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Chromatographic determination of organotin compounds by using spectrophotometric and thermospray ionization… Nwata, Basil Ugwunna 1988

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CHROMATOGRAPHIC DETERMINATION OF ORGANOTIN COMPOUNDS BY USING SPECTROPHOTOMETRY AND THERMOSPRAY IONIZATION MASS SPECTROMETRIC DETECTION By BASIL UGWUNNA NWATA B.Sc. (Hons), U n i v e r s i t y of I l o r i n , N i g e r i a , 1981 M.Sc. U n i v e r s i t y of Ibadan, Nige r i a , 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF CHEMISTRY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA NOVEMBER 1988 ° B a s i l Ugwunna Nwata, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Ctefliisft-Y The University of British Columbia Vancouver, Canada DE-6 (2/88) i i ABSTRACT An assay based on HPLC-UV, HPLC-GFAAS and HPLC-MS was developed f or the chromatographic separation and c h a r a c t e r i z a t i o n of organotin compounds i n some marine invertebrates of B r i t i s h Columbia. To enable HPLC-UV detection, b u t y l t i n complexes of high e x t i n c t i o n c o e f f i c i e n t s were synthesized, and t h e i r chromatographic behavior i s described. This approach was abandoned because of the increase i n i n s t a b i l i t y of the d e r i v a t i v e s . In the marine organisms studied, mainly b i v a l v e s , t r i b u t y l t i n and d i b u t y l t i n compounds were detected by HPLC-GFAAS. The presence of these compounds indicates that the maritime industry or the lumber industry i s the major source of b u t y l t i n p o l l u t i o n i n the areas sampled. Varying amounts of t r i b u t y l t i n compounds i n the range 1 . 1 4 - 4 . 2 9 pg /mL as Sn (wet weight) and d i b u t y l t i n compounds i n the range 0 . 8 1 - 4 . 6 2 pg/g as Sn (wet weight) were found i n the t i s s u e s . The b u t y l t i n content of the s h e l l s of marine organisms was also examined. Varying amounts of t r i b u t y l t i n and d i b u t y l t i n compounds i n the concentration range 6.60-115.60 /ig/g as Sn and 5.20-49.40 Mg/g as Sn r e s p e c t i v e l y were detected by atomic absorption spectrophotometry. i i i TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES ix LIST OF ABBREVIATIONS x i ACKNOWLEDGEMENT x i i CHAPTER 1 1 I Organotin b i o c i d e s 1 1.1 I n t r o d u c t i o n 1 1.2 A n t i f o u l i n g a c t i o n of organotins 3 1.3 T o x i c i t y of t r i b u t y l t i n compounds 6 1.4 E f f e c t of t r i b u t y l t i n on marine l i f e 10 1.5 Governmental r e g u l a t i o n of t r i b u t y l t i n useage . . . . 10 II A n a l y t i c a l methods f o r organotin compounds 11 1.6 Spectrophotometry and S p e c t r o f l u o r i m e t r y 12 1.7 E l e c t r o c h e m i s t r y 13 1.8 Atomic spectrometry 14 1.9 Gas chromatography 15 1.10 L i q u i d chromatography 18 1.11 Objec t ives of the present study 20 i v CHAPTER 2: EXPERIMENTAL 21 2.1 G e n e r a l methods 21 2.2 M a t e r i a l s a n d r e a g e n t s 24 2.3 M e t h o d o l o g y 25 2.4 S y n t h e s i s o f b u t y l t i n o x i n a t e s 25 2.5 S y n t h e s i s o f b u t y l t i n t r o p o l o n a t e s 27 2.6 D e t e r m i n a t i o n o f a b s o r p t i o n w a v e l e n g t h a n d m o l a r a b s o r p t i o n c o e f f i c i e n t s 28 CHAPTER 3: SEPARATION AND DETECTION PROCEDURE 29 3.1 HPLC-GFAAS 29 3.2 O p t i m i z a t i o n o f GFAAS c o n d i t i o n s 32 3.3 S e l e c t i o n o f c h e m i c a l m o d i f i e r s 34 3.4 E s t a b l i s h m e n t o f r e t e n t i o n d a t a 35 3.5 E s t a b l i s h m e n t o f a n a l y t i c a l p r o c e d u r e and r e c o v e r y s t u d i e s 37 3.6 D e r i v a t i z a t i o n and e x t r a c t i o n o f t r i b u t y l t i n a n d d i b u t y l t i n f r o m m a r i n e o r g a n i s m s 38 3.7 Q u a n t i t a t i o n . 40 3 .7 .1 Q u a n t i t a t i o n o f t r i b u t y l t i n c h l o r i d e a n d d i b u t y l t i n d i c h l o r i d e f o r r e c o v e r y s t u d i e s 40 3.7.2 Q u a n t i t a t i o n o f t r i b u t y l t i n c h l o r i d e and d i b u t y l t i n d i c h l o r i d e 42 3.8 Combined h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y and mass s p e c t r o m e t r y 43 3.9 O p t i m i z a t i o n o f HPLC-MS c o n d i t i o n s 48 3.10 V e r i f i c a t i o n o f f r a g m e n t i o n s t o be u s e d f o r HPLC-MS q u a n t i t a t i o n 49 V 3.11 HPLC-MS of b u t y l t i n oxinates and tropolonates . . . . 49 3.12 Analysis of extracts from marine organisms by HPLC-MS 50 CHAPTER 4 : RESULTS AND DISCUSSION 51 4.1 Characterization of b u t y l t i n oxinates and tropolonates 51 4.2 Molar e x t i n c t i o n c o e f f i c i e n t s of b u t y l t i n oxinates and tropolonates 57 4.3 Nature of organotin oxinates and tropolonates . . . . 57 4.4 Chemical modifiers f o r atomic absorption spectrophotometry of b u t y l t i n chlorides 62 4.5 Retention data 68 4.5.1 Retention data f o r b u t y l t i n chlorides . . . . 68 4.5.2 Retention data for b u t y l t i n oxinates . . . . 70 4.6 T r i b u t y l t i n c h l o r i d e , d i b u t y l t i n d i c h l o r i d e : Recovery studies, detection l i m i t and p r e c i s i o n . . . 70 4.6.1 Recovery studies on the ex t r a c t i o n procedure 70 4.6.2 Detection l i m i t and p r e c i s i o n 75 4.7 HPLC-MS . .' 75 4.7.1 Optimization of the thermospray int e r f a c e conditions 75 4.8 Major ions of standard organotin chlorides . . . . 77 4.9 Retention times of organotin compounds i n HPLC-MS 79 4.10 Levels of b u t y l t i n compounds i n the tis s u e s and s h e l l s of marine animals 85 4.11 HPLC-MS of b u t y l t i n oxinates and tropolonates . . . . 99 4.12 Summary 102 v i BIBLIOGRAPHY 103 APPENDIX A : E l e m e n t a l c o m p o s i t i o n o f s t a n d a r d d o g f i s h l i v e r 109 APPENDIX B: * H NMR . s p e c t r a o f b u t y l t i n o x i n a t e s and t r o p o l o n a t e s 112 APPENDIX C : T h e o r e t i c a l i n t e n s i t y p a t t e r n s f o r s t a n d a r d b u t y l t i n c h l o r i d e s and d i p h e n y l d i c h l o r i d e 117 APPENDIX D: HPLC-MS chromatogram and mass s p e c t r a o f t i s s u e e x t r a c t s o f some m a r i n e a n i m a l s 124 v i i L I S T OF TABLES T a b l e s Page 3 . 2 . 1 G r a p h i t e f u r n a c e o p e r a t i n g p a r a m e t e r s 34 3 . 7 . 1 S a m p l e r p a r a m e t e r s f o r s t a n d a r d a d d i t i o n p l o t o f d i b u t y l t i n d i c h l o r i d e 41 3 . 7 . 2 G T A - 9 5 g r a p h i t e t u b e r a t o m i z e r p r o g r a m f o r s t a n d a r d a d d i t i o n p l o t o f b u t y l t i n compounds i n m a r i n e o r g a n i s m s 42 4 . 1 . 1 A n a l y t i c a l d a t a and m e l t i n g p o i n t s o f b u t y l t i n t r o p o l o n a t e s 51 4 . 1 . 2 A n a l y t i c a l d a t a and m e l t i n g p o i n t s o f b u t y l t i n o x i n a t e s 53 4 . 1 . 3 Fragment i o n s o f b u t y l t i n t r i s t r o p o l o n a t e 54 4 . 1 . 4 Fragment i o n s o f d i b u t y l t i n b i s t r o p o l o n a t e 54 4 . 1 . 5 Fragment i o n s o f t r i b u t y l t i n t r o p o l o n a t e 55 4 . 1 . 6 Fragment i o n s o f b u t y l t i n t r i s o x i n a t e 55 4 . 1 . 7 Fragment i o n s o f d i b u t y l t i n b i s o x i n a t e 56 4 . 1 . 8 F r a g m e n t i o n s o f t r i b u t y l t i n o x i n a t e 56 4 . 2 . 1 M o l a r e x t i n c t i o n c o e f f i c i e n t s o f b u t y l t i n t r o p o l o n a t e s 59 4 . 2 . 2 M o l a r e x t i n c t i o n c o e f f i c i e n t s o f b u t y l t i n o x i n a t e s 60 4 . 3 . 1 R e l e v a n t i n f r a r e d d a t a f o r b u t y l t i n t r o p o l o n a t e s 61 4 . 6 . 1 R e c o v e r y s t u d i e s f o r b u t y l t i n c h l o r i d e s 74 4 . 8 . 1 M a j o r f r a g m e n t i o n s o f s t a n d a r d o r g a n o t i n compounds 84 4 . 9 . 1 R e t e n t i o n t i m e s o f o r g a n o t i n compounds i n HPLC-MS 85 v i i i 4 . 1 0 . 1 C o n c e n t r a t i o n o f b u t y l t i n compounds i n whole body o f m a r i n e o r g a n i s m s 86 4 . 1 0 . 2 C o n c e n t r a t i o n o f b u t y l t i n compounds i n the s h e l l s o f m a r i n e o r g a n i s m s 88 4 . 1 1 . 1 Fragment i o n s o f b u t y l t i n o x i n a t e s and t r o p o l o n a t e s 100 ix LIST OF FIGURES Figures Fage 1.3.1 Approximate h a l f - l i v e s for t r i b u t y l t i n degradation 9 3.1.1 Schematic set up of the HPLC-GFAAS system 30 3.1.2 The graphite tube atomizer 33 3.8.1 Schematic configuration of the HPLC-MS system . . . . 45 3.8.2 The thermospray interface 46 4.1.1 Desorption chemical ionization mass spec crura of t r i b u t y l t i n tropolonate 52 4.1.2 n.m. r. spectrum of dibutyl t in bisoxinate 58 4.4.1 Effect of various modifiers on the absorbance of (C^Ho^SnCl 64 4.4.2 Effect of various modifiers on the absorbance of (C^Ha^SnC^ 65 4.4.3 Effect of modifier volume on the absorbance of (C^Hg^SnCl 66 4.4.4 Effect of modifier volume on the absorbance of (C^Ho^SnC^ 67 4.5.1 HPLC-GFAAS chromatogram of butyl t in chlorides . . . . 69 4.5.2 Chromatogram of butylt in oxinates 71 4.6.1 Calibration graph for recovered t r i b u t y l t i n chloride 72 4.6.2 Standard addition plot for recovered dibutyl t in dichloride 73 4.6.3 Estimation of l imi t of detection 76 4.7.1 Effect of variation of vaporization temperature on t r i b u t y l t i n chloride 78 4.8.1 Mass spectrum of t r i b u t y l t i n chloride 80 X 4 . 8 . 2 Mass s p e c t r u m o f d i b u t y l t i n d i c h l o r i d e 81 4 . 8 . 3 Mass s p e c t r u m o f b u t y l t i n t r i c h l o r i d e 82 4 . 8 . 4 Mass s p e c t r u m o f d i p h e n y l t i n d i c h l o r i d e 83 4 . 1 0 . 1 HPLC-MS t o t a l i o n chromatogram o f B e n t - n o s e c l a m t i s s u e e x t r a c t 90 4 . 1 0 . 2 Mass s p e c t r a o f B e n t - n o s e c l a m t i s s u e e x t r a c t . . . . 90 4 . 1 0 . 3 Mass s p e c t r a o f B e n t - n o s e c l a m t i s s u e e x t r a c t . . . . 91 4 . 1 0 . 4 Mass s p e c t r a o f B e n t - n o s e c l a m t i s s u e e x t r a c t . . . . 91 4 . 1 0 . 5 HPLC-MS o f s t a n d a r d " ( C 6 H n ) 3 S n C l " 93 4 . 1 0 . 6 HPLC-MS o f s t a n d a r d " ( C 6 H 1 : L ) 2 S n C l 2 " 93 4 . 1 0 . 7 E l e c t r o n i o n i z a t i o n mass s p e c t r a o f s h e l l e x t r a c t o f t h e B e n t - n o s e c l a m Macoma n a s u t a 95 4 . 1 0 . 8 T h e o r e t i c a l i n t e n s i t y p a t t e r n s f o r ( C ^ H g ^ S n C l , C ^ H g S n , and C ^ g S n C l 96 4 . 1 0 . 9 E l e c t r o n i o n i z a t i o n mass s p e c t r a o f s t a n d a r d d i b u t y l t i n d i c h l o r i d e 97 4 . 1 0 . 1 0 E l e c t r o n i o n i z a t i o n mass s p e c t r a o f t r i b u t y l t i n c h l o r i d e 98 4 . 1 1 . 1 HPLC-MS t o t a l i o n chromatogram o f t r i b u t y l t i n o x i n a t e 101 x i L I S T OF ABBREVIATIONS uv GFAAS HPLC HPLC-GFAAS MS HPLC-MS H P L C - U V ppm O x i n e , OX r . p . m. U l t r a v i o l e t G r a p h i t e f u r n a c e a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y H i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y HPLC i n c o m b i n a t i o n w i t h GFAAS as d e t e c t o r Mass s p e c t r o m e t r y HPLC i n c o m b i n a t i o n w i t h mass s p e c t r o m e t r y as d e t e c t o r HPLC i n c o m b i n a t i o n w i t h u l t r a v i o l e t s p e c t r o s c o p y f o r d e t e c t i o n P a r t s p e r m i l l i o n , a l s o / ig/mL 8 - h y d r o x y q u i n o l i n e r o t a t i o n s p e r m i n u t e T r o p o l o n e , T 2 - h y d r o x y - 2 ,4 , 6 - c y c l o h e p t a t r i e n o n e C 1 8 . ODS W/V AAS GTA NMR M / Z B u t y l THF O c t a d e c y l s i l a n e . b o n d e d p h a s e c o l u m n w e i g h t p e r volume A t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t e r / s p e c t r o p h o t o m e t r y G r a p h i t e t u b e a t o m i z e r N u c l e a r m a g n e t i c r e s o n a n c e Mass t o c h a r g e r a t i o n - b u t y l T e t r a h y d r o f u r a n x i i ACKNOWLEDGEMENT I w i s h t o e x p r e s s my g r a t i t u d e t o P r o f e s s o r W.R. C u l l e n f o r h i s g u i d a n c e a n d i n t e r e s t i n t h i s s t u d y . My g r a t i t u d e a l s o goes t o D r . G . K . E i g e n d o r f and M r . G l e n n B l o c k f o r t h e i r e x p e r t a d v i c e o n mass s p e c t r o m e t r y . I am a l s o g r a t e f u l t o Agyeman K u m a - M i n t a h , Tom O t i e n o , D r . F r e d W i r e k o , Ove P e d e r s e n , T . S . K o k o , and a l l my c o l l e a g u e s i n P r o f e s s o r C u l l e n ' s r e s e a r c h g r o u p f o r t h e i r encouragement and h e l p f u l d i s c u s s i o n s . - 1 -CHAPTER 1 ORGANOTIN BIOCIDES I INTRODUCTION 1 .1 H i s t o r i c a l B a c k g r o u n d O r g a n o t i n compounds have become v e r y s i g n i f i c a n t i n t h e modern s o c i e t y w i t h many i n d u s t r i a l , a g r i c u l t u r a l and m e d i c i n a l a p p l i -c a t i o n s . The s y n t h e s i s o f d i e t h y l t i n d i i o d i d e i n 1849 by F r a n k l a n d , 1 opened the way f o r the e x p l o r a t i o n o f the p o t e n t i a l s o f o r g a n o t i n compounds. R a p i d p r o g r e s s was e n h a n c e d by the d i s c o v e r y o f G r i g n a r d r e a g e n t s w h i c h made i t p o s s i b l e f o r the s y n t h e s i s o f a wide v a r i e t y o f o r g a n o t i n compounds o f the f o r m u l a R4Sn, f r o m w h i c h l o w e r a l k y l o r g a n o t i n compounds c o u l d e a s i l y be d e r i v e d . The G r i g n a r d method i s s t i l l the o n l y method a p p l i c a b l e to the p r o d u c t i o n o f t e t r a p h e n y l t i n . T e t r a -b u t y l t i n and t e t r a o c t y l t i n have b e e n s y n t h e s i z e d by Wurtz s y n t h e s i s , and a l k y l a t i o n by a l k y l a l u m i n u m compounds. The f i r s t i n d u s t r i a l a p p l i c a t i o n o f o r g a n o t i n compounds was made i n 1936, when Yngve o f C a r b i d e and C a r b o n C h e m i c a l C o r p o r a t i o n d i s c o v e r e d the h e a t s t a b i l i z i n g e f f e c t o f o r g a n o t i n compounds on P o l y v i n y l c h l o r i d e ( P V C ) , and o t h e r c h l o r i n a t e d h y d r o c a r b o n p o l y m e r s . 1 I n t h i s r e g a r d , d i b u t y l t i n d i l a u r a t e and d i b u t y l t i n m a l e a t e a r e now m a i n l y u s e d . The d i o c t y l t i n d e r i v a t i v e s a r e u s e d as PVC s t a b i l i z e r s f o r f o o d p a c k a g i n g p o l y m e r s , b e c a u s e o f t h e i r n o n - t o x i c i t y . O r g a n o t i n compounds are a l s o - 2 -used in industry for cold curing of sil icone rubber, and as polymeriza-tion catalysts, e.g. butylchlorotin dihydroxide C^HgSnCl(OH)2. Triorganotin compounds are the most important i n agricultural applications. They exhibit fungicidal , bacter ic idal , and acaricidal a c t i v i t i e s . The t r i e t h y l t i n compounds exhibit acute toxici ty to mammals, the LD50 of t r i e t h y l t i n acetate to rat being 4.0 mg/kg body weight.^ The tr icyclohexylt in compounds are used as miticides. Plictran 3 miticide, made by Dow Chemical Company.contains tricyclohexyl-t i n hydroxide as the active ingredient.^ The t r i b u t y l t i n compounds are active as fungicides and slimicides. As a result of the biological ac t ivi ty of t r i b u t y l t i n compounds, they are incorporated into various forms of antifouling paint formulations, to protect the hulls of ships and boats from fouling by fungi, algae, sponges, molluscs, diatoms, etc. Such fouling has the effect of increasing weight and drag, causing the ship to consume more fuel to maintain i ts speed. The t r i b u t y l t i n compounds also find use, as wood preservatives. When impregnated into wood, they have only slight insect ic idal ac t iv i ty , and are usually formulated with other compounds to broaden the ac t iv i ty . They neither increase flammability, nor impart undesirable colors to the wood. They are also resistant to leaching. The tr iphenyltin compounds also show antifungal a c t i v i t y . T r i -phenyltin acetate and triphenyltin hydroxide are used as agricultural fungicides. Some octahedral organotin dihalides having the formula R 2 SnX 2 L 2 (R = ethyl or phenyl, X = chloride or bromide, L 2 •= O-phenanthroline or 2-(2-pyridyl)benzimidazole) exhibit antitumor a c t i v i t i e s . ^ The same 3 compounds have a l s o b e e n r e p o r t e d to p o s s e s s a n t i - h e r p e s a c t i v i t y i n v i t r o A l s o , some o r g a n o t i n compounds e . g . d i b u t y l t i n d i l a u r a c e are e f f e c t i v e f o r t h e r e m o v a l o f i n t e s t i n a l worms i n p o u l t r y . The m a j o r o r g a n o t i n compounds o f c o n c e r n i n t h e m a r i n e e n v i r o n m e n t a r e t h e t r i b u t y l t i n compounds w h i c h a r e a c t i v e i n g r e d i e n t s i n a n t i -f o u l i n g p a i n t s . The compounds u s e d as a c t i v e i n g r e d i e n t s i n some c o m m e r c i a l a n t i f o u l i n g p a i n t s a r e b i s ( t r i b u t y l t i n ) o x i d e , b i s ( t r i b u t y l -t i n ) d o d e c e n y l s u c c i n a t e , b i s ( t r i b u t y l t i n ) s u l f i d e , t r i b u t y l t i n f l u o r i d e , t r i b u t y l t i n r e s i n a t e , t r i b u t y l t i n m e t h a c r y l a t e , b i s ( t r i b u t y l -t i n ) a d i p a t e . U n f o r t u n a t e l y , t h e s e compounds do n o t r e m a i n l o c a l i z e d and t h e i r s p r e a d t h r o u g h o u t the m a r i n e e n v i r o n m e n t i s c a u s i n g c o n s i d e r -a b l e p r o b l e m s . 