Selenium Speciation by High Performance Liquid ...digitool.library.mcgill.ca/thesisfile55507.pdf · Selenium has been shown to have multiple biochemical effects rangmg from nutrient

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Selenium Speciation by High Performance Liquid Chromatography

- Atomic Absorption Spectrometry

by

Tian Lei

Department offood Science & Agricultural Chemistry

McGiII University, Macdonald Campus, Montreal

April 1994

A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES AND RESEARCH

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

Copyright © 1994 by Tian Lei

7 /.Ir Lél Nome 1 fl J •

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0786 0760

0425 0996

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0370 0372 0373 0388 0411 0345 0426 0418 0985 0427 0368 0415

076S

05M 0300 0992 0567 0350 076') 0750 0982 056<1 034/ 0560

0570 0380

035<1 0381 0571 041)' 057:< 0382 0573 0574 0575

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Stalostlcs

AppIiecl 51CIenc «'5 Applred /I~!!Chanocs Compute. Sc ence

0422

0318 0321 0319 0320 0322 0469

0323

0324 0:126 0:127

0310 0272 0770 0454 0338 0385

0501 0503 0505 0508 0509 0510 0511 0358 0366 0351

0578

0460 0383 0386

0~85 0749 01186 0487 04118 0738 0490 04\11 0494 0495 0754 0405

0605 0986

0606 0608 0748 0607

0798 07~19 0609 0610 07~.2 0756 0611 0463

03.16 0984

SUBJECT CODE

Anclent 0579 Medieval 0581 Modern 0582 Black 0328 Almon 0331 ASIO, Austral,a and Ocean,a 0332 Canad,an 0334 European 0335 loh" Amerocan 0336 M,ddle Eastern 0333 UnIted Siaies 0337

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0700 0344 0709 0999 0453

0537 0538 0539 0540 0541 0542 0543 0544 0348 0545 0546 0547 0794 0548 0743 0551 0552 0549 0765 0554 0790 0428 0796 0795 0994

0621 0384 0622 0620 0t-23 0624 0625 0989 0349 0632 0451

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Suggested short title:

• Selenium Speciation by HPLC-AAS

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ABSTRACT

Selenium has been shown to have multiple biochemical effects rangmg from

nutrient deficiency at low levels to toxicity at high levels. This duaIity of concern has led

to a demand for increased numbers of highly accurate and precise determinations of

selenium in biological materials. A convenient procedure was developed for determining

selenoamino acids by HPLC-THG-AAS, based on the derivatization ofthese analyt(~s with

Sanger's reagent Selenomethionine, selenocystine and selenocysteine (after blocking the

free selenol group with phenylmercuric cation) were converted to their N-2,4-

dinitrophenyl derivatives, and separated on a Nucleosil 5-N02 column with methanolic

mobile phase containing acetic acid and triethylamine. furthermore, an improved HPLC

AAS interface design was modified and optirnized for the detection of selenium in HPLC

column eluate The new design was (i ) compatible with aqueous mobile phases c(mtaining

volatile buffers and (ii.) provided equivalent molar response to analytes containing Se(-II),

Se(+IV) and Se(+VI). A method for simultaneously determination of selenate, !Ielenite,

selenocystine, selenomethionine and selenoethionine was developed by using the HPLC

AAS system with aqueous acetic aeid containing ammonium acetate as eluate solution ()n

the cyanopropyl column. The equivalent low ng limits of detection (1-2 ng as Se) fhr

ditferent oxidation states of selenium analytes were obtained using several different mllbile

phases and/or columns. A phenol extraction procedure for selenate, selenite, selenClcystine,

selenomethionine and selenoethionine was evaluated for the determination of these

selenium an alytes in natural waters and wheat samples. The current HPLC-AAS system

provides an inexpensive alterna.tive ta conventional techniques for the determination of

selenium an alytes in environmental sampI es.

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RÉSUMÉ

Le sélenium possède des effets biochimiques des plus variés. Ceux-ci peuvent se

manit4~ster sous forme de déficience alimentaire ou d'intoxication, selon que la

concentration de l'élément soit plus faible ou plus élevée que celle requise Ce

comportement antagoniste du sélénium justifie cette demande accrue de méthodes

d'analyse précises et exactes. Une procédure d'analyse assez efficace a été dévr:10ppée

pour la détermination des acides séleno-aminés par HPLC-THG-AAS. Sélenomethionine,

sélénocystine et sélénocysteine (après blocage des sites actifs par du phénylmercure), ont

été transformées en leur dérivé N-2,4-dinitrophényl et séparées sur une colonne Nucléosil

