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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 University, 1963 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Chemistry and I n s t i t u t e of Oceanography We accept t h i s thesis as conforming to the required standard The U n i v e r s i t y of B r i t i s h Columbia November, 1966 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 of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y shall, make i t f r e e l y a v a i l a b l e f o r reference and studyo I furt h e r agree that permission-for extensive copying of 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 granted by the Head of my Department or by his representatives. I t Is understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8 , Canada Date ABSTRACT The conversion of ammonia to 2 : 4 - d i n i t r o a n i l i n e by reaction with 1-fluoro, 2:4-dinitrobenzene and the subsequent conversion of d i n i t r o a n i l i n e to a diazo-dye with N-(l-naphthyl)ethylenediamine has been investigated as an a n a l y t i c a l method f o r determining ammonia at the micromolar concentration l e v e l . P a r t i c u l a r emphasis was placed upon the p o s s i b i l i t y of applying t h i s method to the analysis of sea water. D i n i t r o a n i l i n e was formed under a l k a l i n e conditions (pH greater than 8) and required the presence of the f l u o r o -dinitrobenzene as a separate phase f o r i n i t i a t i o n of the r e a c t i o n . The conversion, which was l i g h t s e n s i t i v e , was accelerated by increases i n pH and temperature, but neither of these factors improved the f i n a l y i e l d . The y i e l d of d i n i t r o a n i l i n e had a marked dependence on the amount of fluorodinitrobenzene; i n i t i a l l y increasing with increasing f l u o r o d i n i t r o -benzene content, i t then decreased with higher fluorodinitrobenzene concentrations suggesting further reaction between d i n i t r o a n i l i n e and fluorodinitrobenzene. In both d i s t i l l e d and sea water, the maximum y i e l d of d i n i t r o a n i l i n e from solutions containing ammonia at the micromolar concentration l e v e l was found to be 55-58%. The absorbance of the diazo-dye i n sea water of s a l i n i t y 30.4 %» was only 42% of that observed i n d i s t i l l e d water. Although 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 to sea water analysis, the p r e c i s i o n of the conversion of d i n i t r o a n i l i n e to the diazo-dye i n sea water was poor compared to that achieved i n d i s t i l l e d water. i i i 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 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 Water 9 a) General Procedures 9 b) Sulphite Concentration 9 c) Naphthylethylenediamine Concentration 10 d) Time and Conditions of D i a z o t i s a t i o n 11 e) Acid Concentration 13 f) Summary 15 II . Formation 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 Water 17 a) Amount of FDNB 19 b) pH 21 c) Temperature 21 d) Relationship between the Y i e l d of D i n i t r o a n i l i n e and Ammonia Concentration 21 I I I . A p p l i c a t i o n to Sea Water 25 DISCUSSION 29 CONCLUSION 34 REFERENCES 36 LIST OF TABLES Table Page I The rate of formation and s t a b i l i t y of the diazonium s a l t of 2 : 4 - d i n i t r o a n i l i n e 13 II Comparison of the s e n s i t i v i t y of methods applied to the analysis of ammonia i n sea water. 29 LIST OF FIGURES The spectra of the diazo-dye formed from naphthyl-ethylenediamine 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 reaction of n i t r i t e with naphthylethylenediamine (dashed l i n e ) The v a r i a t i o n i n the absorbance of the diazo-dye with naphthylethylenediamine concentration. D i n i t r o a n i l i n e concentration, 9.11 micromoles/1. The time dependence of the absorbance of the diazo-dye formed i n solutions of varying sulphuric a c i d concentration. D i n i t r o a n i l i n e concentration, 9.11 micromoles/1. The r e l a t i o n s h i p between the absorbance of the diazo-dye 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 concen-t r a t i o n . The e f f e c t of the amount of FDNB on the pH and y i e l d of d i n i t r o a n i l i n e a f t e r 24 hours. Ammonia concentration, 17.3 micromoles/1. Dependence of the y i e l d 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 water a f t e r 24 hours on the i n i t i a l pH. Ammonia concentration, 17.3 micromoles/1. The r e l a t i o n s h i p between the absorbance of the diazo-dye formed i n d i s t i l l e d water and the ammonia concentration. The time dependence of the absorbance of the diazo-dye i n d i s t i l l e d water and sea water ( s a l i n i t y , 30.4%o) . D i n i t r o -a n i l i n e concentration, 9.11 micromoles/1. The r e l a t i o n s h i p between the absorbance of the diazo-dye formed i n sea water ( s a l i n i t y , 30.4%«) and the d i n i t r o -a n i l i n e concentration. ACKNOWLEDGMENTS I wish to thank my supervisor, Dr. E. V. G r i l l , f o r h i s advice and co-operation. I am indebted to the I n s t i t u t e of Oceanography f o r f i n a n c i a l support throughout the course of my studies. INTRODUCTION The purpose of t h i s research was to investigate the use of 1-fluoro, 2:4-dinitrobenzene as an a n a l y t i c a l reagent f o r ammonia. The method studied was the conversion of fluorodinitrobenzene to d i n i t r o a n i l i n e and the subsequent conversion of t h i s substance to a diazo-dye. Of primary i n t e r e s t was the determination of ammonia at the micromolar concentration l e v e l s found i n natural waters, p a r t i c u l a r l y sea water where the ammonia concentration o r d i n a r i l y is"between 0 and 3 micromoles/litre. The Nessler method commonly used f o r the estimation of ammonia i n water i s not, generally, d i r e c t l y applicable to sea water analysis. Although the interference due to the magnesium ion can be avoided by p r e c i p i t a t i o n or complexing, the method lacks p r e c i s i o n because of the c o l l o i d a l nature and i n s t a b i l i t y of the coloured product (Wirth and Robinson, 1933). The methods