e l e v a t e d  the r e a c t i o n t o p r o c e e d at 105°C f o r 2 hours and y i e l d s by  Levy  was  yield  w i t h t h a t o b t a i n e d by o t h e r workers e s p e c i a l l y when the  d i l u t i o n o f the  coupling  t h a t were found t o g i v e r e l i a b l e r e s u l t s .  I n c r e a s e s i n both temperature and pH  favourably  The  a v o i d e d , however, by  the d i n i t r o a n i l i n e from sea water i n t o an o r g a n i c  90%  agent.  diazo-dye formed s t a b l e s o l u t i o n s i n sea water as w e l l as i n  d i s t i l l e d water.  Lockhart  interference  a b s o r b a n c i e s were o b t a i n e d f o r reagent b l a n k s i n b o t h  o f d i n i t r o a n i l i n e i n sea water would not  t h a t the  s o l u t i o n of naphthylethylene-  of temperatures.  allowing  (1960) has  reported  e v a p o r a t i n g an ammonium c h l o r i d e s o l u t i o n , adding potassium  b i c a r b o n a t e and FDNB and h e a t i n g t h e mixture  f o r 2 hours a t 50°C.  The  i n a b i l i t y t o a c h i e v e g r e a t e r than 58% c o n v e r s i o n o f ammonia t o d i n i t r c o a h ' i l i n e c o u l d perhaps be e x p l a i n e d by t h e subsequent r e a c t i o n between the excess FDNB and t h e d i n i t r o a n i l i n e t o form a secondary account  f o r t h e reduced  amine.  This could also  y i e l d s o f d i n i t r o a n i l i n e w i t h i n c r e a s i n g amounts  o f FDNB. The mechanism o f t h e r e a c t i o n between FDNB and ammonia i s unclear.  I t was e v i d e n t t h a t a two phase system was r e q u i r e d f o r i n i t i a t i o n  s i n c e no c o n v e r s i o n was observed i n e t h a n o l o r acetone,  a l t h o u g h both o f these a r e commonly adopted f o r  the a n a l y s i s o f b i o l o g i c a l f l u i d s 1960).  Although  when t h e FDNB was added as a s o l u t i o n  necessary  (e.g. Sanger, 1945; Levy, 1954; Bradbury,  f o r t h e i n i t i a t i o n o f the r e a c t i o n , t h e two  s e p a r a t e phases e x i s t e d f o r o n l y approximately necessary  one q u a r t e r o f t h e time  t o a c h i e v e maximum c o n v e r s i o n o f ammonia t o d i n i t r o a n i l i n e .  the time o f disappearance  o f t h e s e p a r a t e phases, v e r y l i t t l e  had been formed s u g g e s t i n g t h e p r o d u c t i o n o f a r e l a t i v e l y reaction intermediate.  At  dinitroaniline  long-lived  The p a r t i c i p a t i o n o f d i n i t r o p h e n o l i n t h e r e a c t i o n  cannot be excluded, but the presence  o f FDNB i s n e c e s s a r y  s i n c e i t was  found t h a t d i n i t r o a n i l i n e c o u l d not be formed from d i n i t r o p h e n o l and ammonia. Rapp (1963) r e p o r t e d t h a t t h e r e a c t i o n o f FDNB and amino a c i d s was dependent upon t h e h y d r o x y l i o n c o n c e n t r a t i o n . h y d r o x y l i o n was noted  i n the present  case.  The e f f e c t o f the  In t h e range pH 8.0-9.8 t h e  c o n v e r s i o n i n c r e a s e d l i n e a r l y w i t h t h e i n c r e a s e i n pH.  In a l l p r o b a b i l i t y  t h i s i s due t o t h e e f f e c t o f pH as t h e e q u i l i b r i u m between ammonia and the ammonium i o n .  For t h i s equilibrium  [H ] [ N H ] 3  Ka  [NH!]  Ka[OH ] Thus K  [NH ] + [NH ] 3  w  =  10  4  L  =  J  5.68 x 10  -14  .1.76 x 10 ^ Kb  + K [OH ] a  w  -5  10  1  4  8  x [OH ]  + 5.68 x 10  8  x [OH ]  5.68 x 10"  From t h i s r a t i o i t i s e v i d e n t t h a t t h e r e i s n e g l i g i b l e ammonia p r e s e n t at pH l e s s than 8 and t h e p r o p o r t i o n o f m o l e c u l a r  ammonia t o t h e t o t a l  ammonia s p e c i e s i n c r e a s e s r a p i d l y w i t h t h e i n c r e a s e i n h y d r o x i d e i o n c o n c e n t r a t i o n , making more m o l e c u l a r r e a c t i o n w i t h FDNB.  