1.2 A n t i f o u l i n g A c t i o n o f O r g a n o t i n s The g r o w t h o f unwanted m a r i n e o r g a n i s m s on t h e h u l l s o f v e s s e l s has been a m a j o r p r o b l e m i n the m a r i t i m e i n d u s t r y . The g r o w t h o f t h e s e o r g a n i s m s l e a d s t o s e v e r e r e t a r d a t i o n and d r a g i n t h e s h i p ' s p e r f o r -a n c e . S t r u c t u r a l w e a k e n i n g , and d e p r e c i a t i o n i n v a l u e a l s o o c c u r . The c l e a n i n g o f t h e f o u l e d h u l l s i s b o t h e x p e n s i v e and t i m e c o n s u m i n g . One o f t h e e a r l y a p p r o a c h e s t a k e n t o p r e v e n t f o u l i n g i n wooden s h i p s , was t h e u s e o f c o p p e r m e t a l s h e a t h i n g i n t h e c o n s t r u c t i o n o f s h i p s ' h u l l s . T h i s a c h i e v e d modera te s u c c e s s i n t h e c o n t r o l o f f o u l i n g . I n s t e e l s h i p s , t h e use o f c o p p e r m e t a l s h e a t h i n g i s n o t a p p r o -p r i a t e , due t o s e v e r e g a l v a n i c c o r r o s i o n o f s t e e l when i n c o n t a c t w i t h - 4 -copper and sea water. The method of fouling prevention in steel ships is by the use of chemical agents. These chemical agents act by releasing biocides from the paint which k i l l the larvae and spores of any marine animals and p l a n t s attempting to s e t t l e on the s h i p ' s h u l l . Among the early biocides employed for this purpose was cuprous oxide. Cuprous oxide exhibits a wide spectrum of toxici ty to animals, but many plants are resistant to i t . On continuous use, i t forms insoluble greenish salts within the surface layers of the paint f i l m . The bui ld up of these salts on the surface interferes with the controlled release of fresh biocide. This l imits the l i f e time and efficiency of the paint. The search for biocides to boost the performance of cuprous oxide led to the screening of organotin compounds. T r i b u t y l t i n compounds were found to be suitable biocides. The com-pounds possess the following properties: i . low mammalian toxicity i i . lack of color. The low mammalian toxicity ensures human safety in handling paints containing t r i b u t y l t i n as active ingredient. Their lack of color, makes them easily incorporated into brightly colored paints. In the course of searching for ef f ic ient ways of designing effective t r i b u t y l t i n antifouling preparations, the following formulations have become commercially available. i . Contact leachinp antifouling paints. In this design, the anti-fouling system is composed of a tough insoluble film-forming resin such - 5 -as c h l o r i n a t e d r u b b e r , w i t h i n w h i c h t h e t r i b u t y l t i n i s p h y s i c a l l y d i s -p e r s e d i n t h e h a r d m a t r i x . Cn i m m e r s i o n i n w a t e r , the f r e e l y d i s p e r s e d t r i b u t y l t i n compounds n e a r t h e s u r f a c e o f the p a i n t a r e a b l e t o d i f f u s e o u t o f t h e m a t r i x of t h e p a i n t f i l m . As t h e b i o c i d e l e a c h e s o u t o f t h e f i l m , i t l e a v e s b e h i n d m i c r o s c o p i c p o r e s w i t h i n t h e p a i n t m a t r i x . The i n f l o w o f s e a w a t e r i n t o t h e s e m i c r o s c o p i c p o r e s c a u s e s t h e r e l e a s e o f f r e s h t r i b u t y l t i n b i o c i d e f r o m b e n e a t h the s u r f a c e l a y e r s o f t h e f i l m . The s h o r t c o m i n g o f t h i s d e s i g n i s t h a t w i t h the p a s s a g e o f t i m e , the p a i n t f i l m becomes c l o g g e d w i t h i n s o l u b l e m a t e r i a l s m a k i n g i t p r o g r e s s -i v e l y d i f f i c u l t f o r b i o c i d e i n the d e e p e r s t r a t a o f t h e p a i n t m a t r i x to be r e l e a s e d . As a r e s u l t , t h i s d e s i g n o f a n t i f o u l i n g p a i n t works b e s t o n l y d u r i n g t h e e a r l y p a r t o f t h e p a i n t ' s l i f e . When the a n t i f o u l i n g a c t i o n o f the p a i n t f a i l s , a l a r g e amount o f the b i o c i d e i s s t i l l t r a p p e d i n the i n n e r m a t r i x , t h e r e b y c r e a t i n g a s e v e r e p r o b l e m o f p r o p e r d i s p o s a l o f s p e n t a n t i f o u l i n g p a i n t . i i . S o l u b l e m a t r i x / a b l a t i v e f o r m u l a t i o n . I n t h i s d e s i g n , the t r i b u t y l t i n compound i s added t o a s o l u b l e m a t r i x . I n an a b l a t i v e s y s t e m , t h e f i l m m a t r i x i s a m i x t u r e o f i n s o l u b l e and s o l u b l e m a t e r i a l d e s i g n e d t o b r e a k down o v e r t i m e , t h u s a l l o w i n g t h e b i o c i d e p h y s i c a l l y d i s p e r s e d i n t h e p a i n t f i l m t o be r e l e a s e d . ^ A d i s a d v a n t a g e o f t h i s f o r m u l a t i o n i s t h a t i t i s d i f f i c u l t t o c o n t r o l t h e a c t u a l breakdown o f t h e p a i n t f i l m , and t h u s b i o c i d e r e l e a s e , b e c a u s e t h e m a t r i x s o l u b i l i t y a n d r a t e of f i l m b r e a k d o w n a r e a f f e c t e d by w a t e r c o n d i t i o n s and v e s s e l s p e e d . ^ - 6 -i i i . Self-polishing copolymer paints. This design represents the most advanced design in antifouling paint technology. The biocide is chemi-cally bound in the paint film. The release rate of the biocide in this design can be controlled. This allows the biocidal a c t i v i t y of the antifouling paint to last a long time. The film forming resin is a co-polymer of tributyltin methacrylate/ methylmethacrylate, and is also the source of the biocide. The t r i -butyltin methacrylate/methylmethacrylate polymer is hydrophobic, preventing sea water ingress to the depths of the film. At the surface of the paint, sea water interacts with the hydrophobic copolymer, thereby initiating a saponification reaction which cleaves tributyltin from the copolymer backbone, and releases i t into the sea. 1.3 Toxicity of Tributyltin (T.B.T.) Compounds On introduction into the marine environment, tributyltin is mainly removed from the water column by assimilation and metabolism by plants and animals.6 Tributyltin is susceptible to degradation by a variety of organisms and by photolysis. Hydrolysis and volatilization do not 7 ft 9 appear to be major degradative pathways. •°•' Based on experimentally measured values for the sediment-water partition coefficient K ^ , 1 0 - 1 1 tributyltin adsorbs strongly to particulate matter ir. the sediment. The affinity for sediments, makes i t far less bioavailable to organisms in the upper water layer. According to Maguire, 1 1 tributyltin adsorbs so firmly to particulates that under abiotic - 7 -c o n d i t i o n s . t h e r e vas nodesorption o f tributyltin o x i d e f r o m h a r b o r s e d i -m e n t s , o v e r a p e r i o d o f ten months. However, t h e r e was d e g r a d a t i o n by microorganisms resulting in the liberation of butylated and m e t h y l a t e d degradation products. On the contrary, data for San Diego sediment12 have suggested much greater mobility of adsorbed tributyltin f r o m sediment than reported by Maguire. Tributyltin has the tendency to preferentially accumulate in the surface microlayer of natural waters.13,14 This is because tributyl-t i n ' s octanol-water partition coefficient, K o w , and s e d i m e n t - w a t e r p a r t i t i o n coefficient K o c values favor accumulation in the s u r f a c e m i c r o l a y e r . The surface microlayer attracts and sequesters moderately h y d r o p h o b i c chemicals like tributyltin. This preferential a c c u m u l a t i o n i n the surface microlayer is expected to render tributyltin b i o l o g i c a l l y u n a v a i l a b l e to most organisms. However, a variety of o r g a n i s m s a c c u m u l a t e t r i b u t y l t i n to relatively high concentrations b e c a u s e o f i t s m o d e r a t e l y h i g h octanol-water partition coefficient ( K o v , - 2 3 0 0 ) . B a c t e r i a and phytoplankton accumulate tributyltin a t concentrations 6 0 0 t i m e s and 30,000 times respectively, more than their exposure c o n c e n t r a -t i o n s . 1 ^ - 1 ^ A bioaccumulation factor of 4400 has been reported by Evans and Laughlin, 1^ for the hepatopancreas of the mud crab Rhithropanopeus  h a r r i s i i . Organotin compounds exhibit preferential accumulation in certain tissues than others. Ward e_£ aJL-*® observed that the viscera o f sheepshead minnow contained higher concentrations of tributyltin oxide than the cranial or muscle tissues. The reported bioaccumulation factors for tributyltin compounds are high enough to w a r r a n t c o n c e r n - 8 -with regard to their persistence and accumulation in food chains, however, they are degraded i n vivo by bacteria, algae, f i s h and mammals. T r i b u t y l t i n does not appear to be amenable to biomagnification. Macek et a _ l . , ^ have presented data suggesting that chemicals with half-lives less than 40 days jji vivo do not pose a biomagnification problem. T r i b u t y l t i n has a h a l f - l i f e considerably shorter than forty days by aerobic metabolism (Fig. 1.3.1). A variety of organisms can metabolize and excrete t r i b u t y l t i n com-pounds. Degradation rates vary depending on the conditions considered. Microbial degradation under aerobic conditions in some natural environ-ments may be quite rapid. Seligman,20 studied microbial biodegradation of t r i b u t y l t i n using laboratory microcosms, and observed h a l f - l i v e s of 6-13 days under lighted conditions. However, there are contrasting reports about the rate of biodegradation of t r i b u t y l t i n . Vertebrates possess the a b i l i t y to breakdown t r i b u t y l t i n compounds into less toxic metabolites. Ward et a l . ° have reported the rn vivo metabolism of t r i b u t y l t i n to dibutyl t in , monobutyltin and inorganic t i n . These metabolites are less toxic than t r i b u t y l t i n . Maguire et a l . ^ also observed in vivo degradation of t r i b u t y l t i n by a green algae with the major degradation product being dibutyl t in . Photolysis is a major degradation route for t r i b u t y l t i n in the marine environment. Photolysis of t r i b u t y l t i n is affected by the wavelength of the ul t raviolet light. Half lives ranging from less than one day to 100 days have been reported.^•^•22.23 - 9 -2?0 AEROB MT AKAEROB KT FISH KT HYDROL OYS MT PHOTOL Fig. 1.3.1: Approximate average h a l f - l i v e s 6 for t r i b u t y l t i n degraded via aerobic metabolism (AEROB KT), anaerobic metabolism (ANAEROB KT), f i s h (FISH KT), hydrolysis (HYDROL). oysters (OYS KT). and photolysis (FHOTOL). 10 1.4 Effect of Tributyltin (T.B.T.) Toxicity on Marine Life T.B.T. d i s s o l v e d i n water e x h i b i t acute t o x i c i t y to a v a r i e t y of aquatic l i f e . Available data, tend to suggest that fish and larger Crustacea are l e s s s e n s i t i v e to T.B.T. than b i v a l v e s , m o l l u s c s , phyto-p l a n k t o n , and small crustaceans. It has been e s t a b l i s h e d that molluscs are g e n e r a l l y very s e n s i t i v e to organotin compounds.^•25 A p a r t from showing acute t o x i c i t y to c e r t a i n marine l i v e s , T.B.T. e x h i b i t s chronic t o x i c i t y to some aquatic l i v e s at concentrat ions ranging from 10 to less than 0.025 ppb. T.B.T. a lso appears to be e s p e c i a l l y t o x i c to the alga Skeletonema costatum. and to embryonic and l a r v a l stages of the P a c i f i c o y s t e r . 1.5 Governmental Regulat ion of T r i b u t y l t i n Useage F o l l o w i n g the c o r r e l a t i o n between t r i b u t y l t i n , s h e l l malformations and abnormal growth i n oysters , the Government of France i n 1982 banned the use of a n t i f o u l i n g p a i n t s c o n t a i n i n g more than 3% by weight of organot in compounds, f o r the p r o t e c t i o n of h u l l s of boats l e s s than 25 tons . In 1987, a t o t a l ban on the use of organotin p a i n t s on v e s s e l s l e s s than 25 m came into e f f e c t i n France.26 In 1986, England p r o h i b i t e d the r e t a i l s a l e and supply f o r r e t a i l s a l e of a n t i f o u l i n g p a i n t s c o n t a i n i n g t r i b u t y l t i n , i f the t o t a l c o n c e n t r a t i o n of t r i b u t y l t i n i n the d r i e d copolymer p a i n t exceeded 7.5% by weight, or i f the t o t a l concentrat ion of organot in i n other - 11 -0 7 n o n - c o p o l y m e r p a i n t s e x c e e d e d 2.5% by w e i g h t . The u s e o f o r g a n o t i n compounds i n f r e s h w a t e r a n t i f o u l i n g p a i n t s i s p r o h i b i t e d i n Germany and S w i t z e r l a n d . ° I n C a n a d a , t r i b u t y l t i n i s r e g i s t e r e d u n d e r t h e P e s t C o n t r o l P r o d u c t s A c t s f o r u s e as a s l i m i c i d e and f o r g e n e r a l l u m b e r p r e s e r v a t i o n . * I t s u s e as a p r e s e r v a t i v e f o r n e t s i s n o t a l l o w e d . ^ ® I I . A N A L Y T I C A L METHODS FOR ORGANOTIN COMPOUNDS The f i r s t a n a l y t i c a l methods u s e d f o r t h e a n a l y s i s o f t i n w e r e c l a s s i c a l g r a v i m e t r i c o r v o l u m e t r i c p r o c e d u r e s w h i c h gave o n l y t o t a l t i n . O p t i c a l s p e c t r o g r a p h i c a n a l y s i s o f t o t a l t i n was e x t e n s i v e l y u s e d f o r g e o l o g i c a l s t u d i e s b e g i n n i n g as e a r l y as 1931, and e x t e n d i n g i n t o the l a t e 1 9 5 0 ' s and e a r l y 1 9 6 0 ' s . A t t h i s t i m e , more s e n s i t i v e and s p e c i f i c c o l o r i m e t r i c , f l u o r i m e t r i c and n e u t r o n a c t i v a t i o n a n a l y s e s r e p l a c e d t h e s p e c t r o g r a p h i c m e t h o d s . Flame a t o m i c a b s o r p t i o n t e c h n i q u e s were a l s o i n t r o d u c e d , b u t t h e s e r e m a i n e d l e s s p o p u l a r b e c a u s e o f the low s e n s i t i v i t y o f t h e t i n a b s o r p t i o n l i n e s . ^ ® W i t h t h e w i d e u s e o f o r g a n o t i n compounds i n a g r i c u l t u r e and i n d u s -t r i e s , t h e n e e d a r o s e t o m o n i t o r and d e t e r m i n e t h e amount o f o r g a n o t i n r e s i d u e s i n a g r i c u l t u r a l p r o d u c t s and t h e e n v i r o n m e n t . E a r l y methods r e l i e d on t h e c o n v e r s i o n o f t h e o r g a n o t i n s t o i n o r g a n i c t i n u s u a l l y by d i g e s t i o n w i t h m i n e r a l a c i d s , f o l l o w e d by i g n i t i o n . ^ O t h e r methods a p p l i e d t o t h e d i r e c t s p e c i a t i o n o f o r g a n o t i n com-pounds were p o l a r o g r a p h y and gas c h r o m a t o g r a p h y . T h e s e two methods a re 12 a l s o q u a n t i t a t i v e . The g r e a t e m p h a s i s o f a n a l y t i c a l and e n v i r o n m e n t a l c h e m i s t s on q u a n t i t a t i v e a n a l y s e s has r e s u l t e d i n l i t t l e a t t e n t i o n b e i n g g i v e n t o the q u a l i t a t i v e a s p e c t s o f o r g a n o t i n a n a l y s e s . Q u a l i t a t i v e l y , infrared spectrometry, L Mossbauer s p e c t r o m e t r y , ' a n d n u c l e a r m a g n e t i c r e s o n a n c e spectroscopy,34,35,36 have b e e n a p p l i e d t o p r o v i d e i n f o r m a t i o n on m o l e c u l a r structure and f o r t h e c h a r a c t e r i z a t i o n o f o r g a n o t i n compounds . The v a r i o u s t y p e s o f q u a n t i t a t i v e a n a l y t i c a l methods a p p l i e d o v e r t h e y e a r s , f o r t h e d e t e r m i n a t i o n o f o r g a n o t i n compounds a r e d e s c r i b e d i n the f o l l o w i n g s e c t i o n s . 1.6 S p e c t r o p h o t o m e t r y and S p e c t r o f l u o r i m e t r y A l d r i d g e and C r e m e r , were the f i r s t t o u s e d i t h i z o n e f o r the s p e c t r o p h o t o m e t r y d e t e r m i n a t i o n o f o r g a n o t i n compounds . D i e t h y l t i n and t r i e t h y l t i n c h l o r i d e s r e a c t w i t h d i t h i z o n e t o f o r m c o l o r e d c o m p l e x e s . A n a l y s i s o f t h e c o m p l e x e s i s e f f e c t e d f o l l o w i n g p a r t i t i o n i n g between aqueous p o t a s s i u m h y d r o x i d e s o l u t i o n and c h l o r o f o r m . The d i e t h y l t i n s p e c i e s p a r t i t i o n i n t o t h e a l k a l i l a y e r , w h i l e t h e t r i e t h y l t i n s p e c i e s m i g r a t e t o t h e c h l o r o f o r m l a y e r . The s e p a r a t e d o r g a n o t i n compounds c a n t h e n be d e t e r m i n e d b y U V - s p e c t r o p h o t o m e t r y . T h i s method i s q u i t e s e n s i -t i v e and o f f e r s s p e c i f i c i t y . I n 1972, H a v i r a n d Vrestal3& u s i n g a m o d i f i e d d i t h i z o n e m e t h o d , a l s o a c h i e v e d s p e c i f i c i t y b y s e l e c t i v e l y e x t r a c t i n g b i s ( t r i b u t y l t i n ) o x i d e f r o m a l k a l i n e medium u s i n g c h l o r o b e n z e n e i n the p r e s e n c e o f a c o m p l e x i n g 13 a g e n t . S k e e l and B r i c k e r ^ ^ d e v e l o p e d a s p e c t r o p h o t o m e t r i c met ' iod f o r the d e t e r m i n a t i o n o f d i b u t y l t i n d i c h l o r i d e u s i n g d i p h e n y l c a r b a z o n e . The s e n s i t i v i t y of this method is in the microgram range. Other c o l o r i m e t r i c r e a g e n t s u s e d f o r the a n a l y s e s o f o r g a n o t i n s were d i t h i o l , h e m a t o x y l i n , ^ ' 8 - h y d r o x y q u i n o l i n e , p h e n y l f l u o r o n e p y r o c a t e c h o l v i o l e t , 4 2 , 4 3 f l a v i n o l ^ ( 3 - h y d r o x y f l a v o n e ) and q u e r c e t i n ^ ( 3 , 3 ' , 4 ' , 5 , 7 - p e n t a h y d r o x y -f l a v o n e ) . A l l the c o l o r i m e t r i c r e a g e n t s form c o m p l e x e s w i t h i n o r g a n i c t i n and many o t h e r m e t a l s . E f f i c i e n t s e p a r a t i o n o f t i n compounds i s n e c e s s a r y b e f o r e s p e c t r o -p h o t o m e t r y a n a l y s i s b e c a u s e o f the l a c k o f s p e c i f i c i t y o f the c o l o r i -m e t r i c r e a g e n t s . F o r the f l u o r i m e t r i c d e t e r m i n a t i o n o f o r g a n o t i n s , V e r n o n ^ u s e d 3 - h y d r o x y f l a v o n e f o r the d e t e r m i n a t i o n o f t r i p h e n y l t i n s p e c i e s i n w a t e r . A n o t h e r r e a g e n t t h a t has b e e n a p p l i e d t o the a n a l y s i s o f o r g a n o t i n compounds i s m o r i n ( 2 ' , 3 , U ' , 5 , 7 - p e n t a h y d r o x y f l a v o n e ) . Arakawa et a l . ^ u s e d m o r i n as a l i g a n d f o r the f l u o r i m e t r i c a n a l y s i s o f a l k y l t i n and p h e n y l t i n compounds. 1.7 E l e c t r o c h e m i s t r y P o l a r o g r a p h y , a n o d i c s t r i p p i n g v o l t a m m e t r y ^ • ^ a n c \ p o t e n t i o m e t r i c t i t r a t i o n s have b e e n u s e d f o r the d e t e r m i n a t i o n o f o r g a n o t i n compounds i n aqueous and n o n aqueous m e d i a . D i e t h y l t i n d i c h l o r i d e was t h e f i r s t o r g a n o t i n compound whose p o l a r o g r a p h i c r e d u c t i o n b e h a v i o r was r e c o r d e d . ^0 T y u r i n and F l e r o v . - ' l have a l s o p r o v i d e d d a t a on the 14 -polarographic behavior of other organotin compounds. The ease of reduction of organotin compounds in polarography has been found to be a function of the organic moiety on the t i n , the ease of reduction being ethyl > propyl > butyl.^2 Polarography in the d i f f e r e n t i a l pulse mode has also been used in the determination of organotin compounds . • • ->-> Potentiometric t i t r a t i o n in non aqueous medium has also been used in the analysis of organotin compounds.^•^ In one of these methods, b i s ( t r i b u t y l t i n ) oxide reacted with water to produce t r i b u t y l t i n hydroxide which was t i t rated with hydrochloric acid. Although electrochemical methods are able to differentiate between organotin species according to their redox potentials, this method suffers from interference by organic matter. Organic matter present in water coats the electrodes, causing broadening of peaks and shifts in peak potentials . This disadvantage restr ic ts the application of electrochemical techniques in the analyses of natural water. 1.8 Atomic Spectrometry Atomic absorption spectrometry has been applied extensively to the determination of organotin compounds. However, not much information is available on atomic emission spectrometry of organotin compounds. Either a f lame^ or an e l e c t r i c a l l y heated graphite furnace^ can be used for atomization. Since organotin compounds are determined as inorganic t i n by atomic spectrometry, some form of separation of a mixture of organotin compounds is necessary before atomic spectrometry. - 15 -M c K i e , ^ a c h i e v e d s e p a r a t i o n o f t r i b u t y l t i n f r o m d i b u t y l t i n , m o n o b u t y l -t i n , and i n o r g a n i c t i n s p e c i e s by s o l v e n t - s o l v e n t e x t r a c t i o n o f h y d r o -c h l o r i c a c i d t r e a t e d s a m p l e s , p r i o r t o a t o m i c s p e c t r o m e t r y . K o j i m a , ^ a l s o s e p a r a t e d m i x t u r e s o f d i b u t y l t i n and t r i b u t y l t i n s p e c i e s b y s o l v e n t p a r t i t i o n b e f o r e a n a l y s i s b y g r a p h i t e f u r n a c e - a t o m i c a b s o r p t i o n s p e c t r o m e t r y . C o a t i n g t h e g r a p h i t e f u r n a c e i n t e r i o r w i t h a r e f r a c t o r y m e t a l s a l t s u c h as z i r c o n y l a c e t a t e has b e e n shown t o i n c r e a s e a t o m i z a -t i o n e f f i c i e n c y o f t i n . 6 ^ P e e t r e and S m i t h , 6 ^ o b s e r v e d t h a t a r e l a t i o n -s h i p e x i s t s b e t w e e n the a t o m i c a b s o r p t i o n s e n s i t i v i t y and t h e s t r u c t u r e o f o r g a n o t i n compounds. They c o n c l u d e d t h a t s e n s i t i v i t y d e c r e a s e d as the e n e r g y o f t h e a l k y l t i n b o n d d e c r e a s e d . E m i s s i o n s p e c t r o g r a p h y has b e e n a p p l i e d t o the d e t e r m i n a t i o n o f b i s ( t r i b u t y l t i n ) o x i d e . 6 ^ A g a i n , p r i o r s e p a r a t i o n o f a m i x t u r e o f o r g a n o t i n compounds i s n e c e s s a r y b e f o r e the a p p l i c a t i o n o f t h i s t e c h n i q u e . 