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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 o f I l o r i n , N i g e r i a , 1981  M.Sc.  University  o f Ibadan,  N i g e r i a , 1984  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE  REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in  THE  FACULTY OF GRADUATE STUDIES DEPARTMENT OF CHEMISTRY  We a c c e p t t h i s  t h e s i s as conforming  to the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA NOVEMBER 1988 °  B a s i l Ugwunna Nwata, 1988  In  presenting  this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this or  thesis for by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make  it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my 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)  ii  ABSTRACT  An the  a s s a y b a s e d on HPLC-UV, HPLC-GFAAS  chromatographic  separation  and  and HPLC-MS was d e v e l o p e d f o r  characterization  compounds i n some marine i n v e r t e b r a t e s o f B r i t i s h To  enable  described.  This  instability In  approach  was  their  of high  chromatographic  abandoned  because  behavior  tributyltin  i n d i c a t e s t h a t the m a r i t i m e i n d u s t r y o r the lumber  the  source o f b u t y l t i n p o l l u t i o n  Varying  amounts Sn  (wet  of  tributyltin  weight)  and  i n the a r e a s compounds  dibutyltin  and  The p r e s e n c e o f these  compounds  as  is  o f the i n c r e a s e i n  bivalves,  d i b u t y l t i n compounds were d e t e c t e d by HPLC-GFAAS.  pg/mL  extinction  o f the d e r i v a t i v e s .  the marine organisms s t u d i e d , m a i n l y  major  organotin  Columbia.  HPLC-UV d e t e c t i o n , b u t y l t i n complexes  c o e f f i c i e n t s were s y n t h e s i z e d , and  of  industry  is  sampled.  i n the range  compounds  in  1.14-4.29 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 examined. the  butyltin  content  of  the  s h e l l s o f marine organisms was  V a r y i n g amounts o f t r i b u t y l t i n  and  dibutyltin  compounds  also in  c o n c e n t r a t i o n 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 d e t e c t e d by atomic 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 .  iii  TABLE OF CONTENTS  Page  ABSTRACT  ii  LIST OF TABLES  vii  LIST OF FIGURES  ix  LIST OF ABBREVIATIONS  xi  ACKNOWLEDGEMENT  xii  CHAPTER 1  I  II  1  Organotin biocides  1  1.1  Introduction  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 o f t r i b u t y l t i n compounds  6  1.4  Effect  1.5  Governmental r e g u l a t i o n o f t r i b u t y l t i n useage  o f t r i b u t y l t i n on marine l i f e  10 . . . .  10  A n a l y t i c a l methods f o r o r g a n o t i n compounds  11  1.6  Spectrophotometry  12  1.7  Electrochemistry  13  1.8  Atomic spectrometry  14  1.9  Gas chromatography  15  1.10  L i q u i d chromatography  18  1.11  O b j e c t i v e s o f the p r e s e n t  and S p e c t r o f l u o r i m e t r y  study  20  iv  CHAPTER 2:  EXPERIMENTAL  21  2.1  G e n e r a l methods  21  2.2  Materials  24  2.3  Methodology  25  2.4  Synthesis  of b u t y l t i n oxinates  25  2.5  Synthesis  of b u t y l t i n tropolonates  27  2.6  Determination of and  CHAPTER  3:  and r e a g e n t s  molar  absorption  absorption  wavelength  coefficients  28  SEPARATION AND DETECTION PROCEDURE  29  3.1  HPLC-GFAAS  29  3.2  O p t i m i z a t i o n of  3.3  Selection  3.4  Establishment  of  retention  3.5  Establishment  of  analytical  and 3.6  3.7  recovery  chemical  32  modifiers  34  data  35  procedure  studies  37  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 from marine organisms  38  Quantitation  40  3.7.1  3.7.2  3.8  of  GFAAS c o n d i t i o n s  .  Quantitation of t r i b u t y l t i n chloride and d i b u t y l t i n d i c h l o r i d e f o r recovery studies  40  Quantitation of t r i b u t y l t i n and d i b u t y l t i n d i c h l o r i d e  42  Combined h i g h performance chromatography  O p t i m i z a t i o n o f HPLC-MS  3.10  V e r i f i c a t i o n of used f o r  liquid  a n d mass s p e c t r o m e t r y  3.9  HPLC-MS  fragment  chloride  conditions ions  quantitation  to  43 48  be 49  V  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  3.12  A n a l y s i s o f e x t r a c t s from marine organisms by HPLC-MS  50  RESULTS AND DISCUSSION  51  CHAPTER 4 :  4.1  4.2  Characterization of butyltin and t r o p o l o n a t e s  . . . .  49  oxinates 51  Molar 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 and t r o p o l o n a t e s  57  4.3  Nature 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  4.4  Chemical m o d i f i e r s f o r atomic a b s o r p t i o n spectrophotometry o f b u t y l t i n c h l o r i d e s  62  Retention data  68  4.5  4.6  R e t e n t i o n data f o r b u t y l t i n c h l o r i d e s  . . . .  68  4.5.2  R e t e n t i o n data f o r b u t y l t i n o x i n a t e s  . . . .  70  . . .  70  Tributyltin chloride, dibutyltin dichloride: Recovery 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  4.6.2  Recovery s t u d i e s on the e x t r a c t i o n procedure  70  D e t e c t i o n l i m i t and p r e c i s i o n  75  HPLC-MS 4.7.1  . .'  75  O p t i m i z a t i o n o f the thermospray interface conditions  4.8 4.9 4.10  57  4.5.1  4.6.1  4.7  . . . .  Major  ions o f standard organotin c h l o r i d e s  75 . . . .  77  R e t e n t i o n times o f o r g a n o t i n compounds i n HPLC-MS 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  79  and  85  s h e l l s o f marine animals  4.11  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  4.12  Summary  . . . .  99 102  vi  103  BIBLIOGRAPHY  APPENDIX A :  APPENDIX B :  APPENDIX C :  APPENDIX D:  Elemental composition of dogfish liver *H NMR . s p e c t r a and t r o p o l o n a t e s  of  standard  butyltin  109 oxinates 112  Theoretical intensity patterns for 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 diphenyl dichloride  117  H P L C - M S c h r o m a t o g r a m a n d 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  vii  L I S T OF T A B L E S  Tables  Page  3.2.1  Graphite furnace  3.7.1  Sampler parameters plot of d i b u t y l t i n  3.7.2  4.1.1  4.1.2  operating parameters for standard dichloride  34  addition 41  GTA-95 g r a p h i t e t u b e r a t o m i z e r program for standard addition plot of 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  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 b u t y l t i n tropolonates  of 51  Analytical  of  butyltin  data  and m e l t i n g p o i n t s  oxinates ions  53  4.1.3  Fragment  of b u t y l t i n  4.1.4  Fragment  ions  4.1.5  Fragment  ions of  4.1.6  Fragment  ions  of b u t y l t i n  4.1.7  Fragment  ions  of  dibutyltin bisoxinate  56  4.1.8  Fragment  ions  of  tributyltin  56  4.2.1  Molar  of  extinction  tristropolonate  54  bistropolonate  54  dibutyltin tributyltin  tropolonate  trisoxinate  coefficients  oxinate of  4.3.1  59  Molar e x t i n c t i o n c o e f f i c i e n t s oxinates Relevant  i n f r a r e d data  for  of  butyltin 60  butyltin  tropolonates 4.6.1  Recovery  4.8.1  Major  4.9.1  R e t e n t i o n times HPLC-MS  61  studies  fragment  55  butyltin  tropolonates 4.2.2  55  for butyltin  ions of  chlorides  standard  74  organotin  compounds  84 o f o r g a n o t i n compounds i n 85  viii  4.10.1  4.10.2  4.11.1  Concentration of butyltin body o f marine organisms  compounds i n  whole 86  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 marine organisms Fragment i o n s o f b u t y l t i n tropolonates  88  oxinates and 100  ix LIST OF FIGURES  Figures 1.3.1  Fage 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  3.8.2  The thermospray interface  4.1.1  Desorption chemical i o n i z a t i o n mass  . . . .  46  spec crura of t r i b u t y l t i n tropolonate 4.1.2 4.4.1 4.4.2 4.4.3 4.4.4  45  52  n.m. r. spectrum of d i b u t y l t i n bisoxinate  58  E f f e c t of various modifiers on the absorbance of (C^Ho^SnCl Effect of various modifiers on the absorbance of ( C ^ H a ^ S n C ^ E f f e c t of modifier volume on the absorbance of (C^Hg^SnCl  64 65 66  E f f e c t of modifier volume on the absorbance of ( C ^ H o ^ S n C ^  67  4.5.1  HPLC-GFAAS chromatogram of b u t y l t i n chlorides  4.5.2  Chromatogram of b u t y l t i n oxinates  4.6.1  C a l i b r a t i o n graph for recovered t r i b u t y l t i n  4.6.2  chloride Standard addition plot for  recovered  . . . .  69 71 72  d i b u t y l t i n dichloride  73  4.6.3  Estimation of l i m i t of detection  76  4.7.1  E f f e c t of v a r i a t i o n of vaporization  4.8.1  temperature on t r i b u t y l t i n chloride Mass spectrum of t r i b u t y l t i n chloride  78 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  4.8.3  Mass s p e c t r u m o f b u t y l t i n  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  4.10.1  HPLC-MS clam  total  tissue  dichloride  81  trichloride  82  dichloride  i o n chromatogram o f  83  Bent-nose  extract  90  4.10.2  Mass s p e c t r a  o f Bent-nose  clam t i s s u e  extract  .  .  .  .  90  4.10.3  Mass s p e c t r a  o f Bent-nose  clam t i s s u e  extract  .  .  .  .  91  4.10.4  Mass s p e c t r a  of Bent-nose  clam t i s s u e  extract  .  .  .  .  91  4.10.5  HPLC-MS  4.10.6  HPLC-MS o f s t a n d a r d  4.10.7  E l e c t r o n i o n i z a t i o n mass e x t r a c t o f the Bent-nose  4.10.8  4.10.9  4.10.10  4.11.1  of standard  "(C H ) SnCl"  93  " (C H  93  6  6  n  1 : L  3  ) SnCl " 2  2  spectra of shell c l a m Macoma n a s u t a  Theoretical intensity patterns for ( C ^ H g ^ S n C l , C^HgSn, and C ^ g S n C l E l e c t r o n i o n i z a t i o n mass dibutyltin dichloride  spectra  E l e c t r o n i o n i z a t i o n mass tributyltin chloride  spectra  of  95  96 standard 97  HPLC-MS t o t a l i o n c h r o m a t o g r a m t r i b u t y l t i n oxinate  of 98  of 101  xi  LIST  OF ABBREVIATIONS  uv  Ultraviolet  GFAAS  Graphite  furnace  atomic  absorption  HPLC  High performance  liquid  chromatography  HPLC-GFAAS  HPLC  MS  Mass  HPLC-MS  HPLC  i n combination with  mass  HPLC-UV  HPLC  i n combination with  ultraviolet  OX  Tropolone,  .  spectrometry  as  detector  spectroscopy  for  ODS  per m i l l i o n ,  also  /ig/mL  8-hydroxyquinoline  r . p . m.  1 8  detector  spectrometry  Parts  Oxine,  GFAAS a s  detection  ppm  C  i n combination with  spectrophotometry  rotations  T  per  minute  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  O c t a d e c y l s i l a n e . bonded phase column  W/V  w e i g h t p e r volume  AAS  Atomic  GTA  Graphite  NMR  Nuclear  magnetic  M/Z  Mass  charge  Butyl  n-butyl  THF  Tetrahydrofuran  to  absorption tube  spectrophotometer/spectrophotometry  atomizer resonance  ratio  xii  ACKNOWLEDGEMENT  I  wish  guidance  to  express  and i n t e r e s t  My g r a t i t u d e their I  expert am a l s o  also  advice  my g r a t i t u d e  Ove  Cullen's  research  Professor  W.R.  Cullen  for  in this  study.  goes  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  o n mass  grateful  Wireko,  to  Pedersen,  to  for  spectrometry.  t o Agyeman T.S.  group f o r  his  Kuma-Mintah,  Koko,  their  Tom  Otieno,  a n d a l l my c o l l e a g u e s  encouragement  and h e l p f u l  in  Dr.  Fred  Professor  discussions.  -  1  -  CHAPTER 1 ORGANOTIN  I  BIOCIDES  INTRODUCTION  1.1  Historical  Organotin society  Background  compounds  with  many  have  become  industrial,  very  significant  agricultural  and  in  the  modern  medicinal  appli-  cations . The the  synthesis  way  Rapid made  for  it  compounds only  butyltin  potentials  formula be  R4Sn,  the  have  a  from  derived. to  of  discovery of  by F r a n k l a n d ,  of  organotin  variety lower  The G r i g n a r d production  been  of  synthesized  compounds.  Grignard reagents  wide which  opened  1  which  of  organotin  alkyl  organotin  method  is  still  tetraphenyltin. by Wurtz  the Tetra-  synthesis,  and  by a l k y l a l u m i n u m compounds. industrial  when Y n g v e  (PVC),  the  synthesis  applicable  first  heat  the  and t e t r a o c t y l t i n  alkylation The  for  the  of  enhanced by the  could easily  method  1936,  was  possible of  d i e t h y l t i n d i i o d i d e i n 1849  exploration  progress  compounds  the  the  of  of  application  organotin  C a r b i d e and C a r b o n C h e m i c a l  stabilizing effect  and o t h e r  of  of  chlorinated  dibutyltin  dilaurate  and  dioctyltin  derivatives  are  polymers,  because  their  of  organotin  polymers.  d i b u t y l t i n maleate u s e d as  Corporation  compounds  hydrocarbon  are  PVC s t a b i l i z e r s  non-toxicity.  compounds was  In  this  chloride regard,  now m a i n l y u s e d . for  in  discovered  on P o l y v i n y l 1  made  food  O r g a n o t i n compounds  The  packaging are  also  - 2 used i n industry for cold curing of s i l i c o n e rubber, and as  polymeriza-  t i o n c a t a l y s t s , e.g.  2  Triorganotin  b u t y l c h l o r o t i n dihydroxide C^HgSnCl(OH) .  compounds  applications.  They exhibit  activities.  The  are  the  most  fungicidal,  triethyltin  important  bactericidal,  compounds  exhibit  mammals, the LD50 of t r i e t h y l t i n acetate to rat weight.^  The  tricyclohexyltin  in  compounds  and  acute  being are  used  active as fungicides and s l i m i c i d e s . activity  of  tributyltin  As  toxicity mg/kg  as  body  miticides.  The t r i b u t y l t i n compounds are a  result  of  the  biological  compounds, they are incorporated into various  forms of a n t i f o u l i n g paint formulations,  to protect the h u l l s  of  ships  and boats from fouling by fungi, algae, sponges, molluscs, diatoms, Such f o u l i n g has the e f f e c t of increasing weight and drag, ship  to  consume  more  fuel  to  maintain  i t s speed.  compounds also find use,  as wood preservatives.  wood,  slight  they  have  only  insecticidal  When  causing  etc. the  The t r i b u t y l t i n impregnated  activity,  into  and are usually  formulated with other compounds to broaden the a c t i v i t y . increase  to  tricyclohexyl-  3  hydroxide as the active ingredient.^  acaricidal  4.0  Plictran m i t i c i d e , made by Dow Chemical Company.contains tin  agricultural  They  neither  flammability, nor impart undesirable colors to the wood.  They  are also r e s i s t a n t to leaching. The t r i p h e n y l t i n compounds phenyltin  acetate  and  also  show  antifungal  activity.  t r i p h e n y l t i n hydroxide are used as  Tri-  agricultural  fungicides. Some octahedral organotin dihalides having the formula R S n X L 2  ethyl  or  phenyl,  X  =  2-(2-pyridyl)benzimidazole)  chloride exhibit  or bromide, L antitumor  2  2  2  (R =  •= O-phenanthroline or  activities.^  The  same  3 compounds  have  v i t r o A l s o , effective The are  for  the  of  organotin  The  fluoride,  intestinal  compounds o f  compounds  throughout  i n the  active  marine  the  marine  are  environment  ingredients  active  bis(tributyltin)  these  in  in poultry.  concern  as  activity  dilaurace  oxide,  compounds do n o t is  antiin  some  bis(tributyl-  sulfide,  environment  in  ingredients  t r i b u t y l t i n methacrylate,  Unfortunately,  spread  dibutyltin  worms  are  anti-herpes  bis(tributyltin)  t r i b u t y l t i n resinate,  their  possess  used  are  succinate,  adipate.  to  compounds e . g .  antifouling paints  dodecenyl  able  reported  organotin  removal  paints.  commercial  tin)  been  t r i b u t y l t i n compounds w h i c h  fouling  and  some  major  the  tin)  also  tributyltin bis(tributyl-  remain  localized  causing  consider-  problems.  Antifouling  1.2  The been  growth  a major  organisms ance.  One  leads  of  the  of  the  ships'  hulls.  weakening,  fouled hulls early  use This  of  organisms  maritime  severe  retardation  is  both expensive  metal  taken  ships,  due  severe  the  use  galvanic  of  to  sheathing success  of  i n the  and time prevent in  the  i n the  steel  of vessels  growth  i n value  copper metal  corrosion  hulls  The  and drag  and d e p r e c i a t i o n  approaches  copper  on the  industry.  a c h i e v e d moderate  steel to  Organotins  unwanted marine  to  the  was  priate,  of  p r o b l e m i n the  ships,  In  of  Structural  cleaning  Action  of  ship's  also  has  these perfor-  occur.  The  consuming. fouling  i n wooden  construction  control  sheathing when i n  is  of  of  fouling.  not  contact  approwith  - 4 copper  and  sea water.  i s by the  use  of  releasing  biocides  The method of fouling prevention i n steel  chemical from  agents.  These  Among the early biocides Cuprous  employed  act  by  to s e t t l e on the s h i p ' s  for  this  purpose  hull.  was  cuprous  oxide exhibits a wide spectrum o f t o x i c i t y to animals,  but many plants are r e s i s t a n t insoluble  greenish  The b u i l d  up  controlled  agents  the paint which k i l l the larvae and spores of  any marine animals and p l a n t s a t t e m p t i n g  oxide.  chemical  ships  of  salts these  release  of  to  it.  On continuous  use,  it  forms  within the surface layers of the paint f i l m . salts  fresh  on  the  biocide.  surface  interferes  with  the  This l i m i t s the l i f e time and  e f f i c i e n c y of the paint. The search for biocides to boost the performance  of  cuprous  oxide  led to the screening of organotin compounds. Tributyltin  compounds were found to be suitable biocides.  The com-  pounds possess the following properties: i.  low mammalian t o x i c i t y  ii.  lack of c o l o r . The low mammalian t o x i c i t y ensures human safety i n  containing t r i b u t y l t i n as active ingredient.  