Nitrate-selective electrodes with covalently bound sensors.

Transcription

1 Nitrate-selective electrodes with covalently bound sensors. KING, Beverley Ann. Available from Sheffield Hallam University Research Archive (SHURA) at: This document is the author deposited version. You are advised to consult the publisher's version if you wish to cite from it. Published version KING, Beverley Ann. (1985). Nitrate-selective electrodes with covalently bound sensors. Doctoral, Sheffield Hallam University (United Kingdom).. Copyright and re-use policy See Sheffield Hallam University Research Archive

2 I X TELEPEN Sheffield City Polytechnic Library REFERENCE ONLY

3 ProQuest Number: All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if m aterial had to be rem oved, a note will indicate the deletion. uest ProQuest Published by ProQuest LLC(2017). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e Microform Edition ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml

4 A th e s is e n t it le d NITRATE-SELECTIVE ELECTRODES WITH COVALENTLY BOUND SENSORS. presented by BEVERLEY ANN KING in p a r t - f u l f i l l m e n t o f the requ ire m e n ts f o r the degree o f DOCTOR OF PHILOSOPHY o f the COUNCIL FOR NATIONAL ACADEMIC AWARDS Department o f Chemistry S h e ffie ld C ity P o ly te c h n ic Pond S tre e t S h e ffie ld S1 1WB In c o lla b o r a tio n w ith : Kent I n d u s t r i a l Measurements A n a ly t ic a l In stru m e n ts Hanworth Lane C hertsey KT16 9LF September, 1985

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6 ABSTRACT N itr a t e d e te rm in a tio n and io n -s e le c tiv /e e le c tro d e s, w ith p a r t ic u la r emphasis on the n i t r a t e s e le c tiv e e le c tr o d e, are review ed. A new e le c tro d e fo r n i t r a t e has been developed, by c o v a le n t attachm ent o f a p p ro p ria te sensor groups to a ro b u st polymer m a trix, w ith a view to im proving th e l i f e t i m e and the a p p lic a tio n s o f the e le c tro d e to in c lu d e h o s t ile environm ents. The QAS ( I ) were prepared as sensor m a te r ia ls. (1) r «= R" = r = e th y l (2) R» = a l l y l R" = Rm = m eth yl, e th y l, p ro p y l (3) R* = R" = a l l y l RIM = m eth yl, e th y l, p ro p y l, b u ty l (4) R1 = R" = R1" = a l l y l X" = Cl, B r. The p ro d u c tio n o f membranes by c r o s s - lin k in g the sensors ( i ) w ith s ty re n e -b -b u ta d ie n e -b -s ty re n e t r i b l o c k elastom er (SBS) using a fre e r a d ic a l i n i t i a t e d c y c lo p o ly m e ris a tio n i s d e s c rib e d. V arious methods o f membrane p re p a ra tio n have been in v e s tig a te d, and a convenient one-step s o lv e n t c a s tin g procedure i d e n t i f i e d and o p tim is e d. A range o f membranes has

7 been prepared and t h e ir p h y s ic a l and e le c t r o a n a ly t ic a l p r o p e rtie s e va lu a te d. The c r o s s -lin k e d d e n s ity o f th e polym ers was c a lc u la te d from s w e llin g measurements and the F lo ry-r e h n e r e q u a tio n, the e x te n t o f c o v a le n t a tta c h m e n t has been d e riv e d from K je ld a h l a n a lyses. The perform ance o f th e membranes as io n - s e le c t iv e e le c tro d e s i s p resen ted and d iscu sse d. S everal e le c tro d e s showed f a s t response, long l i f e t i m e and N e rn s tia n beh a vio u r in the range mol dm, b u t t h e i r s e l e c t i v i t y was i n f e r i o r to p re se n t com m ercial e le c tro d e s. A number o f m a te ria ls were in tro d u c e d as m e d ia to rs in o rd e r to im prove the s e l e c t i v i t y o f the membranes. A p o s s ib le m e d ia to r ( O - n itr o p h e n y lo c ty l e th e r) was id e n t i f i e d and an analogue (o -n itro p h e n y l-w -u n d e c y le n y l e th e r) prepared which had s u ita b le f u n c t i o n a l i t y f o r co va le n t attachm ent to th e polym er. U n fo rtu n a te ly the in c lu s io n o f t h is m a te ria l d id n o t im prove th e s e l e c t i v i t y to c h lo rid e and the reasons f o r t h i s are discussed.

8 CONTENTS Page No. A b s tra c t. CHAPTER 1. CHAPTER 2. In tr o d u c tio n 1.1 N itr a t e - Cause f o r Concern? Methods o f N it r a t e A n a ly s is D ire c t S p e ctro p h o to m e tryi S p e c tro p h o to m e tric Methods using Chromogenic Reagents The Reduction o f N itr a t e to Ammonia Enzym atic Methods Chrom atographic Methods M isce lla n e o u s Methods Io n -S e le c tiv e E le c tro d e Methods N itr a t e - S e le c tiv e E le c tro d e s P r in c ip le s o f Io n - S e le c tiv e E le c tro d e s The E a r lie s t E le c tro d e s N itr a t e E le ctro d e s uiith L iq u id Membranes Polymer Membrane E le c tro d e s Coated-W ire E le c tro d e s Bound Ion-Exchanger E le c tro d e s Aims o f t h is Work. 39 Choice and P re p a ra tio n o f M a te ria ls and P ro d u ctio n o f Membranes. 2.1 The Polym er The Sensor P re p a ra tio n o f Q uaternary Ammonium S a lts E xp e rim e n ta l Methods T e r t ia r y Amines Q u a te rn is a tio n D ia lly ld ie th y la m m o n iu m C h lo rid e D ia lly ld ie th y la m m o n iu m Brom ide T ria lly lb u ty la m m o n iu m Bromide A lly ltrie th y la m m o n iu m Brom ide. 57

9 Page No C h a ra c te ris a tio n P ro d u ctio n o f Membranes P re p a ra tio n o f Reagents P o ly (s ty re n e -b -b u ta d ie n e -b -s ty re n e ) ( SB S ) ,2 azo b is ( 2- m e t h y lp r o p r io n it r ile ) 'Ca b i n ) te r t- B u ty lh y d ro p e r o x id e (tbhp) T e tra h y d ro fu ra n (THF) M ethanol Q uaternary Ammonium S a lts Methods o f Membrane P re p a ra tio n S olvent C a stin g Hot P re s s in g The Use o f te rt- B u ty lh y d ro p e r o x id e I n i t i a t o r. 75 CHAPTER 3. Techniques f o r Membrane E v a lu a tio n. 3.1 E le c t r o a n a ly t ic a l P r o p e rtie s Membrane P o te n tia ls and th e N ernst E q u a tio n A c t i v i t y and A c t i v i t y C o e ffic ie n ts S e le c t iv i t y and S e le c t iv i t y C o e ff ic ie n t s Mixed S o lu tio n Method Separate S o lu tio n Method E x p e rim e n ta l Equipment E le c tro d e C o n s tru c tio n The 'In - h o u s e 1 Design E le c tro d e The Sensor U n it The Commercial E le c tro d e Reference E le c tro d e s M ete rs A n c illa r y Equipment E xp e rim e n ta l P rocedures. 96

10 . Page No C o n d itio n in g. 96 Standard S o lu tio n s.96 CM CM CM In te rfe re n c e Work C a lib ra tio n Technique C ro s s -lin k e d D e n s ity C ro s s -L in k in g o f Polymer Chains C a lc u la tio n o f C ross-linked D e n s ity The F lory-r e hner E quation E xp e rim e n ta l D e te rm in a tio n o f Swollen W eight. 104 D e te rm in a tio n o f D e n sity. 104 CM C a lc u la tio n o f C ro s s -lin k e d D e n s it y K je ld a h l A n a ly s is E xp e rim e n ta l Reagents Method R esistance Measurements. 108 CHAPTER 4. E v a lu a tio n o f the Membranes. 4.1 S olvent Cast Membranes S olvent Cast Membranes w ith In cre a se d Q uaternary Ammonium S a lt Hot Pressed Membranes Membranes Prepared w ith t e r t - B u ty lh y d ro p e ro x id e (tbhp) I n i t i a t o r Comparison o f D iff e r e n t Q uaternary Ammonium S a lts as N itr a t e Sensors 140 CHAPTER 5. The Use o f M e d ia to rs. 5.1 In tr o d u c tio n Choice o f S o lven t M edia to r D ie le c t r ic Constant S olvent M e d ia to rs used in N itr a t e E le c tro d e s I n i t i a l In v e s tig a tio n s. 153

11 Page No. CHAPTER 6. References L is t o f Tables L i s t o f F igures L i s t o f P la te s Acknowledgements APPENDIX 1. APPENDIX 2. S olvent C asting w ith M e d ia to rs. 160 S e le c tio n and P re p a ra tio n o f a M edia to r f o r Covalent A ttachm ent. 161 E xp e rim e n ta l. 163 P re p a ra tio n o f w -U ndecylenyl p -to lu e n e s u lp h o n a te. 163 P re p a ra tio n o f o -N itro p h e n y l-w - u n d e cyle n yl e th e r. 163 E v a lu a tio n o f o -N itro p h e n y l-w - u n d e cyle n yl e th e r as a M e d ia to r. 165 P re p a ra tio n o f Membranes w ith C o v a le n tly Bound M e d ia to r. 167 Two-Stage S o lven t C a sting. 170 The Response o f Membranes Prepared w ith tbhp I n i t i a t o r to C h lo rid e and S u lph ate. 175 The Response o f Membranes to C h lo rid e. 176 The Response o f Membranes to S u lph ate. 182 C onclusion. 182 The A r a ld it e E le c tro d e. 187 C onclusion and Suggestions f o r Future Work. The C ross-linked D ensity C a lc u la tio n s. 192 The E q u ilib riu m Swollen W eight. 192 The Polymer S o lven t In te r a c tio n Param eter. 193 The Polymer S tru c tu re. 201 S e le c t iv i t y. 212 M e d ia to rs f o r Polymer Membranes. 215 S o lid Ion-E xchangers. 215 L iq u id Ion-E xchangers. 216 Polymer Membranes. 217 C onclusion. 222 BASIC Program f o r th e c a lc u la tio n o f s,v and c ro s s -lin k e d d e n s ity. FORTRAN Program and r e s u lt s f o r th e c a lc u la tio n o f c r o s s -lin k e d d e n s ity when

12 APPENDIX 5. APPENDIX 4. R e su lts f o r the c a lc u la tio n o f c r o s s - lin k e d d e n s ity when / 6= Course o f stu d y. Papers P u b lish e d and P resented.

13 CHAPTER 1 INTRODUCTION 1.1 N itr a t e - Cause f o r Concern? The amount o f n i t r a t e to which the p o p u la tio n i s exposed has ris e n in modern tim e s. The use o f agrochem icals and th e a p p lic a tio n o f sewage sludge to la n d, have c o n trib u te d s u b s t a n t ia lly to t h is in c re a s e ; n i t r a t e is also used in foods as a p re s e rv a tiv e and as an a d d itiv e in d e te rg e n ts. The t o x i c i t y o f n i t r i t e is w e ll re c o g n is e d. I t i s known to cause in f a n t m ethoglobinem ia (b lu e baby syndrome) by o x id a tio n o f haem oglobin. Concern is now being expressed about th e t o x i c i t y o f n i t r a t e. Ingested n i t r a t e may be reduced by b a c te ria to n i t r i t e, which may re a c t in v ivo w ith secondary n itro g e n compounds to form N -n itro s o compounds. N it r a t e is p re se n t in food and d rin k in g w a ter, secondary amines are in g e s te d in f is h and o th e r fo o d s. The re a c tio n is a c id c a ta ly s e d, w ith an optimum ph 2-4,- making the human stomach an id e a l lo c a tio n f o r the p ro d u c tio n o f N -n itro s o compounds. These compounds have been re co g n ise d as p o w e rfu l carcinogens f o r s e v e ra l ye a rs. In view o f the a c tio n o f n i t r a t e in v iv o th e c o n t r o l o f n i t r a t e in d rin k in g w a te r, a u n iv e r s a l source o f n i t r a t e, 2 is now e s s e n tia l. The E.E.C. r e g u la tio n s l i m i t th e maximum c o n c e n tra tio n o f n i t r a t e n itro g e n (NO^.N) in

14 _ 1 p o ta b le ujater to 11.3 mg N 1. The c u rre n t n i t r a t e le v e ls in t h is c o u n try are u jith in the EEC l i m i t s, w ith the p o s s ib le e x c e p tio n o f s m a ll areas w ith n a t u r a lly o c c u rrin g h igh le v e ls o f n i t r a t e. However, the le v e ls o f n i t r a t e in s u rfa c e w aters f lu c t u a t e and may show seasonal maxima which exceed the EEC r e g u la tio n s. At p re se n t w ater a u t h o r it ie s are a ble to b lend w aters w ith high n i t r a t e le v e ls, w ith groun d-w ater s u p p lie s which are u n p o llu te d. The a g r i c u l t u r a l i s t s defend t h e i r use o f chem ical f e r t i l i s e r s on the basis t h a t o n ly s u rfa c e w aters are a ffe c te d, b u t long term m o n ito rin g o f ground w ater s u p p lie s, such as chalk a q u if ie r s, show th a t a steady in c re a s e in n i t r a t e le v e ls is now apparent."^ Thames Water A u th o r ity r e c e n tly p u b lis h e d the r e s u lt s o f t h e i r n i t r a t e s tu d ie s.^ They h ig h lig h t e d th e need to m o n ito r and c o n tr o l th e le v e ls o f n i t r a t e and, u lt im a t e ly, th e source o f the n i t r a t e p o l lu t i o n. A lth o u g h the expense in c u rre d by such a c t i v i t i e s would r e q u ire s u b s ta n tia l in v e s tm e n t, the cost o f p u r if y in g a l l w ater s u p p lie s by the removal o f n i t r a t e should the le v e ls c o n tin u e to r is e, would be enormous in com parison. The c u rre n t e n q u irie s concerning the problem o f a c id r a in h ig h lig h t a new type o f p o llu t io n which in v o lv e s n i t r a t e and re q u ire s a r e li a b l e method o f m o n ito rin g i t.