1 .9 Gas C h r o m a t o g r a p h y (GC)' O r g a n o t i n compounds t o be s e p a r a t e d by gas c h r o m a t o g r a p h y must be v o l a t i l e d u r i n g s e p a r a t i o n . F o r t h e d e t e r m i n a t i o n o f the l e s s v o l a t i l e o r g a n o t i n compounds, d e r i v a t i z a t i o n t e c h n i q u e s t o c o n v e r t them t o v o l a t i l e s p e c i e s a r e u s u a l l y a p p l i e d . D e r i v a t i z a t i o n t o h y d r i d e s o r t e t r a a l k y l t i n compounds a r e two common p r o c e d u r e s : G e n e r a t i o n o f o r g a n o t i n h y d r i d e s . I n the c o n v e r s i o n o f o r g a n o t i n - 16 -compounds t o v o l a t i l e h y d r i d e s , e x c e s s b o r o h y d r i d e i s u s u a l l y u s e d t o c o n v e r t t h e a l k y l t i n s p e c i e s t o h y d r i d e s o f t h e f o r m u l a R ^ S n H ^ ^ . The g e n e r a t e d h y d r i d e s a r e p u r g e d f r o m s o l u t i o n w i t h an i n e r t g a s , and c a n be t r a p p e d c r y o s c o p i c a l l y i n a c o l d t r a p . The t e m p e r a t u r e o f t h e c o l d t r a p i s u s u a l l y i n c r e a s e d , t o r e l e a s e t h e o r g a n o t i n h y d r i d e s o n t o t h e gas c h r o m a t o g r a p h i c c o l u m n . R e a r r a n g e m e n t o r d i s p r o p o r t i o n a -t i o n o f t h e a l k y l g r o u p s o f t h e o r g a n o t i n compounds c a n o c c u r d u r i n g gas c h r o m a t o g r a p h i c a n a l y s i s . ^ i i . C o n v e r s i o n t o t e t r a - a l k v l t i n compounds. C o n v e r s i o n o f o r g a n o t i n compounds t o t e t r a a l k y l t i n compounds i s u s u a l l y a c c o m p l i s h e d b y the r e a c t i o n o f t h e o r g a n o t i n compounds w i t h a G r i g n a r d r e a g e n t . The r e a c t i o n o f m o n o a l k y l t i n , d i a l k y l t i n a n d t r i a l k y l t i n s p e c i e s p r o c e e d s t o c o m p l e t i o n a t v e r y low c o n c e n t r a t i o n s . No r e a r r a n g e m e n t o f t h e o r i g i n a l a l k y l g r o u p s a t t a c h e d t o t h e t i n n u c l e u s i s u s u a l l y o b s e r v e d . ^ The t e t r a a l k y l t i n d e r i v a t i v e s f o r m e d i n g e n e r a l a r e q u i t e s t a b l e i n o r g a n i c s o l v e n t s . ^ > ^ M a g u i r e e t a l . 6 ^ have a p p l i e d t h i s p r o c e d u r e t o t h e d e t e r m i n a t i o n o f t r i b u t y l t i n i n C a n a d i a n h a r b o r s , a f t e r c o n v e r t i n g them t o b u t y l p e n t y l t i n d e r i v a t i v e s . I n gas c h r o m a t o g r a p h y , good s e n s i t i v i t y i s u s u a l l y a c h i e v e d b y t h e u s e o f s e n s i t i v e d e t e c t o r s s u c h as E l e c t r o n C a p t u r e d e t e c t o r ( E C D ) , mass s p e c t r o m e t r i c d e t e c t o r , f l a m e p h o t o m e t r i c d e t e c t o r , and a t o m i c a b s o r p -t i o n s p e c t r o m e t r y . M e t h y l b u t y l t i n s p e c i e s have b e e n d e t e c t e d b y u s i n g a gas c h r o m a t o g r a p h c o u p l e d t o a mass s p e c t r o m e t e r ^ ( G C - M S ) . W a l d o c k and M i l l e r , ^ d e t e r m i n e d t h e t o t a l t i n a n d t r i b u t y l t i n i n s e a w a t e r and o y s t e r s b y G C - M S . - 17 -A flame photometric detector f o r the analyses of organotin compounds was developed by Aue and F l i n n . ^ Flame photometric d e t e c t i o n has b e e n a p p l i e d as a t i n s e l e c t i v e method f o r the gas chromatographic a n a l y s i s of b u t y l t i n species i n w a t e r , 6 ^ s e d i m e n t , ^ a r , d f o r the determinat ion of m e t h y l t i n species i n w a t e r . ^ A l a t e r m o d i f i c a t i o n to the flame photo-metr ic d e t e c t o r , ^ improved i t s d e t e c t i o n l i m i t to 5 x 1 0 " ^ mol . t i n . A disadvantage of the flame photometric detector i s that SnC>2 may accumulate on i n t e r n a l surfaces of the detec tor , causing a decrease i n s e n s i t i v i t y . In a d d i t i o n , Maguire and Tkacz ,^5 reported that the flame photometric detector can e a s i l y be ' p o i s o n e d ' by t ropolone , a l i g a n d sometimes used i n the e x t r a c t i o n of organot in compounds. Coupling the gas chromatograph to an atomic absorpt ion spectrophoto-meter (GC-AAS) appears to be the most popular technique f o r element s p e c i f i c d e t e c t i o n . M e t h y l t i n species i n n a t u r a l water have b e e n determined a f t e r hydride d e r i v a t i z a t i o n by GC-quartz furnace atomic absorpt ion spectrophotometry^ 6 and GC-graphite furnace atomic absorpt ion s p e c t r o p h o t o m e t r y . ^ Analyses of b u t y l t i n compounds as hydrides b y GC-graphite furnace atomic absorpt ion spectrophotometry has been reported by B a l l s . ^ 8 The method features the i n t r o d u c t i o n of the hydrides w i t h the c a r r i e r gas stream i n t o the i n t e r n a l purge i n l e t of the graphite furnace . The GC-AAS technique works well with the very v o l a t i l e methyltin hydrides. For the l e s s v o l a t i l e hydrides of b u t y l t i n and phenyltin species, condensation does occur on the r e l a t i v e l y cool inner surfaces of the graphite furnace assembly before getting into the furnace. 18 Bet ter separa t ion of a mixture of organotin hydrides can be achieved i n gas chromatography by the use of var ious gas chromatographic columns. Wool l ins and C u l l e n , ^ employed a c a p i l l a r y column to separate a mixture of organot in compounds a f t e r c o n v e r t i n g them to v o l a t i l e h y d r i d e s . Complete recovery of the generated hydrides could not be accomplished by helium sparging a lone . A combination of ether e x t r a c t i o n , and sparging had to be employed f o r complete recovery of the t r i b u t y l t i n and t r i p h e n y l t i n hydrides generated i n s o l u t i o n . Recent ly , C l a r k and C r a i g ® ^ e f f e c t e d on-column hydride generat ion of organot in compounds on a gas chromatographic column. Analyses of t e t r a -a l k y l t i n compounds by c o u p l i n g a GC to a plasma emission spectrometer ft 1 has been accomplished by Estes et a l . 1.10 L i q u i d Chromatography (LC) L i q u i d chromatography of organotin compounds does not require the p r e p a r a t i o n of v o l a t i l e species and hence could be u s e f u l f o r the analyses of n o n - v o l a t i l e or h i g h molecular weight organotin compounds. V a r i o u s forms of l i q u i d chromatographic detectors are employed f o r the d e t e c t i o n of organot ins . For h i g h s e n s i t i v i t y , a h i g h performance l i q u i d chromatograph (HPLC) can be coupled to an atomic absorpt ion spectrometer (AAS) or a mass spectrometer (MS). D i r e c t c o u p l i n g of the LC system to a detector system such as the mass spectrometer or the atomic absorpt ion spectrometer i s associa ted with problems such as solvent i n t e r f e r e n c e s . The large amount of 19 -s o l v e n t t h a t goes i n t o t h e d e t e c t o r s y s t e m i s a l s o a m a j o r c o n c e r n . The p r o b l e m o f l a r g e s o l v e n t i n f l o w i n t o the d e t e c t o r c a n be s o l v e d by i n t e r f a c i n g t h e LC and the d e t e c t o r s y s t e m o r by the u s e o f a m i c r o -b o r e c o l u m n . The s m a l l s o l v e n t f l o w r a t e (10-100 / i L m i n " 1 ) i n m i c r o b o r e HPLC has b e e n shown t o be c o m p a t i b l e w i t h d i r e c t e f f l u e n t i n t r o d u c t i o n t o a f l a m e a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t e r . The o r g a n o t i n compounds c a n a l s o be d e r i v a t i z e d t o v o l a t i l e h y d r i d e s a f t e r b e i n g s e p a r a t e d by l i q u i d c h r o m a t o g r a p h y , and t h e n i n t r o d u c e d i n t o t h e a t o m i c a b s o r p t i o n s p e c t r o m e t e r . T h i s method has b e e n a p p l i e d to the ft d e t e r m i n a t i o n o f m e t h y l t i n s p e c i e s . Most work o n l i q u i d c h r o m a t o g r a p h y o f o r g a n o t i n compounds i s p e r f o r m e d on r e v e r s e d phase c o l u m n s , s i z e e x c l u s i o n c o l u m n s , and o t h e r m o d i f i e d columns s u c h as cyano b o n d e d s i l i c a g e l c o l u m n s . O r g a n o t i n compounds a d s o r b s t r o n g l y on u n m o d i f i e d s i l i c a g e l c o l u m n s . L a n g s e t h , ^ s e p a r a t e d a l k y l t i n h a l i d e s on c y a n o p r o p y l - b o n d e d s i l i c a c o l u m n . He u s e d an o n - c o l u m n d e r i v a t i z a t i o n t e c h n i q u e w h i c h e m p l o y e d m o r i n . J e s s e n e t a l . , D J have s t u d i e d t h e a d s o r p t i o n b e h a v i o r o f a l k y l t i n compounds on v a r i o u s c h r o m a t o g r a p h i c c o l u m n s . T h e i r r e s u l t i n d i c a t e d t h a t s i l i c a b a s e d o c t a d e c y l (ODS) and cyano columns a r e n o t s u f f i c i e n t l y i n e r t t o a l k y l t i n h a l i d e s , b u t s i l i c a c o l u m n s p y r o l y t i c a l l y c o a t e d w i t h c a r b o n b l a c k a r e i n e r t t o w a r d s a l k y l t i n h a l i d e s . R e a r r a n g e m e n t o f a l k y l g r o u p s on t i n , w h i c h i s o f t e n a p r o b l e m e n c o u n t e r e d i n GC a n a l y s i s , i s sometimes a l s o e n c o u n t e r e d i n l i q u i d c h r o m a t o g r a p h y , e s p e c i a l l y i f t e t r a a l k y l t i n i s p r e s e n t i n a m i x t u r e o f o r g a n o t i n compounds. - 20 -OBJECTIVES OF THE PRESENT STUDY The known o r g a n o t i n compounds i n aqueous e n v i r o n m e n t s a r e s i m p l e a l k y l t i n compounds ( o x i d e s , 3 3 h y d r o x i d e s , 3 3 o r c a r b o n a t o 1 ^ s p e c i e s ) . T h e s e a l k y l t i n compounds a b s o r b i n the UV r e g i o n b e l o w o r c l o s e t o the c u t o f f p o i n t o f most s o l v e n t s u s e d i n l i q u i d c h r o m a t o g r a p h i c s e p a r a -t i o n s , and a r e t h e r e f o r e n o t e a s i l y amenable t o UV d e t e c t i o n . A l t h o u g h r e f r a c t i v e i n d e x d e t e c t o r s have b e e n p r e v i o u s l y a p p l i e d f o r o r g a n o t i n d e t e c t i o n , the s e n s i t i v i t y i s l o w . H e n c e , the n e e d f o r o t h e r t y p e s o f d e t e c t o r s y s t e m s i n q u a n t i t a t i v e a n a l y s i s . I n t h i s s t u d y the m a i n o b j e c t i v e was t o d e v e l o p a method w h i c h does n o t i n v o l v e t h e f o r m a t i o n o f v o l a t i l e d e r i v a t i v e s f o r t h e a n a l y s i s o f o r g a n o t i n compounds i n the m a r i n e e n v i r o n m e n t . Two s t r a t e g i e s were i n v e s t i g a t e d : i . HPLC s e p a r a t i o n o f d e r i v a t i z e d o r g a n o t i n c o m p l e x e s c o u p l e d w i t h UV d e t e c t i o n i i . HPLC s e p a r a t i o n o f d e r i v a t i z e d o r g a n o t i n c h l o r i d e s c o u p l e d w i t h a t o m i c a b s o r p t i o n o r mass s p e c t r o m e t r i c d e t e c t i o n . The a p p r o a c h t a k e n t o a c h i e v e t h e o b j e c t i v e o f t h i s s t u d y i s as f o l l o w s : i . S y n t h e s i s o f c o m p l e x e s o f t r i b u t y l t i n , a n d i t s m e t a b o l i t e s d i b u t y l t i n and m o n o b u t y l t i n , w h i c h c o n t a i n l i g a n d s o f h i g h m o l a r e x t i n c t i o n c o e f f i c i e n t ( t r o p o l o n e a n d o x i n e ) , t h e r e b y m a k i n g a n a l y s i s b y UV d e t e c t i o n p o s s i b l e . i i . S e p a r a t i o n o f t r i b u t y l t i n and i t s m e t a b o l i t e s p r e s e n t i n m a r i n e o r g a n i s m s b y l i q u i d c h r o m a t o g r a p h y , and t h e q u a n t i f i c a t i o n o f them by LC - G F A A S o r by LC - M S . - 21 -CHAPTER 2 EXPERIMENTAL 2 . 1 I n s t r u m e n t a t i o n 2 . 1 . 1 I n f r a r e d S p e c t r o s c o p y I n f r a r e d s p e c t r a l d a t a were r e c o r d e d i n t h e r a n g e 4000 t o 200 c m " 1 by u s i n g a P e r k i n - E l m e r 598 s p e c t r o p h o t o m e t e r . The compounds were r u n as n u j o l m u l l s e x c e p t where o t h e r w i s e s t a t e d . The s p e c t r a were c a l i b r a t e d r e l a t i v e t o the p o l y s t y r e n e bands a t 1601 cm"^ and 907 c m ' 1 . The i n f r a r e d c e l l was a KRS-5 (58% t h a l l i u m i o d i d e , 42% t h a l l i u m b r o m i d e ) p u r c h a s e d f r o m Harshaw C h e m i c a l Company. 2 . 1 . 2 U l t r a v i o l e t A b s o r p t i o n S p e c t r o p h o t o m e t r y The a b s o r p t i o n w a v e l e n g t h s and m o l a r e x t i n c t i o n c o e f f i c i e n t s were d e t e r m i n e d f r o m d a t a o b t a i n e d on a P e r k i n - E l m e r Coleman 124 d o u b l e beam s p e c t r o p h o t o m e t e r o p e r a t i n g between 200 nm and 400 nm. 2 . 1 . 3 M e l t i n g P o i n t D e t e r m i n a t i o n M e l t i n g p o i n t s were d e t e r m i n e d i n open c a p i l l a r i e s u s i n g a G a l l e n k a m p m e l t i n g p o i n t a p p a r a t u s , and were u n c o r r e c t e d . - 22 2.1.4 Nuclear Magnetic Resonance Spectroscopy (NMR) "^H NMR spectra were obtained using a Varian XL-300 spectrometer operating at 300 MHz. Chemical shifts were measured relative to tetra-methylsilane as external reference. 2.1.5 High Performance Liquid Chromatography (HPLO The HPLC system consisted of Waters Associates' models M-45 and M-510 pumps, and an automated gradient controller . A l l sample injections were manually made via a Waters Associates' U6K injector. Chromophoric groups were detected by using a Waters Associates' Lambda-Max 481 LC spectrophotometer connected to a Waters Associates' Q A - 1 ^ data system. A graphite furnace atomic absorption spectro-photometer was used as the t i n specific detector. The chromatographic column used for a l l HPLC and HPLC-MS separations was a C^g reversed-bonded phase, steel column (/j-Bondapak 3.9 mm (ID) x 30 cm) with a Waters Associates guard column. The column packing material was s i l i c a with part ic le size of 10 microns. Effluents from the HPLC column were collected with the aid of a Gilson microfractionator and transferred manually to the automatic sample delivery system of the graphite tube atomizer. 2.1.6 Low Resolution Mass Spectrometry Low resolution mass spectra using electron impact ionization were 23 -obtained from a Kratos MS50 mass spectrometer. Mass spectra us ing fas t atom bombardment (FAB) were obtained from A . E . I . MS9 mass spectrometer. 2 .1 .7 Graphite Furnace Atomic Absorption Spectrophotometry The apparatus f o r graphite furnace atomic absorption spectrophoto-metry c o n s i s t e d of a Varian 1275 spectrophotometer equipped with a deuterium background corrector, a Varian GTA-95 graphite tube atomizer and a Hewlett -Packard model 82905A printer. The t i n hollow cathode lamp was s u p p l i e d by Hamamatsu Photonics of Japan. The 224.61 nm t i n a n a l y t i c a l l i n e was used f o r a l l analyses . The atomic absorpt ion spectrophotometer was operated at a 1 nm s p e c t r a l s l i t width . The graphite tubes used were V a r i a n Techtron p y r o l y t i c a l l y coated graphite tubes. 2 . 1 . 8 H i g h Performance L i q u i d Chromatography - Mass Spectrometry  (HPLC-MS) The mass spectrometer used f o r HPLC-MS was a Kratos MS80 RFA mass spectrometer wi th a Vestec Kratos thermospray i n t e r f a c e between the HPLC and the mass spectrometer (see Chapter 3, F i g . 3.8.2 f o r the d e s c r i p t i o n of the thermospray i n t e r f a c e ) . The HPLC system i s as descr ibed i n 2 . 1 .5 . above. - 24 2 . 1 . 9 E x t r a c t i o n and Sample P r e p a r a t i o n The c e n t r i f u g e e m p l o y e d d u r i n g t h e e x t r a c t i o n s t e p o f the m a r i n e o r g a n i s m s was a S o r v a l l S u p e r s p e e d RC 2-B a u t o m a t i c r e f r i g e r a t e d c e n t r i f u g e o p e r a t e d a t 2500 r . p . m . , e x c e p t where o t h e r w i s e s t a t e d . A B u c h i R o t a v a p o r - R r o t a r y e v a p o r a t o r was u s e d f o r a l l s o l v e n t e v a p o r a t i o n s , e x c e p t where o t h e r w i s e s t a t e d . The v o r t e x m i x e r u s e d was a V o r t e x - G e n i e model K - 5 5 0 - G ( S c i e n t i f i c I n d u s t r i e s I n c . B o h e m i a , N . Y . , U . S . A . ) , and the m i c r o p i p e t t e was an E p p e n d o r f R e p e a t e r 4780 . 2 .2 M a t e r i a l s and R e a g e n t s T r i b u t y l t i n c h l o r i d e and d i b u t y l t i n d i c h l o r i d e were p u r c h a s e d f r o m V e n t r o n ( A l f a i n o r g a n i c s ) B e v e r l y , M a s s a c h u s e t t s . B u t y l t i n t r i c h l o r i d e and T r o p o l o n e ( 2 - h y d r o x y - 2 , 4 , 6 - c y c l o h e p t a t r i e n o n e ) were p u r c h a s e d f r o m A l d r i c h C h e m i c a l Company, M i l w a u k e e , U . S . A . O x i n e ( 8 - h y d r o x y q u i n o l i n e ) was p u r c h a s e d f r o m BDH C h e m i c a l s . O t h e r r e a g e n t s u s e d were s o d i u m b i c a r b o n a t e ( F i s h e r S c i e n t i f i c c e r t i f i e d ACS g r a d e ) , a c e t i c a c i d ( r e a g e n t g r a d e ) . The f o l l o w i n g r e a g e n t s were a l l r e a g e n t g r a d e : t r i s o d i u m c i t r a t e , L - ( + ) - a s c o r b i c a c i d , c i t r i c a c i d , t a r t a r i c a c i d , D - ( + ) - g l u c o s e . A l l r e a g e n t s were u s e d w i t h o u t f u r t h e r p u r i f i c a t i o n . The s o l v e n t s u s e d f o r s y n t h e s i s were e t h a n o l ( l a b o r a t o r y g r a d e ) , m e t h a n o l a n d c y c l o h e x a n e ( r e a g e n t g r a d e ) . The g r a d e s o f o t h e r s o l v e n t s u s e d a r e s t a t e d where a p p r o p r i a t e . R e c o v e r y s t u d i e s were p e r f o r m e d on s t a n d a r d dog f i s h l i v e r D O L T - 1 , - 25 s u p p l i e d b y N a t i o n a l R e s e a r c h C o u n c i l , Canada (see A p p e n d i x A f o r the e l e m e n t a l c o m p o s i t i o n o f D O L T - 1 ) . A l l s o l v e n t s u s e d as m o b i l e p h a s e s were HPLC g r a d e e x c e p t where s t a t e d o t h e r w i s e , a n d were f i l t e r e d t h r o u g h a m i l i p o r e 0.5 FH f i l t e r p r i o r t o u s e . D e a e r a t i o n o f t h e m o b i l e p h a s e was a c h i e v e d b y vacuum f i l t r a t i o n . 2 .3 M e t h o d o l o g y The o r g a n o t i n compounds were s t u d i e d as o x i n a t e , t r o p o l o n a t e o r c h l o r i d e c o m p l e x e s • A l l r e t e n t i o n d a t a were e s t a b l i s h e d u s i n g w e l l c h a r a c t e r i z e d s t a n d a r d o r g a n o t i n compounds. B a c k g r o u n d c o r r e c t i o n was e m p l o y e d t o remove m o l e c u l a r a b s o r p t i o n when a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r i c d e t e c t i o n was u s e d . 2.4 S y n t h e s i s o f t h e O x i n a t e s 2 . 4 . 1 Sodium O x i n a t e S o d i u m m e t a l ( 1 . 0 0 g , 0 .043 m o l . ) was g r a d u a l l y d i s s o l v e d i n 30 mL o f m e t h a n o l t o f o r m s o d i u m m e t h o x i d e i n s o l u t i o n . O x i n e ( 6 . 3 2 g , 0 .044 m o l . ) d i s s o l v e d i n warm c y c l o h e x a n e was a d d e d t o t h e s o d i u m m e t h o x i d e s o l u t i o n . The m i x t u r e was s t i r r e d f o r 15 m i n and t h e n f i l t e r e d i n t o a 100 mL r o u n d b o t t o m f l a s k . The f i l t r a t e was c o n c e n t r a t e d a t r e d u c e d - 26 p r e s s u r e on t h e r o t a r y e v a p o r a t o r t o g i v e a y e l l o w p r e c i p i t a t e w h i c h was i s o l a t e d by f i l t r a t i o n . The s o d i u m o x i n a t e was c h a r a c t e r i z e d by mass s p e c t r o m e t r y ( M / Z — 167) , u s i n g f a s t atom bombardment ( F A B ) . 2 . 4 . 2 D i b u t y l t i n B i s o x i n a t e Sodium o x i n a t e ( 0 . 9 0 g , 0 .0054 m o l . ) was d i s s o l v e d i n 50 mL o f m e t h a n o l i n a 250 mL r o u n d b o t t o m f l a s k . D i b u t y l t i n d i c h l o r i d e (0 .82 g , 0 .0027 m o l . ) d i s s o l v e d i n 50 mL m e t h a n o l was a d d e d t o t h e m e t h a n o l i c s o l u t i o n o f s o d i u m o x i n a t e . The m i x t u r e was r e f l u x e d w i t h s t i r r i n g f o r 4 h . The s o l u t i o n was f i l t e r e d and c o n c e n t r a t e d u n d e r r e d u c e d p r e s s u r e to g i v e a v e r y v i s c o u s o i l . The v i s c o u s o i l was d r i e d u n d e r vacuum to g i v e a y e l l o w g l a s s y p r e c i p i t a t e . The p r e c i p i t a t e was c h a r a c t e r i z e d by e l e m e n t a l a n a l y s i s , mass s p e c t r o m e t r y and n . m . r . s p e c t r o s c o p y ( C h a p t e r 4 , s e c t i o n 4 . l " , t a b l e s 4 . 