handling  Their lack of color,  paints makes  them e a s i l y incorporated into b r i g h t l y colored paints. In the course of searching for e f f i c i e n t ways of designing effective tributyltin  antifouling  preparations,  the following formulations have  become commercially a v a i l a b l e .  i.  Contact leachinp a n t i f o u l i n g paints.  fouling  In  this  design,  the  anti-  system is composed of a tough insoluble film-forming resin such  - 5 as  chlorinated  persed  rubber,  i n the  w i t h i n which the  hard matrix.  tributyltin  compounds  near  out  matrix  the  of  film,  the  it  inflow fresh The  of  water  shortcoming  ively  becomes  released.  of  trapped  the  of  designed  to  dispersed  i n the  formulation  and  rate  speed.^  part  film,  that  is  film  and thus  the  large thereby  freely  the  are  able  is  biocide  w i t h i n the  deeper of  to  leaches  paint  dispersed diffuse  out  of  the  matrix.  The  pores  causes  the  release  surface  layers  of  the  that with  the  strata  passage  of  the  life.  amount  of  creating  to  best  antifouling  biocide  a severe  the  matrix  works  When t h e  the  film.  progress-  paint  antifouling paint  of  time,  making i t  of  paint's  formulation. to  a  a mixture  of  to  paint  dis-  is  still  problem of  proper  paint.  added  is  it  the  the  design  of  a  is  the  this  down o v e r  paint  i n water,  insoluble materials  in  antifouling  break  is  of  with  matrix,  matrix  pores  design  matrix/ablative  film  physically  As the  from beneath  fails,  compound  the  paint  early  of  is  these microscopic  biocide  inner  spent  tributyltin  the  film.  this  for  paint  Soluble  system,  paint  clogged  the  i n the  disposal  surface  As a r e s u l t ,  during  action  the  into  of  difficult  only  ii.  sea  film  Cn i m m e r s i o n  behind microscopic  tributyltin biocide  paint  be  leaves  of  tributyltin  time, be  In  soluble  matrix.  insoluble  thus  and  a l l o w i n g the  released.^  d i f f i c u l t to  this  A  control  design, In  an  soluble biocide  disadvantage the  biocide  release,  because  f i l m breakdown are  affected  by water  the  actual  the  ablative material  physically of  this  breakdown  matrix  conditions  of  solubility and  vessel  -  iii.  6 -  Self-polishing copolymer paints.  advanced  design in antifouling paint technology.  cally bound in the paint film. design  This design represents the most  can be  controlled.  The biocide is chemi-  The release rate of the biocide in This  this  allows the b i o c i d a l a c t i v i t y o f the  antifouling paint to last a long time. The film forming resin i s a co-polymer of tributyltin methylmethacrylate, butyltin  is  also  the source of the biocide.  methacrylate/methylmethacrylate  preventing  polymer  sea  water  initiating  interacts  with  the  The t r i -  i s hydrophobic,  sea water ingress to the depths of the film.  of the paint, thereby  and  methacrylate/  At the surface  hydrophobic copolymer,  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. of  6  Tributyltin is susceptible to degradation by  a variety  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 particulate matter ir. the  K^,  1 0  -  1 1  tributyltin  adsorbs  strongly  sediment. The affinity for sediments, makes i t  far less bioavailable to organisms in the upper water layer. Maguire,  11  to  According to  tributyltin adsorbs so firmly to particulates that under abiotic  - 7 -  vas nodesorption  c o n d i t i o n s .there ments,  over  a  period of  microorganisms  of  ten months.  resulting  tributyltin However,  oxide  there  from harbor  was  degradation  in the liberation of butylated  On the contrary, data for San Diego  have  greater  much  mobility  of  adsorbed  by  and m e t h y l a t e d  degradation products. suggested  sedi-  sediment  tributyltin  12  from  sediment than reported by Maguire. Tributyltin has the tendency to surface  microlayer  of natural waters.13,14 partition  octanol-water  tin's  coefficient  partition  preferentially  K  o c  coefficient,  values  favor  accumulate  This is because tributylK  o w  , and  accumulation  sediment-water  in the  microlayer.  The surface microlayer attracts and sequesters  hydrophobic  chemicals like tributyltin.  in  to  unavailable accumulate  Bacteria  high  and  and  and  1  organisms.  However,  a variety  to relatively high concentrations  octanol-water  partition  coefficient  phytoplankton accumulate tributyltin  surface  moderately accumulation biologically  of  organisms  because (Kov, -  of  1  600  concentra-  A bioaccumulation factor of 4400 has been reported by  Laughlin, ^  its  2300).  concentrations  at  30,000 times respectively, more than their exposure  tions. ^- ^ 1  most  tributyltin  moderately  times  This preferential  surface microlayer is expected to render tributyltin  the  in the  Evans  for the hepatopancreas of the mud crab Rhithropanopeus  harrisii.  Organotin compounds exhibit tissues  than  others.  preferential  Ward e_£ aJL-*®  accumulation  observed  that  sheepshead minnow contained higher concentrations of than  the  cranial  or muscle  tissues.  factors for tributyltin compounds are high  in  certain  the viscera o f  tributyltin  oxide  The reported bioaccumulation enough  to  warrant  concern  - 8 with  regard  to  their  persistence  and  accumulation  however, they are degraded i n vivo by bacteria, Tributyltin  does  algae,  i n food chains, f i s h and mammals.  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 aerobic metabolism ( F i g . A  variety  pounds.  shorter  than  forty  by  1.3.1).  of organisms can metabolize and excrete t r i b u t y l t i n com-  Degradation rates vary depending on the conditions  Microbial  days  considered.  degradation under aerobic conditions i n some natural environ-  ments may be quite r a p i d . Seligman,20 studied microbial biodegradation laboratory  microcosms,  l i g h t e d conditions.  and  observed  of  half-lives  However, there are contrasting  tributyltin of  6-13  reports  using  days under 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 d i b u t y l t i n , monobutyltin and inorganic t i n . These metabolites are less toxic than also  observed  tributyltin.  Maguire  et  al.^  i n vivo degradation of t r i b u t y l t i n by a green algae with  the major degradation product being d i b u t y l t i n . Photolysis is a major marine  environment.  degradation  Photolysis  of  wavelength of the u l t r a v i o l e t light.  route  for  tributyltin  tributyltin is  in  affected  Half lives ranging from less  one day to 100 days have been reported.^•^•22.23  the  by the than  - 9 -  2?0  AEROB MT  F i g . 1.3.1:  AKAEROB KT  FISH KT  HYDROL  OYS MT  PHOTOL  Approximate average h a l f - l i v e s f o r t r i b u t y l t i n degraded v i a aerobic metabolism (AEROB KT), anaerobic metabolism (ANAEROB KT), f i s h (FISH KT), hydrolysis (HYDROL). oysters (OYS KT). and photolysis (FHOTOL). 6  10  1.4  Effect  T.B.T.  of Tributyltin (T.B.T.) Toxicity on Marine Life  dissolved  aquatic l i f e . Crustacea plankton, are  in  are  less  sensitive  to  and s m a l l c r u s t a c e a n s . very  sensitive  showing acute t o x i c i t y  than 0.025 ppb.  suggest  that  to a v a r i e t y o f  fish  and larger  T.B.T. than b i v a l v e s , m o l l u s c s , p h y t o It has been e s t a b l i s h e d t h a t  to organotin compounds.^•25  t o c e r t a i n marine l i v e s , T.B.T.  t o some a q u a t i c  Skeletonema  e x h i b i t acute t o x i c i t y  Available data, tend to  generally  toxicity  water  l i v e s at concentrations  molluscs Ap  a r  exhibits  t  from  chronic  r a n g i n g from 10 to l e s s  T.B.T. a l s o appears to be e s p e c i a l l y t o x i c to the  costatum.  alga  and to embryonic and l a r v a l s t a g e s o f the P a c i f i c  oyster.  1.5  Governmental R e g u l a t i o n o f 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 , and  abnormal growth i n o y s t e r s ,  shell  malformations  the Government o f France  i n 1982 banned  the use o f 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 organotin tons. less  1986, of  England  antifouling  weight,  3%  by  weight  f o r the p r o t e c t i o n o f h u l l s o f b o a t s l e s s  than 25 m came i n t o e f f e c t  concentration by  than  I n 1987, a t o t a l ban on the use o f o r g a n o t i n  In sale  compounds,  more  than 25  on  vessels  i n France.26  p r o h i b i t e d the r e t a i l paints  paints  of  containing  sale  and s u p p l y f o r r e t a i l  tributyltin,  if  the  total  o f 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% or  if  the  total  concentration  of  organotin  in  other  - 11 non-copolymer The  Acts use  f o r nets  07  i n f r e s h water  i s not  antifouling paints  is  °  tributyltin is registered  as a p r e s e r v a t i v e  under  the Pest  Control  Products  lumber p r e s e r v a t i o n .  *  Its  allowed.^®  A N A L Y T I C A L METHODS FOR ORGANOTIN COMPOUNDS  first  classical tin.  analytical  gravimetric  methods or  geological late  1950's  introduced,  sensitivity  residues relied  analysis  on  were  w h i c h gave  only  total  t i n was e x t e n s i v e l y  used  1931,  time,  fluorimetric  and  neutron  methods.  Flame  atomic  remained l e s s  to monitor products  and e x t e n d i n g  more  sensitive  activation  into and  analyses  absorption  techniques  popular because  o f the low  lines.^® compounds  in agriculture  and determine and the  f o l l o w e d by  a p p l i e d to the d i r e c t  were p o l a r o g r a p h y  tin  this  digestion with mineral acids,  pounds  of total  t h e amount  environment.  the c o n v e r s i o n o f the organotins  O t h e r methods  of  At  of the t i n a b s o r p t i o n  in agricultural  analysis  procedures  1960's.  but these  need arose  the  as  the wide use o f o r g a n o t i n the  for  b e g i n n i n g as e a r l y  the spectrographic  also  tries ,  and e a r l y  colorimetric,  replaced  With  studies  used  volumetric  Optical spectrographic  specific  were  compounds  i n Germany a n d S w i t z e r l a n d .  Canada,  The  the  2.5% b y w e i g h t .  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  II.  for  exceeded  use o f organotin  prohibited In  paints  to  inorganic  and of  Early  indus-  organotin methods  t i n u s u a l l y by  ignition.^ speciation  and gas chromatography.  of These  organotin two m e t h o d s  comare  12 also  quantitative.  chemists given  on q u a n t i t a t i v e  nuclear  The  years,  the  following  on  molecular  types  and  Cremer,  chlorides  react  aqueous  potassium  Mossbauer  partition  of organotin  is  hydroxide into  the  and o f f e r s  achieved  have  been  'and  a p p l i e d to  characterization  methods  applied  over  compounds a r e d e s c r i b e d i n  first  of organotin  with dithizone effected  the a l k a l i  to use d i t h i z o n e compounds.  layer,  and c h l o r o f o r m .  The s e p a r a t e d This  complexes.  partitioning  while the  f o r the  D i e t h y l t i n and  to form c o l o r e d  following  solution  to the chloroform l a y e r .  1972,  spectrometry,  f o r the  analytical  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 .  In  being  analyses.  structure and  were  determination  o f the complexes  tive  attention  and S p e c t r o f l u o r i m e t r y  Analysis  migrate  L  environmental  sections.  spectrophotometry  species  of organotin  of quantitative  Spectrophotometry  triethyltin  in little  spectroscopy,34,35,36  f o r the determination  Aldridge  has r e s u l t e d  and  compounds.  various  the  1.6  resonance  information  organotin  aspects  of analytical  infrared spectrometry,  magnetic  provide  emphasis  analyses  to the q u a l i t a t i v e  Qualitatively,  of  The g r e a t  between  The d i e t h y l t i n  triethyltin  organotin  species  compounds c a n  method i s q u i t e  sensi-  specificity.  Havir  and  Vrestal3&  s p e c i f i c i t y by  u s i n g a m o d i f i e d d i t h i z o n e method,  selectively  extracting  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  bis(tributyltin)  i n the presence  also oxide  o f a complexing  13  agent. Skeel  and B r i c k e r ^ ^  determination  dibutyltin  used for  the  analyses  8-hydroxyquinoline,  (3-hydroxyflavone)  flavone).  All  a n d many  photometry  species  1.7  separation  For  in water.  of  the  et  Another  compounds  a l . ^  The  organotins  reagents  tin  were  dithiol,  colorimetric hematoxylin,^'  form  violet,42,43  ( 3 , 3 ' , 4 ' , 5 ,7-pentahydroxycomplexes  compounds i s  the  lack  the  reagent  that  is  of  with  inorganic  morin  necessary before  specificity  fluorimetric for  used morin  and p h e n y l t i n  of  determination  determination  the of  of  has been a p p l i e d  spectrocolori-  organotins, triphenyltin  to  the  analysis  (2',3,U',5,7-pentahydroxyflavone).  as a l i g a n d  for  the  fluorimetric  analysis  of  compounds.  Electrochemistry  Polarography, titrations in  using diphenylcarbazone.  metals.  used 3-hydroxyflavone  alkyltin  the  and q u e r c e t i n ^  a n a l y s i s because of  organotin  Arakawa  of  colorimetric  reagents.  Vernon^  of  the  other  Efficient  metric  for  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  flavinol^  tin  dichloride  met'iod  of this method is in the microgram range. Other  sensitivity reagents  of  developed a spectrophotometric  have  anodic  stripping voltammetry^ • ^  been used f o r  the  aqueous and non aqueous m e d i a .  organotin r e c o r d e d . ^0  compound Tyurin  whose and  determination Diethyltin  have  n  c  \  also  potentiometric  o r g a n o t i n compounds  dichloride  polarographic  Flerov.-'l  of  a  reduction provided  was  the  first  behavior  was  data  the  on  14 polarographic  behavior  of  other  organotin  compounds.  The  ease of  reduction of organotin compounds i n polarography has been found to be function  of  a  the organic moiety on the t i n , the ease of reduction being  ethyl > propyl > butyl.^2  Polarography i n the d i f f e r e n t i a l  pulse  has also been used i n the determination of organotin compounds . Potentiometric  •  • --  > >  t i t r a t i o n i n non aqueous medium has also been used in  the analysis of organotin compounds.^•^ bis(tributyltin)  mode  oxide  reacted  with  In one water  of  to  these  produce  methods, tributyltin  hydroxide which was t i t r a t e d with hydrochloric a c i d . Although electrochemical methods are able to organotin  species  according  to  their  redox  suffers from interference by organic matter. water peak  coats  the  potentials.  electrodes, This  differentiate potentials,  between  this method  Organic matter present  in  causing broadening of peaks and s h i f t s i n  disadvantage  restricts  the  application  of  electrochemical techniques i n the analyses of natural water.  1.8  Atomic Spectrometry  Atomic  absorption  spectrometry has been applied extensively to the  determination of organotin compounds. available  on  atomic  emission  However, not much information  spectrometry  of  organotin  Either a f l a m e ^ or an e l e c t r i c a l l y heated  graphite  used  compounds  for  atomization.  Since  organotin  inorganic t i n by atomic spectrometry, mixture  of  some  form  of  is  compounds.  furnace^  can  be  are determined as separation  organotin compounds i s necessary before atomic  of  a  spectrometry.  -  McKie,^ tin,  achieved  and  chloric also  separation  inorganic  acid  treated  separated  partition  tin  of  -  t r i b u t y l t i n from d i b u t y l t i n ,  species  samples,  mixtures  before  of  15  by s o l v e n t - s o l v e n t  prior  to  atomic  analysis  by  metal  such  as  z i r c o n y l a c e t a t e has  tion  efficiency  of  tin. ^  ship  exists  of  organotin  the  energy  of  Emission  the  graphite  Peetre  6  atomic  compounds.  has  oxide. ^ is  by  shown t o 6  sensitivity that  solvent  refractory  increase that  atomiza-  a  relation-  and the  sensitivity  hydro-  absorption  with a  and S m i t h , ^ o b s e r v e d  structure  decreased  as  decreased. been  Again,  6  interior  been  absorption  a l k y l t i n bond  compounds  furnace  of  Kojima,^  furnace-atomic  They c o n c l u d e d  spectrography  bis(tributyltin) organotin  the  the  spectrometry.  graphite  Coating  between  extraction  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  spectrometry. salt  monobutyl-  necessary  applied prior  to  the  separation  before  the  determination of  a mixture  application  of  of of this  technique.  1.9  Gas C h r o m a t o g r a p h y  Organotin volatile organotin volatile  (GC)'  compounds  to  be  during separation. compounds, species  tetraalkyltin  are  of  the  usually applied. are  two  organotin  by gas  chromatography  determination  derivatization  compounds  Generation  For  separated  common  hydrides.  techniques  of to  Derivatization  the  less  convert to  must  be  volatile them  to  hydrides  or  procedures:  In  the  conversion  of  organotin  -  compounds convert  the  The and  to  be  the  tion  of  is  gas  the  reaction  of  reaction  alkyl  to  groups  determination  gas  to  the  release  to  R^SnH^^. inert  gas,  The t e m p e r a t u r e  the  organotin  Rearrangement  organotin  tetra-alkvltin  compounds  compounds.  compounds  to  the  derivatives Maguire et of  gas  of  sensitive  or  can  of  hydrides  disproportionaoccur  during  gas  with  a  is  ^ have  usually are  applied  by  the  Grignard reagent.  