15 The development o f a r a p id, v e r s a t ile, a c c u ra te, s e n s itiv e, s e le c tiv e and cheap method o f n i t r a t e a n a ly s is in w ater and food is now o f prim e im portance fo r m o n ito rin g e n viro n m e n ta l c o n d itio n s. 1.2 Methods o f N itr a t e A n a ly s is The d e te rm in a tio n o f n i t r a t e can be accom plished by a number o f methods: d ir e c t sp e ctro p h o to m e try ; s p e c tro - p h o to m e tric methods using chromogenic re a g e n ts; re d u c tio n o f n i t r a t e to ammonia and i t s d e te c tio n ; enzym atic methods; chrom atographic methods; and io n - s e le c t iv e e le c tro d e methods. These methods are review ed in th e fo llo w in g pages D ire c t S pectrophotom etry N itr a t e io n shows a stro n g a b s o rp tio n in the fa r u l t r a - v i o l e t (U. V/.) w ith a peak a t 202nm. In the absence o f substances which also absorb in t h is re g io n n i t r a t e can be determ ined r a p id ly by d ir e c t measurement. N o n -sa lin e w a te rs, low in o rg a n ic m a tte r, and c e r ta in waste waters w ith high le v e ls o f n i t r a t e, are 5 analysed by t h is method. The range o f a p p lic a tio n is _ mg 1 in a sample volume o f 40ml, th e low er l i m i t is dependent on the absence o f i n t e r f e r e n t s. The ra te o f a n a ly s is is a p p ro x im a te ly 10 samples per h ou r. L ig h t s c a tte r by u n d isso lve d p a r t ic le s is a source o f e rro r in the U.V. re g io n. Accurate d e te rm in a tio n s re q u ire c le a r, b r ig h t samples; f i l t r a t i o n is necessary to e lim in a te suspended m a tte r and

16 t u r b i d i t y. N i t r i t e absorbs over a s im ila r wavelength range to n i t r a t e, but is removed by the a d d itio n o f sulpham ic a c id. Carbonate and h y d ro x y l io n s in t e r f e r e, but are e lim in a te d by a c i d i f i c a t i o n. D isso lve d ir o n and o th e r heavy m etals may also cause in te r f e r e n c e. C h lo rid e and brom ide show a b s o rp tio n in the U.V. re g io n o f 200nm and below. The d e te c tio n o f n i t r a t e a t 21Cnm is u s u a lly chosen to m in im ise c h lo rid e in te r f e r e n c e, w h ils t rem a ining clo se to the peak maximum fo r n i t r a t e. A c o r re c tio n fo r U.V. abso rb ing substances o th e r than n i t r a t e can be made by m easuring absorbance o f samples a t 2 w a vele ngths, 210nm and 275nm. N itr a t e should have no s ig n if ic a n t absorbance a t the h ig h e r w avelength i f p re s e n t in s u ita b le c o n c e n tra tio n s f o r t h i s method. A method f o r n i t r a t e d e te rm in a tio n in fre s h and s u ita b le e s tu a rin e w a te rs, by U.V. a b s o rp tio n was proposed by Brown and B e llin g e r. ^ N a tu ra l o rg a n ic p o llu t a n t s, which cause in te r fe r e n c e in the U.V. re g io n were removed by passing the sample thro ugh an ion-exchange r e s in, c h lo rid e le v e ls were c o n tr o lle d by p r e c ip it a t io n w ith s il v e r n i t r a t e, and n i t r i t e was c o n tr o lle d w ith sulpham ic a c id. The method a llo w e d 10 to 15 samples per hour to be analysed and was p re c is e and a c c u ra te in the range m g 1_ ^N0-^.N.

17 A s im ila r a d a p ta tio n iuas used by Rennie e t a l., f o r the d ir e c t d e te rm in a tio n o f n i t r a t e in raw, p o ta b le and waste w a te rs. An a c tiv a te d carbon f i l t e r a t e le v a te d ph was used to e lim in a te in te r fe r e n c e from o rg a n ic m a tte r. The procedure also removed in te r fe r e n c e s from a number o f c a tio n s which were p r e c ip it a t e d. The l i m i t o f d e te c tio n was 0.006mg 1, and the method compared fa v o u ra b ly w ith e s ta b lis h e d autom ated methods f o r a wide range o f sam ples. g Norman and S tu c k i developed a method f o r n i t r a t e d e te rm in a tio n in s o i l by m easuring t o t a l absorbance o f a sample a t 210nm. The n i t r a t e p re s e n t in the sample was then reduced to non^absorbing species using Raney n ic k e l c a ta ly s t in an a c id medium. The U.V/. absorbance was then determ ined, the d iffe r e n c e between the two re a d in g s was a t t r ib u t e d to n i t r a t e c o n c e n tra tio n. N i t r i t e was removed from samples using sulpham ic a c id. This method was s u ita b le -1 fo r IOOyt/g NO^.N g o f s o i l S p e c tro p h o to m e tric Methods using Chromogenic R eagents. N itr a t e can also be determ ined in v a rio u s samples by i t s re a c tio n w ith an o rg a n ic reagent to form a c o lo u re d compound which is then d e te cte d s p e c tr o p h o to m e tr ic a lly. The re a c tio n o f n i t r a t e w ith s u lp h o s a lic y lic a c id produces a y e llo w compound in the presence o f a l k a l i. The absorbance a t 415nm is p r o p o r tio n a l to n i t r a t e c o n c e n tra tio n. T his method is used f o r th e d e te rm in a tio n o f n i t r a t e in raw, p o ta b le and waste w a te rs. The lin e a r

18 _ 1 range o f th e method i s mg 1 on in te r fe r e n c e to ( 0. 2mg 1 _ 'j depending & the ra te o f a n a ly s is is 1-3 hours f o r s ix samples. The main in t e r f e r e n t s are c h lo r id e, o rth o p h o sp h a te, magnesium and manganese ( i l ) S everal methods are re p o rte d in th e l i t e r a t u r e * f o r the d e te rm in a tio n o f n i t r a t e w ith 2, 6- x y le n o l ( 2, 6- d im e th y lp h e n o l). N itr a t e in an a c id ic s o lu tio n re a cts w ith 2,6 - x y le n o l to form 4 - n i t r o - 2, 6- x y le n o l which shows an a b s o rp tio n maximum a t nm. N i t r i t e and c h lo rid e in t e r f e r e w ith the re a c tio n.t h e method is u s e fu l fo r d e te rm in a tio n o f n i t r a t e n itro g e n in the p a rts per m ill io n range (5-29ppm). 10 Andrews a lso used 2,6 - x y le n o l f o r the d e te rm in a tio n o f n i t r a t e. The p ro d u ct o f the r e a c tio n w ith n i t r a t e was e x tra c te d w ith to lu e n e and the absorbance a t 432nm measured. The method was used fo r c o n c e n tra tio n s o f jJLq N03.N I " An a lt e r n a t iv e method f o r d e te rm in a tio n o f n i t r a t e in w ater in v o lv e s th e conversion o f the e x tra c te d phenol to phenoxide b e fo re re c o rd in g the spectrum from 600 to 4G0nm. The absorbance is d e fin e d as the d iffe r e n c e between the 428nm peak and the p la te a u re a d in g between nm caused by t u r b i d i t y. A lin e a r r e la t io n s h ip between absorbance and n i t r a t e c o n c e n tra tio n over the ranqe /jg 1 1 5{Jttg 1 ' was re p o rte d, w ith a l i m i t o f d e te c tio n a t

19 2,4 -X y le n o l has also been used f o r s p e c tro p h o to m e tric d e te rm in a tio n o f n i t r a t e, 12 b u t the re a c tio n p ro d u ct had the disadvantage o f r e q u irin g s e p a ra tio n by d i s t i l l a t i o n. The re a c tio n w ith n i t r a t e produced 6- n i t r o - 2, 4 - x y le n o l in s u lp h u r ic a c id which was then d i s t i l l e d in to an ammoniacal w a te r/is o p ro p a n o l m ix tu re. The in te n s e y e llo w c o lo u r produced was m onitored a t 455nm. In te rfe re n c e s from n i t r i t e and c h lo rid e were e xp e rie n ce d. The method was used fo r samples c o n ta in in g mg MO^.N. A method f o r th e d e te rm in a tio n o f n i t r a t e in p la n ts, 13 s o ils and w aters used th e reage nt 3, 4 -d im e th y lp h e n o l. High to le ra n c e to in t e r f e r e n t s and a p p l i c a b i l i t y to a wide range o f NO^.N values were re q u ire d f o r the a n a ly s is o f such samples. In sta n ta n e o u s n i t r a t i o n o f ' 3, 4 - d im e th y l- phenol was achieved in the presence o f s u lp h u r ic a c id. C h lo rid e in te r fe r e n c e was c o n tr o lle d by p r e c ip it a t io n w ith s i l v e r s u lp h a te. The n i t r a t i o n p ro d u ct was d i s t i l l e d in to a s o lu tio n o f sodium h y d ro x id e in e th a n o l, the absorbance o f t h is y e llo w s o lu tio n was recorded a t 430nm. The method was s u ita b le f o r p la n t tis s u e samples c o n ta in in g _ 'I yCtg g~ MO^.N (d ry w e ig h t), s o i l samples c o n ta in in g /dg g i\m3.i\i. -1 NOj.N, and w ater samples c o n ta in in g 1-20/fg ml

20 The method mas la t e r m o d ifie d f o r the d e te rm in a tio n o f n i t r a t e in ra in -m a te r, 14 by removing the d i s t i l l a t i o n s te p. N it r a t io n o ccu rre d in s ta n ta n e o u s ly a t 0 C in 8 0 ;6 s u lp h u r ic a c id, the p ro d u ct mas e x tra c te d in t o to lu e n e. The e x tra c ta n t mas tre a te d m ith sodium h y d ro x id e to form a co lo u re d p ro d u c t, th e absorbance o f mhich mas recorded _ -i at 432nm. The samples contained # g ml n i t r a t e. The advantages of using phenol as a re a g e n t in the d e te rm in a tio n o f n i t r a t e mere o u tlin e d by E lto n - B o tt; phenol is cheap and r e a d ily a v a ila b le in high p u r i t y, 15 the m olar a b s o r p t iv it y f o r th e n i t r o d e r iv a tiv e o f phenol a t 410nm is h ig h e r, the to le ra n c e le v e l o f th e re a c tio n f o r c h lo rid e is a ls o h ig h e r. Phenol mas used m ith d ilu t e d s u lp h u r ic a c id to produce n itr o - p h e n o Is, p re d o m in a n tly o rth o and para, mhich mere e a s ily d i s t i l l e d. The method mas s u ita b le f o r p la n t m a te ria ls, s o ils and m ater m ith a mide range o f n i t r a t e c o n te n t. 16 Tanaka e t a l., d e scrib e d a procedure f o r the d e te rm in a tio n o f n i t r a t e in ve g e ta b le pro d u cts based on the q u a n tita tiv e n i t r a t i o n o f 2-s e c -b u ty lp h e n o l in s u lp h u r ic a c id. The n i t r a t i o n p ro d u ct mas e x tra c te d, and the absorbance o f the yellom compound formed in a lk a lin e medium mas measured. The c o lo u r re a c tio n mas s e n s it iv e and s ta b le. The absorbance measured a t 418nm obeyed Beers Lam _ 1 betmeen 0.13 to 2.5CjAtg ml NO-^.N m ith a d e te c tio n l i m i t o f 1.3ppm.

21 The method uias not a ffe c te d by normal in t e r f e r e n t s from v e g e ta b le produ cts and showed high to le ra n c e to 17 n i t r i t e. Makamura proposed a method o f n i t r a t e d e te rm in a tio n in which c h lo rid e io n, in the presence o f s u lp h u r ic a c id (15l\l), c a ta ly s e d the re d u c tio n o f n i t r a t e to n i t r i t e. A s e le c tiv e re a c tio n w ith a s o lu tio n o f 4, 5-dih yd ro xyco u m a rin in e th y l a ce ta te produced a coloured compound which was m onitored a t 382nm. The method showed high to le ra n c e to common in t e r f e r e n t s b u t p r io r d e te rm in a tio n o f n i t r i t e was re q u ire d. M itr a te was determ ined r a p id ly in d rin k in g w ater samples in the range ppm NO^.N. 1 8 l/elghe and Claey used 18-19N s u lp h u ric a c id, c o n ta in in g c h lo rid e io n s, to generate the v o l a t i l e n i t r o s y l c h lo rid e. R eaction w ith phenol produced p -n itro p h e n o 1 in e q u ilib r iu m w ith the y e llo w p-benzoquinone monoxime. The coloured compound was m onitored a t 388nm. The re a c tio n tim e was a p p ro x im a te ly 3 min. In te rfe r e n c e s from n i t r i t e, s u lp h a te, iro n ( i l l ) and io d id e were m in im ise d or e lim in a te d. 5 A w id e ly recommended method f o r the d e te rm in a tio n -1 o f n i t r a t e in the range \julq 1 NC^.N, common le v e ls fo r n a tu r a l w a ters, in v o lv e s the heterogeneous re d u c tio n o f n i t r a t e w ith copperised cadmium, z in c, cadmium o r cadmium amalgam fo llo w e d by i t s d ia z o t is a t io n and c o u p lin g w ith a s u ita b le reagent to form a coloured complex. The

22 r e a c tio n is m onitored s p e c tr o p h o to m e tr ic a lly. The p r in c ip le s o f t h i s method have been adapted to continuous flouj apparatus' to form the b a sis o f com m ercial a u to a n a ly s e rs f o r n i t r a t e and t o t a l o x id is e d n itro g e n (TON). An e a rly a u to m a tic method f o r the d e te rm in a tio n o f n i t r a t e and n i t r i t e in fre s h or sea w a ter, and s o i l 19 e x tra c ts was d e scrib e d by H enrikson and S e lm e r-o lse n. N itr a t e was reduced w ith copperised cadmium, the n i t r i t e produced d ia z o tis e d s u lp h a n ila m id e which then coupled w ith N -1 -n a p h th y le th y le n e d ia m in e. The p ro d u c t, a h ig h ly coloured azo-dye was dete cte d a t 520nm by U.\/. s p e c tro photom etry. P re d e te rm in a tio n o f n i t r i t e was re q u ire d. A d ia ly s e r in c o rp o ra te d in to the system prevented c lo g g in g o f the re d u c to r column. The method was used to determ ine -1 n i t r a t e in s o i l e x tra c ts in the range mg 1 NO^.N, and in water samples in the range mg 1 NO^.N. A manual method f o r the d e te rm in a tio n o f tra c e amounts 20 o f n i t r a t e in r iv e r w ater in v o lv e d re d u c tio n by a cadmium-copper column, d ia z o tis a tio n o f p-am inoacetophenone, and co u p lin g w ith m -phenylenediam ine. The f i n a l p ro d u ct ^ was m onitored s p e c tro p h o to m e tric a lly a t 460nm. The method -1 was used to determ ine n i t r a t e a t le v e ls o f < 1mg 1 NO^.N. Sim ultaneous d e te rm in a tio n o f n i t r a t e and n i t r i t e 21 by flo w in je c t io n a n a ly s is was re p o rte d by Gine et a l.