developed f o r the analysis of sea water may be d i f f e r -entiated into two classes. In the f i r s t , ammonia i s ' separated from the sample by d i s t i l l a t i o n (Krogh, 1934; Riley, 1953; G i l l b r i c h t , 1963) or d i f f u s i o n (Riley and Sinhaseni, 1957), captured*in d i l u t e a c i d and f i n a l l y determined by a c o l o r i m e t r i c technique. D i s t i l l a t i o n or d i f f u s i o n circumvents the interferences due to other non - v o l a t i l e substances present i n sea water and provides a method that, because of the p o s s i b i l i t y of concentrating the ammonia, i s generally more s e n s i t i v e than d i r e c t techniques that do not require preliminary separation of ammonia from the sample. Unfortunately, such procedures are time consuming and require equipment not Well adapted to routine use on board ship. The second category consists of co l o r i m e t r i c techniques run d i r e c t l y on the raw sea water samples. The common indophenol-blue t e s t f o r ammonia 2. (Riley, 1953; R i l e y and Sinhaseni, 1957) i s not d i r e c t l y applicable to sea water analysis due to the. p r e c i p i t a t i o n of magnesium and calcium i n the a l k a l i n e pH range at which the reaction occurs. Newell and Dal Pont (1964) avoided t h i s interference by converting the ammonia to quinone chlorimide, extracting with hexanol and completing the formation of the indophenol i n the organic solvent. In an alternate method devised by Roskam and de Langen (1964), magnesium and calcium p r e c i p i t a t i o n was avoided by use of a chelating agent. By s u b s t i t u t i n g thymol f o r phenol, a more stable and more s e n s i t i v e dye was formed than that obtained i n the indophenol-blue method of R i l e y and Sinhaseni (1957). The pyridine-pyrazolone method of Kruse and Mellon (1953) has been modified f o r sea water analysis by Atkins (1957) and St r i c k l a n d and Austin (1959). Richards and Kletsch (1964) described a procedure i n which ammonia'is-oxidised*to n i t r i t e and the n i t r i t e i s subsequently assayed as a diazo-dye. Although reported to have a p r e c i s i o n as good as or bett e r than d i s t i l l a t i o n procedures, the method i s subject to interferences since the amino nitrogen of a number of amino acids was shown to also undergo oxidation to n i t r i t e . 1-fluoro, 2:4-dinitrobenzehe (FDNB) was f i r s t employed by Sanger (1945) f o r the purpose of i d e n t i f y i n g the terminal groups of proteins. Its use has since been extended to the analysis of various nitrogenous compounds such as amino acids (Levy, 1954; Rapp, 1963). and amines (Lockhart, 1956). Palmork (1962) used FDNB to i s o l a t e and i d e n t i f y microgram quantities of amino acids from sea water. Levy (1954) and Lockhart (1956) reported that FDNB reacts with ammonia at about pH 9. By determining the d i n i t r o a n i l i n e formed by absorption 3. spectrophotometry following i t s i s o l a t i o n by paper chromatography, Bradbury (1960) s u c c e s s f u l l y employed t h i s reaction to estimate small amounts of ammonia. Such a procedure, however, would appear to be too cumbersome f o r routine a n a l y t i c a l work. The p o s s i b i l i t y of converting d i n i t r o a n i l i n e to an intensely coloured diazo-dye suggested an a l t e r n a t i v e procedure; one by which the reaction between FDNB and ammonia might be made the basis of a s e n s i t i v e and interference free t e s t f o r ammonia.. It was hoped that such a te s t would prove applicable to sea water an a l y s i s . 4. APPARATUS AND REAGENTS Absorbance measurements were made with a Beckman model DU spectrophotometer, using a s l i t width of 0.01 mm and matched quartz c e l l s of e i t h e r 1 cm. or 5 cm. o p t i c a l path length. Ammonia free water (afterwards re f e r r e d to as d i s t i l l e d water) was obtained by passing d i s t i l l e d water'through a mixed bed ion exchange r e s i n column and f i n a l l y through Dowex 50 immediately before 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 prepared by d i s s o l v i n g 0.020 gm of 2 : 4 - 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 containing 100 ml of concentrated sulphuric a c i d and adjusting the volume to 1 l i t r e . Ten-fold d i l u t i o n gave a working s o l u t i o n containing 9.11 micromoles d i n i t r o a n i l i n e / l i t r e . Naphthylethylenediamine solutions were prepared by d i l u t i n g a stock s o l u t i o n containing 0.30 gm of N-(l-naphthyl)ethylenediamine dihydrochloride i n 100 ml d i s t i l l e d water. The stock s o l u t i o n was stored i n an amber glass b o t t l e i n which i t i s stable over periods of several weeks (Bendschneider and Robinson, 1952). Standard ammonia s o l u t i o n was prepared by d i s s o l v i n g 0.114 gm of ammonium sulphate i n 1 l i t r e of d i s t i l l e d water. The resultant s o l u t i o n contained 1.73 micromoles ammonia/ml. This s o l u t i o n was made up f r e s h l y each two days. Saturated sodium borate s o l u t i o n was prepared from sodium borate and d i s t i l l e d water and was b o i l e d p r i o r to use to expel any ammonia that might have been present. 5. Sodium n i t r i t e s o l u t i o n was prepared by d i s s o l v i n g 1 gm of sodium n i t r i t e i n 100 ml d i s t i l l e d water. 