1 . 2 , 4 . 1 . 7 , F i g . 4 . 1 . 2 ) . 2 . 4 . 3 B u t y l t i n T r i s o x i n a t e Sodium o x i n a t e (1 .82 g , 0 .011 m o l . ) and b u t y l t i n t r i c h l o r i d e (1 .02 g , 0 .0036 m o l . ) were t r e a t e d as i n 2 . 4 . 2 a b o v e , and r e f l u x e d f o r 24 h . The r e s u l t i n g p r o d u c t was c h a r a c t e r i z e d t o be b u t y l t i n t r i s o x i n a t e ( C h a p t e r 4 , s e c t i o n 4 . 1 , T a b l e s 4 . 1 . 2 , 4 . 1 . 6 , A p p e n d i x B , F i g . B - 4 ) . 2 . 4 . 4 T r i b u t y l t i n O x i n a t e T r i b u t y l t i n o x i n a t e was s y n t h e s i z e d a c c o r d i n g t o the method o f - 27 Kawakami et a l . The product was characterized by elemental analysis, mass spectrometry and n.m.r. spectroscopy (Chapter 4 , section 4.1, Tables 4.1.2, 4.1.8, Appendix B, Fig. B - l ) . 2.5 Synthesis of the Tropolonates 2.5.1 Sodium Tropolonate Tropolone (5.00 g, 0.042 mol.) was dissolved in 20 mL of deionized water and the resultant solution added to sodium bicarbonate (3.4 g, 0.04 mol) in 20 mL of deionized water at room temperature. Reaction occurred immediately with effervescence, producing a yellow sol id . After cessation of effervescence, the reaction mixture was concentrated by gentle warming on a hot plate. The sol id product was f i l t e r e d through a Whatman No. 1 f i l t e r paper, and then washed with cold petroleum ether. The washed sol id product was dried at room temperature. The product was.characterized by mass spectrometry (FAB) to be sodium tropolonate (M/Z - 144). 2.5.3 T r i b u t y l t i n Tropolonate A sodium tropolonate (0.38 g, 0.0026 mol.) slurry in 50 mL ethanol was mixed with tributyltin chloride (0.81 g, 0.0025 mol.) dissolved in 50 mL ethanol, in a 250 mL round bottom flask. The mixture was stirred for 1 h at room temperature. At the end of 1 h, the reaction mixture - 28 -was f i l t e r e d t h r o u g h a Whatman N o . 1 f i l t e r p a p e r , t o remove the sodium c h l o r i d e f o r m e d . Th.- f i l t r a t e was c o n c e n t r a t e d u s i n g a r o t a r y e v a p o r a t o r t o g i v e o r a n g e c o l o r e d v i s c o u s l i q u i d . The v i s c o u s l i q u i d was k e p t f o r two d a y s u n d e r vacuum, a n d a p a l e p r e c i p i t a t e was o b t a i n e d . The p r e c i p i t a t e was washed w i t h c o l d m e t h a n o l and d r i e d u n d e r vacuum. I d e n t i f i c a t i o n o f t h i s p r o d u c t was a c c o m p l i s h e d by mass s p e c t r o m e t r y and ^-H n . m . r . s p e c t r o s c o p y ( C h a p t e r 4 , S e c t i o n 4 . 1 , T a b l e 4 . 1 . 5 and A p p e n d i x B , F i g . B - 2 ) . 2 . 5 . 4 B u t y l t i n T r i s t r o p o l o n a t e B u t y l t i n t r i s t r o p o l o n a t e was s y n t h e s i z e d a c c o r d i n g t o t h e method o f Komura e_t a l . , ^  e x c e p t t h a t the r e a c t i o n m i x t u r e was r e f l u x e d f o r 48 h . The s o l i d p r o d u c t was i d e n t i f i e d by e l e m e n t a l a n a l y s i s , m e l t i n g p o i n t and mass s p e c t r o m e t r y ( C h a p t e r 4, S e c t i o n 4 . 1 , T a b l e s 4 . 1 . 1 , 4 . 1 . 3 ) . 2 .6 D e t e r m i n a t i o n o f A b s o r p t i o n W a v e l e n g t h and M o l a r E x t i n c t i o n C o e f f i c i e n t s f o r t h e Complexes A p p r o p r i a t e amounts o f b u t y l t i n o x i n a t e s a n d t r o p o l o n a t e s were p l a c e d i n 15 mL v o l u m e t r i c f l a s k s and made up t o t h e mark t o f o r m s o l u t i o n s i n t h e c o n c e n t r a t i o n r a n g e 1 x 10"^ - 1 x 10"^ M. M o l a r e x t i n c t i o n c o e f f i c i e n t s were d e t e r m i n e d i n m e t h a n o l , a c e t o n i t r i l e , and a c e t o n e i n t h e w a v e l e n g t h r e g i o n 200-400 nm ( C h a p t e r 4 , S e c t i o n 4 . 1 , T a b l e s 4 . 2 . 1 and 4 . 2 . 2 ) . 29 CHAPTER 3 SEPARATION AND DETECTION PROCEDURE 3 .1 H P L C - G F A A S 3 . 1 . 1 I n t r o d u c t i o n C o n s i d e r a t i o n o f e q u a t i o n 1 l e a d s t o the b a s i c i d e a s o f H i g h P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y . H - A U 0 ' 3 3 + B / u + c u 1 H , i s the column p l a t e h e i g h t , A= 2 X d p , where dp i s the d i a m e t e r o f the p a c k i n g p a r t i c l e s , and X i s a p a c k i n g c o n s t a n t . A , r e f l e c t s t h e d i s p e r s i o n from the s p a c e s between the p a c k i n g p a r t i c l e s . B / U i s a term r e p r e s e n t i n g l o n g i t u d i n a l d i f f u s i o n . U i s the m o b i l e phase v e l o c i t y , and CU i s a mass t r a n s f e r t e r m . A , B , and C a r e c o n s t a n t s f o r a g i v e n c o l u m n . I f the r a t e of mass t r a n s f e r between the s t a t i o n a r y and the m o b i l e phase i s s l o w , c i s l a r g e a n d , at h i g h m o b i l e phase v e l o c i t y w i l l d o m i n a t e c a u s i n g a l a r g e p l a t e h e i g h t . 8 7 CU i s e f f e c t i v e l y t h e d i s p e r s i o n t h a t t a k e s p l a c e i n the s t a t i o n a r y zone i n s i d e t h e p o r e s o f t h e p a c k i n g p a r t i c l e s . P e r f o r m a c e c a n be i n c r e a s e d by r e d u c i n g t h e l o n g i t u d i n a l d i f f u s i o n t e r m B / u - T h i s i s a c h i e v e d by u s i n g a s m a l l c o l u m n and s m a l l s i z e d p a c k i n g p a r t i c l e s . The r e d u c t i o n i n the s i z e o f t h e p a c k i n g p a r t i c l e s l e a d s to d e c r e a s e d p e r m e a b i l i t y , and a l s o l e a d s t o r e d u c t i o n i n t h e s p e e d o f the a n a l y s i s . So , i n s t e a d o f a l l o w i n g the m o b i l e phase t o f l o w t h r o u g h the 30 -c o l u m n by d i f f u s i o n as i n o r d i n a r y co lumn c h r o m a t o g r a p h y , the m o b i l e p h a s e i s f o r c e d t h r o u g h the co lumn by means o f pumps. F u r t h e r m o r e , the e f f i c i e n c y o f a l i q u i d c h r o m a t o g r a p h i c s y s t e m can be i m p r o v e d by t h e r e d u c t i o n i n the mass t r a n s f e r t e r m CU. T h i s i s a c h i e v e d by r e d u c i n g the d i s t a n c e t h r o u g h w h i c h the a n a l y t e t r a v e l s i n t h e c o l u m n . T h i s i s a c c o m p l i s h e d by m a k i n g t h e s t a t i o n a r y p h a s e t h i n . A s c h e m a t i c d i a g r a m o f the HPLC-GFAAS s y s t e m e m p l o y e d i n t h i s s t u d y i s shown i n F i g . 3 . 1 . 1 . H e r e , the a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t e r o p e r a t e d i n the g r a p h i t e f u r n a c e mode i s u s e d f o r the d e t e c t i o n o f e l u t e d o r g a n o t i n compounds. FRACTION UV D E T E C T O R C O L L E C T O R C 1 8 C O L U M N L 0 0 P RESTRICTOR INJECTOR A A S S O L V E N T RESERVOIR PUMPS GRADIENT C O N T R O L L E R F i g . 3 . 1 . 1 : S c h e m a t i c s e t up o f t h e HPLC-GFAAS s y s t e m 31 -The c h r o m a t o g r a p h i c c o l u m n c h o s e n f o r t h i s s t u d y i s a r e v e r s e d b o n d e d p h a s e , p r e p a c k e d c o l u m n . A c h r o m a t o g r a p h i c c o l u m n i s t e r m e d r e v e r s e d p h a s e when t h e s t a t i o n a r y p h a s e i s non - p o l a r . When the s t a t i o n a r y p h a s e i s p o l a r , t h e c o l u m n i s r e f e r r e d t o as n o r m a l . I n t h i s s t u d y , t h e s t a t i o n a r y p h a s e o c t a d e c a n e i s c h e m i c a l l y b o n d e d t o the s i l i c a p a c k i n g m a t e r i a l t o f o r m o c t a d e c y l s i l a n e . T h i s h a s t h e a d v a n t a g e o f p r e v e n t i n g t h e s t a t i o n a r y p h a s e f r o m b e i n g washed away b y t h e m o b i l e p h a s e . A f t e r e l u t i o n f r o m t h e c o l u m n , f r a c t i o n s o f the m o b i l e phase a re c o l l e c t e d on a f r a c t i o n c o l l e c t o r a t 0 .5 m i n i n t e r v a l s and m a n u a l l y t r a n s f e r r e d to the a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t e r . The p r i n c i p l e o f a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y i s b a s e d on the measurement o f l i g h t a b s o r b e d by g r o u n d s t a t e a t o m s . The a b s o r p t i o n o f o o l i g h t b y g r o u n d s t a t e atoms f o l l o w s B e e r - L a m b e r t ' s law ( e q u a t i o n 2 ) , ° and c a n be u s e d f o r q u a n t i t a t i o n . * A - PoA e ' K A l c 2 P 0 ^ i s t h e power o f t h e s o u r c e a t w a v e l e n g t h A, P^ i s t h e power o f r a d i a t i o n a t w a v e l e n g t h A a f t e r p a s s a g e t h r o u g h t h e s a m p l e , i s the a b s o r p t i o n c o e f f i c i e n t , 1 i s t h e p a t h l e n g t h t h r o u g h the sample and c , i s t h e c o n c e n t r a t i o n o f the a t o m i c v a p o r . U s u a l l y , t h e g r a p h i t e f u r n a c e mode o f a t o m i c a b s o r p t i o n s p e c t r o -p h o t o m e t r y h a s t h e a d v a n t a g e o f r e a c h i n g f a r h i g h e r a t o m i z a t i o n t e m p e r a t u r e s t h a n a r e a c c e s s i b l e i n f l a m e a b s o r p t i o n s p e c t r o p h o t o m e t r y . M o r e s o , t h e i n s i d e o f t h e g r a p h i t e t u b e has the a b i l i t y t o reduce some r e f r a c t o r y metal o x i d e s to the m e t a l s . - 32 -In the graphite furnace mode, the steps involved are as follows: 1. Sample drying 2. Sample ashing 3 . Sample atomization 4 . Tube cleaning During the drying stage, solvent is removed from the sample in the furnace. At the ashing stage, organic molecules or inorganic materials are removed. At the atomization stage, free atoms are generated within a confined zone. During operation, the incandescent graphite is protected from excessive corrosion by an upward flow of inert gas (argon or nitrogen). In this study, argon was the inert gas. The inert gas flow also sweeps away any ashing product from the l ight path. A diagram of the graphite tube atomizer with a graphite tube in place is shown in Fig . 3.1.2. 3.2 Optimization of GFAAS Conditions The detection capability of the atomic absorption spectrophotometer was optimized using t r i b u t y l t i n chloride solutions in acetone. This was achieved by varying the atomization temperatures, while maintaining the ashing temperature constant, and vice versa. The graphite furnace operating parameters used in this study are shown i n Table 3.2.1. - 33 -Fig. 3.1.2: The graphite tube atomizer 34 Table 3 .2 .1 Graphite furnace operat ing parameters Step Temperature Time Gas Gas Read No. °C Sec. Flow Type Command 1 100 10 3. ,0 Normal 2 100 10 3. .0 Normal 3 100 10 3, .0 Normal 4 700 20 3 .0 Normal 5 700 2, .0 3, .0 Normal 6 700 8. .0 .0 Normal 7 2700 4. .0 .0 Normal 8 2700 4. ,0 .0 Normal 9 2700 2. ,0 3. .0 Normal 10 * steps at which absorpt ion was measured 3.3 S e l e c t i o n of Chemical Modifiers In search of chemical m o d i f i e r s to be used i n the a n a l y s i s of t r i b u t y l t i n c h l o r i d e and d i b u t y l t i n d i c h l o r i d e , 0.5% w/v aqueous s o l u t i o n s of the f o l l o w i n g compounds i n deionized water were examined. 35 i trisodium citrate i i L- (,+)-ascorbic acid i i i c i t r i c acid iv tartaric acid v D-(+)-glucose Each of the above compounds (10 /iL) were mixed with t r i b u t y l t i n chloride or dibutyl t in dichloride (10 fiL) by the automatic sample delivery system of the graphite tube atomizer and analyzed using the graphite furnace parameters shown in Table 3.2.1 above. The effect of these compounds on the absorbances of Sn, in t r i b u t y l t i n chloride and dibutyl t in dichloride is shown in Chapter 4, Figs. 4.4.1 and 4.4.2. 3.4 Establishment of Retention Data 3.4.1 Butylt in Oxinates Solutions of t r i b u t y l t i n oxinate, dibutyl t in bisoxinate, and butyl t in trisoxinate corresponding to 60 jig/mL were made by dissolving appropriate amounts of the well characterized standard compounds in chloroform. Each of the standard solution (15 ^L) was injected into the HPLC, and the retention times were determined by UV detection. Then, a mixture of a l l the standard butyl t in oxinates were injected into the HPLC. Each chromatographic peak was identif ied on the basis of the individual retention times previously established. Detection was a t a 36 fixed wavelength of 265 nm. It was not possible to separate t r i b u t y l t i n oxinate and dibutylt in bisoxinate either under isocratic or gradient elution conditions. Using a gradient elution of 95% ethyl acetate : 5% methanol for 4.5 minutes followed by a change to 20% ethyl acetate : 80% methanol, butyl t in trisoxinate was completely separated from either t r i b u t y l t i n oxinate or dibutyl t in bisoxinate on the C^g reversed phase column (Chapter 4, F ig . 4.5.2). No attempt was made to effect the HPLC separation of the butyl t in tropolonates because t r i b u t y l t i n tropolonate could not be obtained in a pure form, and was therefore not suitable as an analytical standard. However, a HPLC-MS study of the butyl t in tropolonates was undertaken to obtain their fragmentation behavior (Chapter 4, Table 4.11.1). 3.4.2 Butyltin Chlorides The retention data for the butyl t in chlorides were established by detection with graphite furnace atomic absorption spectrophotometry (GFAAS). A solution of dibutylt in dichloride (60 pg/mL) and t r i b u t y l t i n chloride (60 /ig/mL) was prepared by dissolving the appropriate amounts of standard compounds i n acetone. Solutions of each of the standard compounds (20 pL) were injected into the HPLC. The effluent from the HPLC was collected by a fraction collector . The collected fractions (0.5 min interval) were then transferred to the GFAAS. The mobile phase used for elution was 98% (2% acetic acid in acetone) : 2% tetrahydrofuran. Under the conditions of the elution, i t 37 -was not p o s s i b l e to completely separate b u t y l t i n t r i c h l o r i d e from d i b u t y l t i n d i c h l o r i d e . Hence, any b u t y l t i n t r i c h l o r i d e present i n an environmental sample w i l l i n t e r f e r e with the d e t e c t i o n of d i b u t y l t i n d i c h l o r i d e . (Chapter 4, F i g . 4 . 5 . 1 ) . 3.5 Establishment of A n a l y t i c a l Procedure and Recovery Studies Stock s o l u t i o n s (100 /ig/mL) each of t r i b u t y l t i n c h l o r i d e and d i b u t y l t i n d i c h l o r i d e were prepared by d i s s o l v i n g the appropriate amounts of the standard compounds i n acetone, i n a volumetr ic f l a s k (100 mL). Amounts of each of the stock s o l u t i o n s (10 mL) were placed i n two separate volumetr ic f l a s k s (100 mL) and made up to the mark with acetone, to form the working s o l u t i o n (10 pg/mL). A l i q u o t s (0 .5-1 .5 mL) of the working s o l u t i o n of t r i b u t y l t i n c h l o r i d e corresponding to 5-15 /ig of t r i b u t y l t i n c h l o r i d e were placed i n three d i f f e r e n t t e s t tubes. A l i q u o t s corresponding to 5-15 ^g of d i b u t y l t i n d i c h l o r i d e were a lso added i n each of the tes t tubes already c o n t a i n i n g the t r i b u t y l t i n c h l o r i d e , such that each tes t tube contained the same amount of t r i b u t y l t i n c h l o r i d e and d i b u t y l t i n d i c h l o r i d e . The acetone s o l u t i o n s i n the t e s t tubes were evaporated and reduced to about 0.2 mL, by gentle warming i n a water bath . Standard d o g f i s h l i v e r (0.1 g) was added i n t o each of the tes t tubes c o n t a i n i n g the b u t y l t i n mixtures and vortex mixed f o r one minute. Concentrated h y d r o c h l o r i c a c i d (0.5 mL) and sodium c h l o r i d e (0.10 g) were added to each tes t tube, f o l l o w e d by the a d d i t i o n of d i s t i l l e d water (1 mL). The s o l u t i o n s were 38 -v o r t e x m i x e d a g a i n , t o f o r m a homogeneous m i x t u r e . M e t h y l e n e c h l o r i d e (3 mL) was p u t i n t o e a c h t e s t t u b e , v o r t e x m i x e d f o r two more m i n u t e s and l e f t t o s t a n d f o r 15 m i n u t e s . The s o l u t i o n s were f u r t h e r v o r t e x m i x e d f o r 1 m i n u t e , and c e n t r i f u g e d f o r 5 m i n u t e s . A f t e r c e n t r i f u g i n g , the m e t h y l e n e c h l o r i d e l a y e r was removed i n t o a n o t h e r t e s t t u b e . The e x t r a c t i o n p r o c e d u r e was r e p e a t e d , and t h e m e t h y l e n e c h l o r i d e e x t r a c t s o f e a c h t e s t t u b e were p o o l e d t o g e t h e r , and c o n c e n t r a t e d by g e n t l e e v a p o r a t i o n on a w a t e r b a t h . The m e t h y l e n e c h l o r i d e s o l u t i o n was t h e n f i l t e r e d i n t o a V - s h a p e d t e s t t u b e , and e v a p o r a t e d t o d r y n e s s . The c o n t e n t s o f e a c h V - s h a p e d t e s t t u b e were r e c o n s t i t u t e d i n 50 ^ L o f hexane u s i n g a m i c r o p i p e t t e . The hexane s o l u t i o n (25 p L ) was i n j e c t e d i n t o the HPLC (see C h a p t e r A, s e c t i o n 4 . 6 f o r r e s u l t s ) . 3 .6 D e r i v a t i z a t i o n and E x t r a c t i o n o f T r i b u t y l t i n and D i b u t y l t i n f r o m M a r i n e O r g a n i s m s The m a r i n e o r g a n i s m s were removed f r o m the f r e e z e r , where t h e y h a d b e e n s t o r e d and a l l o w e d t o thaw. O y s t e r s and o t h e r b i v a l v e s were s h e l l e d p r i o r t o t h e e x t r a c t i o n s t e p . The e x t r a c t i o n was c a r r i e d o u t on b o t h t h e t i s s u e s and the s o l u t i o n o b t a i n e d by d i s s o l v i n g t h e s h e l l s i n h y d r o c h l o r i c a c i d . 39 3.6.1 E x t r a c t i o n f r o m T i s s u e s P o r t i o n s o f t h e t i s s u e s w e i g h i n g between 34-220 g (wet w e i g h t ) were p l a c e d i n a b l e n d e r , t o g e t h e r w i t h 100 mL w a t e r , a n d h o m o g e n i z e d f o r 3 m i n u t e s . The h o m o g e n i z e d t i s s u e s l u r r y was t r a n s f e r r e d t o a 1 L c o n i c a l f l a s k t o w h i c h 20 g s o d i u m c h l o r i d e a n d 50 mL c o n c e n t r a t e d h y d r o c h l o r i c a c i d were a d d e d . M e t h y l e n e c h l o r i d e (100 mL) was a d d e d t o the t i s s u e s l u r r y and m i x e d b y s h a k i n g . The t i s s u e s l u r r y was f u r t h e r s h a k e n f o r 30 m i n u t e s on a m e c h a n i c a l s h a k e r , and c e n t r i f u g e d f o r 20 m i n u t e s a t 2500 r . p . m . A f t e r c e n t r i f u g a t i o n , the m e t h y l e n e c h l o r i d e l a y e r was r e m o v e d . The r e m a i n i n g aqueous l a y e r was r e - e x t r a c t e d t w i c e more . The m e t h y l e n e c h l o r i d e l a y e r s were p o o l e d t o g e t h e r and e v a p o r a t e d t o d r y n e s s on a r o t a r y e v a p o r a t o r . The r e s i d u e was r e c o n s t i t u t e d i n h e x a n e , l e a v i n g some p r e c i p i t a t e s b e h i n d . The hexane s o l u t i o n was f i l t e r e d i n t o a 50 mL v o l u m e t r i c f l a s k , and made up to the mark w i t h more h e x a n e . The hexane s o l u t i o n (50 /JL) was i n j e c t e d i n t o the H P L C . S e p a r a t i o n o f t h e o r g a n o t i n compounds p r e s e n t i n t h e e x t r a c t was e f f e c t e d w i t h a m o b i l e p h a s e o f 98% (2% a c e t i c a c i d i n a c e t o n e ) : 2% t e t r a h y d r o f u r a n . F r a c t i o n s c o r r e s p o n d i n g t o t h e r e t e n t i o n t i m e s o f t r i b u t y l t i n c h l o r i d e a n d d i b u t y l t i n d i c h l o r i d e p r e v i o u s l y e s t a b l i s h e d u s i n g s t a n d a r d compounds were c o l l e c t e d and d e t e c t e d b y G F A A S . 3.6.2 O r g a n o t i n Compounds i n S h e l l s V a r i o u s p o r t i o n s o f the d r y s h e l l s w e i g h i n g b e t w e e n 12-36 g were - 40 crushed with a mortar and pestle into fine powder. The powdered shell •»7as transferred to a 250 mL beaker, and dissolved in 50 mL of concentrated hydrochloric acid, by gradual addition of the acid. After cessation of effervescence, the solution was transferred into a 100 mL volumetric f lask, and made up to the mark with deionized water. The hydrochloric acid solution of the shells (50 pL) was injected into the HPLC. Fractions corresponding to the retention times of t r i b u t y l t i n chloride and dibutyl t in dichloride were collected and analyzed by GFAAS. 