23 N itr a t e urns reduced to n i t r i t e w ith a copp erised cadmium column, the n i t r i t e uas d ia z o tis e d and coupled w ith N -(1 - n a p h th y l) ethyldiam m onium d ic h lo r id e. The p r e c is io n mas 1.5^ in the range m g 1 n i t r a t e, ujith a _ 'i sam pling ra te o f 90h. The system was la t e r adapted to a standard a d d itio n method f o r the a n a ly s is o f n i t r a t e in 22 p la n t e x tra c ts. The standard a d d itio n method was re q u ire d fo r such samples to overcome m a trix e f fe c ts. A d is c re te a n a ly s e r was used fo r the d e te rm in a tio n o f n i t r a t e in la ke w aters 23 c o n ta in in g high c o n c e n tra tio n s o f d is s o lv e d o rg a n ic m a tte r. The method in v o lv e d re d u c tio n w ith copper and h y d ra z in e, fa llo w e d by r e a c tio n w ith su lp h a n ila m id e and c o u p lin g w ith N -T -n a p h th y le th y le n e diam ine. The copper c a ta ly s t was p ro te c te d from c h e la tio n w ith o rg a n ic m a tte r, by using excess z in c io n s in th e system. The t o t a l a n a ly s is tim e was below 13min. w ith a th ro u g h p u t o f 240 samples per hour in th e range /fg 1 N03.N. A manual method f o r the d e te rm in a tio n o f tra c e amounts o f n i t r a t e and n i t r i t e in w ater was developed 24 in which f r e s h ly prepared cadmium sponge was used f o r the ra p id re d u c tio n o f n i t r a t e to n i t r i t e. At ph2 n itr o u s a c id was formed which d ia z o tis e d w ith p-am inoacetophenone which then coupled w ith N -(1- n a p th y l) - e th y le n e d ia m in e. The azo-dye was e x tra c te d in to b u ta n o l in the presence o f n a p h th o su lp h o n ic a c id and A l(n 03 )^, The absorbance was measured a t 550nm. Sodium metaphosphate was used as

24 a masking agent to p re ve n t in te r fe r e n c e from o th e r io n s. The d e te rm in a tio n o f n i t r a t e in the c o n c e n tra tio n -1 range 2-100ytfg NO^.N 1 by Gaugush and Heath. 25 by manual methods was d e scrib ed The method was s u ita b le f o r s m a ll samples 5ml, and the re a c tio n was c a r rie d out in a te s t tube. The re a c tio n in v o lv e d re d u c tio n w ith cadmium amalgam, re a c tio n w ith s u lp h a n ila m id e and N -( 1- n a p h th y l) - ethyle n e d ia m in e d ih y d ro c h lo rid e and measurement o f the absorbance a t 543nm. A g la s s y carbon, c o lu m n,e le c tro d e was used to reduce n i t r a t e in a flo w e le c t r o ly s is method. 26 N i t r i t e produced on th e column was mixed w ith a s u lp h a n ila m id e and f \ l - (l-n a p h th y l)e th y le n e d ia m in e to produce a c o lo u re d p ro d u c t. The absorbance o f the p ro d u ct was measured a t 540nm. The method was used f o r n i t r a t e d e te rm in a tio n in f i l t e r e d, degassed samples o f r i v e r w ater a f t e r com bin ation w ith a phosphate b u ffe r s o lu tio n c o n ta in in g copper and cadmium io n s. N i t r i t e was prede term ined. Low values o f n i t r a t e were experienced p o s s ib ly as a r e s u lt o f f u r t h e r re d u c tio n o f n i t r i t e by e le c t r o ly s is. The methods discussed above p ro v id e a c c u ra te and s e n s itiv e means f o r the d e te rm in a tio n o f n i t r a t e in v a rio u s enviro nm ental samples. However a l l the methods r e q u ire the use o f one or more unpleasant re a g e n ts ; s k in c o n ta c t w ith

25 I\j«(1 -n a p h th y l)e th y le n e d ia m in e h y d ro c h lo rid e and h yd ra zin e su lp h a te should be avoided ; cadmium m etal and i t s s a lts are t o x ic. Sample p re p a ra tio n and m a n ip u la tio n are im p o rta n t param eters to be considered when s e le c tin g a method f o r r o u tin e ana lyse s. The methods d e scrib e d in v o lv e m u ltis te p re a c tio n s which can be tim e consuming, and c o s tly in re a g e n ts. Some in v o lv e the use o f s p e c ia lis e d equipment which in cre a se s the ra te o f sam pling, b u t does not reduce the o v e r a ll re a c tio n tim e The R eduction o f N itr a te to Ammonia The re d u c tio n o f n i t r a t e to ammonia is an a lt e r n a t iv e method o f n i t r a t e d e te rm in a tio n and is a lso s u ita b le f o r t o t a l o x id iz e d n itr o g e n. 5 The recommended method in v o lv e s the d i s t i l l a t i o n o f ammonia from a sample made a lk a lin e w ith magnesium o x id e. D evarda's a llo y is then added to th e sample and d i s t i l l a t i o n c o n tin u e d. The ammonia produced from the re d u c tio n o f o x id iz e d n itro g e n is absorbed by b o r ic a c id s o lu tio n and t i t r a t e d a g a in s t standard a c id. _ 'i I t is s u ita b le f o r n i t r a t e in the range 12-40mg 1 NO-^.N, the a n a ly s is tim e being 1 hour. The method may o n ly be used f o r n i t r a t e in the absence o f n i t r i t e, or i f n i t r i t e is p re de term ined.

26 1.2.4 Enzymatic Methods The enzyme n i t r a t e reductase s e le c t iv e ly reduces n i t r a t e to n i t r i t e. A lthough the enzyme is s p e c if ic, the re a c tio n c o n d itio n s i t re q u ire s are complex; a s u ita b le c o fa c to r i s re q u ire d f o r the 27 re a c tio n to proceed. The r a d ic a l c a tio n o f 1,1 1- d im e th y l- 4,4 1- b ip y r id in iu m d ic h lo r id e (MU**1") (m ethyl v io la g e n ), which can be generated by th e a c tio n o f [^3 2 5 ^0^ on a cts as an e le c tro n donor - an a r t i f i c i a l c o fa c to r. N itr a t e was reduced to n i t r i t e in the presence o f the r a d ic a l c a tio n and n i t r a t e re d u c ta s e. The n i t r i t e produced by the enzyme was m onitored by re a c tio n w ith s u lp h a n ilia m id e and N -(1-n a p h th y l)e th y le n e d ia m in e d ic h lo r id e to form a coloured azo dye. The r e a c tio n was perform ed in a contin uous flo w system, w ith an im m o b ilise d enzyme and s p e c tro p h o to m e tric d e te c tio n a t 543nm. Samples in the c o n c e n tra tio n range 17ppb to 7ppm o f n i t r a t e were measured w ith freedom from in te r fe r e n c e. 2 8 An a lt e r n a t iv e method in v o lv e d the re d u c tio n of n i t r a t e and n i t r i t e to ammonia using n i t r a t e and n i t r i t e reductases and the r a d ic a l c a tio n MV*+as the e le c tro n donor. The ammonia generated by the re a c tio n was measured w ith an a ir-g a p e le c tro d e. N itr a t e in the -5-2 range x10 f-'l was determ ined by t h is method but n i t r i t e must be e lim in a te d. Common in t e r f e r e n t s

27 do not cause problem s, but copper ( I I ) and m ercury ( I I ) poison the enzymes. The a n a ly s is o f tra c e amounts o f n i t r a t e in w ater mas accom plished ujith NADH (n ic o tin a m id e adenine 29 d in u c le o tid e ) dependent n i t r a t e re d u cta se. NADH acted as the e le c tro n donor f o r the enzyme c a ta ly s e d re d u c tio n o f n i t r a t e. During the re a c tio n NADH was o x id is e d to NAD+, aind i t s disappearance was m onito re d f l u o r o m e t r i c a ll y. The range o f d e te rm in a tio n was 50ppb to 7.5ppm. An enzym atic method f o r the d e te rm in a tio n o f n i t r a t e in meat and f is h p rodu cts was used by Hamano e t a l?*~* f o r the range 10 to 100ppm w ith a d e te c tio n l i m i t o f 0.5ppm. N itr a t e was e x tra c te d from meat p ro d u cts w ith sodium h yd ro xid e and su b je cte d to u l t r a f i l t r a t i o n. The samples were reduced w ith r e s p ir a t o r y n i t r a t e reductase from E. c o l i. The r e s u lt in g n i t r i t e was determ ined by a d ia z o t is a t io n c o u p lin g r e a c tio n. The method was r e l a t i v e l y fre e from in te r fe r e n c e, n i t r i t e d id not a f f e c t the enzymes. 31 Kabos et a l. used a b a c t e r ia l membrane e le c tro d e fo r the d e te rm in a tio n o f n i t r a t e. B a c te ria c o n ta in in g the necessary enzymes to reduce n i t r a t e to ammonia were p o s itio n e d on the s u rfa ce o f an am m onia gas sensor supported by a d ia ly s is membrane. The b a cte riu m A zo tob acte r v in e la n d ii contained the necessary enzymes

28 and c o fa c to rs f o r the re q u ire d re a c tio n s. N itr a t e ujas reduced to ammonia by a two step process in v o lv in g n i t r a t e and n i t r i t e re d u c ta s e. The sensor showed lin e a r -5-4 response between 1x10 and 8x10 FI and a li f e t i m e o f two weeks. The e le c tro d e s showed slow response and recovery tim es because o f t h e ir dependence on the d if f u s io n o f reagents through the membrane la y e rs. A m ajor drawback o f enzym atic methods is the cost o f the enzymes. Im m obilised enzymes can be used many tim es and help to reduce the cost o f such methods. However, the s e l e c t i v i t y o f the re a c tio n depends on the type o f enzyme a v a ila b le and i t s p u r it y. B a c te ria and o th e r c e lls are a u s e fu l source o f enzymes which are e ith e r d i f f i c u l t to is o la t e, or re q u ire complex c o n d itio n s, b u t in te r fe re n c e s may occur from o th e r re a c tio n s which are ta k in g place in the c e lls Chrom atographic Methods Ion chrom atography is a r e l a t i v e l y new method fo r the a n a ly s is o f in o rg a n ic io n s. The in s tru m e n ts c o n s is t o f e f f i c i e n t io n - exchange columns through which an e lu e n t i s pumped. A sample is in tro d u c e d onto the column using an in je c t io n v a lv e. Ions from the sample are re ta in e d by the co lu m n to v a ry in g degrees and emerge a t c h a r a c t e r is t ic r e te n tio n tim e s. Suppressor columns are o fte n used to remove c a tio n s from the samples and so reduce in te r f e r e n c e or in cre a se s e n s i t i v i t y.

29 High performance liq u id chromatography (HPLC) has been used to determ ine tra c e s o f in o rg a n ic io n s. 3 2 W ell-packed a n a ly t ic a l columns, c o n ta in in g s m a ll r i g i d anion-excha nger beads at high p re ssu re, were used to achieve e f f i c i e n t s e p a ra tio n o f s e v e ra l io n s. The beads were covered w ith a m e th a c ry la te r e s in c o n ta in in g q u a te rn a ry ammonium s a lt s. a s tro n g c a tio n exchanger, Suppressor columns c o n ta in in g d iv in y lb e n z e n e copolym er, were used to exchange a l l the c a tio n s in the e lu e n t fo r p ro to n s. D e te c tio n was achieved by a c o n d u c tiv it y c e l l, th e r e fo re removal o f c a tio n s from the sample r e s u lte d in low background c o n d u c tiv ity a t s u ita b le ph. S u ccin a te /h yd ro g e n s u c c in a te o r a d ip a te /h y d ro g e n a d ip a te were s u ita b le e lu e n ts. Phosphate, brom ide, n i t r a t e and su lp h a te io n s were separated in 6m in. D e t e c t io n - lim it s were a p p ro x im a te ly 20/cg 1 fo r a /il sample, and -1 lyjug 1 when 2ml samples were p re c o n c e n tra te d. 33 Okada and Kuwamoto used nonsuppressor io n chrom atography to separate in o rg a n ic ions in e n v iro n m e n ta l samples. The l i m i t o f d e te c tio n f o r n i t r a t e was 15ppb w ith potassium hyd ro xid e e lu e n t and a c o n d u c tiv ity d e te c to r. A problem encountered by t h is system was column o v e r lo a d in g caused by s o lu tio n s w ith high t o t a l io n c o n c e n tra tio n s. The method was th e re fo re u n s u ita b le f o r the a n a ly s is o f e x tra c ts from s o i l and p la n t m a te ria ls prepared by wet a sh in g.

30 HPLC mas used f o r the d e te rm in a tio n o f n i t r a t e and n i t r i t e in e n v iro n m e n ta l mater sa m p le s'^. The tech n iq u e o ffe r e d ra p id a n a ly s is m ith m inim al sample p re p a ra tio n, high s e n s i t i v i t y and fern in te r fe r e n c e s. Samples mere f i l t e r e d p r io r to in je c t io n and elut-ed m ith the m obile phase, aqueous tetram ethylam m onium phosphate. The d e te c tio n l i m i t mas c a lc u la te d as 0.1mg 1 using an absorbance d e te c to r a t 214nm. Chrom atographic methods have also been a p p lie d to the d e te rm in a tio n o f n i t r i t e and n i t r a t e in human 35 plasma. Sample p re p a ra tio n in v o lv e d the p r e c ip it a t io n o f plasma the su p e rn a ta n t mas then in je c te d onto a re ve rse phase precolum n. A p o ly (s ty re n e -d iv in y lb e n z e n e ) basod ion-exchange column mas used to se pa ra te the io ns m ith U.V. d e te c tio n a t 214nm. S e n s it iv i t y o f 0. 01mM and l i n e a r i t y from mM mere e xpe rienced fo r both io n s. Fern in te r fe r e n c e s mere expe rienced because o f the removal o f p ro te in s by p r e c ip it a t io n, o rg a n ic m olecules by a re v e rs e phase precolumn and good r e s o lu tio n o f th e peaks in the chromatogram. The d e te rm in a tio n o f n i t r a t e and n i t r i t e in meat products mas a ls o accom plished using HPLC"^. The meat p ro d u cts mere e x tra c te d, c e n trifu g e d and 20/ t l o f the f i l t e r e d s u p e rn a ta n t mas in je c te d onto th e column. The m obile phase mas a c e to n itrile /te tra p e n ty la m m o n iu m brom ide; the column mas packed m ith n o n -p o la r p o ly (s ty r e n e d iv in y lb e n z e n e ) and d e te c tio n mas s p e c tro p h o to m e tric.