No r e a r r a n g e m e n t  formed i n g e n e r a l 6  accomplished  and t r i a l k y l t i n s p e c i e s  t i n nucleus  organotin  proceeds the  stable  in  procedure  after  to  original  observed.^  quite this  of  The  The organic  to  the  converting  them  derivatives.  detectors  detector,  chromatograph  usually  compounds  al.  Conversion of  t r i b u t y l t i n i n Canadian harbors,  chromatography,  spectrometry.  is  low c o n c e n t r a t i o n s .  attached  In  good s e n s i t i v i t y  is  s u c h as  Capture  Electron  flame photometric  Methylbutyltin species c o u p l e d to  M i l l e r , ^  determined  oysters  GC-MS.  by  formula  i n a cold trap.  column.  organotin  very  butylpentyltin  spectrometric  the  u s u a l l y used  p u r g e d from s o l u t i o n w i t h an  of monoalkyltin, d i a l k y l t i n  solvents.^>^  tion  of  of  is  analysis.^  the  tetraalkyltin  use  hydrides  increased,  tetraalkyltin  c o m p l e t i o n at  to  groups  Conversion to  are  -  excess borohydride  cryoscopically  usually  alkyl  compounds  to  chromatographic  chromatographic  ii.  hydrides  trapped  cold trap  onto  hydrides,  a l k y l t i n species  generated  can  the  volatile  16  the  usually achieved  detector, have  tin  and  (ECD),  atomic  the mass  absorp-  been d e t e c t e d by u s i n g  a mass s p e c t r o m e t e r ^ total  detector  by  (GC-MS).  a  Waldock and  and t r i b u t y l t i n i n sea  water  and  - 17 A flame p h o t o m e t r i c d e t e c t o r  f o r the a n a l y s e s o f o r g a n o t i n compounds  was d e v e l o p e d by Aue and F l i n n . ^ applied of  as  species  metric d e t e c t o r , ^ A  been  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  b u t y l t i n species  methyltin  Flame p h o t o m e t r i c d e t e c t i o n has  disadvantage  in water, ^  sediment,^  6  , d f o r the d e t e r m i n a t i o n of  in water.^  A l a t e r m o d i f i c a t i o n to the flame photo-  improved i t s  detection limit  of  the  flame  photometric  accumulate on i n t e r n a l s u r f a c e s sensitivity.  a r  In a d d i t i o n ,  photometric detector  to 5 x 1 0 " ^  detector  o f the d e t e c t o r ,  mol.  tin.  i s t h a t SnC>2 may  causing a  decrease  in  Maguire and T k a c z , ^ 5 r e p o r t e d t h a t the flame  can e a s i l y be ' p o i s o n e d '  sometimes used i n the e x t r a c t i o n  by  tropolone,  a  ligand  of o r g a n o t i n compounds.  C o u p l i n g the gas chromatograph to an atomic a b s o r p t i o n s p e c t r o p h o t o meter  (GC-AAS) appears  specific  detection.  determined a f t e r  to be the  most  Methyltin  species  hydride  derivatization  absorption spectrophotometry^ spectrophotometry.^ GC-graphite  furnace  r e p o r t e d by B a l l s . ^ 8 hydrides  with  the g r a p h i t e The hydrides.  the  popular  6  atomic  butyltin  absorption  method  carrier  by  natural  for  water  GC-quartz  element have b e e n  furnace  atomic  and G C - g r a p h i t e f u r n a c e atomic a b s o r p t i o n  Analyses of  The  in  technique  features  compounds  as  hydrides  spectrophotometry the  has  introduction  of  by  been the  gas stream i n t o the i n t e r n a l purge i n l e t of  furnace.  GC-AAS  technique  For the l e s s  species, condensation  works  well  volatile  with  hydrides  the v e r y  volatile  of b u t y l t i n  methyltin  and p h e n y l t i n  does occur on the r e l a t i v e l y c o o l i n n e r s u r f a c e s o f  the g r a p h i t e furnace assembly b e f o r e g e t t i n g i n t o the f u r n a c e .  18  Better separation i n gas  chromatography  of a mixture  by the use o f v a r i o u s  W o o l l i n s and C u l l e n , ^ o f o r g a n o t i n compounds Complete r e c o v e r y  to  be  employed  after  converting  them  for  complete  recovery  C l a r k and C r a i g ® ^ e f f e c t e d  has been a c c o m p l i s h e d by E s t e s et  L i q u i d Chromatography  preparation analyses  chromatography of v o l a t i l e  detection  liquid  volatile  forms  of  the  and  sparging  t r i b u t y l t i n and  on-column h y d r i d e g e n e r a t i o n  al.  column.  plasma  A n a l y s e s of  emission  of  tetra-  spectrometer  ft 1  (LC)  of  o r g a n o t i n compounds does not r e q u i r e  species  and  hence  could  o f l i q u i d chromatographic  of organotins.  chromatograph  spectrometer  be  useful  d e t e c t o r s are  For h i g h s e n s i t i v i t y ,  (HPLC)  can  be  coupled  a  for  the the  employed f o r  high  performance  to an atomic  absorption  (AAS) or a mass spectrometer (MS).  D i r e c t c o u p l i n g o f the LC system to a d e t e c t o r system mass  hydrides.  of n o n - v o l a t i l e or h i g h m o l e c u l a r weight o r g a n o t i n compounds.  Various the  to  in solution.  a l k y l t i n compounds by c o u p l i n g a GC to a  Liquid  columns.  h y d r i d e s c o u l d not be a c c o m p l i s h e d by  o r g a n o t i n compounds on a gas chromatographic  1.10  chromatographic  A combination of ether e x t r a c t i o n ,  t r i p h e n y l t i n h y d r i d e s generated Recently,  gas  achieved  employed a c a p i l l a r y column to s e p a r a t e a mixture  o f the generated  helium sparging alone. had  o f o r g a n o t i n h y d r i d e s can be  such  spectrometer  or  the atomic a b s o r p t i o n s p e c t r o m e t e r i s  w i t h problems such  as  solvent  interferences.  The  large  as  the  associated amount  of  19  solvent  that  The by  column.  HPLC h a s  The  the  the  large  detector solvent  LC and the  shown t o  atomic  be  absorption  is  also  inflow into  the  system  flow rate  compatible  compounds  being separated  system  detector  solvent  absorption  organotin  atomic  the  The s m a l l  been  a flame  after  into  problem of  interfacing  bore  to  goes  -  or  a major  concern.  detector by  (10-100  the  c a n be  use  of  a  /iL m i n " )  in  microbore  effluent  introduction  1  with direct  solved micro-  spectrophotometer. can  by l i q u i d  also  be  derivatized  chromatography,  spectrometer.  This  to  volatile  and t h e n  method has  hydrides  introduced  into  been  a p p l i e d to  the  organotin  compounds  is  ft determination Most  work  performed modified  of methyltin  on  on  liquid  reversed  columns  such  as  separated  alkyltin halides  a l . ,  D  have  J  various based  strongly  halides,  black  inert  towards  Rearrangement in  chromatography, organotin  but  of GC  technique  columns.  silica  gel gel  columns.  are  of  columns.  Langseth,^  column.  alkyltin  result not  other  Organotin  which employed m o r i n .  Their  columns  and  He u s e d Jessen  compounds  indicated  that  sufficiently  on  silica  inert  columns p y r o l y t i c a l l y c o a t e d w i t h  et  to  carbon  alkyltin halides.  alkyl  groups  analysis,  especially  compounds.  silica  columns,  on c y a n o p r o p y l - b o n d e d s i l i c a  (ODS) a n d c y a n o  alkyltin  encountered  bonded  exclusion  adsorption behavior  chromatographic  octadecyl  are  the  size  of  on u n m o d i f i e d s i l i c a  derivatization  studied  columns,  cyano  adsorb  on-column  chromatography  phase  compounds  an  species.  if  is  on  tin,  which  sometimes  tetraalkyltin  is  also  is  often  a  encountered  present  in a  problem in  liquid  mixture  of  - 20 O B J E C T I V E S OF THE PRESENT  The  known  alkyltin These cut  tions,  compounds  point  organotin types  of  In  compounds  therefore  detection, detector  involve  organotin  the  index  the  the  used  in  easily  in  is  have  to  chromatographic UV  the  separa-  detection.  been p r e v i o u s l y Hence,  to  the  develop  derivatives  marine  close  applied  need  for  for  other  analysis.  was  of v o l a t i l e  simple  species).  1  to  low.  are  carbonato ^  r  liquid  detectors  environments  UV r e g i o n b e l o w o r  in quantitative  the  o  amenable  main o b j e c t i v e  formation  compounds  i n the  sensitivity  systems  study  absorb  not  i n aqueous  hydroxides,33  solvents  refractive  this  compounds  (oxides,33  o f most  and are  Although  not  organotin  alkyltin  off  STUDY  a method w h i c h  for  environment.  the  does  analysis  of  Two s t r a t e g i e s  were  investigated: i.  HPLC  separation  of  derivatized  organotin  complexes  coupled  with  UV  detection ii.  HPLC atomic  separation absorption  The  approach  i.  Synthesis  to  of  or  derivatized mass  achieve  complexes  dibutyltin  and  extinction  coefficient  analysis ii.  taken  of  Separation organisms  of  objective  of  tributyltin,  by  which  (tropolone  coupled with  of  this and  contain and  study  as  its  ligands  oxine),  is  follows:  metabolites of high  thereby  molar making  possible. and  chromatography,  LC-MS.  chlorides  detection.  the  tributyltin  by l i q u i d  by L C - G F A A S o r  spectrometric  monobutyltin,  b y UV d e t e c t i o n  organotin  its and  metabolites the  present  quantification  in of  marine them  -  21  -  CHAPTER 2 EXPERIMENTAL  2.1  Instrumentation  2.1.1  Infrared  Infrared  Spectroscopy  spectral  data  b y u s i n g a P e r k i n - E l m e r 598 as  nujol  mulls  calibrated The  relative  infrared  bromide)  2.1.2  The  except  cell  purchased  was  otherwise  polystyrene a  i n the  spectrophotometer.  where  the  recorded  KRS-5  stated.  bands  (58%  at  and molar  Melting  to  1601  200  cm"  were  run  spectra  c m " ^ a n d 907 iodide,  42%  1  were cm' . 1  thallium  f r o m Harshaw C h e m i c a l Company.  a b s o r p t i o n wavelengths from data  Melting  4000  The  thallium  Spectrophotometry  spectrophotometer  range  T h e compounds  U l t r a v i o l e t Absorption  determined  2.1.3  to  were  extinction  coefficients  o b t a i n e d on a P e r k i n - E l m e r C o l e m a n 124  o p e r a t i n g between  200  nm a n d 400  were  d o u b l e beam  nm.  Point Determination  points  were  determined  Gallenkamp m e l t i n g p o i n t apparatus,  in  open  and were  capillaries  uncorrected.  using  a  - 22 2.1.4  Nuclear Magnetic Resonance Spectroscopy (NMR)  ^"H  NMR spectra  were obtained using a Varian XL-300  operating at 300 MHz.  Chemical s h i f t s were measured r e l a t i v e to  methylsilane as external  2.1.5  spectrometer tetra-  reference.  High Performance Liquid Chromatography (HPLO  The M-510  HPLC  system  pumps,  injections  and  were  Chromophoric Lambda-Max  an  groups LC  Q A - 1 ^ data system. was  of  automated  manually  481  photometer  consisted  were  made  Waters Associates' models M-45 and  gradient  controller.  phase,  detected  by  using  graphite  particle  column  furnace  to  (/j-Bondapak  the  Associates'  atomic  absorption  3.9  spectro-  The chromatographic  was  a  C^g  reversed-  mm (ID) x 30 cm) with a  The column packing material was  size of 10 microns.  c o l l e c t e d with the a i d of a manually  Waters  used as the t i n s p e c i f i c detector.  Waters Associates guard column. with  a  spectrophotometer connected to a Waters Associates' A  steel  sample  v i a a Waters Associates' U6K i n j e c t o r .  column used for a l l HPLC and HPLC-MS separations bonded  All  Gilson  silica  Effluents from the HPLC column were microfractionator  and  transferred  automatic sample delivery system of the graphite tube  atomizer.  2.1.6  Low Resolution Mass Spectrometry  Low r e s o l u t i o n mass spectra using electron  impact  ionization  were  23  obtained  -  from a K r a t o s MS50 mass s p e c t r o m e t e r .  Mass s p e c t r a  atom bombardment (FAB) were o b t a i n e d from A . E . I .  2.1.7  fast  spectrometer.  Graphite Furnace Atomic Absorption Spectrophotometry  The apparatus f o r graphite furnace atomic metry  MS9 mass  using  consisted  of  d e u t e r i u m background  a  Varian  1275  analytical  line  spectrophotometer graphite  tubes  Hamamatsu was  spectrophotometer  printer.  Photonics  used  for  was o p e r a t e d at  of  all a  Japan.  nm  tube  atomizer  The t i n h o l l o w cathode  analyses. 1  spectrophoto-  equipped w i t h a  corrector, a Varian GTA-95 g r a p h i t e  and a H e w l e t t - P a c k a r d model 82905A  was s u p p l i e d by  absorption  The  224.61  lamp  nm t i n  The atomic a b s o r p t i o n  spectral  slit  width.  used were V a r i a n T e c h t r o n p y r o l y t i c a l l y c o a t e d  The  graphite  tubes.  2.1.8  H i g h Performance L i q u i d Chromatography - Mass Spectrometry (HPLC-MS)  The mass spectrometer spectrometer  with  the  2.1.5.  thermospray above.  MS80  RFA  mass  a Vestec Kratos thermospray i n t e r f a c e between the HPLC  and the mass s p e c t r o m e t e r of  used f o r HPLC-MS was a K r a t o s  (see Chapter 3, F i g . 3 . 8 . 2  interface).  The  HPLC  f o r the d e s c r i p t i o n  system i s as d e s c r i b e d i n  -  2.1.9  The  Extraction  a n d Sample  centrifuge  employed d u r i n g  organisms  was  centrifuge  operated  A Buchi  a  Sorvall at  2.2  rotary  e x c e p t where  and  chloride  inorganics)  Tropolone  Aldrich was  (Alfa  from  grade).  trisodium  citrate,  D-(+)-glucose.  used  (Scientific  Beverly,  Scientific The  following  L-(+)-ascorbic  used  for  and cyclohexane  are stated  marine  stated.  for  The v o r t e x  Industries  and d i b u t y l t i n  A l l r e a g e n t s were  solvents  Recovery  used  the  refrigerated  otherwise  all  mixer  solvent u s e d was  I n c . Bohemia,  was a n E p p e n d o r f R e p e a t e r  N.Y.,  4780.  d i c h l o r i d e were  Massachusetts.  U.S.A.  BDH C h e m i c a l s .  (Fisher  (reagent  methanol  was  stated.  of  automatic  where  studies  certified  acid,  ACS  used without  grade).  from  trichloride  purchased  from  (8-hydroxyquinoline) u s e d were  grade),  were  citric  were  were  reagents  reagents  synthesis  (reagent  Oxine  Other  purchased  Butyltin  (2-hydroxy-2,4,6-cycloheptatrienone)  purchased  The  evaporator  C h e m i c a l Company, M i l w a u k e e ,  bicarbonate  2-B  step  and Reagents  Tributyltin Ventron  RC  e x c e p t where  otherwise  and the m i c r o p i p e t t e  Materials  the e x t r a c t i o n  Superspeed  a V o r t e x - G e n i e model K-550-G U.S.A.),  Preparation  2500 r . p . m . ,  Rotavapor-R  evaporations,  24  a l l acid,  sodium  acetic reagent  acid grade:  tartaric  acid,  further purification.  ethanol  The grades  (laboratory of other  grade), solvents  appropriate. were  performed on standard  dog f i s h  liver  DOLT-1,  -  supplied by National Research elemental All  25  Council,  Canada  (see Appendix  A  for  the  composition of DOLT-1). solvents  used  as  mobile  phases  filtered  through  stated  otherwise,  a n d were  prior  to  Deaeration  use.  w e r e HPLC g r a d e  except  a m i l i p o r e 0.5  o f the m o b i l e phase  FH  where filter  was a c h i e v e d b y v a c u u m  filtration.  2.3  Methodology  The  organotin  chloride well  compounds w e r e  complexes  characterized  •  A l l retention  standard  Background c o r r e c t i o n when a t o m i c  Synthesis  2.4  2.4.1  of methanol mol.)  o f the  organotin  d a t a were  tropolonate  established  or  using  compounds.  was e m p l o y e d t o  spectrophotometric  remove detection  molecular  absorption  was u s e d .  Oxinates  Sodium O x i n a t e  Sodium  metal  mL  (1.00  g,  0 . 0 4 3 m o l . ) was g r a d u a l l y  to form sodium methoxide  dissolved  solution. 100  absorption  s t u d i e d as o x i n a t e ,  in  The m i x t u r e round  bottom  i n solution.  warm c y c l o h e x a n e was s t i r r e d flask.  d i s s o l v e d i n 30 mL  Oxine  (6.32  g,  was a d d e d t o t h e s o d i u m  f o r 15 m i n a n d t h e n  The f i l t r a t e  methoxide  filtered  was c o n c e n t r a t e d  0.044  at  into  a  reduced  -  pressure  on the r o t a r y  isolated  by  filtration.  spectrometry  2.4.2  4 h. to  The sodium o x i n a t e  (0.90  g,  0.0054  i n a 250 mL r o u n d b o t t o m  mol.) dissolved  solution  give  viscous  a yellow glassy  elemental  analysis,  4,  4. l",  section  2.4.3  Butyltin  0.0036  The  resulting  (Chapter  2.4.4  4,  flask.  (FAB).  dissolved  Dibutyltin was  The v i s c o u s  added  4.1.7,  in  50  dichloride to  the  was r e f l u x e d w i t h under  mL  of  (0.82  g,  methanolic stirring for  reduced  pressure  o i l was d r i e d u n d e r v a c u u m  The p r e c i p i t a t e  spectrometry  4.1.2,  (1.82  m o l . ) were product  section  Tributyltin  Tributyltin  b y mass  and n . m . r .  was c h a r a c t e r i z e d spectroscopy  to by  (Chapter  Fig. 4.1.2).  Trisoxinate  Sodium o x i n a t e g,  o i l .  mass  was  and c o n c e n t r a t e d  precipitate.  tables  mol.)  The m i x t u r e  T h e s o l u t i o n was f i l t e r e d a very  w h i c h was  was c h a r a c t e r i z e d  atom bombardment  i n 50 mL m e t h a n o l  o f sodium o x i n a t e .  give  a yellow precipitate  Bisoxinate  Sodium o x i n a t e  0.0027  to give  ( M / Z — 167) , u s i n g f a s t  Dibutyltin  methanol  evaporator  26  g,  0.011  treated was  4.1,  m o l . ) and b u t y l t i n  as i n 2 . 4 . 2  characterized  Tables  4.1.2,  above, to  4.1.6,  be  trichloride  and r e f l u x e d butyltin  (1.02  f o r 24 h . trisoxinate  Appendix B, F i g . B - 4 ) .  Oxinate  oxinate  was  synthesized  according  to  t h e method  of  - 27 Kawakami et a l . mass  The product was characterized by elemental  spectrometry  and n.m.r.  spectroscopy  analysis,  (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 water  and the resultant  solution added to sodium bicarbonate (3.4 g,  0.04 mol) in 20 mL of deionized water occurred immediately with  gentle  warming  on a  through a Whatman No. 1 petroleum  ether.  