5-N02 à l'aide d'une solution méthanolique contenant de l'acide acétique et de la

triéthylamine D'autrepart, une version améliorée de cette interface HPLC-AAS, a été

utilisée pour la détermination de ces acides aminés ainsi que d'autres composés organiques

et inorganiques contenant du sélénium. Cette nouvelle interface, qui a l'avantage de

fonctionner avec des phases mobiles aqueuses, permet d'obtenir des réponses équivalentes

pour différents composés séléniques, indépendamment de leur état d'oxydation Une

méthode permettant la détermination simultanée du séléniate, de la sélénite, sélenocystine,

sélénométhionine et de la sélénoéthionine, a été mise au point. Les différents composés ont

été séparés sur une colonne de cyanure de propyl avec pour éluant, une solution aqueuse

d'acide acétique et d'acétate d'ammonium. Les limites de détection de ces produits ont été

de l'ordre de 1 à 2 ng. Des concentrations du même ordre ont été détectées peu importe la

combinaison de phase mobile ou colonne utilisées. Une procédure d'extraction ml phénol

du séléniate, de la sélénite, la sélénocystine, la sélénomethionine et la sélénoéthionine a été

évaluée pour l'extraction de ces composés dans des échantillons d'eau naturelle et de blé.

Le système HPLC-AAS actuel représente une alternative peu coûteuse aux techniques

conventionnelles pour la détermination du sélénium dans des échantillons environmentaux.

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ACKNOWLEDGMENTS

1 am greatly indebted to Dr. W.D. Marshall, my supervisor, for his invaluable

advice and constant encouragement as weil as financial support during the course of tbis

study.

1 wish to express my sincere appreciation to Dr J.S. Blais and Dr. DJ. Ecobichon

for their constructive suggestion and consistent support.

Many thanks are extended to Ms. G M. Momplaisir, Mr. A Huyghues-Despointes,

and Dr. X Zhao and bis family for their assistance and fiiendsrup, as weil as Dr E Chav~z

who kindly provided plant samples for my experiements.

Finally, my deepest gratitude go es to my husband Zruyt for his love, patience and

understanding, to my lovely daughter Meng (Beryl), and to my parents, sisters and brother

for their moral support .

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• TABLE OF CONTENT

ABSTRACT .................................................................................................................... i

RESUME ....................................................................................................................... ii

ACKN"OWLEDGMENTS ............................................................................................. iii

TABLE OF CONTENT ............................................................................................... iv

LIST OF TABLES ....................................................................................................... vii

LIST OF FIGURES ...................................................................................................... is

Chapter 1 Introduction .................................................................................................. 1

1. 1 Chernical Aspects of Selenium ................................................................................ 1

1 2 Environmental Occurrence of Selenium .................................................................. 3

• 1.2.1 Rocks and Soils ................................................................................................ 4

1.2 2 Plants.. . .......................................................................................................... 5

1.2.3 Water .............................................................................................................. 5

1.2.4 Foods and Feeds ............................................................................................... 6

1.2.5 Selenium Cycling in Nature ............................................................................... 6

1.3 Metabolism of Selenium .......................................................................................... 8

1.4 Detennination and Speciation of Selenium Compounds ......................................... 13

1.5 Research Objectives .............................................................................................. 18

Chapter l Determination of Selenoamino Acids by BPLC-THG-AAS ..................... 19

2.1 Synopsis .............................................................................................................. 19

2.2 Materials and Methods ........................................................................................ 19

2.2.1 Reagents and ChemicaJs ................................................................................. 19

2.2.2 Synthesis of Organoselenium Compound ........................................................ 20

• 2.2.3 Instruments ..................................................................................................... 21

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2.2.4 HPLC-Thermochemical Hydride Generation-ASS Interface . ..

2.25 Preparation ofN-DNP-Selen()amino Acids ...... . .. .

2 2 6 HPLC Conditions . . .. ...... .. .. . . . ...... .

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2.3 Results and Discussion .. ........... ....... .. ....... . .24

2.3.1 Optimization of the Preparation ofN-DNP-Selenoamino Acids.. .. . ........ 24

2.3 .l.1 The pH of the Buffer Solution . ..... . ............ ............. .. . ....... 25

2.3.1 2 Solvents of the Extraction. " . ....... .. . ......... .. .................. 27

2.3.2 The Effect(s) ofPotential Interferences on the Dinitrophenylation Procedure .. 29

2.3.3 Chromatographie Separation ofN-DNP-Selenoamino Acids ...................... 30

2.4 Conclusion.. ............. ... ........ . .......... .................................. 35

Chapter 3 Approaches to the Speciation of Se(-ll), Se(IV) knd Se(VI) Compound!

