SYNTHESIS, CHARACfERIZATION, AND APPROACHES TO THE ANALYSIS BY HPLC-THG-AAS OF TRIMETIiYLSELENONIUM, SELENONIUMCHOLINE AND SELENONIUMACETYLCHOLINE CATIONS.
sv ALEXIS HUYGHUES-DESPOINTES
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A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science
Department of Food Science and Agricultural Chemistry. McGiII university, Macdonald Campus, Montreal, Quebec March,1991 i.. Suggested short title: Synthesis and analysis of selenonium cations by HPLC·THG-AAS. ABSTRAcr
Selenonium cations are electron deficient spedes in which the central selemum atom is bonded to three carbon chains (aryl or alkyl). Trirnethylselenonium iodide was synthesized elther by the methOlJ of Khun et al. (1986) or electrochernically. Se!cnoniumcholine and selenomumacetylchohne were synthesized by reaction of rnethyllithium with metallic selenium to produce methylselenohtlllum wlllch was, in turn, reacted with the appropriate alkylbromide. The selenide thus formed was further methylated at the selenium atom with methyl iodide in methanol in the presence of sodium tetraphcnylborate. Aftcr seve raI recrystallizations the selenonium analytes were charactenzed by AAS. Fr-IR. IH-NMR. 13e_ NMR, F AB· MS and LAMMA spectroscopic techniques and used as standards for analyucal method!> development.
The analysis was performed by high performance Iiquid chromatography wlth.atomlc ab!>orptlOll detection. The chromatography on a cynopropyl silica bonded phase was optlmlzed for mobIle phase composition by response surface analysis. The resulting surface response plots permltted a dltfcrentlatloll between the mechanisms of action of two mobile phase modlfiers: tnethylamme and tnmethybulfonJUm
iodide. The improvement in chromatographie efficiency resulted ln two to three fold dccrea!>c ln the Ilmlt of detection. An extraction procedure WIth Iiquefied phenol was evaluated for the determll1.ltlOn. by HPLC-AAS, of traces of selenonium catIons in biologlcal sampI es. The advantages and !>holtcommg!> of the HPLC-THO-AAS approach are discussed. RESUME
Les cations de sélénonium sont des espèces défi-::itaires en électron03, ou l'atome de sélénium central est lié à trois chaines carbonnées de type aryl ou a1kyI. La méthode de Knun et col. (1986) ainsi qu'un procédé électrochimique furent utilisés pour réaliser la synthèse de l'iodure de tnméthylsélénomum. La sélénoniumcholine et la sélé'loniumacétylcholine furent générées grâce à l'action du méthyllithium sur le sélér.ium métallique donnant du méthylsélénolitium; ce dernier étant par la sUite mis en présence de l'alkylbromide approprié. Le sélénide formé fut ensuite méthylé, dans du méthanol, au nIveau de l'atome de sélémum avec de l'iùdure de méthyle en présence de tétraphénylborate de sodium. Après une série de recrystallisations-cha.actérisations par diverses téchniques spéctroscoplques (SAA, IR-TF, RMN)H, RMN)3C, SM·FAB et LAMMA), ces divers Ions sélénonium furent uhtisés pour le développement de la méthode analytique.
La séparation et la quantification de ces produits furent réalisées par une chromatographie lIquide haute p"rformance (HPLC) couplée à un spectrophotomètre d'absolption atomique (SAA). L'optimisation de la chromatographIe, dont le support était une sIlice gréffée par des groupements cyanopropyl, fut permise grâce à l'étude de réponses de c;urface. Ces analyses statistiques ont également permis d'élUCider le méchanisme d'action de deux substances modifiant la phase mobIle: la tnéthylamme et le triméthylsulfomum IOdide. L'amélioratIOn de l'éfficacité de la chromatograplue a diminué la lImite de détection d'un facteur pouvant varIe; de deux à trois. La procédure d'extraction à partir de phénol liqUide fut évaluée par la quantification de traces de cations de sélénium dans des entités bIOlogiques par le système HPLC·SAA. Les avantages et les inconvenients de cette méthode furent ensUite discutés.
ü ACKNOWLEDGEMENTS
The: The a'\tthor would also like to thanks prof L Van Vaeck for the LAMMA !>pectra and S. MandevIlle for 'lis preciolls help Wlth the FT-IR. The author is particularly gratefui for the help and friendshJp that Georges·Mane Momplalslr, and Tian Lei. fellow-workers, proVlded constantly. iii TABLE OF CONTENT ( Page ABSTRA cr...... ___.. ____ ...... _ ...... _ ...... i RESUME ...... __...... _...... _ ...... - ...... ii ACKN OWLEDG EMENTS _...... _ ...... _..... __ ...... iii TABLE OF CONffiNT .... _ ...... __...... _ ...... _._...... iv LIST 0 F TABLES ...... _. __... __ .. _ ...... _._ ...... _ ...... ix LIST OF FIGURES ...... _ ...... m ...... __ ...... xi CHAPTER: 1: INTRODUcrION ._...... __...... 1 1.1. GENERAL CONSIDERA TIONS ...... __...... _...... 2 1.2. s,ELENJUM COMPOUNPS PRESENT IN FOOPS...... ,3 1.3. METABOLISM OF SELENIUM ...... _ ...... 5 1.2. TOXICITX OF SOME SELENWM COMPOUNPS ...... 6 ( 1.3. BIOA V AILABILITY OF SOME SELENlJ,lM COMPOUNDS AND SeME FOODS AND FEED~ ...... 6 II: SYNTHESIS OF SELENONJUM COMPOUNDS...... 11 2.1. nIE PREPARATION ANDPROPERTIES OFSELENONIUMCOMPOUNDS: A LrrERATURE REVIEW ...... _...... 12 2.1.1. METHODS OF SYNTHESIS ...... M ...... _ ...... 12 2.1.1.1. REACI10N OFDIALKYLSELENlDES (R2SE) wrrn ALKYL HALIDES (RX) ...... 12 2.1.1.1.1. Symmetncal Selenides ...... _ ...... 12 2.1.1.1.1.1. R2Se + RX -----> R3Se+X- ...... 12 2.1.1.1.1.2. R2Se + R'X ----~ R'2R'Se+X· ...... 13 2.1.1.1.2. Unsymmetrical Selenides (RR'§e) ...... 13 ( 2.1.1.1.2.1. R'SeR t RX -----> R2R'Se+X· ...... 13 2.1.1.17_2. R'SeR t R"X ----> RR'R·Se+X· ...... 16 2.1.1.1.3. Cyclic Selemdes with Alkyl Halides ...... 17 iv TABLE OF CONTENT (contmued) Page 2.1.1.2. REACf!ON OF ORGANIC SELENIDES WTIlI OrnER ALKYLA TING AGEN1'S ...... 18 2.1.1.21. Dimethylsulfate...... _ ...... 18 2.1.1.22. Triflate Salts...... 19 2.1.1.2.3. Miscellaneous ...... 19 2.1.1.3. REACTION OF OTHER ORGANIC SE SPECIES ...... 19 2.1.13.1. Diselenides and/or Selenols ...... 19 2.1,1.3.2 Selenoxides ...... 20 2.1.1.4. REACI10N OF INORGANIC SE SPECIES...... 20 2.1.1.4.1. Selenium Halides ...... _ ..... ~_ ...... 20 2.1.1.4.2. ElcmentaJ Selenium ...... 22 2.1.1.4.3. Sodium Selenite ...... 22 2.1.2. REACfIyrrœs OF SELENONIUM COMPOUNDS ...... 22 2.1.2.1. CHEMICAL PROPERTIES ...... 22 2.1.2.1.1. Anion-exchange Reactions ...... 22 2.1.2.1.2. Stability...... _ ...... 24 2.1.2.1.3. Alkylatmg Propertles of Selenomum Compounds ...... 25 2.1.2.1.4. Ylid Formation ...... 25 2.1.2.1.5. OXIdation ...... _ ...... _ ...... 26 2.1.2.2. PHYSICAL PROPERTIES ...... _ ...... 26 2.1.2.2.1. OptlCaJ Resolution ...... 26 2.1.2.2.2. d·Orbltal Resonance ...... 27 2.1.2.2.3. Nuclear Magnetic Resonance (NM R) ...... 28 2.1.2.2.4. X-ray Studies ...... 28 2.1.2.2.5. Infrared Spectroscopy ...... 28 2.1.2.2.6 Mass Spectrometry ...... """ ...... 30 v TABLE OF CONTENT (continued) Page 2.2. SYNTHESIS OF TRIMETHYLSELENONWM. SELENONIUMCHOLINE AND SELENONIYMACETYLCHOUNE SALIS ...... 31 2.2.1. MArnRlALS AND MEJ1-IQill ...... 31 2.2.1.1. REAGENfS ...... m ...... _._ ...... 31 2.2.1.2. INSTRUMENTS ...... _...... 31 2.2.13. SYNTIIESIS OF TRIMErnYLSELENONIUM IODIDE ...... 32 2.2.1.3.1. Chemical Synthesis .... w ...... 2.2.2. RESUL'CS AND OISCUSSION..... _. _____..... _ ...... 34 2.2.2.1. TRIMErnYLSELENONlUM IODIDE ...... 34 2.2.21.1. Chemical Synthesis ...... __ .... _ ...... 34 2.2.2.1.2 Electrochemical Synthesis...... 37 2.2.2.2. SELENONIUMCHOLINE AND SELENQNIUMACE'fYLCHOLINE SALTS ...... 38 2.2.2.2.1. Selenomumcholine Salt trom Dimethyl Selenide ...... 39 2.2.2.2.2 Selenomum Salts from Metallic Selenium ...... 40 2.2.2.3. MASS SPECfROMETRY...... _ .. _ ...... 43 2.2.3. CONCLUSION .... _...... _ ...... _...... _ ...... 50 vi TABLE OF CONTENT (continued) Page III: ANALYSIS OF SELENONIUM COMPOUNDS ...... 51 3.1. ISOLATION, CBARAcrERIZ.'\TION AND DETERMINATION OF SELENONlyM ANALXTES: A LITERATIJRE REVlEW ...... 52 3.1.1 ISOLATIONlCHARACrERlZATION OFTRIMETI-lYLSELENONIUM "'.ND. SE-METIfYLSELENOMETIIIONINE ...... 52 3.1.2 APPROACHES Ta THE ISOLATIONmETERMINP.TION OF SELENONWM COMPOUNPS ...... 55 3.2. ANAL YSIS OF TRlMETHYLSELENONIUM. SELENONIUMCHOL!NE AND SELENONIUMACETYLCHOUNE CATIONS ...... 64 3.2.1. MA TERIALS AND METHODS ...... 64 3.2.1.1. RE..'\GENlS AND STANDARDS .. _...... 64 3.2.1.2. INSTRUMEN1'S ...... 64 3.2.1.3. CALIBRA TION...... _...... 65 3.2.1.4. HPLC·AAS INl'ERFACE ...... _ ...... 65 3.2.1.5. HPLCCOf'ljDmONS ...... 6H 3.2.1.6. OPT1MlZA TION PROCEDURE ._ ...... 6H 3.2.1.7. EXTRACI10NS ...... 6H 3.2.2. RESUL'l'S AND DISCUSSION ...... _ ...... ({j 3.2.2.1. CHROMATOGRAPHIC OPTIMIZATION ...... 69 3.2.2.2. CALffiRA TION AND LIMIT OF DETEcnON (LOD) ...... 93 3.2.2.3. SAMPLE EXTRACI10NS AND ANAL YSIS ...... 97 3.2.3. CONCLUSION ...... _ ...... _ .. _ ...... 105 vii TABLE OF CCNTENT (continued) Page APPENDIX A ...... _ .. _____••••. ""...... ___ ...... _ ...... 106 APPEND IX B ...... _. ______...... " ...... _...... 115 R EFEREN CES ...... _____...... "...... , ..... _ ...... ". ____...... "...... 119 viii LIST OF TABLES Table Title Page Table 1-1: Selenium compounds important in nutntion (Combs and Combs, 1986). Table 1-2: Org Table 1-3: Acute toxicitics of selected selenium compounds (Combs and Combs, 1986). 7 Table 1-4: Biological availability of sorne selenium sources. 9 Table 2-1-1: Properties of sorne tnmethylselenomum salts. 23 Ta ble 2-1-2: Chemical shifts of sorne selenonium compounds (77Se, 13C and lH NMR). 29 Table 2-1-3: Sorne NMR spin-spin coupling constant of selenonium cornpounds. 29 Table 2-2-1: Elemental analyses (percent compositIOn) of the synthe tIC selenomum standards used for chromatography. 36 Table 3-2-1: Instrumental responses3 (Area/height ratIos) for the peak corresponding to selenoniumcholine IOn as a functiOn of mobile pha!)e composition. The data were used as lJlput for the multIple regres!>lon analysis (SAS). 72 Table 3-2-2: The resulting "best fit" polynomial model for the predlcted vanatlon 10 arealheight ratio for selenoniumchoiine as a funetion of mobIle phase compositiOn. 73 Table 3-2-3: Simplified polynomIal expressions for the predlctcd vaflatlon 10 area/height ratio for selenoniumcholine ion as a functlOn of mobIle phase composition with dlethyl ether (PE) fIXed at (a) 20% (v/v): (b) 30% (v/v); or (c) 40% (v/v). 74 ix , , LIST OF TABLES (continued) ( Table Tille Page Table 3-2-4: Observed and predicted capacity factors (k') for selenoniumcholine ion. The polynomial model accounted for less than 95% of the variation in the observed capacity factor resulting from changes in the mobile phase composition. 76 Table 3-2-5: Effect of temperature on reduced retention time and area over height ratio (A/h) for selenoniumcholine (ChoISe) and trimethyiselenonium (TMSe) ions. 91 ( (, x 1 UST OF FIGURES Figure Tille Page Figure 2-1-1: Reactions of dimethyl selenide and diethyl selenide with various bromoalkylcarboxylic acids and their corresponding esters. 14 Figure 2-1-2: Reactions of dimethyl selenide and diethyl selenide with various an''Y1 haiides. 15 Figure 2-1-3: Preparation of selenonium compounds from selenoxides. 21 Figure 2-2-1: Proton Nuclear Magnetic spectra in deuterated dimethylsulphoxlde (eHkDM~O) of: A, trimethylselenonium tetraphenylborate (300 MlL.); D, selenoniumcholine tetraphenylborate prepared from dimethyl selenide and 2-bromoethanol (300 MHz); and C, selenoniumchohne tetraphenylborate prepared from metallic selenium (200 MHz). 35 Figure 2-2-2: Proposed degradation pattern of the selenonium moiety associated with the positive ion mode LAMMA spectrum of selenoniumcholine tetraphenylborate. 45 Figure 2-2-3: Proposed degradation pattern of the tetraphenylborate IOn associated with the positive ion mode LAMMA spectrum of selenomumcholine tetraphenylborate. 46 Figure 2-2-4: Proposed degradation pattern of the selenonium cations associatcd with the negative ion mode LAMMA spectrum of: A, selenoniumcholine tetraphenylborate; B, selenoniumacetylcholinc tetraphenylborate. 47 Figure 2-2-5: Proposed degradation pattern of the tetraphenylborate Ion associated with the negative ion mode LAMMA spectrum of selenoniumcholine tetraphenylborate. 48 Figure 2-2-6: Proposed degradation pattern of the selenonium cations associatcd with the po~itive ion mode LAMMA spectrum of: A, selenoniumacetylcholine tetraphenylborate; D. trimethylselenomum iodide. 49 xi LIST OF FIGURES (continued) ( Figure Title Page Figure 3-2-1: Thermochemical hydride generator: (A) opt;cai tube (12 cm, 9 mm i.d.x 11 mm o.d.); (B) anaiyticai Oame tube (8 cm, 4 mm i.d. x 6 mm o.d.); (C) combustion chamber (3 cm, 7 mm i.d. x 9 mm o.d.); (0) thermospray tube (8 cm, 4 mm i.d. x 6 mm o.d.); (E) silica transfer line; (F) quartz insert to support the transfer Hne; (G) thermoelectric element; (H) modi6ed swagelock fittings; (1) analytical oxygen iniet (15 cm, 2mm i.d. x 3.2 mm o.d.). 66 Figure 3-2-2: Representative HPLC-rnG-AAS chromatograms of selenoniumcholine (a) and trimethylselenonium (c) ions obtained with methanolic mobile phases containing, 29% (v/v) diethyl ether, 1% (v/v) acetic acid, and 70% (vlv) methanol which were supplemented with: A. 0.050% (v/v) TEA, (new column), (Blais, 1989); B, 0.055% (vlv) TEA (3.96 x 10-3 M), (chromatogram recorded one year later); C, 1.52 g/L trimethylsulfonium iodide (1MSI) and 0.63 p.g as Se injected per ( analyte; D, 0.79 g 1MSIIL (3.88 x 10-3 M) and 0.311J.g as Se injected per analyte; E, 0.055% (vlv) TEA and 120 mg TMSIIL and 0.78 IJ.g as Se injected per analyte. 70 Figure 3-2-3: Linear regression analysis to test the concurrence of the observed k' vaiues for the selenoniumcholine cation with predicted values from the model. 77 Figure 3-2-4: Predicted response surfaces for peak AREA for selenoniumcholine (030 IJ.g as Se, plots A, B, and C) and trimethylselenonium (0.150 IJ.g as Se, plots D, E, and F) as a function of the concentration of TEA and TMSI in a mcthanolic mobile phase which contained 1% ace tic acid and; 20% (A, D); 30% (B, E); or 40% (C, F) (vlv) diethyl ether. 78 Figure 3-2-5: Predicted response surfaces for the capaclty factor (A, B, and C) and Area/height (D, E, and F) for selenoniumcholine ion as a function of the concentration of TEA and TMSI in a methanolic mobile phase ( which contained 1% ace tic acid and: 20% (A, D); 30% (B, E); or 40% (c. F) (v/v) diethyl ether. 80 xii LIST OF FIGURES (continued) Figure Title Page Figure 3-2-6: Predicted response surfaces for the C8(.1aclty factor (A, B, and C) and Area/helght (D, E, and F) for trimethylselenonlum ion as a function of the concentration of TEA and l'MSI in a methanolic mobile phase which contained 1% ace tic acid and: A and D 20%; Band E 30%; C and F 40% (v/v) diethyl ether. 81 Figure 3-2-7: HPLC-rnO-AAS chromatograms of (a) selenoniumcholine and (c) trimethylselenonium ions obtained with a methanolic mobile phase which contained 1% acetic acid and either: A. 0.0055% (v/v) TEA, 10 mgIL l'MSI. 40% (v/v) Ether; or B. 0.01% (v/v) TEA, 55 mg/L l'MSI, 20% (v/v) Ether. 84 Figure 3-2-8: Predicted relationship between the normalized area/height ratio for the peak corresponding to trimethylselenonium ion and the capaclty factor for that peak at constant levels of TEA. but variable levels of TMSI (data points 1-7 correspond to 10. 325.43.8.55.0.66.3, 77.5, and 100.0 mgIL respectively) ln the Methanol acetic acid mobile phase. Plots A, D, and 0 correspond to 20% (v/v) ether and 0.01, 0.055 or 0.10% (v/v) TEA respectively; plots B. E. and H correspond to 30% (v/v) ether and 0.01. 0.055 or 0.10% (v/v) TEA respectively; and plots C, F, and I correspl)nd to 40% (v/v) ether and 0.01.0.055 or 0.10% (vlv) TEA. 86 Figure 3-2-9: Schematic representation: (A) of the "c1uster" or "microdroplet" model with zones of high bonding densities and zones of low bonding denslties due to a non homogeneous distribution of silanols at the sllica surface and (8) of our model with an homogeneous distribution of sllanols differing in their accessibilities because of stene factors due not only to the grafted phase but principally to the parous nature of the "naked" silica itself. 89 xiii LIST OF FIGURES (continued) Figure Title Page ( Figure 3·2·10: HPLC-TIlO·AAS chromatograms of a mixture of: A. selenoniumcholine ion (a) and, trimethylselenonium ion (c) or B selenoniumcholine (a), selenoniumacetylcholine (b) and trimethylselenonium (c) ions. The analytes were eluted with an "optimized" solvent system r.Jnsisting of 0.055% v/v TEA, 100 mg TMSI/L and 40% v/v diethyl ether. 94 Figure 3·2·11: Regression analysis for the determination of the Iimit of detection. Peak ares vs quantity af selenoniumcholine ion (15.4 ng to 1540 ng as Se). 95 Figure 3·2·12: Regression analysis for the determination of the limit of detection. Peak ares vs quantlty of trimethylselenonium cation (15.4 ng ta 1540 ng as Se). 96 Figure 3·2·13: Regression ana/ysis for the determinatian of the limit of detection. Peak ares vs quantlty of selenoniumcholine canon (15.4 ng to 154 ng as Se). ( 98 Figure 3·2·14: Regression analysis for the determinatian of the Iirnit of detection. Peak area vs quantlty of trimethylselenonium ion (15.4 ng to 154 ng as Se). 99 Figure 3·2·15: HPLC·TIlO·AAS chromatograms at low levels of selenaniumcholine (a) selenoniumacetylcholine (b) and trirnethylselenonium (c) ions in a methanolic mobile phase containing 1% (v/v) acetic acid and 29% (v/v) diethyl ether: A, 0.055% TEA alone (each ana/yte =31 ng as Se; Blais, 1989); B and D, 0.075% 1EA plus 100 mg TMSI/L (each analyte =33 ng as Se): C and E. 0.075% TEA plus 100 mg TMSI/L (each analyte = 15.4 ng as Se); and F, 0.075% TEA plus 100 mg TMSI/L (each analyte = 7.7 ng as Se). The "s" indicates the solvent front. 100 xiv 1 UST OF FIGURES (continued) Figure Title Page Figure 3-2-16: HPLC-mG-AAS chromatograms resulting from: A. 10 ",L injection containing (a) selenoniumcholine (0.33 ",g as Se) and (c) trimethylselenonium (0.31 p.g as Se) standards; D, 50 ~L injection of control porcine kidney (10 g wet weight) extract; C. 50 ",L mjectlOn from porcine kidney (10 g wet weight) wluch had been splked wllh selenoniumcholine (3.3 ~g as Se) and trimethylselenomum ions (3.1 /-Lg as Se); and D,50 ",L injection of extract from control porcine k1dney (C) spiked, post extraction, Wlth selenoniumcholme and trimethylselenonium ions to result in 0.165 ",g Se and 0.155 J..Lg Se per injection. lOI Figure 3-2-17: HPLC-mG-AAS chromatograms resulting from: A. 10 ",L mjection containing (a) selenoniumcholine (0.33 ",g as Se) and (c) trimethylselenonium (0.31 p.g as Se) standards; 8. 50 ~L injection of control shrimp (10 g wet weight) extract; C. 50 p.L injectlOn from shrimp (10 g wet weight) which had been spiked with selenoniumcholine (6.6 ",g as Se) and trimethylselenomum ion (6.2 J1.g as Se); and D. 50 ~L injechon of extract (C) from shrimp whlch had been spiked, post extraction, (0.4 mL extract + 0.1 mL of standard) wlth selenoniumcholine and trimethylselenonium ions. 102 Figure 3-2-18: The influence of co-injecting mcreasing quantitles (D>C>8>A) of choline chloride on the chromatographie behavior of a mixture of selenoniumcholine and trimethylselenonium ions. 104 FigureA-l: Characterization of the tnmethylselenonium ion: A, fourier transform infrared spectrum of tnmethylselenomum reineckate; B. proton nuclear magne tic (200 MHz) spectrum of tnmethyl:ié)lenomum lodide ln 2H20 with TMS as an external standard; C. fast atom bombardment (positive ion mode, F AB+) mass spectrum of trimethylselenonium lodide in a g1yeerol matriL 107 FigureA-2: LAMMA mass spectra of tnmethylselenonium iodide in both the positive (A) and negative (8) ions mode. lOH LIST OF FIGURES (continued) Figure Tille Page Figure A-3: LAMMA mass spectra of trimethylselenonium tetraphenyll'orate in both the positive (A) and negative (8) ions mode. 109 Figure A-4: Spectral Characterization of selenoniumcholine tetraphenylborate prepared from metallic selenium: A. fourier transfonn infrared spectrum (residual acetone present); B, proton nuclear magne tic Resonance spectrum in eHkDMSO; C, 13e nuclear magnetic resonance spectrum in DMSO. 110 Figure A-5: Spectral e}1 racterization of selenoniumacetylcholine tetraphenylborate prepared from metallic selenium: A. fourier transfonn infrared spectrum (residual acetone present); B, proton nuclear magnetic resonance spectrum in eH]6-DMSO; C, 13e nuclear magne tic resonance spectrum in DMSO. 111 Figure A-6: Spectral information of interest to the Characterization of the selenonium compounds: A. fourier transform infrared spectrum of acetone; B, fourier transfonn infrared spectrum of sodium tetraphenylborate; C, I3e nuclear magne tic resonance spectrum in polysol with TMS as an internai standard. 112 Figure A-7: LAMMA mass spectra of selenoniumcholine tetraphenylborate in both the positIve (A) and negative (B) ions mode. 113 Figure A-8: LAMMA mass spectra of selenomumacety!choline tetraphenylborate in both the positive (A) and negative (8) ions mode. 114 Figure B-l: Linear regression analysis to test the concurrence of the observed k' values for the selenoniumacetylcholine cation with predicted values from the model. 116 xvi UST OF FIGURES (contmued) Figure Title Page Figure B-2: Linear regression analysis to test the concurrence of the observed k' values for the trimethylselenonium cation with predicted values from the moJel. 117 Figure B-3: Predicted response surfaces for the capacity factor (A. B, and C) and Area/helght (D, E, and F) for selenonlumacetylchollne Ion as a function of the concentration of TEA and TMSI in a methanolic mobile phase containing 1% acetic 3Cid and: 20% (A, 0); 30% (B, E); or 40% (C, F) (vlv) diethyl ether. 118 xvii 1 CHAPTERI INTRODUCTION 2 1.1. GENERAL CON~IDERATIONS The metalloid Selenium was discovered in 1817 by Jon Jakob Berzelius. This clement IS found ln the earth's crust principally as selenides of lead, copper, Mercury and salver. One of the world largest accessible supplies, if not the largest, is in Noranda. Ouehec, (Wilher, 1980). Selenium IS a by-product of copper ore refining and is widely used in electronic and electrical components. It IS found IR sorne medicinal shampoos. The amount of selenium present in certain crop foods and feeds was found to depend upon the level of selenium in soils and, to a minor extent, on anthropogemc actlVltles. SelenIte (SeO/> and selenate (SeO/l present in weil aerated alkaline soUs can be Incorporated Into terrestnal plants (Shrift and Ulrich, 1969; and Ulrich and Shrift, 1968). These aniomc selemum specles are also absorbed by aquatic plants (Cutter and Bruland, 1984; Bottino et al., 1984). Inorganic forms of selemum can be methylated by microorganisms to produce volatile selenium species (Doran and Alexander. 1977; Rearner and Zoller, 1980). Selenium rarely poses an industrial pollution problem. However ammals have been known to be poisoned by grazing on forage produced on soils rich in selemum. Plants contaU1Ing In excess of 5 J.l.g/g of selenium are considered to be dangerous to animais. In semiarid reglons of Canada. the U.S. and Mexico, the soils may contain as much as 80 J.l.g/g of selenium. nus 15 not consldered to pose a problem with cultivated crops, but in some selenium-accumulating forage plants selenium levels can exceed 15,000 J.l.g/g dry weight (Wilber, 1980). Plants are not the only concentrators of selenium. Manne fishes and other sea food~ have been found to be effiCIent selenium accumulators (Wdber, 1980). Selenium has been observed In manne tïsh meaIs at levels of 2 J.l.g/g, which is sorne 50,000 times greater than the concentration of Se in sea water (0.09 J.l.g/L of Se). Selenium has long been known for its toxic properties, especially to grazmg animais (Combs and Combs, 1986; Stadtman, 1974, 1979). Furthermore, it has long been considered to be a carcmogen. 1t 1: not until 1957 that selenium was demonstrated to be an essential nutnent (Combs and Combs, 19Hr,: Stadtman, 1974 and 1979). Moreover, selenium was discovered to have antlrnutagemc and anticarcinogenic propertles in ammals (Milner, 1984; Thompson, 1984; Cekan el al.• 1985: L'Abbe el al .• 1939 a. 1989 b; for more references refer to Combs and Combs. 1986) and posslbly humans (Combs and Combs, 1986). However, animais studies, that support the anticarcmogemc effect of selenium also uscd potentially toxic levels of selenium (Burk, 1986). Selenium dietary status appears to have also an effctt not only on immune response but also on prostaglandin synthesis (Combs and Combs, 1986; Sunde, 1984). 3 From the nutritional aspect, it is generally recognized that selenium deticiency poses a serious ( problem and is the cause of appreciable economic losses in the production of farm animais, where supplementation in the diet is considered to he essential. Selenosis can accur in sorne sporadic cases. After absorption by plants, inorganic selenium is metabolized into a variety of organic compounds. Plants, that can survive in highly seleniferous soils, are termed indicator or accumulator plants. They con tain high amounts of "free" organic selenium compounds. Dy contrast, non-accumulators cereal.' and forage crops, con tain selenium mainly in a incorporated form (selenoamino acids) as constituents of pro teins. The normal sources of selenium for farm animais are mainly of plant or marine (tish meals) origin. In the case of humans, cereals, seafoods, Mid animal meats are the major selenium sources in the diet. The selenium content of a widc variety of food~ representing a typical diet consumed in the United States, arranged in decreasmg order, is as follows: organ meats> seafoods > muscle Meats > grains and cereal products (depeonding on origin) > eggs > dairy products > fruits an~ "-.;etables (Levander, 1984). This gencral order may change somewhat depending on whether the food was produced in an area with soils rich or deticient in available selenium (Levander, 1984). Comparisons of the absolute selenium content ingested in the diet represent a rather Iimited view of the complexity of the selemum nutritional problem. 1.2. SELENlyM COMPOyNDS PRESENT IN FOODS Table l-llists sorne of the important selenium compounds in nutrition. Selenium exists in several oxidation states, not only in inorganic forms but also as a component of organic compounds. Table 1·2 iIIustrates further the large number of "free" organic selenium compounds that have been identified in plants. Meats mlght also con tains Many different "free" selenium compounds but in animais, to date, only proteins contaming selenium (Viljoen et al., 1989; Yang et al., 1987; Behne et al., 1988) and an enzyme (glutathione peroxidase) have been identitied (Combs and Corl!hs, 1986; Beilstein and Whanger, 1986; Stadtman, 1974, 1979,1980; Sunde and Hoekstra, 1980; Sunde, 1984). Selenocysteine is considered to be the major selenium compound found in selenoproteins, although selenomethionine has been suggested to be incorporated mdiscriminately in proteins instead of methionine (Combs and Combs, 1986; Bedstein and Whanger, 1986; Stadtman, 1974,1979,1980; Sunde, 1984). It is important to identify the type, and to de termine levels and fate of individual selemum compounds in foods/feeds as each different compound has a unique bioavailability and toxicity. ( Table 1-t: Selenium compounds important in nutrition (Combs and Combs, 1986). Oxidation states of Se Compounds Se2- ~~ (~)l Se (CH )2Se+ Sele~~methionine Sel\!nocysteine Se-methyl-selenocysteine Selenocystathiomne Selenotaurine Selenodiglutathione Amorphous selenium Red selenium (alpha monoclinic) Dark red selenium (beta monoclinic) Gray selenium (hexagonal) Table 1-2 : Organic selenium compounds reported in plants (Combs and Combs, 1986) Dimethyl selenide 3elenocysteine-selemc dCld Dimethyl diselemde Se-methyl-selenocystelne Selenomethionine Se-methylselenometluomne Selenocysteine Se-propenylselenocysteine selenoXlde Selenohomocysteine SelenometluomneselenOJode Selenocystathionine Se-contammg peptJdes Selenowaxes 1 5 1.3. METABOLJSM OF SELENIUM ( Selenium compounas are not o"ly Gompetitors for their sulfur isologs but, more importantly, they also partlclpate ln a vanety of unique biochemical processes (selenoenzymes such as gluthathione peroXidase and others, Stadtman, 1974, 1979, 1980; Sunde, 1984; Combs and Combs, 1986) after which they are tinally eliminated. Selenium after ingestion is excreted principally in the urine (Robinson et al., 1985; Combs and Combs, 1986) or through the lungs, if the organism has been exposed to high doses (Combs and Combs, 1986). Selenium is excreted in the feces to a measurable extent only when co ingested with arsemc (Wilber, 1980). Trimethylselenonium cation is the principal organIc sek:i1lum Metabolite found m the urine (Byard 1969; Pal:ner et al., 1969, 1970). Selemte can be methylated ln presence of glutathione and certain enzymes to fonn dimethyl selenide (Hsleh and Ganther, 1977; Hoffman and McConnell, 1987) and then trimethylselenomum (Mozier et al., 1988; Hoffman and McConnell, 1987). Trimethylselenomum ion has been found to be the urinary end proùuct of several selenium compounds such as selenite (Byard, 1969; Palmer et al., 1969; Kiker and Burie, 1974; NahapetJan et al., 1983), selenoamino acids (palmer et al., 1970; Foster et al., 1986 b; Kraus et al., 1985; NahapetJan et al., 1983), selenobetame [(CH3)2Se+CH2COO-] (Foster et al., 1986 a), selenocyanate (VadhanaVlkit et al., 1987), and selemferous wheat (palmer et al., 1970). It is probable that the selenoamino acids are tirst methylated at the selenium atom (both [Se]-methylselenomethionine and [Se]-methylselcflocysteme have ( been found ln plants) and then degraded to form dimethyl selenide and/or trimethylselenonium (Kraus et al., 1985; Foster et al., 1986 b). The enzymatic degradation of [Se]-methylselenomethionine has been observed in plants by LeWIS et al. (1974), although, a non enzymatic degradatlOn pathway has been suggested (Cooke and Bruland, 1987). Trimethylselenonium ion appears to be rapldly elimmated, vlrtually unchanged, after ingestIOn by anImals (Tsay et al., 1970). In plants (if absorbed from solls; Oison et al., 1976 a) trimethylselenomum ion is Vlrtually not metabolized. However, there are evidences that trimethylselenomum can be, to sorne extent, bJOlogically converted mto volatile compounds (dimethyl selenide) in sOlls and plants (OIson et al., 1976 b.) and posslbly in rats (Obermeyer et al., 1971; Foster et al., 1986 b). Bactena capable of using trimeth;lselenomum ion as a carbon source have been Isolated from soil (Doran and Alexander, 1977). These two compounds [tnmethylselenonium cation , TMSe, (H3C)3Se+; dimethyl selemde, DMSe, (H3C) Se] are general metabohc products of a variety of selemum Z compounds and, since they are rapldly elimmated (in increasmg amount as the amount of selenium ingested increases), and they are relatlvely non tOXle, it has been suggested that they represent the end result of detoXlticatlOn processes. To fully understand the metabolism of selemum compounds especially the metabolism of the selenoammo aClds, It will be neeessary to study the fate of the selenoamino acids when they are ·f admmlstered. at rugh levels, to test animais. At high selenium levels, it might, then, become apparent that one of the means to homeostasls of selenium ln general (not only of selenite) is through selenomum compounds (by blomethylation). 