31 Reversed phase l i q u i d chromatography was used f o r the s e p a ra tio n o f o rg a n ic and in o rg a n ic anions 37 The e lu e n t contained th e o ctyla m in e s a lt o f a m in e ra l a c id which separated in o rg a n ic anions such as brom ide, io d id e, n i t r i t e, io d a te and cyanate. The method was used f o r w a ste-w a te rs and s ila g e. C onventional l i q u i d chromatography apparatus was used w ith a U.V. d e te c to r a t 205nm. The in o rg a n ic io n s showed lin e a r response in the c o n c e n tra tio n range ppm. 38 A law i used HPLC fo r the d e te rm in a tio n o f n i t r a t e _ 'j and n i t r i t e in w ater a t a le v e l o f 5ng m l (p p b ). method was in d ir e c t in comparison to the HPLC methods The p r e v io u s ly d e scrib e d. I t was based on the n i t r a t i o n o f an excess o f phe nol; the O -n itro p h e n o l was e x tra c te d and separated using a reversed phase column, and dete cte d a m p e ro m e tric a lly in the re d u c tio n mode. G a s -liq u id chrom atography (G.L.C.) was used f o r 39 the a n a ly s is o f meat p ro d u cts and cheeses. The -1 d e te c tio n l i m i t was 0.006^ig ml NO^N and the range o f d e te rm in a tio n yag. N itr a te was e x tra c te d from f in e l y ground samples w ith hot sodium h y d ro x id e s o lu tio n. The s o lu tio n was re a cte d w ith 2 -s e c -b u ty lp h e n o 1 in s u lp h u ric a c id to form the n it r a t e d p ro d u c t, 4 - n i t r o - 2-s e c -b u ty lp h e n o 1, which was e x tra c te d w ith to lu e n e. The to lu e n e s o lu tio n was re -e x tra c te d w ith an a l k a lin e s o lu tio n which was then analysed by G.L.C. w ith an e le c tro n capture d e te c to r. In te rfe re n c e s from n i t r i t e

32 and c h lo rid e mere c o n tr o lle d using sulpham ic a c id and sodium s u lp h a te. The use o f chrom atography fo r the d e te rm in a tio n o f ions in e n v iro n m e n ta l samples has a number o f li m i t a t i o n s. The equipment re q u ire d f o r these ana lyses is expensive. P re ca u tio n s must be taken mhen a n a ly s in g r e a l samples to p re v e n t damage to the columns M isce lla n o u s Methods Methods f o r d e te rm in in g o rg a n ic o r in o rg a n ic n i t r a t e in the presence o f o th e r io n s were suggested 40 by Hassan. The methods mere based on th e re d u c tio n o f n i t r a t e m ith cadmium m etal and 0. 1M h y d r o c h lo r ic a c id. Four e q u iv a le n ts o f cadmium io n s mere re le a se d per mole o f n i t r a t e and n i t r i t e n itr o g e n, mhich mas co n ve rte d to n itr o u s o x id e. Four methods f o r d e te rm in in g the cadmium io n s mere compared: atom ic a b s o rp tio n s p e c tr o m etry a t nm; p o la ro g ra p h y ; p o te n tio m e tr ic EDTA t i t r a t i o n a t ph 10 m ith cadmium ISE, and EDTA t i t r a t i o n m ith Erichrom e B lack T in d ic a t o r. O rganic n i t r a t e mas determ ined in th e range m ith o u t in te r fe r e n c e from common anions and c a tio n s, the method mas a c c u ra te and p re c is e. The s tro n g reducing agent chromium ( i l ) mas used 41 in flom in je c t io n a n a ly s is o f n i t r a t e s o lu tio n s.

33 A lthough chromium ( I I ) is u n s ta b le under a tm o s p h e ric, c o n d itio n s o n ly p a r t i a l o x id a tio n occurs in flo w systems, the e x te n t o f o x id a tio n is h ig h ly re p ro d u c ib le. N itr a t e and n i t r i t e mere reduced to ammonia and a p o la ro g ra p h ic d e te c to r a t a w orking p o t e n t ia l o f -1.5 V was used to m o n ito r the change in chromium ( Ill) -E D T A c o n c e n tra tio n. Good l i n e a r i t y was o b ta in e d in sample -5-4 s o lu tio n s o f 2.5x10-10 M. The p ro d u c tio n of h ydrazine or hydroxylam ine by sid e re a c tio n s p re ve n ts the re a c tio n w ith chromium (il)-e D T A and so causes reduced n i t r a t e le v e ls. A complex v o lta m m e tric e le c tro d e was designed 42 f o r the d e te rm in a tio n o f n i t r a t e. An anion-exchange membrane was used to enclose a sm a ll volume e le c t r o ly s is c e l l which in c lu d e d a c o n s tra in e d m ercury column in d ic a t o r e le c tro d e. F i l t e r paper im pregnated w ith 0.1M KC1 and 0.01M ZrOCl^ e le c t r o ly t e as c a t a ly s t, served as a c o n s tra in in g b a r r ie r and spacer f o r the t h in la y e r e le c t r o ly s is chamber. S e le c tiv e t r a n s fe r o f n i t r a t e from the sample to the e le c t r o ly s is chamber, through th e ion-exchange membrane, occurred by Donnan d ia ly s is. C o n tro lle d p o t e n t ia l e le c t r o ly s is a t -1.25V vs Ag/AgCl e le c tro d e p ro vid e d the sensing c u rre n t, the value o f which was p r o p o r tio n a l to the sample c o n c e n tra tio n o f n i t r a t e. The l i m i t o f d e te c tio n using the c u rre n t at 8min. was 6.7x10 ^M N0^. The method was lin e a r over

34 3 decades o f c o n c e n tra tio n. N it r a t e ujas a ls o determ ined v o lta m m e tr ic a lly 43 by Fogg et a l. in a flo w in je c t io n system. R eduction o f n i t r a t e was c a r rie d out on a batch b asis w ith cadmium or o n lin e w ith cadmium w ire. The n i t r i t e produced was then in je c te d in to an a c id ic brom ide e lu e n t* The n i t r i t e was m onitored by the re d u c tio n s ig n a l a t a g la ssy carbon e le c tro d e held a t 0.3V Io n - S e le c tiv e E le c tro d e Methods U n lik e the p re v io u s methods io n - s e le c t iv e e le c tro d e s are s u ita b le f o r the a n a ly s is o f e n viro n m e n ta l samples w ith o u t sample p re tre a tm e n t. Io n - s e le c tiv e e le c tro d e s p ro v id e a f a s t, cheap and r e l a t i v e l y v e r s a t ile method f o r n i t r a t e d e te rm in a tio n. I o n - s e le c tiv e e le c tro d e s measure the a c t i v i t y o f a given io n in the presence o f o th e r io n s, HPLC also measures the a c t i v i t y o f an io n, o th e r methods measure the io n s c o n c e n tra tio n. The p re se n t range o f com m ercial I.S.E. s u f f e r some in te r fe re n c e s from common io n s ; but do not s u f fe r in te r fe r e n c e from t u r b id samples. They are s u ita b le f o r o n - lin e contin uous a n a ly s is. A standard method f o r n i t r a t e d e te rm in a tio n w ith an I.S.E. e x is ts '5. N it r a t e a c t i v i t y is measured p o te n tio m e tr ic a lly in th e tr e a te d sample by means o f a n i t r a t e s e le c tiv e e le c tro d e using a c a lib r a t io n curve or standard a d d itio n method. The standards are tre a te d w ith a reagent to make t h e i r

35 io n ic s tre n g th and ph value u n ifo rm. The range o f -1 a p p lic a tio n i s mg 1 ; the l i m i t o f d e te c tio n _ 1 is 0.05 to 0.5mg 1. Manual sample a n a ly s is tim e is a p p ro x im a te ly 15 m inutes f o r e le c tro d e c a lib r a t io n and a n a ly s is o f a s in g le sample b u t 5 m inutes f o r subsequent samples. N itr a t e e le c tro d e s s u ffe r in te r fe re n c e from s e v e ra l commonly o c c u rrin g a n io n s, s e l e c t i v i t y c o e f f ic ie n t s depend upon the type o f e le c tro d e. C h lo rid e, carbonate and d e te rg e n ts are common i n t e r f e r e n t s. Treatm ent w ith sulpham ic a c id may be re q u ire d to remove high le v e ls o f n i t r i t e. Many examples o f n i t r a t e a n a ly s is by I.S.E. are p re se n t in the l i t e r a t u r e. A b r i e f d is c u s s io n o f a number o f examples w i l l be used to i l l u s t r a t e the v e r s a t i l i t y o f t h is method. 44, Milham e t a l. used an e a rly com m ercial n i t r a t e e le c tro d e (O rion Model )to analyse p la n t, s o i l and w ater samples. A s p e c ia lly prepared b u ffe r s o lu tio n was used to e lim in a te in te r fe re n c e s from c h lo r id e, n i t r i t e and o rg a n ic a n io n s. A lin e a r c a lib r a t io n was o b ta in e d from ppm. The l i m i t o f d e te c tio n f o r w ater samples was 10ppm. The O rion was also used to m o n ito r the 45 b a c t e r ia l growth curves o f E. c o l i. The n i t r a t e le v e ls in the c e ll c u ltu r e were m onitored d i r e c t ly every 6 hours by in s e r tin g the e le c tro d e s. The e le c tro d e s were a ls o used f o r the assay o f n i t r a t e reductase a c t i v i t y. The technique was found to be s u p e rio r to s p e c tro p h o to m e tric methods.

36 H u la n ic k i e t a l. used a more advanced e le c tro d e to m o n ito r n i t r a t e in m ater. Phosphate b u f f e r and s i l v e r s u lp h a te were used to c o n tr o l in te r fe r e n c e s. The e le c tro d e was s u ita b le fo r _5 measurements down to 10 M; i t was used f o r n i t r a t e in ta p -w a te r a t the le v e l 8-10ppm. A Corning n i t r a t e e le c tro d e was used to measure n i t r a t e in grass and c lo v e r. 47 D ried samples were mixed w ith b u ffe re d e x tr a c tio n s o lu tio n and a llo w e d to sta n d, th e n i t r a t e in the s o lu tio n was then determ ined by d ir e c t measurement o f the s o lu tio n. The s im p lic it y o f o p e ra tio n and p o r t a b i l i t y o f 1.5.E. were i l l u s t r a t e d by Bound who used a n i t r a t e E. to m o n ito r n i t r a t e in s o i l pa ste s. The method could be used in th e f i e l d to p ro v id e a map o f n u t r ie n t c o n c e n tra tio n s. The use o f I.S.E. f o r m o n ito rin g n i t r a t e in s o ils was also in v e s tig a te d by Black and W aring. 49 They used a l^sg^ s o lu tio n to e x tr a c t n i t r a t e from a dso rb ing s o ils high in cla y m in e ra ls. An a liq u o t o f e x tra c ta n t was fu r t h e r d ilu t e d w ith anc* n i t r a t e was determ ined by p la c in g the I.S.E. in the s o lu t io n. Sample re a d in g s were o b ta in e d in 15s fo r samples >1m g/- and 30-60s f o r low er c o n c e n tra tio n s. _ 1 lin e a r between mg 1 NO^.N. The c a lib r a t io n was

37 The d e te rm in a tio n o f n i t r a t e in meat p ro d u cts was c a rrie d out using an O rion I.S.E.^*"* Homogenised samples o f meat pro d u cts were e x tra c te d w ith borax b u ffe r (ph9). An a liq u o t o f the e x tra c ta n t was then mixed w ith a s p e c ia lly prepared b u f f e r s o lu tio n which removed in t e r f e r i n g io n s, f i n a l l y the ph was a d ju s te d to 3.3. The method showed good p r e c is io n and agreement w ith s p e c tro p h o to m e tric methods; i t showed lin e a r response -5-2 between 5.6x10-10 M o f n i t r a t e. I o n - s e le c tiv e e le c tro d e s may be used to advantage in flo w in g streams where response and re c o v e ry tim e s fo r the e le c tro d e s are enhanced. Flow I n je c t io n A n a ly s is ( F. I. A. ) is a u s e fu l a d a p ta tio n f o r many a n a ly t ic a l proced ure s. A flo w in g stream is used to c a rry sample and reagents and tim e is a llo w e d f o r re a c tio n s to occur b e fo re the stream passes thro ugh a d e te c to r. S e q u e n tia l a d d itio n o f re a g e n ts, stream s p l i t t i n g and m u ltip le a n a ly s is may be in c o rp o ra te d in to the system. Flowing systems are now used f o r the ra p id p ro ce ssin g o f la rg e numbers o f samples by e s ta b lis h e d methods. N itr a te I.S.E. were used f o r the assay o f n i t r a t e i and n i t r i t e reductase a c t i v i t y in a flo w -s tre a m te c h n iq u e." Ruzicka et a l. used F.I.A. and I.S.E. to m o n ito r s o i l 52-5 e x tra c ts f o r n i t r a t e. The d e te c tio n l i m i t was 1x10 M 53 f o r n i t r a t e. Hansen et a l. used F.I.A. f o r the a n a ly s is o f n i t r a t e in e n viro n m e n ta l samples o f w a ste -w a te rs and s o i l e x tra c ts.

38 N it r a t e in s o i l e x tra c ts ujas determ ined u sin g a 54 n i t r a t e - s e le c t iv e flo w e le c tro d e method and compared w ith a r e d u c t i o n - d i s t i l l a t i o n and a cadm ium -reduction method. The range o f a p p lic a tio n was 0.5-IO O ^gm l _ 'j NO^.N; 20 samples per hour were processed. E q u iv a le n t r e s u lt s were o b ta in e d using a l l th re e methods. 55 T ro ja n o w icz and Lewandowski used a flo w -th ro u g h system f o r the contin uous p o te n tio m e tr ic d e te rm in a tio n o f c h lo r id e, f lu o r id e, n i t r a t e and ammonia in n a t u r a l w a te rs. The method was used f o r samples in th e range 5-20ppm n i t r a t e. 1.3 N itr a t e - S e le c t iv e E le ctro d e s P r in c ip le s o f Io n -S e le c tiv e E le ctro d e s Io n - s e le c tiv e e le c tro d e s are devices which enable th e a c t i v i t y o f a given io n, in a s o lu tio n to be determ ined p o t e n t io m e t r ic a lly. They c o n s is t o f a s e le c tiv e membrane, an in t e r n a l re fe re n c e e l e c t r o l y t e ( f i l l i n g s o lu tio n ) and an in t e r n a l re fe re n c e e le c tro d e, which form a h a l f - c e l l. An e x te rn a l re fe re n c e e le c tro d e com pletes the c i r c u i t. When the io n - s e le c t iv e membrane is immersed in a s o lu tio n o f io n s a p o t e n t ia l d iffe r e n c e is generated between the in t e r n a l f i l l i n g s o lu tio n and the sample s o lu tio n, across the membrane. The p o t e n t ia l (E) v a rie s w ith io n a c t i v i t y a ccording to the N e rnst e q u a tio n :

39 E = E' * + RT log a. zf 0 1 E* = standard p o t e n t ia l R = gas cons ta n t T = a b s o lu te tem p era ture z a. 1 = charge oin the io n = a c t i v i t y When, as is custom ary, th e r e la t io n s h ip is expressed as m i l l i v o l t s per decade change in io n decadet1 a c t i v i t y, AF is clo se to 60 ml/^at 25 C fo r m onovalent ions - N e rn s tia n response. The membrane components o f I.S.E. vary c o n s id e ra b ly and may be used to d e fin e the type o f e le c tro d e. The development o f I.S.E. is o u tlin e d below w ith p a r t ic u l a r emphasis on n i t r a t e re sp o n sive e le c tro d e s The E a r lie s t E le ctro d e s U n t i l re c e n t years io n - s e le c t iv e e le c tro d e s were lim ite d to c la s s ic a l g la ss e le c tro d e s and c e r ta in s o lid s ta te e le c tro d e s which con ta in e d non-porous membranes; u s u a lly a la rg e c r y s t a l or a d is c composed o f e le c t r o - a c tiv e m a te ria l. The s o lid s ta te e le c tro d e s are somewhat lim it e d because o f the s tr in g e n t re q u ire m e n ts which the membrane \ m a te ria l must s a t is f y. I t must be h ig h ly in s o lu b le, non-porous and have a s u f f i c i e n t m echanical s tre n g th. However, the most l i m i t i n g re q u ire m e n t is the c o n d u c tiv it y