3.7 Quantitation 3.7.1 Quantitation of Tr ibutyl t in Chloride and Dibutylt in Dichloride  for Recovery Studies At the 5-15 ug level of study, t r i b u t y l t i n chloride was quantitated by a normal calibration graph (Fig. 4.6.1). Solutions from which data for normal cal ibration were obtained were prepared by dissolving appropriate amounts of t r i b u t y l t i n chloride in acetone to form a stock solution (100 ng/mL). Aliquots of the stock solution (2-10 mL) were placed into volumetric flasks (100 mL) and made up to the mark with acetone to form working solutions in the concentration range 2-10 /ig/mL. Each of the working solutions (20 /iL) and ascorbic acid (15 /iL) used as a chemical modifier were injected into the GFAAS by the automatic sampler. The dibutyl t in dichloride was quantitated by a different method. - 41 -The standard a d d i t i o n method was used f o r the q u a n t i t a t i o n of d i b u t y l t i n d i c h l o r i d e because b e t t e r l i n e a r i t y of g -'aph was obtained by standard a d d i t i o n than f o r normal c a l i b r a t i o n . The standard a d d i t i o n c a l i b r a t i o n was accomplished by u s i n g a standard s o l u t i o n of d i b u t y l t i n d i c h l o r i d e (2 ng/mL) i n acetone, which was dispensed by u s i n g the automated standard a d d i t i o n program on the GTA-95 graphite tube atomizer (Table 3 . 7 . 1 ) . Volumes of the standard d i b u t y l t i n d i c h l o r i d e (2-18 pL) corres -ponding to 1.2 x l O " ^ - 1.08 x 10" 1 ug were mixed with the appropriate HPLC f r a c t i o n (10 pL) and the m o d i f i e r (10 ^ L ) . This mixture was then i n j e c t e d i n t o the GFAAS and analyzed. A t y p i c a l standard a d d i t i o n p l o t f o r d i b u t y l t i n d i c h l o r i d e i s shown i n Chapter 4, F i g . 4 . 6 . 2 . Table 3 .7 .1 : Sampler parameters f o r standard a d d i t i o n p l o t of d i b u t y l t i n d i c h l o r i d e S o l u t i o n Standard Sample Blank M o d i f i e r Volume (^L) Volume (pL) Volume (pL) Volume (^L) Blank - - 30 10 A d d i t i o n 1 2 10 18 10 A d d i t i o n 2 6 10 14 10 A d d i t i o n 3 10 10 10 10 A d d i t i o n 4 14 10 6 10 A d d i t i o n 5 18 10 2 10 - 42 -3 .7 .2 Q u a n t i t a t i o n o f T r i b u t y l t i n C h l o r i d e and D i b u t y l t i n D i c h l o r i d e  i n M a r i n e O r g a n i s m s B o t h t r i b u t y l t i n c h l o r i d e a n d d i b u t y l t i n d i c h l o r i d e p r e s e n t i n m a r i n e o r g a n i s m s were q u a n t i t a t e d by t h e s t a n d a r d a d d i t i o n m e t h o d . The s t a n d a r d a d d i t i o n p r o g r a m u s e d i s shown i n T a b l e 3 . 7 . 2 . No c h e m i c a l m o d i f i e r was u s e d , b e c a u s e i n t h i s c a s e , b e t t e r l i n e a r i t y was o b t a i n e d w i t h o u t t h e m o d i f i e r . T a b l e 3 . 7 . 2 : G T A - 9 5 G r a p h i t e Tube A t o m i z e r Program f o r S t a n d a r d A d d i t i o n p l o t o f B u t y l t i n Compounds i n M a r i n e O r g a n i s m s S o l u t i o n S t a n d a r d Sample B l a n k M o d i f i e r Volume Volume Volume Volume (ML) (/iL) (ML) (MD B l a n k - - 20 A d d i t i o n 1 1 10 9 A d d i t i o n 2 3 10 7 A d d i t i o n 3 5 10 5 A d d i t i o n 4 7 10 3 A d d i t i o n 5 9 10 1 Sample - 1 0 10 - 43 F o r t h e q u a n t i t a t i o n o f t r i b u t y l t i n c h l o r i d e , a s t a n d a r d s o l u t i o n o f t r i b u t y l t i n c h l o r i d e (0 .74 p g / m L as Sn) was u s e d . A s t a n d a r d s o l u t i o n o f d i b u t y l t i n d i c h l o r i d e i n a c e t o n e ( 0 . 7 9 ^ g / m L as Sn) was u s e d f o r t h e q u a n t i t a t i o n o f d i b u t y l t i n d i c h l o r i d e i n m a r i n e o r g a n i s m s . The s t a n d a r d a d d i t i o n was e f f e c t e d by u s i n g t h e a u t o m a t e d s t a n d a r d a d d i t i o n p r o g r a m o f the G T A - 9 5 g r a p h i t e t u b e a t o m i z e r . 3 . 8 " Combined H i g h P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y and Mass S p e c t r o m e t r y (HPLC-MS) 3 . 8 . 1 I n t r o d u c t i o n HPLC-MS i s an o n - l i n e i n s t r u m e n t a l c o m b i n a t i o n o f HPLC and MS. T h i s c o m b i n a t i o n u t i l i z e s t h e e f f i c i e n t s e p a r a t i o n c a p a b i l i t y o f l i q u i d c h r o m a t o g r a p h y and t h e s p e c i f i c i t y o f a mass s p e c t r o m e t r i c d e t e c t o r . I t a l s o d e c r e a s e s t h e number o f manual i n t e r v e n t i o n s w h i c h a r e o t h e r w i s e i n v o l v e d i n t h e a n a l y s i s o f s a m p l e s by s e p a r a t e HPLC and MS t e c h n i q u e s . HPLC-MS has t h e a d v a n t a g e o f c o n v e n i e n c e and s p e e d o f a n a l y s i s o f m u l t i c o m p o n e n t m i x t u r e s , and i n most c a s e s does n o t r e q u i r e c h e m i c a l d e r i v a t i z a t i o n o f s a m p l e s . To p e r f o r m H P L C - M S , an i n t e r f a c e i s r e q u i r e d t o a d m i t t h e HPLC e f f l u e n t i n t o t h e mass s p e c t r o m e t e r . The s t r i n g e n t r e q u i r e m e n t s e x p e c t e d o f t h e i n t e r f a c e a r e as f o l l o w s : i . h i g h y i e l d ( t r a n s f e r o f sample e n t e r i n g t h e mass s p e c t r o m e t e r ) i s r e q u i r e d , a n d t h e a n a l y t e s h o u l d be i o n i z e d w i t h o u t b e i n g 44 -c h e m i c a l l y m o d i f i e d i n an u n c o n t r o l l e d manner . i i . C h r o m a t o g r a p h i c peak b r o a d e n i n g i n the i n t e r f a c e s h o u l d be m i n i m a l , a n d the i n t e r f a c e s h o u l d n o t pose l i m i t a t i o n s on the o p e r a t i n g c o n d i t i o n s o f t h e HPLC o r t h e mass s p e c t r o m e t e r . i i i . a b i l i t y t o v a p o r i z e a n a l y t e s o f low v o l a t i l i t y i v . t h e p e r f o r m a n c e s h o u l d n o t depend on t h e m o l e c u l a r mass , v o l a t i l i t y o r t h e r m a l s t a b i l i t y o f t h e s a m p l e . I n p r a c t i c e t o d a t e , no LC-MS i n t e r f a c e h a s b e e n a b l e t o f u l f i l l t h e s e r e q u i r e m e n t s . P o o r m a t e r i a l t r a n s f e r i s s t i l l a p r o b l e m i n a l l i n t e r f a c e s d e v e l o p e d so f a r . Two d i f f e r e n t i n t e r f a c i n g methods a r e most commonly u s e d : a . D i r e c t L i q u i d I n t r o d u c t i o n I n t e r f a c e : I n t h i s i n t e r f a c e , the e f f l u e n t e m e r g i n g f r o m the HPLC co lumn i s i n t r o d u c e d i n t o the i o n s o u r c e . The s o l v e n t i s removed by some a p p r o p r i a t e p r o c e s s s u c h as n e b u l i z a t i o n , t h e r m o s p r a y o r e l e c t r o s p r a y . b . T r a n s p o r t I n t e r f a c e : I n t h i s i n t e r f a c e , the e f f l u e n t f r o m the HPLC c o l u m n i s d e p o s i t e d on a m o v i n g b e l t o r m o v i n g w i r e w h i c h t r a n s p o r t s a c h r o m a t o g r a p h i c f r a c t i o n f r o m the e x t e r n a l h i g h p r e s s u r e r e g i o n o f t h e HPLC u n i t , t h r o u g h an i n t e r f a c e i n t o t h e h i g h vacuum r e g i o n o f t h e mass s p e c t r o m e t e r , where i o n i z a t i o n i s e f f e c t e d . I n t h i s s t u d y , t h e i n t e r f a c e e m p l o y e d i s t h e t h e r m o s p r a y i n t e r f a c e , a m o d i f i c a t i o n o f t h e t y p e o r i g i n a l l y d e s c r i b e d b y V e s t a l e t a l . ^ 9 A s c h e m a t i c c o n f i g u r a t i o n o f t h e HPLC-MS as e m p l o y e d i n t h i s s t u d y i s shown i n F i g . 3.8.1. The t h e r m o s p r a y i n t e r f a c e i s shown i n F i g . 3 . 8 . 2 . - 45 -SAMPLE—>-Eluent COLUMN BY-PASS HPLC SYSTEM 0.2%Trifluoroacetic acid in H2O THERMOSPRAY INTERFACE MS Y EXCESS I MOBILE PHASE Fig. 3.8.1: Schematic configuration of the HPLC-MS system 46 JET T.C. S O U R C E B L O C K T . C . E L E C T R O N BEAM FOR " F I L A M E N T O N " MODE PUMPING L I N E TO L I Q U I D N I T R O G E N T R A P P R O B E T . C . L . C , N E F F L U E N T ( T . C . = T H E R M O C O U P L E ) Fig . 3 .8 .2 : The thermospray interf ace - 4 7 -In HPLC-MS using thermospray ionization, an aqueous solution o f an electrolyte is used to effect ionization. In this study, the electrolyte is 0.2% trif luoroacetic acid in water. The electrolyte is allowed to mix with the eluent after the HPLC column (Fig. 3.8.1). A mixture of the column effluent and tr if luoroacetic acid at a total flow rate of 1 mL/min enters the vaporizer (Fig. 3.8.2) through an e l e c t r i c a l l y heated stainless capi l lary . The rapid heating of the effluent results i n the production of a supersonic je t of vapor and aerosol containing a mist of fine droplets or par t ic les . Some o f the droplet population in the aerosol beam are e l e c t r i c a l l y charged, the number of positive droplets being equal to the number of negative droplets. As the droplets travel through the channel in the block, they are further heated. As the size of the charged droplet is reduced, the electr ic f i e l d at the l i q u i d surface increases u n t i l ions present in the l i q u i d phase are ejected from the droplets. The ions are extracted through an exit aperture into the mass analyzer. Both positive and negative ions are produced, but in this study, only posit ively charged ions were analyzed. The ion source also has the capability of producing electron beams for normal chemical ionization, when the thermospray interface is operated in the 'filament on' mode. In this study, the thermospray interface was operated in the 'filament o f f mode because of higher sensi t iv i ty and higher mobile phase flow rate which could then be achieved. Ions can be produced in the thermospray ionization process by direct evaporation of a sample ion, or in a two-step process analogous to - 48 conventional chemical i o n i z a t i o n whereby an ion of the e l e c t r o l y t e e jec ted from a drople t reacts with an analyte molecule i n the gas phase and generates an analyte i o n that i s mass a n a l y z e d . ^ Thermospray, i n any of i t s i o n i z a t i o n modes provides very m i l d i o n i z a t i o n such that l i t t l e fragmentation informat ion i s p r o v i d e d , ^ 1 and i s thus the method of choice when s l i g h t fragmentation of molecules i s d e s i r e d . Two types of mass spectrometers have been a p p l i e d i n LC-MS: i . The double f o c u s s i n g mass spectrometer with combined magnetic and e l e c t r o s t a t i c analyzers i i . The quadrupole mass analyzer The former operates at a much higher ion source vol tage (>3000V) and higher vacuum (10"^-10"^) t o r r ) than the l a t t e r . The low voltage and higher opera t ing pressures of the quadrupole mass spectrometer make i t more s u i t a b l e f o r i n t e r f a c i n g with l i q u i d chromatographic system. 3.9 O p t i m i z a t i o n of HPLC-MS Condit ions The thermospray i n t e r f a c e c o n d i t i o n s f o r the organot in c h l o r i d e s were optimized by u s i n g a s o l u t i o n of t r i b u t y l t i n c h l o r i d e i n acetone (36.5 jig/mL as Sn) . This was achieved by keeping the temperature of the b l o c k constant , and v a r y i n g the v a p o r i z a t i o n temperature (Chapter 4, F i g . 4 . 7 . 1 ) . 49 -3.10 V e r i f i c a t i o n o f Fragment Ions t o be U s e d f o r HPLC-MS Q u a n t i t a t i o n S o l u t i o n s e a c h o f t r i b u t y l t i n c h l o r i d e (36 .5 / ig /mL S n ) , b u t y l t i n t r i c h l o r i d e (42 ng/mL as S n ) , d i b u t y l t i n d i c h l o r i d e (39.1 / ig /mL as S n ) , and d i p h e n y l t i n d i c h l o r i d e (34 .5 / ig /mL Sn) were p r e p a r e d i n 100 mL v o l u m e t r i c f l a s k s by d i s s o l v i n g t h e a p p r o p r i a t e amounts i n a c e t o n e , a p a r t f r o m d i p h e n y l t i n d i c h l o r i d e w h i c h was d i s s o l v e d i n 10 mL o f t e t r a h y d r o f u r a n (THF) and t h e n made up t o t h e mark w i t h a c e t o n e . E a c h o f t h e s o l u t i o n s (20 /iL) was i n j e c t e d i n t o t h e t h e r m o s p r a y - M S . The mass s p e c t r a o f t h e s t a n d a r d o r g a n o t i n c h l o r i d e s a r e g i v e n i n C h a p t e r 4, F i g s . 4 . 8 . 1 . , 4 . 8 . 2 . , 4 . 8 . 3 . and 4 . 8 . 4 . The m o b i l e p h a s e was 60% m i x t u r e o f [98% (2% a c e t i c a c i d i n a c e t o n e ) + 2% THF] a t a f l o w r a t e o f 0 .6 m L / m i n : 40% (0.2% t r i f l u o r o a c e t i c a c i d i n w a t e r ) a t a f l o w r a t e o f 0 . 4 m L / m i n . 3 .11 HPLC-MS o f B u t y l t i n O x i n a t e s and T r o p o l o n a t e s HPLC-MS f r a g m e n t a t i o n p a t t e r n o f t h e b u t y l t i n o x i n a t e s and t r o p o l o n a t e s was o b t a i n e d f o r t r i b u t y l t i n t r o p o l o n a t e , d i b u t y l t i n b i s -t r o p o l o n a t e , b u t y l t i n t r i s t r o p o l o n a t e , t r i b u t y l t i n o x i n a t e , d i b u t y l t i n b i s o x i n a t e , and b u t y l t i n t r i s o x i n a t e , u s i n g a m o b i l e p h a s e o f 80% (5% a c e t i c a c i d i n m e t h a n o l ) a t a f l o w r a t e o f 0 .6 m L / m i n : 2% ( 0 . 1 M aqueous ammonium a c e t a t e ) a t a f l o w r a t e o f 0 . 4 m L / m i n . The i o n i z i n g e l e c t r o l y t e (aqueous ammonium a c e t a t e ) m i x e d w i t h t h e e l u e n t a f t e r the c h r o m a t o g r a p h i c c o l u m n . The f r a g m e n t i o n s o b t a i n e d a r e g i v e n i n C h a p t e r 50 -4, T a b l e 4 . 1 1 . 1 . 3.12 Analysis of Extracts from Marine Organisms by HPLC-MS The e x t r a c t s a n a l y z e d b y HPLC-MS were t h e same ones a n a l y z e d by H P L C - G F A A S as d e s c r i b e d i n s e c t i o n s 3 . 6 . 1 and 3 . 6 . 2 a b o v e , e x c e p t t h a t t h e h y d r o c h l o r i c a c i d s o l u t i o n o f t h e s h e l l s was e x t r a c t e d i n t o m e t h y l e n e c h l o r i d e . The e x t r a c t i o n o f o r g a n o t i n compounds i n t h e s h e l l s i n t o m e t h y l e n e c h l o r i d e was n e c e s s a r y t o e x c l u d e any h y d r o c h l o r i c a c i d w h i c h i s n o t c o m p a t i b l e w i t h t h e mass s p e c t r o m e t e r s y s t e m . A l l t h e e x t r a c t s were e v a p o r a t e d t o d r y n e s s on a r o t a r y e v a p o r a t o r . The r e s i d u e was p l a c e d i n 5 mL v o l u m e t r i c f l a s k s , and made up to the mark w i t h a s o l u t i o n o f d i p h e n y l t i n d i c h l o r i d e ( 3 4 . 5 / ig /mL as Sn) i n a m i x t u r e o f a c e t o n e a n d THF ( 9 : 1 ) . The d i p h e n y l t i n d i c h l o r i d e was the i n t e r n a l s t a n d a r d . A m i x t u r e o f t h e i n t e r n a l s t a n d a r d and t h e e x t r a c t (20 /xL) was i n j e c t e d i n t o t h e H P L C - M S . The u s e o f t h e i n t e r n a l s t a n d a r d e l i m i n a t e s t h e e f f e c t o f the v a r i a t i o n s i n i n s t r u m e n t p a r a m e t e r s o n the a n a l y t e , i f r a t i o s o f i o n c u r r e n t o r i n t e n s i t i e s o f t h e i n t e r n a l s t a n d a r d and t h e a n a l y t e a r e u s e d f o r q u a n t i t a t i o n . 51 -CHAPTER 4 RESULTS ANr DISCUSSION 4 . 1 Characterization of the Oxinate and Tropolonate Complexes T r o p o l o n e , a n a t u r a l l y o c c u r r i n g l i g a n d forms c o m p l e x e s w i t h o r g a n o -t i n compounds . T r i m e t h y l t i n t r o p o l o n a t e ^ and b u t y l t i n t r i s t r o p o l o n a t e ^ 3 a r e known a n d h a v e b e e n c h a r a c t e r i z e d . I n t h i s s t u d y , d i b u t y l t i n b i s t r o p o l o n a t e and b u t y l t i n t r i s t r o p o l o n a t e a f f o r d s a t i s f a c t o r y e l e m e n t a l a n a l y s e s ( T a b l e 4 . 1 . 1 ) . However , t r i -b u t y l t i n t r o p o l o n a t e does n o t show s a t i s f a c t o r y e l e m e n t a l a n a l y s i s , even on r e p e a t e d s y n t h e s e s , b u t the m o l e c u l a r i o n i s o b t a i n e d by mass s p e c t r o m e t r y i n the d e s o r p t i o n c h e m i c a l i o n i z a t i o n mode a t M/Z 412 ( F i g . 4 . 1 . 1 ) . T a b l e 4 . 1 . 1 : A n a l y t i c a l d a t a and m e l t i n g p o i n t s of b u t y l t i n t r o p o l o n a t e s % C H 0 M.P. (°C) B u t y l t i n t r i s t r o p o l o n a t e C a l c d . 55. .69 4, .49 17. .80 ( c 2 5 H 2 4 ° 6 S n > MW=539.151) f o u n d 55. .49 4, .49 238-240 D i b u t y l t i n b i s t r o p o l o n a t e C a l c d . 55. .61 5. .94 13, .47 ( c 2 2 H 2 8 ° 4 S n - MW-475.152) f o u n d 55. .62 6 .00 13, .30 94-96 T r i b u t y l t i n t r o p o l o n a t e C a l c d . 55. .50 7 .85 7, .78 < c 1 9 H 3 2 ° 2 S n ' MW=411.153) f o u n d 54. .63 7, .94 -100 412 50 i n 0 1 1 1 1 1 1 1 254 2 9 ? T y n i i*| 11 11 i 11 • 100 150 200 250 300 350 400 500 T r T " 550 i i j 11 i 11 i 111 j 1111111 111111 i 600 630 700 M / Fi&- 4.1.1 : D e s o r p t i o n c h e m i c a l I o n i z a t i o n mass spectrum of t r i b u t y l t i n tropolonate 53 T a b l e 4 . 1 . 2 : A n a l y t i c a l d a t a and m e l t i n g p o i n t s o f b u t y l t i n o x i n a t e s % C H 0 M . P . ( ° C ) B u t y l t i n t r i s o x i n a t e C a l c d . 61 .21 4 . 47 6 .91 ( c 3 1 H 2 7 N 3 ° 3 S n - MW-608.265) f o u n d 61 .06 4 . 31 6 .87 220-222 D i b u t y l t i n b i s o x i n a t e C a l c d . 59 .91 5. 80 5 .37 ( C 1 6 H 3 0 N 2 O 2 S n , MW-401.118) f o u n d 59 .74 5. .34 5 .25 136-138 T r i b u t y l t i n o x i n a t e C a l c d . 58 .09 7 , .66 3 .23 c 2 1 H 3 3 N O S n - MW-439.191) f o u n d 58 .05 7. .63 3 .36 -The mass s p e c t r a l d a t a f o r the o t h e r b u t y l t i n t r o p o l o n a t e c o m p l e x e s i n the e l e c t r o n i o n i z a t i o n mode a r e g i v e n i n T a b l e s 4 . 1 . 3 , 4 . 1 . 4 , and 4 . 1 . 5 . The f r a g m e n t a t i o n p a t t e r n shows the l o s s o f a b u t y l g roup f rom the m o l e c u l a r i o n , b u t the m o l e c u l a r i o n i s n o t d e t e c t e d . The b u t y l t i n t r i s t r o p o l o n a t e and d i b u t y l t i n b i s t r o p o l o n a t e show peaks a t M / Z 483 and 419 r e s p e c t i v e l y . A l l t h e ' b u t y l t i n t r o p o l o n a t e s show an i n t e n s e f r a g m e n t i o n c l u s t e r e d a t M/Z 241 , c o r r e s p o n d i n g t o [ S n - t r o p o l o n a t e ] + . A l s o c h a r a c t e r i s t i c o f the f r a g m e n t a t i o n p a t t e r n o f the b u t y l t i n t r o p o l o n a t e s , i s the c o l l a p s e o f the s e v e n membered t r o p o l o n e r i n g i n t o a s i x - m e m b e r e d r i n g t o fo rm a [ S n - p h e n o x i d e ] + f r a g m e n t a t M/Z 213. The b u t y l t i n . o x i n a t e s - a f f o r d good e l e m e n t a l a n a l y t i c a l r e s u l t s ( T a b l e 4 . 1 . 2 ) . The mass s p e c t r a i n the e l e c t r o n i o n i z a t i o n mode a r e g i v e n i n T a b l e s 4 . 1 . 6 , 4 . 1 . 7 and 4 . 1 . 8 . L i k e the t r o p o l o n a t e c o m p l e x e s , the m o l e c u l a r i o n s a r e n o t o b s e r v e d , b u t f ragment i o n s c o r r e s p o n d i n g to 54 -Table 4 . 1 . 3 : Fragment ions of b u t y l t i n t r i s t r o p o l o n a t e Fragment mass (M/Z) I n t e n s i t y (% base peak) Assignment 483 9.8 + SnT 3 419 60.2 + C4H9SnT2 362 11.3 + SnT 2 241 100.0 + SnT 213 8.1 [Sn p h e n o x i d e ] + 122 52.0 T + T + = t ropolonate ion Table 4 .1 .4 : Fragment ions of d i b u t y l t i n b i s t r o p o l o n a t e Fragment mass (M/Z) I n t e n s i t y (% base peak) Assignment 419 22.4 + C4HoSnT2 355 15.9 ( C A H 9 ) 2 S n T 241 83.1 + SnT 213 6.6 [Sn p h e n o x i d e ] + 122 52.0 T + T = tropolonate i o n 55 -T a b l e A . 1 . 5 : Fragment i o n s o f t r i b u t y l t i n t r o p o l o n a t e F r a g m e n t mass ( M / Z ) I n t e n s i t y (% b a s e p e a k ) A s s i g n m e n t 355 4 2 . 8 ( C 4 H 9 ) 2 S n T 241 100 .0 + SnT 213 8.2 [Sn p h e n o x i d e ] + 122 9 .4 T + T + = t r o p o l o n a t e i o n T a b l e 4 . 1 . 