6 1.4. TOXICI'IY OF SOME SELENIUM COMPOUNDS The acute toxicity of selenium to animais depends upon the animal species. the route of administration and, more importantly, on the chemical forms of the element as iIIustrated by the examples in Table 1·3. Thus. for a given organism and route of administratIon, it can be shown that toxicity is a function of the chemical form of the selenium. "The relative chronic toxicities of individual selenium compounds are of interest wlth regard to the selection of sources of selenium for use in the supplementanon of animal feeds and human foods". "However, no studies have been conducted to date ln wruch a senes of selenium compounds have becn evaluated toxicologically in a chronic exposure design" (Combs and Combs, 1986). ThIs comment 18 stIll valid today as only sporadic studics on the chronic or sub-chromc toxicity of selemum compounds have. to our knowledge. been reported (a few of these are: Heinz et al., 1988; Das et al., 1989; Hoffman et al .• 1989). Interestingly, in presence of 4 mg of As (as sodium arsenite)/kg body welght the i.p. LDso of trimethylselenonium ion was reduced trom 49.4 mg Se!kg to 2-3 mg/kg (Obermeyer et al .• 1971). At intermediate levels, the presence of arsenite in the diet shghtly Increased the tOXIClty ot trimethylselenonium chloride (Obermeyer et al., 1971) whereas it IS generally considered that arsenic protects agajnst the toxicity ~f selenium (Combs and Combs, 1986). The protectIve action of arscnl~ amI mercury on selenium poisoning (and vice versa) is not always the rule. The toxicity of dimethyl selemde and trimethylselenonium ion is enhanced by several order of magnitude in the presence of morgamc forms of arsenic and mercury (Wilber, 1980). It would appear that the tOXIClty of selelllum depends not only on the chemical forrus present but also on other dietary factors. As a source of selemum for diet supplementation it woulrJ seem that the least tOXIC selenium compound would be preferable. However, tbis is unlikely to be the case since the selemum compounds which are less toxic, such as trimethylselenonium canon, tend to be the ones whlch are least blOavaIlable. 1.5. BIOAVAILABILITY OF SOME SELENIUM COMPOUNDS AND SOME FOODS AND FEEDS The bioavailability, to an organism, of the selenium ln foods and feeds depends pnmanly on the type of selenium compounds present and, secondly, on interactIons Wlth other nutnents, pnncipally metals. 7 I!J!I! 1:J: Acute toxicities of sc.iected selenium compounds (Combs and Combs, 1986). a Se compounds Specie Route LDSO (mg/kg) b NazSe03 Chick embryo Aireell O.3 O.se Rat Oral 3.2 (10 days)d Oral 4.S-6.oi i.v. S.7~ 1 SoC. 3.2 b NazSe04 Chaek embryo Aireell O.13 Aireell 1.S-2.OC Elemental Se Rat Oral 6700 (10 days) ~ SeS2 Rat Oral 76 (10days) ~~ Dlmethyl selenide Rat i.p. 1600 (24 hr)d Mouse j.p. 1300 (24 hr)d Trimethylselenonium Chiek embryo Aireell lS.7b chloride Rat i.p. 49.4 (4hr)d Dimethyl selenoxide Chiek embryo Aireell 6.s3b DL-Selenomethionine Chiek embryo Air eell O.13b Rat j.p. 4.25 (48 hrJd Mouse i.v. 8.9 (24hr) DL-Selenocystine Chick embryo Aireell O.64b Rat j.p. 4.0 (48 hr)d DL-Selenocysteine Chiek embryo Aireell 0.57b a.Route of admimstration: i.p .• inuatJCrironea1; s.e .• subculaneous; i.v.• inh'a venous b.administered 4 days aClcr inc:ubation by injection throup e8PheU ta surfac.e of ail' ccli. c.adminislcred 14 days aCter incubation by injection throup egsheU ta surface of air ccli. d.pcnod of incubation indlcated in parentheSIS. • 8 Table 1-4 compares the bioavailability of sorne selenium compounds found in foods and feeds. As .... it cao be seen, not ail the selenium contained in certain foods is fully bioavailable. "The wide range m selenium availability values obselVcd with these foods raises questions about the validity of assessing human selenium status mercly by mcasuring dietary selenium intake" (Levander, 1984). Oearly bioavailability depends on the selenium compound(s) presentes) in each food or feed as each of these selenium compounds has a specific bioavailability. Furthermore, bioavailability can be modified by other constituents of the food/feed. Many other nutritional factors cao affect the selenium availability (Combs and Combs, 1986). One of these factors is the presence of hea\')' metals in the food in question. Selenium, in mushrooms, is relatively unavailable (Levander, 1984). Mushrooms are a1so known to accumulate hea\')' metals and interactions between these metals and selenium, prior to or arter ingestion, may accO\lnt for this low availability (Levander, 1984). The chemical nature of selenium in mushrooms is unknown. Fungi can methylate selenium to form dimethyl selenide (Fleming and Alexander, 1972). The possible formation of trimethylselenonium ion is not to be underestimated as an explanatlon for the low availability of selenium in mushrooms (Levander, 1984). The availability of selenium in seafoods (tuna, crabs and oysters) is relatively low (Douglass et al .. 1981) and may a1so be explained by the presence of heavy metals (especially mercury). It is presumed that the cationic heavy metals combine with selenides to form insoluble products. On the other hand, it bas been demonstrated that the availability of selenium ID certam seafoods (crab, oyster, shrimp and Baltic herring) is a function of the biological criterion chosen, the level of selenium supplied, and other unlrnown dietary factors (Mutanen et al., 1986). Some selenium compounds might be absorbed and found in plasma or liver but they might have no biological actlvJties. It is weil known that marine systems can create exotic organic forms of trace elements, as is the case with arsenic. Arsenobetaine [(~C)3As+~COO-] was first isolated from rock lobsters (Edmonds et al., 1977; Edmonds and Francesconi. 1981), and has since been found in Many other aquatic orgamsms. invertebrates such as crayfish, prawn, scallop, squid (Maher, 1985) and vertebrates such as fresh water or marine fishes (Lawrence et al., 1986; Maher, 1985), the blue pointer and the whltetip shark (Hanaoka and Tagawa, 1985). A tetramethylarsonium salt [(H3C)4As+] was a1so identified in a clam specie (ShlOml et al., 1987) and arsenocholine [(H3C)3As+~CH20H] was found in shrimp (Lawrence et al., 1986). Sincc selenium, Iike arsenic, often cecurs in methylated forms, Ganther (1984) considered that selenomum isologs of arsonium compounds (arsenobetaine) might he found in tuna and mlght explam the low biological availability of selenium in this food. A portion of the tuna juice selenium, that had the solubility properties of a selenonium cation, was isolated. When compared to synthetic selenobetame by thin-layer chromatography and elcctrophoresis the "onium" selenium fraction from tuna did not migrate to the same position as the selenonium standard. Whereas, the most widely found methylated arsemc specles IS arsenobetaine, trimethylselenonium ion is, probably, the most stable of the selenonium compounds. Actually selenobetaine undergoes spontaneous dccomposition to trimethylselcnomum cation on standlllg 9 Iabl~ 1-4: Biological availability ofsome selenium sources. .. Relative availability (%)a Se source Speeie L.N.& EDe N.P.A.a G.PAe Elemental selenium Rat -0 Chiek 7 Sodium selenide Chiek 42 Sodium selenite Rat 100 Chiek 100 Chick 100 Rat 100 Selenium dioxide Rat 69 Sodium selenate Rat 122 Chiek S8,89 DL-Selenocysteine Rat 92 DL-Selenocystine Rat 96 Chiek 69,78 Chiek 121-133 dl-Selenomethionine Rat 96 Chiek 18,32 61 Chiek 348-377 Selenocystathionine Rat 110 1 2-Selenouracil Rat <4 " 6-Selenopurine Rat 76 . Chiek 20 Wheat Chiek 71 Chiek 360 Whole wheat bread Rat 142 Wheat, boiled Rat 83 Berring meal Chiek 2S Tunameal Chiek 22 Chiek 47 Tuna, water-canned Rat S7 Tuna, oil-canned Rat 49 Shrimp, boiled Rat 73 Oysters, boiled Rat 38 Crab, boiled Rat S8 Meat and bone meal Chiek IS Beet kidney. cooked Rat 97 Mushrooms Rat 4 il. Biological availablilty relative 10 that oC selenite for each blOUSa)'. b. as determined by the prevention of üvcr Necrœis in vitamin E-deficient ram (from Combs and Combs, 1986). c. as determmed by the prevention of Exudalive Diathesas ln vitamin E-deficient chicken (from Combs and Combs, 1986). d. as determmed by the prevention of Nutntlonal Pancreatlc Atrophy in Vltamin E·fed chicken (Co!!!b~ and C~bs, 1986). e. as measured by increased G1utathlone Pcroxidasc Actlvity in livcn of Se-depleted rats (from Lcvander, 1984) r 1 10 (Ganther, 1984). Trimethylselenonium cation might thus, have been the "onium" compound present in tuna. Furthermore, trimethylselenonium ion represents a poorly bioavailable form of selenium. It is excreted rapidly via the kidneys and is ineffective (Iow biological availability) in preventing the necrogenic syndrome in the rat (Tsay et al., 1970). Moreover, there are evidences that selemum is methylated to selenonium compounds [(H3C)2Se+R] in aquatic systems (Cooke and Bruland, 1987). The objectives of this rescarch was to develop a method of analysis for sclenonium compounds in foods and especially in marine foods. Such an analytical method was bound to provide us with more data on the metabolism of selenium and the relative importance of selenonium coml'ounds. For the development of the desired analytical tool, selenonium standards were required, and the synthesis of trimethylselenoniur.1, selenoniumcholine [(H3C)2Se+CH2~OHJ and selenoniumacetylcholine [(~C)2Se+~~OC(O)CH3) cations is described in chapter two of this thesis. Chapter t1uee examines the actual attempted method of analysis for these analytes. [Note: it was the author's desire that the selenonium isolog of arsenocholine he called selenocholine. However. a literature search showed that selenocholine already existed in the literature but described another compound [(~C)3W~~SeH]. TItus, the name selenoniumcholine and selenoniumacetylcholine were given to [(H3C)2Se+~~OHl and [(H3C)2Se+CH2CHz0C(O)CH3) respectively). 11 CHAPTERII ( SYNTHESIS OF SELENONIUM COMPOUNDS 1 12 2.1. THE PREPARATION AND PROPERTIES OF SELENONIUM CQMPOUNDS: A LlTERATYRE REVIEW Several definitions of selenonium compounds have been given in the literature. For this discussion, selenonium compounds are organic selenium compounds in which three alkyl and/or aryl groups are Iinked to a selenium atom which is formally deficient in electron density. They are the selenium isologs of oxonium (0) and sulfonium (S) ions. Their chemistry is also somewhat similar to that of their arsonium (As) and ammonium (N) cousin .... 1beir general structure,!.. is as follows (X· is any suitable counter ion, and Rt, ~ and R3 are any alkyi or aryI poups): Rt' • /Se-R3 1 RZ Several reviews have already been published on the subject (Brad t, 1934; Rheinboldt, 1955; Shine, 1973). In tbis review, only the synthesis of alkylselenonium or nlixed a1kylarylsclenonium compounds was considered. l'be synthesis of triarylselenonium compounds was, of less intcrest since the traditional synthetic approach was somewhat different. 2.1.1. METHODS OF S\'N'[HESIS 2.1.1.1. REAcrION OF DIALKYL SELENIDES (R2Se) WITH ALKYL HALIDES (RX) The most common method for the preparation of a1kylselenonium compounds IS the rcaction of a dialkyl selenide with an alkyl halide. This high yield reaction was carried out wlth or wlthout solvcnt. 2.1.1.1.1. Symmetrlcal Selenldes 2.1.1.1.1.1. R~ + RX -----> R~+X One of the earliest reported symmetrical trialkylselenonium compound to have been preparcd by the simple reaction of a symmetrical selenide with the corresponding alkyl halide was triethylsclenonium iodide (Pieverling, 1876 and 1877). Trimethylselenonium iodide was synthesized accord mg to thls rnethod only much later. 1bc direct action (in the absence of solvent) of dimethyl selenide on mcthyl iodlde was fast (1 h) and virtually quantitative (92 %) al room tempe rature (Hashimoto. et al. 1967; Yamauchl. 1979).1bis reaction was also performed under reftUl (palmer, et al. 1969; Tsay el al. 1970; Yamauchi 1979; Janghorbani et al. 1982; Nahapetian et al. 1983; B10tcky et al. 1985, 1987, 1988). The Yleld for the 13 synthesis of triethylselenonium iodide by the addition of ethyliodide to diethyl selenide was much lower ;.. (54% for a reaction time of ten days at room temperature) according to Hashimoto et al., (1967). Trimethylselenonium cllIoride wu prepared by the action of methyl chloride on dimethyl selenide in the absence of a solvent, in a sealed tube for one week (Wynne and George, 1969). This reaction of a selenide with an alkyi halide was also carried out in the presence of a solvent. Mcfarlane prepared trimethylselenonium iodide trom dimethyl selenide and methyl iodide in ether (Mcfarlane, 1967). In the case of the tripropylselenonium and the tributylselenonium cations, the reaction between the dialkyl selenide and the corresponding halide in ethylene dichloride appeared to be more efficient in presence of silver tetraftuoroborate or silver perchlorate (Hashimoto, et al. 1967). 2.1.1.1.1.2. R~ + R'X ••••••• > R~'St+JC Brom"alkylc:arboxylic acid 50eh as 2·bromoacetie acid, 2· or 3·bromopropionic acid, as weil as their esters, added to dimethyl or diethyl selenide to form, in Most cases, the corresponding selenetine (selenium analogs of betaines) compounds or their corresponding esters; but this was not a1ways the case. Representative examples are presented in Figure 2-1-1. An illustration of this reaction is the formation of selenobetaine [dimethylselenetine, (carboxymethyl)dimeîhylselenonium inner salt,lI resulted trom the addition of 2·bromoacetic acid to dimethyl selenide. Whereas sorne authors were able to prepare these selenetine compounds, others found that, instead of the desired product, trialkylselenonium cations were formed. This discrepancy wu probably the result of differences in the reaction conditions. Actually, Foster et al. reported that if the reaction was c:arried out at low temperature (4·10°C) with a very large excess of alkyl halide then the selenetine compound .i was formed whereas, at room temperature or higher, the reaction favored the formation of the trialkylselenonium ion (Foster et al., 1985; 1986 a). These authors devised a two phase system (aqueouslorganic) in which the selenetine, upon formation. was irnmediately extracted into the aqueous layer in order to proteet it trom the attack of the lipophilic dialkyl selenide.1be formatiorl of the trialkylselenonium ion was, thus, prevented. Dirnethyl selenide and diethyl selenide reacted fairly efficiently with rnany other a1kyl halides as was suggested by the representative examples of Figure 2·1-2. 2.1.1.1.2. Vnsymmetrlcal selenldes (RR'Se) 2.1.1.1.2.1. R'SeR + RX ••••••• > R~'St+JC r Decylmethyl selenide reacted with methyl iodide to give the corresponding selenonium ion (von Braun et al., 1929). However, if the sflenide was present in excess, trimethylselenonium was formed as a side product: • 14 Figure 2-1·1: Reactions of dimethyl selenide and diethyl selenide with various bromoalkylcarboxylic acids and their corresponding esters. 1 ~0/eH3N02 Br· '+~OH 'Sc Br~OH ...loGe Foster et al., 1985, 1986 a "", • ,.,,:;c 2 0 0 Br· Foster et al, 1985 :>~O~ 'Sc Br~O-...../ ... 8ird and Challenger, 1942 "", • 0 0 r1 :>.~ OH 8ulman and Jensen, 1936 o 51.14910 Br· 'Sc • .JyOH "", :>/ 0 < 47.86'" 100°C Br· 8111man and Jensen, 1936 , :>/ "",Sc IIcJyO ...... • 0 0 0 Br~ 'Sc Foster el al., 1985 "", • 8r~OH ... :> OH 0 0 Br~ Foster cr al., 1985 :> O/"'. 'Sc "", • Br~o~ ... Br· Foster et al., 1986 a 'Sc Br~O' ... :>ry0'- "", • 0 0 Br- ~ + Br~OH y,"OH Carrara, 1894 ---""",Sc ... 0 0 y B~ OH r1 ~ 030% Blilman and Jensen, 1936 ~Se + IIcJyOH Br- 0 < y/'.. 70% <'" 15 Fleure 2·1·2: Reactions of dimethyl selenide and diethyl selenide with various alkyl halidés. i \." Baker and Morne, 1930 • Br~ • Hughes and Kurt yan, 1935 29a ,.Br~o Lotz and Gosselck, 1973 + Br /Se o o F F Bahner et al., 1952 + Br o o ------ 16 2.1.1.1.2.2. R'SeR ... R·X ------> RR'R-slx From this discussion it may seem that ail organic selenides reacted with alkyl halides. That this was not always the case was exernplified by the difficult reaction of methylphenyl selenide and methyl iodlde (Baker and Moffit, 1930). The apparent reduC'.ed activity of a1kylaryl selenide for alkyl halides (compared Wlth dialkyl selenide) was greatly irnproved if a silver salt, such as silver perchlorate, was added to the reaction mixture. An a1kylphenyl selenide upon reaction with an alkyl halide in the presence of silver perchlorate in acetonitrile at room temperature for three to four days gave the corresponding selen"nium salt (Kobayashi et al.. 1986). However. these authors did not mention the yield of the selenonium compcund formed, and in their text they mentioned the reaction of ethylphenyl selenide Wlth methyl iodide whereas the reaction was depicted as "Me·Se·Ph + Et! _•• > ". A sirnllar ex ample of reduced activity was the reported lack of reaction between 2- phenylselenoacetic acid [C6HsSe~COOH] and methyl bromide during 50 days at room tempe rature (Edwards et al., 1928). However. methylselenoacetic acid [CH3Se~COOH. (carboxymethyl)methyl selemde), selennmethionine, J, and [Se)·methylselenocysteine. !. were found to be methylated by methyl lodide in ace tic acid, formie 3Cid mixtures to give [Se]-methylselenomethionine, ~ and [Se] dimethylselenocysteine. ~ (Foster et al. 1985. 1986 a; Foster and Ganther, 1984): NU NH 2 2 ~ .l.. .. ~ CH]I ./"0.... ~ .. ~ 'Sc" ~ lr OH • >- -:- l OH 3 0 5 0 'Sc~OH 4 0 Thus. one could suggest that a1kylaryl selenides are less reactive toward a1kyl halide than are the dialkyl selenides. Again, this is not a1ways the case as is iIIustrated with the next examples. The addition product (carboxymethyl)methylphenylselenonium bromide (pher.ylmethylselenetine bromide, 1) was actually synthesized by the addition of 2-bromoacetic acid to methylphenyl selenide (pope and Neville 1902; Edwards et al. 1928): ©l",> + 17 In the same way, 2-bromoacetic acid added to (4-methylphenyl)methyl selenide to givc the appropriate selenetine compound •• (Ldwards tllll.. 1928): + 2-ch1oroacetic acid also reacted with an alkylaryl selenide, 2. to yield se!enoniu m ions H (Dallacker et al., 1965). However. if the halide was present in excess and the reaction mixture heatcd at 100°C for 24 hr, displacement of a methyl group occurred to produce (66 % yield) a selenonium dicarboxylate :0; 11:::a: H C1CH C;OH ~ H 2 \0 + -CH COOH < / a" 2 g /CICH2COOH 10 24 h /l00oC <0 ~ H o 1 + -CH COOH ~ 2 :0::Cl" 1 Il CH2COOH Thus, it is fairly safe to say that no generalization cao he made towards the reactivity of alkylaryl selenide relative to that of dialkyl selenides. Long chain dialkyl selenides were also found to rcacr inefficiently with long chains alkyl halides and did 50 only in the presence of silver salts (Hashimoto et al., 1967). 2.1.1.1J. Cycllc selenldes with Ilkyi hal1,j~" Selenium, when part of an aliphatk ring system, was observed to react with alkyl halides to form selenonium compounds. If the ring system was not appreciably strained, then the corresponding selenonium ion was formed without ring cleavage. As an example selenOOochroman, .u. in presence of an a1kyl halide, formed the corrcsponding selenoLrochromanium haJide,.u and H. according to the following reactions (Holliman and Mann, 1943 and 1945): CO Br - ('(" 41 CC) ~S:-~ ." ~~~CI 12 Se 13 14 0 ~ Br o Hagelberg (1929) syntbesized several cyclic selenonium compounds, U. .16. and l.1. from the appropriate cyclic selenides and alkyi halides (methyl iodide or 1.4-dibromobutane): 18 ~/ D/ D~·s(] 15 16 17 However. if the ring containing the selenium was strained. then ring opening occurred. and a noncyclic selenonium cation. 18. 12. or :9.. was formed as shown by the following exampk~s (Backer and Winter. 1937): ~ Se CH]I. XCH11 .." 18 /'v CH2Se(CH3)1 ~Se CH31 • C)(CH11. 1" 19 ~ CH2Se(CH3)2 One of the strangest reaction ever observed between a cyclic selenide and an alkyl halide was perhaps that reported by Truce and Emrick for a selenacycloheptane system. They suggested that two molecules of methyl iodide added without ring opening to give the di"positive selenonium ion li: 2,7- dihydro-1.1-dimethyl-3,4-5,6-dibenzoselenepinium düodide (Truce and Emrick, 1956). 1" ( ... ~ I 1 " ~ 21 2.1.1.2. REAcnON OF ORGANIC SELENIDES WITH OmER ALKYLATING AGENTS In cases for which the a1kyl halide was not very reactive towards the alkylaryl seJenide other alkylating agents were used. 2.1.1.2.1. Dimethylsulfate Dimethyl sulphate was found to be a better methylating agent than methyl iodide for the formation of selenonium salts trom alkylaryl selenides (Baker and Moffit, 1930). Alk, CH 1 Ait, + 1" 3 (Low yicld) Se • ~Sc -CH3 Ar/' 6lJoC Ar;' AIt, AIt, + Se /Sc -CH] (Quantitative) Ar/' Ar CH30S(O)20 - { ... Dimethylphenylselenonium methylsulphate was prepared by the action of dimethyl sulphate on the corresponding selenide (methylphenyl sclenide) (Baker and Moffit. 1930. Gilow and Walker. 1967 and Gilow el al., 1968). The 0-, m- and p-dimethylnitrophenylsclenonium methylsulphates were also 19 prepared from the corresponding methyl(p-nitrophenyl) selenide and dimethyl sulphate (Baker and Moffit, 1930). 2.1.1.2.1. Trlftate salts Trimethylselenonium triOate and diethylmethylselenonium triDate were prepared from the corresponding dialkyl selenide and methyl triflate in Freon-113 and, in identical fashion, ethyl triflate was used for the synthesis of triethylselenonium triflate (Laali et al., 1987). Methyl triflate (20 % excess), in dichloromethane, has been used to methylate several arylmethyl selenides in high yields (85·96%) by reaction under reflux to form the corresponding (4-chlorophenyl)dimethylselenonium triflate, (4- cyanophenyl)dimethylselenonium triflate, and dimethyl(4-nitrophenyl)selenonium triflate (Lewis et al., 1985). 2.1.1.2.3. MlsceUaneous Parikh et al., (1985) synthesized radioiodinated benzylselenonium salts. The syntheses of (0- e2SI]iodobenzyl)-dibenzylselenonium tetratluoroborate (a triarylselenonium salt) and of (0- e2SI]iodobenzyl)benzylmethylselenonium tetratluoroborate are of interest to trus discussion as they involved two alkylating agents not in general use for the synthesis of selenoniums: a) benzyl tosylate ln presence of silver tetratluoroborate and b) trimethyloxonium tetratluoroborate. 2.1.1.3. RFAcnON OF OmER ORGANIC Se SPECIES 2.1.1.3.1. Dlselenldes and/or selenols Trimethylselenonium iodide and benzyldimethylselenonium iodide were both formed upon the interaction of dibenzyl diselenide and methyl iodide (Jackson, 1874). Dimethyl diselenide [CH3Se-SeCH3]. in heptane, added to an aqueous solution of 2-bromoacctlc acid in presence of sodium borohydride at room tcmperature, formed the methylselenoacetate [H3CSeC~COO'Na+] in one hour. 11le latter. after partial purification, was methylated with methyl iodide in a water, acetic acid, formie acid (1:1:1 v/v/v) mixture to form selenobetaine (l, Figure 2-1-1; Foster et al., 1986 a). Selenocystine, .no was reduced with sodium borohydride and then methylated with methyl iodide to give Se-methylselenocysteine. This Se-methylated selenocysteine was converted, at low tempe ratures (4°q, to its selenonium salt by the action of iodomethane in an acidic media (500:250 v/v of a formic/atetic acid mixture; Foster and Ganther. 1984; Foster el al., 1985, 1986 a). If the reactlon wa.., a1lowed to proceed for a longer time at room temperature and in presence of an excess of methyl iodide, ....------_._-- 20 trimethylsclen(\nium was formed quantitatively (Foster and Ganther 1984, Foster el al. 1985, 1986 a). Thcse synthetic methods have becn useful, especially, for the preparation of radioactive ['SSe]_ selenonium compounds because of the losses which occur when dealing with the hi&hlY volatile [7SSeJ- dimethyl selenide. HOyC~ _~-{OH o 22 :z ' .. youo NHz 2.1.1.3.2. Selenoxldes Condensation of selenoxides with trimethylsilyl-cyclopentadiene, gave selenoniocyclopentadienides, Zi (Hartke and Wendebourg. 1989). In presence of trifluoroacetic anhydride, 3 equivalent of dimethylselenoxide reacted with trimethylsilyl-cyclopentadiene to give a tri selenonium salts, U. With indole the 3-selenonium salt, 26. was formed whereas with pyrrole the 2- and 3-selenonium salts, n. and A were formed (Hartke and Wendebourg. 1989). The selenoxide was also prepared in situ by the action of N-chlorosuccinamide on the corresponding selenide (Hartke and Wendebourg, 1989). Refer to Figure 2-1-3. 2.1.1.4. RF.AcrION OF INORGANIC Se SPECIES This section will review synthetic routes to selenonium compounds which were based on inorganic selenium species as starting material. Reactions which proceeded via an organic selenide which in lorn, was reacted with an alkyi halide in situ (without isolation of the selenide) are also considered. 2.1.1.4.1. Selenium halldes Selenium tetrachlocide was obselVed by Rathke to react with diethylzinc in ether to give triethylselenonium tetrachlorozincate and zinc chlocide (Rathke, 1869). Selenium monobromide reacted with ethylmagnesium bromide and ethyl bromide to give triethylselenonium hydroselenide according to Pieroni and Coli (1914). \ r~ l 21 Figure 2·1·3: Preparation of selenonium compounds from selenoxides. RI R2 =0 Mc Mc Ph Ph 1.TFAA >=0 ~ TFAA • TriFluoroAcetic Anhydride , 1.TFAA - ~ 1 + + Sc =0 .. ."" O:J 0:::(~ CIO .. - 1 1 H H 26 1.TFAA Rt' Sc =0 RI Q + ~."" 1 1 ~ RI Se R3 1 ~ ...... , Q-Sc + CIO.. - 27 +,~ 1 28 1 C10 .. - RJ RJ r Rt' Q + .""Sc ~ CF3C02" -t. ~ 2.UCIO.. 1 RJ 22 2.1.1.4.2. Elemental selenium Elemental Se was heated to 180°C with methyl iodide in a sealed tube ta yield trimethylselenonium triiodide as a black oil (Scott. 1904). The oil was dissolved in ethyl acetate and the product precipitated by ether addition ta result in dark purple seales melting at 39°C (Scott 1904; Emeleus and Heal, 1946; Doering and Hoffmann. 1955; Lequan et al., 1984). The dHodide of trimethylselenonium iodide was readily converted to trimethylselenonium iodide by adding water to a solution of the triiodide in ethyl acetate and bubbling hydrogen sulfide through the resulting solution until the color was discharged (Scott, 1904; Emeleus and Heal. 1946). A very interesting reaction was that of metallic selenium with methyllithium in anhydrous tetrahydrofuran (THF) to form methylselenolithium (MeSeLi) which then was reacted in situ with two equivalent of methyl lodlde to form trimethylselenonium iodide which was then isolated simply by filtration (Kuhn el al. 1986) 2.1.1.4.3. Sodium selenite It was possible to reduce sodium selenite (N~Se03) (Byard, 1969; Foster el al., 1986; Hoffman ( and McConnel, 1987; and elemental selenium, Klayman and Griffin, 1973) with sodium borohydride in water or ethanol. To the selenide (NaSeH), thus formed, methyl iodide was added to pro duce trimethylselenonium iodide. If the reaction was performed in methanol with just enough of sodium borohydride ta give a red solution, even with a large cxcess of methyl iodide, the yield was extremely low: close to one percent (Byard. 1969). On the other hand, yields were much better (70%) If sodIUm borohydride was added in large excess in an aqueous solution of the selenite (Foster et al., 1986; Hoffman and McConnel, 1987). These findings were in agreement with those of Klayman and Griffin (1973) who reported that: "the borohydride decomposed very rapidly in methanol, and as a consequence, large excesses were necessary to completely dissolve the selenium". Water and especially ethanol have been reported to be excellent choices for this reaction (Klayman and Griffin, 1973). 2.1.2. REACfMTIES OF SELENONJUM COMPOUNDS 2.1.2.1. CHEMICAL PROPERTIES 2.1.2.1.1. Anion·exchange reactions As wIll be dlscussed in the next sections, selenonium compounds are ionic in character, and thus .