temperature.  at  effervescence,  After cessation of effervescence, by  deionized  The  washed  temperature.  producing  a  Reaction  yellow s o l i d .  the reaction mixture was concentrated  hot p l a t e . filter  room  paper, solid  The s o l i d product was f i l t e r e d and then product  washed  with  cold  at  room  was dried  The product was.characterized by mass  spectrometry (FAB)  to be sodium tropolonate (M/Z - 144).  2.5.3  A  T r i b u t y l t i n Tropolonate  sodium  tropolonate (0.38 g, 0.0026 mol.) slurry i n 50 mL ethanol  was mixed with tributyltin chloride (0.81 g, 0.0025 mol.) dissolved 50  mL ethanol, in a 250 mL round bottom flask.  for 1 h at room temperature.  At the end of 1 h,  in  The mixture was s t i r r e d the  reaction  mixture  -  was  filtered  chloride  formed.  evaporator was  kept  The  through  to  precipitate  Identification ^-H n . m . r . B,  was  colored  under vacuum,  was  paper,  concentrated  viscous  and a pale  product  spectroscopy  4,  Section  precipitate  4.1,  a  rotary  The v i s c o u s  liquid  was o b t a i n e d .  and d r i e d under  was a c c o m p l i s h e d by mass  (Chapter  the sodium  using  liquid.  washed w i t h c o l d methanol  of this  t o remove  vacuum.  spectrometry  Table 4.1.5  and  and Appendix  F i g . B-2).  2.5.4  Butyltin  Butyltin Komura The  filtrate  orange  f o r two d a y s  -  a Whatman N o . 1 f i l t e r  Th.-  give  28  a n d mass  2.6  tristropolonate  e_t a l . , ^  solid  Tristropolonate  except  product  was s y n t h e s i z e d  that  the r e a c t i o n  mixture  was i d e n t i f i e d b y e l e m e n t a l  spectrometry  (Chapter  4,  Section  Coefficients  placed  f o r the  amounts  solutions  in  extinction  coefficients  acetone Tables  in 4.2.1  the  the  butyltin  flasks  concentration  and  Tables  f o r 48 h .  melting  4.1.1,  and M o l a r  oxinates made  range  were d e t e r m i n e d  wavelength  and 4 . 2 . 2 ) .  was r e f l u x e d  of  point  4.1.3).  Extinction  Complexes  of  i n 15 mL v o l u m e t r i c  t o t h e method  analysis,  4.1,  Determination of A b s o r p t i o n Wavelength  Appropriate  according  region  1  up  and to  x 10"^  i n methanol,  tropolonates  the -  mark  to  1 x 10"^ M. acetonitrile,  2 0 0 - 4 0 0 nm ( C h a p t e r  4,  Section  were form Molar and 4.1,  29  CHAPTER 3 SEPARATION AND DETECTION PROCEDURE  3.1  HPLC-GFAAS  3.1.1  Introduction  Consideration Performance  of  Liquid  equation  is  the  packing  column p l a t e particles,  dispersion  CU  is  a  column. mobile  mass  phase  that  packing  B  /u-  packing The  to  the  basic  ideas  of  High  A=  is  the  diffusion. A,  mass is  /u  c  B,  dp i s  the  constant.  the  and  C  diameter A,  m o b i l e phase are  between  at  high  a  large  plate  height.  takes  place  in  stationary  the  1  where  U is  and,  u  packing p a r t i c l e s .  transfer  large  +  packing  between  of  B  2Xdp,  a  term.  c  +  3 3  B/U is  CU zone  for  is  a  a  the the term  velocity,  stationary  m o b i l e phase  8 7  reflects  constants the  of  and  given  and  the  velocity  will  effectively  the  inside  the  pores  of  particles.  Performace term  slow,  causing  dispersion the  is  rate  0  X  spaces  transfer the  AU '  height,  longitudinal  If  dominate  -  and  from the  representing  leads  Chromatography.  H H,  1  can be  This  i n c r e a s e d by r e d u c i n g the  is  achieved  in  the  by u s i n g  a small  longitudinal  diffusion  column and s m a l l  sized  particles. reduction  decreased  permeability,  analysis.  So,  instead  of  size  and a l s o allowing  of  the  leads the  to  packing  particles  r e d u c t i o n i n the  m o b i l e phase  to  flow  leads  to  speed of  the  through  the  30  column phase  by is  diffusion  forced  through  Furthermore, be  improved  a c h i e v e d by the  A is  the  by  This  schematic  shown i n  operated eluted  Fig.  in  is  the  ordinary  of  reduction the  column chromatography,  c o l u m n b y means a liquid in  distance  the  of  3.1.1.  Here,  graphite  the  through  atomic  furnace  chromatographic  the  the  term CU.  stationary  system employed  is  mobile  system  analyte  absorption  mode  the  pumps.  which  HPLC-GFAAS the  of  mass t r a n s f e r  a c c o m p l i s h e d by making  diagram  the  organotin  in  efficiency  the  reducing  column.  as  -  used for  can  This  travels phase in  this  is in  thin. study  spectrophotometer the  detection  of  compounds.  AAS UV  DETECTOR  FRACTION COLLECTOR  SOLVENT RESERVOIR  C  1  8  GRADIENT CONTROLLER  COLUMN  L  0  0  P  RESTRICTOR  INJECTOR  Fig.  3.1.1:  Schematic  PUMPS  set  up o f  the  HPLC-GFAAS  system  31  The  chromatographic  bonded  phase,  reversed  phase  column chosen  prepacked when  the  stationary  phase  study,  the  stationary  silica  packing material  column.  for A  stationary  is polar,  of p r e v e n t i n g the  -  to  study  phase  is  is  to  reversed  column i s  This  termed  When  the  In  this  to  the  as n o r m a l .  chemically  form o c t a d e c y l s i l a n e . phase  a  non - p o l a r .  referred  octadecane  stationary  is  chromatographic  the column i s  phase  this  bonded has the  advantage  f r o m b e i n g w a s h e d away b y t h e  mobile  phase. After collected  elution  from  on a f r a c t i o n  transferred  to  the  collector  the atomic  of  light  at  absorption  The p r i n c i p l e o f a t o m i c measurement  column,  fractions 0.5  min  of  the m o b i l e phase  intervals  and  are  manually  spectrophotometer.  absorption spectrophotometry  absorbed by ground s t a t e  atoms.  i s based  on the  The a b s o r p t i o n  of  oo light  by  ground state  and c a n be u s e d f o r  atoms  follows Beer-Lambert's  quantitation. *A  P ^ 0  is  t h e power o f t h e s o u r c e  radiation  at  absorption  coefficient,  is  wavelength  the c o n c e n t r a t i o n Usually,  photometry  has  temperatures Moreso,  the  refractory  the  -  PoA e '  at  wavelength  A after  1 is  o f the atomic  graphite the  of  i n s i d e o f the graphite oxides  to the  A  2  l c  A,  P^  is  the  power  of  is  the  through the sample,  length through  the sample  and  c,  vapor.  furnace  advantage  K  passage  the path  than are a c c e s s i b l e  metal  law ( e q u a t i o n 2 ) , °  mode o f a t o m i c reaching  far  absorption higher  i n flame a b s o r p t i o n tube  metals.  has  the  spectro-  atomization  spectrophotometry.  ability  to  reduce  some  - 32 In the graphite furnace mode, the steps involved are as follows:  During furnace. are  1.  Sample drying  2.  Sample ashing  3.  Sample atomization  4.  Tube cleaning  the  drying stage, solvent i s removed from the sample i n the  At the ashing stage, organic molecules or inorganic  removed.  materials  At the atomization stage, free atoms are generated within  a confined zone. During excessive  operation,  the  incandescent  is  protected  from  corrosion by an upward flow of inert gas (argon or nitrogen).  In this study, argon was the inert gas. away  graphite  The inert gas flow also  any ashing product from the l i g h t path.  sweeps  A diagram of the graphite  tube atomizer with a graphite tube in place i s shown in F i g . 3.1.2.  3.2  Optimization of GFAAS Conditions  The detection c a p a b i l i t y of the atomic absorption  spectrophotometer  was optimized using t r i b u t y l t i n chloride solutions i n acetone. achieved by varying the atomization temperatures, ashing temperature The  graphite  constant, furnace  shown i n Table 3.2.1.  This was  while maintaining  the  and vice versa. operating  parameters used i n this study are  - 33  F i g . 3.1.2:  -  The graphite tube atomizer  34  Table  3.2.1  Graphite  Step No.  Temperature °C  1  f u r n a c e o p e r a t i n g parameters  Time Sec.  Gas Flow  Gas Type  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  Read Command  10  *  3.3  steps a t which a b s o r p t i o n  was measured  S e l e c t i o n of Chemical Modifiers  In s e a r c h o f c h e m i c a l m o d i f i e r s tributyltin  chloride  and  to  dibutyltin  be  used  in  dichloride,  the 0.5%  analysis w/v  of  aqueous  s o l u t i o n s o f the f o l l o w i n g compounds i n d e i o n i z e d water were examined.  35  Each  of  i  trisodium c i t r a t e  ii  L- (,+)-ascorbic acid  iii  c i t r i c acid  iv  t a r t a r i c acid  v  D-(+)-glucose  the  above  chloride or d i b u t y l t i n delivery  system  of  compounds dichloride  the  (10 /iL) were mixed with t r i b u t y l t i n (10  fiL)  by  the  automatic  graphite tube atomizer and analyzed using t h e  graphite furnace parameters shown i n Table 3.2.1 above. these  compounds  on  sample  The  effect  of  the absorbances of Sn, i n t r i b u t y l t i n chloride a n d  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 s . 4.4.1 and 4.4.2.  3.4  Establishment of Retention Data  3.4.1  B u t y l t i n Oxinates  Solutions butyltin  of  tributyltin  trisoxinate  dibutyltin  bisoxinate,  well  characterized  standard  compounds  HPLC.  of  all  the  retention  Then,  a  standard b u t y l t i n oxinates were injected into the  Each chromatographic peak was i d e n t i f i e d  individual  in  Each of the standard solution (15 ^L) was injected into the  HPLC, and the retention times were determined by UV detection. mixture  and  corresponding to 60 jig/mL were made by dissolving  appropriate amounts of the chloroform.  oxinate,  on  times previously established.  the  basis  of  the  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 d i b u t y l t i n  bisoxinate either under i s o c r a t i c or gradient e l u t i o n conditions. a  gradient  elution  of 95% ethyl acetate : 5% methanol f o r 4.5 minutes  followed by a change trisoxinate  Using  to  20%  ethyl  acetate : 80% methanol,  butyltin  was completely separated from either t r i b u t y l t i n oxinate or  d i b u t y l t i n bisoxinate on the C^g reversed phase column (Chapter 4,  Fig.  4.5.2). No  attempt  was made to effect the HPLC separation of the b u t y l t i n  tropolonates because t r i b u t y l t i n tropolonate could not be obtained i n a pure  form,  and was  therefore not suitable as an a n a l y t i c a l standard.  However, a HPLC-MS study of the b u t y l t i n tropolonates was undertaken  to  obtain their fragmentation behavior (Chapter 4, Table 4.11.1).  3.4.2  B u t y l t i n Chlorides  The  retention  detection with (GFAAS).  data  graphite  for the b u t y l t i n chlorides were established by furnace  atomic  absorption  A s o l u t i o n of d i b u t y l t i n dichloride (60 pg/mL) and t r i b u t y l t i n  chloride (60 /ig/mL) was prepared by d i s s o l v i n g the of  standard  compounds  in  acetone.  was  collected  by  appropriate  amounts  Solutions of each of the standard  compounds (20 pL) were injected into the HPLC. HPLC  spectrophotometry  The e f f l u e n t  a fraction collector.  from  the  The c o l l e c t e d fractions  (0.5 min i n t e r v a l ) were then transferred to the GFAAS. The mobile phase used acetone) : 2%  for  tetrahydrofuran.  elution  was  98%  (2%  acetic  acid  in  Under the conditions of the e l u t i o n , i t  37  was not  possible  dibutyltin  dichloride.  (Chapter  Stock  acetone,  to  three d i f f e r e n t dibutyltin  prepared  test  detection  of  and Recovery  of  from  dibutyltin  by  dissolving  stock s o l u t i o n s (100  mL)  mL)  of  to 5-15 tubes.  and  (10  the  Studies  tributyltin  compounds i n a c e t o n e ,  chloride  the  appropriate  i n a volumetric (10  flask  (100  mL) were p l a c e d i n  two  made  up  to the mark w i t h  pg/mL).  working  solution  of  tributyltin in  Aliquots  of  corresponding  added i n each o f the  t r i b u t y l t i n chloride,  to  5-15  ^g  t e s t tubes  such t h a t each t e s t tube  already  contained  same amount 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 .  acetone s o l u t i o n s mL,  i n the t e s t tubes were evaporated  by g e n t l e warming i n a water b a t h .  g) was added i n t o  each  and  acid  mL) and sodium c h l o r i d e  followed  mixed  the  mixtures (0.5  vortex  of  and  /ig o f t r i b u t y l t i n c h l o r i d e were p l a c e d  d i c h l o r i d e were a l s o  c o n t a i n i n g the  the  each  form the working s o l u t i o n  c h l o r i d e corresponding  trichloride  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 an  with  /ig/mL)  flasks  (0.5-1.5  butyltin  4.5.1).  were  standard  volumetric  Aliquots  0.2  Fig.  Amounts o f each o f the  separate  the  interfere  (100  dichloride  o f the  any  o f A n a l y t i c a l Procedure  solutions  dibutyltin  mL).  4,  separate  Hence,  sample w i l l  Establishment  amounts  completely  dichloride.  environmental  3.5  to  -  test  tubes  f o r one m i n u t e . (0.10  and reduced to  Standard d o g f i s h l i v e r containing Concentrated  the  about (0.1  butyltin  hydrochloric  g) were added to each t e s t  by the a d d i t i o n o f d i s t i l l e d water (1 mL).  The  tube,  The s o l u t i o n s were  38  vortex  mixed a g a i n ,  (3  mL)  was  and  left  to  mixed the  for  test  contents  of  the  HPLC  tube,  was  was  tube,  each  test  (see  shelled the  tube  The hexane  C h a p t e r A, s e c t i o n  organisms  and a l l o w e d  prior  to  tissues  hydrochloric  and  4.6  of  tube.  chloride  chloride  The  extracts  by  gentle  s o l u t i o n was to  (25 p L )  then  dryness.  reconstituted  solution for  centrifuging,  concentrated  evaporated were  vortex  The  i n 50 ^ L  was  of  injected  results).  Tributyltin  and D i b u t y l t i n  from  Organisms  marine  stored  and  further  test  methylene  The m e t h y l e n e  a V-shaped test V-shaped  and the  minutes  After  another  chloride  two more  were  5 minutes.  removed i n t o  together,  a water bath.  Methylene  solutions  for  repeated,  pooled  mixture.  vortex mixed f o r  The  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  The  both  layer  using a micropipette.  Marine  been  test  and c e n t r i f u g e d  chloride  on  into  3.6  each  tube were  filtered  into  f o r m a homogeneous  15 m i n u t e s .  procedure  evaporation  hexane  for  1 minute,  methylene  each  into  stand  extraction of  put  to  -  the  removed from the  thaw.  extraction  and the  acid.  to  were  step.  Oysters  and  freezer, other  The e x t r a c t i o n  was  where  they  bivalves carried  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 the  had were  out  shells  on in  39  Extraction  3.6.1  Portions placed  Tissues  tissues  w e i g h i n g between  to  acid  were  s l u r r y was  w h i c h 20 g s o d i u m c h l o r i d e added.  on  a  weight)  and homogenized  transferred  ( 1 0 0 mL) was  The t i s s u e  mechanical  (wet  to  a  a n d 50 mL c o n c e n t r a t e d  Methylene c h l o r i d e  and mixed by s h a k i n g .  minutes  34-220 g  t o g e t h e r w i t h 100 mL w a t e r ,  The h o m o g e n i z e d t i s s u e  flask  30  the  i n a blender,  minutes.  slurry  of  from  shaker,  s l u r r y was  1 L  for  3  conical  hydrochloric  added to further  and c e n t r i f u g e d  were  the  tissue  shaken  for  for  20 m i n u t e s  was  removed.  at  r.p.m.  2500  After  centrifugation,  remaining chloride rotary some  aqueous layers  solution  flask,  effected  of  with  a  tetrahydrofuran. tributyltin  3.6.2  The behind.  the  Fractions and  was  the  into  of  the  twice  of  more.  evaporated  to  reconstituted  s o l u t i o n was  the  The  The  methylene  dryness  i n hexane,  filtered  mark w i t h more  into  on  a  leaving a 50 mL  hexane.  The  hexane  the  extract  HPLC.  98%  in  (2% a c e t i c  corresponding  to  the  acid  i n acetone)  retention  dibutyltin dichloride previously  compounds w e r e c o l l e c t e d  portions  layer  compounds p r e s e n t  phase  O r g a n o t i n Compounds i n  Various  and  The hexane  organotin  mobile  chloride  re-extracted  residue  injected  chloride  standard  was  a n d made up t o  ( 5 0 /JL) was  Separation  methylene  were p o o l e d t o g e t h e r  precipitates  using  layer  evaporator.  volumetric  the  and d e t e c t e d by  times  was :  2% of  established  GFAAS.  Shells  dry s h e l l s  weighing  between  12-36  g  were  - 40 crushed •»7as  with  a mortar and pestle into fine powder.  transferred  concentrated  to  a  250  and  dissolved  in  mL  flask,  and  Fractions  of  After  the solution was transferred into a 100 mL  made  up to the mark with deionized water.  hydrochloric acid solution of the s h e l l s (50 pL) was injected HPLC.  50  hydrochloric a c i d , by gradual addition of the a c i d .  cessation of effervescence, volumetric  mL beaker,  The powdered s h e l l  corresponding  to  into  The the  the retention times of t r i b u t y l t i n  chloride and d i b u t y l t i n dichloride were c o l l e c t e d and analyzed by GFAAS.  3.7  Quantitation  3.7.1  Quantitation of T r i b u t y l t i n Chloride and D i b u t y l t i n Dichloride for Recovery Studies  At the 5-15 by for  a  ug l e v e l of study, t r i b u t y l t i n chloride was  normal c a l i b r a t i o n graph ( F i g . 4 . 6 . 