1-fluoro, 2:4-dinitrobenzene was added to the samples as the pure l i q u i d (m.p. 25-27°C). EXPERIMENTAL The method of analysis r e l i e s on two separate steps; f i r s t l y , the conversion of ammonia to 2 : 4 - d i n i t r o a n i l i n e and, secondly, the quantitative estimation of d i n i t r o a n i l i n e by the development of a diazo-dye. Before i n v e s t i g a t i o n of the f i r s t step could proceed, i t was necessary to develop adequate methods f o r carrying out the second step. The coupling agent selected was N-(1-naphthyl)ethylenediamine. This reagent had previously been employed as a coupling agent by Bendschneider and Robinson (1952) and Richards and Kletsch (1964) and was considered s u i t a b l e f o r t h i s work since i t forms intensely coloured dyes and couples under the strongly a c i d i c conditions required to d i a z o t i s e d i n i t r o a n a l i n e . Phenolic agents, which couple under mildly a l k a l i n e conditions, were found to be less s a t i s f a c t o r y because of the d i f f i c u l t y of n e u t r a l i z i n g the i n i t i a l l y strongly a c i d i c d i a z o t i s a t i o n mixture to a reproducible pH. The dye formed from d i n i t r o a n i l i n e and naphthylethylenediamine i s rose-red, i s stable under strongly a c i d i c conditions and has an absorption maximum at 525 my which i s s u f f i c i e n t l y intense to r e a d i l y d i f f e r e n t i a t e between micromolar concentrations of d i n i t r o a n i l i n e (Fig. 1). To obtain quantitative conversion of d i n i t r o a n i l i n e to i t s diazonium s a l t and ensure that the y i e l d i s independent of the n i t r i t e concentration, 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 presenceof an excess 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 during the subsequent coupling reaction since naphthylethylenediamine not only reacts with diazonium s a l t s , but also with n i t r i t e (Benschneider and Robinson, 1952) forming, i n the l a t t e r case, a mauve coloured material with an absorption peak at 565 my (Fig. It was found that sodium sulphite reduced t h i s mauve product to a near-colourless material with no appreciable absorbance at 525 my and that the 7. 0.400 _ 0 1 400 WAVELENGTH, Millimicrons Fig. 1. The spectra of the diazo-dye formed from naphthyl-ethylenediamine and dinitroaniline (solid line) and the compound produced by the reaction of n i t r i t e with naphthylethylenediamine (dashed l i n e ) . 500 600 8. sulphite could be added i n s o l u t i o n along with naphthylethylenediamine without i n t e r f e r i n g i n the formation of the dizao-dye. Before attempting to apply t h i s procedure to sea water, the optimum conditions f o r carrying out the reactions 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 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 Water (a) General Procedures The optimum conditions f o r d i a z o t i s a t i o n and coupling were determined using a standard s o l u t i o n containing 9.11 micromoles/1 d i n i t r o a n i l i n e . Sulphuric acid- e i t h e r concentrated or d i l u t e d 1:1 with d i s t i l l e d water- was added by p i p e t t e to 10 ml of the standard 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 glass-stoppered graduated c y l i n d e r and the f i n a l volume was brought to 20 ml with d i s t i l l e d water. The heat generated by the addition of the acid was d i s s i p a t e d by holding the samples under running tap water. D i a z o t i s a t i o n was effected by adding 2 drops of a 1% sodium n i t r i t e s o l u t i o n and coupling by the addition of 1 ml of an aqueous s o l u t i o n containing N-(l-naphthyl)ethylenediamine dihydrochloride and sodium s u l p h i t e . (b) Sulphite Concentration The optimum sulphite concentration preventing interference from the side reaction between n i t r i t e and the coupling agent was determined by taking 15 ml of d i s t i l l e d water and adding 5 ml of concentrated sulphuric acid. A f t e r cooling to about 10-15°C, 2 drops of 1% sodium n i t r i t e s o l u t i o n were added followed by 1 ml of a s o l u t i o n containing 0.01% of naphthyl-dethylenediamine and varying amounts of sodium su l p h i t e . This second sol u t i o n was added by means of a syringe pipet-te. The samples were mixed a f t e r each addition by i n v e r t i n g the glass stoppered cylinders i n which they were contained. The a f f e c t of sodium sulphite concentrations ranging between 0.1% and 5% was observed. It was found that a 2% sodium sulphite s o l u t i o n gave a near colourless blank which faded i n a few seconds to y i e l d a pale 10. green so l u t i o n with no appreciable absorbance at 525 my. With lower sulphite concentrations the mauve coloured product faded less r a p i d l y . In a l l cases, the mauve material could be regenerated through a e r i a l oxidation when the samples were agitated, but the remaining sulphite again bleached out t h i s colour when the samples were allowed to stand. Using a 2% sulphite s o l u t i o n , i t was found that f i l l i n g the spectrophotometer c e l l s at least 10 minutes before readings were to be made ensured complete removal of the interference. Higher sulphite concentrations offered no improvement and were, i n any case, undesirable since they might endanger the success of the method by reduction of the diazonium s a l t . The coupling 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 sulphite and a stock s o l u t i o n of N-(l-naphthyl)ethylenediamine. c) Naphthylethylenediamine Concentration The naphthylethylene diamine concentration necessary to produce maximum colour, development was determined by taking 10 ml aliquots of the standard 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 to 20 ml with sulphuric a c i d d i l u t e d 1:1 with d i s t i l l e d water. The solutions were cooled to about 10-15°C and 2 drops of 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 standing f o r 10 minutes, 1 ml of a s o l u t i o n containing 2% sodium sulphite and naphthylethylenediamine i n concentrations ranging from 0.005% to 0.08% was admitted by means of a syringe p i p e t t e . Time series observations established that the dye had obtained maximum development a f t e r about 15 minutes and was stable f o r periods of at least an hour. To compare the a f f e c t of varying the naphthylethylenediamine concentrations on the f i n a l colour i n t e n s i t y , readings were taken against a d i s t i l l e d water o p t i c a l blank at 525 mu i n a 1 cm c e l l a f t e r the colour had been allowed to develop f o r 30 minutes. These readings were compared with reagent blanks prepared 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 the d i n i t r o a n i l i n e . The r e s u l t s , depicted i n F i g . 