( f their amons are readdy exchangeable. It is a valuable property since in metabolism studies the chloride or " hydroxide form may be preferred. As an example, trimethylselenonium cation is considered (refer to Table 2+1). The iodide salt was converted to the hydroxide by the action of silver mode (Emeleus and 23 Table 2-1-1: Properties ofsome trimethylselenonium salts. Anion M.pt.a Properties6 References Fluoride 124 very deliquescent vs water and ethanol ss aeetone and ether Emeleus and Heal. 1946 Chloride deliquescent Byard,1969 184 Hashimoto et al., 1967 183-184 ns chloroform vss diehloromethane vss nitrometane smethanol hygroscopie Wynne and George, 1969 Bromide 198 Hashimoto et al., 1967 187-198 vs water ss eold ethanol BiJlman and Jensen, 1936 Iodide 151-153 Imai et al., 1988 150-151 Doering et al., 1955 162-163 Hashimoto et al., 1967 153-155 Foster et al., 1985 Reineckate non hygroscopie Byard,1969 Triiodide 39 s ethyl acetate Scott, 1904 Tetrafluoroborate 230 HashJmoto et al., 1067 Tetrachloroborate 168-170 ss dichloromethane Wynne and George, 1969 Fluorosulphate 83·85 Olah et al., 1973 Silicofluoride 300 vss ethanol swater not deliquescent Emeleus and Heal, 1946 à. decomposition meltmg points m Celsius degrees b. s: soluble; vs: very soluble; vss: vcry spanngly solublc; ss: sllghtly soluble 24 Heal. 1946, Yamauchi et al., 1979; Butte, 1980) and tbis hydroxide was then converted, by the simple additIOn of hydrochlol'ic acid (Yamauchi et al., 1979), hydrogen fluoride, or hydrotluosilicic acid (Emeleus and Heal, 1946) to the cbloride, fluoride, or silicofluoride salts respective!:; Silver acetate and sllver oxide (Butte, 1980). and silver tetrafluoroborate (Hashimoto et al., 1967) have been used for the direct convertion of trimethylselenonium iodide into the desired salt (hydroxide, acetate, and tetrafluoroborate. respectively). Anion exchange resins. in the hydroxyl or the cbloride form (Dowex-l and AG-2) have also been used especially if dealing with minute amounts (Byard, 1969; Palmer et al., 1969; Nahapetian et al., 1983). It was possible to modify appreciably the properties (stabiIity, hygroscopie character, solubility, melting point, etc.) of a given selenonium compound by just changing its anion. 2.1.2.1.2. StablIIty During the preparation of unsymmetlical selenonium compounds, several authors have observed the formation of trialkylselenonium cations as side products. Biilrnan and Jensen (1936) found that the reaction of 2-bromopropionic 3Cid with dimethyl selenide or diethyl selenide produced 48% and 70% respectively, of the trialkylselenonium ion. In the same manner, ethyl 2-bromopropanoate [CH3C~(Br)C(O)OC~CH3] in presence of dimethyl selenide gave 74% of tnmethylselenonium bromide (Biilman and Jensen 1936). Foster et al. (1985; 1986 a), Foster and Ganther (1984), and Ganther ( (1984), reported the extreme instability of (carboxymethyl)dimethylselenonium inner salt (dimethylselenetine; selenobetaine, [(œ3)2Se+~C001) and of Se-dimethylselenocysteine. The synthesis of these compounds were achieved only at low temperature. Selenobetaine was extremely unstable and underwent "spontaneous decomposition to the trimethylselenonium cation" Ganther (1984). Decarboxylation might have been favored because two stable end products, (trimethylselenonium ion and carbon dioxide) were produced. Similarly, Se-dimethylselenocysteine, in the presence of excess methyl lodide and a formic/acetic acid mixture, produced trimethylselenonium iodide, at room ternperature. Foster and Ganther (1984). in an attempt to explain the instability of Se dunethylselenocysteme,suggested that "dimethyl selenide was eliminated from Se-dimethylselenocysteme ''. to forrn dehydroalamne which was then reduced to alanine as a result of formate oxidation to CO2 nus disproportionation reaction was not confined to selenonium compounds having a carboxylate rnoiety. Trirnethylselenonium iodide was formed. as a by product. upon the addition of methyl iodide to decylmethyl selenide (von Braun et al. 1929). Dibenzyl diselenide, when reacted with an excess of methyl iodide did not yield only pure ben..ryldirnethylselenonium ion but also trimethylselenoniurn cation as a by-products (Jackson, 1874). Fluorenyl-9-dimethylselenonium bromide (~ Figure 2-1-2) was converted to dimethyl selenide and 9-fluorenyl a1cohol upon refluxing in an aqueous solution for 30 r rnmutes (Hughes and KUCl)'an, 1935). Upon heating, Most of selenonium halides were converted to a selenide and an alkyl halide. (Carboxymethyl)methylphenylselenonium brornide and 2S (carboxymethyl)methyl( 4-methylphenyl)selenonium bromide were degraded to their corresponding arylselenoglycolic: acid, JI, and a and methyl bromide (Edwards et al.. 1928): rô' Br- _ CH,1Ie • ~.. "OH ~"OH o .6 30 ° Triethylselenonium iodide dec:omposed at 8O-1260 C into diethyl selenide and ethyl iodide (von Pieverling, 1877). Trimethylselenonium iodide upon heating in an inert atmosphere formed methyl iodide and dimethyl selenide; the later product tan he trapped and used for the synthesis of other selenonium Gompounds (Foster et al.. 1985). Oosely related to the stability of selenonium compounds are their alkylating properties. 2.1.2.1.3. Alkylatlng propertles of selenonlum compounds The intra-molec:ular rearrangement of selenonium compounds (Hartke and Wendebourg, 1989; Kobayashi el al.. 1986) or inter-molecular alkylation with selenonium salts (Butte, 1980; Yamauchi el al., 1979) were generally carried out in basic solutions with the simultaneous formation of the corresponding selenide. The selenonium countet ion plays an important role in their reactivity (alleylative properties) as has been demonstrated for trimethylselenonium salts: the hydroxide being more reactive than the acetate (Butte, 1980). 2.1.2.1.4. Ylld tormatlon Selenonium ylids were produc:ed by the reaction of a base which removed an hydrogen ion trom the alpha position of the selenonium ion in question. Generally. this reaction was etfected in a non aqueous solvent. Auorenyl-9-dimethylselenonium bromide (12, Figure 2-1-2), produced a black ylid (29a. Figure 2-1-2) in aqueous alkali (Hughes and Kuriyan, 1935). Selenonium compounds have heen useful organic synthetic reagents. The utility of dimethylphenacylselenonium ylid. J.Z. prepared with sodium hydroxide in chJoroform/ethanol, has been demonstrated to he a powerful reagent for the synthesis of cyclopropane systems (Lotz and Gosse1ck, 1973). Selenonium compounds have been used for the synthesis of olefins with potassium t-butoxide (t BuOK) or potassium hydroxide (KOH) in nIF or in DMSO at 2(fC (Halazy and Krief, 1979, 1980). An excellent review has becn published on -synthetic methods using alpha-heterosubstituted organometallics" in which selenonium ions were discussed (Knef. 1980). Other reviews on 26 organoselenium chemistry (incluoing selenonium ions) have been published (Oive, 1978: Liotta. 1987). Epoxides have been prepared stereoselectively from selenonium compounds having a beta-hydroxyl group by reaction with bases such as KOH (10%) in diethyl ether or in CP""l02' sodium hydride (NaH) in nIF and t-BuOK in DMSO (Oive, 1978; K.rief, 1980; Liotta. 1987). Oamma-hydroxymethylselenides were methylated with neat methyl iodide to form the selenonium ions which upon reaction with a base produced homoaJlylaicohols regioselectively and in quantitative yields (Oive, 1978: Krief, 1980: Liotta. 1987). -rôl )~~ 032 2.1.2.1.5. Oxfdation Trimethylselenonium chloride was found to be fairly stable to wet oxidation with a mixture of HNO) and Hz02 (in Ha) whereas a mixture of HNo) and HOO.. (in Ha), devised by Janghorbani et al., (1982), resulted in a quantitative convertion of trimethylselenonium chloride to selenite. Para tolylmethylselenetine bromide, in the presence of bromine in carbon tetrachloride, lost the methyl group to give p-tolylselenoglycolic acid dibromide W>: and, ifbromine was in excess. p-tolylselenium tribromide C,W and bromoacetic acid were produced (Edwards et al., 1928): 'fi B Br2 'fi Br ~. ~OH --. ~ '/"V 0H ..,.> Il CCI.. Sc Il H 010 Br"U° Br 33 ° 2.1.2.2. PHYSICAL PROPERTIES 2.1.2.2.1. Optical resolutlon The first indication that selenonium ions with three different substituents could exist as a pair of optically active isomers was reported in 1902 by Pope and Neville (1902). These authors attempted to resolve (carboxymethyl)methylphenylselenonium bromide cr. methylphenylselenetine bromide) with silver d-bromocamphorsultonate. The silver salt was used in order to precipitate the bromide ion. By fractional recrystallization it was possible to separate the two selenonium stereoisomers into stable 1- or d· fonns. The bromocamphorsulfonate salts were, then. converted to the opticaJly active platinum chlorides whereas the attempt to make optically active mercuric·iodide saJts failed (pope and Neville, 1902). That 27 racemisation occurred upon formation of mercuri-iodide salts of methylphenylselenetine was confirmed by Balfe and Phillips (1933) but was attributed to the instability of the selenetine base and not to a "symmetrical structure" of selenonium compounds in general. The second selenonium ion to be resolved into optically active d-bromocamphorsulfonate salts was the 2-p-chlorophenacylseleno-iso-chromanium system (W of Holliman and Mann (1943, and 1945). 1be optically active salt (I-selenonium d brQmocamphorsulfonate) which had been recovered by fractional recrystallization from a1cohol was converted to the corresponding picrate or into the mercuric tri-iodide salts. The picrate salt was optically stable in acetone at room tcmperature but was racemized upon boiling. In contrast to the finding of Pope and Neville (1902), these authors reported that thcir d- and 1-2-p-chlorophenacylseleno-iso-chromanium mercuri tri-iodide, were optically active in acetone; but "underwent slow racemization even at room temperature" (Holliman and Mann, 1943, and 1945). This confirm the findings of Balfe and Phillips (1933) on the instability of the methylphenylselenetine base. The d-selenonium I-bromocamphorsulfonate was also prepared and converted to the picrate (Holliman and Mann, 1945). It iS only much later that the absolute configuration of an optically active selenonium ion was established (Kobayashi et al., 1986). Their conclusions were based on asymmetric induction and on circular dichroism (CD) spectra which both supported the assumption that the absolute configuration of d-selenonium salt was (S) (Kobayashi et al., 1986). 1.1.1.2.1. d-Orbltal resonance T:te only deuterium exchange study, to have been reported. indicated an appreciable d-orbital resonance for trimethylselenonium iod~de (Ooering and Hoffmann, 1955). This study reported a decrease in the rate of exchange for "onium" isologs going down a periodic group of the periodic table. Trimethylsulfonium (98.0% deuterium atom exchanged) was more reactive than trimethylselenomum (13.2%) which in tum was more reactive than trimethyltellunum (0.45%). These authors attnbuted the decrease in exchange rate not to the contribution of d-orbitals, whicn was reportedly the samc. but rather to an increase in bond length. Group V followed the same trend but the exchange rate was less than for the group VI isologs. For example the exchange reaction for tetramethylarsonium (7.44%) was le~ important than for the trimethylselenonium cation. Hyperconjugation of the low-lying vacant "d" orbitais of the selenium atom was considered responsible for the increased stability of trimethylselenonium iodide (McDaniel. 1957). H+ ~C= Sc -CH) 1 CH) The catalytic activity of selenonium compounds in the liquid-phase oxidation of hydrocarbons has been explained, in considerable detail. by the occupancy or rather the "partial occupancy" of the d orbitais (Ohkubo, 1971; Ohlcubo and Kanaeda, 1971). ------_._-_._- 28 2.1.2.2.3. Nuclear magnetlc resonance (NMR) The trimethylselenonium ion has been studied, in some depth by NMR. Trimethylselenomum chloride was charactenzed by a 1H chemical shift of 2.96 ppm in methanol (Wynne and George, 1969). In D 0, reported 1H chemical slufts for trimethylselenonium chloride were 2.7 ppm (Palmer et al., 1969), 2 ~ 2.72 ppm (Khun el al., 1986), and 3.25 ppm (Byard, 1969), relative to an extemal standard of tetramethylsilane. The 77Se, 13e , and 1H NMR spectral characteristics for sorne selenonium compounds are summarized in Tables 2-1-2, and 2-1-3. According to Laali el al. (1987): "the most significant observation (lH ~~R) was that a consistent trend of increased shielding in "onium" ions could be established [R30+ > R3S+ > R3Se+ > R3Te+], reflecting charge delocalization and shielding by heavier atoms". It appeared that this was true also for the 13e NMR spectra of these compounds. The coupling constants 13e.1H of alpha-methyls in R3Se+ were large (142-148 Hz) whereas that of the beta-methyls (130-131 Hz) were close to that of normal methyls (125-130 Hz) (Laali el al., 1987). 2.1.2.2.4. X-ray study To date only one X-ray study performed of a1kyl selenonium ions has been reported. ( Trimethylselenonium iodide had an orthorhombic unit cell with a Pnma space group and cell dimensions O O O of a = 14.078 A , b = 8.000 A , and c = 6.177 A with four formula units in the cell (Hope, 1966). The C Se distances were found ta be between 1.946 and 1.962 A 0. Since a weak bond (3.776 A 0) existed between the selenium and the iodide atoms it was probable that trimethylselenonium iodide was a charge transfer complex wÎth the selenonium cation being the acceptor and the iodide anion being the donor (Hope, 1966). Since the bonds angles between the three methyl groups of trimethylselenomum selenonium lodlde were closer to 90° (97.9°, and 99.1°) than 120° (Hope, 1966), and since the reduced NMR couplmg - - constants K13C 77Sc and KlH 77Sc were negative and positive respectively (Jameson, 1969), it can be conclurJed that the hybridization of selenium, in this case, was doser to a "p3" than to a "sp3n hybndizatlOn. 2.1.2.2.5. Inrrared sp~ctroscopy One of the earliest studies of trialkylselenonium cations by infrared, was performed by Wynne and George (1969) on trimethylselenonium cllloride and trimethylselenonium chloride-boron tnchloride adduct. In tins study, it was found that trimethylselenonium chloride, "possessing approximate e3v symmetry", showed no absorptIOn in the 300-400 cm-1 region where the selenium-chlorine covalent b~md ( normally absorbs (Wynne and George, 1969). Moreover, the IR spectrum of, the boron trichloride aclduct dlsplayed the characteristic absorptions for the tetrachloroborate ion (Wynne and George, 1969). Z9 Table 2·1·2: Chemical shifts of some selenonium compounds ("Se, 13e and tH NMR). Compound Solvent "Se 13C IH Reference [(CH )3Se +][cf] CH 0H 3 3 2.96 Wynne and George, 1969 [(CH )3Se +][FS0 -] 5°2 2.70 Olah ct al , 1973 3 3 a +][FS0 -] 3.20(q ),CH [(CH3C~)3Se 3 5°2 2 1.40(t),CH Olah ct al., 1973 3 [(CH3)3Se +][01Î] SOi-4(PC 255 21.78 Laali et al, 1987 [(CH3)3Se +][OTf] CF S0 H/rt 254 Laah et al., 1987 3 3 Se CF S0 H/rt 35.8, SeCH [(CH3)2 +C~CH3][OTn 3 3 28S 2 19, SeCH 3 8.8, SeC~CH3 Laah et al, 1987 + - b DCCI 7.26-7.89(m) [(CH3)2Se C HC(O)C6H5] 3 4.74, SeCH l 2.64,SeCH Lott and Gosselck, 1973 3 CF S0 H/rt 328 35.5, SeCH [(CH3CH2)2Se +CH3][01Î] 3 3 2 16.7, SeCH 3 9.9, SeC~Ctl3 Laah el al., 1987 CF S a. Where s means smglet, t means triplet, q means quarte t, and m means multiplet b. an ylid Table 2·1·3: Some NMR spin-spin coupling constant of selenonium compounds. Compound Solvent 13C·lH 77Se.1H 13C·77Se Reference [(CH )3 +][f] 145.8 9.3 -50 McFarlane, 1967 3 Se ~O [(CH3)3Se +][OTf] SOi-40°C 147 51.1 Laah et al , 1987 Se CF S0 H/rt 52.7, SeCH [(CH3)2 +C~CH3][OTn 3 3 146,SCC~ 3 146,SeCH 48.7,SeCH 3 2 131, SeC~Ctl3 LaaIJ et al , 1987 [(CH CH )2Se +CH ][OTc"] CF S0 H/rt 55, SeCH 3 2 3 3 3 146,SeC~ 3 142, SeCH 49.5, SeCH 3 2 130, SeC~Ctl3 Laali ct al., 1987 .....or, [(CH CH )3Se +][OTf] CF S0 H/rt 148, SeCH 52.5 3 Z 3 3 2 130,SeCH Laa" et al , 1987 3 30 This evidence confirmed the appreciable ionic c:haracter for the trimethylselenonium cation when the anion was an halide or even tetrachloroborate. The infrared spectNm of trimethylselenonium Reineckate has also becn published (Palmer et QI.. 1969). This salt is interesting because it is not soluble in water. The vibrational spectra of trimethylselenonium iodide and its per-deuteurated analog was studied in detail by infrared and raman spectroscopy (Imai et al.. 1988). According ta the se authors' calculations. the Se-c bond was not weakened upon formation of the selenonium ion from the selenide as was the case for the sulfonium and specially the oxonium compounds. Both X-rays and infrared analysis support the ionic formulation of selenonium compounds. 2.1.2.2.6 Mass spedrometry Byard (1969) is probably one of the tirst and few workers to have studied a selenonium compound (trimethylselenonium chloride) by mass spectrometry. A direct probe (heated to 175·180°C) introduction technique was used. If the probe was not heated above 17cfc, no peaks were observed. The molecular ion could not be observed as the selenium-carbon bonds are broken before the selenonium ion becomes volatile (Byard, 1969). The breakdown products to be detected were dimethyl selenide, methylselenol and hydrogen selenide (Byard, 1969). Although, Foster and Ganther (1984) found similar mass fragments for trimethylselenonium iodide (mie = 142 [CH]I]. 127 [1],110 [CH3SeCH]], 95 [CH3Se] with the fragments at 110 and 95 showing the characteristic isotopie pattern for selenium), they had to heat the probe to a temperature of 115°C only. Still. they were not able to observe the molecular ion. r 31 ,-,.. 2.2. SYNTHttSIS OF TRIME'IlMSELENONIUM. SELENONJUMCUOyNE. AND SELENONIUMACEl)'LCHOLJNE SALIS It was considered that selenonium eompounds were probably important intermcdiates III selenium metabolism and that they might he present in food systems. Arsonium isologs have bccn observed in aquatic biologica1 matrices and trimethylselenonium cation has been reported in urine. ln order to develop a suitable analytical method for the determination of the analytes JO biologieal samplcs. analytical standards were required. The following section reports the preparation. purification and the spectroscopie characterization of trimethylselenonium, selenoniumcholine and selenoniumacctyJcholmc salts. 2.2.1. MATERIALS AND METHODS 2.2.1.1. REAGENTS Tetrahydrofuran, acetone, and benzene (CaJedon Inc•• Georgetown. Ont.) and methanol (BDfI Inc., Montreal, Que.) were "distilled in glass" grade. Tetrahydrofuran nominally contained 0.03% water and was used without further modification. Diethyl ether (anhydrous). dimethylformamide (I3DIf Ine .• Montreal. Oue.), sodium tetraphenylborate (Anachernia Canada Ine., Ville St·Pierre. Que.) wcrc ccrtlltcd ACS reagent grade and methyllithium. 1.4 M in diethyl ether, was salt free (Aldrich Chemlcals Co., Milwaukee. Wis.). Selenium powder, ·100 rnesh, 99.5%, was from Aldrich Chemicals Co., and !>elcflIum pellets puriss. pearl. 99.995%. rectifier quality, was from Fiuka (Fluka Chemieal Corp., Ronkonkoma, New York). Ali other chemica1s were reagent grade or better (Aldrich Chemicals Co., Milwaukee, WIS.). Water was double-distilled and deionized. 2.2.1.2. INSTRUMENTS Nuclear magnetic resonance spectra were recorded on XL 200 Varian (IH :. 200 MHz; \3C = 50.3 MHz) or XL 300 Varian (IH = 300 MHz; 13C = 75.4 MHz) instruments. The FAB mass ~pectra were recorded from a glycerol matrix on with a fast atom bombardment source (ZAB 2FIIS). The la!>cr ablation microprobe mass analyses (LAMMA) were recorded on a LAMMA 500 Instrumcnt (lcybold Hereaus, Koln. Germany). The details of the operating parameters have been descnbcd e1scwhere (Van Vaeck et al.• 1989). 1be structural assignrnents were performed by prof. L Van Vacck. Ulllver~Jtalre Instelling Antwerpen. Infrared spectra were recorded with a fourier transform instrument (FT·IR Nlcolet 82100il analyzer; Nicolet, Chicago, USA) operated at 4 cm-I. The an alytes. dissolved in a<.etone, wcrc introduced in the ATR ccII, and the solvent was allowed to evaporate slowly at room tempcraturc tu 32 produce a very fine evenly distributed film of the compounds. Sixty four interferograms were co-added ( before fourier transformation and boxcar apodization. Melting point determinations were performed on a Fisher-Johns hot stage apparatus (Fisher Scientific Company, Ottawa, Ont.) and were uncorrected. The elementaJ analyses were performed by Guelph Chemical Laboratories (Guelph, Ont.). Rectified current, controlled with a power supply, was applied across the electrodes and the potentiaJ was controlled during the electlOchemicaJ synthesis. 2.2.1.3. SYNTHESIS OF TRIMETHYLSELENONIUM IODIDE 2.2.1.3.1. Chemlcal synthesls Trimethylselenonium iodide was prepared by reaction of selenium powder with methyllithium to form methylselenolithium which was then methylated with methyl iodide according to the method of Kuhn el al. (1986). The crude product was recovered by filtration, redissolved in a minimum of warm methanol (40°C), vacuum filtered, and the filtrate was allowed to cool to room temperature in a large test tube. This tube was then placed in an Erlenmeyer flask containing diethyl ether, the tlask was stoppered • and the assembly was left to stand for 20 h. The test tube was then, removed trom the assembly, stoppered, and left to stand ovemight at -2(fC. The crystals that separated were filtered and dried over phosphorus pentoxide at room temperature and under vacuum. A second and third crop of crystals were obtained by the ftlfther addition of ether to the methanolic filtrate. Trimethylselenonium tetraphenylborate and trimethylselenonium Reineckate were obtained by adding sodium tetraphenylborate or ammonium Reineckate {NH4[Cr(NH3)2(SCN)4]} to a solution of trimethylselenonium iodide in methanol. The precipitate was tiltered, dissolved in acetone and recrystallized with the slow addition ofbenzene (or methanol). 2.2.1.3.2. Eledrochemlcal synthesls An electrochemical synthesis of trimethylselenonium iodide was investigated. The selenium electrode was prepared as follows. Selenium pellets (1 g) were placed inside a Teflon TFE tube (8 cm x 6 mm o.d .• 4.48 mm i.d.; Cole-Parmer Instrument Company, Chicago, III.) which had been heat sealed at one cnd. The tube and contents were placed in a boiliog g1ycerol bath until the selenium (melting pomt: 21'flC) înelted. At this point, a copper wire (27 gauge, 10 cm long) which had been polished with emery paper (gr,lde 2/0) was inserted into the molten selenium taking care that the end was 3 mm from the bottom and did not touch the inner walls of the Teflon tube. The assembly was removed from the g1ycerol bath and aJluwed to cool. After solidification of the selenium, the Teflon tube was removed with a razor blade and tt~e resulting electrode weighed. This electrode was, then, inserted through a rubber septum ( (Suba-SeaJ NQ 45, Aldrich Chemical Company,lnc.) into a 100 mL round bottom tlask containing 1% NaCI04 in dim.t:thylformamide (50 mL) tating care that the exposed upper section of the copper wire did 33 not contact the Iiquid. Methyl iodide was then added in a large excess (10 mL). A silver Wlre (26 gauge, 10 cm), inserted into the electrolytic solution through the same septum, served as the anode. A small Tenon (PFA) tube (2 cm, 1 mm id x 2.2 mm od; Cole-Parmer Instrument Company, Chicago, Ill.) was also inserted through the septum sa that the head space of the reaction tlask was open to the atmosphere. TIle septum served onlyas a holder for the anode and cathode. Twenty volts were then apphed, across the electrodes. After 4 h of reaction at room tempe rature, the power was disconnected and the septum-dual electrode assembly was removed. The dark red mixture was maintained at room tempe rature, wlth mlXll1g, for a further 2 h. The reaction ftask was then fitted with a distIllation head, a condenser, a recclving assemblyand a heating mantle. When the temperature of the distillation vapors reached 42-600 C, the excess methyl iodide was observed to distill (and could have been reused m subsequent reaCtlons). Then. as the temperature of the distlliate rose slowly from 80°C ta beyond 140oe, a transparent hqUld, havmg the characteristic odor of dimethyl selenide, was collected into the receiver tlask (contammg 10 mL of THF) which was immersed in a ice-salt bath (-19 ta -15°C). This distillate slowly crystalhzed in both, the condenser and the receiving ftask. The resulting distillate was allowed ta stand, at room temperature, to complete crystallization. 2.2.1.4. SYNTHESIS OF SELENONIUMCHOUNE AND SELENONIUMACETYLCHOLINE SALTS 2.2.1.4.1. Selenoniumcholine salt from dimethyl selenide Dimethyl selenide (0.5 g) was added to 2-bromoethanol (6 g) at OoC wlth stlHing and the reaction was then allowed ta proceed at room T under mtrogen for 24 h. Sodium tetraphenylboralc (1.5 g) in methanol (10 mL) was added to preclpitate the crude selenomumcholme product. The resultmg fine precipitate was then filtered under vacuum (Whatman ~ 1 paper), washed wlth methanol (3 x 10 mL) and dried for 12 hours, under vacuum, at 56°C in the presence of KOH. 2.2.1.4.2. Selenoniumcholine and selenoniumacetylchollne from metalllc selenium and methylllthium 2 Ta metallic selenium (gray, powder; 4.8 g, 6.08 x 10. mol), suspended. wlth vigorous !>tJrnng, ln 100 mL of tetrahydrofuran (lHF) under dry nitrogen and at OOC, was slowly added 31.H2 g (6.07xHr2 mol) of methyllitluum solution (welghed by dlfference). The reaction was contmued at oOe for 0.5 h. the appropriate alkyl bromide (2-bromoethanol, 8.60 mL, 6.07xlO·2 mol or bromoethyl ace tale. 6.70 mL. 6.07xlO-2 mol), was then added slowly to the c1ear yellowish liquld at O°e. When the addition Wa1> 34 complete, solution "A" was allowed to s10wly warm to room temperature. After a turther 3 h of reaction, solution "A" was con~ntrated to approximately 10 ml (under vacuum with a rotoevaporator placed in a weil ventilated fume hood). The concentrate, diluted with 20 mL diethylether, was transferred into a separa tory tunnel. The reaction tlask was rinsed twice with 20 ml diethyl ether and the washings added to the separatory funnel to result in a total volume of, approximatively. 70 mL The mixture was washed four times with 20 mL of double-distilled diionized water. The aqueous washes were discarded. The organie layer was then dried over calcium chloride, tiltered, and concentrated to 10 mL under vacuum. Sodium tetraphenylborate (6.08 x 10-2 mol) and methyl iodide (12.16 x 10-2 mol) în methanol (30 mL) were added , to the concentrated solution. The reaction was allowed to proceed, under N2 with constant stirring for 24 h at room temperature. The resulting precipitate was recovered by filtration (Whatman N° 1 paper) and redlssolved in a minimum of boding acetone. Fine crystals of tetraphenylborate salts were obtained by the slow addition of benzene (at room temperature). Alternately, very large crystals were obtained by the slow evaporation of the solvent, at room temperature. 2.2.2. RESULTS AND DISCUSSION 2.2.2.1. TRIMETHYLSELENONIUM IODIDE 2.2.2.1.1. Chemlcal synthesis The yield of erude trimethylselenonium iodide, following the method of Kuhn and eo-workers (1986). was in excess of 80 % if proper care was taken. The IH-NMR of trimethylselenonium iodlde in 2H20 is presented in Figure A-l, B of appendix A (any Figure whose number starts with an "A" will be found in appendix A). The chemieal shift of the nîne equivalent protons of this product was observed at 2.75 ppm relative to the external standard tetramethylsiJane (l'MS), whereas the chemical shIft of the methyl protons of trimethylselenonium tetraphenylborate in deuterated DMSO was observed at 2.63 ppm (Figure 2-2-1, A). The fast atom bombardment mass spectrum (FAB+) of trimethylselenonium iodide in a glycerol matrlX is glven 10 Figure A-l, C. The fragment at mlz 125 is the molecular ion (BOSe) and the fragment at mlz 217 is the moleeular ion plus glycerol. The laser ablation microprobe mass analysis (LAMMA) positive and negative ion mass spectra, of trimethylselenonium iodide and trimethylselenomum tetraphenylborate are reproduced in Figures A-2, and A-3 and the infrared spectrum of trimethylselenomum Reineckate is given in Figure A-l, A and is similar to that recorded by Palmer et al. (1969). Elemental analyses are reeorded in Table 2-2-1. Ali of the element were assayed independently. The elemental analysis was unaceeptable. The spectroscopie data corroborated that trimethylselenonium iodide was indeed the compound prepared. That it was also relatively pure was shown by the HPLC-mO-AAS chromatographie system in 35 Figure 2·2-1: Proton Nuclear Magnetic spectra in deuterated dimethylsulphoxide (eHkDMSO) of: A, trimethylselenonium tetraphenylborate (300 MHz); D, selenoniumcholine tetraphenylborate prepared trom dimethyl seleruJe and 2-bromoethanol (300 MHz); and C, selenoniumcholine tetraphenylborate prepared from metallic selenium (200 MHz). ~O A ASSIONMI!H1'S. a (H,C>,sc· • ..2.61". DMSO 7.0 6.0 ------5.0 10 1.0 0.0 B ASSIGNMI!.N1'S: Ha° • b c cl (H,C)~'~Ola°H a •• 161ppm b • undcr Ihc walcr peak c· d· - 7.162ppm ? DMSO --r--:____ ...... d 7.0 •.0 5.0 •. 0 3.0 1.0 0.0 a c ASSIONMENTS: • b c d (H,C)~'OlaCH,OH •• 166 ppm b. UDdc, UIc WlIU ~ c.lAI .... d .5.52 ppII RaO ~--- b d DMSO i~""'.iii~iiii , , , , , 1:.:. • , i , , , • .1 36 Table 2·2·1: Elemental analyses (percent composition) of the synthetic selenonium standards used for chromatography. Element TMSeII [CHOLSe ][TPhB]b [AcCHOLSe ][TPhB]C Carbon 15.86 (14.36)d 71.84 (71.05) 69.91 (69.92) .. Hydrogen 3.29 (3.61) 6.72 (6.60) 6.48 (6.45) Î Oxygen -- -- 1.68 (3.38) 2.34 (6.21) Boron -- -- 3.34 (2.28) 2.97 (2.10) Iodide 50.23 (5056) ------Selenium 30.15 (31.47) 17.11 (16.68) 17.25 (15.32) Total 99.53 (100.00) 100.69 (100.00) 98.95 (100.00) a. TMSeI = trimethylselenonium iodide b. (CHOLSe][TPhB] =selenomumcholine tetraphenylborate c. [AcCHOLSe][TPhB] = selenoniumacetylcholine tetraphenylborate d. The calculated theoretlcal values are in parenthesis 37 which only a single peak appeared in chromatogram recorded using a variety of mobile phase compositions. Moreover, as discussed in a later section of this thesis, the response of the HPLC-THG AAS system was virtually identical for injections of equivalent amounL _[ each of the three synthetic standards. The unacceptable results of the elemental analysis was probably due to the nature of the selenonium compounds themselves and the lack of expertise of the techmcian(s) m this area. Selt:nomum compounds are not common compounds and their analysis is not performed roulmely (selemum. anù boron are not elements that are routinely analyzed for). 2.2.2.1.2. Electcochemlcal synthesls Greyelemental Sex was reduced electrochemically to Se/won platmum (grid), glassy c~lfbon (c1oth), and on Mercury (pool) in aprotic solvents (Degrand and Nour, 1985; Gaulheron and Dcgrand. 