6 : Fragment i o n s o f b u t y l t i n t r i s o x i n a t e Fragment mass (M/Z) I n t e n s i t y (% b a s e p e a k ) A s s ignment 552 0 .8 + S n 0 X 3 465 24 .2 + C 4 H 9 S n 0 X 2 408 20 .6 + S n O X 2 264 100 .0 + SnOX 145 72 .5 0 X + 0 X + •= o x i n a t e i o n 56 -T a b l e 4 . 1 . 7 : Fragment Ions o f d i b u t y l t i n b i s o x i n a t e F r a g m e n t mass ( M / Z ) I n t e n s i t y (% b a s e p e a k ) A s s i g n m e n t 465 58 .6 + C 4 H 9 S n 0 X 2 408 10 .2 + S n 0 X 2 378 12 .6 + ( C 4 H 9 ) 2 S n 0 X 264 100 .0 + SnOX 145 6 8 . 1 0 X + 0 X + = o x i n a t e i o n T a b l e 4 . 1 . 8 : Fragment Ions o f t r i b u t y l t i n o x i n a t e Fragment mass ( M / Z ) I n t e n s i t y (% b a s e peak) A s s i g n m e n t 378 87 .2 + ( C 4 H q ) 2 S n 0 X 264 100 .0 + SnOX 145 8 8 . 3 0 X + 0 X + = o x i n a t e i o n 57 -t h e l o s s o f one b u t y l g r o u p f r o m t h e m o l e c u l a r i o n a r e a p p a r e n t . A l l a s s i g n e d f r a g m e n t i o n s a r e f o u n d t o match c l o s e l y w i t h t h e t h e o r e t i c a l i n t e n s i t y p a t t e r n s f o r s u c h f r a g m e n t s . F u r t h e r c h a r a c t e r i z a t i o n o f t h e o x i n a t e a n d t r o p o l o n a t e c o m p l e x e s i s p r o v i d e d b y n . m . r . s p e c t r o s c o p y . R a t i o o f i n t e g r a t e d p e a k a r e a s o f t h e l i g a n d a n d b u t y l t i n p r o t o n s a r e u s e d t o d e t e r m i n e t h e number o f l i g a n d s p r e s e n t i n t h e c o m p l e x e s . F i g . 4 . 1 . 2 shows t h e n . m . r . s p e c t r u m o f d i b u t y l t i n b i s o x i n a t e . n . m . r . s p e c t r a o f t h e o t h e r b u t y l t i n c o m p l e x e s a r e g i v e n i n A p p e n d i x B . S i m i l a r r i n g p r o t o n n . m . r . s i g n a l s o f o x i n e c h e l a t e s have p r e v i o u s l y b e e n r e p o r t e d b y W e s t l a k e and M a r t i n . ^ 4 .2 M o l a r E x t i n c t i o n C o e f f i c i e n t s o f B u t y l t i n O x i n a t e s a n d T r o p o l o n a t e s The b u t y l t i n o x i n a t e s a n d t r o p o l o n a t e s shown i n T a b l e s 4 . 2 . 1 and 4 . 2 . 2 have h i g h m o l a r e x t i n c t i o n c o e f f i c i e n t s w h i c h make t h e a p p l i c a t i o n o f HPLC w i t h UV d e t e c t i o n f e a s i b l e f o r t h e i r a n a l y s e s . 4 . 3 N a t u r e o f O r g a n o t i n O x i n a t e s and T r o p o l o n a t e s O x i n e a n d t r o p o l o n e f o r m b i d e n t a t e c h e l a t e c o m p l e x e s w i t h many m e t a l s . A l t h o u g h t h e c o m p l e x e s f o r m e d b y t h e s e l i g a n d s a r e commonly s i x c o o r d i n a t i o n , s e v e n c o o r d i n a t i o n has b e e n r e p o r t e d f o r o x i n e ^ and F * 8 - 4 . 1 . ? : * l l NMR s p e c t r u m o f d i b u t y l t i n bisoxinate 59 Table 4.2.1: Molar extinction coefficients of butylt in tropolonates Compound Methanol Acetonitri le Acetone Anm € (Lmol'^cm"1) Amn « (Lmol'^cnT 1) Anm € (Lmol"^cm"-1) (C 4 H 9 ) 3 SnT 232 2, .8 X 104 231 2, .7 X 104 375 5. ,6 X 103 322 9 .8 X 103 328 1. .2 X 104 384 7. .2 X 103 373 6. .4 X 103 374 4, .5 X 103 395 6. .5 X 103 393 5. .4 X 103 (C 4 H 9 )SnT 2 242 1. .0 X 104 242 1. .5 X 104 375 9. .5 X 103 314 2, .4 X 103 256 3. .2 X 104 370 3 .5 X 103 C4H9SnT3 234 7 .0 X 104 238 1. .7 X 106 320 3, .9 X 104 325 9, . 3 X 105 373 2, .1 X 104 372 4. .2 X 105 372 1. ,4 X 104 - 60 -Table A.2.2: Molar e x t i n c t i o n c o e f f i c i e n t s of b u t y l t i n oxinates Compound Methanol A c e t o n i t r i l e Acetone Anm e(Lmol^cm"1) Amn £(Lmol^cm" 1) Anm t (Lmol^cm" 1) ( C 4 H 9 ) 3 S n 0 X 241 2, .0 X 10 4 240 1. 8 X 10 4 326 2. .7 X 103 254 1, .0 X 10* 253 3. 2 X lO* ( C 4 H 9 ) 2 S n 0 X 2 243 2. .9 X 10 4 240 4. ,2 X 10 4 375 4. .1 X 103 252 3, .5 X 10 4 255 6, .5 X 10 4 C 4 H 9 S n O X 3 232 1. .1 X 103 240 5, .4 X 10 4 375 4 .0 X l O 3 254 1. .0 X 10 4 255 5 .4 X 10 4 tropolone. J Five coordination has also been reported f o r t r i p h e n y l t i n o x i n a t e . ^ I t i s anticipated, that oxine and tropolone would exhibit seven, s i x , and f i v e coordination i n t h e i r monobutyltin, d i b u t y l t i n , and t r i b u t y l t i n complexes r e s p e c t i v e l y . Relevant i n f r a r e d data i n Table 4.3.1 support t h i s view f or the tropolonate complexes. The carbonyl s t r e t c h i n g frequency^ 6 which i n the free tropolone ligand i s at 1615 cm"1 i s s h i f t e d to lower frequency (Table 4.3.1). This i s evidence that tropolone i s coordinated to t i n , through i t s carbonyl group. The absence of - O H s t r e t c h i n g v i b r a t i o n i n the range 3500-3100 cm"1 i n d i -cates that there i s no free - O H group i n the tropolonate complexes. On - 61 -Table A . 3 . 1 : Relevant i n f r a r e d data f o r b u t y l t i n t ropolonates Compound i/C=0 (cm" 1) ( C 4 H 9 ) 3 S n T 1592 ( C 4 H 9 ) 2 S n T 2 1592 1562 C 4 H 9 S n T 3 1589 1576 1572 t h i s b a s i s , t r i b u t y l t i n t ropolonate , d i b u t y l t i n b i s t r o p o l o n a t e , and b u t y l t i n t r i s t r o p o l o n a t e are f i v e , s i x , and seven coordinate r e s p e c t i v e l y as expected. For the oxinates , the c o o r d i n a t i o n could not be a s c e r t a i n e d because of the d i f f i c u l t y encountered i n a s s i g n i n g N->Sn s t r e t c h i n g v i b r a t i o n s . Numerous weak bands are present i n the frequency range A06-387 c m " 1 and about 395 c m " 1 which P o l l e r ^ and Okawara et a l . ^ - * r e s p e c t i v e l y have ass igned to N-»Sn s t r e t c h i n g v i b r a t i o n s . The absence of —OH s t r e t c h i n g v i b r a t i o n s i n the oxinate complexes i s evident from the i n f r a r e d s p e c t r a . - 62 -A. A Chemical Modifiers f o r Atomic Absorption Spectrophotometry of B u t y l t i n Chlorides In atomic absorption spectrophotometry, high s e n s i t i v i t y i s u s u a l l y obtained by the removal of substances that suppress the absorption s i g n a l of the analyte. This i s u s u a l l y achieved by matrix modification. The matrix of the analyte i s d e l i b e r a t e l y a l t e r e d by the addition of chemicals (chemical m o d i f i e r s ) . The chemical modifiers achieve matrix m o d i f i c a t i o n i n some of the following ways: 1. The chemical modifier forms more r e f r a c t o r y oxides or carbides than the analyte, thus making the analyte a v a i l a b l e for atomization. 2 . The chemical modifier reduces r e f r a c t o r y compounds of the analyte to the metal. 3. The chemical modifier forms complexes of high b o i l i n g point with the analyte and thus permits higher drying and charring temperatures that w i l l remove matrix compounds which w i l l otherwise i n t e r f e r e with detection. Various chemical modifiers are used for the electrothermal atomic absorption spectrophotometry of various elements. For example, aqueous s o l u t i o n of N i C l 2 i s a s u i t a b l e chemical modifier f o r the analysis of As, Au, B i , and Te. For the electrothermal atomic absorption of t i n , a mixture of ammonium phosphate and magnesium n i t r a t e has also been found to be a good chemical modifier.^® Tominaga and Umezaki,^ have inv e s t i g a t e d the a b i l i t y of various compounds to suppress interferences i n the electrothermal atomic absorption spectrophotometry of t i n . Ascorbic a c i d was found to be e f f e c t i v e i n enhancing the absorption 63 -s i g n a l o f t i n . I n t h i s s t u d y , t h e a b i l i t y o f t r i s o d i u m c i t r a t e , a s c o r b i c t c i d , c i t r i c a c i d , t a r t a r i c a c i d , and g l u c o s e to s e r v e as c h e m i c a l m o d i f i e r s f o r t h e g r a p h i t e f u r n a c e a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y o f b u t y l t i n compounds i s i n v e s t i g a t e d . A m i x t u r e o f t h e c h e m i c a l m o d i f i e r and t r i b u t y l t i n c h l o r i d e o r d i b u t y l t i n d i c h l o r i d e was i n j e c t e d i n t o t h e g r a p h i t e f u r n a c e , and the a b s o r b a n c e o f t i n was m e a s u r e d . The e f f e c t o f the v a r i o u s c h e m i c a l m o d i f i e r s on t h e a b s o r b a n c e o f Sn i n t r i b u t y l t i n c h l o r i d e and d i b u t y l t i n d i c h l o r i d e i s shown i n F i g s . 4 . 4 . 1 and 4 . 4 . 2 . The a b s o r b a n c e o f Sn i n t r i b u t y l t i n c h l o r i d e i s g r e a t l y e n h a n c e d by a s c o r b i c a c i d . The a b s o r b a n c e o f Sn i n d i b u t y l t i n d i c h l o r i d e shows l i t t l e v a r i a t i o n w i t h t h e t y p e o f c h e m i c a l m o d i f i e r , b u t t a r t a r i c a c i d and c i t r i c a c i d p r o d u c e d the g r e a t e s t enhancement o f i t s a b s o r b a n c e . I t i s s u r p r i s i n g t o f i n d t h a t a s c o r b i c a c i d c o u l d g r e a t l y enhance the a b s o r b a n c e o f the Sn i n t r i b u t y l t i n c h l o r i d e w i t h o u t d o i n g t h e same f o r the Sn i n d i b u t y l t i n d i c h l o r i d e . The e f f e c t o f t h e volume o f t h e c h e m i c a l m o d i f i e r on the a b s o r b a n c e s o f t h e Sn i n t r i b u t y l t i n c h l o r i d e and d i b u t y l t i n d i c h l o r i d e i s shown i n F i g s . 4 . 4 . 3 a n d 4 . 4 . 4 . The a b s o r b a n c e o f S n , i n d i b u t y l t i n d i c h l o r i d e shows l i t t l e s e n s i t i v i t y t o t h e volume o f m o d i f i e r u s e d . 64 ABSORBANCE 0.I6H 0.14-0.12-0.10-0.08-0.06-0.04-0.02-l l O b to o CD O CC < o o o 2 o o o r o < £ < < U CO o o r > _ i o MODIFIER F i g . 4.4.1: E f f e c t of various modifiers on the absorbance o'f ( C 4 H 9 ) 3 S n C l - 65 -ABSORBANCE 0.08-0.07-0.06-0.05-0.04-0.03-0.02 O.OH Q Of o E O Q OQ O CC< O U 2 gs o O Q co MODIFIER o o => —J o F i g . 4.4.2: E f f e c t of various modifiers on the absorbance of (C^Hcj^SnCl^ 0.07-ABSORBANCE 0.06-0.05-0.04-0.03" 0.02-0.01-1 — — i 1 1 1 1 —i • 5 10 15 20 25 30 35 0.5% ASCORBIC ACID ( M L) F i g - 4 . 4 . 3 E f f e c t o f m o d i f i e r volume on the a b s o r b a n c e o f ( C ^ q ^ S n C l ABSORBANCE O.I2-I o . i o H 0.08H <J3 0.06H 0.04H 0.02H n 1 1 1 1 1 i 5 10 15 20 25 30 35 0.5%ASCORBIC ACID (/*L) Fig . 4.4.4: E f f e c t of modifier volume on the absorbance of ( C 4 H 9 ) 2 S n C l 2 - 68 -4 . 5 R e t e n t i o n D a t a 4 . 5 . 1 R e t e n t i o n d a t a f o r b u t y l t i n c h l o r i d e s The r e t e n t i o n t i m e o f e a c h b u t y l t i n c h l o r i d e was d e t e r m i n e d , by c h r o m a t o g r a p h i n g a s o l u t i o n o f t h e b u t y l t i n c h l o r i d e u s i n g a m o b i l e p h a s e o f 98% [2% a c e t i c a c i d i n a c e t o n e ] : 2 % T H F . The c o l u m n e f f l u e n t s c o l l e c t e d e v e r y 0 .5 m i n . i n t e r v a l were a n a l y z e d by t h e GFAAS, to d e t e r m i n e t h e r e t e n t i o n t ime o f t h a t compound. T h e n , a m i x t u r e o f the b u t y l t i n c h l o r i d e s whose i n d i v i d u a l r e t e n t i o n t i m e s h a d b e e n d e t e r m i n e d was c h r o m a t o g r a p h e d and d e t e c t e d by the G F A A S . The G F A A S - c h r o m a t o g r a m o f t r i b u t y l t i n c h l o r i d e and d i b u t y l t i n d i c h l o r i d e i s shown i n F i g . 4 . 5 . 1 . T r i b u t y l t i n c h l o r i d e has a r e t e n t i o n t i m e o f 3 . 5 - 4 . 5 m i n s , w h i l e d i b u t y l t i n d i c h l o r i d e e l u t e d as a b r o a d b a n d w i t h a r e t e n t i o n t i m e o f 5 - 6 . 5 m i n s . The e l u t i o n o f d i b u t y l t i n d i c h l o r i d e as a b r o a d b a n d i s i n d i c a t i v e o f a d s o r p t i o n e i t h e r on the co lumn o r t h e c o n n e c t i n g t u b i n g s o f the HPLC s y s t e m . A n o t h e r m o b i l e p h a s e c o m p o s i t i o n c a p a b l e o f s e p a r a t i n g t r i b u t y l t i n c h l o r i d e f r o m d i b u t y l t i n d i c h l o r i d e i s 80% [1% a c e t i c a c i d i n a c e t o n e ] : 2 0 % p e n t a n e . A p r e l i m i n a r y i n v e s t i g a t i o n o f the r e t e n t i o n t i m e o f b u t y l t i n t r i c h l o r i d e shows t h a t b u t y l t i n t r i c h l o r i d e c o e l u t e d w i t h d i b u t y l t i n d i c h l o r i d e . H e n c e , any b u t y l t i n t r i c h l o r i d e p r e s e n t i n e n v i r o n m e n t a l s a m p l e s w o u l d i n t e r f e r e w i t h the d e t e c t i o n o f d i b u t y l t i n d i c h l o r i d e . CTi ABSORBANCE 0.05i 0.04-0 0 3 -0.02-0.01 0 1 I" (C*H9)2SnCl2 (C4H9)3SnCI 3 4 n 1 1 7 8 9 RETENTION TIME (MIN) F i g . 4.5.1-" HPLC-GFAAS chromatogram of b u t y l t i n c h l o r i d e s 70 -4 . 5 . 2 R e t e n t i o n d a t a f o r b u t y l t i n o x i n a t e s By u s i n g a g r a d i e n t e l u t i o n o f 95% e t h y l a c e t a t e : 5 % m e t h a n o l f o r 4 . 5 m i n . f o l l o w e d b y a change t o 20% e t h y l a c e t a t e : 8 0 % m e t h a n o l , t h e c h r o m a t o g r a m i n F i g . 4 . 5 . 2 was o b t a i n e d . T r i b u t y l t i n o x i n a t e and d i b u t y l t i n b i s o x i n a t e c o e l u t e d a t 3 .41 m i n , w h i l e b u t y l t i n t r i s o x i n a t e e l u t e d a t 1 0 . 4 0 m i n . 4 . 6 T r i b u t y l t i n C h l o r i d e , D i b u t y l t i n D i c h l o r i d e : - R e c o v e r y S t u d i e s , D e t e c t i o n L i m i t , and P r e c i s i o n 4 . 6 . 1 R e c o v e r y s t u d i e s on t h e e x t r a c t i o n p r o c e d u r e E q u a l c o n c e n t r a t i o n s o f t r i b u t y l t i n c h l o r i d e and d i b u t y l t i n d i c h l o r i d e (5-15 ^ g / 0 . 1 g d o g f i s h l i v e r ) were s p i k e d i n t o s t a n d a r d d o g f i s h l i v e r , and t h e n e x t r a c t e d i n t o m e t h y l e n e c h l o r i d e as d e s c r i b e d i n s e c t i o n 3 . 5 . The m i x t u r e o f t r i b u t y l t i n c h l o r i d e and d i b u t y l t i n d i c h l o r i d e e x t r a c t e d i n t o m e t h y l e n e c h l o r i d e was s e p a r a t e d b y HPLC and d e t e c t e d b y G F A A S . Q u a n t i t a t i o n o f t h e e x t r a c t e d t r i b u t y l t i n c h l o r i d e was e f f e c t e d by a n o r m a l c a l i b r a t i o n p r o c e d u r e . The a b s o r b a n c e o f t h e compound was compared w i t h t h e a b s o r b a n c e o f v a r i o u s known c o n c e n t r a t i o n s o f s t a n d a r d t r i b u t y l t i n c h l o r i d e s o l u t i o n i n a c e t o n e ( F i g . 4 . 6 . 1 ) . D i b u t y l t i n d i c h l o r i d e was q u a n t i t a t e d b y t h e s t a n d a r d a d d i t i o n method ( F i g . 4 . 6 . 2 ) . A m i x t u r e o f t h e s e p a r a t e d d i b u t y l t i n d i c h l o r i d e and the v a r i o u s c o n c e n -A B S O R P T I O N T r i b u t y l t i n o x i n a t e and D i b u t y l t i n b i s o x i n a t e 3.41 J B u t y l t i n t r i s o x i n a t e 10.40 ~T~ 15 0 5 10 1< K T K N I - I O N T I M E < m i n > F i g - 4.5.2: Chromatogram o f b u t y l t i n o x i n a t e s - 72 -ABSORBANCE 0.08 H 8 10 12 (C 4Hg) 3SnCI (/xg/mL) Fig. 4.6.1: Calibration graph for tribut y l t i n chloride ABSORBANCE O.I6t o.2-ld.8 -8.4 -6.0 -3.6 -1.2' 1.2 3.6 6.0 8.4 Fig. 4.6.2 Standard addition plot for recovered dibutyltin dichloride 10.8 13.2x10"2 (C«H9)2SnCl2 («g - 74 tions of the standard dibutylt in dichloride in acetone was injected into the GFAAS, and their absorbances determined. A l l cal ibrat ion graphs were drawn using the least square method. At the level of study (5-15/ig/O.l g standard dogfish l i v e r ) , t r i b u t y l t i n chloride and dibutyl t in dichloride afford good recoveries (Table 4.6.1). Table 4.6.1: Recovery studies for butylt in chlorides Level of study Compound % Recovery* Regression Correla-(/ig/0.1 g std. Extracted Equation for tion dogfish l iver) Calibration Coeffi-Graph cient 5 (C 4 H 9 ) 2 SnCl2 89.20 ± (C 4 H 9 ) 3 SnCl 97.30 ± 10 ( C 4 H 9 ) 2 S n C l 2 - 86.00 ± (C 4 H 9 ) 3 SnCl 83.30 ± 15 ( C 4 H 9 ) 2 S n C l 2 91.60 ± (C 4 H 9 ) 3 SnCl 90.00 ± 0.01 Y-1.9x + 0.0268 0.9378 0.04 Y=0.38x + 0.0060 0.9934 0.03 Y=1.8x + 0.0448 0.9051 0.05 Y=0.0054x + 0.0032 0.9846 0.02 Y-1.6x + 0.0685 0.9951 0.04 Y-2.1x + 0.0077 0.9694 Precision is expressed at the 95% confidence level 75 -A . 6 . 2 Detection l imi t and precision The limit of detection is defined as the analyte concentration which gives a signal equal to the signal of the blank, plus thrice the standard deviation of the blank. A method of determining this l imi t by using the intercept and the standard deviation of the y-residuals has been described elsewhere.^0 The l imi t of detection was obtained by plot t ing the absorbance of various concentrations of t r i b u t y l t i n chloride i n acetone against concentrations (Fig. 4.6.3). By using the intercept of the graph of Fig . 4.6.3, as found by the regression equation, and the standard deviation of the y-residuals , the l imit of detection is estimated to be 0.3 ng/mL as Sn. The precision of the atomic absorption measurements was determined by repeated analyses of an acetone solution of t r i b u t y l t i n chloride (10 mg/mL as Sn). The relative standard deviation (R.S.D.) for ten determinations is 2.8%. 4.7 HPLC-MS 4.7.1 Optimization of the thermospray interface conditions The thermospray interface conditions were optimized to obtain e f f i c i e n t ionization of the analyte . T r i b u t y l t i n chloride was used for this optimization. An acetone solution of t r i b u t y l t i n chloride (36.5 Mg/mL as Sn) was injected into the thermospray-MS, at a constant thermospray 76 -Fig. 4.6.3: Estimation of limit of detection (regression equation Is y = 15.62x + 0.0454, r - 0.9963) - 77 -interface block temperature of 225°C which was found to be ootimum for tributyltin chloride. The vaporization temperature was varied during each injection of tributyltin chloride and the mass spectra was recorded. The mobile phase was 60% I ( 2 % acetic acid in acetone)+ 2% THF)] at a flow rate of 0.6 mL/min:40%(0.2%trifluoroacetic acid in water) at a flow rate of 0.4 mL/min. The effect of the variation of vaporization temperature on the intensity of the base peak M / Z 349 is shown in Fig. 4.7.1. The intensity increased sharply as the vaporization temperature increased, reaching a maximum at 185°C. Further increase in the vaporization temperature leads to a very sharp decrease in intensity. On the basis of this information, the following conditions were selected for the thermospray-MS analysis: Vaporization temperature 1826C Probe temperature 117°C Block temperature 225°C Jet temperature 213-215°C 4.8 Major Ions of Standard Organotin Chlorides The mass spectra of standard organotin chlorides obtained by the injection of acetone solutions of tributyltin chloride, dibutyltin dichloride, butyltin trichloride, and diphenyltin dichloride (as described in Chapter 3, section 3.10) into the thermospray-MS are shown - 78 -ABSOLUTE INTENSITY < »rbltr»rv unit! > xlO 5 5 -4 -3-2 -I-165 170 175 180 185 190 2 0 0 °C VAPORIZATION TEMP. Fig. 4.7.1: Effect of variation of vaporization temperature on the intensity of tr i b u t y l t i n fragment ion at M/Z 349 -79 -i n F i g s . A . 8 . 1 , 4 . 8 . 2 , 4 . 8 . 3 and 4 . 8 . 4 r e s p e c t i v e l y . The m a j o r f r a g m e n t i o n s a r e g i v e n i n T a b l e 4 . 8 . 1 . The f r a g m e n t i o n s o b t a i n e d i n d i c a t e t h a t t h e b u t y l t i n c h l o r i d e s f o r m compounds w i t h components o f t h e m o b i l e p h a s e . A l l a s s i g n e d f r a g m e n t i o n s were compared w i t h t h e o r e t i c a l i n t e n s i t y p a t t e r n f o r s u c h f r a g m e n t s g e n e r a t e d b y c o m p u t e r s i m u l a t i o n and f o u n d t o m a t c h c l o s e l y ( A p p e n d i x C ) . Same f r a g m e n t i o n s a r e a l s o o b t a i n e d i n HPLC-MS when t h e o r g a n o t i n c h l o r i d e s a r e a l l o w e d t o p a s s t h r o u g h t h e c h r o m a t o g r a p h i c c o l u m n . When u s i n g t h e t h e r m o s p r a y i n t e r f a c e c o n d i t i o n s o p t i m i z e d f o r d e t e c t i n g t r i b u t y l t i n c h l o r i d e , the mass s p e c t r o m e t e r e x h i b i t e d v e r y low s e n s i t i v i t y t o w a r d s b u t y l t i n t r i c h l o r i d e . A n a c e t o n e s o l u t i o n o f b u t y l t i n t r i c h l o r i d e (10 / i g / m L ) i n j e c t e d i n t o the t h e r m o s p r a y - M S was n o t d e t e c t e d . 