2, indicated that maximum colour i n t e n s i t y could be achieved when the coupling s o l u t i o n contained 0.03% N-(1-naphthyl) ethylenediamine dihydrochloride. This concentration, i n conjunction with a 2% sodium sulphite concentration, was employed i n subsequent studies. (d) Time and conditions of d i a z o t i s a t i o n The a f f e c t of the time of d i a z o t i s a t i o n upon the f i n a l colour i n t e n s i t y of the diazo-dye was studied f o r solutions prepared by the addition of 10 ml of 1:1 sulphuric a c i d to 10 ml of the 9.11 micromolar d i n i t r o a n i l i n e s o l u t i o n . The samples were cooled to about 10-15°C and 2 drops of 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 of the coupling s o l u t i o n was added and the absorbance of the dye was measured at 525 mu i n c e l l s of 1 cm o p t i c a l path length a f t e r the colour had developed f o r 30 minutes. The r e s u l t s , shown i n the table below, indicated that the diazonium s a l t was formed quickly and was stable under the experimental conditions f o r periods up to 20 minutes. To ensure maximum colour development, i t was decided to add the coupling s o l u t i o n 10 minutes a f t e r the addition of the n i t r i t e s o l u t i o n . It was observed that the absorbance did not increase s i g n i f i c a n t l y i f the d i a z o t i s a t i o n and coupling were c a r r i e d out at 0°C. 12. 0.250. 0.02 0.04 0.06 0.08 % NAPHTHYLETHYLENEDIAMINE Fig- 2.-The variation in the absorbance of the diazo-dye with naphthylethylenediamine concentration. Dinitroaniline concentration, 9.11 micromoles/1. 13. Table I. The rate of formation and s t a b i l i t y of the diazonium s a l t of 2 : 4 - d i n i t r o a n i l i n e . Time of 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 (e) Acid Concentration The s t a b i l i t y of the diazo-dye was found to be dependent upon the f i n a l a c i d concentration of the s o l u t i o n . Tests were conducted by adding varying amounts of concentrated sulphuric a c i d to 10 ml of the standard d i n i t r o a n i l i n e s o l u t i o n and bringing the samples to a f i n a l volume of 20 ml with d i s t i l l e d water. D i a z o t i s a t i o n was affe c t e d by the addition of 2 drops of 1% sodium n i t r i t e s o l u t i o n and the coupling s o l u t i o n was added a f t e r 10 minutes. The development of the diazo-dye was followed with time by measuring i t s absorbance at 525 my i n a 1 cm c e l l versus a d i s t i l l e d water o p t i c a l blank. The r e s u l t s are shown i n F i g . 3. When less than 3 ml of concentrated sulphuric a c i d were present, the absorbance of the s o l u t i o n r a p i d l y achieved a maximum and then faded. As the acid TIME, Minutes Fi g . 3. The time dependence, of the absorbance of the diazo-dye formed in solutions of varying sulphuric acid concentration. D i n i t r o a n i l i n e concentration, 9.11 micromoles/1 15. concentration increased, the colour was formed less r a p i d l y , but remained more stable. In the case where 5 ml. of acid were present, maximum colour i n t e n s i t y was obtained i n less than 20 minutes whereas with 10 ml of acid maximum development was achieved only a f t e r 160 minutes. It was v e r i f i e d f o r a l l a c i d concentrations that: a) reagent blanks i n which d i s t i l l e d water was substituted f o r the 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) that d i a z o t i s a t i o n was accomplished within a few minutes at 10-15°C and that diazonium s a l t was stable f o r periods of up to 20 minutes. These r e s u l t s indicated that acid concentrations approximately 5 ml of concentrated sulphuric a c i d i n a t o t a l volume of 20 ml would be su i t a b l e f o r furth e r work provided that the absorbance was recorded 30 minutes a f t e r the addition of the coupling agent. f) Summary Concentrations of d i n i t r o a n i l i n e i n the range 0-10 micromoles/1 can be estimated by the following procedure. To 10 ml of the d i n i t r o a n i l i n e s o l u t i o n to be analyzed add 10 ml of 1:1 sulphuric acid and cool to 10-15°C. Add 2 drops of a 1% sodium n i t r i t e s o l u t i o n and, a f t e r 10 minutes, 1 ml of a s o l u t i o n containing 0.03% N-(1-naphthyl)ethylenediamine dihydrochloride and 2% sodium sulphite. Measure the absorbance at 525 mu i n a 5 cm c e l l a f t e r colour development has proceeded f o r 30 minutes and compare with a reagent blank. The spectrophotometer c e l l s should be f i l l e d at least 10 minutes before readings are to be made. The s e l e c t i o n of a 10 minute, i n t e r v a l between the addition of the n i t r i t e and coupling 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 the 16. consistency of 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 of the dye produced and the amount of d i n i t r o a n i l i n e present was studied using the method of analysis described above (recording the absorbance i n 5 cm c e l l s ) and was found to 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 of the diazo-dye 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. 18. II . The Formation 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 Water Previous work (Levy, 1954; Palmork, 1962) indicated that FDNB reacts with nitrogenous compounds under mi l d l y a l k a l i n e conditions (pH 8-10). In the following experiments t e s t samples were made up by d i l u t i n g 1 ml of standard ammonia s o l u t i o n to 100 ml with d i s t i l l e d water giving a so l u t i o n 17.3 micromolar i n ammonia, and adding 2 ml of saturated sodium borate s o l u t i o n to buf f e r the s o l u t i o n to pH 9.3. FDNB has generally been employed by the addition of eit h e r an acetone or ethanolic s o l u t i o n (Sanger, 1945; Bradbury, 1960). This p r a c t i c e was followed i n the i n i t i a l experiments, adding 1 ml of a 2% sol u t i o n of FDNB i n acetone or ethanbl to 100 ml of t e s t sample contained i n 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 . The samples were agitated at room temperature e i t h e r by a magnetic s t i r r e r or a Burrel wrist action shaker and 10 ml aliquots were withdrawn at various times f o r d i n i t r o a n i l i n e a n a l ysis. Using the previously described procedure, no d i n i t r o a n i l i n e