1984). Symmetncal dialkyl selenides and diselenides were formed competitively if the catholyte contamed an alkyl halide (Degrand and Nour, 1985: Gautheron and Degrand, 1984). The ratio of selenuje 10 diselenide depended principallyon the type of electrode used (platmum, carbon, or Mercury). Ultrasound was also used to mduce (increase in the rate of the electrolysis) the electrochemlcal synthesis of Sc 22" and Se2" (Gautheron et al., 1985). A selenium electrode was prepared by allowing Iiquitied elemental metallic selenium to sohdJfy around a tbin copper wire. A silver wire served as the anode. A voltage of 20 v was appllcd across thc electrodes, IJ"lmersed in a 5% Na004 dimethylformamide solution contaimng methyllodide. After 4 h of electrolysis. the electrodes were removed and the reachon was a1lowed to proceed for a further 2 h. llIcn, the reaction mixture was distIlled and a hJgh boiling fraction (800 e to 140°C) was trapped 10 niF and allowed to crystalhze. The yields. in teern of how much selenium reacted were generally low (of the order of onc percent). On the other hand, about 80% of the selenium which reacted was converted to trirnethylselenonium iodide. The melting palOt was 155-15SoC m a "sealed tube" [hlerature M. pt. tor authentic trirnethylselenonium iodide: refer ta Table 2-1-1J The electrochemical synthesls proved to be an easy and lOexpenslVe route to (H3C)3Sc· as mo~t of the materials and reagents were reusable. Moreover, dlmethyl selenide (a foui smellmg, low bOlll/1g toxic Iiquid) was not directly mampulated and the use of methyllithium (a fire hazard) was aVOIded. The low quantity of water in the dlmethylformamide solvent did not affect the reactlon perceptlbly. If the voltage was mcreased above 20 volts, an appreclable gas production was observed. ft IS recommend<::d to have a me ans of venting the reaction charnber as gases were produced even wlth an applJed potentwl of ., 20 volts. Unfortunately, the constructIOn of the electrode proved to be difficult; ~mall cavltles ln the electrode surface were unavoidable. These cavities were attacked preferentlally resultmg ID an uneven consumptlOn of the selenium which generated an unwanted contact between the copper wlre and the L-______38 solution. At this point, large bubbles formed at the copper selenium interface and disintegration (of the selenium coating) of the electrode became rapid. Control experiments were performed in which no voltage was applied across the electrodes. Several observations suggested that there was no reaction. If no voltage was applied, the characteristic dark red cloud which formed just at the surface and especially in the cavities of the selenium cathode was not observed. Additionally, there was no formation of an orange-red cloud in the immediate vicinity of the anode surface. It is known that metallic selenium reacts with methyl iodide only at high temperature and pressure (Scott, 1904; Emeleus el al., 1946). Over the course of several electrolytic runs the temperature of the reaction solutions never exceeded 3(fC. If a uniform and thin surface of selenium can be deposited on a conducting material, this method will find applications. The principal limitation is that metallic selenium is only a moderately good electncal conductor (electncal comJuctance = 0.08 microhoms·1). However selenium, being a semiconductor, will absorb electromagnetic energy (if it is applied above a certain critical frequency) whtch can knock out electrons from their valence site. These "tree" electrons (free to wander in the crystal) will in tum increase the conductivity of the metallic selenium. This effect is called photoconductivity. In the same manner thermal energy can be absorbed by the semiconductor crystal. nus is termed the thermal excitation effect and will also increase the conductivity of the crystal (Gibbons, ( 1966). Accordingly, the electrical conductivity of the selenium electrode can be increased (thus increasing the yleld) by shinning the appropriate light energy on the electrode surface and/or by increasing the temperature (difficult with methyl iodide whose boiling point is 42's°C). The use of trimethyloxonium tetratluoroborate as a methylating agent instead of methyl iodide might permit higher reactlon (emperatures, Dlmethyl selenide can be produced by this method. The reaction mixture could have been heated and the product vapors entrained trom the crude reaction solution with a continuous stream of mtrogen. The entramed vapors could then have be~n bubbled into water to remove volatile by-produets (methyl iodide) and a1lowing ~he hydrophobic vapors [(H3C)2Se] to be carried into a second trap eontaining a cold orgamc solvent (Foster el al•• 1985). 2.2.2.2. SELENONIUMCHOUNE AND SELENONIUMACETYLCHOUNE SALTS The easiest and more direct preparation of the tltle compounds was the reaction of dimethyl selenide wlth an alkyl halide (bromide). However this route suffers from several drawbacks. Not only is dimethyl selenide a foui smelling. volatile and toxie liquid, it is also dlfficult to prepare. Methods which have been reported for the synthesis of (H3C)2Se (Bird and Challenger, 1942; Parizek and Benes, 1973), were inefficient (Foster et al., 1985). Moreover, the reaction of (H3C)~e and an a1kyl halide is prone to a ( side reaction leading to the formatIon of trimethylselenonium cation [(H3C)3Se+] as an impurity. 11ùs side reactlon was considered to result trom the attack (demethylation) on the deslred product by (H3C)2Se, and was mlmmized by maintaining the a1kyl halide in a vast excess. However, for a gram scale 39 synthesis. such an excess of a1ky1 halide was, for practical purposes, difficult to achieve. Foster el al. (1985) reported that the addition of sufficient water to the organic reoction mixture to ob tain a two phase reaction medium, appreciably decreased the formation of (H3C)3Se+ by extracting and stabilizing the desired selenonium product as it was formed and makmg it unavailable to attuck by (H3C)2Se which remained in the organic phase. 2.2.2.2.1. Selenonlurnchollne salt from dlmethyl selenlde A first approach to the synthesis of these selenonium compounds was simply to react purchascd (H3C)ZSe with flte appropriate alkyl bromide. This simple reaction was investigated with a ten·fold cxccss of 2-bromoethanol in order to form selenoniumcholine Ion in Methanol. Diethyl ether was then addcd to precipitate the positively charged selenoniumcholine as the eorresponding bromide salt. The dcslrcd product oUed out rather than crystallizing. If the oil was kept for a long period of time at -16()C a fcw crystals appeared. However, upon filtration of the cold mixture, the crystals melted very rapidly. 1\vo explanations of tbis behavior were possible: 1) Selenoniumcholine bromlde was a hqUld at room temperature and/or 2) tbis product was extremely deliquescent. Furthermore, it was obscrved that. on contact with air or water, the origmally yellow oil turned orange. This undeslrable feature suggcstcd an oxidation and indicated that tbis salt would be unacceptable as an analytical standard. Stable non hygroscopie crystallinc analytes are preferred as standards sil1Ce they are easier to manipulate and give rise to less variability when preparing standard solutions. In order to stabilize the product, a method of counter-ion exchange was investigated since the properties of selenonium salts are bighly dependent on the anion (cf. section 2-1-2-1·1). A counler Ion which increased the melting point of the product while making it less hygroscopie and more reslstant to oxidation was required. Sodium tetraphenylborate is a weil known reagent for the determlllation 01 potassium and other positively eharged organic species such as ammonium salts (Crane. 1956). Il wa!-> possible to precipitate the selenoniumcholine cation as the tetraphenylborate salt by simply addmg a saturatr,rl sodium tetraphenylborate solution to an aqueous or methanolic (if large quantilles of the selenonium analytes were present) solution of the selenonium halide of interest. A white very fine powllcr was thus obtained. The IH-NMR spectrurn. in deuterated DMSO. of selenoniumcholme tetraphenylborate produced by tbis method is ieproduced in Figure 2-2-1, B. The probable peak asslgnrncnts are prc!>Cntcll on the figure. From the integration of the signais in tbis spectrum, the peaks "d", "b" and "a" wcre III the proportion of 6:2:1 as would be t'xpected for the selenoniumcholine cation. The "COI protons wcre Ju!->t below the water peak wbich appeared at 3.34 ppm in the deuteurated DMSO solvent. However, the theoreticaJ ratio of 20 to 6 for the 6 equivalent protons of the two methyl groups of sclenoOJumcholmc ion and the 20 protons of the tetraphenylborate moiety was not observed. Furthermore, if MX equlYalent protons were represented by an integration of 2.fJ cm then 20 protons should, in turn. have bccn 40 represented by 8.67 cm and not the observed 12.05 cm. Thus, 3.38 cm of the aryl resonance signai corresponded to tetraphenylborate complexed to an impurity. This impurity also contained selenium since a IH_77Se coupling was also observed. As indicated above, other researchers have postulated that this impurity was (H3C)3Se+. The lH-NMR spectrum of trimethylselenonium tetraphenylborate in DMSO was also recorded (Figure 2·2·1, A). The nine equivalent hydrogen of (H3C)3Se+ were observed to resonate at 2.63 ppm wruch is very close to the 2.62 ppm of the impurity signal in Fig. 2·2·1, B. If the ;mpurity was (H3C)3Se+, then, the observed integration ratio of 3.38 cm over 1.5 cm should have been equal to 200ver 9. The observed value, 2.25 ppm, was close enough to the theoretical value (20/9) that it was judged Iikely that the impurity was, indeed, the trimethylselenonium selenonium cation. Repeated efforts to prepare selenoniumcholine ion by the action of dimethyl selenide on 2·bromoethanol, under the conditions mentioned earlier, consistently resulted in a mixture containing about 30% (on a molar base) of trimethylselenonium cation as an impurity. Since it proved to be extremely difficult to separate selenoniumcholine ion from trimethylselenonium cation by either recrystallization or preparative ion exchange, a new preparative route to selenoniumcholine was sought in which trimethylselenonium ion would not be formed as a SI de product. ( 2.2.2.2.2. From metalllc selenium One possible way to prevent the formation of trimethylselenonium ion was to precipitate the desired products (sclenoniumcholine and selenoniumacetylcholine ions) from the reaction medium as they were for "'led. This was accomplished by the co-addition of sodium tetraphenylborate to the methanol-ether reaction solution at the same time as the a1kyl halide was added. This precipitation procedure could have been used for the reaction of dimethyl selenide with an alkyl bromide instead of the recommended bi-phasic system (Foster et al., 1985) but it was not easy or pleasant to work with dimethyl selenide for the reasons described ab ove. Thus, another route of formation for the desired compounds was explored. Metallic selenium is known to react (in an inert atmosphere) with methyllithium (MeLi) in anhydrous THF to form methylselenohthium (Kuhn et al., 1986; Plenevaux et al., 1987). It was further shown that, upon reaction of y -bromo- a-aminobutyric acid (plenevaux et al., 1987), with methylselenolithium (MeSeLi), the desired organic selenide (selenomethionine) was formed Wlthout the confounding formation of a selenonium cation as was the case for the reaction of methylselenolithlUm with methyl iodlde (Kuhn et al., 1986). It is unfortunate that these authors did not stress the importance of adding only one equivalent of MeLi as any excess of trus reagent will attack the alkyl halide directly. This is especlally true in the case of Plenevaux et al. where a 25 fold excess of MeLi and only a two fold excess of the alkyl bromide were added. lt IS somewhat surprising that they were able to obtain yields in excess of 80%. These authors did state that yields were low for larger scale preparations and this was 41 attributed to oxidation. Kuhn et al., (1986), on the other hand, reported that the addition of methyllithium should be stopped when a c1ear yellow solution is obtained (an indication of the stochiometry of the reacnon). The continued addition of methyllithIum ln excess of the stochlometnc requirement, resulted in a transparent, then, a whitish c10udy solution If more methylhthlUm was addcd and inevitably an excess resulted in lower yields. The deSlfed seienides were obtained by reacting MeSeLi in THF Wlth a senes of alkyl bromidcs added in an exact molar ratio of 1:1 to prevent the formation of any selenomum side products. TIle reaction of MeSeLi with an a1kyl bromide proved to be 50 fast cornpared to the reactlOn rate of the formed selenide with the a1kyl bromide reagent that vutually no selenomum Impuntles were produccd. When a 1:1 ratio was used it was essential that no excess of methyllithium be added. The methylluluum was, also, added slowly as it took sorne time (few minutes) before the reagent attacked the sohd phase (selenium partlcles). The reaction of methyllithium With selenium resulted in the formation of methylselenuhthlUm which was then reacted Wlth an alkyl halide in a "one pot reaction". The resulting crude reactlOn mIXture, solution "Ait, contained the appropriate organic selenide; by simply adding excess methyl lodlde tht' desired selenonium compound was obtained. To assure a c1ean and pure end produet, It was deemed necessary to remove any selenonium compounds (such as [MeSe+R2UB(]) or unreacted MeSeLI prlor to the addition of CH3I. Trimethylselenonium iodide would have been formed upon additIOn of CH)I to unreacted MeSeLi. Thus, a washing procedure was included in the preparative sequence. The erude reaction mixture "A" containing the orgamc selemde was concentrated under a reduced pressure, dlluted with ether and transferred to a separatory funnel. The ether phase was then washed wlth double-dlstilled deionized water. MethylselenohthIum (excess) in contact with water was converted to methybelemdc anion/methylselenol (CH Se-/CH SeH) which is very soluble in aqueous phases. Aftcr the washmgs, no 3 3 selenonium ions should !lave been present and unreacted MeSeLi should have been completcly removed (as MeSe-). One mir,ht have thought that it would have been prudent ta carry out these c1eanup stcps under an inert atmosphere but it proved to be unnecessary. The advantages of this synthetic scheme were that 1) the reagents were easdy obtamed, 2) the use of dlmethyl selenide was avoided, 3) the reactlons were rapld and 4) a yleld of 60-70% was obtamcd consistently. More importantly, tnmethylselenonium cation was, at most, a very mmor Impunty a~ evidenced by the absence of a detectable signal at 2.63 ppm 10 the IH-NMR speetrum of Figure 2-2-J, C. Additlonally, the desired products were isolated as non-hygroscopie, au and heat stable, sohds. HowC!ver, exposure of the products to :acids, even weak ones, must be avoided as the tetraphenylborate mUlcty IS sensitive to these reagents. These selenonium products were dlssolved ln aeetone and dlluted wlth methanol ta form stable primary standards. Alternately, the eounter Ion can be change by treatmg an acetone:water (50:50, v/v) solution of these compounds wlth sllver nitrate. The !>clenomum 42 tetraphenylborate salts were very soluble in this mixed solvent w~reas silver tetraphenylborate (Crane, ( 1956) was not. The infrared spectra of selenoniumcholine and selenoniumacetylcholine tetraphenylborate presented in Figures A-4, A and A-S, A respectively. were characteriz.ed by prominent absorption bands at 3500.5 cm-l and 1214.3 cm-l characteristic of an a1cohol and ester functiollality respectively. Unfortunately in both spectra acetone. whose spectra is represented in Figure A-6. ft.. was present and sorne peak overlapping could not be avoided. The infrared spectrum of sodium tetraphenylborate is reproduced on Figure A-6. B. The tH-NMR spectra of selenoniumcholine and selenoniumacetylcholine in deuterated DMSO are iIIu' .' ated in Figures A-4. B and A-S. B respectively. Figures A-4. C and A-S. C represent their respective 13C-NMR spectra. Peak assignments are shown on each figure. For comparison the 13C-NMR spectrum of sodium tetraphenylborate is given in Figure A-6, C (Sadtler research laboratories. subsidiary of Block Engineering. 3316 Spring Garden street. Phi .• PA. 19104). In the IH-NMR spectrum of selenoniumacetylcholine tetraphenylborate an impurity at 2.62 ppm (presumably trimethylselenonium tetraphenylborate) was evident. It was possible to eliminate this impurity by successive recrystallizations since it was present in low amount. 1be LAMMA mass spectra (separate positive and negative ion spectral of both selenoniumcholine tetraphenylborate and selenoniumacetylcholine tetraphenylborate are ( presented in Figures A-7 and A-8. This spectral information was recorded to corroborate the identity of each compound. The purity of the selenonium compounds is best assessed byelemental analysis (Table 2-2-1). However, the results of these analyses were unacceptable. For example. with selenoniumacetylcholine, the percentage of carbon and hydrogen was very close to the calculated values but for the rest of the elements, the obselVed values were far off the calculated percentages. The chromatograms recorded (HPLC-mG-AAS; refer to section 3.2.) with various mobile phase compositIons contained only a single peak per selenonium analyte. ThIs system can easily detect 10 ng as Se for each selenonium salt. With injections of up to 1 p.g as Se for each salt. there was no evidence for any selenium containing impurities. Thus, the amount of possible selenium containing impurities present in cach selenonium standards was less than 1%. Additionally. the separate injection of equivalent (as Se) amounts of each of the tbree selenonium standards directly into the interface (column removed) produced identical responses from the AAS detector. Thus, there was no evidence of impurities (which did not contaifl any Se) in these synthetic standards. Selenium dioxide "gold label" similarly inJected directly lOto the interface a1so produced the same response per mole of analyte (Blais, 1990). Attempts to synthesize selenoniumbetaine [(H3C)2Se+~C02-] via the reaction of rnethylselenolithJUm with 2-bromoacetic acid were unsuccessful. This is not necessarily due to the method. as several factors may have come into play. 'The instability of selenobetaine is weil documented in 43 the Iiterature (Ganther, 1984; Foster and Ganther. 1984; Foster et al.• 1985). The tempe rature al whlch the reaetions were performed may have been too high. Il IS alsa important to note that ln titis C.ISC sodium tetraphenylborate was not used as it would have been slowly destroyed by the carboxyhc acid. Thus, ammonium Reineckate was used to precipitate and proteet the desired selenobetame. Moreover. the precipitation of zwitter ions with ammonium Reineckate can only be achleved ln an aCldlc medIUm (G1iek, 1944). The methylation of methylselenoacetate was, thus. performed in a mIXture of methanol and glacial acenc acid. Il has been shown that such acidie media were excellent chOlce for the methylatlon of several selenides (Foster et al., 1985). Unfortun&tely, only tnmethylselenomum cahon was Isalated. However. the relative instabllity of selenobetaine makes it an unlikely candidate as a stable constituent of foods. The synthe sis of tbis compound was not pursued in greater detall. 2.2.2.3. MASS SPECfROMETRY Laser ablation microprobe mass analysis (LAMMA) proved ta be an excellent tool tor the characterization of the synthe tic products since molecular ions as weil as severa! fragment ions were present in the resulting spectra. The proposed degradation patterns were found to be sufficlcntly interesting to warrant their consideration ID sorne detaiJ. For the actual spectra please refcr ta Appendix A. It was found that the FAB source was not "sufficiently energetic" to desorb the tetraphenylborate salts of the different selenoDlum compounds; thus, only the analysis of trimethylselenonium iodide was partially successful. By contrast, the laser ablatIOn techmque appeared to be the method of choice in trus case, lJrobably because of the presence of the phenyl groups of the tetraphenylborate anion. It is weil known that aromatic molecules absorb strongly in the UV. ThiS charactenstic presumably explained the effiCient energy transfer from the laser beam ta the molccules. For [(H3C)3Se+][f], a further advantage of the LAMMA over the FAB lomzatlOn techmque wa~ the production of severa! fragmentation ions in addition ta the parent Ion (Figure A-l, C vs Figure A-2). These fragmentatIOn patterns can be ratlOnalized by the assumptlon that preformed Ions can dcsorb mtact but, to sorne extent, can also undergo thermal degradation. The desorbed specles can he, subsequently, converted to ions by elcctron and/or adduct lomzatlons (Van Vaeck. 1990; Van Vaeck el al., 1989). In the following discussion, severaJ abbreviations are used:  T, thermal degradatlon; e.e.l., eleetron capture IOnizatlon (Ieading to M-'); ej., eleetron ionization (gJVmg M+'); a.i., adduct JOnJzatlon (yielding [M+H]+, [M+Na]+, etc.); "fish hook" arrows are used to represent a ane-clcctran ~hJtt; two slded arrows represent the rnovement of an electron pair; [mJz ... 1, brackets mdlcate th.Jt thl~ am IS nat detected. The reported mJz values for The mass spectrum of selenoniumcholine tetraphenylborate, recorded in the positive ion mode, is reproduced in Figure A-7. The preformed cation (mlz 155) is the Most abundant signal (base peak). The different fragments were relatively weak but were easily detected. The ions at mlz 105 and 91 were not specifie to the structure and May have been generated by less weil understood degradation and/or plasma interactions. The probable decomposition route has been suggested to be as shown in Figure 2-2-2 for the selenoniumcholine moiety and as reproduced in Figure 2-2-3 for the tetraphenylborate anic'n (Van Vaeck. 1990). In the negative ion mode, the structural characterization of selenoniumcholine tetraphenylborate was as represented in Figures 2-2-4, A and 2-2-5 for the selenoniumcholine and the tetraphenylborate moietles respectively. The isotopie pattern at m/z 58-59 supported this interpretation. Ions with mlz values of 25,49 and 73 are disintegration cIusters from the central region of the laser spot, Le. the region wlth the hlghest power density. These ions are not specifie to the structure. The structural interpretation of the positive mass spectrum of selenoniumacetylcholine tetraphenylborate was as shown in Figure 2-2-6, A for the sdenonium cation. The first c1eavage (mlz 197 - .> 87) is characteristic (Van Vaeck et al., 1988) and dominates over the usual alpha cleavage (Mc Lafferty rearrangement leading to mlz 59/60). The formation of mlz 43 could be e,."lained by thermal degradatlOn followed by charge localization in the subsequently ionized product. For the tetraphenylborate mOiety, the asslgnment was as described for the selenoniumcholine analog. ln the negatlve Ion mode, the probable identities of the characteristic fragment Ions from selenomumacetylchohne cation is reproduced in Figure 2·2-4, B. It should be noted that the Isotope pattern at mlz 180/181 and 58/59 was c1early different from the corresponding pattern 10 the spectrum of selenomumchohne cation. This is why a dlfferent interpretation of the structures has been proposed. although a partIal contributIOn of (Ph)2BO' and B0 - to the SIgnai mtenslty of m/z and was not 3 181 59 excluded. Because the product was desorbed by appreciable less energy, disintegratlon clusters were vlrtually noneXlstent in the spectrum of selenomumacetylcholine. The fragments due ta the tetraphenylborate anion are consldered to result in the same manner as they did for the selenoniumchohne compound. 45 Figure 2-2-2: Proposed degradation pattern of the selenonium moiety associated with the positive ion mode LAMMA spectrum of selenoniurncholine tetraphenylborate. -CH 0n , +/'...,/OH 3 Se ..J • 'S:/. ~ ~ -s:::j I l!-H mlz 155 1 mlz 123 mlz 123 ~ ;." t ,., ~- ~ - (CH3)2Se + , + /'...,/OH H C'-..../On Se0 ~ 2 • [::OH 1 mlz ISS mlz45 m/z45 , +/'...,/OH ll.T , a.l. ,. Se • /Se • /Se -Ii 1 (neutral mlz ISS mlz III lntermediate) " 46 Figure 2·2-3: Proposed degradation pattern of the tetraphenylborate ion associated with the positive ion mode LAMMA spectrum of selenoniurncholine tetraphenylborate. , 1 ! 1 f, t 1f î:t 8 L ~~ ~ ~ +V mlz 241 mil 165 mil 165 m/z 163 < }-B=OH m/II05 m/191 .. ------,. , 47 Figure 2·2·4: Proposed degradation pattern of the selenonium cations associated with the negatlve Ion mode LAMMA spectrum of: A. selenoniumcholine tetraphenylborate: 8, selenomumacetylcholine tetraphenylborate. A '." e.c.1. . 'S:/".../OH 'Se/".../OH èS;~OH 1 -CH' 1 - Se /"'../ OH ...41- ___l__ ..J mlz 1%5 (vel')' weak signai) B AT 's~~oy 'Se~oy e.c.I. ~ 'se~oru 1 0 o 0._ [mlz 182) r)~q(0- 'Se,.)(H ....41---~~ 'se~oy, ....4~ __-..;;.;H;.,.· __....J 0._ mlz 181 mlz 181 L mlz59 48 '- Figure 2·2·5: Proposed degradation pattern of the tetraphenylborate ion associated with the negative Ion mode LAMMA spectrum of selenoniumcholine tetraphenylborate. Se Se- Il 1 o-B--S? <> B- B 0 (J'O .. • U'a t, m/z319 mlz 245 : (base peak) 0 0- - Il 1 B B- B (J'O (J'O 4 .. (J'a m/z165 mlz 181 (very weak signal) mizS9 m/z73 mlz49 mJz2S , -- - -_ ..... ------~. -----.------(deslntegratlon clusters) , i 49 Figure 2-2-6: Proposed degradation pattern of the selenonium cations associated with the positive Ion mode LAMMA spectrum of: A. selenoniumacetylcholine tetraphenylborate; Bt tnmethylselenomum lodide. ------1 A .1' fOY .. I( 'S: '--.? LY0 • IV + .0 mlz 197 mlzS7 m/z43 'S:~oy • '~(] 0 ~H mlz 197 mlz 123 B Ô,T e.l. .+/ -H· 'S:/ ~ Se/ ~ Se ~ =S:, 1 1 l) H m/z12S mlz 110 m/zl09 (CHfSe-CHJ)2·(CH3)JSe + mlz375 + or CH3-Se-Se(CHJ)2 m/z20S 50 'The only ion characteristic of the selenonium moiety in the positive mass spectra of trimethylselenonium tetraphenylborate was the molecular cation. This was probably due to the lesser laser energy required to desorb tbis compound and hence to the lower internai energy of the released species. For the same reason, very Uttle fragmentation was observed in the negative ion mode with the base peak being the tetraphenylborate anion. 1be positive ion LAMMA mass ~ctrum of trimethylselenonium iodide was, on the other hand, very interesting. This salt required more energy than the tetraphenylborate analog for desorption. Thus, fragmentation and other peculiar phenomena were observed (Figure 2-2-6, B). Recombination of neutral moieties W1th the preformed cation was also observed (mlz 205 and 375). At mlz 375 a "trimeric" adduct resulting from a recombination of the cation and its degradation produet was indicated by the isotopie pattern. The detection of the radical moleeular ion of thermal decomposition product (mlz 110) was noted in tbis spectrum but was practically absent in the positive spectra of the tetraphenylborate analog. The presence of r appeared to enfavor the detection of ~. and fragments. The negative ion spectrum contained the following ions: r (mlz 127); ~- (mlz 254), Ii (mlz 381), and NaI.r (mlz 277). The interpretation of the very small signais at mlz 379 and 377 remains unclear. Thcse peaks are a!ways observed for I-containing organic compounds and appear to be independent of the structure (Van Vaeek, 1990). The proposed degradation patterns are somewhat similar to those observed for ammonium salts (Van Vaeck el al., 1984). 2.2.3. CONCLUSION Ar. electrochemical synthesis of trimethylselenonium iodide was investigated and the advantages of this synthetic scheme are considered to warrent further research in this area (Huyghues-Despointes and Marshall, 1991). l'Wo new sclenonium compounds, i.e. selenoniurncholine and selenoniumacetylcholine salts, were synthesized. A new method for the synthesis of (H3C)2Se+R cations was devised in which the use of dimethyl selenide was avoided, and the formation of trimethylselenonium cation as a side produet was rnmimized (Huyghues-Despointes and Marshall, 1991). The usefulness of the relatively new laser ablation microprobe mass analysis (LAMMA) technique for the eharaeterization of selenonium tetraphenylborate eompounds was demonstrated (Huyghues-Despointes and Marshall, 1991). Although elemental analysis provided unacceptable results, according to Fr-IR, NMR, LAMMA, and AAS, these selenonium salts, after rigorous recrystallization, were fully charaeterized, relatively pure and were fit for use as chromatographie standards (Huyghues-Despointes and Marshall, 1991). 51 CHAPTERIII ANALYSIS OF . SELENONIUM COMPOUNDS 52 J.l. I§OlATlON. CHARACfERIZADON AND DETERMINATION OF SEI.ENONIUM ANALITES IN BIOWGIÇAL MATRICES: A LITERATyRE RE\1EW ln preparing this section it was recognized (1) that relatively little work had been reported in terms of analytical methods for selenoniurn compounds, (2) that the reported methods were rather dlfferent in their approach, and (3) that there was no comprehensive or critical review of these procedures in the literature. In consequence separate approaches are considered in sorne detail. J.