40 o f the membrane. The u s e fu l s o lid s ta te e le c tro d e s in e x is te n c e a l l e x h ib it abn orm ally high membrane c o n d u c tiv it ie s. The f i r s t e le c tro d e system respon sive to anions ujas developed by S o l l n e r ^, the membranes mere produced by im p re g n a tin g c o llo d io n m ith protam ine. These membranes mere e le c tr o p o s it iv e and could be used to d e te c t s e v e ra l a n io n s. The e le c tro d e s mere used m a in ly in e le c tr o m e tr ic t i t r a t i o n s because they mere s e n s itiv e b u t not s e le c tiv e. A s o lid - s t a t e membrane e le c tro d e fo r n i t r a t e mas prepared from compressed s il v e r d ie th y ld ith io c a r b a m a te 57 pomder. A fte r c o n d itio n in g in n i t r a t e s o lu tio n the e le c tro d e shomed N e rn stia n response to n i t r a t e in the range M o f. n it r a t e io n ; the s e l e c t i v i t y c o e f f ic ie n t _2 fo r n i t r a t e over c h lo rid e io n mas 3.0x N itr a t e E le ctro d e s m ith L iq u id Membranes The f i r s t io n - s e le c t iv e e le c tro d e s a v a ila b le co m m e rcia lly mere of the l i q u i d io n-e xhang er ty p e. These e le c tro d e s co n siste d o f a porous membrane mhich supported a mater im m is c ib le s o lu tio n o f io n -e xch a n g e r and s o lv e n t. The f i r s t e le c tro d e s mere prepared using 57 an O rion calcium e le c tro d e by re p la c in g th e in t e r n a l s o lu tio n s m ith a s u ita b le exchanger f o r n i t r a t e The o r ig i n a l ion-e xchang er used by O rion f o r n i t r a t e, mas a t r i s ( s u b s t i t u t e d 1,10 p h e n a n th r o lin e ) N i( II ) 59 complex. L a te r O rion and Beckman e le c tro d e s c o n ta in e d

41 te tra -a lkyla m m o n iu m c a tio n s. Corning used do tridecylhexadecylam m onium n i t r a t e in n - o c t y l- p - n itr o p h e n y l e t h e r ^. The q u a te rn a ry ammonium s a l t, m eth yl t r ic a p r y ly l ammonium n i t r a t e in 1-d e ca n o l uias used by Coetzee and F re is e r *. A re c e n t l i q u i d membrane was c o n s tru c te d using a 4, 4 1- d ip h e n y l- 2, b ip y r id in e complex in 2-n itro -p -c y m e n e This neuj type o f e le c tro d e was a c o n s id e ra b le improvement on the e a rly p ro ta m in e -c o llo d io n membranes and gave N e rn s tia n response over a u s e fu l w orking ra n g t. However, these e le c tro d e s also showed poor s e n s i t i v i t y and g ra d u a l changes in both s e n s i t i v i t y and s e l e c t i v i t y. The m ajor drawback was the cumbersome c o n s tru c tio n o f the e le c tro d e s. The porous membranes h o ld in g the w a ter im m is c ib le m ix tu re o f io n-e xchang er and s o lv e n t a llo w co n sta n t leakage o f the e le c tr o a c tiv e li q u i d to o cc u r. The in t e r n a l arrangem ent o f the e le c tro d e re q u ire d re s e r v o ir s f o r the exchanger which must be se pa ra te d from the in n e r e l e c t r o ly t e. T his lead to co m plicated e le c tro d e c o n s tru c tio n and d i f f i c u l t y when r e p le n is h in g e le c tro d e components. A f u r t h e r c o m p lic a tio n was caused by p re ssu re and tem p era ture f lu c t u a t io n s which in flu e n c e the b a r r ie r formed by the o rg a n ic l i q u i d in the membrane pores. These e le c tro d e s also showed poor re s is ta n c e to p re s s u re, which may make them u n s u ita b le f o r use in con tin u o u s flo w systems which undergo p ressure f lu c t u a t io n s.

42 1.3.4 Polymer Membrane E le ctro d e s An im p o rta n t advance in I.S.E. te ch n o lo g y was the in tr o d u c tio n o f non-porous p o ly ( v in y l c h lo r id e ) PVC membranes by Moody e t a l. 64 in N itr a t e bb resp o n sive membranes were produced by adding Corning or O rion n i t r a t e li q u i d io n -e xch a n g e rs to a s o lu tio n o f PV/C in THF. The s o lv e n t u/as a llo w e d to evaporate producing a s o lid membrane. The c o n s tru c tio n o f e le c tro d e s was s im p lif ie d and t h e ir li f e t i m e improved by the use o f these new membranes. The c h a r a c t e r is t ic s o f these new membranes were dependent on the io n -e xch a n g e r. The PVC based membranes responded to n i t r a t e in the c o n c e n tra tio n -1-4 range M, th e main in t e r f e r e n t was io d id e. N ie lse n and H a nsen^ used the new typ e o f membrane and lo n g -c h a in q u a te rn a ry ammonium s a lts (QAS) to c o n s tru c t a s e n s itiv e n i t r a t e e le c tro d e. T h e ir choice o f e le c tr o a c tiv e m a te ria l was based on the s o l u b i l i t y o f the membrane components. The PVC e le c tro d e s are e s s e n tia lly li q u i d exchange membranes, bu t the porous membrane was repla ced by a s o lid polymer su p p o rt in which th e e le c tro c a tiv e m a te ria l is e n ta n g le d. The perform ance o f the e le c tro d e is dependant on the i n s o l u b i l i t y o f the membrane m a te ria ls in the t e s t s o lu tio n s. N ie ls e n and Hansen chose s e v e ra l Q.A.S. f o r exam ination as sensors: tetradodecyjbmmonium n i t r a t e, trio ctylm e th yla m m o n iu m

43 n i t r a t e, and tetraheptylam m onium n i t r a t e. The new e le c tro d e s were s u p e rio r in s t a b i l i t y and s e n s i t i v i t y to any p re vio u s e le c tro d e s, the l i m i t o f _7 d e te c tio n was a p p ro x im a te ly 7.9x10 fiand the s e l e c t i v i t y c o e f f ic ie n t f o r n i t r a t e over c h lo rid e was 5x10 ^. The new membrane, tetradodecylam m onium n i t r a t e, P. W.C. and d ib u ty lp h th a la te p l a s t i c is e r is used in s e v e ra l com m ercial e le c tro d e s. The io n a s s o c ia tio n complex o f b is (n e o c u p rio n e ) copper ( 1 ) and n i t r a t e was re p o rte d to show b e t t e r s e l e c t i v i t y towards io d id e and su lp h a te than the 67 O rion exchanger when used in a P.W.C. l i q u i d membrane C oated-w ire E le ctro d e s C oate d-w ire e le c tro d e s are a v a r ia t io n on the P.W.C. membrane e le c tro d e ; the membrane is in d ir e c t co n ta ct w ith the in t e r n a l re fe re n c e. The c o n s tr u c tio n o f these e le c tro d e s i s r e l a t i v e l y sim ple as no in t e r n a l f i l l i n g s o lu tio n is re q u ire d. The e le c tro d e s are produced by repeated d ip p in g o f a prepared m etal w ire in to a s o lu tio n o f polym er and io n -e xch a n g e r. A c o a te d -w ire e le c tro d e was developed using A liq u a t 336S in p o ly (m ethyl m e th a c ry la te ) on p la tin u m w i r e ^. A liq u a t 336S (m ethyl t r i c a p r y l y l ammonium c h lo rid e ) has been used in s e v e ra l l i q u i d membrane e le c tro d e s.

44 Coated - w ire e le c tro d e s o fte n show u n s ta b le sta n d a rd p o t e n t ia ls. The e le c tro c h e m ic a l process a t the metal-membrane in te r f a c e is n o t f u l l y und e rsto o d. The e f f e c t o f oxygen and the s ta te o f the p la tin u m c o n ta c t on the p o t e n t ia l s t a b i l i t y were in v e s tig a te d f o r the n i t r a t e e le c t r o d e ^. The e le c tro d e s tu d ie d was c o n s tru c te d from P V/C and t r i s (b a th o p h e n a n th ro lin e ) n i c k e l ( I I ) n i t r a t e in 2- n itr o p h e n y l phenyl e th e r Bound Ion-exchanger E le ctro d e s Polymer membrane e le c tro d e s are now w e ll e s ta b lis h e d in s e v e ra l areas o f a n a ly t ic a l c h e m is try. However, improvement in the p r o p e rtie s o f the membranes could extend t h e ir a p p lic a tio n s c o n s id e ra b ly. Poly ( v in y lc h l o r id e ) P\JC is a commonly used m a trix m a te r ia l, b ut has poor m echanical q u a li t i e s, and very l i t t l e re s is ta n c e to ch e m ic a lly h o s t ile enviro nm ents. S ilic o n e rubbers e x h ib it s u p e rio r re s is ta n c e to h o s t ile environm ents when c ro s s lin k e d, b u t a d d it io n a l p ro c e s s in g is then re q u ire d. However, even i f the m a trix m a te r ia l is ro b u s t, the io n-exchanger is u s u a lly v u ln e ra b le to p h y s ic a l or chem ical d e t e r io r a t io n. A m ajor weakness o f entang led exchangers is the ease by which they can be removed from the polym er su p p o rt by le a c h in g. C ovalent attachm ent o f the io n-e xchang er to the m a trix m a te r ia l would improve the lif e t im e o f the e le c tro d e s. C ovalent attachm ent to a polymer m a trix o f s u ita b le p r o p e r tie s would improve the chem ical re s is ta n c e o f the e le c tro d e s.

45 r

46 A b r i e f d isc u s s io n o f the d if f e r e n t approaches to t h is problem is in c lu d e d belouj. a ) The Work o f D o bb le ste in and D ie h l In the la te 1960 s D o b b le ste in and D ie h l a c c id e n t a lly d iscovered a membrane system u/hich was 70 re sp o n sive to n i t r a t e. They were w orking on membranes im pregnated w ith n ic k e l dim ethylglyd xim ate in o rd e r to produce an e le c tro d e f o r n ic k e l. Problems o f s o l u b i l i t y caused them to t r y a s o lu tio n o f n ic k e l 4,4 ' d ih y d ro x y b e n z ild io x im a te in B a k e lite A polym e r; t h i s was polym e rised to B a k e lite C using ammonia as a c a t a ly s t. The id ea behind the choice o f reagent was to o b ta in an even d is p e rs io n th ro u g h o u t the membrane by in v o lv in g the p h e n o lic groups in the polymer# The membranes were ordered c r y s t a l li n e m a te r ia ls. They responded to u n iv a le n t anions and hydrogen. The s t r u c t u r e was examined by X -ray powder d i f f r a c t i o n and the r o le o f each reagent was in v e s tig a te d. The s t r u c t u r e (F ig u re 1.1 ) shows th a t the n ic k e l is complexed by th e p o ly m e ric fram ework. T h is is not a c o v a le n tly bound sensor because li k e o th e r complexes i t s s t a b i l i t y depends on c o n d itio n s and p r e f e r e n t ia l com plexation o f o th e r m eta l io n s may occur changing th e p r o p e rtie s o f th e membrane.

47 b ) The Work o f 3yo Oyo and coiuorkers developed a new type o f a n io n - 71 s e le c tiv e e le c tro d e.the membrane was an a n io n - exchange resin, im pregnated w ith n itro b e n z e n e and the exchange s it e was a hydrop hobic q u a te rn a ry ammonium group w ith a long a l k y l ch a in. This anion exchange re s in was prepared by the c h lo ro m e th y la tio n o f A m b e rlite, a c ro s s -lin k e d s ty re n e -d iv in y lb e n z e n e copolym er, and the subsequent q u a te r n is a tio n o f the c h lo ro m e th y la te d re s in w ith d im e th y lte tra d e c y la m in e. The membrane was then c o n d itio n e d and im pregnated w ith n itro b e n z e n e. The low er l i m i t o f N e rn s tia n response f o r t h is e le c tro d e was 1Q ^ - 10"^M. 72 A la t e r membrane was based on a homogeneous c ro s s -lin k e d p o ly s ty re n e w ith c o v a le n tly a tta c h e d b e n zy ltrio c ty la m m o n iu m io n s. The membrane showed a cce p ta b le e le c t r c a n a ly t ic a l p r o p e rtie s b u t was hard and m e c h a n ic a lly weak. The p re p a ra tio n o f the membrane was la t e r m o d ifie d to produce a more ro b u s t 73 polym er. An o le o p h ilic anion exchange r e s in r e in fo r c e d w ith p o ly p ro p y le n e was prepared by c r o s s - lin k in g in a m u ltis te p r e a c tio n. The membrane was tre a te d w ith chlorom ethane, q u a te rn is e d w ith t r io c t y la m in e, and im pregnated w ith n itro b e n z e n e. The new membranes showed near N e rn stia n response to n i t r a t e wi';h a low er l i m i t o f -3 response being 5x10 M and the s e l e c t i v i t y c o e f f ic ie n t

48 f o r n i t r a t e over c h lo rid e was 7.9x10 ^ in mixed s o l u t i o n. c) O ther Ideas The p ro d u c tio n o f polym ers w ith c o v a le n tly bound sensors was in v e s tig a te d by s e v e ra l groups o f w o rkers. These u/ere s y s te m a tic in v e s tig a tio n s o f new m a te r ia ls, r a th e r than a d a p ta tio n s o f e x is tin g m a te ria ls o r a c c id e n ta l d is c o v e rie s o f m a te ria ls w ith s u ita b le p r o p e r tie s. K e il e t a l used a v in y l a lc o h o l/ v in y l c h lo rid e copolym er, a l k y l phosphate groups were g r a fte d to the polym er a t the h y d ro x y l s it e s. The polymer was prepared fo r use in a calcium io n - s e le c t iv e e le c tro d e. The io n - s e le c t iv e polymer was thsn entangled in a p o l y ( v i n y l c h lo r id e ) PVC supp ort m a trix and used in an io n - s e le c t iv e e le c tro d e. The e le c tro d e showed a f a s t and steady response to calcium but an improvement in l i f e t i m e was not observed. 75 C a tio n ic s u rfa c ta n t e le c tro d e s were made by su lp h o n a tin g PVC durin g p o ly m e ris a tio n to produce 75 a n io n ic groups in the polym er. A n io n ic s u r fa c ta n t e le c tro d e s were prepared by chain te rm in a tio n o f PVC w ith an amine, and q u a te rn is a tio n o f the f i n a l p o ly m e ric m a te ria l to produce p o s it iv e ly charged s it e s. The li f e t i m e o f these s u r fa c ta n t e le c tro d e s was extended in comparison w ith l i q u i d ion-e xchang er s u r fa c ta n t e le c tro d e s - the l i f e t i m e was lim it e d by the le a c h in g o f the p l a s t i c is e r.