1 ) . normal  appropriate  calibration amounts  s o l u t i o n (100 ng/mL). placed  into  were  obtained  were  quantitated  Solutions from which data prepared  by  dissolving  of t r i b u t y l t i n chloride i n acetone to form a stock Aliquots of the stock  volumetric  flasks  (100  solution  (2-10  mL)  were  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  dibutyltin  dichloride  was  quantitated by a d i f f e r e n t 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  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 o f g 'aph was  obtained  -  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 s t a n d a r d  was a c c o m p l i s h e d by u s i n g a s t a n d a r d s o l u t i o n o f (2  ng/mL)  in  acetone,  which  was  dispensed  standard  a d d i t i o n program on the GTA-95 g r a p h i t e  3.7.1).  Volumes o f the s t a n d a r d  HPLC  fraction  injected  A typical  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,  Table 3 . 7 . 1 :  Sampler parameters f o r s t a n d a r d dibutyltin  Solution  dichloride  by  2  u s i n g the  tube  automated  atomizer  (Table  (2-18 pL) c o r r e s the  appropriate  T h i s mixture was then standard  addition  plot  F i g . 4.6.2.  a d d i t i o n p l o t of  dichloride  Standard Volume (^L)  Blank Addition 1  dibutyltin  (10 pL) and the m o d i f i e r (10 ^ L ) .  i n t o the GFAAS and a n a l y z e d .  standard  calibration  ug were mixed w i t h  1  by  addition  dibutyltin dichloride  ponding to 1.2 x l O " ^ - 1.08 x 1 0 "  of d i b u t y l t i n  Sample Volume (pL)  Blank Volume (pL)  Modifier Volume (^L)  -  30  10  10  18  10  Addition 2  6  10  14  10  Addition 3  10  10  10  10  Addition 4  14  10  6  10  Addition 5  18  10  2  10  -  3.7.2  Quantitation in  Both marine  modifier without  was the  3.7.2:  and D i b u t y l t i n  chloride  used,  and  dibutyltin  dichloride  q u a n t i t a t e d by the standard  program  used  because  i s shown  i n this  in  case,  in  addition  method.  3.7.2.  No  chemical  was  obtained  Table better  present  linearity  The  modifier.  GTA-95 G r a p h i t e Addition  Solution  plot  Tube A t o m i z e r  of Butyltin  Standard Volume  Sample Volume  Blank  Program f o r  Compounds  -  Blank Volume (ML)  -  20  10  9  Addition  1  Addition  2  3  10  7  Addition  3  5  10  5  Addition  4  7  10  3  Addition  5  9  10  1  1  -  1  0  Standard  i n Marine  (/iL)  (ML)  Sample  Dichloride  Organisms  tributyltin  addition  -  of T r i b u t y l t i n Chloride  o r g a n i s m s were  standard  Table  Marine  42  10  Organisms  Modifier Volume (MD  -  For  the  tributyltin  quantitation chloride  A standard as  Sn)  standard  3.8  (0.74  was  a standard  quantitation  of  The s t a n d a r d  a d d i t i o n was  effected  a d d i t i o n program of  the  GTA-95  Liquid  solution  of  used.  d i b u t y l t i n d i c h l o r i d e i n acetone  " Combined H i g h Performance  3.8.1  Sn)  the  Spectrometry  (0.79  ^g/mL  dibutyltin dichloride in by  graphite  using tube  the  marine  automated  atomizer.  Chromatography and Mass  (HPLC-MS)  Introduction  HPLC-MS  is  an o n - l i n e  combination u t i l i z e s chromatography  and the  also  the  decreases  involved  i n the  HPLC-MS has  the  multicomponent derivatization  the  effluent expected  into of  instrumental efficient  specificity  analysis  of  samples  advantage  of  mixtures,  and  of  a mass  spectrometric  interventions  which  of  liquid  detector. are  It  otherwise  b y s e p a r a t e HPLC a n d MS t e c h n i q u e s .  convenience in  capability  This  most  and  cases  speed does  of  analysis  not  require  to  admit  of  chemical  samples.  the  an i n t e r f a c e  mass  interface  are  high y i e l d  (transfer  of  and  the  is  required  spectrometer.  the  required,  c o m b i n a t i o n o f HPLC a n d MS. separation  of  number o f m a n u a l  To p e r f o r m HPLC-MS,  i.  tributyltin chloride, p g / m L as  s o l u t i o n of  was u s e d f o r  organisms.  of  43  as  The  stringent  the  HPLC  requirements  follows:  sample analyte  entering should  the  mass  spectrometer)  be  ionized  without  is  being  44  chemically ii.  m o d i f i e d i n an u n c o n t r o l l e d  Chromatographic and  the  i i i .  a b i l i t y to vaporize  iv.  the performance  In these  a.  different  Direct  to  date,  is  thermospray  Interface:  mass  unit,  transfer  mass,  has been  is s t i l l  schematic  a r e most  Interface:  volatility  a  able  to  problem  fulfill in  all  commonly u s e d : In  interface,  through an i n t e r f a c e  the i n t e r f a c e  interface,  introduced  into  appropriate  the  the  process  the e f f l u e n t  o r moving wire  from the e x t e r n a l  where  this  ion  such  as  electrospray.  on a moving b e l t  spectrometer, study,  or  In t h i s  fraction  into  ionization is  high  from the  which  pressure  transports region  of  effected.  o r i g i n a l l y described by V e s t a l  The thermospray  HPLC  t h e h i g h vacuum r e g i o n o f  employed i s the thermospray  c o n f i g u r a t i o n o f t h e HPLC-MS  shown i n F i g . 3.8.1.  3.8.2.  interface  i s r e m o v e d b y some  a m o d i f i c a t i o n o f the type A  spectrometer.  f r o m t h e HPLC c o l u m n i s  is deposited  HPLC  this  no LC-MS  Introduction  a chromatographic  In  operating  sample.  material  The s o l v e n t  Transport  the  on the  o f low v o l a t i l i t y  i n t e r f a c i n g methods  emerging  nebulization,  the  o f the  Poor  Liquid  source.  column  limitations  d e v e l o p e d so f a r .  effluent  b.  pose  s h o u l d be m i n i m a l ,  s h o u l d n o t depend on the m o l e c u l a r  requirements.  Two  not  analytes  stability  practice  interfaces  should  o f t h e HPLC o r t h e mass  thermal  manner.  peak b r o a d e n i n g i n the i n t e r f a c e  interface  conditions  or  -  interface,  et a l . ^ 9  as employed i n t h i s  interface  is  shown  in  study Fig.  -  Eluent  45  -  0.2%Trifluoroacetic acid in H2O  SAMPLE—>COLUMN BY-PASS  HPLC SYSTEM  THERMOSPRAY INTERFACE  MS  Y EXCESS I MOBILE PHASE  Fig. 3.8.1:  Schematic configuration of the  HPLC-MS  system  46  J E T T.C. SOURCE  BLOCK T . C .  PUMPING LIQUID  E L E C T R O N B E A M FOR " F I L A M E N T O N " MODE  PROBE T . C .  L.C,  N  (T.C.  EFFLUENT  Fig.  3.8.2:  The  thermospray  =  THERMOCOUPLE)  interf  ace  LINE  TO  NITROGEN  TRAP  -  In  HPLC-MS  47 -  using thermospray i o n i z a t i o n , an aqueous solution o f an  electrolyte  is  used  to  effect  ionization.  In  electrolyte  is  0.2% t r i f l u o r o a c e t i c acid i n water.  this  rate  1  mL/min  electrically  enters  heated  the  stainless  vaporizer  aerosol  containing  a  droplets.  positive  droplets  liquid  3.8.1).  A  The rapid  supersonic  through  are  being  heating of the  jet  of  electrically  equal  to  an  vapor and Some o f the  charged,  the  the number of negative  As the droplets travel through the channel i n the block, they  further heated.  electric  3.8.2)  mist of fine droplets or p a r t i c l e s .  droplet population i n the aerosol beam of  (Fig.  capillary.  effluent results i n the production of a  are  (Fig.  of the column effluent and t r i f l u o r o a c e t i c acid at a t o t a l flow  of  number  the  The e l e c t r o l y t e i s  allowed to mix with the eluent a f t e r the HPLC column mixture  study,  As the size of the charged droplet i s reduced, the  f i e l d at the l i q u i d surface increases u n t i l ions present i n the phase  are  ejected  from  through an exit aperture into the negative  ions  the droplets. mass  The ions are extracted  analyzer.  Both  p o s i t i v e and  are produced, but i n this study, only p o s i t i v e l y charged  ions were analyzed. The ion source also has the c a p a b i l i t y of producing for  normal  chemical  ionization,  when  the  In  this  operated i n the 'filament on' mode. interface  was  operated  in  s e n s i t i v i t y and higher mobile  the  electron  thermospray study,  beams  interface i s  the  thermospray  'filament o f f mode because of higher  phase  flow  rate  which  could  then be  achieved. Ions can be produced i n the thermospray i o n i z a t i o n process by direct evaporation of a sample i o n , or  in  a  two-step  process  analogous  to  -  conventional ejected  chemical  ionization  of  little  its  fragmentation  Two types The  modes  of  the  molecule  electrolyte  i n the  gas  double  The former  fragmentation  focussing  Thermospray,  and i s  1  o f molecules  is  thus  the  mass spectrometer w i t h combined magnetic  a much h i g h e r  i o n source v o l t a g e  (10"^-10"^) t o r r ) than the l a t t e r .  for  and  analyzer (>3000V)  and  The low v o l t a g e  and  i n t e r f a c i n g w i t h l i q u i d chromatographic  thermospray  interface  conditions  f o r the  were o p t i m i z e d by u s i n g a s o l u t i o n o f t r i b u t y l t i n jig/mL as  block constant, Fig.  method  make  it  system.  O p t i m i z a t i o n of HPLC-MS C o n d i t i o n s  The  (36.5  that  desired.  h i g h e r o p e r a t i n g p r e s s u r e s o f the quadrupole mass s p e c t r o m e t e r  3.9  in  analyzers  o p e r a t e s at  vacuum  more s u i t a b l e  phase  p r o v i d e s v e r y m i l d i o n i z a t i o n such  information i s p r o v i d e d , ^  The quadrupole mass  higher  ion  o f mass s p e c t r o m e t e r s have been a p p l i e d i n LC-MS:  electrostatic ii.  an  i o n t h a t i s mass a n a l y z e d . ^  ionization  o f c h o i c e when s l i g h t  i.  whereby  from a d r o p l e t r e a c t s w i t h an a n a l y t e  and g e n e r a t e s an a n a l y t e any  48  4.7.1).  Sn).  chloride  T h i s was a c h i e v e d by k e e p i n g the  and v a r y i n g the  vaporization  organotin in  chlorides acetone  temperature o f  temperature  (Chapter  the 4,  49  3.10  V e r i f i c a t i o n of  Solutions trichloride and  each  (42  apart  of  the  Figs.  the  4.8.2.,  mobile  was  the  60%  and  Sn)  which  made up t o  organotin  p h a s e was  the  into  chlorides  mixture  of  [98%  a  flow rate  of  0.6  m L / m i n : 40%  i n water)  at  a  flow rate  of  0.4  mL/min.  of  HPLC-MS tropolonates tropolonate, bisoxinate,  Butyltin  was  obtained  butyltin and b u t y l t i n in  acid  aqueous  ammonium a c e t a t e )  column.  pattern  at  at  /ig/mL as  amounts  i n 100  in  dissolved  given  i n 10  (2%  acetic  mL o f  acetone.  in  mL  acetone,  thermospray-MS.  are  Sn),  Each  T h e mass  Chapter  4,  a  of  the  butyltin  tributyltin  ammonium a c e t a t e )  acetone) acid  rate of  0.4  of  ions  dibutyltin  phase  0.6  obtained  eluent  are  given  bis-  80% 2%  The  the  and  dibutyltin  of  mL/min :  mL/min.  mixed w i t h  oxinates  oxinate,  using a mobile  a flow rate  in  trifluoroacetic  tropolonate,  flow  The f r a g m e n t  (0.2%  acid  Tropolonates  tributyltin  trisoxinate,  methanol)  (aqueous  chromatographic  for  and  tristropolonate,  acetic  electrolyte  Oxinates  fragmentation  (39.1  butyltin  4.8.4.  at  HPLC-MS  /ig/mL S n ) ,  mark w i t h  the  Quantitation  were p r e p a r e d  was  2% THF]  3.11  HPLC-MS  (36.5  appropriate  injected  4.8.3.  Used f o r  /ig/mL  dichloride  /iL)  be  chloride  (34.5  and then  standard  4.8.1.,  The +  of  (20  to  dibutyltin dichloride  by d i s s o l v i n g  (THF)  solutions  spectra  Sn),  diphenyltin  tetrahydrofuran  Ions  tributyltin  dichloride  flasks  from  of  ng/mL a s  diphenyltin  volumetric  Fragment  -  (0.1  (5% M  ionizing after in  the  Chapter  50  4,  Table  3.12  4.11.1.  A n a l y s i s o f E x t r a c t s from Marine Organisms by HPLC-MS  The  extracts  HPLC-GFAAS the  as  described  methylene  which  is  All  not the  mark w i t h a  compatible  of  internal  standard.  The  acetone  was  of  into  the  the  of  to  intensities  of  the  was  to  exclude  dryness  except  that  extracted  into  compounds  i n the  shells  any h y d r o c h l o r i c  spectrometer  acid  system.  on a r o t a r y  flasks, (34.5  evaporator.  a n d made u p t o /ig/mL as  Sn)  internal  standard  and  the  the  in  T h e d i p h e n y l t i n d i c h l o r i d e was  standard  parameters the  above,  a n a l y z e d by  a the  extract  HPLC-MS.  internal  current  quantitation.  organotin  i n 5 mL v o l u m e t r i c  A mixture  i n instrument  for  mass  same o n e s  shells  diphenyltin dichloride  variations or  of  the  and 3.6.2  the  a n d THF ( 9 : 1 ) .  injected  use  of  were e v a p o r a t e d  placed  were  3.6.1  necessary  w i t h the  solution of  mixture  /xL)  solution  c h l o r i d e was  was  HPLC-MS  The e x t r a c t i o n  extracts  residue  by  i n sections  acid  chloride.  into  (20  analyzed  hydrochloric  methylene  The  -  on the  internal  eliminates analyte,  standard  the  effect  of  the  if  ratios  of  ion  and the  analyte  are  used  51  -  CHAPTER 4 RESULTS ANr  4.1  DISCUSSION  Characterization of the Oxinate and Tropolonate Complexes  a naturally  tin  compounds.  Trimethyltin tropolonate^  are  known a n d h a v e  been  In  dibutyltin bistropolonate  this  afford  satisfactory  butyltin on  study,  tropolonate  repeated  spectrometry  ligand  complexes  and b u t y l t i n  with  organo-  tristropolonate^  3  characterized.  elemental does  syntheses, i n the  occurring  forms  Tropolone,  not but  analyses  and b u t y l t i n  (Table  show s a t i s f a c t o r y the  molecular  tristropolonate  4.1.1).  However,  tri-  elemental  analysis,  even  ion  is  o b t a i n e d by  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  mass (Fig.  4.1.1) .  Table  4.1.1:  A n a l y t i c a l data  and m e l t i n g p o i n t s  %  C  of  butyltin  H  0  tropolonates  M.P.  (°C) Calcd. found  55. .69 55. .49  4, .49 4, .49  17. .80  Dibutyltin bistropolonate ( 22 28°4 - MW-475.152)  Calcd. found  55. .61 55. .62  5. .94 6 .00  13, .47 13, .30  Tributyltin  Calcd. found  55. .50 54. .63  7 .85 7, .94  7, .78  Butyltin ( 25 24°6 c  H  c  < 19 c  H  H  tristropolonate S n  >  MW=539.151)  S n  32°2  S n  tropolonate '  MW=411.153)  238-240  94-96  -  100 412  in  50  254  0  1111111  100  150  Fi&-  4.1.1 :  200  29? T y n  250  Desorption  i i*| 11  Tr  11 i 1 • 1  300  chemical  350  500  400  Ionization  mass  spectrum  T"  550  i i j 11 i 11 i 111 j 1111111 111111 i  600  of t r i b u t y l t i n  630  700  tropolonate  M  /  53  Table  4.1.2:  Analytical  data  and m e l t i n g  points  %  of  butyltin  0  H  C  oxinates  M.P.  (°C)  MW-608.265)  Calcd. found  61.21 61.06  4 . 47 4 . 31  6.91 6.87  220-222  Dibutyltin bisoxinate ( C H 3 N O S n , MW-401.118)  Calcd. found  59.91 59.74  5. 80 5. .34  5.37 5.25  136-138  Tributyltin  Calcd. found  58.09 58.05  7 ,.66 7. .63  3.23 3.36  -  Butyltin  trisoxinate  ( 31 27 3°3 c  H  N  0  1 6  c  21 33 H  2  -  S n  2  N O S n  -  oxinate MW-439.191)  The mass s p e c t r a l in  the  electron  4.1.5. the  The  ionization  fragmentation  molecular  ion,  tristropolonate 419  Also  characteristic is  the  a six-membered r i n g The (Table given  butyltin. 4.1.2).  of  the' M/Z the  to  form a  oxinates-  ions are  not  given in  ion  is  butyltin 241,  not  detected.  to  pattern  good the  and 4 . 1 . 8 .  observed, but  +  fragment  elemental electron Like  4.1.4, group  The  at  show  the  fragment  and from  butyltin  M / Z 483 an  and  intense  [Sn-tropolonate] . +  of  the  s e v e n membered t r o p o l o n e  [Sn-phenoxide] afford  a butyl  show p e a k s  corresponding  the  of  tropolonates  fragmentation  complexes  Tables 4.1.3,  loss  bistropolonate  T h e mass s p e c t r a i n 4.1.7  b u t y l t i n tropolonate  shows t h e  molecular  collapse of  in Tables 4.1.6,  molecular  mode a r e  and d i b u t y l t i n All  the o t h e r  pattern  the  i o n c l u s t e r e d at  tropolonates,  the  but  respectively.  fragment  data for  at  M/Z  butyltin ring 213.  analytical  ionization tropolonate  into  results  mode  are  complexes,  ions corresponding  to  54 -  Table 4 . 1 . 3 :  Fragment i o n s o f b u t y l t i n  Fragment mass (M/Z)  T  Table  +  Assignment  (% base peak)  483  9.8  419  60.2  362  11.3  241  100.0  213  8.1  122  52.0  + SnT  3  + C4H SnT2 + SnT 9  2  + SnT [Sn p h e n o x i d e ] T  +  +  = tropolonate ion  4.1.4:  Fragment ions 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  Fragment mass (M/Z)  T  Intensity  tristropolonate  Intensity  (% base peak)  Assignment  419  22.4  + C4HoSnT2  355  15.9  (C H ) SnT  241  83.1  + SnT  213  6.6  122  52.0  = tropolonate i o n  A  9  2  [Sn p h e n o x i d e ] T  +  +  55  Table  A.1.5:  F r a g m e n t mass  T  Table  Fragment  ions of  (M/Z)  -  tributyltin  Intensity  (%  tropolonate  Assignment  base peak)  (C H ) SnT  355  42.8  241  100.0  213  8.2  [Sn p h e n o x i d e ]  122  9.4  T  =  +  tropolonate  4.1.6:  Fragment  mass  9  2  + SnT  +  ion  Fragment  (M/Z)  4  ions  of b u t y l t i n  Intensity  (%  trisoxinate  base peak)  Ass ignment  + Sn0X  552  0.8  465  24.2  + C H Sn0X  408  20.6  + SnOX  264  100.0  + SnOX  145  72.5  0X  +  •= o x i n a t e  ion  4  0X  3  9  +  2  2  +  56 Table  4.1.7:  Fragment  mass ( M / Z )  Ions  of d i b u t y l t i n  Intensity  (%  bisoxinate  Assignment  base peak)  465  58.6  + C H Sn0X  408  10.2  + Sn0X  378  12.6  + (C H ) Sn0X  264  100.0  145  68.1  0X  Table  Fragment  +  = oxinate  4.1.8:  Fragment  0X  Fragment Ions  mass ( M / Z )  of  4  2  2  9  2  + SnOX 0X  +  t r i b u t y l t i n oxinate  Intensity  (%  87.2  264  100.0  145  88.3  = oxinate  9  ion  378  +  4  ion  base peak)  Assignment  + (C Hq) Sn0X 4  + SnOX 0X  +  2  57 the  loss  of  one b u t y l  assigned  fragment  intensity  patterns  Further provided the  group  ions for  from the  are  such  found  n.m.r.  of  the  present  spectrum  of  butyltin  complexes  4.2  the  dibutyltin  Similar been  in  ring  reported  Molar  match  apparent.  