4 . 9 R e t e n t i o n T i m e s o f O r g a n o t i n Compounds I n HPLC-MS The r e t e n t i o n t i m e s o f the- s t a n d a r d o r g a n o t i n c h l o r i d e s e s t a b l i s h e d b y u s i n g t h e HPLC-GFAAS c o n d i t i o n s , a r e n o t a p p l i c a b l e t o t h e HPLC-MS c o n d i t i o n s b e c a u s e o f t h e d i f f e r e n c e s i n f l o w r a t e a n d e l u t i o n volume i n t h e two m e t h o d s . The r e t e n t i o n t i m e s o f s t a n d a r d t r i b u t y l t i n c h l o r i d e , d i b u t y l t i n d i c h l o r i d e , and d i p h e n y l t i n d i c h l o r i d e u n d e r HPLC-MS c o n d i t i o n s were e s t a b l i s h e d by i n j e c t i n g a n a c e t o n e s o l u t i o n o f a m i x t u r e o f t r i b u t y l t i n c h l o r i d e ( 2 1 . 9 / ig /mL as S n ) , d i b u t y l t i n d i c h l o r i d e (23 .4 / ig /mL as Sn) , a n d d i p h e n y l t i n d i c h l o r i d e (34 .53 ug/mL as Sn) i n t o t h e H P L C - M S , and - 80 -I N ' T E N S I T V 65 sa 100-1 S0 17S 1(1 M 7 ' — r — i — i i i r - r - r - r -2 7 S - i—i— " " i —•—i r—r-| I I I — i — p r — I — I — r 3 2 S 3 S B 3 7 g F i g - 4.8.3: Mass spectrum of butylt in tr ichloride «0g M / Z 2 \ N - 84 -Table 4.8.1: Major ions of standard organotin compounds Compound Major Ion M/Z Assignment Cone. of Compound ppm Relative Inten-s i t y * (C 4H 9) 3SnCl 349 (C4H9)3SnC0(CH3)2 100 42267a (MW-325.19) 327 (C 4H 9) 3SnCl 4227 291 (C 4H 9) 3Sn 24092 (C A H 9 ) 2 SnCl 2 351 (C 4H 9) 2Sn[C0(CH 3) 2] 2 + H 100 78856 (MW=303.69) 327 + (C4H9)2SnClC0(CH3)2 - H 125017 293 + (C4H9)2Sn00CCH3 153716b 269 + (C 4H 9) 2SnCl - H 43040 C 4 H 9 SnCl 3 363 + C4H9Sn00CCF3C4Hg0 + H 1000 11678 (MW-282.19) 327 + C4H9SnOOCCF3Cl + H 7108 305 + C4H9SnCl(0H)200CCH3 - H 25386c 268 + C4H9SnClC0(CH3)2 + H 3724 (C 6 H 5 ) 2 SnCl 2 391 + (C6H5)2Sn00CCH30C(CH3)2 100 6922 (MW=343.69) 367 + (C6H5)2SnC10C(CH3)2 - H 25950 333 + (C6H5)2SnOOCCH3 14273 309 + (C 6H 5) 2SnCl - H 8220 175 - 64875d Relative intensity is the intensity of each peak relative to the base peak base peak of fragments arising from (C 4H 9) 3SnCl base peak of fragments arising from (C4H9)2SnCl2 base peak of fragments arising from C 4H 9SnCl 3 base peak of fragments arising from (Cgl^^SnC^ a b c - 85 detecting the eluted compounds by using total ion current monitoring. The retention times of the standard compounds are shown in Table 4.9.1. Table 4.9.1: Retention times of organotin compounds in HPLC-MS Compound Retention time (min) (C 4 H 9 ) 3 SnCl 8.81 ( C 4 H 9 ) 2 S n C l 2 12.48 ( C 6 H 5 ) 2 S n C l 2 10.68 4.10 Levels of Butyltin Compounds in the Tissues and Shells of Marine Animals 4.10.1 Analysis by HPLC-GFAAS The methylene chloride extract of the marine organisms obtained after extraction of the organotin compounds as chlorides was evaporated to dryness, and then reconstituted in hexane (Chapter 3, section 3.6). The hexane solution of the extracts was chromatographed and the eluted organotin compounds were detected by graphite furnace atomic absorption spectrophotometry. The identity of the eluted organotin compounds was - 86 -e s t a b l i s h e d by c o m p a r i s o n w i t h r e t e n t i o n t i m e s o f s t a n d a r d t r i b u t y l t i n c h l o r i d e and d i b u t y l t i n d i c h l o r j . d e . The l e v e l o f b u t y l t i n compounds p r e s e n t i n the t i s s u e o f m a r i n e o r g a n i s m s i s g i v e n i n T a b l e 4 . 1 0 . 1 . T a b l e 4.10.1: Concentration of butyltin compounds in whole body (soft tissue) of marine organisms / i g / g (wet w e i g h t ) " as Sn O r g a n i s m O r i g i n T r i b u t y l t i n D i b u t y l t i n P a c i f i c o y s t e r C r a s s o s t r e a g i p a s Fanny Bay 4, .29 + 0. .36 2, .11 + 0 .19 S h r i m p s 3 Howe Sound 4. .14 + 0 .28 1 .34 + 0. . 12 B u t t e r C lam Saxidomus p i p a n t e u s P a t r i c i a Bay 1. .67 + 0 .11 0, ,81 + 0, .12 B e n t - n o s e Clam Macoma n a s u t a P a t r i c i a Bay 3, .52 + 0, .12 4, .62 + 0, .60 P u r p l e p i c k l e s e a cucumber M o l p a d i a i n t e m e d i a - 1. .37 + 0, .05 0. .87 + 0. ,15 S o f t - s h e l l e d C lam Mva a r e n a r i a P a t r i c i a Bay 2 .46 + 0, .04 3, .09 + 0. .08 S o f t - s h e l l e d C l a m Mva a r e n a r i a C o l e ' s Bay 1. .54 + 0. ,11 0. ,91 + 0. 07 B a s k e t C o c k l e C l i n o c a r d i u m n u t t a l l i i P a t r i c i a Bay 3. ,23 + 0. .05 4 . .08 + 0. 32 B l u e m u s s e l M v t i l u s e d u l i s Head of H a s t i n g s Arm 1. ,14 + 0. 09 1. 91 + 0. 03 S h r i m p s b e l o n g t o the f a m i l y P a n d a l i d a e P r e c i s i o n e x p r e s s e d a t the 95% l e v e l o f c o n f i d e n c e 87 C o m p a r a t i v e d a t a on the l e v e l s o f o r g a n o t i n compounds i n the t i s s u e s o f v a r i o u s u iar ine o r g a n i s m s i s o l a t e d f rom t h e i r n a t u r a l h a b i t a t are s c a r c e , b u t d a t a a r e a v a i l a b l e f o r the l e v e l s o f b u t y l t i n compounds i n the t i s s u e s of o y s t e r s . R a p s o m a n i k i s and H a r r i s o n 1 ^ 1 have r e p o r t e d l e v e l s o f t r i b u t y l t i n and d i b u t y l t i n o f 0 . 0 2 7 - 1 . 6 6 7 ug/g d r y w e i g h t and 0 . 0 1 2 - 0 . 4 0 2 M g / g d r y w e i g h t r e s p e c t i v e l y on the o y s t e r s ( C r a s s o s t r e a  g i g a s ) o f E n g l a n d . Wa ldock and M i l l e r 1 * ^ have a l s o r e p o r t e d t r i b u t y l t i n l e v e l s o f up t o 4 . 5 ng/g d r y w e i g h t f o r some o y s t e r s o f E n g l a n d . T h a i n and W a l d o c k l u J have r e p o r t e d t r i b u t y l t i n l e v e l s o f 0 . 4 - 1 . 3 5 M g / g wet w e i g h t f o r some o y s t e r s o f E n g l a n d . R i c e e t a l . , 1 ^ 4 a l s o r e p o r t e d t r i b u t y l t i n l e v e l s o f 0 . 0 6 - 1 . 5 7 M g / g w e t w e i g h t f o r the o y s t e r C r a s s o s t r e a v i r g i n i c a . The l e v e l o f t r i b u t y l t i n i n the o y s t e r C r a s s o s t r e a g i g a s i n t h i s s t u d y i s much h i g h e r ( 4 . 2 9 M g / g w e t w e i g h t ) t h a n the v a l u e s r e p o r t e d by T h a i n and W a l d o c k , l u 3 and R i c e e t a l . 1 ^ f o r some o y s t e r s o f E n g l a n d . No c o m p a r a t i v e d a t a on the l e v e l s o f t r i b u t y l -t i n i n o y s t e r s f r o m the C a n a d i a n m a r i n e e n v i r o n m e n t a r e a v a i l a b l e . F o r the o t h e r o r g a n i s m s a n a l y z e d , no c o m p a r a t i v e d a t a a r e a v a i l a b l e . To o b t a i n the l e v e l s o f b u t y l t i n compounds p r e s e n t i n the s h e l l s o f m a r i n e o r g a n i s m s , a d i l u t e h y d r o c h l o r i c a c i d s o l u t i o n o f the s h e l l s was i n j e c t e d i n t o t h e HPLC and c h r o m a t o g r a p h e d . The e l u t e d b u t y l t i n compounds were d e t e c t e d by GFAAS ( T a b l e 4 . 1 0 . 2 ) . The l e v e l s o f t r i b u t y l t i n and d i b u t y l t i n f o u n d i n the s h e l l s a r e much h i g h e r t h a n the l e v e l s f o u n d i n the t i s s u e s . T h e r e seems t o be a r e l a t i o n s h i p be tween the l e v e l s of b u t y l t i n compounds i n t h e t i s s u e s and the s h e l l s . The m a r i n e o r g a n i s m s t h a t show h i g h l e v e l s of t r i b u t y l t i n and d i b u t y l t i n compounds i n t h e i r t i s s u e s a l s o show h i g h l e v e l s o f the - 88 -T a b l e A . 1 0 . 2 : C o n c e n t r a t i o n o f b u t y l t i n compounds i n t h e s h e l l s o f m a r i n e o r g a n i s m s T r i b u t y l t i n 3 D i b u t y l t i n 3 O r g a n i s m O r i g i n fig/g ( d r y wt) ng/g ( d r y wt) as Sn as Sn P a c i f i c oyster Fanny Bay 85.20 ± 0.27 A9.A0 ± 0.08 Crassostrea gieas Butter Clam P a t r i c i a Bay 10.10 ± 0.13 6.60 ± 0.09 Saxidomus giganteus Bent-nose Clam P a t r i c i a Bay 115.60 ± 0.58 19.00 ± 0.09 Macoma nasuta S o f t - s h e l l e d Clam Coles Bay 15.80 ± 0.15 5.20 ± 0.06 Mya a r e n a r i a S o f t - s h e l l e d Clam P a t r i c i a Bay 6.60 ± 1.60 27.30 ± 0.17 Mya a r e n a r i a 3 P r e c i s i o n i s e x p r e s s e d a t the 95% l e v e l o f c o n f i d e n c e same compounds i n t h e i r s h e l l s , e . g . t h e P a c i f i c o y s t e r , and the B e n t - n o s e C l a m . However , t h e a v a i l a b l e d a t a on t h e l e v e l s o f o r g a n o t i n compounds i n t h e s h e l l s o f m a r i n e o r g a n i s m s a r e s p a r s e , f o r p u r p o s e s o f c o m p a r i s o n . 89 -4.10.2 HPLC-MS of marine organisms (i) Tissue Extracts The hexane solutions of the tissue extracts of the marine organisms were evaporated to dryness and then reconstituted in an acetone solution of the chosen internal standard, diphenyltin dichloride. A mixture of the tissue extract and the internal standard was injected into the HPLC-MS, and the eluted compounds detected by their total ion current and their mass spectra. The characteristic polyisotopic intensity pattern of tin should be diagnostic in identifying tin compounds. In a l l the tissues analyzed, no fragment ion having M/Z value corresponding to that of any of the standard butyltin fragment ions (Table 4.8.1) was observed. The HPLC-MS of the tissue extracts show fragment ions at M/Z 303 and M/Z 369, whose intensity pattern bear some resemblance to that of tin (Figs. 4.10.2 and 4.10.4). These fragment ions at M/Z 303 and 369 were + + suspected to be (CgH^^SnOH and (CgHj^^Sn respectively. To ascertain + + i f these fragment ions actually belong to (CgH^^Sn and (CgH^ )^2Sn0H, authentic samples of (CgH^i^SnB^ and (CgHn^SnBr were each spiked into standard dogfish liver (0.5 g/0.1 g dogfish liver) , derivatized to their chlorides and extracted into methylene chloride using the extraction procedure described in Chapter 3, section 3.6.1. The methylene chloride was evaporated to dryness and the extracted standard cyclohexyltin compounds were reconstituted in acetone and injected into the HPLC-MS. The mass spectra are shown in Figs. 4.10.5 and 4.10.6. 90 -F i g . 4 . 1 0 . 1 : HPLC-MS total ion current chromatogram of Macoma nasuta (Bent-nose clam)tissue extract.A, B, C , correspond to the retention times of tributyltin chloride, diphenyltin dichloride and dibutyltin dichloride respectively 19b I T I C - l I3?1««. I»fl-Z1»B56] E l r 35» M / Z Fig. 4.10.2: Mass spectra of position A of Fig. 4.10.1 - 91 -J42 [ T l C - l M t l S . I I I M I I I ! ] El > h H 01 z U h z H 3 » M / Z Fig. 4.10.3: Mass spectra of position B of Fig. 4.10.1 J55 [TIC-186956. IMX-7K64] El ' I 1 1 1 1 4 » H w z u h H 1 1 * 1 i i i i i i i i i i i i i I i l-r ±i_ ' T' i' i i i i' I V i I i i i j1 i - M / I 1 1 1 1 4»» Fig. 4.10.4: Mass spectra of position C of Fig. 4.10.1 - 92 The mass spectrum of the standard dicyclohexyltin compound did not show the peak at M/Z 303 (Fig. 4.10.6). This indicates that the peak at M/Z 303 found in the mass spectra of the tissue extracts of marine organisms is probably not due to a dicyclohexyltin compound. The mass spectrum of the standard tr icyclohexylt in (Fig. 4.10.5) shows a fragment ion at M/Z 369, and another one at M/Z 427 + + (CgH^^)3Sn0C(CH3)2, which is the base peak. If ( C g H ^ ^ S n was present in the extracts, the fragment ion at M/Z 427 (the base peak) should have been prominent in the mass spectra of the tissue extracts, but this is not the case. On this basis, the presence of cyclohexyltin compounds is ruled out. The "isotopic" pattern of the peaks at M/Z 303 and 369 varies with the sample, which is further evidence against their being associated with t i n compounds. The HPLC-MS of some tissue extracts of marine organisms analyzed are given in Appendix D. The fragment ion at M/Z 367 (Fig. 4.10.3) is due to the internal standard, and was or ig inal ly intended to be used for quantitation (see Chapter 3, section 3.12) . i i . Shell extracts of marine organisms The dilute hydrochloric acid solutions of the shells was extracted into methylene chloride solutions, using the extraction procedure of Chapter 3, section 3.6.1. The methylene chloride extract was reduced in volume and injected into the HPLC-MS, and the spectra were recorded. No butyl t in compounds are observed in the mass spectra obtained by thermospray ionization. On analysis of the shell extracts by mass - 93 -[ T I C - 7 3 4 4 H . l » I - l l l » m E l H » W t " W 3 . h Z » H i i 11 i i i i | i i i i i I"I i' i i i i 11 11 i 11 i 11 1 1 1 1 1 1 1 'l1 I I I I I I I I I I I I 4S« " « Figs . 4.10.5: HPLC-MS of standard " ( C 6 H 1 1 ) 3 S n C l " [ T I C - 1 3 9 7 4 0 , 1 M X - 2 2 8 3 S ) E l h H W W h H * ' i i i i i i i i | i i i i i i i i i | i i i i i ' ' i ' | ' I ' 2 M »SI »»» ISf I 1 I I I I I I I F i g . 4.10.6: HPLC-MS of standard " ( C 6 H 1 1 ) 2 S n C l 2 " - 94 -spectrometry using electron ionization, weak peaks which indicate the presence of t i n compounds are obtained for the shel l extracts of the Macoma nasuta.Bent-nose clam(Fig. 4.10.7). The fa i lure to observe these peaks in the HPLC-MS is probably due to matrix effects . The fragment ion at M/Z 269 (Fig. 4.10.7) i s assigned + to (C^Ho^SnCl. The other peaks at M/Z 213 and 177 are assigned to C^HgSnCl and C^gSn respectively. The isotopic pattern of these frag-ment ions f i t s well with the calculated isotopic pattern (Fig. 4.10.8). Although these fragment ions are capable of arising either from dibutyl-t i n dichloride or t r i b u t y l t i n chloride, comparison of the intensities of the peaks at M/Z 269 and 177 arising from the shel l extract (Fig. 4.10.7) with same peaks i n the mass spectra of the standard dibutyl t in dichloride (Fig. 4.10.9) and t r i b u t y l t i n chloride (Fig. 4.10.10), reveals that the fragment ions obtained in the shel l extract of the Macoma nasuta Bent-nose clam originate mainly from t r i b u t y l t i n chloride. It is surprising to f ind that the HPLC-MS does not detect fragment ions of butyl t in compounds i n the other shel l extracts, or tissue extracts, when the HPLC-GFAAS detects high levels of these species. The Macoma nasuta Bent-nose clam in whose shel l extract the mass spectro-meter detected butyl t in compounds also gives the highest concentration of butyl t in compounds as detected by HPLC-GFAAS (Table 4.10.2). This observation tends to suggest that the l imi t of detection of the HPLC-MS method is not low enough to detect these compounds. The HPLC-MS method as found i n this study has a detection l imit of 0.3 ng/mL as Sn (Chapter 4, section 4.6.2). The detection l imi t of the HPLC-MS is estimated by replicate injections of various concentrations C T I C - 1 7 6 5 3 4 0 8 , 1 0 0 X - 1 9 4 0 6 0 ] E I 100 ^ 9B (-1 8* H " W " z w h z H 40 30 20 10 247 I I'M I | II 240 I I'M | I I 260 269 212 |291 .1.1. • .1. LJC I | II II II M l | II I I I I I I I | III I I I I I I | I I II II I I I | M M I I I I I | I I M I II M | I II I I 280 300 320 340 360 380 400 O N h H 10 z w h Z H 100 90 80 70 60 60 40 30 20 10 58 76 I I I82 l i ' i ' i ' i ' i ' i ' l ' l ' i ' l ' h i n 1 ! ^ T i I'I'I'I'I'I I I ri'l'l'l'i I Tl ' l ' l i i i I'l'l I'I'I'I'I'I-1 1 1 I'l'l'l'l'l'l'l l l l ' i i l l'l'| I I'l'l'l'l'l'l'i I I'l I I I 100 120 140 160 1B0 200 220 M / Z 4. ii 121 1SS ii 177 213 1* 60 80 Fig. 4 . 1 0 . 7 : E l e c t r o n i o n i z a t i o n mass s p e c t r u m o f s h e l l e x t r a c t o f the B e n t - n o s e c l a m Macoma n a s u t a - 96 -TOTAL ABUNDANCE 1 0.99935 AVERAGE MASS = 268.426350 MOST ABUNDANT PEAK= 269.011439 1 MASS EXACT MASS INTENS 261 . 261 .014519 2 . 27 262 . 262 .017874 0, . 20 263 . 263 .012190 2 , .29 264 . 264 .013557 1 . .02 265. 265 .011323 34 , . 66 266 . 266 .012937 21 . 58 267 . 267 .010951 70, .21 268. 268 .012632 32 . , 73 269 . 269 .011439 100. ,00 270. 270 .012739 1 5 . .67 271 . 271 .010525 37 . , 49 272 . 272 .013643 3. . 40 273 . 273, .014013 18. .05 274 . 274 , .017362 1 . , 66 275. 275, .012189 4 . 64 276 . 276. .015468 0. 42 277 . 277 , .018731 0. .02 (a) TOTAL ABUNDANCE= 0.99961 AVERAGE MASS = 211.309814 MOST ABUNDANT PEAK= 211.940879 1 MASS EXACT MASS INTENSITY 204 . 203 .944092 2 .31 205 . 204 , .947447 0, . 10 206 . 205, .94 1798 2 . 32 207 . 206 , .942903 0, . 93 208 . 207 , .941043 35 , . 1 3 209 . 208, .942361 20. .27 2 10. 209 . 940486 70. . 26 211. 210. .942037 29 . 94 212. 2 11. .940879 100. ,00 213. 212. .941515 1 1 . 25 214. 213. .939908 37 . ,43 215. 2 14. 943294 1 . 71 2 16. 215. 943582 18. 21 217. 216. 947028 0. 84 2 18. 217. 94 1613 4 . 66 219 . 218. 944949 0. 21 (b) TOTAL ABUNDANCE= 0.99971 AVERAGE MASS= 175.857509 MOST ABUNDANT PEAK= 176.972680 NOM MASS EXACT MASS INTENSITY ( c ) 1 69 . 168. 975239 2 . 85 170. 169 . ,978594 0. , 13 171 . 170. 973190 1 . , 95 172 . 171 . 973995 1 . 1 1 173 . 172. .972156 42. .70 174. 173. .973590 24 . . 65 175 . 174 , 972067 73 . ,00 1 76 . 1 75 , .973908 29 . 05 177. 176. . 972680 100. .00 178. 177. .976078 4 . 59 179 . 178. ,973886 1 4 . . 18 180. 179. .977288 0, . 65 181 . 180. . 975692 17, . 92 182. 181 . 979126 0, .83 183 . 182, .982401 0, .01 Fig. A.10.8: Theoretical mass spectral intensity pattern for + + + (a) (C 4H 9) 2SnCl; (b) CAH9SnCl (c) C4H9Sn; L3E379.11 CTIC-1086208, 100X-274160] EI 60 100 > 9 0 [4 80 H 70 (/) Z (-1 40 2 30 H 2 0 10 0 247 j I I I I I I I I l | ' l 2t0 240 T ' I ' I ' I I 1 1 f 269 260 11 I 3 0 4 1 1 ''1 1 I 1 1 1 ri'Pfh 1 I 1 1 1 1 1 1 1 1 j 1 1 1 1 1 |, 1 , • 1 , 280 300 320 340 r r r 1 1 1 1 I ' 'I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 j 1 1 1 , , 360 380 400 M / Z 100 90 > " h ^ H 6 0 I/) 50 Z 4 0 u h Z 2 0 H 1 0 30 $7 41 i 1 1 1 1 ! " 1 1 1 1 1 1 M 111111111111111 iTh 1111111 1111111 4 0 w 03 iaa 120 155 140 t • | M I I I I I ' l ' l | ' | I I I I I 1 1 1 1 • i 1 |'| 212 160 T 180 200 M / ^ 220 F i g - 4 . 1 0 . 9 : E l e c t r o n i o n i z a t i o n mass s p e c t r u m o f s t a n d a r d d i b u t y l t i n d i c h l o r i d e 100 90 80 70 60 50 40 30 20 10 0 L32378.3 tTIC-561824. 100X-37088] EI 269 l i i i i i ' l ^ i l i i i i n V i ' i ' i ' i ' i 220 240 260 2 ? 9 ?13 1 1 ' I 1 1 1 1 1 1 1 1 1 | 1 1 ' i'l'l i'l'l | ' I I M I M I | I I I I I | M'l I I I I I I I | | | I | | | | | | | | , , 2 8 0 3 " ™ * 340 360 3 ' „ \ „ M / Z 100 90 80 70 60 50 40 30 20 10 0 41 r t r k 57 TT .213 155 ,69 40 60 ''I l ' l ' l ' | I I I I I | | | ' | | | | | | | | | ' | 80 19 »77 100 'I'l'l'l" I I I'l'l I t • I'l'l'lM'I'l'l1!'! I • l^l'l 120 140 \ca 140 160 180 Fig. 4.10.10: Electron ionization mass spectrum of tributyltin chloride I'l'l'l H I M I'l'l'l I'l'l f T 200 220 M / Z - 99 -o f t r i b u t y l t i n c h l o r i d e i n a c e t o n e i n t o the t h e r m o s p r a y - M S . S o l u t i o n s o f c o n c e n t r a t i o n s l e s s t h e n 3 Mg/mL as t r i b u t y l t i n c h l o r i d e o r 1.09 Mg/mL as Sn a r e n o t d e t e c t e d by the mass s p e c t r o m e t e r . A t t h i s l i m i t o f d e t e c t i o n ( a b o u t 1 .09 Mg/mL as Sn) f o r t h e HPLC-MS m e t h o d , t h e mass s p e c t r o m e t e r s h o u l d have d e t e c t e d t h e t r i b u t y l t i n compounds a t t h e l e v e l s q u a n t i t a t e d by HPLC-GFAAS m e t h o d . T h e r e was a t i m e l a p s e b e t w e e n t h e HPLC-GFAAS e x p e r i m e n t , and the HPLC-MS e x p e r i m e n t , b e c a u s e the s a m p l e s h a d t o w a i t t h e i r t u r n f o r the HPLC-MS e x p e r i m e n t . D u r i n g t h i s w a i t i n g p e r i o d (2-3 w e e k s ) , the samples were s t o r e d i n t h e f r e e z e r . Whereas the s t a n d a r d t r i b u t y l t i n c h l o r i d e i n a c e t o n e o r hexane s o l u t i o n was f o u n d t o be s u i t a b l e as an a n a l y t i c a l s t a n d a r d f o r o v e r a p e r i o d o f t h r e e w eeks , i t c a n n o t be s a i d w i t h c e r t a i n t y w h e t h e r the e x t r a c t e d t r i b u t y l t i n c h l o r i d e s w o u l d be s t a b l e i n the p r e s e n c e o f b i o l o g i c a l m a t r i x f o r a l o n g p e r i o d . F u r t h e r s t u d i e s a r e n e c e s s a r y t o v e r i f y what e f f e c t t h e w a i t i n g p e r i o d f o r HPLC-MS e x p e r i m e n t c o u l d have on the HPLC-MS r e s u l t s o f t h e o r g a n o t i n compounds. A . 