49 1.2 1,4 diph0nyl-3,-phenylimino-1,2,4 tria zo lin e

50 7 Chiou et a l. attem pted to s y n th e s is e a n i t r a t e s e le c tiv e polymer by in c o rp o r a tin g a n it r o n group (1,4 d ip h e n y l - 3, -p h e n y lim in o -1,2,4 t r i a z o l i n e ) (F ig u re 1.2) in to the polym er backbone* The polym er was re p o rte d to have reasonable s e l e c t i v i t y b u t I o u j c a p a c ity as an ion-exchange r e s in. In c re a s in g the number o f n it r o n groups in the polymer backbone r e s u lte d in a reason ably s e le c tiv e polymer fo r the rem oval o f o x id is in g anions such as NO^", ND TaO^~, M o 0 ^~, Cr > MnO^. The r e s in ujas s u ita b le f o r removing n i t r a t e and n i t r i t e from w ater s u p p lie s - these would be the o n ly o x id is in g anions p re se n t in a p p re c ia b le c o n c e n tra tio n. R egeneration o f the re s in was re q u ire d a f t e r each ion-exchange process. 77 Lawton and Yacynych describ e d the p re p a ra tio n o f g ra p h ite e le c tro d e s coated w ith m o d ifie d p o lym e rs. S everal polym ers were used, in c lu d in g p o ly m e ric q u a te rn a ry ammonium s a lts. The membranes were prepared from v in y lb e n z y l c h lo rid e monomer which a f t e r p o ly m e ris a tio n was m o d ifie d by n u c le o p h ilic s u b s t it u t io n. The n i t r a t e e le c tro d e was made from p o ly t r i e t h y l ( v i n y l b e n z y l ) ammonium c h lo rid e. The polymer membranes are n o t c ro s s -lin k e d and d is s o lv e in a number- o f o rg a n ic s o lv e n ts. The e le c tro d e showed s u b -N e rn s tia n response -2-5 to n i t r a t e in the range 1x 10-1x 10 M.

51 7 8 In an e a r li e r re se a rch p r o je c t in t h i s la b o r a to ry, an io n - s e le c t iv e e le c tro d e f o r calcium was prepared which had a s u p e rio r l i f e t i m e, compared to PVC membranes, and was capable o f o p e ra tin g in h o s t ile environm ents. The s e le c tiv e membrane was prepared u sin g t r ia lly lp h o s p h a t e groups as senso rs, and a ro b u s t polym er, s ty re n e -b u ta d ie n e - s ty r e n e. The phosphate groups were c o v a le n tly a tta ch e d to the c r o s s -lin k e d polym e r Aims o f t h is Work The e s s e n tia l requ ire m e n ts f o r io n - s e le c t iv e e le c tr o n s are o u tlin e d below. They should: ( i ) be o f sim ple c o n s tru c tio n and easy to han dle ; ( i i ) be easy to c a lib r a t e and g iv e re p ro d u c ib le and m e a n in g fu l r e s u lt s w ith in the everyday range -2-5 o f io n a c t i v i t i e s M; ( i i i ) have a f a s t response tim e so th a t a c o n s ta n t e.m.f. is a tta in e d w ith in m inutes a t m ost; ( iv ) have l i t t l e o r no memory e f f e c t, in o rd e r to a llo w v a s tly d if f e r e n t a n a ly te s o lu tio n s to be measured r a p id ly ( e s s e n tia l f o r o n - lin e a p p lic a t io n s ) ; (v) be h ig h ly s e le c tiv e, th a t i s measure the c o r r e c t a c t i v i t i e s o f the io n o f in t e r e s t ir r e s p e c t iv e o f the presence o f o th e r io n s - in te r fe r e n c e should be in s i g n i f i c a n t.

52 I t is also u s e fu l i f the e le c tro d e shows N e rs tia n response over the norm al w orking range, b u t i t is n o t e s s e n tia l. The e le c tro d e should also be ro b u s t a llo w in g i t to be used in h o s t ile p h y s ic a l and chem ical environm ents. I t should also r e q u ire l i t t l e m aintenance and have a li f e t i m e o f s e v e ra l months when in co n sta n t use. The aim o f t h is work was to develop a n i t r a t e - s e le c tiv e e le c tro d e w ith a c o v a le n tly bound sensor group. Such an e le c tro d e would be expected to have an \ im proved li f e t i m e compared to l i q u i d and polym er enta n g le d e le c tro d e s because the e le c tr o a c tiv e m a te r ia l would n o t leach from th e membrane.

53 CHAPTER 2 CHOICE AND PREPARATION OF MATERIALS AND PRODUCTION OF MEMBRANES 2.1 The Polymer The in tr o d u c tio n o f polym ers as su p p o rt m a te ria ls f o r io n - s e le c t iv e membranes was a s ig n if ic a n t advance in e le c tro d e te c h n o lo g y, s im p lify in g c o n s tru c tio n and im proving li f e t i m e s. The m a jo r ity o f com m ercial, polym er membranes are made from p o ly (v in y l c h lo rid e ) PVC. However t h is polymer has s e v e ra l l i m i t a t i o n s. H o s tile chem ical environm ents cause poor response and a c c e le ra te 79 the d e t e r io r a t io n o f the membrane. PVC re q u ire s a p l a s t i c is e r to low er the g la ss t r a n s it io n te m p e ra tu re o f the polym er and f a c i l i t a t e charge tr a n s p o r t. U n p la s tic is e d PVC is a hard r e s i l i e n t m a te r ia l, s u ite d to use in the c o n s tru c tio n in d u s tr y. Polymer membranes may co n ta in as much as 70^ W/W o f p l a s t i c i s e r, such membranes are s o f t, g e la tin o u s and prone to d is t o r t io n by p re ssu re. The s h e lf l i f e o f a PVC e le c tro d e is a ffe c te d by the presence o f a p l a s t i c is e r. Slow e va p o ra tio n o f the p l a s t i c is e r may occur causing th e membrane to harden and the response o f the e le c tro d e to d e te r io r a te.

54 A number o f o th e r polym ers have been examined f o r t h e i r s u i t a b i l i t y as polym er membranes f o r io n - s e le c t iv e e le c tro d e s. P o ly ( v in y l is o b u ty l e th e r) 80 u/as used to prepare a calcium s e le c tiv e membrane The polymer ujas n o t expected to re q u ire a p l a s t i c i s e r because the presence o f b u lk y is o b u t y l groups produced a low g la ss t r a n s it io n te m p e ra tu re. However the polymer membranes showed good response o n ly when the sensor m a te ria l had p l a s t i c is i n g p r o p e rtie s. S ilic o n e rubber was used as a m a trix m a te ria l f o r a 81 potassium e le c tro d e, the sensor v a lin o m ycin was entangled in the c r o s s -lin k e d rubbe r m a trix. The e le c tro d e operated s u c c e s s fu lly w ith o u t a p l a s t i c i s e r. 82 A potassium e le c tro d e w ith a long li f e t i m e was prepared from the b lo ck copolym er p o ly (b is p h e n o l - A carbon ate ) and p o ly (d im e th y ls ilo x a n e ) w ith v a lin o m y c in as the sensor. The b lo ck copolym er was a two phase system; the p o ly s ilo x a n e b lo c k s formed a co ntin uous amorphous phase through which m o le c u la r tra n s p o r t o ccu rre d, and the p o lyca rb o n a te b lo c k s formed a d is c o n tin u o u s c r y s t a llin e phase c r o s s - lin k in g the s t r u c t u r e. The entanglem ent o f the sensor m a te ria l in a ro b u s t c r o s s -lin k e d polymer such as s ilic o n e rubber would improve the p h y s ic a l p r o p e rtie s o f the membranes, and t h e ir re s is ta n c e to h o s t ile environm ents. However the li m i t i n g fa c to r in the l i f e t i m e o f an e le c tro d e membrane, is the r a te a t which the e le c tr o a c tiv e m a te ria l is

55 leached from the s u p p o rtin g polym er. The le a c h in g o f e le c tr o a c tiv e m a te r ia l is a c c e le ra te d when an e le c tro d e i s used f o r con tin u o u s m o n ito rin g in a flo w in g stream o f a n a ly te. Covalent attachm ent o f the e le c tr o a c tiv e m a te ria l to an im proved polym er m a trix would p re ve n t i t le a ch in g from th e membrane. These s p e c ific a t io n s r e s u lte d in the choice o f a t r i b lo c k elastom er p o ly ( s ty re n e -b -b u ta d ie n e - b -s ty r e n e ) SB5, as a s u ita b le m a trix polymer f o r t h is work. The 7 8 polymer was used p r e v io u s ly by t h is group in the p re p a ra tio n o f a n o ve l e le c tro d e. T r ia lly lp h o s p h a te groups were c o v a le n tly a tta ch e d to the polym er membrane to produce a calcium e le c tro d e. The polymer SBS has the re q u ire d p h y s ic a l and m echanical p r o p e r tie s, i t is easy to work w ith, w e ll documented and r e a d ily a v a ila b le. The polym er becomes c r o s s -lin k e d v ia the b u ta d ie n e u n s a tu ra tio n, to form an in s o lu b le, tough polym er. The c r o s s - lin k in g re a c tio n may be used to in c o rp o ra te o th e r u n its in to the polym er by g r a f t c o p o ly m e ris a tio n. S everal methods f o r a tta c h in g sensor groups to s u p p o rtin g polym ers were discussed in S ection C u tle r et a l. used a sensor which acted as a chain te rm in a to r in the p o ly m e ris a tio n o f the supp ort polym er. The p ro d u c tio n o f u s e fu l polym ers by t h is method re q u ire s the m a n ip u la tio n and c a r e fu l c o n tr o l o f th e p o ly m e ris a tio n re a c tio n. Syo and coworkers 9

56 m o d ifie d e x is tin g polym ers by chem ical r e a c tio n a t s u ita b le s it e s, to form sensor groups. Polymers which have the re q u ire d chem ical c h a r a c t e r is t ic s f o r J y o s method may not have the re q u ire d p h y s ic a l p r o p e r tie s. 79 The method pioneered in t h is group in c o rp o ra te s co v a le n t attachm ent o f the sensor and the c r o s s - lin k in g re a c tio n s o f the polym er, in one step. 2.2 The Sensor N itr a t e s e le c tiv e e le c tro d e s u s u a lly c o n ta in a q u a te rn a ry ammonium s a l t o r t r i s ( s u b s t it u t e d - o p h e n a n t h r o lin e ) n ic k e l( I I ) as the e le c tr o c a tiv e m a te r ia l. 70 D o b b le ste in and D ie h l produced a p o ly m e ric n ic k e l complex using B a k e lite polym er. Ikeda and 83 Hamato prepared p o ly (p -c h lo r o s ty re n e ) supp orted p h e n y l( d ip y rid y l)n ic k e l c h lo rid e f o r use as a c a t a ly s t in p o ly m e ris a tio n r e a c tio n s. However, such n ic k e l complexes do not y ie ld c o v a le n tly bound sensors, because o n ly the lig a n d s are c o v a le n tly a tta c h e d to the polym er. The n ic k e l io n is e s s e n tia l to the a c tio n o f the se n so r, b u t under s u ita b le c o n d itio n s the n ic k e l io ns could be re pla ced by o th e r m etal io n s. Displacem ent o f the n ic k e l io n s may r e s u lt in lo s s o f response f o r n i t r a t e.

57 INITIATION PROPAGATION INTRAMOLECUL (2) GROWTH. ZCH < (3 ) N < X R PROPAGATION BY INTRAMOLECULAR GROWTH. ALTERNATIVE REPETITION OF (2) + (3) POLYMER. F ig.2.1 Proposed mechanism for polymerisation of diallyldialkylammonium halides.

58 Q uaternary ammonium s a lt s mere chosen fo r t h i s work because- they w i l l y ie ld a tru e c o v a le n tly bound system. In th e la te 1950's and e a rly 1960's B u tle r e t a l p u b lish e d a s e rie s o f papers, on the s y n th e s is * and p o ly m e ris a tio n o f q u a te rn a ry ammonium s a lt s. The q u a te rn a ry ammonium s a lts co n ta in e d u n s a tu ra te d groups such as a l l y l, d ic h lo ro p ro p e n e, v in y lo x y e th y l or p ro p a rg y l and formed lin e a r o r c r o s s -lin k e d polym ers. The polym ers were s u ita b le f o r use as ion-exchange re s in s, but had very poor m echanical p r o p e rtie s because o f t h e i r low m o le cu la r w e ig h t. Q uaternary ammonium s a lts w ith two u n sa tu ra te d groups produced lin e a r polym ers which were water s o lu b le. I f the q u a te rn a ry ammonium s a l t co n ta in e d th re e or fo u r u n s a tu ra te d groups, w ater in s o lu b le polymers were produced by c r o s s - lin k in g o f polym er chains in a th re e d im e n sio n a l n e tw o rk. The p o ly m e ris a tio n o f these q u a te rn a ry ammonium s a lts was a t t r ib u t e d to a chain p ro p a g a tio n mechanism which in v o lv e d a lte r n a te in tr a m o le c u la r - in te r m o le c u la r 92 growth steps. The proposed polym er s tr u c tu r e was a lin e a r chain o f r e c u r rin g n - s u b s titu te d p ip e r id in iu m h a lid e u n its a lte r n a tin g along the chain w ith m ethylene groups, F igure 2.1. L a te r work has shown th a t the polym er c o n s is ts 93 o f both f iv e and s ix membered r in g s

59 The s tr u c tu r e and p r o p e rtie s o f these q u a te rn a ry ammonium s a lts makes them s u ita b le f o r co v a le n t bonding to the SBS framework by r a d ic a l i n i t i a t i o n. The p o ly m e ris a tio n and c y c lo p o ly m e ris a tio n p r o p e rtie s o f these q u a te rn a ry ammonium s a lts are beyond th e re q u ire m e n ts o f t h is p r o je c t because we wish to a tta c h r e l a t i v e l y is o la te d q u a te rn a ry n itro g e n s to the backbone o f another polymer# However t h e ir s u s c e p t i b il it y to r a d ic a l i n i t i a t i o n w ith o u t breakdown of. the q u a te rn a ry s tr u c tu r e is an extre m ely v a lu a b le p ro p e rty. The q u a te rn a ry ammonium s a lts o f in t e r e s t are w h ite c r y s t a l li n e s o lid s o f a ve ry h yg ro sco p ic n a tu re. The in te rm e d ia te t e r t i a r y amines are c le a r c o lo u rle s s liq u id s which would be e a s ie r to work w ith, b u t do n o t undergo p o ly m e ris a tio n. The p o ly m e ris a tio n r e a c tio n is o n ly achieved i f a q u a te rn a ry n itro g e n is p re s e n t; t e r t i a r y amines a ct as r a d ic a l scavengers, quenching p o ly m e ris a tio n r e a c tio n s. N ie lse n and H a n se n ^ showed th a t q u a te rn a ry ammonium s a lts w ith long a lk y l chains improved th e s e n s i t i v i t y o f n i t r a t e e le c tro d e s. These e le c tro d e s, m entioned e a r lie r in S ection , were o f the PVC membrane ty p e, w ith an entangled exchanger. The in flu e n c e o f the s o l u b i l i t y c o e f f ic ie n t o f the QAS on the s e n s i t i v i t y o f n i t r a t e ISE was examined. Q uaternary ammonium s a lts w ith long a lk y l chains were le s s s o lu b le