in Natural Water and Wheat by BPLC-AAS ......................................... 36

3.1 Synopsis.. ......................... . ........... ..................................................... 36

3.2 Materials and Methods.. .............. ...... ...... .... ..... ........... ............. . . .. 37

3.2.1 Reagents and Chemicals . .. ..... .... . ............ ........ .......... .... .. ...... 37

3.2.2 Syntheses ofOrganoselenium Compounds ................................................. 37

3.2.3 HPLC-ASS Interface ............................................................................. 39

3.2.4 Optimization Procedure ............................................................................... 42

3.2.5 HPLC Conditions .................................................................................... 42

3.2.6 Calibration.............................. ........ ......... .............................. . .................. 43

3.2.7 RecoverylStability Trials ............................................................................... 43

3.3 Results and Discussion..... ....... ............................... . ..................................... 45

3.3.1 Optimization of the HPLC-ASS Interface .................................................... .45

3.3.2 Chromatographie Separation of Selenium Compounds .................................. 53

3.3.3 Calibration and Limit of Detection (LODs) .................................................. 62

3.3.4 Sample Extraction and Analysis .............................................................. 69

3.3.5 Suggested Future Experiments. " .. .............. .......... ............. .. ................ 81

3.4 Conclusion ......................................................................................................... 81

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• Appendices .•..•..•••••..........•••••• e •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 84

References ........................... ~ ......................................................................................... 94

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LIST OF 'fABLES

Table 1-1 Sorne chemical and physical properties of selenium 2

Table 1-2 Organic selenium compounds which have been reported in plants.. . ... ..... 3

Table 2-1 The influence of extracting solvent on the yield ofN-DNP-selenornethionine28

Table 2-2 The yields ofN-DNP-selenoamino acids .. .28

Table 2-3 The effect of potential interference on yield of N-DNP-selenomethionine .... 29

Table 3-1 Observed and predicted peak area for selenomethionine as a function of

the flow rates of mobile phase, oxygen and hydrogen .. 47

Table 3-2 The statistial results of analysis of variance and regression estimates for

selenomethionine as a function of the flow rates of mobile phase, oxygen

and hydroge~ .. .. . .48

Table 3-3 Simplified polynomial expression for the predicted peak area for

selenomcthionine (30 ng as Se) as a function of the flow rates of oxygen

and hydrogen to the pyrolysis charnber The flow rate of mobile phase

fixed at ( a) 0 5 ml/min, (b) 0 7 mL/min, or (c) 0 8 mL/min ..... ... 51

Table 3-4 Relative AAS responses of selenium compounds ..... 54

Table 3-5 Estimates of the chromatographie limits of detection* (ng of Se) for

selenium analytes in HPLC eluate ... . .. .. . .. 68

Tab!e 3-6 The reeoveries of selenium compounds from water extract ... ...... .. .. 71

Table 3-7 The recoveries of selenium compoullds from phenol extract for water and

wheat samples . ..77

Table A-t Observed and predicted peak area for selenate as a function of the flow

rates of mobile phase, oxygen and hydrogen ... 84

Table A-2 The statistial results of analysis of variance and regression estimates for

selenate as a function of the flow rates of mobile phase, oxygen and

hydrogen ... ....... .......... ... .. . ...... .......... .. . .. 85

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Table A-3 Simplified polynomial expression for the predicted peak area for selenate

as a function of the flow rates of oxygen and hydrogen with the flow rate of

mobile phase fixed at (a) 0.5 mVmin; (b) 0.7 mL/min, or (c) 0.8 mL/min .... 86

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LIST OF FIGURES

Figure 1-1 The cycling of selenium in nature ........................... ........ ..... .. . . ... .... . .. 7

Figure 1-2 Proposed mechanism for the methylation of selenium ................................. 1 1

Figure 1-3 Proposed mechanism for the methylation of selenium.............................. .. 1 1

Figure 1-4 Proposed metabolic events that would result in the fonnation of selenoamino

acids and the eventuaJ elimination of selenium from plants ......................... 12

Figure 2-1 HPLC-THG-AAS interface consisting of (a) optical tube (9 mm i.d. x II

mm o.d. x 12 cm)~ (h) analytical flame tube (4 mm i.d. x 6 mm 0 d. x 8 cm)~

(c) combustion chamber (7 mm i.d. x 9 mm o.d. x 4 cm)~ (d) thennospray

tube (4 mm i.d. x 6 mm o.d. x 8 cm); (e), (f) oxygen and hydrogen inlets

(2 mm i.d. x 3.2 mm o.d. x 5 cm, 2.5 cm apart), (g) deactivated capillary

silica transfer line (50 ~m i.d. x 20 cm); (h) quartz guide tube (2 mm i.d

x 3.2 mm o.d. x 10 cm)~ (j) analytical oxygen quartz tube intet (2 mm i.d. x

3.2 mm o.d x 15 cm) ........................................................................... 22