w i t h the  are  and t r o p o l o n a t e  Ratio  used  complexes.  to Fig.  bisoxinate.  are  are  closely  oxinate  spectroscopy.  l i g a n d and b u t y l t i n protons  ligands  to  ion  All  theoretical  fragments.  characterization  by  molecular  of  integrated  determine 4.1.2  n.m.r.  complexes  is  peak areas  of  the  shows  spectra  number  the of  of  n.m.r. the  other  g i v e n i n Appendix B.  proton n.m.r.  by Westlake  Extinction  and  signals  of  oxine  chelates  have  previously  Martin.^  C o e f f i c i e n t s of  B u t y l t i n Oxinates  and  Tropolonates  The b u t y l t i n o x i n a t e s 4.2.2 of  4.3  have  high molar  extinction  HPLC w i t h UV d e t e c t i o n  Nature  Oxine metals.  of  shown  coefficients  in  for  their  Organotin Oxinates  and  Tropolonates  Although  the  seven  form  bidentate  complexes  coordination  chelate  been  the  4.2.1  and  application  analyses.  formed by these has  Tables  w h i c h make  feasible  and t r o p o l o n e  coordination,  and t r o p o l o n a t e s  complexes  ligands  reported  are for  with  many  commonly  six  oxine^  and  F  *8-  4.1.?:  * l l NMR  spectrum of  dibutyltin  bisoxinate  59 Table 4 . 2 . 1 :  Compound  (C H ) SnT 4  9  3  (C H )SnT 4  9  C4H SnT3 9  2  Molar extinction c o e f f i c i e n t s of b u t y l t i n  Anm  Methanol € (Lmol'^cm" )  232  2,.8 X 10  4  231  2, .7 X 10  4  375  5.,6 X 10  3  322  9 .8 X 10  3  328  1..2 X 10  4  384  7. .2 X 10  3  373  6. .4 X 10  3  374  4, .5 X 103  395  6. .5 X 10  3  393  5..4 X 103 375  9..5 X 10  3  372  1.,4 X 10  4  1  Acetonitrile Amn « (Lmol'^cnT )  tropolonates  1  242  1..0 X 10  4  242  1..5 X 10  4  314  2,.4 X 10  3  256  3..2 X 10  4  370  3 .5 X 103  234  7 .0 X 10  4  238  1..7 X 10  6  320  3,.9 X 10  4  325  9,. 3 X 10  5  373  2,.1 X 10  4  372  4. .2 X 10  5  Acetone Anm € (Lmol"^cm"- ) 1  -  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 o f b u t y l t i n  Compound Anm  (C H ) Sn0X 4  9  3  (C H ) Sn0X2 9  4  2  C H SnOX 4  9  3  tropolone.  -  60  Methanol e(Lmol^cm" )  Amn  1  Acetonitrile £(Lmol^cm" ) 1  240  1. 8 X 1 0  1,.0 X 10*  253  3.  243  2..9 X 1 0  4  240  4.,2 X 1 0  4  252  3,.5 X 1 0  4  255  6,.5 X 1 0  4  232  1..1 X 103  240  5,.4 X 1 0  4  254  1..0 X 1 0  255  5 .4 X 1 0  4  241  2,.0 X 1 0  254  4  4  2 X  4  Anm  Acetone t (Lmol^cm" )  326  2..7 X 103  375  4..1 X 103  375  4 .0 X l O  lO*  It  is  a n t i c i p a t e d , t h a t oxine  tributyltin  complexes  4.3.1 s u p p o r t  1  respectively.  t h i s view f o r the  frequency^  6  which  tropolonate in  i s s h i f t e d t o lower frequency  tropolone  is  coordinated  to  Relevant  The  i n Table carbonyl  i s a t 1615  T h i s i s evidence  that  i t s c a r b o n y l group.  The  absence o f - 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 cates  i n f r a r e d data  the f r e e t r o p o l o n e l i g a n d  t i n , through  exhibit  d i b u t y l t i n , and  complexes.  ( T a b l e 4.3.1).  3  triphenyltin  and t r o p o l o n e would  seven, s i x , and f i v e c o o r d i n a t i o n i n t h e i r m o n o b u t y l t i n ,  cm"  1  F i v e c o o r d i n a t i o n has a l s o been r e p o r t e d f o r  J  oxinate.^  stretching  oxinates  3500-3100  cm"  1  indi-  t h a t t h e r e i s no f r e e - O H group i n the t r o p o l o n a t e 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 r o p o l o n a t e s  Compound  i/C=0 ( c m " ) 1  (C H ) SnT 4  9  (C H ) SnT 4  9  1592  3  2  1592  2  1562  C H SnT 4  9  1589  3  1576 1572  this  basis,  butyltin  tributyltin  tristropolonate  r e s p e c t i v e l y as For  tropolonate,  dibutyltin  are  six,  five,  and  Numerous  weak bands are p r e s e n t  about 395  cm"  assigned  to  spectra.  in  coordinate  the c o o r d i n a t i o n c o u l d not be a s c e r t a i n e d  o f 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  vibrations  seven  and  expected.  the o x i n a t e s ,  1  bistropolonate,  which P o l l e r ^ and  oxinate  vibrations.  i n the f r e q u e n c y range A06-387 c m " Okawara  N-»Sn s t r e t c h i n g v i b r a t i o n s . the  stretching  because  complexes  is  et  al.^-*  respectively  The absence evident  1  and have  o f —OH s t r e t c h i n g  from  the  infrared  -  A. A  62  -  Chemical M o d i f i e r s f o r Atomic A b s o r p t i o n Butyltin  Spectrophotometry of  Chlorides  I n atomic a b s o r p t i o n spectrophotometry, h i g h s e n s i t i v i t y i s obtained  by  the  removal  s i g n a l o f the a n a l y t e . The  matrix  chemicals  of  the  of  substances  t h a t suppress the  usually  absorption  T h i s i s u s u a l l y a c h i e v e d by m a t r i x m o d i f i c a t i o n . a n a l y t e i s d e l i b e r a t e l y a l t e r e d by  (chemical m o d i f i e r s ) .  The  chemical  the a d d i t i o n o f  modifiers  achieve  matrix  m o d i f i c a t i o n i n some o f the f o l l o w i n g ways: 1.  2.  3.  The  chemical  or c a r b i d e s  than  the a n a l y t e ,  thus making the a n a l y t e a v a i l a b l e f o r  The  chemical  m o d i f i e r reduces r e f r a c t o r y compounds o f the a n a l y t e  to  the  metal.  The  chemical  m o d i f i e r forms complexes of h i g h b o i l i n g p o i n t w i t h  the  a n a l y t e and  thus permits  that  remove  will  with  higher  matrix  drying  and  atomization.  charring  compounds which w i l l  temperatures  otherwise  interfere  detection.  Various absorption  chemical  Au,  m o d i f i e r s are used f o r  the  spectrophotometry o f v a r i o u s elements.  s o l u t i o n of N i C l 2 As,  m o d i f i e r forms more r e f r a c t o r y oxides  i s a s u i t a b l e chemical  B i , and Te.  electrothermal  atomic  For example, aqueous  modifier for  the  analysis  of  For the e l e c t r o t h e r m a l atomic a b s o r p t i o n o f t i n , a  m i x t u r e o f ammonium phosphate and magnesium n i t r a t e has  a l s o been  found  to  Umezaki,^  have  be  a  good  chemical  modifier.^®  Tominaga  and  i n v e s t i g a t e d the a b i l i t y  o f v a r i o u s compounds to suppress  in  atomic  the  electrothermal  A s c o r b i c a c i d was  found to be  absorption effective  in  interferences  spectrophotometry enhancing  the  of  tin.  absorption  63  signal  of t i n .  In  this  citric for  study,  acid,  is  dibutyltin  The  but its  was  to serve  absorption  citrate, as  ascorbic  chemical  spectrophotometry  tcid,  modifiers of butyltin  The  chemical  and  into  tributyltin  the graphite  little  of  acid.  variation  and c i t r i c  modifiers  dibutyltin  absorbance  by a s c o r b i c  acid  and  chloride  furnace,  or  and the  measured.  chloride  shows  modifier  injected  o f the v a r i o u s  enhanced  tartaric  dichloride in  the type  produced  is  shown  tributyltin  The a b s o r b a n c e  with  acid  Sn  on t h e a b s o r b a n c e  of  o f Sn  i n Figs.  chloride  is  o f Sn i n d i b u t y l t i n chemical  the g r e a t e s t  modifier,  enhancement  of  absorbance.  the  is  surprising  absorbance  to f i n d  effect  4.4.3  shows  little  ascorbic  o f the volume  and 4 . 4 . 4 . sensitivity  acid  could  chloride  greatly  without  enhance  d o i n g t h e same  dichloride.  t h e Sn i n t r i b u t y l t i n  Figs.  that  o f t h e Sn i n t r i b u t y l t i n  t h e Sn i n d i b u t y l t i n The  of  dichloride  and 4 . 4 . 2 .  It  for  atomic  chemical  o f t i n was  dichloride  of trisodium  and glucose  o f the  effect  greatly  acid,  furnace  tributyltin  4.4.1  ability  investigated.  A mixture  absorbance  the  tartaric  the graphite  compounds  in  -  o f the chemical  chloride  modifier  on the  and d i b u t y l t i n d i c h l o r i d e  The a b s o r b a n c e to the volume  of Sn, i n of modifier  dibutyltin used.  absorbances is  shown i n  dichloride  64  ABSORBANCE  0.I6H 0.140.120.10-  0.080.060.040.02-  ll  Ob to o  F i g . 4.4.1:  o  CD O CC < o o  2  o  or < £ <  o  <  o  U CO o o r> _i o  E f f e c t o f v a r i o u s m o d i f i e r s on the absorbance o'f (C H ) SnCl 4  9  3  MODIFIER  - 65 -  ABSORBANCE  0.080.070.060.050.040.030.02 O.OH Q Of  oE  Fig.  4.4.2:  OQ OQ O CC< O U  2  gs o  OQ  E f f e c t o f v a r i o u s m o d i f i e r s on the absorbance o f (C^Hcj^SnCl^  co o o =>  —J  o  MODIFIER  0.07ABSORBANCE  0.060.050.040.03" 0.020.011——i  5  10  1  1  1  1  15  20  25  —i  30  35  0.5% ASCORBIC ACID  Fig-  •  ( L) M  4 . 4 . 3  Effect  of  m o d i f i e r volume on the  absorbance  of  (C^q^SnCl  ABSORBANCE  O.I2-I  o.ioH 0.08H <J3  0.06H 0.04H  0.02H n  5  Fig.  4.4.4:  1  10  1  15  1  20  1  25  1  i  30  35  0.5%ASCORBIC ACID (/*L)  E f f e c t o f m o d i f i e r volume on the absorbance o f ( C H ) S n C l 4  9  2  2  -  4.5  Retention  4.5.1  of  98% every  determine  the  butyltin  of  5-6.5  i n acetone]:2%  whose  time  mobile  chloride  from  acetone]:20%  and  dichloride.  were  that  eluted  has  retention  by  times  time  of  of  dibutyltin dichloride  composition dichloride  column or  capable is  the  of  80%  the  by  mobile  effluents GFAAS,  a mixture  to  of  the  determined  The G F A A S - c h r o m a t o g r a m  a broad band w i t h a  on the  a  had been  dichloride  a retention  using  Then,  GFAAS.  determined,  The c o l u m n  as  either  phase  THF.  analyzed  the  was  chloride  compound.  dibutyltin  dibutyltin  is  shown  in  Fig.  3.5-4.5 mins,  while  retention as  time  a broad band  connecting  separating  [1%  of is  tubings  tributyltin  acetic  acid  in  pentane.  preliminary  trichloride  of  individual  adsorption  Another  samples  interval  elution  system.  butyltin  and d e t e c t e d by  chloride  HPLC  A  acid  min.  The  of  butyltin chloride  acetic  dichloride  indicative  each  chlorides  the  Tributyltin chloride  mins.  the  of  retention  tributyltin  butyltin  s o l u t i o n of  0.5  chlorides  dibutyltin  of  a  chromatographed  4.5.1.  for  time  [2%  collected  was  data  retention  chromatographing phase  -  Data  Retention  The  68  shows  investigation that b u t y l t i n  Hence,  any  would i n t e r f e r e  of  the  retention  trichloride  butyltin  with  the  coeluted  trichloride  detection  of  present  time  of  with in  butyltin  dibutyltin  environmental  dibutyltin dichloride.  ABSORBANCE  0.05i  (C*H )2SnCl2 9  0.04CTi  (C4H ) SnCI  003-  9  3  0.020.01  1  0  I"  3  4  n  7  1  1  8  9  RETENTION TIME (MIN) Fig.  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  Retention  By min.  chromatogram dibutyltin eluted  at  by  in  change  to  4.5.2  20%  was  coeluted  ethyl  obtained.  at  acetate:5% methanol  for  4.5  acetate:80% methanol,  the  Tributyltin  and  oxinate  3.41 m i n , w h i l e b u t y l t i n  trisoxinate  10.40 m i n .  Chloride, Dibutyltin  Limit,  Recovery  Equal  studies  (5-15  liver,  section  on the e x t r a c t i o n  dichloride  of  ^g/0.1  and then  3.5.  The  extracted  Dichloride:  - Recovery  Studies,  and P r e c i s i o n  concentrations  dichloride dogfish  oxinates  e l u t i o n o f 95% e t h y l  bisoxinate  Tributyltin  4.6.1  a  Fig.  Detection  in  for butyltin  using a gradient followed  4.6  data  -  procedure  tributyltin  g  dogfish  extracted mixture  chloride  liver)  into  were  methylene  and  spiked into  chloride  of tributyltin chloride  i n t o methylene  dibutyltin  as  standard described  and d i b u t y l t i n  c h l o r i d e was s e p a r a t e d  by  HPLC  and  was e f f e c t e d  by a  d e t e c t e d b y GFAAS. Quantitation normal  o f the e x t r a c t e d  calibration  procedure.  compared w i t h the absorbance tributyltin dichloride A mixture  chloride  o f the separated  The  absorbance  of various  solution  was q u a n t i t a t e d  tributyltin chloride  in  of  the  known c o n c e n t r a t i o n s  acetone  by the standard  (Fig.  4.6.1).  a d d i t i o n method  compound of  was  standard  Dibutyltin (Fig. 4.6.2).  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  concen  -  A B S O R P T I O N  Butyltin  trisoxinate  10.40 T r i b u t y l t i n oxinate and Dibutyltin bisoxinate  3.41  J 0  5  ~T~  10  15  1< K T K N I -  Fig-  4.5.2:  Chromatogram o f b u t y l t i n  I O N  oxinates  T  I  M  E  < min  >  - 72 -  ABSORBANCE  0.08 H  10 12  8  (C Hg) SnCI (/xg/mL) 4  Fig. 4.6.1:  3  C a l i b r a t i o n graph f o r t r i b u t 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 10.8 13.2x10"2 (C«H9)2SnCl2 («g  F i g . 4.6.2  Standard addition plot for recovered dibutyltin dichloride  - 74 tions of the standard d i b u t y l t i n dichloride i n acetone was injected the GFAAS, and their absorbances  determined.  A l l calibration  into  graphs  were drawn using the least square method. At  the  level  of  study  (5-15/ig/O.l  g  t r i b u t y l t i n chloride and d i b u t y l t i n dichloride  standard afford  dogfish l i v e r ) , good  recoveries  (Table 4 . 6 . 1 ) .  Table 4 . 6 . 1 :  Recovery studies f o r b u t y l t i n chlorides  Level of study (/ig/0.1 g s t d . dogfish l i v e r )  5  Compound Extracted  % Recovery*  Regression Equation f o r Calibration Graph  (C H ) SnCl2  89.20 ± 0.01  Y-1.9x + 0.0268  0.9378  (C H ) SnCl  97.30 ± 0.04  Y=0.38x + 0.0060  0.9934  (C H ) SnCl -  86.00 ± 0.03  Y=1.8x + 0.0448  0.9051  (C H ) SnCl  83.30 ± 0.05  Y=0.0054x + 0.0032 0.9846  91.60 ± 0.02  Y-1.6x + 0.0685  0.9951  90.00 ± 0.04  Y-2.1x + 0.0077  0.9694  4  4  10  4  4  15  9  9  9  9  2  3  2  3  (C H ) SnCl 4  9  2  (C H ) SnCl 4  9  2  3  2  P r e c i s i o n i s expressed at the 95% confidence l e v e l  Correlation Coefficient  75 A.6.2  Detection l i m i t and p r e c i s i o n  The l i m i t o f detection i s defined as the analyte concentration which gives a signal equal to the  signal  o f the  blank,  plus  thrice  the  standard deviation of the blank. A  method  of  determining this l i m i t by using the intercept  standard deviation of the y - r e s i d u a l s has been described The l i m i t of detection was obtained by p l o t t i n g various  concentrations  concentrations Fig.  4.6.3,  of t r i b u t y l t i n  (Fig. 4.6.3). as  found  by  deviation of the y - r e s i d u a l s ,  the  chloride  in  By using the intercept the  regression  and the  elsewhere.^0 absorbance  acetone  of  the  of  against graph  of  equation, and the standard  the l i m i t of detection i s 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 mg/mL  as  Sn).  determinations  4.7  The r e l a t i v e  standard  deviation  (R.S.D.)  (10  for ten  i s 2.8%.  HPLC-MS  4.7.1  The  Optimization of the thermospray interface  thermospray  interface  conditions  were  conditions  optimized  to  obtain  e f f i c i e n t i o n i z a t i o n of the a n a l y t e . T r i b u t y l t i n chloride was used for this optimization. as  Sn)  An acetone solution of t r i b u t y l t i n chloride  (36.5  Mg/mL  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  tributyltin chloride and the mass phase  was  60% I (  varied during each injection of  spectra  was  recorded.  The mobile  acetic acid in acetone)+ 2% THF)] at a flow rate of  2%  0.6 mL/min:40%(0.2%trifluoroacetic  acid in water) at a flow rate of  0.4  mL/min. The  effect  of  the  intensity of the base intensity  increased  peak  leads  of  M / Z 349  vaporization temperature on the is  shown  in  Fig.  4.7.1.  The  sharply as the vaporization temperature increased,  reaching a maximum at temperature  variation  185°C.  Further  the  vaporization  to a very sharp decrease in intensity.  On the basis  of this information, the following  increase  conditions  in  were  selected  for  the  thermospray-MS analysis: Vaporization temperature  182 C  Probe temperature  117°C  Block temperature  225°C  Jet temperature  213-215°C  6  Major Ions of Standard Organotin Chlorides  4.8  The  mass  spectra  injection of  acetone  dichloride,  butyltin  of  standard organotin chlorides obtained by the  solutions  of  trichloride,  described in Chapter 3, section 3.10)  tributyltin and  chloride,  dibutyltin  diphenyltin dichloride  into the thermospray-MS are  (as  shown  - 78 -  ABSOLUTE INTENSITY  < »rbltr»rv unit! >  xlO 55  4-  32I-  165  170  175  180  185  190 2 0 0 °C VAPORIZATION TEMP.  Fig. 4.7.1:  E f f e c t of v a r i a t i o n of vaporization temperature on the i n t e n s i t y of t r i b u t y l t i n fragment ion at M/Z 349  -79 in  Figs.  ions  A.8.1,  are given The  ions  with  were  by  and 4 . 8 . 4  respectively.  The m a j o r  fragment  4.8.1.  obtained  components  compared  generated  4.8.3  i n Table  fragment  compounds ions  4.8.2,  with  indicate  the b u t y l t i n c h l o r i d e s  o f the mobile phase.  theoretical  computer  that  intensity  A l l assigned  pattern  form  fragment  f o r such  fragments  closely  (Appendix  s i m u l a t i o n and found to match  C). Same  fragment  chlorides When detecting  by  using  thermospray  the  tributyltin towards  butyltin  the  the chromatographic  interface  t h e mass  column.  conditions  spectrometer  optimized  exhibited  t r i c h l o r i d e . A n acetone i n t o the thermospray-MS  organotin  for  v e r y low  s o l u t i o n of  butyltin  was n o t d e t e c t e d .  