} ISOIAIIONICHARACl'ERIZATION OF IRIMETH\'LSELENONIUM AND SE-METUVL SELENOMETHIONINE Paper chromatography has been used to determine the urinary excretion pattern of selenium compounds from rats, after a single intraperitoneal injection (2 IJ.g or 0.8 mg Se/kg b.wt.) or an oral administration (15 ppm Se in diet + 20 ,",C [75Se]-selenite) of[75§e ]-selenite. Palmer et al. (1969) observed a single major spot (in addition to several minor products) on autoradiograms from two dimensional paper chromatograms of the urine. The relative mobility (Rf) ùf the major product (code-named U-1) was 0.92 in phenol-water (73:27;w/W) and 0.42 an butanol-acetlc acid-water (4:1:1;v/v/v). To identlfy thls compound, Palmer et al. (1969) isolated a large amount of trus metabohte by concentratmg 3 liters of pooled rat urine JO vacuo at 5cfc. The residue was dissolved ln ethanol, filtered, and the filtrate was evaporated to dryness. The ethanol soluble residue was dissolved in water (150 mL) and separated on a cation-exchange column (5.2 cm x 45 cm; AG 50W-X8 resin, H+, 20-50 mesh). The column was eluted successlVely with 2.4 L portions of water. 0.05 M HCI, 0.5 M HCI, 1.5 M HCI and 4.0 M HCI. The 4.0 M HCI eluate , which contained U-l, was evaporated and the resldue (3-5 mL) was again &Llbjected to cation-exchange chromatography (2 cm X 150 cm; AG SQ-XlO resin, 200-400 mesh; equilibrated with pyndme-acetate buffer, pH 3.1). Elutlon at 1.4 mUmin was performed wlth a pH gradJent consisting of pyndine-acetate buffeTS adJusted to pH 3.1 for 11 h, to pH 4.0 for 10 h, and pH 5.3 until the complete elution of the labelled metabolite was acrueved. A total of 2.4 L was required. After separation by Ion exchange chromatography and concentration of the fraction containing the eSSe]-metabohte m vacuo at 50DC. the metabolite, V-l, was recovered by precipitation from water with ammonium Reineckate, then punfied by repeated recrystallization (five times) from acetone-water (1:4, v/v). The solubility of the metabolite Remeckate was 1 mg/mL in acetone (Palmer et al., 1969). Around the same time, Byard (1969) alsa reported the isolation of a major urinary selenium S containing metabolite (which he termed Xl) from rats which had recelved eSe]-H2Se03 in their diet (10 ppm Se in their drinking water). Pooled urine was acidified with HO to remove carbonates as CO2 and to precipitate proteins. The supernatant was passed through a cation-exchange column (8 x 50 cm; Dowex 53 50, Ir form). Elution with water, then with 2 M NHPH was sufficient to remove most of the impuritlcs. Basic solutes including Xl were then recovered from the column with 6 M HCI. After evaporalloll, al room tempcrature, of the solvent from this fraction and addition of dilute NH40H (to readJust Ihe 1'1110 10), 2% ammonium Reineckate in methanol was added to precipitate basic substances. The RC1l1cd.. llc salts which had been recovered by filtration, washed with propanol, and dlssolved 111 S()% uqUl'OUS acetone were passed through Dowex 1 (OH' form) to regenerate the free base of Xl. The prollucts wlllch eluted were choline (-95%) and Xl (-5%). Choline and Xl were separated with an ammo .Icld an,tlyzcr using 0.2 M sodium citrate buffer (pH 3.28, at a tlow rate of 30 mllh) and a 18 cm x 0.9 cm (i.d.) COIUlllfl or a 18 cm x3.9 cm preparative column. From the basic properties of Xl or U-l, the ~stimated molecular welght (Byard, \9(9), lhe electrophoretic behavior at different pH's and the work of Byard and Baumann (1967, 1965), thclC W:t! strong evidence that the major unnary selenium metabolite (Palmer el al., 1969 and Byan.J, 19(,9), W,I! trimethylselenonium cation which was corroborated by comparmg the mobihty on paper chromatography and spectroscopie properties (NMR, IR, MS) of the isolated matenal wilh those of synthetlc Co·chromatography of the isolated major selenium metabollte from rat unnc wlth ~ynlhctll. trimethylselenonium standard was observed on (Whatman N° 1) descenllmg paper chromatography u'>lng 70% aqueous ethanol (v/v) or pyridine·formic acid·water (150:10:350. v/v/v) (Byard, 1969) or on two dimensional paper chromatography using the followlng pairs of solvents: phenol·water (73:27 w/w), \. butanol-acetic acid-water (4:1:1, v/v/v); chloroform-methanoI-17% NH40H (2:2:1, v/v/v), I·hulanol· ace tic acid-water (3:1:1, v/v/v); lsopropanol·formic aCld-water (3.5:0.5:1. v/v/v), ethanol-tert. buthanol· NH 0H-water (é:2:0.5:1.5, by vo!.) (Palmer el 1969). The synthetlc tnmethybelenol1lum W.I'> 4 al., visualized on the paper with the Munier and Mé.'cheboeuf modIficatIon of the DragondOlf1> rcagcnt (Palmer et al., 1969) or, If labelled, by autoradiography followed by countmg ln a gamma coulller (Palmer et al., 1969 and Byard, 1969). Byard (1969) anJ Palmer et al. (1969) reported that the IH-NMR spectra of the l1>olated metabolite (chloride) and the synthetic standard were charactenzed bya Single resonance ln 1)20 at 325 ppm (Byard, 1969) or 2.7 ppm (Palmer et al. 1969). Byard (1969) also compared the mass spcctrum of tlu<; metabolite (chloride) with that of synthetic trimethylselenonium chloride. Mass spectra were dctcrmlllcd with a double-focusing mass spectrometc:r usmg a direct probe heated to 175-180ue. The author dlll Ilot obseIVe a molecular ion but rather observed mass fragments at mlz 110, 95, and HO corre~pondll1g 10 dimethyl selenide, methyl selenol, and hydrogen selenide, respectlvely. for bath the metabollte and synthetic trimethylselenonium Ion. Palmer el al. (1969) recorded the infrarcd spcctrum of trimethylselenonium Reineckate and compared Jt with the mfrared spectrum of the unnary metabohtc Reineckate salt. The IWO spectra were Identical. Further proof was obtamed by co·cry1>talllzatlOrJ of trimethylselenonium Reineckate and the urinary metabolite (U-1) Reineckate salt. The 75Sc speclflt 54 actlvlty remained the same though two recrystallizations from acetone-water (palmer et al. 1969. and Palmer el al. 1970). Se-methylselenomethionine, [(CH3)2Se+CH2CH(~)C02H] was identified in plants which had received [75Se).SeO/- by several research groups (peterson and Butler, 1962; Virupaksha and Shrift, 1965) includmg the roots of red c1over, white c1over. and ryegrass. Roots were separated trom the shoots, finely chopped, and extracted for 15 min with 100 mL ofboiling 80% (v/V) aqueous ethanol (Peterson and Butler, 1962). ExtractIOn W1th fresh solvent was repeated four times. 'The combined ethanolic extract was then concentrated under reduced pressure at 400C. The resulting concentrate was fractionated by paper electrophoresls (3,4 hours at 20 V/cm) usmg a phosphate-citrate buffer (pH 2.7). "When its position corresponded wlth marker S-methylmethionine (sulfonium chloride)", the appropnate fraction was recovered and subJected to paper chromatography with n·butanol·pyndine-water (1:1:1 by vol.) (Peterson and Butler, 1962). Portions of the ethanol extracts were also subjected to two dimensionaI paper ehromatography usmg two solvent systems: 1) n-butanol-pyridine-water (1:1:1 by vol.) and 2) n-butanol aeetlc aCld-water (25:6:25 by vol., upper phase). "Peales were obsetVed m the expected pOSitIOns" compared to that of the reference ~ulphur lsolog (peterson and Butler, 1962). Other eharactenstic ehromatograms were also obtamed on DEAE-cellulose using 0.01 M acetate buffer, (pH 4.7) contammg 0.001 M disodlum ethylenedJanune-tetraacetate (peterson and Butler, 1962). It IS unfortunate that these authors dld not use synthetlc Se-methylselenometluonine as reference standard instead of the sulfomum Isolog. Virupaksha and Shrlft, (1965) used Se-methylselenomethionine as a reference standard m thelr study on selemum accumulator and non-accumulator Astragalus species. The leaves of plants grown ln the presence of [75Se)-selemte were excised and extracted with cold 5% trichloroacetic aCld. The aCld was removed from the cru de extract by washing with ether; the aqueous phase was flash evaporated and fractlOnated on a Dowex 50 (~ form) column. Se-methylselenomethiomne was eluted from the column wlth 4 M HCI. The eluate was subJected to paper chromatography/autoradiography usmg n-butanol-acetlc aCld-water (60:15:25 by vol.) as the developmg solvent. The fraction wluch co-chromatographed wlth authentlc [75Se]·Se-methylselenomethionine was recovered with water and subjected to a second paper electrophoretlc separatIOn in pyridine-acetate buffer (pH 6.5) at 40 V/cm for 30 mm. The lsolate had the sa me catlomc moblhty as authentlc Se-methylselenomethionine. TIus band was again eluted from the e1ectropherogram and chromatographed on DEAE-ceUulose paper with 0.02 M acetate buffer (pH 4.7; 0.001 M EDTA). The unknown mlgrated with an Rf value of 0.85 (the same as for authentic Se methylselenometluonme) and gave a purple color with ninhydnn "which is characteristic of the methionme group of amine acids on DEAE-cel1ulose paper" (Virupaksha and Shrlft. 1965). 1 55 3.1.2 APPROACHES 10 mE ISOlADON!DmRMINATlON OF SELENONIUM COMPOUNDS Several of the methods for the determination of selenonium compounds are based. mamly. on paper chromatography and/or ion exchange procedures developed origmally by Pal mer and co-workers (1969. 1970) and Byard (1969). OIson and co-workers (1976) studied the "absorption of trimethylselenomum IOn by pl.mts". Parts of the plants were eut in ta short pieces. added to 20 volumes of 80% aqueous cthanol and mechanlcally ground with a pestle and mortar. The mIXtUre was then tiltered, washed wlth 80% ethanol, and the filtrate was concentrated under reduced pressure (OIson el al., 1976). The resllJuc was dlssolvcd in 1.0 mL of ethanol and 0.4 mL of water; a 25 J.J.L a1iquot was lheu chromatographed on papcr accordll1g to the method of Palmer el al. (1969, 1970) or on a column of AG 50W-X8 (H+) as descnbed by P.llmer et al. (1969) (OIson et al., 1976). Nahapetmn et al. (1983), ID a study of the urinary excretlon of tnmethylselenomum Ion by rats, used the procedure of Byard (1969) Wlth certain modificatIOns. The filtrate of aCldlfied unne (pH 2,4 M HC!), 2 mL, was apphed to a 2.5 x 10 cm AG 50W-XlO (20-50 mesh, hydrogen form) callOn-cxchange resin and eluted sequentlally with 200 mL water, and 200 mL of 6 M HCI. Concentrated duate!> (together with standards of eSSe]-tnmethylselenomum chJonde and eSSe]-selemte) were spotted (100 jlL) on paper sheets (3.5 X 57 cm) and chromatographed, m a descend mg manner, accordlllg to a modltïcatlOn ot the method of Pa.lmer et al. (1969) for 17 hours usmg butanol-acetic aCld-water (4:]: 1 v/v) a, the mobile phase. Detection was performed by autoradiography for at least one month and/or by gamma ray spectrometry Wlth the assumptlOn that ail the radioactlVlty m the 6 M HCI resulted from (7'iSe 1- trimethylselenonium Ion alone (NahlpetJan el al., 1983). Nahapetlan et al. (1984) subsequently reported that if a source of (7SSe}-tnmethylsc1cnonlUm cation such as the 6 M Hel eluate (from Dowex 50 in the H+ form) of 75Se from rat unne wa.~ mlxcd wlth 25 mL of human unne and ce-chromatographed on a catIOn exchange (AG 50W-X8) column (2.5 x 10 cm) the recovery of label in the aCldic eluate was only 10%. These authors actually reported that thcu quanritatlOn was performed on the "water eluate from ln vlvo-Iabelled rat urine"; thls was ~ub!>C4ucl1tly mterpreted by Kraus et al. (1985) as bemg the 6 M Hel eluate. It seems more Iikcly that Nahapetlan el al. (1984) used the 6 M HCI eluate smce they were mterested. princlpally, m the analysis and recovcry 01 tnmethylselenomum Ion which cannat be eluted from a column of Dowex 50W wlth pure water. Ta resolve the problem of low recoveries in human unne, Nahapetian el al. (1984) developed an amon-cation dual-column system. The urine (25 mL) was first passed through the amon exchanger (1.5 x 3 cm of AG 2-XlO resm, cr form), and then through a 1.5 x 3 cm AG 50W·X8 resm (H+ form), the catIOn exchangcr. The dual column system was eluted Wlth 200 mL of water and the columns werc then dlsconncctcd. "The catlon-exchange coIumn was then eluted with 200 mL of 2 M ammomum hydroxlde, followcd by 200 mL ofwater and tinally 100 mL of 6 M HCI". TIns final e1uate was concentrated ta dryness undcr vacuum at 56 SO°e. The residue was repeatedly redissolved in water (50 mL) and re-evaporated to dryness until no more HCI was detected. The final residue was suspended in 20 mL of concentrated nitnc acid, digested .. with 10 mL of concentrated perchJo.ic acid and prepared for neutron activation analysis (Nahapetian et al., 1984). For a complete description of the digestion procedure please refer to the articles by Janghorbani el al. (1982) and Nahapetian el al. (1983, and 1984). Even with distilled water (2 L) contaimng 10 ng of trimethylselenonium salt and 300 J.l.g of copper Ion (added as an Interferent), low recoveries of the selenonium analyte were observed using the Ion exchange method of Palmer et al. (1969) (Oyamada and Ishizaki. 1986). The method was subsequently modlfied for the determination of trimcthylselenomum ion in environmental water samples. Water samples (2 L) and IS mL of 0.1 M sodium thiosulfate (as a masking agent for metals) solutIOn were mlXed and applied to a cation exchange column (1 x 10 cm; Dowex 50W-X8, H+ form). The resm was eluted sequentially Wlth water (20 mL) and HO (0.1 M, 20 mL; 0.5 M, 20 mL; and 1.0 M, 20 mL). Trimethylselenomum cation was recovered with 25 mL of 4 M HCI. Aliquots (15 mL) of the tnmethylselenomum fraction were transferred to a pressure decomposition vessel and heated at 200°C for 80 mm. Hydroxylamll1e hydrochloride (2 mL of 20% solution to reduce Se (VI) to Se(IV» was. then, added and the mIXtUre bolled for 10 min. Selenite Ion was extracted trom the mIXture with dltluzone (1 mL of 0.02%) m carbon tetrachlonde. TIuosulfate was added 50 that other cation such as copper would be washed from the column pnor to the elution of trimethylselenomum with 4 M HCl. The method afforded lecoveries ln excess of 86% with coeffiCients of variation less than 11% for levels of trimethylselenomum ranglng from 12.5 to 25.0 ngIL of river and ground water (Oyamada. and Ishizald . 1986). Cooke and Bruland (1987) also found evidence of selenium biomethylation 10 aquatlc systems. However. thelr dimethylated selenonium compound was suggested to be Se-methylselenometluonine and not the trimethylselenomum ion. Collected water samples were immediately filtered thrùugh a 0.3 IJ.m polycarbonate mt!mbrane fil ter wlth a Teflon filter sandwich. Aliquots were aCldified to pH 2 with HCI to stabllize the dlmethylselenonium ion and each a1iquots was stripped of the volatile methylated selenium compounds by purgmg Wlth mtrogen gas. The analytical procedure requlred at least four aliquots for selenium speciatlon. The first one was mlXed with 4 N HCI and 3% NaBH4 was added. The mIXtUre was purge wlth Nig) and the new volatIle selenium specles were trapped and analyzed. This represented a rncasure of selemte and a1kylselenium ions. A second ahquot underwent essentially the same treatment as the first one, but followlng the addition of 4 N Ha, the mixture was heated for 20 minutes. This was performed In order to reduce selenate to selenite and thus obtain a measure of the selenate ion. Total nonvolatde dissolved selenium was obtained by oxidizing ail of the nonvolatlle dissolved selemum species \VIth potassIUm persulfate. The selenate, thus formed, was reduced to selemte (hot 4 N Hel) and converted to the hydnde with NaBH4 for analysis. Cationic selenium species (dimethylselenonium Ions) were separated trom dimethyl selenone and dimethyl selenoxide in a fourth aliquot by cation exchange on 57 • AG-50, and the 4 N Ha eluate was again reduced with NaBH4 According to these authors. the NaBH" reduction was successful for the dimethylselenonium ion(s) leading ta dimethylselemde whde orgamc selenides such as selenomethionine were not. The cryogenie trappmg (liquld nllrogen) was performcd with 5% av 3 on Chromosorb WHP (80-100 mesh) and the trapped selenides wcre selectively dlstlllcd with a temperature program from -196 to 100 Oc over 4 minutes. The liberated selemdcs were. thcn. detected by gas-phase tlame atomic absorption speetrometry. ThIs analytlcal procedure lor selemum speciation deterrnination was mostly based on chemical procedures rather than on chrornatography. IsolatIOn procedures for (CH3)3Se+ from rat or human unne based on Remeckate preCipitation have been described (Foster et al., 1986 b; Ganther, 1984; Ganther et al., 1987). Unne samples wcre separated on a SP-Sephadex (H+ form) cation-exchange column Wlth a maximum of 1 mL ot urme (lcr mL of resin (Foster et al., 1986 b; Ganther et al., 1987). The column was washed sequentJally wlth 0.01 M HCI (50 mL) , water (25 mL), and 0.25 M ammonium formate (pH 4.0,25 mL). Selenomum compounds were subsequently recovered with 25 mL of 0.5 M ammonium formate (pH 4.0) and 25 mL of 1 M ammonium formate (pH 4.0). To each of the two resultmg fractIOns (pH adjusted to 6-7 'Nlth aqucous ammoma) 100 ~L of 1% trimethylsulfonium iodide carrier were added followed by 25 mL of 2 % ammonium Reineckate in order to precipitate the trimethylselenonium ion (Fosterel al., 1986 b; Ganther et al., 1987). After standing at 4°C for 18 hours. products were recovered by filtratIon ami wa~hcd wlth 5 mL of 1% ammonium Reineckate and assayed for actlvity with a Gamma counter (Foster el al., 1986 b, Ganther et al., 1987). Tnmethylselenonium was precipltated (> 90% recovery) whereas selenomethlOnllle and selenocysteme were not precipltated to any appreclable extent «2%) (Foster et al.. 1~86 b). Selenonium compounds havmg a carboxylate mOlety such as Se-dlmethylseienocystelllc and Sc· methylselenomethionine were recovered to sorne extent (11% and 30% respectlvely) ln the preClpltate (Foster el al., 1986 b). These latter compounds may thus have represented possIble Irnpunlles ln the Reineckate precipitates. However, It was demonstrated that Se-methylselenomethlOnme was cornplctcly metabolized by rats ",hen administered as a single dose at 2 mg Se/kg b.wt.; none of the toxlcant wa~ recovered unchanged in the rat urine (Kraus el al., 1985). For unlabeled samples. the Reaneckatc precipltate was dissolved in a small volume of 50% ethanol and the Remeckate amon removed by applymg the solution first to a QAE-Sephadex (acetate form) column and clutang the trimethylselenomum callOn W1th water an\~ then applymg the appropnate fractIon of column cluate to a SP-Sephadex colurnrl to Immobllize the tnmethylselenonium cation and remove the rcsldual Reancckate anion (Ganther et al., 1987)_ Several selenonium compounds in unne have been separated by HPLC (Krau~ el al., 19H5; Ganther el al., 1987). This method is, to our knowledge, the only one that separates !)Cveral sclcnomum compounds efficiently. Urine (15 mL) was mvœd wlth ten volumes of absolute ethanol and chllled wlth COfacetone for 15-20 min. After centrifugation. the ethanol supernatant was iccovercd by decantatlOlI and dried. This procedure was repeated twice. The resldue from the combmed supcrnatant~ wa~ dls~olvcd 58 ln 300 /JL of 50% aqueous ethanol and 100 ",L a1iquots were analyzed by HPLC using a strong cation ( exchange column (Nucleosd 5-,lJrnJSA, 4 x 200 mm). A mobile phase, 0.003 M ammonium phosphate (pH 4.0) for eleven minutes followed by a hnear gradient of 0.003 to 0.33 M of ammonium phosphate (pH 4.0) over 71 mm, was used to separate Se-dimethylselenocysteine, Se-methylselenomethionine, and tnmethylselenonium cations (in order of elunon). The elution of ethylselenetine [(CH3)2Se+CH2C(O)OCH2CH3] cation required a change from 0.33 to 0.5 M ammonium phosphate for 28 mm as it was the most basiC of this group of selenonium analytes. For a tlow rate of 0.8 mUmin, the total separation tlme was 110 min and trimethylselenonium ion has a retention Ume of about 60 min. However, the retentlon time of trimethylselenonium was somewhat variable [55-68 min for the standard, and 60-70 mm for desalted rat urane (Ganther et al., 1987)]. This method was characterized by excellent resolution smce Se-dimethylselenocysteine and Se-methylselenomethionine were completely separated even though theu structure differ by only one methylene group. Recoveries were also very good for selenonium compounds; more than 90 % of these analytes were recovered 10 the ethanol extract after the desalting step, and recoveries from the column were greater than 85 %. This method was later used to study the metabolism of selenocyanate ln rats (Vadhanavikit et al., 1987). The retentlon time of radiolabeled trimethylselenonlum ion ln desalted urine was 42 min (wlth a band W1dth of 38 to 55 mm total) on a 4 x 200 mm 5 I-'m SA strong cation exchange column and a flow ( rate of 1.0 mUmm (VadhanavJltit et al., 1987). A modification of the method by Kraus et al. (1985) was used to study the "dose-response in unnary excretion of tnmethylselenonium m the rat" (Zeisel et al., 1987). Rat urme, 50 /JL, which had been filtered through a 0.5 p.m regenerated cellulose tilter was analyzed by HPLC using also a 200 x 4 mm 5SA NucleosJl column and a bmary gradient of 0.01 M ammonium phosphate (pH = 4.0; buffer A) and 1 M ammonium phosphate (pH = 4.0: buffer B) as the mobile phase at a flow rate of 1.0 mUmin (Zelsel et al., 1987). Because the specifie activity of the radiolabeled analytes was high, there was no need for large volumes of unne sample. Il was not necessary to desalt the unne and up to 100 J.l.L of urine could be dlrectly applied on the column with high recoveries (Zeisel et al., 1987). For the first time an on-line radlometric detector was used. Two unknown retamed peaks (30-35 min) were eluted pnor to the tnmethylselenonium cation (retention time 50-58 minutes). According to these authors one of these unknown peaks mlght be the same unknown peak which appeared at 30-35 mm in the chromatogram of the urme of rats InJected wlth [7SSe].dimethylsclenocysteine reported by Krauss el al. (1985). The~e suggestIOns are rughly speculative smce the treatments of the rat unne samples prior to chromatography were appreclably dlfferent and the tlow rates used were also different. This is weil demonstrated when one compares the retentlon tJmes found by Krauss et al., 1985, (60-70 min) and Zeisel et al., 1987, (50-58 min) ( for the tnmethylselenomum IOn in rat urine. Unfortunately, the method developed by Kraus el al. (1985) ... which appears to work fairly weil for rat urine (and has found general acceptance) presents sorne problems when applied to human un ne (Kraus et al., 1985; Ganther et al., 1987). "The analysis of human 59 urine using this procedure results in lower recovencs of tnmethylselenonium Ion than expected. When eSSe)-trimethylselenonium was added to human urine, recovery of tnmethylselenonium in the desaltmg - .~ proc.edure was satisfactory (85%), but du ring the subsequent HPLC the (SSe)-tnmethylselenonium was eluted as a very broad band, rather than a peak, and earlier than would be expected for the pure compound" (Ganther et al., 1987). The effect of overloading on SP-Sephadex (W) was then studietl. For a constant bed volume of 22 mI.., 25 mL or less of spiked ([7SSe)-trimethylselenonlum) human unne resulted in recoveries not lower than 98%. Recoveries were .nuch lower (44% and 24%) If the hum.m urine volumes were 50 and 100 mL respectively (Kraus et al., 1985). Improvements were observetl It the human urine (10 mL) was de33lted, taken up in 6 mL of water and passed though a Sep-Pak C1S cartndgc. The eluate and the washmgs (10 ml water) were combmed, and evaporatcd to dryness; the residues were dissolved in 0.8 mL of 20% ethanol and 0.5 mL were inJected lOto the HPLC !.ystcm (Ganther et al., 1987). Trimethylselenonium ion was finally identdied Wlth certamty m human urine by combining the method of Reineckate preCIpitatIOn followed by HPLC separation. For the isolation of trimethylselenonium ion in human urine, Ganther (1984, 1987) proposcd the followmg procedure. The urine was aCldified to pH 2 and applied on a catlOn-exchange column (SI' Sephadex, H+ form). The non-retained fraction was discarded and the retamed fraction (52% of the total Se) was eluted wlth 0.5 M of ammonium formate (pH 4.0). The eluted catlomc fraction (38% of total Sc) was spiked with 75Se+(CH3)3 and tnmethylsulfomum (added as a camer), neutraltzed (pH 6) and trcatcd with 1% aqueous ammonium Remeckate to precipitatc the tnmethylselenomum Ion. The supcrnatant wao!> then acidified (pH 2) to precipitate selenomum compounds having a carboxylate group followlllg the procedure of Glicl( (1944) for the selective recovery of hetaine and choline. However. no preclpltalc was observed. This implies that half of the "cationlC" selenium in unne is not preclpltated at ail by Remcckate. The first (and only) precipitate which contained trimethylselenonium ion (11% of ortglllai total Sc) was dissolved in alcohol and applied to cation (SP-Sephadex) and anion (OAE-Sephadex) exchanger rcslI1s to remove the Reineckate anion. This punfied tnmethylselenonium fractIOn was then subJcctcd to HPLC (cation exchange column, following the method of Krauss et al., 1985 and Ganther, 1987) wlth aqucous ammomum phosphate as the eluent. The retentlon tirre of this "catlomc" selemum contaulIng traction corresponded to the retention bme of authentic 75Se" (CH )3' This method may be effective for human 3 urine but it requires precipitatIOn of the analyte Wlth Remeckate and seems to work only for tnmethylselenomum cation. It was also noted that 40% of the selemum in urine was rctamcd on the OAE-Sephadex anion exchanger and could he recovered Wlth 0.05 M Hel (Ganther, 1984). Detection was performed off-fine using a gamma-counter. Hoffman and McConnell (1987) reported the catlOn-exchange HPLC separation of several selenite metabolites, including trimethylselenomum ion, in the liver or urine of rats whlch had bccn injected W1th [75Se]-selenite. Each rat Iiver was homogemzed in 3 mL of cold 5% perchlonc aCld, and the resulting suspensIOn was centrifuged. Supernatant (50 ~L) was then tnjected mto the chromatograph 60 (described below). Urine samples were mixed with equal volumes of 10% perchloric acid, clulled, and centrifuged. Supernatant (50 #.'L) was then separated on a 4.6 x 250 mm Sepralyte SCX cation-exchange column using a bmary gradient elution program. "Solvent A was prepared by adding 10 mM NH40H to 5% acetonitnle m water and adjustmg the pH to 3.5 with formic acid. Solvent B was prepared by ad ding 0.4 M (NH4)2S04 to solvent A and readjusting the pH back to 3.5 with H2SO 4' The elution program (2 mUml.l) conslst~d of 100% A for 6 min, a gradient to 100% B at 16 min, and continumg 100% B to 20 mm" (Hoffman and McConnell, 1987). The system required re-equilibration for 7 min with 100% A between injections. One milliliter fractions were collected and assayed, off Hne, for 75Se Wlth a gamma counter. Most of the work on the determination and separation of selenonium compounds has been performed by ion exchange chromatography based, mainly, on the pioneering work (ion-exchange) of Byard (1969) and Palmer et al. (1969). The first and only approach to reverse phase HPLC for the separation of several seleno-compounds (selenite, selenoethionine, selenomethionine, selenocystine) from trimethylselenomum Ion was not very successfuI. Different columns (635 x 250 mm) were tested: Partisil 10 00S-2 (C18 reversed phase), PartJsII-10 (C8 reversed phase), and Partisil 5 (sllica gel). The best results were obtamed with the Partisll 10 00S-2 column at room temperature. The elutlOn was performed isocratically with a water-acetonitnle mixture (100:3 v/v) as mobile phase at a rate of 1 mUmin. Surpnsingly, tnmethylselenonium cation, which appears to be a rather strong base (6 M HClls generally used for its elution from strong cation-exchangers), was found to be eluted as a "sharp peak between 1.5 and 2.5 mL of elution solution regardless of the column employed" (Blotcky et al., 1985). From the number of separate fractions (x 3) which were assayed for actiVlty it is difficult to judge whether appreciable tailing occurred Wlth these conditions. It is surprising that trimethylselenomum Ion eluted from a Partlsll 5 (sllica gel) column as a sharp peak in only 2.5 min with a water-acetonitnle (100:3 v/v) mobile phase. It IS mterestmg to note that none of the authors who worked with derivatized silica stationary phases such as the Partlsil-l0 00S-2 (Blotcky et al., 1985) or the Sep-Pak C18 cartridge (Ganther et al., 1987) has commented on the strong affinity of trimethylselenomum cation for free sllanols groups (refer to the result section). Because the separation on reverse phase packings was not considered satlsfactory, Blotcky et al. (1985) returned to the more conventional ion exchange approach. Cation exchange separation was studied on a 0.7 x 10 cm borosilicate glass column filled with 6 g of AG-50W-X8 in tht hydrogen form. The optimum eluent was found to be aqueous lithium nitrate (0.5 M). Presumably litluum was used instead of sodium because of the mterference of sodium in the neutron activation detection process. Chlonde represents a simllar source of interference. Ali radioactivatable ions (seleno-compounds: selemte and selenoamino acids, sodium and chloride ions) must be separated trom the tnmethylselenomum catIOn If quantitative measurements are to be performed. This was possible by the catIOn exchange method since these interfering ions were eluted pnor to the trimethylselenonium cation 61 in the following general order (Iess retained to more retained): chlonde, selemte, sodium. selenocystme. selenomethionine and finally trimethylselenonium ion. The relative selectivities (order ot elutlOn) .lppear to depend on the sample matrix (aqueous or hydrolyzed urine). The mcomplete resolutJon of certain peaks was alsa a problem but less 50 in the hydrolyzed urine. Recoveries were very hlgh for ail selello compounds trom water (93-100%). However, in the hydrolyzed unne matnx, recovertes for trimethylselenonium cation were highly variable and generally low (28-84%). Losses May have resulted trom the hydrolysis procedure (5 mL urme, 3 mL 6 M Ha, 2 mL glacial acetic aCld. and 0.5 mL 1 M mercaptoethanol sealed in glass tube and heated at 1000C for 5 h) and/or overloadmg prohlems. A hydrolysis procedure using HN0 where tnmethylselenonium IS known to be stable (Janghorballl el al .• 3 1982: Blotcky et al.