Increases  ammonia a v a i l a b l e f o r n u c l e p p h i l i c  i n pH t o v a l u e s g r e a t e r than 9.8  had no f u r t h e r e f f e c t upon the r a t e o f f o r m a t i o n  apparently  of d i n i t r o a n i l i n e .  34.  CONCLUSION The of  i n v e s t i g a t i o n showed t h a t FDNB r e a c t e d w i t h  ammonia i n the micromolar  solutions  c o n c e n t r a t i o n range i n both d i s t i l l e d  sea water t o form 2 : 4 - d i n i t r o a n i l i n e i n 55-58% y i e l d .  I t was  and  found  t h a t by c o n v e r t i n g ammonia t o d i n i t r o a n i l i n e , f o l l o w e d by the f o r m a t i o n of  a d i a z o - d y e w i t h N - ( 1 - n a p h t h y l ) e t h y l e n e d i a m i n e , i t was  determine water. for  ammonia i n the c o n c e n t r a t i o n range 0-12  The  s c a t t e r observed i n attempts  p o s s i b l e to  micromoles/1  t o produce  in distilled  a Beers's Law  the a n a l y s i s o f d i n i t r o a n i l i n e i n sea water o f 30.4%o  plot  salinity  indicated  t h a t , as such, the method would be u n r e l i a b l e f o r the a c c u r a t e d e t e r m i n a t i o n of  the s m a l l q u a n t i t i e s o f ammonia found i n sea  water.  D i a z o t i s a t i o n o f the d i n i t r o a n i l i n e o c c u r r e d q u i c k l y i h n b o t h distilled  and s e a water and the diazonium  s t a b l e f o r p e r i o d s l o n g e r than 20 minutes examined. was  The  I t formed  q u i c k l y , was  and had maximum absorbance  a c i d had been added t o the sample.  formed was  found t o be  under the c o n d i t i o n s o f a c i d i t y  dye produced by c o u p l i n g w i t h  highly coloured.  60 minutes  salt  N-(1-naphthyl)ethylenediamine s t a b l e f o r p e r i o d s o f over  when an equal volume o f 1:1  sulphuric  In p e r f o r m i n g the c o u p l i n g r e a c t i o n ,  more c o n s i s t e n t r e s u l t s were o b t a i n e d when the c o u p l i n g s o l u t i o n added by means o f a s y r i n g e p i p e t t e t h a n by a g r a v i t y p i p e t t e .  was This  was  accounted f o r by the more r a p i d m i x i n g a c h i e v e d by i n t r o d u c i n g the c o u p l i n g s o l u t i o n i n the former manner. distilled  water by c o o l i n g the a c i d i f i e d samples t o about  f o r m a t i o n o f the dye was 0-5°C.  R e p r o d u c i b l e r e s u l t s were o b t a i n e d i n 10-15°C:  not enhanced by m a i n t a i n i n g the temperature  the at  To q u a n t i t a t i v e l y convert salt,  an excess o f n i t r i t e was  from a c o l o u r e d n i t r i t e and  d i n i t r o a n i l i n e to the  required.  The  diazonium  interference resulting  compound produced by a s i d e r e a c t i o n between the  the c o u p l i n g  agent was  s u c c e s s f u l l y suppressed by  sodium s u l p h i t e to the n a p h t h y l e t h y l e n e d i a m i n e s o l u t i o n . occurred  r e a d i l y i n d i s t i l l e d water, but  p e r s i s t e n t i n sea water. was  achieved  by  In the  photometer c e l l s were f i l l e d  adding  Bleaching  the i n t e r f e r e n c e was  more  l a t t e r case, however, complete  i n c r e a s i n g the s u l p h i t e c o n c e n t r a t i o n . 25 minutes b e f o r e  t h i s p e r i o d o f time a l l o w e d complete b l e a c h i n g  readings  excess  The  bleaching  spectro-  were t o be  taken;  o f the i n t e r f e r e n c e from  the sample, i n c l u d i n g t h a t r e g e n e r a t e d by a e r i a l o x i d a t i o n on f i l l i n g  the  cells.  %«.  was  The  absorbance o f the diazo-dye i n sea water o f s a l i n i t y 30.4  found t o be  about 42%  of that i n d i s t i l l e d  water.  36.  