60 in w ater and leached more s lo w ly from the membrane. The s e n s i t i v i t y is in flu e n c e d by le a c h in g because a p o in t is reached when the amount o f m a te ria l being leached from the membrane in to a d i lu t e s o lu tio n in flu e n c e s the response o f the e le c tro d e. The lo n g -te rm a f f e c t o f le a ch in g is to d e p le te the membrane o f e le c tr o a c tiv e m a te ria l causing a steady d e t e r io r a t io n in the e le c tro d e s response. I f th e e le c tr o a c tiv e m a te r ia l is c o v a le n tly bound to the polym er, i t s s o l u b i l i t y c o e f f ic ie n t w i l l n o t in flu e n c e the s e n s i t i v i t y o f the e le c tro d e. N ie lse n and H a n se n ^ a lso c o r re la te d in c re a s in g s e l e c t i v i t y f o r n i t r a t e w ith in c re a s in g chain le n g th o f the a l k y l s u b s titu e n ts, b u t th e r e la t io n s h ip was n o t i l l u s t r a t e d by examples. The q u a te rn a ry ammonium s a lts s e le c te d f o r i n i t i a l in v e s tig a t io n in t h is work were d ia lly ld ia lk y la m m o n iu m c h lo rid e s ; X R1= R " = a th y l,b u ty l, h e x y l, o c t y l

61 A range o f q u a te rn a ry ammonium s a lts was chosen in o rd e r to in v e s tig a t e the in flu e n c e o f the s tru c tu r e s on e le c tro d e s e l e c t i v i t y. The le n g th o f the a l k y l s u b s titu e n ts w i l l be in flu e n c e d by the c o v a le n t attachm ent o f the q u a te rn a ry n itro g e n to a polym er ne tw o rk, which w i l l p ro v id e s u b s titu e n ts o f i n f i n i t e le n g th. The c h lo rid e s were chosen because they are more s u s c e p tib le to p o ly m e ris a tio n than the brom ides o r io d id e s. N egi, Harada and Is h iz u k a 94 a t t r ib u t e d t h i s to the ease o f o x id a tio n o f the h a lid e s which decreases I~ ^ 0 r V C l. T h e re fo re brom ide io n s are more e a s ily o x id is e d than c h lo r id e, g e n e ra tin g brom ine which a c ts as an i n h i b i t o r to p o ly m e ris a tio n by consumption o f s t a r t in g r a d ic a ls and te rm in a tio n o f growing ch a in s P re p a ra tio n o f Q uaternary Ammonium S a lts B u t le r 's work covered the p re p a ra tio n o f a number o f a l l y l s u b s titu te d q u a te rn a ry ammonium s a lt s, b u t the d ia lly ld ih e x y la m m o n iu m and d ia lly ld io c ty la m m o n iu m d e r iv a tiv e s were n o t in c lu d e d. The p re p a ra tio n o f t e r t i a r y amines and c r y s t a l li n e q u a te rn a ry ammonium s a lts in v o lv e d one or two step r e a c tio n s, and sh o u ld be a p p lic a b le to a wide range o f d e r iv a tiv e s. The s y n th e s is o f a wide range o f p o ly m e ris a b le q u a te rn a ry ammonium s a lts was o u tlin e d by S c h u lle r and 94 Thomas and in c lu d e d :-

62 / Y R1 R" The p re p a ra tiv e d e t a ils are sparse, but i t appears th a t the q u a te rn a ry ammonium s a lts were n o t is o la t e d as monomers, but prepared and polym e rised in s it u, as an aqueous s o lu tio n. L i t t l e d i f f i c u l t y was envisaged in the p re p a ra tio n o f the chosen q u a te rn a ry ammonium s a lts because o f the e x te n s iv e docum entation o f t h is cla ss o f compounds. In t h is work d ia lly ld ie th y la m m o n iu m bromide and c h lo rid e were prepared using p u b lis h e d 95 methods, b u t these methods were u n su cce ssfu l f o r the s y n th e s is o f the b u ty l, h e x y l and o c t y l d e r iv a tiv e s. Many a tte m p ts were made to q u a te rn is e t e r t i a r y d i a l k y l a l l y l - amines under d if f e r e n t c o n d itio n s. In most p re p a ra tio n s an orange o i l was produced, however under c e r ta in c o n d itio n s (no s o lv e n t, ic e cooled) a w h ite s o lid was o b ta in e d. U n lik e the d ia lly ld ie th y la m m o n iu m h a lid e s, these s o lid s were not h yg ro sco p ic and had a g ra n u la r appearance. The d ib u t y l and d ih e x y l d e r iv a tiv e s were w ater s o lu b le. The d io c t y l d e r iv a tiv e was in s o lu b le in w a te r. The in fr a r e d s p e c tra o f these p r e c ip it a t e s showed th a t the s tru c tu r e s contained no a l l y l s u b s titu e n ts. The n.m.r. sp e ctra showed s ig n a ls f o r the two a lk y l groups a tta c h e d to n itro g e n, bu t no o th e r s ig n a ls. The w h ite p r e c ip it a t e s

63 mere considered to be secondary amine a a lt s. X R1 = R" = b u t y l, h e x y l, o c t y l H H This s tr u c tu r e is c o n s is te n t w ith the in fr a r e d and n.m.r. da ta. The absence o f n.m.r. s ig n a ls f o r th e p rotons is p ro b a b ly due to exchange w ith D2O s o lv e n t. M ole cu la r models o f d ia lly ld ia lk y la m m o n iu m s a lt s in d ic a t e s t e r ic crowding o f the s u b s titu e n ts. This crow ding may cause i n s t a b i l i t y ( in s a lts c o n ta in in g b u lk y a l k y l g ro u p s), le a d in g to lo s s o f the a l l y l groups on q u a te r n is a tio n. The fo llo w in g q u a te rn a ry ammonium s a lts were made by q u a te rn is in g an a ik y ld ia lly a m in e w ith a l l y l brom ide which is more r e a c tiv e than a l l y l c h lo rid e R= m eth yl e t h y l p ro p y l b u t y l a l l y l

64 The tr ia lly la lk y la m m o n iu m s a lts appear to be le s s s t e r i c a l l y crowded, le s s h yg ro sco p ic and e a s ie r to handle than the d i a l l y l d e r iv a tiv e s. The homologous s e rie s was not extencted beyond b u t y l. T ria lly lis o p ro p y la m m o n iu m brom ide was also prepared b u t was s u s c e p tib le to deco m p osition. The n.m.r. and in f r a - r e d s p e ctra o f tria lly lis o p ro p y la m m o n iu m brom ide'showed the presence o f some a l l y l c h a ra c te r, b u t some secondary ammonium s a lt was also p re s e n t. F u rth e r r e c r y s t a l li s a t i o n in cre a se d deco m p osition. Decom position o f t h is q u a te rn a ry ammonium s a l t may a lso be in flu e n c e d by s t e r i c c o n s id e ra tio n s because the is o p ro p y l group is b u lk y compared to the o th e r a l k y l s u b s titu e n ts E xp erim e ntal Methods The q u a te rn a ry ammonium s a lts were prepared from s u ita b le t e r t i a r y amines, the t e r t i a r y amines were prepared from com m e rcially a v a ila b le p rim a ry o r secondary amines T e r tia r y Amines a) A lly ld ia lk y la m in e s The p re p a ra tio n o f a lly ld ie t h y la m in e w i l l i l l u s t r a t e the procedure.

65 A secondary amine (d ie th y la m in e, 26g) was placed in a 3-necked f la s k w ith 25 cm^ o f d i s t i l l e d w a te r. Then a l l y l c h lo rid e (26.20g) and sodium h yd ro xid e s o lu tio n (25-50cm^; 14g o f sodium h y d ro x id e ) were added dropw ise, s e p a ra te ly, from two fu n n e ls, w ith s t i r r i n g. A fte r the a d d itio n was com plete the m ix tu re was heated f o r 3-5 hours between C w ith s t i r r i n g. A fte r c o o lin g the o i l y la y e r was sepa ra ted, d rie d o v e rn ig h t using sodium h yd ro xid e p e lle t s and d i s t i l l e d using a f r a c t io n a t in g sid e arm (th e h ig h e r amines re q u ire d vacuum d i s t i l l a t i o n ). This method was used to prepare a l ly ld ie t h y la m in e, a lly ld ib u t y la m in e, a lly ld ih e x y la m in e and a l ly ld io c t y la m in e. A l l were c le a r, c o lo u rle s s liq u id s. They were c h a ra c te ris e d by n.m *r. and ij?. sp e ctro sco p y, b ) D ia lly la lk y la m in e s The p re p a ra tio n o f d ia lly le t h y la m in e w i l l i l l u s t r a t e th e proced ure. D ia lly la m in e (25g) was placed in a 3-necked f la s k w ith 25cm^ o f d i s t i l l e d w a te r. Bromoethane (28g) was added dropw ise, and then sodium h yd ro xid e s o lu tio n (25-50cm^, 10-30g o f sodium h y d ro x id e ) was a lso added dropw ise w ith s t i r r i n g * A fte r the a d d itio n was com plete the m ix tu re was heated f o r 3-5 hours between 6Q-80 C w ith s t i r r i n g. The o i l y la y e r was tre a te d as above.

66 T h is method was used f o r the p re p a ra tio n o f d ia lly le t h y la m in e, d ia lly lb u t y la m in e, d ia lly lh e x y la m in e and d i a ll y lo c t y la m i n e. These were a l l c le a r c o lo u rle s s liq u id s Q u a te rn is a tio n The q u a te rn a ry ammonium s a lts o f a lly la m in e s are extre m ely h y g ro s c o p ic. A l l y l c h lo rid e, a l l y l brom ide, acetone and the t e r t i a r y amines were a l l d rie d and d i s t i l l e d im m e d ia te ly b e fo re r e a c tio n D ia lly ld ie th y la m m o n iu m C h lo rid e A l l y l t e r t i a r y amine, was mixed w ith the e q u iv a le n t amount o f f r e s h ly d i s t i l l e d a l l y l c h l o r i d e in d ry a cetone. The w h ite c r y s t a l li n e s o lid o f q u a te rn a ry ammonium s a l t g ra d u a lly p r e c id ita te d from the s o lu tio n a t room te m p e ra tu re. The s o lu tio n was a llo w e d to stand f o r a minimum o f two days, and the liq u o r was then decanted. The c r y s ta ls were re p e a te d ly washed w ith co ld acetone and d rie d by v/acuum. The c r y s ta ls were n o t c o lle c te d bv f i l t r a t i o n because o f t h e i r h yg ro s c o p ic n a tu re. T his method proved u n su cce ssfu l f o r o th e r q u a te rn a ry ammonium s a lt s D ia lly ld ie th y la m m o n iu m Bromide A l l y l bromide was added dropw ise to th e t e r i a r y amine in acetone. The m ix tu re was s t ir r e d and th e apparatus p ro te c te d by a d ry in g tu b e. I t was necessary

67 to co o l the re a c tio n ve sse l in ic e because o f th e exotherm ic n a tu re o f the r e a c tio n. The q u a te rn a ry ammonium s a l t p r e c ip it a t e d r a p id ly, and was c o lle c te d by f i l t r a t i o n, washed and d rie d under vacuum. P o rtio n s were r e c r y s t a l li e d from hot acetone c o n ta in in q s m a ll q u a n titie s o f a b s o lu te e th a n o l. S e vera l v a r ia t io n s on these methods have been t r i e d in o rd e r to prepare o th e r q u a te rn a ry ammonium s a lt s. These in c lu d e : g e n tle r e flu x in g o f th e r e a c tio n m ix tu re a f t e r a d d itio n o f re a g e n ts; th e use o f d i f f e r e n t s o lv e n ts (TH'F, acetoph enon e); or c o o lin g o f r e a c tio n v e s s e l. In the m a jo r ity o f p re p a ra tio n s no p r e c ip it a t e was observed and decom position o f e ith e r a lly lb r o m id e or the t e r t i a r y amine began to o ccu r. W hite p r e c ip it a t e s were c o lle c te d from th e r e a c tio n s o f the t e r t i a r y amines w ith a l l y l brom ide in th e absence o f a s o lv e n t. These p r e c ip it a t e s were r e c r y s t a l li s e d and examined. There was no evidence o f a l l y l groups in e ith e r i r o r nmr s p e c tra o f the p ro d u c ts T ria lly lb u ty la m m o n iu m Bromide A l l y l brom ide (4.8 4 g ) was added dropw ise to a s t ir r e d s o lu tio n o f d ia lly lb u ty la m in e (7.9 g ) in dry acetone (5cm^) cooled in an ic e -b a th. A p r e c ip it a t e developed s lo w ly over s e v e ra l days. The c r y s ta ls were

68 Table 2.1 B o ilin g P o in ts o f T e r t ia r y Amines and M e ltin g P o in ts o f Q uaternary Ammonium S a lts. T e r t ia r y Amine bp/ C observed ( l i t ; Q uaternary Ammonium Bromide mp/ C observed ( l i D ia lly lm e t b y l 109 T r ia lly lm e t h y l 89 (91 D i a l l y l e t h y l 123 (130) T r i a l l y l e t h y l 158 (159 D ia lly lp r o p y l 147 T r i a l l y l p r o p y l D ia lly lis o p r o p y l 145 D i a l l y l b u t y l 170 (170) T r i a l l y l b u t y l 177 (175 T r i a l l y l 149 (149.5) T e t r a a ll y l 181 (185 A lly ld im e t h y l 62 (62-63) A l l y l d i e t h y l 111 ( 110) D i a l l y l d i e t h y l 148 (155 A l ly l d ip r o p y l 153 (150) A l i y l t r i e t h y l (202 a decomposes a t 200 C