• 1985) could not have been used since the selenoamino aClds are uns table under thcse conditions. Further studies would be required to determine whether these low recovenes were the result of the hydrolysis procedure or because of overloading of the column. ThIs simple cation exch,mge metllod was finally abandoned by the authors who developed a dual anion-cation (in tandem) exchange system. The anion exchange column (0.7 x 4 cm packed with 15 g of AG 2-X8 in the chlonde form. 200-400 mesh) was incorporated ln the chromatographie procedure to trap the radloactivatable amons (peptide Iinked seleno amine acids, non·protein-bound seleno amine aClds, selemte and the chlonde Ion). The trimethylselenomum cation would pass through the anion exchanger to the cation exchange column where It is separated trom the radioactlVatable cations (Na+). The catIOn exchange column was the same as the one descnbed previously by the authors (0.7 x 10 cm borosdicate glass column contammg 6 g ot AG 50W-X8. W form, 200-400 mesh). Raw unne (65 mL) was added to the dual column system whICh . was eluted with 97 ml. of 0.5 M LIN03 Trimethylselenomum IOn was retamed on the catIOn column efficiently (>90%) for spikIng levels of 0.1 and 55 p,g/mL urine).1t 15 unfortunate that the authors dld not attempt to spike with lower concentrations of anlyte such as the ones normally encountered ln unnc (0.01-0.06 p,g/mL). Since more than 125 mL of eluting solvent are reqUIred to recovcr the trimethylselenonium ion trom the cation exchange resm followed by a lengthy concentratlOn-evaporatlon step prior to the analysis by neutron activation, Blotcky et al. (1985) studied the direct actIvatIOn of rcsm which did not contam intrinsic selemum. Blotcky et al. (1987) were able to selectively d:termme tnmethylselenomum IOn and SeO) 2· 111 urine by anion exchange chromatography and molecular neutron activation analysls. Two types of amon exchange resin (AG-1-X8 in the acetate form, 100-200 mesh. and AG-2·X8 In the chlonde form, 200-40(J mesh, in 0.7 x 10 cm borosdicate column) were evaluated. The eluent was 0.5 M LIOH. One mL fractIOns of the chromatographie eluate were coUected m vials which were then Irradlated wlth neulrons, and radioassayed for 77mSe actlvity. The timlt of detectlon was 10 ng of Se as tnmethylsclenonJum or SeO) 2· per mL. Prior to chromatography ail urine samples were made alkaline (pH of 10-11) to dl!>~oclate frcc selenoamino acids trom possible protem·biding sites and to denature protems (rendenng them marc soluble) thus, allowing ready passage through the resin columns. The authors observed that the AG-2-X8 resm in the cWoride form (200-400 mesh) resulted in a more effiCIent separatIon and a hlgher recovery. 62 However, tJus separatIOn technique could not be used for the selenoamino acids. Selenomethionine was retained appreciably on both columns whereas selenocystine and selenocysteine were irreversibly 2 adsorbed by both resins. Since the recoveries for trimethylselenonium and Se03 - were quantitative, the method of standard additions was used to determine their respective concentrations in urine. More recently, Blotcky et al. (1988) optJmized therr procedure for the determination of trimethylselenonium and selemte not only m urine but a1so in serum. They expanded thelf anion exchange chromatography and molecular activation analysis technique so that total selenoamino acids could also be estimated. The best results were obtamed with a 0.7 x 10 cm borosilicate column loaded with AG2-X8 resm in the nitrate form. Trimethylselenomum and selenite in urine were analyzed as described earlier (Blotcky et al., 1987). If the total selenium was less than 40 ng/ml, the analysis was not performed. The same method was also applicable to serum. Total selenoamino acids in urine was determined by derivatlzing the amino acids with o-phthalaldehyde (OPA) and 2-mercaptoethanol. The derivatized amino acids were retamed on the resm whereas trimethylselenonium and selenite eluted. The resin were then irradiated and radioassayed for selenium content. If non detectable levels of selenite were present and that the total selenium was greater or equal to 40 ng/ml, then the procedure was relatively straight forward_ However, If selenite could be detected, then, a precolumn precipitation/coprecipitation of Se0 2- with Ba(N0 )2 and 3 3 (NH4)2S0 4 followed by derivatization and anion exchange chromatography was necessary. Selemte was found to rcact wlth the OPN2-mercaptoethanol reagent solution and the product was partially retamed on the column. In the presence of selenite the total selenium concentration had to be hIgher than 80 ng/mL for the analysls to be successful. Decarboxylated Se-adenosylselenomethionine and especially Se-adenosylselenomethionine. selenonium compounds, were observed to be important selenomethionine metabolites in K-562 cells (KaJander et al., 1989). Supernatants of cultured cells (absolute amounts were not mentioned) mixed with HCl04 (0.4 M final concentration), stored in an ice bath for 15 min and centnfuged at 3000 g for 10 minutes, were vlgorously mixed Wlth an equal volume of Alamine-Freon and re-centnfuged. The upper phase,20 J,l.L, was added to a 2.1 x 100 mm Hypersil ODS HPLC column and eluted (0.5 mUmm, 37°C) Wlth a gradient of 10 to 85% of buffer B in buffer A (0-2 min 10%,2-17 min hnear gradient to 85%, 17- 19 mm 85% and 19-20 min from 85% to 10%). Buffer B was 70% 100 mM KHzP04 (pH 3.2,8 mM sodIUm octane sulfonate) and 30% acetomtrile (v/v) whereas buffer A conslsted of 96% 100 mM KH2P04 (pH 2.5,8 mM sodium octane sulfonate) and 4% acetonitrile (v/v). Detection was performed on Une by UV absorption at 257 nm (detection Iimit of 20 pmol per compound). Efficient chromatographie separations (separation of the selenium analytes trom the ~ulphur isologs) permitted the use of a non selective detector such as an UV absorptIon spectrophotometer. ,,. "One of the most neglected areas of selenium research is the identification of forms of this trace element in foods" (Ganther et al" 1984)_ Manne fishes such as runa are nch in selenium, but are charactenzed by a low selemum bIOloglcai availabIlity (Douglass et al., 1981). Marine systems can create 1 63 novel organic chemical fonns of certain trace elements. For example, in marine foods, arsenic is found in organic forms such as arsenobetaine (Edmonds et al., 1977), tetramethylarsonium. (Shiomi et al" 1987) and arsenocholine (Lawrence et al., 1986). Since selenium also often occurs in methylated forms, Ganther et al. (1984) considered that "selenonium compounds such as the selenium Isolog cf arsenobetame mlght be a form of selenium in tuna". They found that "a substantial portion of selenium in canned tuna (packed in water) was present in the juice in low-molecular-weight forms, and that the concentration of selenium in juice exceeded the concentration in tuna Meat (about 8 ppm, dry basis. vs 3 ppm)", These authors chuse ie work with the juice from "one case of tuna", TIus Juice was filtered. extracted with ether and the aqueous phase acidified (pH 2). The details of the procedure were not given, The procedure involved cation exchange chromatography (Sp-Sephadex and CM-cellulose), ammonium Reincckate precipitation and gel filtration (Sephadex G-10). Following the procedure they were able to Isolate a portion of the tuna selenium which had the characteristic properties of a selenon\lun compound. However, the behavior of this isolate on thin layer chromatography and electrophoresis was dltferent from authentic (CH )2Se+CH COO· (made from dimethyl selenide and bromoacetic aCld), Thus tlus 3 z selenonium compound was not the expected selenetine. The "selenonium" fraction contamed about 350 1J.g of selenium whereas the starting juice from one case of tuna contamed 2247 /.Lg of selenIUm that IS about 15% of the selenium in the tuna juice was present probably as selenomum compoumJ(s), TI1is would explain why these authors found phenol ta extract only 10-27% of the selenium ongmally present in the tuna jUlce, whereas nearly ail of the arsenic in lobster was extracted by phenol. 3.2. ~~~o!!ilL!::Y=S=ES==O=F=T=R=IM~ET=HYL==:a:S=E=L=EN=O===N=IU=Mi:ô!'::l!iS:::E=L=EN=O~N;.,;,I=U==M==Cl!:!lH=O~L=I~NO:::;!E AND SELENONIUMACETVLCHOLINE CATIONS..; The airn of this research was to develop an analytical method whlch would permit the routme determination (separation and detection) of selenonium compounds in bio:IJ~ir.al sJmples. Hlgh performance Iiquid chromatography (HPLC) is known for its separatlve abIliUl!~ whereas atoml\""; absorption spectrophotometers (AAS) are considered to be higWy selective and SI!nsltlVe ,;jetectors for metals and sorne metaIloids. It was believed that intetfacing those two Instruments, HPLC and AAS. would combine the separatIon power of HPLC without compromlsmg the sensit1'w'ity of AAS. A sclelllum hydnde (HzSe) generator quartz interface was developed and optimlzed (Blais, 1990; BiaIS et al., 1990). However, because the chromatography was poor (excessive band broademng) the \tmlt of detection was limited by the chrornatography rather than by the interface Itself. Ir was antlclpatcd that Improving the chromatographie efficiency would lower the mstrumentallimlts cf rl~tectlOn (LODs). The applicatIOn ta blOlogical extracts was also explored not only in terms of the C:ètector response (mtertace compatibility Wlth sample extracts and mobile phase compositions) but aIso hl terms of chromatographlc reproduciblllty and dependence on extract composition. 3.2.1. MATERIALS AND METHODS 3.2.1.1. REAGENTS AND STANDARDS Ali solvents were "distIlled In glass" or "HPLC" grade (Caledon, Inc., Georgetown, Ont.). Hydrochloric acid and acetic aCld were certified ACS reagent grade. Watei 'l'las double·dlstilled and deionized. Triethylamine was purified "gold label" grade (Aldrich Chemicals Co., MIlwaukee. Wis.). Ali other chemicals 'vere reagent grade or better (Aldrich Chemicals Co., Milwaukee, WIS.). TIle syntheses. purification and praof of shucture of the synthetic standards were descnbed In section 2.2 of thlS thesis. 3.2.1.2. INSTRUMENTS The instruments for this study cOltsisted of an HPLC system (Beckman model 100 A pump, and model 401 program module), an autosampler (LKB, model 2157) and an atomlC absorptIOn spectrometer (Phillips, PU9100 set at 196.2 nm) which was equipped ·;nth a high euergy selenium hollow cathode lamp " (Photron super lamp system, Australia). The atomic absorptIOn spectrometer was eqUlpped wlth deuterium arc background correction. Since it mcre:~d the background nOIse of the detector appreciably, the correCTIon system was used only for confirmation and only when extracts were analyzed. 65 It should be emphasized that there was no way to have the recordmg mtegrator (HP 33~0 A, Hewlett Packard) calculate peak areas without adding "tiek marks" to the resultmg chromatogram. These "tick marks" should not be mterpreted as noise spikes. Narrow-bore stainless·steel tubing (0.007 cm i.d.) was used post-in je ct or. The 50 m i.d. silica transfer Hne was conneeted to the HPLC rubing through a capJllary reducing umon (Chromatographie Specialties, BrockYiUe Ont.). 3.2.1.3. CALIBRATION The calibration models for bath the selenoniumcholine and trimethylselenonium cations were obtained by analyzmg (25 p.1 injections) sequential dilutions of a fresh standard solution containing both analytes. The Iimlts of deteetlon (LODs) were determined from the calibration curves (linear regresslons) by using a first-order error propagation model with base-Hne noise normally distributed (Foley and Dorsey, 1984). 3.2.1.4. HPLC-AAS INTERFACE The HPLC-AAS interface has been described elsewhere (Blais et al., 1990). Diagrams of this thermoehemical hydnde generator (mG) interface are reprodueed in Figure 3-2·1. The main body (ali quartz; LaSalle SClentlfie, Ine., Ont.) consisted of: an optical tube (A, 12 cm, 9 mm i.d. x 11 mm o.d.); an analytical f1ame tube (B, 8 cm, 4 mm i.d. x 6 mm a.d.) and a si de arm which contained a combustion chamber (C,3 cm, 7 mm Ld. x 9 mm o.d.) and met the analytical tlame tube at an angle of 45 Oc. The combustIon chamber was fitted with an oxygen and hydrogen inlet tubes (2 mm i.d., 3.2 mm o.d., 5 cm, 2.5 cm apart) W1th the oxygen inlet bemg upstream from the hydrogen inlet; a thermospray tube (D, 8 cm, 4 mm I.d. x 6 mm o.d.). The thermospray assembly consisted of a deaetivated caplliary si/ica transfer line (E, 50 m I.d. x 20 cm; Chromatographie Specialties, Brockville, Ont.) connected to the HPLC outlet and centered wlthm the thermospray tube by a quartz guide tube (F, 10 cm, 2 mm i.d. x 3.2 mm o.d) wluch had been restncted to approximately 2 mm at the outlet; and a coil of resistance wire (G, 22-guage Chromel 875 alloy, 40 cm, Hoskins Alloys, Toronto, Ont.). The heatmg coil was insulated with refractive wool (Flberfrax, The Carborundum Co., Niagara Falls, NY) and encased in a shaped firebrick casing held together by a screw-c1ip. The he3ting element was energIzed Wlth a CUITent of 4 to 5 amps (2 A on standby) maintained by an ac. varIable transformer and momtored with a standard ampmeter. 1\vo stamless steel modified Swagelock (Forsyth and Marshall, 1985) assemblies (H) were used to pOSition the slhca gUIde tube mto the thermospray tube (D) and the analytical oxygen quartz tube inlet (l, 15 cm, 2 mm I.d. x 3.2 mm o.d.) WIthin tl.e analytical tube (B). The outlet edge of the analytical oxygen mlet (1) was vltrified Wlth a torch and posltioned approximately 0.5 cm trom the optical tube (A)/analytical tlame tube so as ta mamtain the t1p of the analytical tlame just below the entrance of the optical tube and thus of the optical beam. 66 Fi"ure 3-2-1. ThermochemlcaJ hydride generator: (A) opticaJ tube (12 cm, 9 mm i.d. x 11 mm o.d.); (B) analytical tlame tube (8 cm, 4 mm i.d. x 6 mm o.d.); (C) combustion chamber (3 cm, 7 mm i.d. x 9 mm o.d.); (0) thermospray tube (8 cm. 4 mm i.d. x 6 mm o.d.); (E) silica transfer line; (F) quartz insert to support the transfer Hne; (G) thermoelectric el.:ment; (H) modified sWagelock fittings; (1) analytical oxygen inlet (15 cm, 2 mm i.d. x 3.2 mm o.d.). H 0 2 2 L , -A B A 1 -i> F ©,~ J E H,/' ~~~ l ~ HPLC ELUATE ~ 67 The capIHary guide tube was tixed Wlthm the heated reglon of the thermospray tube (one ccntlmeter below the downstream end of the heatlPg element). Thermospray oxygen and hydrogen as weil as the analytlcaJ oxygen were separately controlled with flowmeters (Matheson. Toronto, Ont). ConnectIons between the flowmeters for the thermospray oxygen and hydrogen were made of tnfluoroethyiene (TFE) tubing (2.48 mm I.d. x 4 mm o.d .• Cole-Parmer Co., Chlcago, Il.) whlch were heat ~hrunk on the quartz tube ga'i mlets, whereas the analytIcal oxygen was connected to the analytlca! tlame tube tllrough the Swagelock assembly. The optIcal tube was secured m an aluminIUm casmg by firebnck h~lf disks and refractlve waal at both ext;emes only leaving most of the optlcal tube expcsed The thermochemlcal hydnde generator (mG) mterface was Igmted as fol!ows: a) the t'lcrmospray heatmg element current was mcreased to 5 A; b) after about 2 mm, the thermospray oJ0'gen ftow rate was adJusted to 650 mUmm; c) 5 mm after, the caplliary transfer lme was pushed mldway Into the heated portion of the thermospray tube and d), as soon as possible. the HPLC eluent (100% methanol) was rapldly Increascd to 0.65 mU:;nm (in 0.1 mm); e) the hydrogen t10w rate was then adJusted ta 1.7 Umm; 0 the excess hydrogen was then ignited at both end of the optlcal tube; g) the analytlcal oxygen flow rate was then adJusted to 170 mUmm or more untJl the analytlCal flame was Igmted; h) the hydrogen flow rate was then decreased untll the flames at each cnd of the optlcal tube were extlOgUished (wlthour eX!mctIOn of the analytlt:al flame) and subsequently returned to ItS origInal value (no flames should be present at the ends of the optical tube); i) the pOSitIOn of the capillary was subsequently adJusted to produced the thermospray effect WIth an homogeneous transparent t1ame accompamed wlth a chari.'':tenstlc "spray" sound; j) the mobile phase was then changed (as neces"ary) and the pOSitIOn of the capillary transfer line was readjusted, If necessary, for maJOmum thermospray (Oz + --> HzO) elfect. Water vapor was formed H2 Wlthm the Interface and was observed V1sually to condensed at the outlet of the OptlCal tube (dlfference 111 air densItIes). 11us was not a problem as long as a good hood was placed ab ove the THG mterface. It was observed the air suctlOn should be relatlvely stable as fluctuatIOn ofvapor del1S1tles 10 the optIcal path Increased background nOises apprecwbly. It was Important to igmte the Interface v,1th pure methanol as other fuel SJurces may not burn as efflclently (partlcularly when the quartz assembly was not hot enough) resultmg m carbon deposlts on the quartz surface. Upon such an event, It was found useful ta increase the thermospray vÀ]'gen tlow rate. [CAUTION: m cases where the quartz assembly was not hot enough upon mtroductlOn of the methanol. nOlsy explOSIOns occurred. Pnol to the addition of the eluent, the whole asscmbly must be bnght orange (not red) othefWIse methanol may not Igmte IInmediately and the accumulatIOn of thls methanol vapors Cati <:ause violent explOSions.] Shut-down of the interface was perfonned smoothly by followmg this procedure in reverse sequence. ft was preferable ta wlthdraw the capillary transfer line from the thermospray tube as soon as the HPLC pump was shut off. r.. 68 3.2.1.5. HPLC CONDITIONS ... The selenomum standarfis and the extracts from biological standards were separated on a ~ihca based cyanopropyl bonded phase (5 #.Lm &dica support, 0.46 mm I.d. x 15 cm, Le-eN, Supdco, Ille., Bellefonte, PA) with methanolic mobile phases containmg 1% acetlc 3Cld (vlv) and vanous amounl ot diethyl ether (pE, P stands for percent), tnethylamine (TEA), and lnmet~ylsuifolllum 10llJd~ (TMSI). 3.2.1.6. OPTIMlZATION PROCEDURE ChromatographIe Optlmizatlon, by definmg an optimum mobile phase compOl>itloll, WOlS performed with a statistacal experimenta! design (DeBaun's c.ùboctahedron desIgn). wlth l'creent ethcr [PE; 20,30, or 40%, (v/v)], tnethylamme [TEA: 0.D10. 0.055, or 0.100% (vlv)l, and trimclhylsullol11um iodide [TMSI; 10, 55, or 100 mg/L] as the three mdependent varIables. The detector rcsponsc to two selenomum sl.andards (with dlffelent amounts of Se) or ta eqUivalent amounts of thrcc SdClllllllUIll standards was recorded for each of 12 separate combinatlOns of mobile phJM: compo~lllon. Tw('('.;c expenmental p01l1t~ plus three rephcates of the center pomt were recorded. Arealhelght and peak .lfC;! were taken directly from the rp~order prïntout whereas capaclty factors was calculated accordmg to the lh foUowing formula: k'= (t. - to)/t wilh tn = (t ~ to) bemg the reduced elutlon time of the I compon~nt. t o l l the actual elution tlme for "i", and to the elutlOn of an unretained compound (acctonc) The :-e!>pon:-e~ were modeled u~ing the RSREG procedure (least-SQuares multIple regresslOn) of the SAS ::,tatl~llcal software (SAS instItutc, Cary, NJ). Effects and mteractlOns were studlcd visually by uSlIlg lhe predldcd response surface plots. 3.2.1.7. EXTRACTIONS Biologlcal samples (porcine kidney, and shrimps meat) were purchased from local grocery ~lorcl> and wcre immediately homogenized then fiozen at -20°C 111 separa te 10 grams portIOns Sclcnolllum compounds were recovered from thawed sampi es (10 g fresh welght) Wltl! three scqucnllal 15 ml. methanolic washes. After centrifugation, the methanohc extracts were combmed, then WIlLcntr.ltcLi Ylrtually to dryness under reduced pressure at room temperature. The reslduc wa~ solubJlIll'd In 10 mL double-dlstilled dClomzed water and aCldlfied to pH 2 wllh concentrated HCI The .HjUCOUl> ph.l\C W,l'> extraeted wlth IiqUlfied phenol (1 x 10 mL + 3 x 5 mL). The phenohc phases were comblllcrJ, wa"hcll wllh water (3 x 5 mL), dlluted wlth 75 mL of dlethyl ether, then back-extracted Wllh water (1). 5 mL). '1 he aqueous extracts were eombmed and evaporated to df}'ness under reduced pres,ure at 37°C. The rC\lout: was solubihzed In methanol (3 X 3 mL) and the resuitmg solution was reduced 10 1 mL by llu"hll1g undcr a gentle stream cf dry mtrogen at 37°C. This final extract was then centnfuged, and 50 #.LL or 1 (JO #.LI. of the supernatant was mjeeted lOto the HPLC system. 69 3.2.2. RESULTS AND orSÇUSSIQN .. 3.2.2.1. CHROMATOGRAPHIe OPTIMIZATION Several experimental observations have corroborated the hypothesls that a senes of oxicio reductlon steps take place m the "th 1991). Upon entenng the combustion chamber of ~he interface the selenonium an alytes, wcre pyrolyzed and oxirlized 10 aIl oxygen rich atmosphere. These selenium oxidatlOn products then were converted to the correspondmg hydnde(s) In the hydrogen nch regIon. Presumably, because of the c?talytlc propertJes of the quartz surface, the hydrogen gas was converted, at rugh temperatures, to hydrogen radlcals wluch srrved as the reducmg specles. Just at the analytical tlarne tube (B)/optical tube (A) JunctJon hydrogell Sf"lenide was converted (10 the oxygen analytical flame) ta the atomic specie (SeO) which absorbed a portIOn of the energy (1%2 nm) from the hollow cathode lamp. The principal lImitation of the prototype "thermochemlcal hydride generatlon" mterface was the requirement 10r an organic nch mobile phase to act as a fuel for the pyrolysls flame. 'The objectives of the current studies were: 1) to reduce the Iimlt of detection (LOr) for selenomumcholine and tnmethylselenonium cations by optimizing the chromatography of the an alytes, and 2) ·0 define conditions for the determmation of these analytes in blOlogical samp!es. The efficient chromatogr:}phlc separation of selenonium ,:ompounds with efficient on-hne detection by atcmic absorptlor. spectrometry (AAS) proved ta be fairly difficult because of the ver} strong mteractions of these analytes Wlth active silanol groups on slhca bonded phase HPLC columns. The <.:yanopropyl bonded phase seemed ta offer more selectivity than other silica based bonaed phases (BIaIs, 1990). With pure methanol as the mobile phase, !lelenonium analytes were totally (but reverslbly) retamed demonstratmg a very strong bmding of these analytes to the stationary phase. It ..vas found that tnethylamine 10 a methanolic mobile phase con tain mg one percent ace tiC aCld was capable of elutmg these products wlth sufficient selectiVlty. However. trimethylselenonium Ion was excesslvely retamed and was charactenzed by a degraded and asymmetnc peak shape e\ en at hIgh tnethylamme concentratIOn (l mUL of mobIle phase). Increased concentrations of the amme Ylelded appreclably reduced selectlvlty; a concentratloli of 0.50 to 0.55 mL TEAJL represented an acceptable compromise (BiaiS, 1990). The optimum compositIOn of the mobile phase was determmed to be (v/v): 70% of methanol, 29% dlethyl ether. 1% glacial acetlc acid, and 0.055 % of trie thyi amme. A typlcal chromat' gram 1S presented In Figure 3-2-2 A Over the course of the current studies the charactenstics of the HPLC colu:nn changed apprecl3bly as cVldenced by the chromatogram B of Figure 3-2·2 wluch was obtamed one year later using VIrtually an Identlcal mobIle phase. Tne change was not considered a detenoratlon of performance smce the analytes were more retamed and selectlVlty was better. The mcreLllie In selectlvlty can be beneficlal (peak sharpemng bemg stIll possible)...... ------70 FiKure 3-2-2 Representative HPLC-THO-AAS chromatograms of selenoniurncholine (a) and trirnethylselenonium (c) ions obtained with methanolic mobile phases containing, 29% (v/v) diethyl ether, 1% (v/v) acetic acid, and 70% (v/v) methanol wluch were supplemented WIth: A, 0.050% (v/v) TEA, (new column), (Blais, 1990); 8, 0.055% (v/v) TEA (3.96 X 10-3 M). (chromatogram recorded one year later); C, 1.52 gIL trirnethylsulfomum iodide (TMSI) and 0.63 J..I.g as Se injected per analyte; D, 0.79 g TMSI/L (3.88 x 10.3 M) and 0.31 fJ.g as Se Injected per analyte; E. 0.055% (v/v) TEA and 120 mg TMSIIL and 0.78 J..Lg as Se Injected per analyte. a o c;,;N A B a c S o~ en,.. .,..cd c M o 3 c o E a c a e lOlO CD~ CDN C'i0) CD· oci~ ID s .,.. s N M ,..CD • 71 It mlght have been pO~lble to explOit the mcrease In selectlVlty If the apparl nt chromatograpluc 1 cfticlency of the tnmethylselenonium IOn could have been Improved. The addltJOn of dlcthyl ether to the '14i metnanohc mobile pha:..e Improved the ~lectIV1ty sllehtly, but It was not possible to decrease the apprectable tallmg of the tflmethylselenomum ion by varymg the relative proportIOn of ether, TEA, and acetlc aCH] JJ1 the mobile phase wlthout compromlsmg the selectlvlty. What was needed wa:. a "magic" modlfler tha! would help JO sharpenmg the peak~ (espec13l1y that of tnmethybelenomum catIOn) wlthout reducmg the selectlvlty Jt was observed (hat tnmethylsulfomum lodlde apprcclably sharpened the peaks (especJally the tnmethylsclcnonlUm peak) but at the expense of reso!utIOn (Figure 3-2-2 C), In an attempt to Improvr the re~olutlon, the concentratIOn of TMSI was decreased conslderably, Unfortunately, even at lower TMSI cflDcentrJtlons. base-hne resolutlon was bare/y acllleved and then. only If the concentration of the selenonlUm anJlytes were rcduced (Figure 3-2-2 D). Still smaller roncel1tratIOns of TMSJ lJ1 the mobile phase mlght have been used. However. It was consIdcred that the resu1tlJ1g chromatography would have been dJftïcult to reproduced and would have been apprCClably mfluenced by co-extractives From the blOloglCal sampi es. Subsequent optlmizatlOn ~tudles were thus based on a combmatlon of TMSI and TEA m the mobile phase 111 an effort ta optlmlze chromatographIe efficIenc.y whlle retammg a baseline resolutIOn of the analyte~ and keepmg JO mmd possible applIcatIOns for blOlogleal extraets. The Optlmlzatlon of the mobIle phase compositIOn was performed wIth a ~~atlstlcal expenmencal drsign (DeBaun's cuboetahedron deSIgn) Wlth percent ether (PE), tnethy1amme CrEA). and tnmethylsulfol1lum IOdlde (TI.1SI) concentratIOns as the three JOdependent vanables. Response surface methodologles are appreclably more cumbersome th an other approaches (such as sImplex OptlmlzatJon proccdure~) to predlctmg conditIOns for an optimal response. Nonetheless, It was antlclpated that the mtormutlon gamed by thls approach woul.d proVlde addcd 1I1sIght mto the mechamsm(s) of chromatographie separation of selenanium compounds. The multiple regresslOn analysls procedure calculates d "best fit" mathematieal expressIOn (a polynomial) whIch related the dependent vanable (peak area, ::..realhelght ratIO, Capac.lty factor, etc.) to the mdependent v,lfIables (m thIs case mobIle phase compOSitIOn). Arbltranly, the OptlmlzatlOn (a lelteratlve procedure) was IImlted ta a quadratlc expressIOn for each mdependent vanable as weil as cross produets (lI1ter..lctlOn tcrms). ThiS "best fit" polynomial equatIon thus took the torm: Response '" b + o b (PE) + b (TEA) + b (TMSI) + b 11 (PE)2 + b TEA )2 + b (TIv1SI)2 + b1iPE)(TEA) + b (PE)(TMSI) l 2 3 2i 33 13 + b ..1( TEAllTMSl), where b IS the estlmatec1 mtercept. b , b , and b are the estlmated coeffiCient 2 o l 2 3 a~socJated wlth each mdependent vanable, bn , b22, and b33 are the coeffiCIents a$soelated wlth thelr quadr.ltlc terms and b , b 13' and b those assoclated Wlth thelr crass products (IP'eractlOn terms), 12 23 A representatlve example of the statIstlcal mput and output (SAS) of the expenment (10 thls ca ... e: Nh for seienonJUmcholme IOn) IS gIVen JO Tables 3-2-1 and 3-2-2. The resultmg regressJOn equatlOn W..lS then turthc( slmpltfied by flXlng one vanable (percent ether, PE) of the predlcted equatlOn sa that re~ponses vaned ,KCOrÙJI1g ta only twa vanables: TEA. and TMSI (Table 3-2-3). ----~--~~~- 72 Table 3-2-1 : Instrumental responsesa (Ared/height ratios) for the peak corrèspondmg to selenomumcholine ion as a functlon of mobile phase composition. The data were used as lI1put for the multiple regresslOn analysls (SAS). Pomt PEb TEAc TMSld Response (%, v/v) (%. v/v) (mgIL) 01 20 (-1) 0.010 (-1) 55 (0) 0.433 02 20 (-1) 0.055 (0) 10 (-1) 0.315 03 20 (-1) 0.100 (1) 55 (0) 0209 04 20 (-1) 0.055 (0) 100 (1) 0.220 05 30 (0) 0.010 (-1) 10 (-1) 0.685 06 30 (0) 0.010 (-1) 100 (1) 0.390 07 30 (0) 0.100 (1) 10 (-1) 0.267 08 30 (0) 0.100 (1) 100 (1) 0.232 09 30 (0) 0.055 (0) 55 (0) 0.287 10 30 (0) 0.055 (0) 55 (0) 0.274 11 30 (0) 0.055 (0) 55 (0) 0.279 12 40 (1) 0.010 (-1) 55 (0) 0510 13 40 (1) 0.055 (0) 10 (-1) 0.420 14 40 (1) 0.055 (0) 100 (1) 0.302 15 40 (1) 0.100 (1) 55 (0) O.2S5 a The coded matnx values whIch appear in parentheses were used to calculate the regressIOn equatlOn lor the model (refer to Table 3-2-2) b PE = percent dlethyl ether C TEA = triethylamme d mSI = trimethylsulfonium iodlde 73 ... 