could be detected, even a f t e r the experiments had been allowed to proceed f o r as long as 48 hours. Further experiments i n which the i n i t i a l pH of the sample containing borate was adjusted over the range of 7.5 to 12 by the addition of sodium hydroxide or hydrochloric acid, r e s p e c t i v e l y , i n d i c a t e d that no d i n i t r o a n i l i n e was formed when the FDNB was added i n so l u t i o n . When, however, pure l i q u i d FDNB was added so that a two phase system was produced, slow conversion of ammonia to d i n i t r o a n i l i n e was obtained. The two phase system existed f o r only about one quarter of the time necessary to obtain maximum development of d i n i t r o a n i l i n e . By su b s t i t u t i n g 1 ml of a 2% aqueous dinitrophenol s o l u t i o n f o r l i q u i d FDNB and conducting the experiment under i d e n t i c a l conditions, 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 that FDNB was necessary f o r the reaction to proceed. As observed i n other work with t h i s compound, the reaction was found to be s e n s i t i v e to l i g h t (Peraino and Harper, 1961; Pataki, 1964). When c a r r i e d out i n amber glass b o t t l e s , conversion to d i n i t r o a n i l i n e occurred; no d i n i t r o a n i l i n e was formed when the reaction was conducted i n cle a r glass b o t t l e s . At room temperature, with a sample containing 0.1 ml of FDNB per 100 ml of tes t s o l u t i o n and pH adjusted to 9.3 by the addition of 2 ml saturated sodium borate, maximum conversion was achieved a f t e r a period of 32 hours. Based on the absorbance of solutions of known d i n i t r o a n i l i n e content, 55% of the ammonia o r i g i n a l l y present had been converted to d i n i t r o a n i l i n e . It was cons i s t e n t l y found that 45% conversion was obtained a f t e r 24 hours. a) Amount of FDNB The a f f e c t of varying the FDNB content on the y i e l d of d i n i t r o a n i l i n e a f t e r 24 hours at pH 9.3 was studied. As shown by F i g . 5, the amount of d i n i t r o a n i l i n e formed increased i n approximately l i n e a r proportion to the FDNB up to a maximum of 0.1 ml FDNB/100 ml sample. Further increases i n the amount of FDNB resul t e d i n a decreased y i e l d of d i n i t r o a n i l i n e . The addition of 0.1 ml FDNB to 100 ml of the tes t s o l u t i o n c o n s i s t e n t l y gave 45% conversion a f t e r 24 hours, and thus, t h i s volume of the reagent was used f o r subsequent t e s t s . Further observations indicated that the pH of the test s o l u t i o n decreased as the reaction proceeded, presumably due to the hydrolysis of FDNB to dinitrophenol. Tests were c a r r i e d out to determine the af f e c t of adding more sodium borate i n an attempt to maintain the pH of the te s t s o l u t i o n . It was observed that the amount of d i n i t r o a n i l i n e formed was not af f e c t e d by increasing the amount of borate. 20. c o cc o ml FDNB/100 ml sample Fig- 5. The effect of the amount of FDNB on the pH and yield of dinitroaniline after 24 hours. Ammonia concentration, 17.3 micromoles/1. 21. b) pH Tests were conducted to observe the a f f e c t of pH on the reaction. 2 ml of saturated sodium borate s o l u t i o n were added to 100 ml of d i s t i l l e d water and the pH of the so l u t i o n was adjusted by the addition of hydrochloric acid or sodium hydroxide, r e s p e c t i v e l y . 0.1 ml of FDNB and 1 ml of the standard ammonia so l u t i o n were added and the reaction was allowed to proceed f o r 24 hours with shaking. A f t e r t h i s period 10 ml samples were withdrawn and analyzed f o r d i n i t r o a n i l i n e . In each case a reagent blank was run. As shown i n F i g . 6, below pH 8, the amount of conversion was i n s i g n i f i c a n t ; above pH 8, the y i e l d increased with pH u n t i l a maximum was reached at about pH 9.5. Further increase i n pH did not improve the y i e l d . By allowing the r e a c t i o n to go to completion, i t was observed that 55% conversion of ammonia to d i n i t r o a n i l i n e was achieved f o r a l l samples with an i n i t i a l pH greater than 8.0. At pH 10 maximum conversion was obtained a f t e r 28 hours, 4 hours quicker than at pH 9.3. c) Temperature Increasing the temperature produced a marked acceleration i n the rate of formation of d i n i t r o a n i l i n e . Tests were conducted by shaking the react i o n vessels i n a thermostatically c o n t r o l l e d water bath. At higher temperatures the same maximum percentage conversion, 55%, as at room temperatures was con s i s t e n t l y obtained. At 50°C and pH 9.3 the reaction reached completion a f t e r about 8 hours; at pH 10 the period necessary to achieve maximum conversion was reduced to less than 3 hours. d) Relationship between the Y i e l d of D i n i t r o a n i l i n e and the  Ammonia Concentration. A series of standard ammonia solutions was prepared 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 F i g . 6. INITIAL pH The dependence of the y i e l d 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 water a f t e r 24 hours on the i n i t i a l pH. Ammonia concentration, 17.3 micromoles/1. 23. water and 100 ml samples were treated at 50°C with 2 ml of saturated sodium borate and 0.1 ml FDNB. A f t e r 10 hours, 10 ml aliquots were withdrawn f o r d i n i t r o a n i l i n e analysis. The r e s u l t s are depicted i n F i g . 7. Absorbancies were measured i n 5 cm c e l l s at 525 my. The l i n e a r r e l a t i o n s h i p between concentration and absorbance indicated that the y i e l d of d i n i t r o a n i l i n e was independent of ammonia concentration f o r solutions up to at l e a s t 12 micromolar i n ammonia concentration and, thus, that small quantities of ammonia can be detected and d i f f e r e n t i a t e d by t h i s procedure. Moreover, the method appeared to be 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 to sea water. 1.000 24. F i g . 7. The r e l a t i o n s h i p between the absorbance of the diazo-dye i n d i s t i l l e d water and the ammonia concentration. 