1 1 HPLC-MS o f B u t y l t i n O x i n a t e s and T r o p o l o n a t e s The f r a g m e n t i o n s o f b u t y l t i n o x i n a t e s and t r o p o l o n a t e s o b t a i n a b l e f r o m s o l u t i o n s b y u s i n g t h e r m o s p r a y i o n i z a t i o n a r e g i v e n i n T a b l e 4 . 1 1 . 1 . S u c h f r a g m e n t i o n s m i g h t be o f u s e i n f i n g e r - p r i n t i n g b u t y l t i n compounds , e s p e c i a l l y the l i g a n d i f t r o p o l o n e i s u s e d as an e x t r a c t a n t f r o m , f o r example t i s s u e s a m p l e s . However , t h e c o m p l e x e s decomposed on t h e c h r o m a t o g r a p h i c co lumn w i t h t h e l i g a n d and t h e b u t y l t i n m o i e t y 100 -Table 4..11.1: Fragment ions of butyltin oxinates and tropolonates Compound Mobile Phase M / Z Relative Assignment Intensity Ionizing Electro-lyte (C 4H 9) 3Sn0X ( C 4 H 9 ) 2 S n O X 2 C 4 H 9 S n O X 3 ( C 4 H 9 ) 3 S n T ( C 4 H 9 ) 2 S n T 2 C4H9SnT3 80% (5% acetic acid 435 43821 in methanol) + 20% 378 189688 (0.1M aqueous NH40Ac) 350 179435 308 247828a 291 107661 146 1340672 80% (5% acetic acid 378 599380b in methanol) + 20% 146 1071232 (0.1M aqueous NH40Ac) 80% (5% acetic acid 465 76084c in methanol) + 20% 146 1071040 (0.1M aqueous NH40Ac) 80% (5% acetic acid 412 57644 in methanol) + 20% 350 295424d (0.1M aqueous NH40Ac) 308 187791 291 232119 123 610112 80% (5% acetic acid 355 993655e in methanol) + 20% 293 496827 (0.1M aqueous NH40Ac) 123 1227456 80% (5% acetic acid 539 10454f in methanol) + 20% 123 1254528 (C4H9)3Sn0X (C4H9)2Sn0X (C4H9)3Sn0Ac (C4H9)SnOH (CAH9)3Sn OX* + H ( C A H 9 ) 2 S n O X 0.1 OX? + H N H A 0.1 M N H A O A C M 40Ac C4H9SnOX2 H O X + + 0.1 M NH40Ac (C4H9)3SnT 0.1 M (C4H9)3SnOAc NH40Ac (C4H9)3S^!0H (C 4H 9) 3Sn T^ + H (C 4H 9) 2S*T 0.1 M (C4H9)2SriOAc NH40Ac T + + H CAHqShT3 - H 0.1 M -.Attganj r + H (0.1M aqueous NH40Ac) NH40Ac OX+ - oxinate ion; OAc - acetate ion; T + - tropolonate i on a.b.c.d.e.f = base peaks for (C4H9)3SnOX, (C4H9)2SnOX2, C4H9SnOX3, (C4H9)3SnT, (C 4H 9) 2SnT 2 and C4H9SnT3 respectively 101 -e l u t i n g a t d i f f e r e n t r e t e n t i o n t i m e s ( F i g . 4 . 1 1 . 1 ) . A s s i g n m e n t o f the o b s e r v e d f r a g m e n t i o n s i s made by c o m p a r i s o n w i t h t h e t h e o r e t i c a l s p l i t t i n g p a t t e r n s f o r s u c h i o n s . - T I M E Cmin> TIC | , « 7 f.17 till «•»» •••» F i g . 4 . 1 1 . 1 : HPLC-MS t o t a l i o n chromatogram o f t r i b u t y l t i n o x i n a t e - 102 -4 .12 Summary The r e s u l t s o f t h e p r e s e n t s t u d y show t h a t h i g h l e v e l s o f b u t y l t i n compounds a r e p r e s e n t i n some m a r i n e o r g a n i s m s o f B r i t i s h C o l u m b i a . The t y p e o f o r g a n o t i n compound f o u n d i s an i n d i c a t i o n o f t h e s o u r c e o f p o l l u t i o n . The p r e s e n c e o f t r i b u t y l t i n compounds i n t h e m a r i n e o r g a n i s m s i m p l i e s t h a t f o u l i n g i s due t o e i t h e r m a r i t i m e a c t i v i t i e s o r f r o m t h e l u m b e r i n d u s t r y , s i n c e t r i b u t y l t i n i s a l s o r e g i s t e r e d i n Canada u n d e r t h e P e s t C o n t r o l P r o d u c t s A c t f o r g e n e r a l l u m b e r p r e s e r v a t i o n . The d e t e c t i o n o f d i b u t y l t i n i n t h e s e m a r i n e o r g a n i s m s i s e x p e c t e d i f t r i b u t y l t i n compounds a r e p r e s e n t i n t h e o r g a n i s m s b e c a u s e d i b u t y l t i n i s an e s t a b l i s h e d m e t a b o l i t e o f t r i b u t y l t i n . F o l l o w i n g r e p o r t s t h a t b u t y l t i n compounds p e r t u r b t h e c a l c i f i c a t i o n mechanisms i n o y s t e r s ^ 0 c a u s i n g a b n o r m a l s h e l l t h i c k e n i n g , an i n v e s t i g a t i o n was c a r r i e d o u t on t h e p r e s e n c e o f b u t y l t i n compounds i n t h e s h e l l s o f m a r i n e o r g a n i s m s b e c a u s e s u c h i n f o r m a t i o n m i g h t l e a d to a b e t t e r u n d e r s t a n d i n g o f t h e r o l e o f t h e s h e l l i n d e t o x i f i c a t i o n . The p r e s e n c e o f t r i b u t y l t i n and d i b u t y l t i n i n t h e s h e l l s a n a l y z e d c a l l s f o r more i n v e s t i g a t i o n i n t o t h e f o r m i n w h i c h t h e s e t i n compounds e x i s t i n t h e s h e l l s . 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Statist ics for Analytical Chemistry, E l l i s Horwood L t d . , England, p. 99, (1984). 101. Rapsomanikis, S., Harrison, R.M. Appl. Organomet. Chem., 2(2). 151, (1988). 102. Waldock, M . J . , M i l l e r , D. Marine Environmental Quality Committee Report CM 1983/E:12, Ministry of Agriculture, Fisheries and food, Fisheries Laboratory, Remembrance Avenue, Burnham-on-Crouch, Essex CMO 8HA, U . K . , (1983). 103. Thain, J . E . , Waldock, M.J. Wat. S c i . Technol. (Plymouth), 18, 193, (1986). 104. Rice, C D . , Espourteille, F . A . , Huggett, R . J . Appl. Organomet. Chem., 1, 541, (1987). 109 -APPENDIX A ELEMENTAL COMPOSITION OF STANDARD DOGFISH L I V E R I l l Coding The coding refers only to the ultimate method of analyte determination. No mention is made here regarding the various methods of sample preparation, decomposition and possible analyte separation prior to determination within each coded method. c - Cold vapour atomic absorption spectrometry. d - Inductively coupled plasma mass spectrometry. f - Flame atomic absorption spectrometry. g • Graphite furnace atomic absorption spectrometry. h - Hydride generation atomic absorption spectrometry. i - Inductively coupled plasma atomic emission spectrometry. n - Instrumental neutron activation analysis. p • Isotope dilution inductively coupled plasma mass spectrometry. t - Trtrimetry. v • Vapour phase chromatography. This reference material is primarily intended for use in the calibration of procedures and the development of methods used for the analysis of marine animals and materials with a similar matrix. The materia] should be kept tightly closed in the original bottle and should be stored in a cool location, away from any intense radiation sources such as ultraviolet lamps and sunlight. The bottle should be well mixed by rotation and shaking prior to use, and tightly closed immediately thereafter. A teflon ball is included with each sample. It should be inserted into the bottle the first time it is opened. This aids in mixing the material which may tend to cake on prolonged standing. 112 APPENDIX B H NMR SPECTRA OF BUTYLTIN OXINATES AND TROPOLONATES J j j .,,77 1 1 1 ' i i • 1/ | i i i i i i i i n i i i i | 2 1 0 PPM Fig- B -2 : 1H NMR s p e c t r u m of tributyltin tropolonate Irl 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 9 8 7 i i i i | i i i i | I I i i | i I I i | i i i i | I I • ' I ' i | i 6 5 4 3 i i i i I i i i i I i i i i | i i i ' I i ' ' i | ' • i 1 2 1 0 P P M Fig. B-3: 1H NMR spectrum o f dibutyltin bistropolonate 117 APPENDIX C THEORETICAL MASS S P E C T R A L INTENSITY PATTERN FOR STANDARD BUTYLTIN CHLORIDES AND DIPHENYLTIN DICHLORIDE 118 T O T A L A B U N D A N C E ' 0 . 9 9 8 8 4 AVERAGE M A S S = 3 4 8 . 1 7 0 1 2 1 MOST A B U N D A N T P E A K = 3 4 9 . 1 5 5 6 0 2 1 M A S S E X A C T M A S S I N T E N S 341 . 341 . 1 5 7 9 5 8 2 . 7 3 3 4 2 . 3 4 2 . 1 6 1 3 6 4 0 . 47 3 4 3 . 3 4 3 . 1 5 5 9 6 1 1 . 9 0 3 4 4 . 3 4 4 . 1 5 7 1 7 0 1 . 2 9 3 4 5 . 3 4 5 . 1 5 4 8 2 1 41 . 0 5 3 4 6 . 3 4 6 . 1 5 6 7 7 1 2 8 , . 8 4 3 4 7 . 3 4 7 . 1 5 4 9 8 0 7 3 , . 3 2 3 4 8 . 3 4 8 . 1 5 7 0 8 5 3 6 , . 9 9 3 4 S . 3 4 9 . 1 5 5 6 0 2 1 0 0 . . 0 0 3 5 0 . 3 5 0 . 1 5 8 8 6 5 16 , . 9 1 351 . 3 5 1 . 1 5 7 1 19 1 5 . . 0 6 3 5 2 . 3 5 2 . 1 6 0 1 0 5 2, , 44 3 5 3 . 3 5 3 . 1 5 8 4 6 3 1 7 . . 38 3 5 4 . 3 5 4 . 1 6 1 8 2 2 2 . , 98 3 5 5 . 3 5 5 . 1 6 4 9 4 7 0 . . 28 3 5 6 . 3 5 6 . 1 6 8 4 7 5 0 . . 0 1 + Fig. C - l : Theoretical mass spectral intensity pattern for (C^Hg^SnCOCCl^^ TOTAL ABUNDANCE- 0.99874 AVERAGE MASS" 326.505809 MOST ABUNDANT PEAK- 327.053230 NOM MASS EXACT MASS INTENSITY 319. 319.056384 2 .25 320. 320.059807 0 .29 321 . 321.054017 2 .27 322. 322.055560 1 .08 323. 323.053279 34 . 25 324 . 324.054852 22 .50 325. 325.052900 70 .13 326. 326.054790 34 .77 327. 327.053230 100 .00 328. 328.055004 18 .99 329. 329.052389 37 .78 330. 330.055570 4 . 6 7 _ 331 . 331.055924 18 .03 332 . 332.059344 2 .25 333. 333.054154 4 .67 334. 334.057297 0 .57 335. 335.060036 0.04 Fig. C - 2 : Theoretical mass spectral intensity pattern for + ( C A , H 9 ) 2 S n C l C 0 ( C H 3 ) 2 119 T O T A L A B U N D A N C E " A V E R A G E MASS= MOST ABUNDANT PEAK = NOM MASS E X A C T MASS 0 . 9 9 9 1 0 3 2 5 . 5 4 2 3 7 9 3 2 6 . 0 8 1 7 B 0 3 1 8 . 3 1 9 . 3 2 0 . 321 . 3 2 2 . 323 . 324 . 325 . 326 . 327 . 328 . 329 . 3 3 0 . 331 . 332 . 333 . 334 . 318 . 3 1 9 . 320 . 321 . 322 . 323 . 324 , 325 . 326 . 327 , 328 , 329 , 330 , 331 , 332, 333 , 334, 0 8 4 9 4 5 0 8 8 4 0 4 0 8 2 6 7 8 0 8 4 1 6 4 0 8 1 9 9 9 083421 0 8 1 4 2 7 0 8 3 3 4 6 0 8 1 7 8 0 0 8 3 6 4 0 0 8 0 8 1 4 0 8 4 1 2 6 084504 0 8 7 8 6 3 0 8 2 7 5 7 0 8 5 8 8 5 0 8 9 1 5 7 I N T E N S I T Y 2 . 2 4 0 . 3 1 2 . 2 7 1 . 10 3 4 . 17 22 . 83 7 0 . 1 4 _ 35 . 45 1 0 0 . 0 0 " 2 0 . 0 1 _ 3 7 . 7 0 5 . 0 7 1 7 . 9 6 " 46 65 0 . 6 3 0 . 0 4 F i g . C - 3 : T h e o r e t i c a l mass s p e c t r a l i n t e n s i t y p a t t e r n f o r ( C 4 H 9 ) 3 S n C l TOTAL A B U N D A N C E -A V E R A G E M A S S -MOST ABUNDANT PEAK 0 . 9 9 9 1 9 2 9 0 . 0 8 9 6 6 1 2 9 1 . 1 1 3 6 2 3 1 MASS E X A C T MASS INTENS 2 8 3 . 2 8 3 . 116092 2 . 76 284 . 284 . 119523 0 . 38 285 . 2 8 5 . 1 14 200 1 .91 286 . 2 8 6 . 115197 1 .24 2 8 7 . 287 . 113004 41 , . 58 288 . 288 . 114531 27 , .77 2 8 9 . 2 8 9 . 113137 73 . .27 2 9 0 . 2 9 0 . 114965 34 . .87 291 . 291 . 113623 100. , 00 292 . 2 9 2 . 116990 13. , 56 293 . 2 9 3 . 115040 14 . , 55 294 . 294 . 118168 1 . 93 2 9 5 . 2 9 5 . 116584 17. ,51 2 9 6 . 2 9 6 . 120038 2 . 42 2 9 7 . 297 . 123254 0 . 15 + F i g . C - 4 : T h e o r e t i c a l mass s p e c t r a l i n t e n s i t y p a t t e r n f o r ( C ^ H t j ^ S n 120 T O T A L A B U N D A N C E 3 0 . 9 9 8 9 6 A V E R A G E M A S S = 3 4 9 . 1 3 4 3 0 5 MOST A B U N D A N T P E A K 3 3 5 0 . 1 2 6 9 9 6 MASS E X A C T M A S S I N T E N 342 . 3 4 2 . 1 2 9 3 9 6 2 . 73 3 4 3 . 3 4 3 .1 3 2 8 4 1 0 . 44 344 . 344 . 1 2 7 4 4 6 1 . 9 1 345 . 3 4 5 . 1 2 8 5 7 4 1 . 27 346 . 3 4 6 . 1 2 6 2 4 5 41 , . 1 4 347 . 3 4 7 . 1 2 7 9 5 1 28 . . 44 348 . 3 4 8 . 1 2 6 4 0 7 7 3 . . 2 7 349 . 3 4 9 . 1 2 8 3 4 6 36 . . 3 1 3 5 0 . 3 5 0 1 2 6 9 9 6 1 0 0 . . 0 0 351 . 35 1 . 1 3 0 3 2 3 15 . . 9 0 352 . 3 5 2 . 1 2 8 5 5 8 15 . . 1 3 353 . 3 5 3 . 1 3 1 5 8 3 2 . . 3 1 354 . 3 5 4 . 1 2 9 8 8 7 17 . .41 355 . 3 5 5 . 1 3 3 3 3 6 2 . . 8 0 356 . 3 5 6 . 1 3 6 0 8 7 0 . , 28 357 . 3 5 7 . 1 3 7 4 4 9 0 . .01 F i g . C-5 : Theoretical mass spectral i n t e n s i t y pattern f o r + ( C A H 9 ) 2 S n [ C O ( C H 3 ) 2 ] 2 T O T A L A B U N D A N C E 3 0 . 9 9 8 7 4 A V E R A G E M A S S = 3 2 6 . 5 0 5 8 0 9 MOST A B U N D A N T P E A K = 3 2 7 . 0 5 3 2 3 0 MASS E X A C T M A S S I N T E N : 3 1 9 . 3 1 9 . . 0 5 6 3 8 4 2 . . 2 5 3 2 0 . 3 2 0 . . 0 5 9 8 0 7 0 . . 29 321 . 321 , . 0 5 4 0 1 7 2 . . 27 322 . 3 2 2 , . 0 5 5 5 6 0 1 . . 0 8 323 . 3 2 3 , . 0 5 3 2 7 9 34 . . 25 324 . 324 . . 0 5 4 8 5 2 22 . . 5 0 3 2 5 . 3 2 5 . . 0 5 2 9 0 0 7 0 . . 1 3 326 . 3 2 6 , . 0 5 4 7 9 0 34 . . 7 7 327 . 3 2 7 , . 0 5 3 2 3 0 1 0 0 . . 0 0 328 . 3 2 8 . . 0 5 5 0 0 4 1 8 . . 9 9 3 2 9 . 3 2 9 . 0 5 2 3 8 9 3 7 , . 7 8 3 3 0 . 3 3 0 . 0 5 5 5 7 0 4 , . 6 7 331 . 331 . 0 5 5 9 2 4 18 . . 0 3 332 . 3 3 2 . 0 5 9 3 4 4 2 . . 25 3 3 3 . 3 3 3 . 0 5 4 1 5 4 4 , . 6 7 334 . 3 3 4 . 0 5 7 2 9 7 0 . . 5 7 3 3 5 . 3 3 5 . 0 6 0 0 3 6 0 . . 0 4 F i g . C-6: Theoretical mass spectral i n t e n s i t y pattern f o r + ( C 4 H 9) 2SnClCO(CH 3 ) 2 121 TOTAL A B U N D A N C E " 0 . 9 9 9 0 2 A V E R A G E M A S S " 2 9 2 . 0 1 7 9 6 4 MOST ABUNDANT P E A K " 2 9 3 . 0 5 6 5 4 3 NOM MASS E X A C T MASS I N T E N S I T Y 2 B 5 . 2 8 5 . 0 5 8 9 6 9 2 . 78_ 2 8 6 . 2 8 6 . 0 6 2 3 2 4 0 . 31 2 8 7 . 2 8 7 . 0 5 6 9 7 2 1 . 9 3 _ 2 8 8 . 2 8 8 . 0 5 7 9 9 6 1 . 21 2 8 9 . 2 8 9 . 0 5 5 9 8 9 41 . 74 2 9 0 . 2 9 0 . 0 5 7 4 8 5 2 6 . . 96 2 9 1 . 2 9 1 . 0 5 5 9 4 6 7 3 . . 17 2 9 2 . 2 9 2 . 0 5 7 8 6 4 3 3 . 47 2 9 3 . 2 9 3 . 0 5 6 5 4 3 1 0 0 . 0 0 2 9 4 . 2 9 4 . 0 5 9 8 5 0 1 1 . , 48 2 9 5 . 2 9 5 . 0 5 7 8 7 1 14 . , 75 2 9 6 . 2 9 6 . 0 6 1 0 9 2 1 . ,64 2 9 7 . 2 9 7 . 0 5 9 4 6 0 1 7 . ,61 2 9 8 . 2 9 8 . 0 6 2 7 8 4 2 . 0 3 _ 2 9 9 . 2 9 9 . 0 6 5 1 5 1 0 . , 17 Fig. C -7 : Theoretical mass spectral intensity pattern for + (C4H9)2SnOOCCH3 > T O T A L A B U N D A N C E 3 0 . 9 9 9 3 5 A V E R A G E MASS= 2 6 8 . 4 2 6 3 5 0 MOST ABUNDANT PEAK= 2 6 9 . 0 1 1 4 3 9 1 MASS E X A C T MASS INTENS 261 . 261 . 0 1 4 5 1 9 2 .27 2 6 2 . 262 . 0 1 7 8 7 4 0, . 20 263 . 263 . 0 1 2 1 9 0 2 .29 264 . 264 . 0 1 3 5 5 7 1 , .02 2 6 5 . 265 . 0 1 1 3 2 3 34 , . 66 2 6 6 . 266 . 0 1 2 9 3 7 21 , . 58 2 6 7 . 267 . 0 1 0 9 5 1 70, .21 2 6 8 . 268 . 0 1 2 6 3 2 32 . . 73 269 . 269 . 0 1 1 4 3 9 100. , 00 2 7 0 . 270 . 0 1 2 7 3 9 15 , . 67 271 . 271 . 0 1 0 5 2 5 37 , . 49 272 . 272 . 0 1 3 6 4 3 3. . 40 273 . 273 . 0 1 4 0 1 3 18 . .05 274 . 274 . 0 1 7 3 6 2 1 . 66 2 7 5 . 275 . 0 1 2 1 8 9 4 . , 64 2 7 6 . 276 . 0 1 5 4 6 8 0 . , 42 2 7 7 . 277 . 0 1 8 7 3 1 0 . .02 Fig. C-8: Theoretical mass spectral intensity pattern for 122 TOTAL ABUNDANCE3 0.99919 AVERAGE MASS= 267.952422 MOST ABUNDANT PEAK= 269.020047 MASS EXACT MASS INTEN: 261 . 261 .022582 2 . 80 262 . 262 .025937 0. .22 263 . 263 .020531 1 . .93 264 . 264 .021482 1 . . 1 5 265 . 265 .019578 42 . 10 266 . 266 .020938 25 . 79 267 . 267 .019523 73 . 04 268 . 268 .021441 31 . 36 269 . 269 .020047 100. .00 270. 2 70 .023536 8 . 26 271 . 271 .021434 14 . 78 272 . 272 .024767 1 . . 1 9 273 . 273 .023044 1 7 . . 76 274 . 274 .026581 1 . . 45 275 . 275 .027958 0. , 16 F i g . C - 9 : T h e o r e t i c a l mass s p e c t r a l i n t e n s i t y p a t t e r n f o r + ( C A H 9 ) S n C l C O ( C H 3 ) 2 TOTAL ABUNDANCE3 0.99910 AVERAGE MASS= 325.542379 MOST ABUNDANT P EAK= 326.081 780 1 NOM MASS EXACT MASS INTENSITY 318. 318 .084945 2 .24 319 . 3 I 9 .088404 0 .31 320. 320 .082678 2 . 27 321 . 321 .084164 1 . 10 322 . 322 .081999 34 . 1 7 323 . 323 .083421 22 . 83 324 . 324 . 081427 70, . 14 325 . 325 , .083346 35 , . 45 326 . 326 . 081780 100, .00 327 . 327 .083640 20, .01 328 . 328 , .080814 37 , . 70 329 . 329 , .084126 5 . 07 330. 330. .084504 1 7 . . 96 33 l . 33 1. .087863 2 . 46 332 . 332 , .082757 4 . 65 333 . 333 . 085885 0. .63 334 . 334 . 089157 0. ,04 F i g . C-10: T h e o r e t i c a l mass s p e c t r a l i n t e n s i t y p a t t e r n f o r + (C AH 9 ) S n O O C C F 3 C AH eO 123 TOTAL ABUNDANCE= AVERAGE MASS= MOST ABUNDANT PEAK = 0.99949 331.998936 332.993940 NOM MASS EXACT MASS INTENS 325 . 324 . 996368 2 . 74 326 . 325 . 999723 0. .43 327 . 326 . 994494 1 . .91 328 . 327 . 995545 1 . . 27 329 . 328 . 993193 4 1 . , 1 7 330. 329 . 995148 28 . 37 331 . 330. .993390 73 . 27 332 . 331 . 995454 36 . , 1 5 333 . 332 . ,993940 100. ,00 334 . 333 . 997230 15 . , 66 335 . 334 . 995477 15. , 1 1 336 . 335. , 998515 2 . 28 337 . 336 . 996869 1 7 . . 43 338 . 338 . 00017 3 2 . 76 339 . 339 , .002896 0. ,.27 340. 340. .004421 0. .01 F i g . C - l l : T heoretical mass sp e c t r a l i n t e n s i t y pattern f o r )^SnOOCC^ TOTAL ABUNDANCE= 0.99961 AVERAGE MASS= 308.406746 MOST ABUNDANT PEAK= 308.948685 NOM MASS 301 . 3)02 . 303 . 304 . 305 . 306 . 307 . 308 . 309 . 310. 3 11. 3 12. 3 13. 314. 315. 316. 317. EXACT MASS INTENSITY 300. 301 . 302 . 303 . 304 . 305 . 306 . 307 . 308 . 309. 310. 311. 3 12. 313. 314. 315. 316. 951917 955272 949748 951136 948860 950315 948489 950209 948685 950581 947652 951 1 15 951447 954751 949763 952774 956129 2 . 24_ 0. 30 2.27 1.11 34.20_ 22.76_ 70.13_ 3 5.30_ 100.00_ 19.78_ 37.68_ 4 . 99_ 1 7 . 9 6_ 2 . 4 1_ 4 . 66 0.61 0.04 F i g . C-12 : Theoretical mass spectral i n t e n s i t y pattern f o r (c^H^S^C 1 124 APPENDIX D HPLC-MS CHROMATOGRAM AND MASS SPECTRA OF TISSUE EXTRACTS OF SOME MARINE ORGANISMS - 125 -T I M E ( m i n ) 1 B 0 X - 1 6 8 3 9 0 4 23.48 Fig. D-1:HFLC-KS total ion current chromatogram of oyster tissue extract 1 9 3 [ T 1 C = 1 1 7 6 9 6 8 , 1 8 8 X « 6 4 3 7 5 ] E I z u H 3 B A 1 3 5 0 M / Z Fig. D-2: Mass spectra of position * in Fig. D-l * corresponds to position on the horizontal axis of Fig. D-l 126 -227 [T IC -516016 . 1 0 0 X » 3 7 2 6 6 1 EI ~ i — i — i — i — r " T ~ i — i — r — i — r * 200 250 300 35J0 M / Z F i g . D - 3 : Mass s p e c t r a o f p o s i t i o n * i n F i g . D - l . 278 t T I C - 5 9 4 6 2 4 , 1O0X-78876] EI n—i—i—i—i—rn—i—pi—i i i—i—i—I—i—i—[• zee 250 - | — i — [ i j T — r \ — i — i — r 36B M / Z Fig. D - 4 : Mass spectra of position * in Fig. D - l . * c o r r e s p o n d s t o p o s i t i o n on t h e h o r i z o n t a l a x i s o f F i g - 127 400 45 F i g . D - 5 : H P L C - K S total ion current chromatogram of Butter clam tissue extract 198 [T IC- I 158336. 100* .102140] EI > 8 h H 7 I/) u h •W Fig. D -6: Mass spectra of position * in Fig. D-5 * c o r r e s p o n d s t o p o s i t i o n on t h e h o r i z o n t a l a x i s o f F i g . D-5 M / Z 128 2\% [ T I C - S 8 7 0 8 8 . 100X-18874] EI v r 1 ! i i i i"imi i i 400 f^l / ^ 4 5 e Fig. D-7: Mass spectra of position * in Fig. D-5 248 [ T I C - 3 3 1 0 5 6 , 100X-57335] EI 205 231 I I I 1 1 1 I | I- I I I1 I I "I I I | I l' 1 1 1 1 I I I | I I 350 -| I I I I I I I I I | 4 " M / Z 4 5 A Fig. D-8: Mass spectra of position * in Fig. D-5 * corresponds to position on the horizontal axis of Fig. D-5 - 129 -100X-243088 1 *e 180 IS* 2 80 258 388 Fig. D-9:HFLC-MS total ion current chronatogram of purple "sea cucumber tissue extract > h H (fl z w 198 [TIC-166528, I00X-15585) EI 175 ]—I T I I I 200 i i r i i | I —r • i 'i i • i' 300 | i i I i i—i i i I—| I i I—i i i i i i | 350 400 1^ 1 / 450 Fig. D-10: Mass spectra of position * in Fig. D-9 * corresponds to position on the horizontal axis of Fig. D-9 - 130 -209 [ T I C - 2 4 3 « 8 8 . ie f lX -41758] EI (I) w Z r l — i — r - i — i — r | i i i i I i i i i | Fig. D - l l : Mass spectra of position * in Fig. D-9 * corresponds to position on horizontal axis of Fig. D-9 - 131 -T I M E ( m i n ) 11:49 14:12 16:35 100X-1982144 F i g . D-12:HFLC-KS total ion current chromatogram of basket cockle tissue extract 194 [T IC -255156 . 100X'20012] EI 175 h H 14 z w h z H i—I r i 200 355 ,377 ~ i r n r ^ - M r r , | , , , l— r r 'i—I'T i ' i— i • j • i — i ' ' i " [ ' i ' Y ± 250 300 350 J S ^ / *Z 400 Fig. D-13: Mass spectra of position * in Fig. D-12 * corresponds to position on the horizontal axis of Fig. D-12 132 -212* I T I C - l 982 1 44 . 100X-647168] EI 2 1 3 , 1260 278 309 ,327 | i'1"!-1 1 J - i - T T ^ - n - r ^ r V - r - r - i - V i i I' | ' i i 1 ^ y I 1 1 1 1 F i g . D - 1 4 : Mass s p e c t r a o f p o s i t i o n * i n F i g . D-12 T I M E : C m i n > 1 1 : 49 14:12 16:3 1001-1216064 50 100 150 f 200 250 300 Fig. D-15:HPLC-MS t o t a l i o n current chroaatogran of Blue mussel M v t l l u s edulls t i s s u e ex t rac t 190 CTIC-1216064 . 100X-116236) EI 133 -T 300 LxiJL i i i r r i r 350 M / Z F i g . D - 1 6 : Mass s p e c t r a o f p o s i t i o n * i n F i g . D-15 204 IT IC-433488 . 100X-17500] EI ,203 181 , | ,215 i i i—i r i" V i '| I 'n—I i " i — r " n — 1 11 [ 200 250 I I T 'I I | I ~f 300 r i — | i V ' I—I' i T I Y 11" i— i i — r 350 f V j / Z^l 400 Fig. D-17: Mass spectra of position * in Fig. D-15 * corresponds to position on horizontal axis of Fig. D-12 - 134 -TIC 100 100X=514224 r T I M E ( m i n ) 11:49 14:12 , 50 100 150 200 , 2S0 300 F i g . D-18:HPLC-XS total ion current chromatogram of soft-shelled clam (Coles Bay) tissue extract 198 [T IC -514224 . 100X-30G43] EI 100 176 b H 6 0 w u ^ 30 H 200 ' I 1 1 250 n— I I I I I I 1 1 1 1 400 Fig. i)-iy: Mass spectra of position * in Fig. D-18 - 135 -240 [ T I C - 8 9 4 2 0 . 100X=20512] EI 208 t i — i — r ~ i — i — r • T—I—i—i—i—r - i r~] r ~ r 300 ~\—i—i—i—i—i r 350 M / Z Fig. D-20: Mass spectra of position * in Fig. D-18 248 [T IC -101616 , 100X-34807] EI 400 - | — n — T T " ~ i — i r i — i — i — I — r n — r ~ n — r ~ 1 — i r ~ | — i — r 200 250 300 H— i—i—|—i— i— r I i — i — i | i i—i—r 350 M / Z *BB Fig. D-21: Mass spectra of position * in Fig. D-18 * corresponds to position on horizontal axis of Fig. D-20 

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