69 c o lle c te d by d e c a n ta tio n, and washed w ith dry THF to remove the orange o i l from which they had p r e c ip it a t e d, then r e c r y s t a l li e d from hot acetone and a b s o lu te e th a n o l (9+ 1). T his method was also used to prepare t r i a l l y m e t h y l - ammonium brom ide, tria lly le th y la m m o n iu m brom ide and tria lly lp ro p y la m m o n iu m brom ide. T etraallyla m m onium brom ide was prepared from t r ia lly a m in e and q u a te rn is e d w ith a l l y l brom ide A lly ltrie th y la m m o n iu m Bromide This q u a te rn a ry ammonium s a lt was prepared by q u a te r n is a tio n o f tr ie th y la m in e w ith a l l y l brom ide in dry acetone. The q u a te rn a ry ammonium s a l t was chosen to com plete the range o f a l l y l s u b s titu te d q u a te rn a ry ammonium s a lt s. The same m a te ria l was a lso purchased from Lan caste r S ynthesis L td C h a ra c te ris a tio n The t e r t i a r y amines were c le a r, c o lo u rle s s l i q u i d s and the q u a te rn a ry ammonium s a lts were w h ite o r c o lo u r le s c r y s ta ls. The b o ilin g p o in ts o f the amines and m e ltin g p o in ts o f the q u a rte rn a ry ammonium s a lts are g ive n in Table 2.1. The c h a r a c t e r is t ic i r and nmr s p e c tr a l data f o r the t r i a l l y l homologous s e rie s i s i l l u s t r a t e d by the b u ty l d e r iv a tiv e s (Tables 2.2 and 2.3 ). The i r and

70 Table 2.2 I n fr a r e d S p e c tra l C h a r a c te r is tic s o f Amines and S a lts Sample Sampling Method Band/cm In te rfe r e n c e D ia lly lb u ty la m in e l i q u i d f ilm 1642 C=C a l l y l 1000 ) ) c h a r a c t e r is t ic o f a l l y l 920) T r i a l l y l b u t y l ammonium brom ide KBr d is c 1642 C=C a l l y l

71 Table 2.3 C h a r a c te r is tic s o f T ria lly lb u ty la m m o n iu m Bromide H N.m.r. S/ppm H atom r e la t iv e in t e n s it y d 3.20 t t H c H H. a b H, a S o lven t D2O R1= a l l y l Hr T ria lly lb u ty la m m o n iu m Bromide

72 Table 2.4 I n f r a - r e d and N.m. r. S p e c tra l C h a r a c te r is tic s of Decomip o s itio n P roduct I n f r a - r e d band/cm" 1 In fe re n c e 1600 secondary amine s a l t mh gp. o f s ig n a ls secondary amine s a l t nh2+ N.m.r. 3 <J/ppm H atom r e l a t i v e in t e n s it y 2.70 t 1.30 q 1.05 m Ha 4 Hb 4 Hc t Hd 6 a S o lven t D2O R =butyl N R H Decom position p ro d u ct o f a lly ld ib u ty la m in e q u a te rn is e d w ith a lly lb r o m id e.

73 and nmr s p e c tr a l data o f the decom position p ro d u c t, a secondary amine s a l t, do not e x h ib it a l l y l s ig n a ls (Table 2.4 ). 2.3 P ro d u c tio n o f Membranes The s t a r t in g m a te ria ls f o r t h is neu/ type o f io n - s e le c t iv e membrane were chosen f o r t h e ir s u i t a b i l i t y to a one -step c o p o ly m e ris a tio n r e a c tio n. An i n i t i a t o r is re q u ire d to s t a r t chain growth r e a c tio n s in the polym er, which w i l l r e s u lt in c ro s s - lin k in g and c o v a le n t attachm ent o f the q u a te rn a ry n itr o g e n s. The a z o - n i t r i l e 2,2 a z o b is ( 2- m e t h y l p r o p r io n i t r il e ) ABIN was used in the p ro d u c tio n o f membranes w ith 78 c o v a le n tly bound sensors f o r calcium. R a d ica ls were generated by th e rm a l or photochem ical ru p tu re o f a l i p h a t i c 84 a z o - n i t r i l e s. B u tle r and co-w orkers used th e i n i t i a t o r te r t- b u ty lh y d r o p e r o x id e (tbhp) to p o lym e rise a l l y l s u b s titu te d q u a te rn a ry ammonium s a lt s. This p e ro x id e i n i t i a t o r form s r a d ic a ls by th e rm a l decom position from C. In o rd e r to produce a homogeneous polym er, a m ix tu re o r s o lu tio n o f th e s t a r t in g m a te ria ls is re q u ire d. The o rg a n ic s o lv e n t te tra h y d ro fu ra n (THF) is a good s o lv e n t f o r SBS and was a s u ita b le medium f o r fre e r a d ic a l fo rm a tio n. The q u a te rn a ry ammonium s a lt s o f in t e r e s t were in s o lu b le in THF but were s o lu b le in

74 uiater and m ethanol. The q u a te rn a ry ammonium s a lt s were prepared as e ith e r the c h lo rid e or brom ide; th e n i t r a t e form o f th e s a l t is re q u ire d f o r the e le c tro d e. The q u a te rn a ry ammonium s a lts were used in the o r i g i n a l form because o f t h e ir h yg ro sco p ic n a tu re. C onversion to the n i t r a t e form by ion-exchange would have made re co ve ry o f the c r y s t a l li n e m a te ria l d i f f i c u l t. The r e a c t i v i t y o f these u n s a tu ra te d ammonium compounds is r e la te d to t h e ir c o u n te r-io n, the r e a c t i v i t y o f n it r a t e s is unknown. Thus the q u a te rn a ry ammonium h a lid e s were used fo r the p re p a ra tio n o f membranes, and the h a lid e s w i l l be re p la ce d by n i t r a t e by ion-exchange when the sensor is c o v a le n tly a tta ch e d to th e polym er P re p a ra tio n o f Reagents P o ly ( s ty r e n e -b - b u ta d ie n e -b - s ty re n e ) SBS The m a te r ia l used was the t r i b lo c k e la s to m e r, C a rifls x S tyrene Butadiene Rubber, s u p p lie d by S h e ll Chem icals, London. S everal types o f SBS are a v a ila b le w ith d i f f e r e n t p ro p o rtio n s o f s ty re n e and b u ta d ie n e. SBS.TR 1101 and TR 1102 were used f o r t h is p r o je c t. % Butadiene % S tyrene H igher M o le cu la r W eight.p olym er 28 Lower M o le c u la r W eight. Polym er.

75 I t was necessary to remove s t a b il is e r s which r e ta rd the proposed f r e e - r a d ic a l re a c tio n. The polym er was d is s o lv e d in THF (24 hou rs) and p r e c ip it a t e d in to r a p id ly s t i r r e d, ic e -c o o le d m ethanol. The r e s u lt in g w h ite p r e c ip it a t e was f i l t e r e d and d rie d under vacuum. I t was s to re d in a dark g la ss c o n ta in e r in the r e f r ig e r a t o r ,2 a z o b is ( 2 - m e t h y lp r o p r io n it r ile ) (ABIN) The i n i t i a t o r was s u p p lie d by A ld ric h Chem icals. (G illin g h a m, D o rs e t). I t was r e c r y s t a llis e d from h o t m ethanol. The r e s u lt in g w h ite, n e e d le -lik e c r y s ta ls were s to re d in the r e f r ig e r a t o r te r t- B u ty lh y d ro p e r o x id e (tbhp) Used as s u p p lie d by K o ch -L ig h t L a b o ra to rie s, L td. (C olbrook, B e rk s h ire ). (70^ + 30% d i - t e r t - b u t y lp e r o x id e ) T e tra h y d ro fu ra n (THF) T e tra h y d ro fu ra n ( p u r is s.p.a. F luka, Fluorochem. L t d., G lossop, D e rb y s h ire ) was r e flu x e d over potassium m etal f o r s e v e ra l hours and d i s t i l l e d. I t was c o lle c te d in a dark g la ss c o n ta in e r and used im m e d ia te ly M ethanol Used as s u p p lie d by A ld r ic h Chemicals (G illin g h a m, D o rs e t). S p e c tro p h o to m e tric grade (Gold Label ^ p u re ) Q uaternary Ammonium S a lts The c r y s ta ls were d rie d and s to re d in a vacuum d e s s ic a to r.

76 uv a. g la s s p la te b. em broidery r in g c. c e llo p h a n e f ilm d. g la s s r in g (95 mm i. d. ) e. u je ig h t/m irro r su p p o rt f. f r o n t - s il v e r e d m ir r o r g. w e ig h t h. c a s tin g polym er s o l u t i F i g. 2.2 C asting Apparatus

77 2.3.2 Methods o f Membrane P reparation S everal methods f o r the p re p a ra tio n o f c ro s s - lin k e d membranes w ith c o v a le n tly bound sensors were e v a lu a te d S o lven t C asting The f i r s t method o f membrane p re p a ra tio n to be eva lu a te d was s o lv e n t c a s tin g. T his method was used by 64 Moody et a l. to produce PVC membranes, f o r io n - s e le c tiv e e le c tro d e s. The method was adopted f o r t h is work because i t is e f f e c t iv e and re q u ire s ve ry l i t t l e s p e c ia lis e d equipm ent. The c a s tin g apparatus is i l l u s t r a t e d in F igure 2.2. In o rd e r to cast a homogeneous membrane a s o lu tio n or suspension o f a l l the membrane components was re q u ire d. The polym er and QAS s o lu tio n s were found to be in c o m p a tib le. The SBS polym er was d is s o lv e d in THF s o lv e n t, the QAS in m ethanol, but m ethanol caused the p r e c ip it a t io n o f the polym er from s o lu tio n. In o rd e r to o b ta in a homogeneous s o lu tio n th e m e th a n o l/q u a te rn a ry ammonium s a l t s o lu tio n was made w ith the minimum amount o f s o lv e n t and added dropw ise to the SBS/THF s o lu tio n, which was s t i r r e d r a p id ly f o r s e v e ra l m inutes. In most membrane p re p a ra tio n s a c le a r s o lu tio n was o b ta in e d. O c c a s io n a lly, w ith hig h le v e ls o f q u a te rn a ry ammonium s a lts and th e re fo re m ethanol, a c o llo id a l suspension was o b ta in e d.

78 a ) Method of Membrane p re p a ra tio n by Solvent Casting S ty re n e -b -b u ta d ie n e -b -s ty re n e (SBS) (2g-4g) ujas d isp e rse d in a s u ita b le amount o f fr e s h ly d i s t i l l e d THF (4g to 50 cm^). The fla s k was sealed, flu s h e d w ith argon o r n itro g e n and s to re d a t sub-zero te m p e ra tu re s, in darkness f o r 24 hours, to a llo w the SBS to d is s o lv e. The c a s tin g s o lu tio n was prepared by adding the re q u ire d amount o f i n i t i a t o r (ABIN) and monomer (QAS) d is s o lv e d in THF and m ethanol r e s p e c t iv e ly, to the f la s k. The s o lu tio n was s t ir r e d f o r a p p ro x im a te ly 15 m inutes and then poured in to the c a s tin g a p p a ra tu s. The s o lu tio n was contained in a 95mm i. d. r in g supported by a pure cellop hane f ilm (W.E. g la s s Cannings L im ite d, B r i s t o l ), which was s tre tc h e d between wooden em broidery r in g s to form a t i g h t se a l on the g la s s r in g and p re ve n t leakages (F ig u re 2.2 ). U l t r a - v i o l e t ir r a d i a t i o n was used to p h o to c h e m ic a lly decompose ABIN and re le a s e r a d ic a ls in the s o lu tio n. In o rd e r to d ir e c t the U.V. l i g h t onto the c u rin g polym er a f r o n t s ilv e r e d m ir ro r was placed over the assem bly. The U.V. disch a rg e source was an Osram ME/D medium p re ssu re m ercury (Osram/GEC, London), which was s itu a te d between 0.5m and 1.0m from the g la ss ring.. The s o lu tio n was ir r a d ia t e d o v e rn ig h t^ a p p ro x im a te ly 15 hou rs. The membrane was then s o lid, and was removed from th e apparatus, but no t the g la s s r in g. The c e llo p h a n e was removed by p e e lin g i t from the membrane, i t was

79 0 T D DC 0 C - P -H CO *H H I TD P P P n C J P - P Q 0 * - in a m cn in ON ON CO m cn rn ON 1 r - CD CD * m tn m rn LA <± CM CM m Membranes prepared by U.V. Irr a d ia tio n and Solvent C asting cn -p c 0 E cc CM c m m O ON m m CD CD m fa O IA I VO co o CM O CD CM VO O O CD a. CD CD la ON!> O CD CD CD CD CD CD CD 0 cc cc c cc cc cc c C CD CL L J L d L d L d t L d L d L d CC ca >, Q Q Q Q l - a Q Q Ld cc f - cc CC cc c t cc cc cc cc cc Q O Q Q O O O h- h- cn O n m VO VO CD CD m CD CD m a r in < ± * la la CD CD ON CD H c a ^ CD a a a a CD a CD CC CD CD a a o CD a o CD a cn CD CO CD CD CD 03 a CD CD CD CD m m CD CD LA ON CD a CD a CM a CD CD C*- in «- CM CM CM CO CD CD cc CT VO Zl ' *» 0 0 ID 0 H TD E H 0 O E TD P H JD P E JD O E - p D E JD H D C. o c E D E H 0 E c r 1 0 >% r-h J Z > s E - p J Z P r I H 0 >. T 3» 1 i i i 1 >N < i > x I 1 rh 0 I 1 0 P 0 H - P H P 0 a f - h ~ I I I 0 TD O. CJ u CM CJ m C J CM o fa Q L d CM L u LA Li_ CD cc CD L d cc CD L d CC a : cc cc Q 1 -

80 sometimes necessary to m oisten the cellophane b e fo re removing i t. The membrane iuas then heated in a vacuum oven a t 5 5 C to remove any tra c e s o f s o lv e n t. Table 2.5 g ive s d e t a ils o f the m aster membranes, prepared by t h is method, b ) M o d ific a tio n o f C asting Apparatus The membranes produced in the s in g le g la ss r in g (95mm i. d. ) mere o fte n uneven in te x tu re and appearance. T his mas a t t r ib u t e d in p a rt to the d i f f e r e n t e v a p o ra tio n ra te s o f the tmo s o lv e n ts, m ethanol and THF. E va poration o f the s o lv e n ts appears to have a chrom atographic e f f e c t on the membrane components causing some areas to be cloudy- or.c o n ta in c r y s t a ls, and o th e rs to be c le a r. The o r ig i n a l c a s tin g apparatus mas m o d ifie d by the use o f th re e s m a lle r g la ss r in g s (40 mm i. d. ) in p la ce o f the s in g le la rg e r in g. The s m a lle r r in g s mere i n d iv i d u a l ly sealed m ith cellop hane and used in the m eighted m ir ro r arrangem ent P la te 2.1. The membranes produced using t h is apparatus mere more even in appearance (Table 2.6 and 2.7 ). The m aster membrane M6 produced a number o f p rom ising e le c tro d e s. Table 2.7 l i s t s s e v e ra l membranes produced as v a r ia tio n s o f the M6 fo rm u la tio n by v a ry in g the amount o f monomer b u t m a in ta in in g the amounts o f

81