25. II I . A p p l i c a t i o n to Sea Water Tests were conducted to determine the a p p l i c a b i l i t y of the procedures developed to sea water systems. In order to investigate the ef f e c t of sea water upon the d i a z o t i s a t i o n and coupling steps, stock d i n i t r o -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 with P a c i f i c Ocean water of 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 resultant s o l u t i o n had a d i n i t r o a n i l i n e concentration of 9.11 micromole/1 and a s a l i n i t y of 30.4%» . Blanks were prepared 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 the d i n i t r o a n i l i n e s o l u t i o n . 10 ml of 1:1 sulphuric a c i d were added to 10 ml of these solutions. Tests with the blank indicated that the colour r e s u l t i n g from the reaction of the naphthylethylenediamine with n i t r i t e was dissi p a t e d much more slowly i n sea water than i n d i s t i l l e d water. This colour was dispersed within 35 minutes using a 2% sulphite s o l u t i o n and within 20 minutes when the sulphite concentration was increased to 6%. The absorbance of the blank at 525 my was small and consistent when the coupling s o l u t i o n contained 6% sulphite and the spectrophotometer c e l l s were f i l l e d 25 minutes before reading. D i z a o t i s a t i o n of the d i n i t r o a n i l i n e occurred quickly i n water of t h i s s a l i n i t y and the diazonium s a l t was found to be stable over a period 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 obtained by adding 1 ml of a s o l u t i o n containing 0.03% naphthylethylenediamine and 6% sodium s u l p h i t e 10 minutes a f t e r the addition of 2 drops of 1% sodium n i t r i t e to samples cooled to about 10-15°C. The absorbance of the dye was read at 525 my a f t e r the colour had developed f o r 30 minutes. P a r a l l e l t e s t s conducted i n d i s t i l l e d water indicated that coupling was not affe c t e d by the increase i n sulphite concentration, but the presence of sea water s a l t s considerably reduced the colour i n t e n s i t y of the diazo-dye (Fig. 8). 26. rt o o LO CN LO w CJ < cc: o CO ca < 0.250-5 0.'200. c ex. -O—H3 O — O G O O — - O O C D — 0.150_ 0.100J -© -Q &-0.050. O •-' D i s t i l l e d Water Q - Sea Water i (30.4 %o s a l i n i t y ) — r 10 T" 20 30 40 TIME, M i n u t e s 50 60 Fig- 8. The t i m e dependence o f t h e 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 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 % o ) . 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 . The absorbance of the sample was not increased e i t h e r by increasing the proportion of sulphuric acid i n the sample or by the amount of naphthyl-ethylenediamine i n the coupling s o l u t i o n . Although the colour i n t e n s i t y of the dye i n sea water of s a l i n i t y 30.4%o was reduced to only about 42% of that i n d i s t i l l e d water, sensible r e s u l t s could s t i l l be obtained i n sea water systems by using o p t i c a l c e l l s of 10 cm path length. A series of d i n i t r o a n i l i n e solutions i n the concentration, range 0-rl7 micromoles/1 was prepared i n sea water so as to have a f i n a l s a l i n i t y of 30.4%o . When analyzed as described above, considerable s c a t t e r occurred about the best s t r a i g h t line through the points (Fig. 9) suggesting that, under the conditions used, the procedure would not be r e l i a b l e i n sea water. Reductions i n the amount of n i t r i t e i n the d i a z o t i s a t i o n step and the addition of sulphite a f t e r the coupling agent d i d not resolve these d i f f i c u l t i e s . Tentative r e s u l t s were obtained concerning the conversion of FDNB to d i n i t r o a n i l i n e i n sea water of s a l i n i t y 30.4% 0 . 100 ml of sea water to which 17.3 micromoles/1 ammonia had been added was treated with 0.1 ml of FDNB. The s o l u t i o n was buffered by the addition of 2 ml of a saturated sodium borate s o l u t i o n and sodium hydroxide. At room temperature conversion was more ra p i d at pH 10 than at pH 9 and appeared to reach completion a f t e r 24 hours. By comparing the absorbance corrected f o r a sea water blank with that of 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 of the same s a l i n i t y , i t was found that there was approximately 58% conversion of ammonia to d i n i t r o a n i l i n e . 28. 0.700 0.600 0.500 _ 0.400 0.300 0.200 -0.100 O 0 • + • represent the r e s u l t s from three d i f f e r e n t runs 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 of the diazo-dye formed i n sea water ( s a l i n i t y , 30.4% o) and the d i n i t r o a n i l i n e concentrati 29. DISCUSSION The s e n s i t i v i t y of a spectrophotometry method of analysis i s defined by Sandell (1950) as: "...the number of micrograms of the element, converted to the coloured product, which i n a column of s o l u t i o n having a cross section of one square centimetre shows an e x t i n c t i o n of 0.001." The s e n s i t i v i t y of the 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 of small quantities of ammonia i n aqueous solutions i n the table below. Table I I . Comparison of the s e n s i t i v i t y of methods applied to the analysis of ammonia i n sea 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 - sea water 2.48 Richards and Kletsch (1964) 0.504 Roskam and de Langen (1964) 1.3 Newell and Dal Pont (1964) - d i s t i l l e d water 0.321 Newell and Dal. Pont (1964) - sea water 0.512 R i l e y and Sinhaseni (1957) 2.91 Although not as s e n s i t i v e as the methods of Richards and Kletsch (1964), Newell and Dal Pont (1964) and Roskam and de Langen (1964), the FDNB technique, because of 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 to sea water analysis i f the p r e c i s i o n of the d i n i t r o a n i l i n e determination could be improved. The procedure of Newell and Dal Pont (1964), although very 30. s e n s i t i v e , appears to be too elaborate f o r routine shipboard use. The methods devised by Roskam and de Langen (1964) and Richards and Kletsch (1964) require strongly basic conditions - pH greater than 11- to oxidise the ammonia. Richards and Kletsch (1964), by running comparisons