the  times  o f the- s t a n d a r d  HPLC-GFAAS  because  conditions,  o f the d i f f e r e n c e s  organotin  chlorides  are not a p p l i c a b l e i n flow rate  established  t o t h e HPLC-MS  and e l u t i o n volume i n  two m e t h o d s . The  retention  times  of  standard  dichloride,  and d i p h e n y l t i n d i c h l o r i d e  established  by i n j e c t i n g  chloride and  i n HPLC-MS when  T i m e s o f O r g a n o t i n Compounds I n HPLC-MS  retention  using  through  chloride,  /ig/mL)injected  Retention  conditions the  obtained  to pass  t r i c h l o r i d e (10  The  are also  are allowed  sensitivity  4.9  ions  (21.9  an acetone  tributyltin chloride, under  solution  HPLC-MS  diphenyltin  dichloride  (34.53  conditions  of a mixture  /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 ug/mL a s  (23.4  Sn) i n t o  dibutyltin  of  were  tributyltin  /ig/mL  as  Sn) ,  t h e HPLC-MS, and  - 80 -  I N'T E N S I T V  65 sa  100-1  17S  S0 1(1  M 7  '—r—i—i  i  i  r-r-r-r2 7 S  F i  g-  4.8.3:  -i—i—  ""i  3 2 S  —•—i  r—r| I 3SB  II—i—pr—I—I—r 3  7  Mass spectrum of b u t y l t i n t r i c h l o r i d e  g  «0g  M / Z  2 \ N  - 84 Table 4.8.1:  Compound  Major ions of standard organotin compounds  Major Ion  Relative Cone. of Compound Inten-  Assignment  ppm  M/Z  (C H ) SnCl  349  (C H ) SnC0(CH )2  (MW-325.19)  327  (C H ) SnCl  291  (C H ) Sn  351  (C H ) Sn[C0(CH ) ]  327  + (C H ) SnClC0(CH ) - H + (C H ) Sn00CCH + (C H ) SnCl - H + C H Sn00CCF C H 0 + H + C H SnOOCCF Cl + H + C H SnCl(0H) 00CCH - H + C H SnClC0(CH ) + H + (C H ) Sn00CCH 0C(CH ) + (C H ) SnC10C(CH ) - H + (C H ) SnOOCCH + (C H ) SnCl - H -  4  9  3  (C H ) SnCl A  9  2  2  (MW=303.69)  293 269  4  9  4  9  4  4  9  4  9  4  9  363  3  327  (MW-282.19)  305 268 (C H ) SnCl 6  5  2  (MW=343.69)  2  391 367 333 309 175  a b c  9  4  4  9  3  2  +H  2  100  125017  2  153716  b  43040  2  3  4  g  2  1000  5  2  6  5  2  25386  c  3724  2  3  3  3  5 2  2  100  14273 8220  2  of each  peak  (C H ) SnCl (C H ) SnCl2 C H SnCl (Cgl^^SnC^ 4  4  4  9  9  3  9  2  3  6922 25950  2  3  Relative intensity is the intensity base peak base peak of fragments arising from base peak of fragments arising from base peak of fragments arising from base peak of fragments arising from  11678 7108  3  3  5  78856  3  6  6  2  3  9  6  3  2  9  4  a  24092  2  9  4  42267 4227  3  9  4  100  3  3  9  4  C H SnCl  3  sity*  64875  d  relative  to the  - 85 detecting  the  eluted  compounds by using t o t a l ion current monitoring.  The retention times of the standard compounds are shown i n Table  Table 4 . 9 . 1 :  Retention times of organotin compounds i n HPLC-MS  Compound  Retention time (min)  (C H ) SnCl 4  9  8.81  3  (C H ) SnCl  2  12.48  (C H ) SnCl  2  10.68  4  6  4.10  4.9.1.  9  5  2  2  Levels of B u t y l t i n Compounds i n the Tissues and Shells of Marine Animals  4.10.1  The  Analysis by HPLC-GFAAS  methylene  chloride  extract  of  the marine organisms obtained  a f t e r extraction of the organotin compounds as chlorides was to  dryness,  and then reconstituted  evaporated  i n hexane (Chapter 3, section  The hexane solution of the extracts was chromatographed and the organotin  3.6). eluted  compounds were detected by graphite furnace atomic absorption  spectrophotometry.  The i d e n t i t y of the eluted organotin  compounds was  - 86 established chloride present  Table  by  comparison with  and d i b u t y l t i n in  the  tissue  4.10.1:  retention  dichlorj.de.  of  marine  The  organisms  times level is  of  standard  of  given  tributyltin  butyltin in  Table  compounds  4.10.1.  Concentration of b u t y l t i n compounds i n whole body (soft tissue) of marine organisms  Organism  Origin  Pacific oyster Crassostrea gipas  Fanny  Shrimps  Howe  3  / i g / g (wet w e i g h t ) " as Sn Tributyltin Dibutyltin  Bay  4, .29  +  0. .36  2, .11  +  0 .19  Sound  4. .14  +  0 .28  1 .34  +  0. . 12  B u t t e r Clam Saxidomus p i p a n t e u s  Patricia  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  Patricia  Bay  3, .52  +  0, .12  4, .62  +  0, .60  1. .37  +  0, .05  0. .87  +  0. ,15  2 .46  +  0, .04  3, .09  +  0. .08  1. .54  +  0. ,11  0. ,91  +  0. 07  3. ,23  +  0. .05  4 . .08  +  0. 32  1.,14 +  0. 09  1. 91  +  0 . 03  -  P u r p l e p i c k l e s e a cucumber Molpadia intemedia S o f t - s h e l l e d Clam Mva a r e n a r i a  Patricia  S o f t - s h e l l e d Clam Mva a r e n a r i a  Cole's  Basket Cockle Clinocardium n u t t a l l i i  Patricia  Bay  Blue mussel Mvtilus edulis  H e a d of Hastings  Arm  Shrimps b e l o n g Precision  to  the  expressed  at  family the  Bay  Bay  Pandalidae  95%  level  of  confidence  87  Comparative of  various  scarce, the  but  of  data  levels  Mg/g  of  up  to  for  some  tributyltin  than  the  some o y s t e r s in  the  other  injected  The  from  organisms  the  into  than  tributyltin  the  levels  the  The m a r i n e  and  shells.  dibutyltin  the  (Table  levels  environment data  acid  the  that  their  weight  tributyltin  oyster  et  et  levels  solution  oyster  the w  wet  reported  the  in  are  Thain  Mg/g  also  4  Mg/g  are  and  weight) a l .  of  ^  1  for  tributyl-  available.  For  available.  in  the  shells  of  of  the  shells  was  The  eluted  butyltin  4.10.2).  tissues.  of b u t y l t i n  organisms  ^  and R i c e  on the  and d i b u t y l t i n  found i n  compounds i n  data  chromatographed.  b y GFAAS  1  reported  (Crassostrea  for  compounds p r e s e n t  hydrochloric and  a l . ,  (4.29 l u 3  have  1  England.  tributyltin  and W a l d o c k ,  in  0.4-1.35  weight  no c o m p a r a t i v e  of  between  et  much h i g h e r  of b u t y l t i n  HPLC  relationship  is  w  et  compounds  reported  of  Rice  are  dry  of  tissues  habitat  oysters  also  levels  of  Canadian marine  a dilute  detected  levels  much h i g h e r  the  analyzed,  the  level  study  ug/g  some o y s t e r s  Mg/g  No c o m p a r a t i v e  levels  organisms,  for  the  natural  butyltin  on the  have  England.  by T h a i n  England.  oysters  compounds were  this  reported  of  To o b t a i n marine  in  of  0.027-1.667  tributyltin  The  their  1  1  0.06-1.57  compounds i n  and H a r r i s o n ^  respectively  of  virginica.  values  tin  of  levels  of  dry weight  oysters  gigas  the  from  and M i l l e r * ^  reported  levels  Crassostrea Crassostrea  have  l u J  weight  ng/g  organotin  Rapsomanikis  Waldock  4.5  for  and d i b u t y l t i n  dry  England.  and W a l d o c k weight  oysters.  of  isolated  available  tributyltin  of  levels  organisms  are  of  0.012-0.402 gigas)  on the  uiarine  tissues  levels  data  in  There  compounds i n  show h i g h  tissues  found  also  levels  the  shells  seems t o  be  are a  the  tissues  of  tributyltin  show h i g h  levels  of  and  the  -  Table  A.10.2:  Concentration marine  88 -  o f b u t y l t i n compounds  i n the shells  organisms  Tributyltin fig/g ( d r y w t ) as Sn 3  Organism  Pacific  Origin  oyster  Crassostrea Butter  85.20 ± 0.27  A9.A0 ±  0.08  P a t r i c i a Bay  10.10 ± 0.13  6.60 ±  0.09  115.60 ± 0.58  19.00 ±  0.09  15.80 ± 0.15  5.20 ±  0.06  giganteus Patricia  S o f t - s h e l l e d Clam Mya a r e n a r i a  C o l e s Bay  S o f t - s h e l l e d Clam Mya a r e n a r i a  P a t r i c i a Bay  Precision  same c o m p o u n d s Bent-nose compounds  3  Fanny Bay  B e n t - n o s e Clam Macoma n a s u t a  3  Dibutyltin ng/g ( d r y w t ) as Sn  gieas  Clam  Saxidomus  of  i s expressed  in  Clam.  their  i n the s h e l l s  comparison.  6.60  a t t h e 95% l e v e l  shells,  However,  Bay  of  ± 1.60  confidence  e.g.  the  Pacific  the available  data  on the l e v e l s  o f marine  organisms  27.30 ± 0.17  are sparse,  oyster, of  and the organotin  f o r purposes  of  89 -  4.10.2  (i)  HPLC-MS of marine organisms  Tissue Extracts  The hexane solutions of the tissue extracts of the marine  organisms  were evaporated to dryness and then reconstituted i n an acetone solution of the chosen internal standard, diphenyltin dichloride. the  tissue  extract  and the  internal  their  mass  spectra.  of  standard was injected into the  HPLC-MS, and the eluted compounds detected by their and  A mixture  The characteristic  total  ion current  polyisotopic intensity  pattern of t i n should be diagnostic i n identifying t i n 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 (Figs.  4.10.2 and 4.10.4). + suspected to be (CgH^^SnOH i f these fragment authentic into their  of t i n  These fragment ions at M/Z 303 and 369 were + and (CgHj^^Sn respectively. To ascertain + +  ions actually belong to  samples of  that  (CgH^^Sn and (CgH^^)2Sn0H,  (CgH^i^SnB^ and (CgHn^SnBr were  each spiked  standard dogfish liver (0.5 g/0.1 g dogfish l i v e r ) , derivatized to chlorides  extraction  and extracted  procedure  described  into  methylene  i n Chapter  3,  chloride section  using  the  3.6.1.  The  methylene chloride was evaporated to dryness and the extracted cyclohexyltin the HPLC-MS.  standard  compounds were reconstituted in acetone and injected into The mass spectra are shown in Figs. 4.10.5 and 4.10.6.  90 -  Fig.  H P L C - M S 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  4.10.1:  19b I T I C - l I3?1««. I»fl-Z1»B56] E l  r 35»  Fig. 4.10.2:  M / Z  Mass spectra of position A of Fig. 4.10.1  - 91 J42 [ T l C - l M t l S .  IIIMIII!]  El  >  h H  01  z U h  z H  3»  Fig. 4.10.3:  M / Z  'I  1  1  1 1  4»  Mass spectra of position B of Fig. 4.10.1  J55 [TIC-186956.  I M X - 7 K 6 4 ] El  H  w u z  h  H  1  1  *  1  i i i i i i i i i i  i  i  i  I  i  l-r  ' T'  i'  i i  i  i' I  ±i_ V  i  I  -  Fig. 4.10.4:  i  i  i  j  1  i  M /  Mass spectra of position C of Fig. 4.10.1  I  1  4»»  1  1 1  - 92 The  mass spectrum  of the standard d i c y c l o h e x y l t i n compound did not  show the peak at M/Z 303 M/Z  303  found  in  the  ( F i g . 4.10.6). mass  This indicates that the peak at  spectra of the tissue extracts of marine  organisms i s probably not due to a d i c y c l o h e x y l t i n compound. The mass spectrum of shows  the  standard  tricyclohexyltin  a  fragment ion at M/Z 369, and + (CgH^^)3Sn0C(CH3)2, which i s the base peak.  i n the extracts, the fragment ion at M/Z 427  another  (Fig.  4.10.5)  one  at M/Z 427 + I f ( C g H ^ ^ S n was present  (the base peak) should have  been prominent i n the mass spectra of the tissue extracts, but not the case. ruled out. varies  On this b a s i s ,  the  peaks  at  The HPLC-MS of some tissue  organisms analyzed are given i n Appendix D.  M/Z 367  (Fig.  originally  M/Z  303  and  369  sample, which i s further evidence against their being  associated with t i n compounds. marine  is  the presence of cyclohexyltin compounds is  The " i s o t o p i c " pattern of the  with  this  4.10.3)  is  due  to  the  internal  extracts  of  The fragment ion at standard,  and  intended to be used for quantitation (see Chapter 3,  was  section  3.12) .  ii.  Shell extracts of marine organisms  The d i l u t e hydrochloric acid solutions of the s h e l l s into  methylene  Chapter 3,  chloride  section 3.6.1.  solutions,  was  extracted  using the extraction procedure of  The methylene chloride extract was reduced i n  volume and injected into the HPLC-MS, and the spectra were recorded.  No  butyltin  by  compounds  are  thermospray i o n i z a t i o n .  observed  in  the  mass  spectra  obtained  On analysis of the s h e l l extracts by mass  - 93 [TIC-7344H.  l » I - l l l » m El  H »  W t  "  W h  3.  Z  »  H 'l  1  i i 11 i i ii i i|i i  F i g s . 4.10.5:  I"I  i' i i i i 11 11 i  11 i  11  1 1 1 1 1 11  I  I  I  I  I  I  I I I  I  I  4S«  HPLC-MS of standard  [TIC-139740,  I  " «  "(C H ) SnCl" 6  11  3  1MX-2283S) E l  h  H  W W h H  *  'i i i ii i i i | i i i i i i i i i | i i i i i ' ' i '  F i g . 4.10.6:  2M  »SI  HPLC-MS of standard  | ' I »»»  '  I1  ISf  "(C H ) SnCl2" 6  11  2  I  I  I  I  I  I I  - 94 spectrometry using electron i o n i z a t i o n , weak peaks presence  of  tin  compounds  which  indicate  the  are obtained for the s h e l l extracts of the  Macoma nasuta.Bent-nose clam(Fig. 4.10.7). The f a i l u r e to observe these peaks i n the HPLC-MS i s probably due to matrix  effects. +  to (C^Ho^SnCl.  The fragment ion at M/Z 269 ( F i g . 4.10.7) i s assigned The other peaks at M/Z 213  C^HgSnCl and C ^ g S n  respectively.  and 177  are  assigned  to  The i s o t o p i c  pattern of these frag-  ment ions f i t s well with the calculated i s o t o p i c  pattern ( F i g . 4.10.8).  Although these fragment ions are capable of a r i s i n g either from d i b u t y l t i n d i c h l o r i d e or t r i b u t y l t i n c h l o r i d e , comparison of  the  peaks  at  (Fig.  the  intensities  M/Z 269 and 177 a r i s i n g from the s h e l l extract ( F i g .  4.10.7) with same peaks i n the mass spectra of the dichloride  of  4.10.9)  and t r i b u t y l t i n  reveals that the fragment ions obtained i n  standard  chloride the  shell  dibutyltin  (Fig.  4.10.10),  extract  of  the  Macoma nasuta Bent-nose clam originate mainly from t r i b u t y l t i n c h l o r i d e . It  is  s u r p r i s i n g to f i n d that the HPLC-MS does not detect fragment  ions of b u t y l t i n compounds  in  the  other  shell  extracts,  or  tissue  extracts, when the HPLC-GFAAS detects high levels of these species. Macoma nasuta Bent-nose clam i n whose s h e l l extract meter  detected  mass  spectro-  b u t y l t i n compounds also gives the highest concentration  of b u t y l t i n compounds as detected by HPLC-GFAAS observation  the  The  (Table  4.10.2).  This  tends to suggest that the l i m i t of detection of the HPLC-MS  method i s not low enough to detect these compounds. The HPLC-MS method as found i n t h i s study has a detection 0.3  ng/mL as Sn (Chapter 4, section 4 . 6 . 2 ) .  limit  of  The detection l i m i t of the  HPLC-MS i s estimated by r e p l i c a t e injections of  various  concentrations  CTIC-17653408, 100X-194060] EI 100 ^  9B  (-1  8*  H  "  W  "  z  w  40  h  30 20  H  10  z  269 212 I I'M I | II 240  100  ON  247  |291  LJC  I | II II.1.1. II •M.1. 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  I I'M | I I 260  280  300  320  340  360  380  400  58  90  h H 10  80 70 60  z  60  h  30  w  Z  H  40  76  20 10  1SS 121 I I I 4. l i ' i ' i ' i ' i ' i ' l ' l ' i ' l ' h i n ! ^T i I'I'I'I'I'I I I ri'l'l'l'i I T l ' l ' l i i i I'l'l I'I'I'I'I'I  ii  82  60  1  80  100  120  140  177  213 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 160 1B0 200 220 -1 1 1  ii  M / Z Fig.  4.10.7  :  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 t h e B e n t - n o s e c l a m Macoma n a s u t a  - 96 -  TOTAL ABUNDANCE 0.99935 AVERAGE MASS = 268.426350 MOST ABUNDANT PEAK= 269.011439 1  1 MASS  EXACT MASS  261 . 262 . 263 . 264 . 265. 266 . 267 . 268. 269 . 270. 271 . 272 . 273 . 274 . 275. 276 . 277 .  261 .014519 262 .017874 263 .012190 264 .013557 265 .011323 266 .012937 267 .010951 268 .012632 269 .011439 270 .012739 271 .010525 272 .013643 273, .014013 274 ,.017362 275, .012189 276. .015468 277 ,.018731  INTENS 2 .27 . 0,. 20 2 .29 , 1 .02 . 34 ,. 66 21 . 58 70, .21 32 ., 73 100. ,00 1 5 .67 . 37 ., 49 3. . 40 18. .05 1 ,. 66 4 .64 . 0. 42 0. .02  (a)  TOTAL ABUNDANCE= 0.99961 AVERAGE MASS = 211.309814 MOST ABUNDANT PEAK= 211.940879 1 MASS  EXACT MASS  INTENSITY  204 . 205 . 206 . 207 . 208 . 209 . 2 10. 211. 212. 213. 214. 215. 2 16. 217. 2 18. 219 .  203 .944092 2 .31 204 ,.947447 0, . 10 205, .94 1798 2 . 32 206 ,.942903 0, . 93 207 ,.941043 35 ,. 1 3 208, .942361 20. .27 209 ..940486 70. . 26 210. .942037 29 ..94 2 11..940879 100. ,00 212. .941515 1 1 .25 213. .939908 37 .,43 2 14. 943294 1 . 71 215. 943582 18. 21 216. 947028 0 . 84 217. 94 1613 4 . 66 218. 944949 0 . 21  (b)  TOTAL ABUNDANCE= 0.99971 AVERAGE MASS= 175.857509 MOST ABUNDANT PEAK= 176.972680 NOM MASS 1 69 . 170. 171 . 172 . 173 . 174. 175 . 1 76 . 177. 178. 179 . 180. 181 . 182. 183 .  EXACT MASS  INTENSITY  ( ) c  168. 975239 2 . 85 169 .,978594 0 . , 13 170. 973190 1 ,. 95 171 . 973995 1 .1 1 172. .972156 42. .70 173. .973590 24 . 65 174 ,,972067 73 .,00 1 75 .973908 , 29 ..05 176. . 972680 100. .00 177. .976078 4 . 59 178. ,973886 1 4 . .18 179. .977288 0, . 65 180. . 975692 17, . 92 181 ..979126 0, .83 182, .982401 0,.01  Fig. A.10.8: Theoretical mass spectral intensity pattern for + + + (a) (C H ) SnCl; (b) C H SnCl (c) C H Sn; 4  9  2  A  9  4  9  L3E379.11  CTIC-1086208,  100X-274160]  EI  100 >  9  0  [4 80  H  70  (/)60 Z (-1  40  2  30  H  247  2 0 269  10 0  j I I I I I I I I l|'l  2t0  T'I'I'II  240  1 1 f  260  11 11  I  3  0  4  rrr  I 1 ' 'I, 1•11 1, 1 1 1 1 1 1 1 1 1 1 1 1 1 j ''1 1 I 1 1 1 ri'Pfh 1 I 1 1 1 1 1 1 1 1 j 1 1 1 1 1 |, 280  300  320  1 1 1 , ,  1 1 11  340  360  380  400  M / Z 100 $7  90  > h H  " ^  6  0  I/)  50  Z  40  u  41  212  30  h  Z  H  2  0  155  10  i 4  1  1  1  1  !  " 1  1  w  0  Fig-  4.10.9:  1  1 1 1 M 111111111111111 03 iaa Electron  iTh  11111111111111  120  140  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  t • | M I I I I I'l'l |'| I I I I I 1 1 1  160 dibutyltin  180 dichloride  1•i  200  M  1 |'| /  ^  T  220  L32378.3 tTIC-561824. 100X-37088] EI 100  269  90 80 70 60 50 40 30 20 10 0  2  liiiii'l^iliiiinVi'i'i'i'i 220  240  1 1  260  'I 2  1 1 1  8  1  1  1  ?  ?13  9  1 1 1  0  |  1 1  3  ' 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 | | | | | | | | , "  ™*  340  360  3  ' „  ,  \ „  M / Z 100 41  90 80 70  57  60  .213  50 40  155  30  »77  20  0  19  ,69  10  rtrk  TT 40  ''I l ' l ' l ' | I I I I I | | | ' | | | | | | | | | ' | 60  F i g . 4.10.10:  80  'I'l'l'l" I I I'l'l I • I'l'l'lM'I'l'l !'! I • l^l'l I'l'l'l H I M I'l'l'l I'l'l 1  t  100  120  140 140  \ca 160  180  Electron ionization mass spectrum of tributyltin chloride  200 M / Z  fT  220  -  of  tributyltin  of  concentrations  Mg/mL a s At  Sn a r e  this  method,  at  There HPLC-MS  a  stored  acetone  certainty  whether  the  are  necessary  A.11  The  the  Such  on t h e  Oxinates  for  detected  the  1.09  the  HPLC-MS  tributyltin  from,  example  samples.  