REFERENCES  A t k i n s , W. R. G. 1957. The d i r e c t e s t i m a t i o n o f ammonia i n s e a water, w i t h notes on n i t r a t e , copper, z i n c and sugars. J . c o n s e i l permanent i n t e r n , e x p l o r a t i o n mer. 2_2, 271-277. Bendschneider, B. and R. J . Robinson. 1952. d e t e r m i n a t i o n o f n i t r i t e i n s e a water. Foundation) 11, 87-96.  A new spectrophotometry J . Mar. Research (Sears  Bradbury, J . H. 1960. A d i n i t r o p h e n y l a t i o n method f o r t h e m i c r o d e t e r m i n a t i o n o f amide n i t r o g e n . A n a l . Chim. A c t a 22, 444. G i l l b r i c h t , M. 1961. D e t e c t i o n o f ammonia i n sea water. Wiss. Meeresuntersuch. 8^ 58-67.  Helgolaender  Krogh, A. 1934. Method f o r d e t e r m i n a t i o n o f ammonia i n water and a i r . B i o l . B u l l . 67, 126-131. Kruse, J . M. and M. G. M e l l o n , 1953. C o l o r i m e t r i c d e t e r m i n a t i o n o f ammonia and cyanate. A n a l . Chem. 25_, 1188-1192. Levy, A. L. 1954. A paper chromatographic method f o r t h e q u a n t i t a t i v e e s t i m a t i o n o f amino a c i d s . Nature 174, 126. L o c k h a r t , I. M. i956. Paper chromatographic i d e n t i f i c a t i o n o f 2:4d i n i t r o p h e n y l d e r i v a t i v e s o f a l i p h a t i c amines. Nature 177, 393. Newell,  B. and G. D a l Pont. 1964.  Ammonia i n s e a water.  Nature 201, 36-37.  Palmork, K. H. 1962. The use o f 2 : 4 - d i n i t r o , 1-fluorobenzene i n the s e p a r a t i o n and i d e n t i f i c a t i o n o f amino a c i d s from s e a water. Acta Chem. Scand. 37, 1456. P a t a k i , G. 1964. T h i n l a y e r chromatography f o r t h e sequence a n a l y s i s of peptides. I I I . N o n - d e s t r u c t i v e d e t e c t i o n o f amino a c i d s on t h i n l a y e r chromatograms. J . Chromatog. Ii5, 541-543. P e r a i n o , C. and A. E. Harper. 1961. Q u a n t i t a t i v e paper chromatography o f plasma amino a c i d s , m o d i f i c a t i o n o f the d i n i t r o p h e n y l a t i o n procedure o f Levy. A n a l . Chem. 33, 1863-1865. Rapp, R.  1963.  Determination  o f serum amino a c i d s .  C l i n . Chem. 9, 27-30.  R i c h a r d s , F. A. and R. A. K l e t s c h , 1964. The s p e c t r o p h o t o m e t r y d e t e r m i n a t i o n o f ammonia and l a b i l e amino compounds i n f r e s h and s e a water by o x i d a t i o n t o n i t r i t e . Recent Res. F i e l d s Hydrosphere. Atmosphere ' N u c l . Geochem. 65-81. R i l e y , J . P. 1953. The s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n o f ammonia i n n a t u r a l water w i t h p a r t i c u l a r r e f e r e n c e t o s e a water. A n a l . Chim. A c t a 9, 575-589.  R i l e y , J . P. and P. S i n h a s e n i . 1957. The d e t e r m i n a t i o n o f ammonia and t o t a l i o n i c i n o r g a n i c n i t r o g e n i n sea water. J . Mar. B i o l . A s s o c . U.K. 36, 161-168. Roskam, R. Th. and D. de Langen. 1964. A simple c o l o r i m e t r i c method f o r the d e t e r m i n a t i o n o f ammonia i n sea wtaer. A n a l . Chim. A c t a 56-59. S a n d e l l , E. B. 1950. I n t e r s c i e n c e , New  C o l o r i m e t r i c determination York. 673 pp.  Sanger, F. 1945. 507-515.  f r e e amino groups o f i n s u l i n .  The  30,  of traces of metals.  Biochem. J . '39,  S t r i c k l a n d , J . D. H. and H. K. A u s t i n . 1959. The d i r e c t e s t i m a t i o n o f ammonia i n sea water w i t h notes on " r e a c t i v e " i r o n , n i t r a t e s and i n o r g a n i c phosphorus. J . C o n s e i l permanent i n t e r n , e x p l o r a t i o n mer 24, 446-451. W i r t h , H. E. and R. J . Rohinson. 1933. Photometric i n v e s t i g a t i o n o f N e s s l e r r e a c t i o n and W i t t i g method f o r d e t e r m i n a t i o n o f ammonia i n sea water. Ind. Eng. Chem., A n a l . 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