74 Table 3·2-3: Simplified polynomial expressions for the predicted variation in area/helght ratio for selenoniurncholine ion as a function of mobile phase composition with diethyl ether (PE) tLœd at (a) 20% (v/v); (b) 30% (v/v); or (c) 40 % (vlv). a) PE = 20%. [Figure 3·2-5 (D)] AtH = 0.2375- 0.1279(fEA)· 0.0621(fMsl) + 0.0792(fEA~ + 0.0342(IMSI~ + OI)650(fEA)(fMSI) b) PE = 30%. [Figure 3-2-5 (E)] AtH =0.2800 • 0.1281(fEA)· 0.0679(fMsl) + O.0792(fEA~ + O.0342(fMsl~ + 0.0650(fEA)(fMsl) c) PE = 40%. [Figure 3-2-5 (F)] A/H = 0.3225 - 0.1284(fEA)· 0.0736(fMSI) + 0.0792(fEA~ + O.0342(fMsl~ + O.0650(fEA)(rMSI) 1 75 Since there were three levels of ctiethyl ether (PE), each predicted surface response can be represented by three graphs, one for each level of ether. If the lack of fit was sigmricant (model accounts for less than 95 % of the variation), predicted values were calculated from the non simphfied regresslOn equation (Table 3 n 2-4) and plotted against the observed values (refer ta Figures 3-2-3, B-l, and B-2). [NOTE: figu-es, with numbers starting by B, will be found in appendix B]. In these cases, according ta the regression of predicted versus observed (slope of one and mtercept zero), the model fitted the experimental data reasonably weil. It is believed that extremel:' small vanations in retentlOn times at the center of the design were the cause of this significant lack of fit. Furthermore. the distribution of the residuals from the regresslOns indicated that these errors were not biased. In ail other cases there was no slgnificant lack of fit between the predicted and experimental values at the 95% level of confidence (and often at the 99% level of confidence). Area/height and peak area were taken directly from the recorder prmtout whereas capacity factors were calculated according ta the following formula: k'= (t - to)/t with l o tn = (t. - to) bemg the reduced elution time of the "i" cornponent, ti the actual elution time for "i", and to the elutlOn of unretamed compound. The predicteci surface response curves were derived (except for the area re5ponse surface curves; refer ta the next paragraph) tram a standard solution containing three selenomum compou"ds (selenoniumcholine, selenoniumacetylcholine, and trimethylselenonium cations; 310 ng each as Se) \'Iluch was injected under various mobile phase compositions. In general, the following discussion was based primarily on selenoniumcholine and tnrnethylselenonium cations since selenomumacetylchohne behaved very simllarly to the selenoniumcholine ion. SelenoniumacetyJchoIine was mcluded In the study as a more CritlCal test of the resolving power of the chromatographic system. It should be pointed out that e'Ven thought these surface response curves accurately predlcted the general trends, the uncertainties assocJated with the model May be of the same arder as small variations ln the surface (especIally at the extremes of the model). As had been observed previously (Blais, 1990; Blais, et al., 1990), the peak area for each of the three analytes vaned slightly Wlth the composit~on of the mobile phase (the resllit of changing detector sensiuVItles). In order ta mlmmize errors due io improper integration, at low resolutions, a separate expenment was performed Wlth only two selenonium an alytes [selenoniurncholine (310 ng Se/injection) and tnrnethylselenonium (150 ng Se/mjection) cations]. Furthermore, selenoniumacetylchoIine was somewhat unstable in solutIOn and hydrolysis of tlus compounds during the experiment might have introduced a systemahc error. Figure 3-2-4 presents the predicted response surfaces for the vanatIOn of peak area for selenomurncholine (A, B, and C) and trimethylselenonium (D, E, and F) cations as a functlOn of TEA and TMSI content at various level of ether in the methanol-acettc acid mobile phase. The mfluence of the ether content in the mobile phase can be seen by comparing plots A vs B vs C and D vs E vs F. 76 Table 3-2-4 : Observed and predicted capacity factors (k') for selenoniumcholine Ion. The polynomial model accounted for less than 95% of the variation in the observed capaclty factor resultmg from changes in the mobile phase composition. FAcroRS RESPONSE POINT PE TEA TMSI OBSERVED PREDlCfED DEVIATION %(v/V) %(v/V) ppm(w/V) % 1 20.00 0.010 55 339 3.48 2.65 2 20.00 0.055 10 132 1.55 17.32 3 20.00 0.100 55 0.74 0.62 15.99 4 20.00 0.055 100 0.96 0.76 21.01 5 30.00 0.010 10 .5.21 4.89 6.13 6 30.00 0.010 100 2.82 2.93 3.95 7 30.00 0.100 10 1.02 0.91 10.91 8 30.00 0.100 100 0.85 1.17 37.60 9 30.00 0.055 55 1.34 1.31 2.72 10 30.00 0.055 55 1.27 1.31 2.90 11 30.00 0.055 55 131 1.31 0.02 12 40.00 0.010 55 3.93 4.05 3.00 13 40.00 0.055 10 1.97 2.17 10.24 14 40.00 0.055 100 1.49 1.26 15.36 15 40.00 0.100 55 1.26 1.17 7.11 1", " 77 ... Figure 3-2-3: Linear regression analysis to test the concurrence of the ob~IVed k' values for the selenomurncholine cation with predicted values tram the model. -, Regression AnalyslS - unear model Y· a+bX Dependent vanable: OBSERVED; Indc:pcndent variable' PREDICTED Standard T Prob. 1"1 Parame ter Estima te Error Value Leve 1 "., Intercept 9.07065E-7 00889681 101954E-S 0.99999 Siope 1 0.0386709 25.8593 000000 AnalYSls of Vanance Source Sum of Squares Of Mean Square F-Rallo Model 23.75204 1 Z3.75204 668 7016 Error 0.461755 13 0035520 Total (Corr.) 24.213798 14 Correlatlon CoeffiCient = 0990419; R-squared = 9809%, Stnd Error of Est!male " 0.188467 Regression of OBSERVED on PREDICTED 6 5 Q 4 w a::> w 3 film 0 2 1 0 ! 1 0 1 2 3 4 5 PREDICTED RESIDUALS for the Regression of OBSERVED on PREDICTED 0.48 0.28 fil ...1 c( :::» Q 0.08 en w a:: -0.12 -0.32 , 1 o 1 2 3 4 5 PREDICTED 78 Fif.wre 3-2-4: Predicted response surfaces for peak AREA for selenoniumcholine. (0.30 J.l.g as Se. plots A, B. and C) and trimethylselenonlum (0.150 J.l.g as Se, plots D, E, and F) as a funcnon of the concentration of TEA and TMSI in a methanolic mobIle phase which contained 1% acetic acid and: 20% (A, D); 30% (B, E); or 40% (C, F) (v/v) diethyl ether. 0 A 111 24 -§'CII (22.... ,.. ~2O ~n ~ ~ 5" ~a ,. n 0.0' 0·01 0.04 .... O.CI TEA ('" Vlv) B E "0 fOi ,..'00 ~ .. :i1O ~ .. ,. 10 00' O.~ 0.04 0.01 0 CIl 0.0. 0.01 0.01 .... O.CI 0.'0 TEA ('" Vlv) TEA (", V/v) C F 21 120 ~ ~ ~,. ~1I -, 11 79 The detector response (peak areas) to each of these two selenomum analytes were reduced modestly by mcreasmg amounts of tnethylamine (TEA) and diethyl ether (PE) m the mobIle phase (Figure 3-2-4). The influence of the concentration of tnmethylsulfomum lodlde (TMSI) on the peak area was more complex. At low levels of TEA, peak areas were in generaJ moderately increased (presumably a result of irnproved chromatography) whereas at htgh TEA content, TMSI appeared to have Iittle effect (selenomumcholme; Figure 3-2-4 A. 8, and C) or a a moderately negative mfluence (tnmethylselenomum Ion; FIgure 3-2-4 D, E, and F) on the detector response (increasmg the TMSI content decreased the peak area). For the enVlsaged application to trace analysis the variatIon In area «25%) was Jtldged to be relatlvely unimportant (detector response bemg relatively msensitive to mobile phase compositlOn) proVlded that an Isocratlc ~olvent system could be used for the analysis. In contrast to peak area, the capaclty factor (k') and the area to height ratio (A/h) were both greatly mfluenced by the composition of the mobile phase. It is interesting to note that peak retention for the selenomum analytes, as measured by the capaclty factor (Figure 3-2-5 A. 8, C, Figure 3-2-6 A, 8, C. and Figure 8-3 A. B, C) was appreclably mtluenced by the triethylamme (TEA) content of the mobile phase and, at low levels of TEA, only moderately by tnmethylsulfnnium lodlde (TMSI). [NOTE: Figure 8-3 IS the thud figure of appendix B]. The greater the content of TEA the less reramed were the cornpounds; and. at low TEA contept, the greater the TMSI content the shorter the retentIOn on the column. The dlethyl ether content of the mobile phase (A vs 8 vs C of each Figure) has only a minor mfluence on peak: retention (the more ether the longer the retention). The stronger effect of TEA cornpared to that of TMSI on retentlon can be explained, in part at least, by the relative proportions of 4 3 5 each of these additives In the mobile phase CrEA =7.2 x 10- to 7.2 X 10- M, and TMSI = 4.9 x 10- to 4.9 4 X 10- M). At hlgher TEA levels the effect of TMSI was masked. At low concentration of TEA (0.01 %), Ath was reduced (peak shape was Improved) 2 fold by mcreasmg TMSI content from la to 100 mlIL TItis was true for both the selenomurncholme (Figure 3-2- 5 D, E, F) and the selenoniumacetylchohne Ions (Figure 8-3 D, E, F). Interestingly tnmethylselenomum Ion (Figure 3-2-6 D, E, F) behaved abnormally. In trus case the reduction in AJh as TMSI was mcreased (at low TEA content) four fold and the capaclty of TMSI ta decrease the Nb remamed appreclable even at hlgher concentratIOns of TEA In the mobIle phase. The fact that lomc compounds strongly interact with silica based stationary phases (either norrndl or reverse phases) is weIl docurnented In the lite rature (Miller et al., 1988; Foti et al., 1989; Bayer anc Paulus, 1987; Kohler et al., 1986; Moats, 1986; DaJgnault et al., 1989; Nahum and Horvath, 1981; Nawrocki and BuszewskI, 1988). When sllica based bonded-phase packings for HPLC are prepared by bond mg an organic layer enta a slhca support, it has been estimated that 50% or less of the avaIlable 511anols on the sIiica surface are actually denvatized dunng the preparation process (Unger et al., 1976). Stenc factors timlr fu:·ther reactlOns. For efficient chromatography, the bonded layer should completely caver the surface and mask the residual sllanols (Moats, 1986). HO " Fil:ure 3-2-5: Predicted response surfaces for the capaclty fador (A. B. and C) and Area/height (D, E, and F) for selenoniumcholine ion as a function of the concentration of TEA and TMSI in a methanollC mobile phase which contained 1% acetic ac.id and: 20% (A, D); 30% (H, E); or 40% (C. F) (v/v) diethyl ether. A o a: • o.• 4 ~ ua ~ CJ ~ a ~ a U J: cf ~ 0.21 u 0 D.t. 0.02 0.04 D.Ot Il CIl 0.04 CI.OI 0.,- 0.10 a.oe 0,- TEA (". V/v) TEA (%, Vlv) B E a: • o.n 0 4 ~ 0.11 lJ CJ ~ :1 ~o.u ~ U a ~ 0.41 ~UI U~ 0 0.02 0.04 0.01 O.CII CI .04 a.oe 0,- CI.OI 0.'- CI, '0 TEA (". Vlv) TEA (%, Vlv) C F a: • 0.71 • ~ o.. CJ cC~ 4 ~ ~ :1 1:: ~ 2 ~ ooa ~ U 0 G.2I lLD1 O.QI .... 0.01 ~ 7EA (%, VIv) 81 Figure 3-2-6: Predicted response surfaces for the capacity factor (A, B, and C) and Area/height (D, E, and F) for trimethylselenonium Ion as a function of the concentratIon of TEA and TMSI ln a methanolic mobile phase which contained 1% acetic acid and: A and D 20%; Band E 30%; C and F 40% (v/v) diethyl ether. A o 1.5 a: 8 !i: 1.2 eU 8 [ijO.9 ~ "::c i= 4 ~O.8 Ü a: <4: 0.3 ~ 2 CJ 0 0.0 0.01 00, 004 0.01 0.01 0011 TëA (%, V/V) B E te 1 ~ (J 1 !i:1.2 ~ "[ij i= 4 ~0.8 o , ~0.4 - f 2 'ldil "rI'.}-~ .• fi',L'o.' ~ 0.0 ~-""---.... __~~.. - tP n" o o~ 0 .M .002 a 04 • ",C) ~'" o 'lI 0.02 0.04 ~ 0.01 001 T O,oe 0011 010 ..,Cl_~ TEA (%, Vlv) ëA (%, V/V) ~~~ C F a: 8 1.5 !i:1.2 ~ 1 ~ [ij0.9 ~ 4 " o ~0.8 ~ 2 ~0.3 (J 0.0 a Of o~ 004 0.01 0011 TEA (%, V/V) 82 However, certain solutes, especially basic compounds, chromatograph very poorly on bonded 511icas because of interactions with residual surface silanols. Many techniques have been devised to 1 reduce/elimmate the:;e undesÎ.Jble interactions by decreasing the number of residual active sites. Different pre-treatments of the starting silieas sueh as rehydroxylation have been sugg'!sted (Mauss and Engelhardt, 1986; Kohler and Kirkland, 1987), more reactive silica derivatizing agents have been found, new sildation methods have been devised, and various end-eapping techniques have been introduced to reduce resldual sIlanols and/or to increase the shielding of the residL1a1 surface silanols (Marshall et aL, 1986. 1987; Buszewski et aI., 1990; Schomburg et al., 1983). Despite these efforts, for basic an alytes, mteraction with slhca can be the dominant chromatographi;: mode, especiaUy in water-Iean mobile phases. The best way to ellminate silanol interactions is to use an aIl organic po!ymeric phase. Unfortunately, the chromatography on 5uch a phase, often, is characterized by poor selectlvlty for polar compounds. A second effective way to clrcumvent these silanophilic mteractions Wlthout destroying the selectivity IS to dynamkally modlfy the active sites on the stationary phase with amines (Bayer and Paulus, 1987; Wahlund and Sokolowski. 1978). Several studies have suggested that these silanol interactions are the result of a relatlvely few isolated, non-hydrogen-bridged, highly acidic silanol groups (Kohler et al., 1986; Kohler and Kirkland, 1987). That a major contributor to the deterioration of peak shape is the mteraction(s) with a small number of active sIlanols is suggested by the smaIl amounts of TEA needed to improve the chromatography of basic analytes with different brands of reverse phase packings. These dlfferent ~hases were charactenzed by very different behaviors in the strength of their sIlanol interactions (Bayer and Paulus, 1987). [Note: the effects of silanol blocking age!1ts and ion-painng agents are not the same (Moats, 1986) J. Moreover, It has been observed that the addition, to the mobile phase (already containing TEA), of up to a 500 fold (rn/m) excess of ammonium acetate did not influence the chromatography of selenomum Ions perceptibly and that the substitution of n-hexylamine for TEA resulted in a degraded chromatographIe rerformance. It has been observed, by other researchers (Wahlund and Sokolowski, 1978), that protonated tertiary amines and quatemary ammonium ion!! are generally more retained than are secondary or pnmary amines on siliea based columns (presumably the results of differences in pK ). b Differences in retentlOn have been correlated with the relative masking abIlitles of ammes (Wahl und and Sokolowskl, 1978). It has also been reported that, for normal phase separations, a cyanoalkyl bonded phase packing usually behaves as a weakly retelltive silica column with relatively few speelfic mteractions (Welser et al., 1984). This is especmJ'" true if the alkyl chain bearing the cyano group is short (propyl) 10 whlch case residual silanols are not "screened" (covered) by the a1kyl chain (Smith and MIller, 1989). The observations reported ab ove can be explained, in part at least, by a cation exehange mechanism III wluch onium Ions (triethylammOidum and tnmethylsulfomum iodide) ID the mobile phase compete Wlth the selenonium analytes for active silanol sites on the stationa'1' phase. Differences in retentlOn of the selenomum analytes on the column are the result of ,iJfferences in the strengths of 83 interactions b·!tween the analytes and the surface silanols. However, only a portion of the decrease JO area/height ratio could be accounted for by the decrease in the retention time of the analyte. If the omum additIves in the mobile phase compete with the selenonium analytes for actives sites on the stationary phase, then an increa:;ed onium additives concentration is predicted to resuIt in reduced analyte retention. If the principal contnbutor to an increased peak Wldth is longitudmal diffusion of analyte Ions from zones of hIgh concentration to zones of low concentration then reduced retention will result 10 less on-column band spreading (sharper peaks). Longitudinal diffusion is generally consldered to be rclatlvely unimportant in HPLC.1t was anticipated that, in the absence of other effects, the shape of the predlcted response surface for the variation in area/height ratio with mobile phase compositIOn should be slmllar ta that for the variation in the capacity factor provided the overall scales (if not the umts) are the same [Figure 3-2-5 (A vs D, 8 vs E, and C vs F), Figure B-3 (A vs D, 8 vs E, and C vs F), and 3-2-6 (A vs D, B vs E, and C vs F)]. By comparing plots 8 vs E (30 % ether) and C vs F (40% ether) It is apparent that, at low TEA content in the mobile phase, the area/height ratio IS reduced appreciably by added TMSI (E and F) and that only a portion of this reduction can be accounted for by the corresponding dccrease 10 the capacity factor (8 and C). This was especial1y true for the trimethylselenomum cation (Figure 3-2-6). The effect of TMSI on sharpening the peak of the trimethylselenomum cation is not only a result of a decrease in peak retentlOn but IS also the result of other unknown factors. These plots also mdlcated that added 1EA decreased the Nh more rapidly than would have been predicted from the capaclty factor; but that at high levels it also masked the effect ofNSI. Comparing the predicted surface response plots for the capaclty factor (A. B, and C) to those ot the arealheight (D, E, and F respectively) in the case of selenomumcholine (Figure 3-2-5) and selenoniumacetylcholine (Figure 8-3), it was observed that these surfaces were nearly supcnmposablc (t!xcept at low TEA content as discussed above). This would tend to support the fact that retentlon tlmc and peak shape are related and, provided TEA was present in sufficient amount, that longitudinal diffusion was the major factor intluencing peak shape. On the other hand, in the case ot the trimethylselenonium cation (Figure 3-2-6) the predicted surface response plots for the capaclty factor (A, B, and C) and the area/height (D, E, arJd F respectively) were not superimposable even at rc1atlvely hlgh concentration of TEA. The polarities and basicities of selenoniumchohne, selenomumacetylcholme, and tnmethylselenonium catIons are anticipated to be very slmIlar espec13J1y JO the case of the lattcr two Ions. The surprismg dlfferences in chromatographic behavlOr relatIve to the other two analytes was mtngumg and warranted ftuther study. The chromatograms in Figure 3-2-7 corroborate thJs conclusion as chromatogram 8 15 characterized by much better peak shape for trimethylselenonium ion eventhough retentlOn for thls analyte is greater than in chromatogram A. 84 '. Figure 3·2·7: HPLC·TIIG·AAS chromatograms of (a) selenoniumcholine and (c) trimethylselenonium ions obtained with a methanolic mobile phase which contained 1% acetic acid and ether: A, 0.055% (v/v) TEA, 10 mg/L lMSI, 40% (v/v) Ether; or B, 0.01% (v/v) TEA,55 mg/L TMSI. 20% (v/v) Ether. , a ,', ~ ....~'~ A -d c s B '\-. 85 The results. using the cyanopropyl colunul were consistent with the fact that this silica based bonded-phase has appreciable ion exchange properties. It is postulated that, for water lean and acidic mobIle phases used ln these expenments. active silanols acted as exchange sites (exchangmg onium ions for W). However. the behaVlor of the trimethylselenonium cation suggested that another elutlon mechanism was also operative In which, the influence of trimethylsulfonium iodide (TMSI) in sharpening the trimethylseleuonium peak was not due ta "its influence on the retention time alone". Since the detector response (peak area) varied somewhat Wlth the composition of the mobile phase, and our interest was primanly in the resulting peak width, area/height ratios were normalized for = + area changes. The effect of area on peak WIdth was made constant by the folJowing formula: A/hN AJh l )/A ], Nb [(A - A avc. avc. Wlth A/hN being the nonnalized area over helght ratio (or peak width at half height); A IS the area; and A av.c. IS the average area at the center of the experimental deSign. To gam further msight lOto the effects of the mobile phase composition on the chromatography of tnmethylselenonium cation Figure 3-2-8 was generated by fixing two vanables (TEA and PE) and ) plottmg, for varlous level of TMSI, the predicted normalized area ta height ratio (AlhN versus the predicted eapacity factor. For this plot, tbree levels of triethylamine (TEA; 0.01, 0.055, and 0.1 %), and three levels of dlethyl ether (PE; 20, 30, and 40%) were arbitrarily chosen, then at constant TEA and constant PE the predlcted NhN for trimethylselenonium ion was plotted versus the predicted k' for each \. of seven levels of trimethylsulfonium iodide (1MSI). Accordingly, curves ft.. B, and C, of Figure 3-2-8 correspond ta 0.01. 0.055 and 0.1% (vtv) of TEA respectively in a methanol-ace tic acid mobile phase containing 20% (v/v) diethyl ether. Simllar plots resulted when the ether content in the mobile phase was increased ta 30% (eurves D, E. and F corresponding to 0.01. 0.055 and 0.1% TEA respectively) and to 40% (curves G. H, and 1 corresponding ta 0.01,0.055 and 0.1% TEA respectively). The seven data pomts (1-7) of each plot correspond ta 10.0, 32.5,43.8, 55.0, 66.2, 77.5, and 100.0 mg TMSIIL m the mobile phase. As anticlpated, bath the predicted k' and the predicted decreased as the TMSI content was A/hN mcreased. However. tlus decrease was not monotonie: in eaeh case, the initial addition of TMSI caused a greater decrease 10 bath A/h and in k' than did subsequent additions (the influence of added TMS! was not !inear ). An mteresting aspect of these plots was that, at higher TEA content, the predicted influence of TMSI did not result in a linear relationship between normalized area/height ratio and capacity factor as was the case for low levels of TEA (ft.. D, and G). At intermediate levels of TEA, the influence of TMSI resulted in slightly curvilinear plots (8, E, and F) whereas at lugh TEA content the effect was distinctly curvilinear (plots: C. F, and 1). It was predlcted that, at high TEA content (0.1%). :ncreasing levels of '1, TMSI appreciably reduced the peak Wldth at half-height while causmg virtually no change in peak retention as measured by the capaclty factor. 86 Fil:ure 3-2-8: Predicted relationship between the normalized area/height ratio for the peak corresponding ta trimethylselenonium ion and the capacity factor for that peak at constant levels of TEA. but variable levels of lMSI (data points 1-7 correspond to 10, 32.5,43.8, 55.0, 66.3, 77.5, and 100.0 mg/L r.:spectively) in the rnethanol acetic acid mobile phase. Plots A, D, and G correspond ta 20% (v/v) ether and 0.01, 0.055 or 0.10% (v/v) TEA respectlvely; plots B, E, and H correspond ta 30% (v/v) ether and 0.01, 0.055 or 0.10% (v/v) TEA respectively; and plots C. F, and 1 correspond to 40% (v/V) ether and 0.01, 0.055 or 0.10 % (v/v) TEA. 1.5 1.4 - 1 A 0 G 1.3 - ~ ~ 1 • 1 1.2 - 1 " / " " H 1 " " 1.1 - E " / " B 0 " 1 " • 1.0 - A 2 " " ./ rf " E / • , 1 - / , .. 0.9 - 3 / " 0 ,:;1 Z 0.8 - • 1 4 " /" " ..... C ~ " 4 21 0.7 - ~;"c / 1 ::J: " 1 0.6 - 5 1 ~ ,Ii!! a • AC ' 6 / "/f ,21 0.5 - a Ad U." . / / 0.4 - • A ' ", ' " l , " !!I 0.3 - • 7rJ 1 ~ 0.2 - 0.1 - 0 1 1 r 0 2 4 8 8 CAPACITY FACTOR (K') 87 The shght increase in capacity factor (k') with inereasing TMSI (from 48.3 ta 100.0 mg/l) at high 1 TEA content is unusuaJ. However. It must be pointed out that these predictions resulted from two predicted regrescion equation and that appreciable errors can arise especially at the extremes of the models. The effect of the diethyl ether (PE) for the same level of TEA and TMSI was anticipated ta decrease the solubihty of the selenonium cation in the mobile phase resulting in increase retention. This was mdeed the case (compare plots A vs D vs G of Figure 3-2-8). However, if longItudinal diffuSion of the analytes contributed apprecJably ta on-column broadening then an increase in the capacity factor should have been accompanied by an mcrease in peak Wldth. There was. however. no evidence for mcreasing peak Wldth wlth increased % ether at low levels of 1EA and TMSI (plots A vs D vs G. points 1 or 2). indlcatmg agam that longItudmai diffusion was not appreciable. On the other hand, as the level of TEA was mcreased (A. D, and G vs B, E. and H vs C. F, and 1) added ether caused a more rapld increase in peak width compared ta that of the increase in the corresponding eapacity factor. Higher levels of TMSI m the mobile phase also inereased the rate of change in A/hN Wlth added ether. From the chromatographie data, it is c1ear that selenonium ions were strongly (but reverslbly) adsorbed on the cyanopropyl bonded silica phase (if no 1EA and/or: TMSI In presence of acetic aeid were added). It IS possible to explam these results by assuming that the elutlon mechanism was mainly the result of silanophilic interactions: a process of cation exehange between the selenonium cations in the mobile phase and exposed residual surface sdanols on the stationary phase. To observe appreciable band broan~!1!!"!b the statlonary pha:;c must have a variety of appreciably different silanol sites I.e., one can enVIsage an a1most contInUOUS distributIon of silanol acidities (the more acidic, the more retentive). Assumml!, that there is a broad distribution of surface silanol acidlties and that the analyte interacts preferentially with the most active sites, a small quantity of selenC'n!um an alytes can overload the very small populatIOn of most active silanols and cause band broademng. Other omum additives in the mobile phase, If present in sufficlent amounts, can then compete Wlth the analytes for these active sites. In the limit, the net effect of omum additives is ta mask the residual silanols and to prevent thelr mteraction(s) wlth the analytes. The accepted view is that there are several types of unreaeted silanols and that only a very small proportion of these silica hydroxyls are actually reactive toward basIC compounds (NawrockI and BuszewskI, 1988; Pt1eiderer and Bayer, 1989; Nawrocki. 1987). 'The Simple cationic exchange elution mechanism alone, does not explain the observed behavior of the tnmethylselenonium cation in terms of band broadening compared to that of the two other c10sely related selenomum analytes (selenoniumcholine and selenoniumacetylcholine) which were not appreclably band broadened. The differences between the basicities of the three selenonium analytes are ,f' antlclpated to be minimal. 88 Severa! authors have stressed that ste rie factors may be responsible for the relative strength of several amines as silanol blocking agents. Dimethylalkylammoniums and trimethylalkylammomums seems to be more effective than trialkylammomum or tetraalkylammoniums if the all..1'1 group IS more than three carbons long (Wahlund and Sokolowskl, 1978; Sokolowski and Wahlund, 1980; BIJ et al., 1981). The geometry around the nitrogen atoms IS most Important (GIll et al., 1982). l'wo effects h.lVe beell suggested to dictate the effectlVeness of an amine blocking agent: a) how weil the amme can react wlth a sterically hindered silanol (baslcity and ste rie factor) and b) how weil it can, at the same tlme, "shleld" other neighboring silanol groups (SokolowskI and Wahlund. 19HO). It has been reported thar, for normal phase separatIOns, a cyanoalkyl bonded phase packing usually behaves as a weakly retentlve slhca column with few specifie interactions (Welser et al., 1984). 11us IS true if the alkyl cham carrymg the cyano group IS short (propyl) 10 which case, resldual silanols are not "screened" (covered) by the alkyl cham (Sn1lth and Miller, 1989). Spectroscopie studies have indlcated a non uniform distnbution of the bonded phases on the silica surface. For pyrene bondcd phases, "the majonty of the bound chams eXlsts ln hlgh denslty regions W1th average nng spacing less than 4.5 A and the mean spatial distance between remammg groups wluch exceeds 8 A" (Lochmuller et al., 1983; Gilpin, 1984). Cyanoalkyl bonded phases have been found to have cyano groups present in different envlfonments (hydrogen bonded or non-hydrogen bonded); thls was explained by the bonding density variations of the grafted phase and the "c1ustenng effect". In reglon of high bonding denslty, the alkyl chains are ordered perpendicularly to the slhca support and extend straight into the mobile phase (Figure 3·2-9 A). On the other hand. in regIons of lower bond mg density or at the edged of htghly bonded areas, the orientation of the alkyl cyano group is more random because the spacer arm is -more free :0 move (Lanin et al., 1989; Suffolk and GIIpm, 1985). In such cases cyano groups are able ta come into contact with free silanols and form hydrogen bonds (Figure 3·2·9 A). Thus there are regions of high bonded denslty and areas of low bondmg density. This is generally referred as the "mlcrodroplet" model which is based on the assumption that the dlstnbutlOn of sllanols on a tully hydroxylated silica surface is non-homogeneous and consequently bnngs about clustering of covalently bond stationary phase (Lochrnuller et al., 1983; 1984). This model has been recently re-exammed (Falln and Avnir 1987; Avmr, 1987). These authors suggested that the sllanols groups are, on the contrary. homogeneously distnbuted but that various curvatures of the slhca surface may result lfI dltterent accesslbIllties of these sllica hydroxyls. The anomalous chromatographie behavior of trimethylselenomum Ion relative to the other two selenonium Ions (selenoniurncholine and selenoniumacetylcholine) and the dlfferences ln the actions ot TEA and TMSI mlght be an indication of an ion c:xcluslon effect. A packmg (amorphous, porous slhca) which IS heterogeneous with respect to pore shape and size (a distnbutlon of pores slze) ~rves as the basis for tlus model (Unger, 1979). 1 89 Figure 3-Z-9: Schematic representation: (A) of the "cluster" or "microdroplet" model with zones of high bon ding density and zones of low bonding density due to a non homogeneous dIstribution of silanols at the silica surface and (B) of. our model with an homogeneous distribution of silanols differing in their accessibilities because of sterie factors due not only to the grafted phase but also to the poroos nature of the "naked" silica itself. A B o Cyano group Unreacled sHanol • Si alom 777 Silica surface C/anopropyl group hnked lo a !ulica hydrolyl on lhe silica surface 90 It is possIble that trimethylselenonium ion was able to enter pores WIthin the padang and to mteract wlth sllanols Viithm the~ caVlties whereas both selenoniumcholine and selenomumacetylcholine were sterically excluded from these pores. By the sarne token the triethylammonium ion may have been tao big to approach certam sterically hindered adsorptIon sItes which the smaller trimethylselenomum catIOn, on the other hand can access more easlly. It is suggested that the tnmethylsulfomum cation more c1ûsely resemble (baslcity and size) trimethylselenonium ion than does the tnethylammomum cation and accordmgly is able to enter the same caVIties as Joes the tnmethylselenonium Ion In arder to compete for these "stencally hllldered" highly aCldic silanols) (Figure 3·2·9 B). TIus is not to say that the mfluence of the hetero-atom should be disregarded. Our observatIons supported the idea of an homogeneous distnbutlOn of the slla'1ols on the sllIca surface but dlffenng in their accessibility. Foti and co-workers (1989) proposed a more complex model In whlch "the unreacted siIanols are ail sterically Iundered 50 that thelr adsorption actlVlty largely depends on the size of the ad!