tissue  column w i t h  experiment, wait  the  if  the  their  it  as  waiting  an  cannot  be  the  samples chloride  said  with  be  stable  Further  studies  period  the  the  analytical  would  a long period.  of  the  tributyltin  chlorides  results  and  turn for  weeks),  suitable  weeks,  for  HPLC-MS  o r g a n o t i n compounds.  and T r o p o l o n a t e s  oxinates  and t r o p o l o n a t e s  ionization  i o n s m i g h t be o f use ligand  chromatographic  for  thermospray  e s p e c i a l l y the  the  to be  three  HPLC-MS  compounds, for  c h l o r i d e or  Sn)  standard  tributyltin  ions of b u t y l t i n using  the  found  of  extracted  fragment  have  HPLC-GFAAS  Whereas  period  Butyltin  from s o l u t i o n s by  Mg/mL a s  w a i t i n g p e r i o d (2-3  effect  fragment  4.11.1.  this  v e r i f y what  of  1.09  samples had to  matrix  c o u l d have  HPLC-MS  the  s o l u t i o n was a  tributyltin  Solutions  spectrometer.  should  of b i o l o g i c a l  to  mass  (about  freezer.  over  presence  experiment  During  or hexane  thermospray-MS.  q u a n t i t a t e d by HPLC-GFAAS m e t h o d .  because  i n the  for  by the  l a p s e between the  experiment.  the  Mg/mL as  spectrometer  time  -  into  3  detection  levels  standard  in  of  experiment,  HPLC-MS were  was  then  detected  mass the  i n acetone  less  not  limit  the  compounds  in  chloride  99  tropolone However, ligand  are  given  obtainable in  Table  in finger-printing butyltin is the and  u s e d as  an  complexes the  extractant  decomposed on  butyltin  moiety  100 Table 4..11.1:  Fragment ions of butyltin oxinates and tropolonates  Relative Intensity  Assignment  435 378 350 308 291 146  43821 189688 179435 247828 107661 1340672  (C H ) Sn0X 0.1 M (C H ) Sn0X N H A O A C (C H ) Sn0Ac (C H )SnOH (CAH ) Sn OX* + H  80% (5% acetic acid in methanol) + 20% (0.1M aqueous NH 0Ac)  378 146  599380 1071232  OX +  80% (5% acetic acid in methanol) + 20% (0.1M aqueous NH 0Ac)  465 146  76084 1071040  80% (5% acetic acid in methanol) + 20% (0.1M aqueous NH 0Ac)  412 350 308 291 123  57644 295424 187791 232119 610112  (C H ) SnT 0.1 M (C H ) SnOAc NH 0Ac (C H ) S^!0H (C H ) Sn T^ + H  80% (5% acetic acid in methanol) + 20% (0.1M aqueous NH 0Ac)  355 293 123  993655 496827 1227456  (C H ) S*T 0.1 M (C H ) SriOAc NH 0Ac T + H  80% (5% acetic acid in methanol) + 20% (0.1M aqueous NH 0Ac)  539 123  10454 1254528  -.Attganj C HqShT - H 0.1 M NH 0Ac H  Mobile Phase  (C H ) Sn0X 4  9  3  80% (5% acetic acid in methanol) + 20% (0.1M aqueous NH 0Ac) 4  (C H ) SnOX2 4  9  2  Ionizing Electrolyte  M/Z  Compound  4  9  3  4  9  2  4  a  9 3  4  9  9 3  (CAH ) SnOX  b  9  2  ?  H  0.1 M  0Ac  NHA 4  4  C H SnOX 4  9  3  C H SnOX O X + H  c  4  9  0.1 M  2  NH 0Ac  +  4  4  (C H ) SnT 4  9  3  4  (C H ) SnT 4  9  2  2  4  C H SnT 4  9  3  4  d  e  9  3  4  9 3  4  9 3  4  9  4  4  9  4  3  2  9 2  4  +  f  A  r+  4  3  4  OX - oxinate ion; OAc - acetate ion; T - tropolonate i on +  +  a . b . c . d . e . f = base peaks for (C H ) SnOX, (C H ) SnOX , C H SnOX , (C H ) SnT, (C H ) SnT and C H SnT respectively 4  4  9  3  4  9  9 3  2  4  2  4  9  9  2  2  3  4  9  3  101  eluting  at  observed  fragment  splitting  different  patterns  retention  ions  is  f o r such  times  made  by  -  (Fig. 4.11.1). comparison  with  the  - T I M E |,«7  4.11.1:  f.17  HPLC-MS t o t a l  o f the  theoretical  ions.  TIC  Fig.  Assignment  till  « • » »  i o n chromatogram  Cmin>  • • • »  of tributyltin  oxinate  -  4.12  -  Summary  The  results  compounds The of  102  are  type  of  present of  from  the  under  lumber  The  Pest  tributyltin  mechanisms  was  of  calls exist  more  i n the  Although  due  Act  an  are  present  is  of  maritime also  source  i n the  marine  registered  in  or  Canada  preservation.  organisms  is  because  expected  if  dibutyltin  is  tributyltin.  carried  out  on the  organisms the  the  activities  lumber  organisms  t h a t b u t y l t i n compounds  of  butyltin  B r i t i s h Columbia.  compounds  general  i n the  of  indication of  either  for  causing  of  to  tributyltin  0  understanding  for  since  of  tributyltin  oysters^  marine  The p r e s e n c e  organisms  d i b u t y l t i n i n these marine  reports in  shells  fouling is  metabolite  investigation  better  of  compounds  Following  of  Control Products  an e s t a b l i s h e d  the  that  show t h a t h i g h l e v e l s  compound f o u n d i s  presence  industry,  detection  study  i n some m a r i n e  The  implies  the  present  organotin  pollution.  organisms  the  role  abnormal presence  because of  perturb  the  such  into  the  shell  calcification thickening,  an  compounds  in  of b u t y l t i n  i n f o r m a t i o n might  shell  in  t r i b u t y l t i n and d i b u t y l t i n i n  investigation  the  lead  to  a  detoxification. the  shells  form i n which these  tin  analyzed compounds  shells. the  mass  HPLC-MS,  this  organotin  compounds.  spectrometer  technique  is  still  is not  a well  very  specific  adapted  for  the  detector analysis  in of  103 -  BIBLIOGRAPHY  1.  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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, andtightlyclosed 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 B U T Y L T I N OXINATES AND TROPOLONATES  J  j  .,,77  j  1  1 1  ' i i • 1/ | i i i i i i i i n i i i i |  2  Fig-  B-2:  1  H  NMR  s p e c t r u m of t r i b u t y l t i n  tropolonate  1  0  PPM  Irl  111111111111111111111111111  9  8  Fig. B-3:  7  1  H  i i i i | i i i i | 6  NMR  I I  i  i|  i  I I  5  spectrum o f  i | i  i  i  i| 4  I I  • ' I ' i  | i 3  i i i i  I  2  i i i i I i i i i | i i i ' I i ' ' i | ' • i 1 1 0 PPM  dibutyltin bistropolonate  117  APPENDIX  T H E O R E T I C A L MASS S P E C T R A L  C  INTENSITY  CHLORIDES AND D I P H E N Y L T I N  PATTERN FOR STANDARD B U T Y L T I N  DICHLORIDE  118  TOTAL ABUNDANCE' 0.99884 AVERAGE MASS= 348.170121 MOST A B U N D A N T PEAK= 349.155602 1 MASS  EXACT  341 .  341 . 1 5 7 9 5 8 342 . 161364 343 . 155961 344. 157170 345. 154821 3 4 6 . 156771 347. 154980 348 . 157085 349. 155602 350. 158865 351 . 1 5 7 1 19 352 . 160105 353 . 158463 354 . 161822 355 . 164947 356. 168475  342 . 343 . 344 . 345 . 346 . 347 . 348 . 34S . 350. 351 . 352. 353 354 355 356  MASS  . . . .  INTENS 2 .73 0 . 47 1 . 90 1 .29 41 . 0 5 28, .84 73, .32 36, .99 1 0 0 .. 0 0 16 ,. 9 1 15. .06 2,, 44 17. . 38 2 ., 9 8 0 . . 28 0 . .01  +  Fig. C - l : Theoretical mass spectral intensity pattern  for (C^Hg^SnCOCCl^^  TOTAL ABUNDANCE0.99874 AVERAGE MASS" 326.505809 MOST ABUNDANT PEAK327.053230 NOM MASS 319. 320. 321 . 322. 323. 324 . 325. 326. 327. 328. 329. 330. 331 . 332 . 333. 334. 335.  Fig. C - 2 :  EXACT MASS  INTENSITY  319.056384 2 .25 320.059807 0 .29 321.054017 2 .27 322.055560 1 .08 323.053279 34 . 25 324.054852 22 . 5 0 325.052900 70 .13 34 .77 326.054790 3 2 7 . 0 5 3 2 3 0 100 . 0 0 328.055004 18 .99 329.052389 37 .78 330.055570 4 .67_ 331.055924 18 .03 332.059344 2 .25 333.054154 4 .67 334.057297 0 .57 335.060036 0.04  Theoretical mass spectral intensity pattern + (CA,H ) SnClC0(CH )2 9  2  3  for  119  TOTAL ABUNDANCE" 0.99910 A V E R A G E MASS= 325.542379 326.0817B0 MOST ABUNDANT PEAK = NOM MASS 318. 319. 320. 321 322. 323 324 325 326 327 328 329 330. 331 332 333 334 Fig.  . . . . . . . . . . . .  EXACT 318 . 319. 320. 321 . 322 . 323. 324 , 325. 326. 327 , 328 , 329, 330, 331 , 332, 333, 334,  INTENSITY  MASS  084945 088404 082678 084164 081999 083421 081427 083346 081780 083640 080814 084126 084504 087863 082757 085885 089157  2.24 0.31 2.27 1 . 10 3 4 . 17 22 . 83 70.14_ 35 . 45 100.00" 20.01_ 37.70 5 .07 17.96" 46 65 0.63 0.04  C - 3 : T h e o r e t i c a l mass  spectral  intensity  pattern  for(C4H9) SnCl 3  TOTAL ABUNDANCE0.99919 AVERAGE MASS290.089661 MOST ABUNDANT PEAK 291.113623  Fig.  1 MASS  EXACT  283. 284 . 285 . 286 . 287. 288 . 289. 290. 291 . 292 . 293 . 294 . 295. 296. 297.  283. 284 . 285. 286. 287 . 288 . 289. 290. 291 . 292. 293. 294 . 295. 296. 297 .  MASS  INTENS  116092 2 . 76 119523 0 . 38 1 14 2 0 0 1 .91 115197 1 .24 113004 41 ,. 58 114531 27 ,. 7 7 113137 73. .27 114965 34 .. 8 7 113623 100. , 0 0 116990 1 3 . , 56 115040 14 ., 55 118168 1 . 93 116584 1 7 . ,51 120038 2 . 42 123254 0 . 15  C - 4 : T h e o r e t i c a l mass  spectral  intensity  pattern  for  +  (C^Htj^Sn  120  TOTAL A B U N D A N C E 0.99896 A V E R A G E MASS= 349.134305 MOST A B U N D A N T P E A K 350.126996 3  3  MASS 342 . 343.  EXA CT  344 . 345 . 346 . 347 . 348 . 349 . 350.  342 343 344 345 346 347 348 349 350  351 352 353 354 355 356 357  35 1 352 353 354 355 356 357  Fig.  . . . . . . .  MASS  .129396 .1 3 2 8 4 1 .127446 .128574 . 126245 .127951 .126407 .128346 126996 .130323 .128558 .131583 .129887 .133336 .136087 .137449  INTEN 2 . 73 0 . 44 1 .91 1 . 27 41 ,. 1 4 2 8 .. 4 4 7 3 .. 2 7 3 6 .. 3 1 1 0 0 .. 0 0 15 .. 9 0 15 .. 1 3 2 .. 3 1 17 .. 4 1 2 .. 8 0 0 . , 28 0 . .01  C - 5 : 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 ) Sn[CO(CH3)2]2 A  9  2  TOTAL A B U N D A N C E 0.99874 A V E R A G E MASS= 326.505809 MOST A B U N D A N T P E A K = 327.053230 3  MASS 319. 320. 321 . 322 . 323 . 324 . 325 . 326 . 327 . 328 . 329 . 330. 331 . 332 . 333 . 334 . 335 .  Fig.  EXACT  MASS  319. .056384 3 2 0 .. 0 5 9 8 0 7 3 2 1 ,. 0 5 4 0 1 7 3 2 2 ,. 0 5 5 5 6 0 3 2 3 ,. 0 5 3 2 7 9 3 2 4 .. 0 5 4 8 5 2 3 2 5 .. 0 5 2 9 0 0 3 2 6 ,. 0 5 4 7 9 0 3 2 7 ,. 0 5 3 2 3 0 3 2 8 .. 0 5 5 0 0 4 329 . 0 5 2 3 8 9 330 . 0 5 5 5 7 0 331 . 0 5 5 9 2 4 332 .059344 333 .054154 334 . 0 5 7 2 9 7 335 . 0 6 0 0 3 6  I NTEN: 2 .. 2 5 0 .. 29  2 .. 2 7 1 .. 0 8 3 4 .. 2 5 2 2 .. 5 0 70. . 1 3 3 4 .. 7 7 1 0 0 .. 0 0 1 8 .. 9 9 3 7 ,. 7 8 4 ,. 6 7 18. . 0 3  2 .. 2 5 4 ., 6 7 0.. 5 7 0.. 0 4  C-6: 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 + (C4H ) SnClCO(CH )2 9  2  3  121  TOTAL A B U N D A N C E " 0.99902 AVERAGE M A S S " 292.017964 MOST ABUNDANT P E A K " 293.056543 NOM MASS 2B5. 286. 287. 288. 289. 290. 291. 292. 293. 294. 295. 296. 297. 298. 299.  Fig.  E X A C T MASS 285.058969 286.062324 287.056972 288.057996 289.055989 290.057485 291.055946 292.057864 293.056543 294.059850 295.057871 296.061092 297.059460 298.062784 299.065151  INTENSITY 2 . 78_ 0 . 31 1 . 93_ 1 . 21 41 . 74 26. .96 7 3 . .17 3 3 . 47 100. 0 0 1 1 ,.4 8 14 ., 7 5 1 ., 6 4 1 7 . ,61 2.03_ 0 .,17  C - 7 : Theoretical mass spectral intensity pattern for +  (C H ) SnOOCCH > 4  9 2  3  TOTAL ABUNDANCE 0.99935 A V E R A G E MASS= 268.426350 MOST ABUNDANT PEAK= 269.011439 3  1 MASS  EXACT  261 . 262. 263 . 264 . 265. 266. 267. 268. 269 . 270. 271 . 272 . 273 . 274 . 275. 276. 277.  261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277  Fig.  MASS  .014519 .017874 .012190 .013557 .011323 .012937 .010951 .012632 .011439 .012739 .010525 .013643 .014013 .017362 .012189 .015468 .018731  INTENS 2 .27 0,. 20 2 .29 1 .,0 2 34 ,. 66 21 ,. 58 70, .21 3 2 . . 73 100. , 0 0 15 ,. 67 37 ,. 49 3. . 4 0 18 .. 0 5 1 .66 4 ., 64 0 . , 42 0 ..02  C-8: Theoretical mass spectral intensity pattern for  122  TOTAL ABUNDANCE 0.99919 AVERAGE MASS= 267.952422 MOST ABUNDANT PEAK= 269.020047 3  Fig.  MASS  EXACT MASS  INTEN:  261 . 262 . 263 . 264 . 265 . 266 . 267 . 268 . 269 . 270. 271 . 272 . 273 . 274 . 275 .  261 .022582 262 .025937 263 .020531 264 .021482 265 .019578 266 .020938 267 .019523 268 .021441 269 .020047 2 70 .023536 271 .021434 272 .024767 273 .023044 274 .026581 275 .027958  2 . 80 0..22 1 .93 . 1 .. 1 5 42 . 10 25 . 79 73 . .04 31 .36 . 100..00 8 .. 26 14 . 78 1 .. 1 9 1 7..76 1 .. 45 0., 16  C - 9 : T h e o r e t i c a l mass s p e c t r a l + (C H )SnClCO(CH ) A  9  3  intensity  pattern  for  2  TOTAL ABUNDANCE 0.99910 AVERAGE MASS= 325.542379 MOST ABUNDANT P EAK= 326.081 780 1 NOM MASS EXACT MASS INTENSITY 3  318. 319 . 320. 321 . 322 . 323 . 324 . 325 . 326 . 327 . 328 . 329 . 330. 33 l . 332 . 333 . 334 .  Fig.  318 .084945 2 .24 3 I 9.088404 0 .31 320 .082678 2 . 27 321 .084164 1 . 10 322 .081999 34 . 1 7 323 .083421 22 . .83 324 . .081427 70,. 14 325 , .083346 35 . , 45 326 . .081780 100,.00 327 .083640 20,.01 328 , .080814 37 . , 70 329 , .084126 5 .07 . 330..084504 1 7..96 33 1..087863 2 .46 . 332 , .082757 4 . 65 333 . .085885 0..63 334 . .089157 0.,04  C-10:  T h e o r e t i c a l mass s p e c t r a l  +  (C H )SnOOCCF3C H O A  9  A  e  intensity  pattern  for  123  TOTAL ABUNDANCE= 0.99949 AVERAGE MASS= 331.998936 MOST ABUNDANT PEAK = 332.993940 NOM MASS 325 . 326 . 327 . 328 . 329 . 330. 331 . 332 . 333 . 334 . 335 . 336 . 337 . 338 . 339 . 340.  Fig.  EXACT MASS  INTENS  324 .996368 2 . 74 325 .999723 0..43 326 .994494 1 .91 . 327 ..995545 1 .. 27 328 .993193 4 1 ,. 1 7 329 .995148 28 . .37 330..993390 73 . 27 331 . .995454 36 . , 15 332 . , 993940 100.,00 333 . 997230 15 , . 66 334 .. 995477 15., 1 1 335., 998515 2 . 28 336 . .996869 1 7..43 338 . .00017 3 2 .76 . 339 , .002896 0.,.27 340..004421 0..01  C - l l : 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  for  )^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.  Fig.  EXACT MASS  INTENSITY  300. 951917 2 . 24_ 301 .955272 0. 30 302 .949748 2.27 303 .951136 1.11 34.20_ 304 .948860 22.76_ 305 .950315 70.13_ 306 .948489 3 5.30_ 307 .950209 308 .948685 100.00_ 19.78_ 309. 950581 37.68_ 310. 947652 4 . 99_ 311. 951 1 15 1 7 . 9 6_ 3 12. 951447 2 . 4 1_ 313. 954751 4 . 66 314. 949763 0.61 315. 952774 0.04 316. 956129  C-12 : 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  for (c^H^S^C  1  124  APPENDIX D  HPLC-MS CHROMATOGRAM  AND MASS SPECTRA  OF T I S S U E  OF SOME MARINE ORGANISMS  EXTRACTS  - 125 T I M E  (min) 23.48  1B0X-1683904  Fig. D-1:HFLC-KS total ion current chromatogram of oyster tissue extract 193  [T1C=1176968,  188X«64375]  EI  z  u H  3B  A  1 350  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  [TIC-516016.  ~i—i—i—i—r  200  EI  "T~i—i—r—i—r*  Fig. 278  100X»372661  tTIC-594624,  D-3:  250  Mass  1O0X-78876]  spectra  300  of  35J0  position  *  in Fig.  M / Z  D-l.  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  M / Z  36B  Fig. D - 4 : Mass spectra of position * in Fig. D - l . *  corresponds  to  position  on t h e  horizontal  axis  of  Fig  - 127  400  Fig.  D-5:HPLC-KS  198  total ion current chromatogram of Butter clam tissue extract  [ T I C - I 158336.  100*.102140]  EI  > 8  h H  7  I/)  u h  •W Fig. D-6: *  45  M / Z  Mass spectra of position * in Fig. D-5  corresponds to p o s i t i o n  on the h o r i z o n t a l  axis  of F i g .  D-5  128 2\%  [TIC-S87088.  100X-18874]  EI  v r ! i i i i"i i i i 400 f^l / ^ 1  Fig. D-7: 248  [TIC-331056,  1 1 I |  I-  I I  4 5 e  Mass spectra of position * in Fig. D-5  100X-57335]  205  I  m  EI  231 I  1  I  I  I "I  I  I I  1  |  I  l'  1  111  I  I  I  |  I I  350  Fig. D-8: Mass spectra of position * in Fig. D-5  -| 4  I  "  I  I  I  I  I  I  M / Z  * corresponds to position on the horizontal axis of Fig. D-5  I  I  | 4 5 A  - 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 198 [TIC-166528,  I00X-15585) EI  175  >  h H  (fl  z w  ]—I 200  T  I  I I  i i  r  i  i  |  I —r • i 'i  300  i • i'  |  i  i  I  350  i  i—i  i  i  I—|  I  i  400  Fig. D-10: Mass spectra of position * in Fig. D-9 * corresponds to position on the horizontal axis of Fig. D-9  i  I—i  1^1  /  i  i  i  i  | 450  - 130 -  209  [TIC-243«88.  ieflX-41758]  EI  (I)  w Z  rl—i—r-i—i—r  |  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  I  i  i  i  i |  - 131 ( min  T I M E 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  [TIC-255156.  100X'20012]  EI  175  h  H 14  z w h  z H  355  i—I r i 200  Fig.  D-13:  ~i 250  r n r ^ - M r r | l—r ,  , , ,  300  ,377  r 'i—I'T i ' i — i • j • i — i ' ' i "[' i ' Y ± 350  JS^  /  *Z  400  Mass spectra of position * i n 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  213  ,  i' "!1  Fig.  D-14:  Mass  1  1260  278  309  ,327  |  1 J - i - T T ^ - n - r ^ r V - r - r - i - V i i I' | ' i i  spectra  of  position  *  in Fig.  1  ^  I  y  1  1  TIME:  Cmin> 14:12  1001-1216064  f 200 250 300 D-15:HPLC-MS t o t a l i o n c u r r e n t c h r o a a t o g r a n o f B l u e mussel M v t l l u s edulls t i s s u e e x t r a c t  Fig.  100  150  1  D-12  1 1 : 49  50  1  16:3  133 -  190  CTIC-1216064. 100X-116236)  EI  LxiJL i i i r  T  350  300  Fig. 204  D-16:  ITIC-433488.  181  i  ,  ,203 |  Mass  100X-17500]  of p o s i t i o n  *  in Fig.  D-15  EI  ,215  i i—i r i" V i '| I 'n—I 200  spectra  r i r  M / Z  i "i—r"n—1  1[ 1  250  I  I T  'I  I  |  I ~f  300  ri—|  i V ' I—I' i T I Y 1" 1  fVj /  Z^l  Fig. D-17: Mass spectra of position * i n Fig. D-15 350  i—i 400  * corresponds to position on horizontal axis of Fig. D-12  i—r  - 134 T I M E  TIC  11:49 100  14:12  r  100X=514224  ,  (min)  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 198 100  [TIC-514224.  100X-30G43]  EI  176  b H  60  w u ^  30  H  200  Fig. i)-iy:  ' I  1  250  1  n—II  Mass spectra of position * in Fig. D-18  I I I  I  1  400  1 1  1  - 135 240  [TIC-89420.  208  Fig. 248  t  100X=20512]  • T—I—i—i—i—r - i  i—i—r~i—i—r  r~]  r~r  300  ~\—i—i—i—i—i  r  M / Z  350  400  D-20: Mass spectra of position * in Fig. D-18 [TIC-101616,  1 0 0 X - 3 4 8 0 7 ] EI  - | — n — T T " ~ i — i  Fig.  EI  200  D-21:  r i — i — i — I — r n — r ~ n — r ~ 1 — i 250  r ~ | — i — r 300  H—i—i—|—i—i—r 350  M  Mass spectra of position * i n F i g . D-18  I  i — i — i  /  Z  * corresponds to position on horizontal axis of Fig. D-20  |  i  *BB  i—i—r  

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