of t h i s method on natural sea water samples against the d i s t i l l a t i o n method devised by R i l e y (1953), showed that under these conditions there was a s i g n i f i c a n t error i n the ammonia estimated due, presumably, to the degradation of amino acids and amines. Further, i n the Roskam and de Langen (1964) method c a r e f u l preparation of the sample i s required i n order that a l l the magnesium and calcium ions present should be chelated with cyclo-hexyl trans 1:2 diaminetetraacetic acid. I f even s l i g h t p r e c i p i t a t i o n i s allowed to occur, low r e s u l t s are obtained. The p o s s i b i l i t y of interference from nitrogen containing compounds such as amino acids 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 investigated. I t i s u n l i k e l y , however, that these would s i g n i f i c a n t l y contribute to the amount of d i n i t r o a n i l i n e recovered. Levy (1954) and Lockhart (1956) indi c a t e d that v i r t u a l l y quantitative y i e l d s were obtained with primary and secondary amines and amino acids suggesting that l i t t l e c ontribution of d i n i t r o a n i l i n e would be l i k e l y from t h i s source. Sanger (1945) and Bradbury (1960) found that there was no appreciable breakdown of the dinitrophenyl derivatives of amino acids and amines even when refluxed with 20% hydrochloric a c i d f o r 8 hours; with h i s t d i n e , however, the imidazole group reacted slowly with FDNB so that, under the conditions of hydrolysis described above, some 2:4-d i n i t r o a n i l i n e was obtained. Under the experimental conditions employed i n t h i s study t h i s i s an u n l i k e l y source of error. The addition of sodium sulphite to the s o l u t i o n of naphthylethylene-diamine used f o r coupling appeared to s u c c e s s f u l l y remove the interference from the side reaction between n i t r i t e and the coupling agent. Low and consistent absorbancies were obtained f o r reagent blanks i n both d i s t i l l e d water and sea water and, thus, the lack of p r e c i s i o n i n the determination of d i n i t r o a n i l i n e i n sea water would not appear to be caused by interference from t h i s side r e a c t i o n . Furthermore, the r e s u l t s shown i n F i g . 8 indicate that the diazo-dye formed stable solutions i n sea water as well as i n d i s t i l l e d water. The reason f o r the poor p r e c i s i o n of the d i n i t r o a n i l i n e determination i n sea water, therefore, i s obscure. The apparent affect of sea s a l t s on the reaction could p o s s i b l y be avoided, however, by extracting the d i n i t r o a n i l i n e from sea water i n t o an organic solvent, re-extracting i n t o a c i d i f i e d d i s t i l l e d water and conducting the d i a z o t i s a t i o n and coupling under conditions that were found to give r e l i a b l e r e s u l t s . Increases i n both temperature and pH s i g n i f i c a n t l y accelerated the reaction between FDNB and ammonia, but neither of these factors was found to influence the ultimate y i e l d of d i n i t r o a n i l i n e . The y i e l d compares favourably with that obtained by other workers e s p e c i a l l y when the extreme d i l u t i o n of the ammonia solution, i s considered. Levy (1954) found that at 40°C aqueous solutions of amines gave 100% conversion to the dinitrophenyl derivatives within 80 minutes, but could only obtain 25% conversion of ammonia to d i n i t r o a n i l i n e even by heating f o r 7 hours at elevated temperatures. Lockhart (1956) has obtained 50% conversion to d i n i t r o a n i l i n e by allowing the reaction to proceed at 105°C f o r 2 hours and Bradbury (1960) has reported 90% y i e l d s by evaporating an ammonium chloride s o l u t i o n , adding potassium bicarbonate and FDNB and heating the mixture f o r 2 hours at 50°C. The i n a b i l i t y to achieve greater than 58% conversion of ammonia to dinitrcoah'iline could perhaps be explained by the subsequent r e a c t i o n between the excess FDNB and the d i n i t r o a n i l i n e to form a secondary amine. This could also account f o r the reduced y i e l d s of d i n i t r o a n i l i n e with increasing amounts of FDNB. The mechanism of the reaction between FDNB and ammonia i s unclear. I t was evident that a two phase system was required f o r i n i t i a t i o n since no conversion was observed when the FDNB was added as a s o l u t i o n i n ethanol or acetone, although both of these are commonly adopted f o r the analysis of b i o l o g i c a l f l u i d s (e.g. Sanger, 1945; Levy, 1954; Bradbury, 1960). Although necessary for the i n i t i a t i o n of the reaction, the two separate phases existed f o r only approximately one quarter of the time necessary to achieve maximum conversion of ammonia to d i n i t r o a n i l i n e . At the time of disappearance of the separate phases, very l i t t l e d i n i t r o a n i l i n e had been formed suggesting the production of a r e l a t i v e l y l o n g - l i v e d reaction intermediate. The p a r t i c i p a t i o n of dinitrophenol i n the reaction cannot be excluded, but the presence of FDNB i s necessary since i t was found that d i n i t r o a n i l i n e could not be formed from dinitrophenol and ammonia. Rapp (1963) reported that the reaction of FDNB and amino acids was dependent upon the hydroxyl ion concentration. The e f f e c t of the hydroxyl ion was noted i n the present case. In the range pH 8.0-9.8 the conversion increased l i n e a r l y with the increase 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 to the e f f e c t of pH as the equilibrium between ammonia and the ammonium ion. For t h i s equilibrium Ka [H ][NH 3] [NH!] Thus [NH3] + [NH4] Ka[OH ] K + K [OH ] w a L J w = 10 -14 .1.76 x 10 Kb -5 ^ = 5.68 x 10" 5.68 x 10 8 x [OH ] 10 1 4 + 5.68 x 10 8 x [OH ] From t h i s r a t i o i t i s evident that there i s n e g l i g i b l e ammonia present at pH less than 8 and the proportion of molecular ammonia to the t o t a l ammonia species increases r a p i d l y with the increase i n hydroxide ion concentration, making more molecular ammonia av a i l a b l e f o r n u c l e p p h i l i c reaction with FDNB. Increases i n pH to values greater than 9.8 apparently had no further e f f e c t upon the rate of formation of d i n i t r o a n i l i n e . 