>orbate". "Only a neghgJble proportIOn of the sllanols is accessIble Wlthout bemg sterically hmdered". Accordmg to the theory of Foti et al. (1989) there are not two types of unreacted sIlanols (fi !e and hmdered) but rather ail free silanols are at one time or another more aVaJlable than others because of dynamic "tllpping" of the bonded alkyl phase. According ta these extremes : a) each resldual sllanol IS more or less accessible a certain amount of time owmg to the Vlbrational motIon of the graft (a dynamlc model; Fotl et al., 1989); and/or b) a certain percentage of the sil~jol groups IS sltuated at sItes wlth more or less easy access (a fixed model; Farin and Avmr 1987; Avmr, 1987). Regardless of how the resldual sllanols becomes sterically shielded the phenomena can be very Important. Our own observatIOns tend to cOIToborate its importance. At low level of TEA In the mobIle phase (Figure 3·2-8) It IS not possIble to separate out the mteractions of l'MSI Wlth accessIble sIlanols from the Interactions wHh hmdered sIlanols. At the other extreme, for lugh TEA concentrations, most of the non-stencally hmdered resldual sl1anols are blocked by TEA and then the action of TMSI on stencally hindered sdanol groups can be more c1early recognized. The retentlOn of tnmethylselenonium and selenomumcholine ions increased with an Increase in temperatures (Table 3-2-5). TIus behaVlor IS consIstent Wlth the concept of a dynamic motIon of the graft wlth more exposure of resldual sIlanols (as a functJon of tlme) owing to an increase in vibratlOnal motIOns of the graft as temperature increased. The fact that an Improvement in peak shape for tnmethylselenomum Ion was accompamed with slightly poorer peak shape for selenomumcholine ion mtght be explamed by a more even dIstribution of silanol aClditles (a decrease in the numbers of "stencally hmdered sllanols"). The "stenc hindrance" of certain silaIlOls was apparently decrease whereas the acttvtty of other "free sIlanols" was mcreased. 91 Table 3·2·5: Effect of temperature on reduced retention time and area over height ratio (A/h) for selenoniumcholine (CholSe) and trimethylselenonium (TMSe) ions. Temperature tra (min) Nha (oC) CholSe TMSE CholSe TMSE 22 4.09 5.78 0.269 0.405 50 4.50 6.20 0.276 0.366 a = • values, the average of two separate determinations, were calculated from tr (ti ta)' 92 This model would tend ta corroboratc that of Foti and co-workers (1989), although, it is considered that there are other steric factors whieh are a1so important. These other ste rie factors arise pnmarily because of Lhe heterogeneity and/or porosity of the bare silica material itself. TIle screening factor of the bonded phase would also eontribute (and probably would do so in areas of the bare silica which are already sterically hindered). Our proposed model is a combmatlon of both the models of Farin and Avntr and that of Fou and co-workers. It is believed that, within small pores, silanol groups are not easlly avallable for reaction with the bonded phase which explains the observation that the bonded phase eXlst ln zones of high density and low densities on the SiliC3 support. These zones of low bonding denslty would be found III sm ail unaceessible pores. In these pores the number of "free" silanols would be greater than expected and wOùld be available only to small molecules able ta enter these pores. By grafting a phase on the sllica, the pores dlameter actually decreases, but with increasing molecular motion of the graft, the me an diameter of the pores IS constantly changing. Over a given penod of tlme, if the eqUlhbrium IS fast, probability tells us that the actual mean pore diameter will be larger than normally experienced by solute molecules and additives and that peak shapes for sm ail an alyte s (tnmethylselenomum) will be improved whereas the peak shapes of the larger analytes (selenoniumacetylcholine, selenonium(;holine) will be slightly deteriorated. At high temperatures the Ianetlcs of the Ion exchange mechamsm should be fast, resulting in better chromatographie efficiencies. However, trus Increase in efficiency would be somewhat reduced by the kinetics for the availability of "free sllanols". That the presence of iodide did not influence the chromatography efficiency was demonstrated M). by replacmg, in the mobile phase, lMSI by NH4I (equivalent molar concentrations, 5.0 x 10'4 The fact that the type of counter anions has no apparent effects on the chromatography of aromatic amines on LIChrosorb RP-18 ln both methanol-Iean or methanol rieh eluents has aIso 'Jeen reported (Papp and Vigh, 1983a, and b), These experimental results support the idea that the silica support is not inert, and that tbis actIvity is the result not only of especially active sllanols but also of the accessibility of the analytes to these unreacted tughly aCldic silanols. In our view not only the Mean pore diameter and me an pore volume, but also the dlstnbution of each one of these and the actual shape of these pores are important factors. Another Important influence 1S the screening effect of the bonded phase which varies with temperature and solvent polarity. This discussion did not try to speculate on the actual origin of the increase in acidlty of certain unreacted silanols but rather attempted to explain, rationally, the availability or rather the non-availability of sorne of these highly acidic silica hydroxyls centers. 93 With a methanolic mobile phase containing two onium additives, it was possible to obtain very good chromatographie efficiencies (with base line resolution). Peak shape improvement was not necessarily obtained at the exp=nse of selectivity. The chromatograms (Figure 3-2-10) which resulted from an optimized mobile phase composition attest to tbis. TMSI was more effective than TEA at blocking the most active surface silanols. By contrast, the less efficient masking by TEA resulted ln a greater sclectlVlty of the chromatographie system. Overall, the combined influence of TMSI and TEA resulted in a powerful chromatographie system for resolving selenonium analytes which very strongly bind to slhca bonded stationary phases. This system may be applied to other chromatographic applications where alkylammonium modifiers alone are not efficient enough. An a1temate approach (ion pairing) to reduce interaction of the selenonium cations with the residual surface silanols was attempted but without success. Several ion-pairing agents such as methane, ethane, p-toluene, and pieryl sulphonic acid were ail ineffective. 3.2.2.2. CALIBRATION AND LIMIT OF DETECfION (LOD) The Iimits of detection (LODs) for the selenonium analytes were estimated using a mobile phase with high levels of TEA and TMSI, to mimic conditions thought to be required for the analysls of extracts rieh in co-extractives. According to the response surface regression analysis for selenonium ions, peak area (detector response) was somewhat dependent on the mobile phase composition. In general, the higher the ocganic content the lower the response. Actually at high carbon content in the mobile phaM!, the calibration curve proved to he curvilinear over a hundred fold concentration range of selemum (Figures 3-2-11, and 3-2-12). This May have resulted from the fact that the higher selel1lum Icvel standards were the last injected. Over the course of the experiment sorne 15 chromatograms (3 repl icale injections of each of 5 standard solutions) were recorded. It is possible that, during that time, thcre was li continued deposition of pyrolysis products on the inner walls of the interface WhlCh, 111 tum decrcased the catalytic activity of the quartz surface. To venty this postulate, the oxygen tlow rate to the combustion chamber and to the analytical tlame were both increased substantially. The black carbon deposits became red hot and were actually seen to MOye and volatilize. After the oxygen tlow rates had becn returned to their original values, injection of the same high selenium level standards resulted 10 an apprecaable increase (-20%) in response (peak area) compared to peak areas obtained praor to the trcatment. Thu!>, for very "dirty" extracts, for which carbon deposits in the interface are observed, It would be neccs~ary ta included a cleaning cycle (the oxygen tlow rates would be mcreased) of the interface betwcen sam pie analyses. It should noted that, in contrast to carbon deposits, deposition of halides, especaally of chlofldc, irreversibly decreased the response. 94 1 Figure 3-2-10: HPLC-TIIG-AAS chromatograms of a mixture of: A, selenoniumcholine ion (a) and, trimethylselenonium ion (c) or B, selenoniumcholine (a), selenoniumacetylcholine (b) and trimethylselenonium (c) ions. 1be analytes were eluted with an "optimized" solvent system consisting of 0.055% v/v TEA, 100 mg lMSI/L and 40% v/v diethyl ether. a A c ~ o.... ab C CDCDtD N .... WO:; cDGtO B .... S N C'l CIl) v. 95 l Figure 3·2·11: Regression analysis for the detennination of the limit of detection. Peak area vs quantlty of selenoniurncholine ion (15.4 ng to 1540 ng as Se). 1 Regression Analysis • Unear model: Y • a+bX • For selenomurnchohne tetraphenylboron (0.D154-1.54 ,uS Se) Dependent variable' AREA; Independent variable AMOUN'f Standard T Prob Parameter Estimate Error Value Leve 1 Intercept 18905 650052 290822 01221 Slopc 284531 9220.49 308585 OOOJO AnalyslS of Variance Source Sumor Of Mean F Squares Square RallO Madel 4.003IEll 1 4.0031Ell 9.522E02 Error 5.46S0E09 13 4.2039EOS Total (Corr.) 4.0578Ell 14 Correlation CoefficIent" 0.993243; R-squared = 98.65%. Stnd. Error of Esumate " 20503 4 Regression of Peak AREA on AMOUNT 5 4 3 1~ 2 1 o o 0.4 0.8 1.2 1.6 AMOUNT (ug) RESIDUALS for the Regression of AREA on AMOUNT 38 . § 18 . ~ ~ 0 ...1 ·2 < ~ Q .' in w CC -22 -42 o 0.4 0.8 1.2 1.6 AMOUNT (ug) 1 Fil'ure 3-2-12: Regression analysis for the determination of the limit of detection. Peak area vs quantlty of trimethylselenonium cation (15.4 ng to 1540 ng as Se). ------l Regression AnalyslS - Lmear model: Y .. a+bX - For Trlmethylselenonlum lodlde (00154-154 )Jg Se) Dependent vanable. AREA; lndependent variable AMOUNT Standard T Prob Parameter Estlmate Error Value Level Intercept 17709.4 é85609 258302 002273 Slope 321374 9614.11 334273 000000 Analysls of Variance Source SumoC or Mean F squares Square ratio Model 5.2253Ell 1 S.2253Ell 1.1178 Error 60792E09 13 46763EOS Total (Corr.) S.2861Ell 14 Correlation Coefficient =0994233. R-squared = 9885%, Stnd Error of Estlmate = 216Z4 9 Regression of Peak AREA on AMOUNT o 0.4 0.8 1.2 1.6 AMOUNT (ug) RESIDUALS for the Regression of AREA on AMOUNT 64 .... ~24 Q~ 4 ffi IX: -16 -36 o 0.4 0.8 1.2 1.6 AMOUNT (ug) • 97 Choosmg the Iinear regression parameters calculated for 15.4 ta 1540 ng of selenium Wlth five ( dlluted standard solutions (Figures 3-2-11 and 3-2-12) ta calculate the LODs resulted in limits of detections of 64.2 and 68.9 ng as Se for trimethylselenonium and selenoniumchotine ions respectively. Usmg the Imear reglon of the calibration curve (three first points, between 15.4 ng and 154 ng of Se, Figures 3-2-13 and 3-2-14) the Iimits of detection were found ta be 7.5 ng and 7.7 ng as Se for tnmethylselenomum and selenoniumcholine ions respectively. The later choice is more relevant, as suggested by Figure 3-2-15, since amounts as low as 7 ng of Se can be easIly discriminated from the background nOIse. The objectives of this phase of the research were met as the optirnization of the chromatography considerably reduced the timits of detectIon for the trimethylselenonium ion [Figure 3-2- 2 (A vs B)]. 3.2.2.3. SAMPLE EXTRACTIONS AND ANALYSIS In preliminary trials, extraction procedures based on Reineckate salt precipitation were found ta be somewhat irreproduclble at the levels tested (0.1 to 1 p,g/g wet weight of sample), whereas catIOn exchange isolation steps were found ta be tlme consuming. Sorne other simple technique such as solvent extraction followed by a solvent partition/purification step would be simpler, more rapld, and equally effective for a vlrtually specifie detector such as AAS. Unfortunately, it IS very dlfficult to extract ( selenonium IOns efficiently mto an organic S(\lvent even in presence of a complexing agent or an Ion painng agent. Several common reagents including dithizone, sodium diethyldlthiocarbarnate, ammonium pyrrolydmedlthiocarbamate, potassium ethylxanthate, and sodium heptanesulphonate ail proved ta be meffectlve. However, the arsemc isolog, arsenobetaine, has been reported (Edmonds et al., 1977) to be extracted efficlently into liquified phenol. Extraction of selenomurnchohne and tnmethylselenomum standards from pure water (1 J.Lg each as Se In 10 mL) lOto phenol (1 x 10 mL extract followed by three 5 mi.. extracts) and subsequent washing of combmed phenolic extracts with water (3 x 5 mL) followed bya back-extractlOn of the selenonmm cations in ta water (3 x 5 mL) after addition of 75 mL of ether to the phenollC phase resuited 10 a Vlrtually quantitative recovery (>98%) of bath selenomumcholme and tnmethylselenomum cations. However, when this technique was attempted on more complex matrices such as porcme ktdney, and shnmp Meat, recoveries were much lower. In the case of the kidney (Figure 3- 2-16). recovenes were 28.3% +/_ 0.7% and 41.5% +/_ 1.8% for the selenoniumchohne ion (3.3 J.1.g Se/lO g), and trimethylselenonium Ion (3.1 J.1.g SeIlO g), respectively. For shrirnp (Figure 3-2-17), recoveries were 27.2% +/. 10% and 42.7% +/. 5.2% for selenomurncholine ion (6.6 J.Lg SellO g) and tnmethylselenonium catIOn (6.2 J.1.g Se/l0 g) respectlvely. Statlstlcs are based on two replicate determinatlOns for each matlIX. For the shnmp extracts the problem in reproduclbility (selenomurncholine ion) is Most probably the ( result of peak shape degradation caused by unknown factors. • j Fiaure 3-2-13.: RegressIOn analysis for the determination of the limit of detection. Peak area vs quantity of selenoniumcholine cation (15.4 ng to 154 ng as Se). Regression Anal)'SlS - Lmear model: y:: a+bX - for selenonlumchohne tetraphcnylboron (0 0154-D 154}lg Se) Dependent variable: AREA; Independent variable AMOUNT Standard T Prob Parame ter Estlmate Error Value Leve! Intercept 845639 1230.71 0.687117 51412 Siope 488617 13525.1 36.1268 O()(JOO) Analysts of Vanance Sumof or Mean F Source Squares Square Ratio Model 8.2462E9 1 8.2462E9 1 JOSEJ Error 44227474 7 6318211 Total (corr.) 8.2904E9 8 Correlation Coefficlcnt:: 0.997329; R-squared:: 99.47%; Stnd Error of Esumatc = 2513 61 Regression of Peak AREA on AMOUNT 0 0 0.04 0.08 0.12 0.16 AMOUNT (ug) RESIDUALS for the Regression of AREA on AMOUNT 33 23 -8 9"" 13 ~ en ~ 3 ::::» Q ën -7 w a: -17 ·27 0 0.04 0.08 0.12 0.16 AMOUNT (ug) 99 Firmre 3-2-14: Regression analysis for the determination of the Iimit of detection. Peak area vs quantity of trimethylselenonium ion (15.4 ng to 154 ng as Se). Regression Analysls· Lmear model: Y = a+bX· For Tnmethylselenomum lodlde (00154-0154 flg Se) Dependent variable: AREA. Independent variable AMOUNT Standard T Prab Parame ter Estlmate Error Value: Level Intcrcept 1697.68 1207 29 14062 20247 Siope 496n7 13116.6 378739 000000 Analysls of Variance Source Sumof Of Mean F Squares square RJlIO Model 8.7214E9 1 87214E9 14J4EJ Error 42S601R8 7 6080027 Total (corr.) 8. 7639E9 8 Correlation CoeffiCient = 0.997569; R-squared" 99.51%; St:!·..:. Error of Esumate " 2465 77 Regression of Peak AREA on AMOUNT 10 ... 2 o o 0.04 0.08 0.12 0.16 AMOUNT (ug) RESIDUALS for the Regression of AREA on AMOUNT 47 8... 27 ~ 7 :;)~ c ën w a: -13 .., -33 0 0.04 0.08 0.12 0.16 AMOUNT (ug) lOO FiKure 3-2-15: HPLC-THG-AAS chromatograms at low levels of selenoniumcholine (a) selenoniumacetylcholine (b) and trimethylselenonium (c) ions in a mcthanolic mobile phase contammg 1% (v/v) ace tic acid and 29% (v/V) diethyl ether: A, 0.055% TEA alone (each an alyte ;: 31 ng as Se; Blais, 1990); B and D, 0.075% TEA plus 100 mg TMSI/L (cach analyte = 33 ng as Se); C and E, 0.075% 1EA plus 100 mg TMSIIL (each analyte =15.4 ng as Se); and F, 0.075% TEA plus 100 mg lMSIIL (cach analyte = 7.7 ng as Se). The "s" mdicates the solvent front. B c A a c s 5 a C a s c o 3 S (min) o 3 6 (mIn) o 3 e (mIn] D E F a c s a c ~ t' . . .. o 3 S ("un) 3 S (min) o 3 o ..,1 101 FiKure 3-2-16: HPLC-THG-AAS chromatograms resulting from: A, 10 IJ.L injection contaimng (a) selcnoniumcholine (0.33 J.l.g as Se) and (c) tnmethylselenonium (0.31 Jjg as Se) standards; Dt 50 IJ.L Injection of control porcine kidney (10 g wet weight) extract; C 50 J.l.L injection t'rom porcine kIdney (10 g wet wcight) which had been splked with selenomurncholine (3.3 Jjg as Sc) and tnmethylselenomum ions (3.1 JJ.g as Se); and D,50 jJL injectIOn of extract from control porcme kidney (C) spiked, post extraction, with selenomurncholine and tnmethylselenomum Ions to result in 0.165 Jjg Se and 0.155 Jjg Se per injectIon. A a m ":c B c o a c 'P- 0 ..... ~ cD m c S s a N ,...... N N CD. Cf) Cf). ~• Cf) CD en , 102 Figure 3-2-17: HPLC·TIIG-AAS chromatograms resulting tram: A. 10 J.l.L injection contaming (a) selenomumcholine (0.33 J.l.g as Se) and (c) trimethylselenonium (0.31 J.l.g as Se) standards; B, 50 J.l.L mjection of control shrimp (10 g wet weight) extract; C, 50 J.l.L InJe :tion from shnmp (10 g wet welght) which had been spiked with selenoniumcholine (6.6 J.l.g as Se) and trimethylselenonium ion (6.2 J,J.g as Se); and D, 50 J,J.L injection of extract (C) from shrimp whlch had been spiked. post extraction, (0.4 mL extract + 0.1 mL of standard) wlth selenomumchohne and tnmethylselenomum ions. A a Ji. Cf) ~ c CD ca B CD. • CD • 0 &Wi B. Cf) D c a c ,..:~ -caCD • 8 8 Cf)• ~ a &Wi ~ cD • 103 Recoveries mlght also have been reduced Dy the influence of co-extractives on the extraction performance. If t/us is the case, an increase in solve nt volumes for extraction, an Incorporation of a cleaning sequence pnor to phenol extraction (for example solid phase extractions), a washing of the phenol layer with aCldlC solutions (O.OlM HN0 ) instead of pure double-dlstJlled diiomzed water, nught 3 Improve the recovenes. To tackle t/us problem it would be helpful to know at what stage of the Isolation procedure these losses occurred. It is considered that a good way to approach this problem would be to spike the biological matnx Wlth radiolabeled [14C]-tnmethylselenoniurn cation and to monitor the efficiency of each step of the isolation procedure by liquid scintillation counting. To this end, [14C]_ tnmethylselenomum bromlde was synthesized from dimethyl selenide and [14C]-methyl bromide accordmg to the method of Palmer et al (1969); however, time constraints dld not permit the completion of trus study. The effect of co-extractives on the detet:tor response (peak are a) was not appreciable « 10%) per mdividuaJ Injections. However, reductlon of the detector response was observed (with time) upon repetitive mjectlOns because of carbon deposits. This can he solved by including a c1eaning cycle as mentioned prevlOusly. An important side effect of the co-extractIves was their influence on the chromatography of the selenomum analytes. In the presence of either shnrnp or porcine Iadney extract, selenomurncholine Ion was less retained whereas trimethylselenoniurn was rnuch more retained than when standards alone were injected (Figures 3-2-16 ar!,J 3-2-17, A vs C). TIIe interestmg part of trus observation IS that the selectivity between the analytes was increased considerably indlcating that the action of these co-extractlv!(s) on selenoniurncholine ion and tnmethylselenonium ion was dlfferent. To mimlc the effect of possible co-extractive(s) that might have caused such behavior, choline chlonde in large excess was co-inJected Wlth the standards (Figure 3-2-18). As the quantlty of co-mjected choline was mcreased the capaelty factor of the selenoniurncholine cation decreased whereas that of tnmethylselenomum catIOn mcreased resulting in increased selectlVlties. A second aspect of the mt1uence of co-extractives on the chromatograp/uc behavior of selenonium ions was the apprecl3ble decrease ln area/helght ratio for the peak corresponding to the trirnethylselenonium cation (Figure 3-2-18). On the other hand, peak degradatlon for the selenomurnchohne ion became apparent. 11us effeet was. agam, mlmleked by the additIOn of choline cblonde to standards. To mimmlze the effect of co-extractives on the chromatography It mlght be possible to amend the mobile phase with a choline salt (not ehlonde since it decreased irreverslbly the detector sensltiVlty by affectmg the efficlency of the therrnochernlcal hydride benerator mterface) to assure that peak retention becomes mdependent of the extract composition. If this does not work, then, the extract can be spiked \Vith hlgh level of a choline salt in methanol 50 as to swamp out mlnor dlfferences ln the co-extractives compositIOn on the chrornatography. • '1 \j •, 104 .., Fil:ure 3-2-18: The influence of coinjec~ng increasing quantities (D>C>B>A) of choline chloride on the chromatographie behavior of a mixture of selenoniurncholine and trimethylselenonium ions. • a c:. C A U!- 0l1li' B cD a c o a c N N N N g cd 0; cD • J 105 3.2.3. CONCLUSION A chromatographie method for the analysis of selenonium compounds has been developed usmg an HPLC-AAS on line system. (BI rus et al., 1991). The optimization of this method prùvided insight on the mechanism of retention of selenonium compounds on a cyanopropyl silica based packing (Huyghues Despomtes et al., 1991). Trimethylsulfonium iodidc was used for the first time as a silanol blocking agent. Hs effectlveness was compared to that of triethylarnine. Tnmethylsulfonium iodide was found to be superior to tflethylamine as a maskmg agent but reduced, by the same token, the selectivity. Carefut control of the relative concentratIon of both additives resulted in an efficient chromatography and accordmgly In lowcr Hmlts of detectlon (Huyghues-Despomtes et al., 1991). A phenol extraction method for setenomum Ions in biological samples was IOvestigated and warrants further study. TIus is. to our knowledge, the frst attempt ta analyze routinely levels of selenonium cations in foods. It is believed that this analytical IT ethod has the potential of becoming a routine method for the analysis of selenonium compounds in blological systems. 106 APPENDIXA 107 Figure A·t: Characterization of the trimethylselenonium ion: A, fourier transform infrared spectrum of tri!TIethylselenonium Reineckate; B, proton nuclear magne tic (200 MHz) spectrum of tnmethylselenonium iodide in 2~O with TMS as an extemal standard; C, fast atom bombardment (positive ion mode. FAB+) mass spectrum oftrimethylselenonium lodide in a glycerol matrlX. ------A .. 10 •• p 00 le: 42: lI7 •.. • ... •.. •o 1 N ,," ••C'f a..~ ...L. • a.0. o Ca Z D (W o .. =•'f Q ~ o f ~ __ ----~----~~----~------~~--~~----~--~---+~ ___ -~- ~ ~ooo. 0 ...72 •• 20~~. ~ .... UI. 7 l.~~. 4 1880.8 I~UI. 7 uea. Il IiUII!I.8Q eRe .00 W.v.nulllCl." (011- U B ASSIGNMENTS: (H)C),Sc • • • _2.75 ppm H,O MeOH .1" '1""" 00,'"'''''',''''' "1 "Of" "'" .. ""'"'''~''''' " c ~."'''''1I~~.''''''''''''2!I-''''.~'''''.-a..+.-.,...--.... Figure A-2: LAMMA mass spectra of trimethylselenonium iodide in both the positive (A) and negative (8) IOns mode. l! 1 sI II -, 1 '.' ! i ~ i i i .. 1 ! • 1 D li , • If 1 lit t ~ • 1 -1 I! • 1 • ! 1.. l 1 • ! 1 ! ! 1 1 ! a.:. J 1 1 I! 1 1 1 1 ~ ! ~ 1 ~ , Il.. • 1 ~ 1 , • 1 , 1 , (_AIa_I~• (--'--I~ (..... '--'-- ('-'--I~ al l! 1 ~ 1 II ., 1 ! i i i ; ~ 1 ! • 1 D li ., 1 ! 1 D Il ; ! 1 • • ! • 1 • ~ ! • ! 1 • ! 1 ! 1 • .!. 1 1 ~ !! • ! M' t ·, ~j Il.. 1 ! 1 !! • Il ! • 1 ! (_-'--,• ", (_ ","-1....-- ... (..... ~-- (.-,--,-- 109 Figure A'3: LAMMA mass spectra of tnmethylselenonium tetraphenylborate in bath the positive (A) and negatlve (8) Ions mode. ! 1 II II 1 1 1 1 1 1 , i ! , ! 1 -! • 1 -, • li , • -, , , , ï ~, •1 1 1 1 • ~ 1 t 1 ~ 1 Il 1 1 1 ! , • 1 ! • Il , (~W-' __ (...,"--'• (...,~I-- ...... (.... ~-- ID ! 1 1 1 1 1 ~ 1 -1 1 1 1 1 ! -1 • • 1 -R Il • -1 • ! , ! 1 • . - 1 , ~ ~ • ij 1 1 • 1 ~ ' t J i ~ 1 !I Il 1 1 ! • ! 0 1 ! Il ! l..n"--l __ (...,,,--,--• (..,~I-- I~~I-- ct , l 1lO Figure A·4: Spectral Charactenzatlon of selenoniumcholine tetraphenylborate prepared from metallic selemum: A, fourier transform mfrared spectrum (residual acetone present); B. proton nuclear magnetic Resonance spectrum In [2H]6-DMSO; C. l3C nuc1ear magne tic resonance spectrum in DMSO. ... 0 G• A ... .I!!CHOLINI!!-TP8 07 ••" 80 20: O.: 1. .,1'1.. ., ... • .. .j. ., • CI III ..,N G o.. ... 1'1• .j. ...U., , 0 e.,C .. CI ,QO ~ ... L • .. .. . 0" + .. ,1-., • ,QG... ., ... ..,.. o C., 0 .. 1'1 ... 0 .. o CI CI ~i .. 0 + 1'1 .,,.. .. .,1'1•.. i .. o ~ .. . •.. O.j...... _ ~ ~ .000.03800.03200.02800.02.00.02000.01.00.01800.0 ".0001200 0 1000.0 878.00 .... v.nulllO.,. CCIII-Il B ASSIGNMI!NTS. 1 b c d (HJq~'~~OH •• 2.66 ppm b - under the .IIU peM c-J.81ppm d- '.52ppm ~o DMSO '~ " , , l , ' , , , , " " ' .. , il " i , , " 'Il' , . '1;";''''' • c ASSIGNMENTS (H,qzSc'CH1CHzOH • b c • ·20200 b - 43871 e - S6 171 DMSO J • , , , 'JI' • • • • , , , '.lIt' , , , i " ',"'" i , , , 'l" ,, , i , ' .. ' , , , i , , , 'JI' , , , r , , , 'l' , , , , , . , 'III '~' , i ' , , '.' 111 Figure A-5: Spectral Charactenzation of selenoniumacetylcholine tetraphenylborate prepared from metallic selenium: A. fourier transform mfrared spectrum (resldual acetone present); B, proton nuclear magnetic resonance spectrum ID eHkDMSO; C. l3C nuclear magne tic resonance spectrum In DMSQ. 1 0... Il A • 10 Sep .0 17: 2.3: a7 N 0 N Il• • . •.. .,.. • 1 •, ...• CI O·• .u.- - ~ l, c- ..0 A.•• 0"-, .,.. "o- . , .a•• .. .. .N ~ ~" .,.-"".., Il, • ON 0 l'CI ... CI, -"• R ... "0 0 1 ~----4-- 1 • "000.0,.. ., •••••_-_-4".424' •• " '.44,"'8.0.814'. . . •. " ·US •.••••.•••• S 00 ....v.nUIllD.,. (08-1) B l-, ASSIGNMEN1'S' b cd. (HJC)~·CKaCH.pqO)CHJ •• 2.07 ppm b. 2.71 ppm c.348ppm • d ••.•2 ppm 7.2.62 ppm ,i _ t ~~~~~~~~~i~,~i~"~i~'i~i~i~il~i~"~i~i~,,~,,~,~i~"~i~ii~i~'~'~~'~"~i~I'~'~'~il~'~"~i~"~".~ c ASSIGNMan'S: • c cS c b (H,C)~'CH,œ,oqO)CHJ b • • or b • 20.39 ppm b on • 20 66 ppm c· 311.66 ppm d. S908ppm e • 170.00 ppm 1 f f f 40 JO 22 20 DMSO j J • , , , , ....., UIO 140 1210 100 20 o 1 1 112 Figure A·6: Spectral information of mterest to the CharactenzatlOn of the selenomum compounds: A, founer transform mfrared spectrum of acetone; B, fourier transform infrared spectrum of sodium tetraphenylborate; C, I3C nuclear magnetic resonance spectrum in polysol Wlth TMS as an mternal standard. A "ClETo.. ... O! !i ;:, ! •..,-on -,.. .' ~lr- Il ....U. ~ CCI .0 .QO ~ L • ON a"... ·0 •... CIl ".. ~ ~ \1 ...... Il \1 •CI 0 ... • .. 0 1 CI CI 0.. o1 _ • -- 1 1 l , , , , .'000.03"72,2 a ..... , .. a"'8. 7 I."~." se.o.8 ' .. le. '7 s U12, •••• , •• e.8. 00 "'evenullllt.,. (0"'- U B .. TN CI 0 .," ...1'1 ., Il" .... ,..o uO .. c .,• .'ICI L ' 0" .. C'tI Il a.." Il 1'1 \1 .., CI II! CIl Il •• .. N 'N N 0 liN ' .. ,0 •...... CI CIl' Nil! ~ " • (III " Il •0 ' .. .. ., ., ... . ".. .. CI " ~ "II ... 0 • Il .. 0 1'" ." .. • .,N 1 .1 "CIl Or=~~~~~~~~~~~~~ __ -= ____ ~ __ --~--~~ "000.0 3800,0 3aoo. 0 a.oo. 0 a .. oo. 0 aooo, 0 S .. oo. 0 saoo 0 1000.0 .78. C wevenuIII"er c_ ... - - - •• - - •• - - • "SSICiNIIENTS • A 121. 6 H U~!I 0 ~ 1 ~ L c• lJ~ Il ~ ., 0 163 Z Q ~~ 'Q'Je~~ i) , C t ,. .. M• u s COL800 G T ,1 "'\ 0 \1"". J " U C ~ 1 -..;: ~ e , 1 .. C ~ C J W • • "~ y 1 .. Z "l, - ,. ,. ,.. ,. •• ,.. •• 'N ... •• • • .. • • • • • • 4111 C 113 Figure A-7: LAMMA mass spectra of selenoniurncholine tetraphenylborate in bath the positive (A) and negative (B) ions mode. 114 " -.. Figure A·8: LAMMA mass spectra of selenoniumacetylcholine tetraphenylborate in both the positive (A) and negatlve (8) Ions mode. ! 1 II 1 1 1 1 ; i i 1 i II! ! .. -1 1 1 1 ! 1 1 1 1 1 • 1 , 1 J 1 - 1 • li , • 1 , 1 ! 1 1 .11 1 If.. 1- ! 1 ,• 1, -; 1 ..J 1 0 1 ! 1 ! /1 1 1 ~ (~"'--) ...... (--.~--- (--.~-- (~~ .... al ~ 1 II 1 1 ; ; i ! -1 ~ ------= - 1 , --' 8- ...... , 1 ~ ! ~ 1 1 -' . ;; 1 , 1 1 1 - 1 ! 5 , - - • 1 -, . i 1 .t .i 1 , 1 1 I! i .. - 1 • ~ t 1.i - ~ Il.. 1 1 0 J 1 ! /1 ~ 1 ! (~~)--- (--.~)-- (~~~ L 115 APPENDIXB 116 Figure B- t. Lmear regresslOn analysis ta test the concurrence of the observed k' values for the selenomumacetylcholme catIon Wlth predicted values from the mode!. REGRESSION ANALYSIS - Lmear model y" a+bX Dependent vanable: OBSERVED; Independent variable: PREDICfEO Standard T Prob. ~ Parame!~r Esllmate Error Value Level -.;;a:.- Intercept -8.20121E-4 009028S7 -908362E-3 0.99289 Slope 0850554 00333716 25 4874 O.!XXXXJ AnalyslS of Variance Source Sum of Squares Of Mean Square F-RJtlO Model 23.738740 1 23.738740 6496054 Error 0.475063 13 0036543 Total (Corr.) 24213800 14 Correlallon CoefficIent =0990142; R-squared" 98.04 %, Stnd Error of Esumate " 0.191163 Regression of OBSERVED on PREDICTED 6 5 Q 4 w ~ w 3 la o 2 1 o o 1 2 3 4 5 8 PREmCTED RESIDUALS for the Regression of OBSERVED on PREDICTED , i , i 1 i , •• 1 • , i il' i i '-r 0 ...... ,- 0.3 ~ 0( ~ Q 0.1 ên w a: -0.1 -0.3 o 1 2 3 4 5 6 PREDICTED .. " 117 " Figure 8-2: Lmear regresslOn analysIs ta test the concurrence of the observed k' values for the tnmethylselenomum catIon with predlcted values frpm the model. RegressIon Analysls - unear mode!. Y z a+bX Dependent variable. OBSERVED; Independent variable. PREDICTED Standard T Prob Parameter Estlmale Error Value Level Intercept 7.03286E-7 0.151327 464747E-6 1.0CXXXl Slope 00492163 2U3185 o(XX)()() AnalyslS of Variance Source Sum of Squares Of Mean Square F·RaI!O Model 39.01596 3901596 4128405 Error 1228580 13 009450b Total (Corr ) 40244543 14 Correlatton CoefficIent" 0 984618. R-squared" 96.95 % Stnd. Error of Esumate .0307419 Regression of OBSERVED on PREDICTED 8 6 Q w w~ 4 o oID 2 o 02468 PREDICTED RESIDUALS for the Regression of OBSERVED on PREDICTED 0.6 0.4 o 0.2 Q~ 0 Ci) W a: -0.2 -0.4 -0.6 o 2 4 6 8 PREDICTED 118 Fil:ure 8-3: Predlcted response surfaces for the capacity factor (A, B, and C) and Area/height (D, E, and F) for selenoniumacetylcholine ion as a functlon of the concentration of TEA and TMSI In a methanolic mobile phase containing 1% ace tiC aCld and: 20% (A, D); 30% (B. E); or 40% (C. F) (v/v) diethyl ether. 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