34. CONCLUSION The i n v e s t i g a t i o n showed that FDNB reacted with solutions of ammonia i n the micromolar concentration range i n both d i s t i l l e d and sea water to 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 . It was found that by converting ammonia to d i n i t r o a n i l i n e , followed by the formation of a diazo-dye with N-(1-naphthyl)ethylenediamine, i t was possible to determine ammonia i n the concentration range 0-12 micromoles/1 i n d i s t i l l e d water. The s c a t t e r observed i n attempts to produce a Beers's Law pl o t f o r the analysis of d i n i t r o a n i l i n e i n sea water of 30.4%o s a l i n i t y indicated that, as such, the method would be u n r e l i a b l e f o r the accurate determination of the small quantities of ammonia found i n sea water. D i a z o t i s a t i o n of the d i n i t r o a n i l i n e occurred quickly ihnboth d i s t i l l e d and sea water and the diazonium s a l t formed was found to be stable f o r periods longer than 20 minutes under the conditions of a c i d i t y examined. The dye produced by coupling with N-(1-naphthyl)ethylenediamine was highly coloured. I t formed quickly, was stable f o r periods of over 60 minutes and had maximum absorbance when an equal volume of 1:1 sulphuric acid had been added to the sample. In performing the coupling reaction, more consistent r e s u l t s were obtained when the coupling s o l u t i o n was added by means of a syringe pipette than by a gravity p i p e t t e . This was accounted f o r by the more ra p i d mixing achieved by introducing the coupling s o l u t i o n i n the former manner. Reproducible r e s u l t s were obtained i n d i s t i l l e d water by cooling the a c i d i f i e d samples to about 10-15°C: the formation of the dye was not enhanced by maintaining the temperature at 0-5°C. To q u a n t i t a t i v e l y convert d i n i t r o a n i l i n e to the diazonium s a l t , an excess of n i t r i t e was required. The interference r e s u l t i n g from a coloured compound produced by a side reaction between the excess n i t r i t e and the coupling agent was s u c c e s s f u l l y suppressed by adding sodium sulphite to the naphthylethylenediamine s o l u t i o n . Bleaching occurred r e a d i l y i n d i s t i l l e d water, but the interference was more per s i s t e n t i n sea water. In the l a t t e r case, however, complete bleaching was achieved by increasing the sulphite concentration. The spectro-photometer c e l l s were f i l l e d 25 minutes before readings were to be taken; t h i s period of time allowed complete bleaching of the interference from the sample, including that regenerated by a e r i a l oxidation on f i l l i n g the c e l l s . The absorbance of the diazo-dye i n sea water of s a l i n i t y 30.4 %«. was found to be about 42% of that i n d i s t i l l e d water. 36. REFERENCES Atkins, W. R. G. 1957. The d i r e c t estimation of ammonia i n sea water, with notes on n i t r a t e , copper, zinc and sugars. J . c o n s e i l permanent int e r n , exploration mer. 2_2, 271-277. Bendschneider, B. and R. J . Robinson. 1952. A new spectrophotometry determination of n i t r i t e i n sea water. J . Mar. Research (Sears Foundation) 11, 87-96. Bradbury, J . H. 1960. A dinitrophenylation method f o r the microdetermination of amide nitrogen. Anal. Chim. Acta 22, 444. G i l l b r i c h t , M. 1961. Detection of ammonia i n sea water. Helgolaender Wiss. Meeresuntersuch. 8^  58-67. Krogh, A. 1934. Method f o r determination of 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. Mellon, 1953. Colorimetric determination of ammonia and cyanate. Anal. Chem. 25_, 1188-1192. Levy, A. L. 1954. A paper chromatographic method f o r the quantitative estimation of amino acids. Nature 174, 126. Lockhart, I. M. i956. Paper chromatographic i d e n t i f i c a t i o n of 2:4-dinitrophenyl d e rivatives of a l i p h a t i c amines. Nature 177, 393. Newell, B. and G. Dal Pont. 1964. Ammonia i n sea water. Nature 201, 36-37. Palmork, K. H. 1962. The use of 2:4-dinitro, 1-fluorobenzene i n the separation and i d e n t i f i c a t i o n of amino acids from sea water. Acta Chem. Scand. 37, 1456. Pataki, G. 1964. Thin layer chromatography f o r the sequence analysis of peptides. I I I . Non-destructive detection of amino acids on t h i n layer chromatograms. J . Chromatog. Ii5, 541-543. Peraino, C. and A. E. Harper. 1961. Quantitative paper chromatography of plasma amino acids, modification of the dinitrophenylation procedure of Levy. Anal. Chem. 33, 1863-1865. Rapp, R. 1963. Determination of serum amino acids. C l i n . Chem. 9, 27-30. Richards, F. A. and R. A. Kletsch, 1964. The spectrophotometry determination of ammonia and l a b i l e amino compounds i n fresh and sea water by oxidation to n i t r i t e . Recent Res. F i e l d s Hydrosphere. Atmosphere 'Nucl. Geochem. 65-81. R i l e y , J . P. 1953. The spectrophotometric determination of ammonia i n natural water with p a r t i c u l a r reference to sea water. Anal. Chim. Acta 9, 575-589. R i l e y , J . P. and P. Sinhaseni. 1957. The determination of ammonia and t o t a l i o n i c inorganic nitrogen i n sea water. J . Mar. B i o l . Assoc. 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 determination of ammonia i n sea wtaer. Anal. Chim. Acta 30, 56-59. Sandell, E. B. 1950. Colorimetric determination of traces of metals. Interscience, New York. 673 pp. Sanger, F. 1945. The free amino groups of i n s u l i n . Biochem. J . '39, 507-515. Str i c k l a n d , J . D. H. and H. K. Austin. 1959. The d i r e c t estimation of ammonia i n sea water with notes on " r e a c t i v e " i r o n , n i t r a t e s and inorganic phosphorus. J . Conseil permanent i n t e r n , exploration mer 24, 446-451. Wirth, H. E. and R. J . Rohinson. 1933. Photometric i n v e s t i g a t i o n of Nessler reaction and Wittig method f o r determination of ammonia i n sea water. Ind. Eng. Chem., Anal. Section, 5_, 293-296. 

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