Figure 2-2 The influence of the buffer pH on the yield ofN-DNP··selenomethionine ..... 26

Figure 2-3 HPLC-THG-AAS chromatogram ofN-DNP-selenomethionine (a) and

N,N-di-DNP-selenocystine (h) separated on a Nucleosil 5 N02 column with

using a methanolic mobile phase containing acetic acid and triethylarnine

(0.05 and 0.8 J1LImL, respectively) delivered at 0.65 mUmin ..................... 31

Figure 2-4 HPLC-THG-AAS chromatograms ofN-DNP-selenomethionine (a),

N,N-di-DNP-selenocystine (h), and N-DNP-Ph-Hg-selenocysteine (c)

obtained on Nucleosil 5-N02 column with a methanolic mobile phase

containing 6 ~UmL tetrabutylâJ1lJl1onium nitrate and 0.9 J.1UmL of

triethylamine, delivered at 0.65 roUmin .................................................... 33

Figure 2-5 HPLC-THG-AAS chromatogram ofN-DNP-selenomethionine (a),

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N,N-di-DNP-selenocystine (b) and N-ONP-Ph-Hg-selenocysteine (c)

separated on a Nuc1eosil 5-N02 column with a methanolic mobile phase

containing acetic aeid and triethylamine (0.05 and 08 J.1L/mL respeetively)

delivered at 0.65 mUmin ............ ... ................ .. ...................................... 34

Figure 3-1 HPLC-AAS interface consisting of (a) optical tube (7 mm i.d. x 9 mm o.d.

x 12 Hcm); (b) pyrolysis chamber (9 mm i.d. x Il mm o.d. x 4 cm); (e)

thermospray tube (4 mm i.d. x 6 mm o.d. x 6 cm); (d), (e) quartz guide

tubes for gas inlets (4 mm i.d. x 6 mm o.d. x 7 cm); (t) a deactivated

capillary silica transfer line (50 J..lm i.d. x 20 em); (g) quartz guide tube

(2 mm i.d x 3.2 mm o.d. x 8 cm); 0) oxygen quartz tube intet (2 mm i.d.

x 3.2 mm o.d x 10 cm); (h) hydrogen quartz tube inlet (2 mm i.d. x

3.2 mm o.d x 10 cm) ................................................................................. 40

Figure 3-2 Simple regression analysis orthe observed Peak Areas for selenomethionine

with predieted values from the model .......................................................... 49

Figure 3-3 Predicted response surfaces for peak area for selenomethionine (30 ng as Se)

as a function of the flow rates of oxygen and hydrogen with the flow rate of

mobile phase fixed at (A) 0.5 mL/min; (8) 0.7 ml/min; (C) 0.8 mUmin ..... 52

Figure 3-4 HPLC-AAS chromatograms ofselenocystine [RT, 4.52 min (A); 4.86 (B)],

selenomethionine[RT, 5.32 (A); 5.85 (B)] and selenoethionine [RT, 5.96

(A); 6.93 (B)] (0.125 ~g as Se for each compound) separated on the

Nucleosil 5-SA column with O.S mUmin of mobile phase eonsisting of (A)

aqueous aeetic acid (0.1% v/v) eontaining 0.05% (v/v) triethylamine or (B)

aqueous ammonium acetate (0.12% w/v) .................................................... S6

Figure 3-5 HPLC-AAS chromatograms of selenocystine [RT, 3.00 (A); 3.0S (B)],

selenomethionine [RT, 4.47 (A); 4.41 (8)] and selenoethionine [RT, 7.93

(A); 7.97 (B» (0.3 ~g as Se for eaeh compound) separated on the NueleosiI

C18 column with 0.6 rnUmin ofmobiIe phase consisting of(A) aqueous

aeetic acid (0.05% v/v) containing 0.2% (v/v) tetramethylammonium

hydroxide or (8) aqueous ammonium acetate (0.06% w/v) ......................... 57

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Figure 3-6 HPLC-AAS chromatograms of(A) methaneseleninic aeid [RT, 6.80] and

methaneseleronic aeid [RT. 7.86] separated with aqueous ammonium

carbonate (0.02% w/v) and (B) methaneseleninic aeid [RT, 4.54],

methaneselenonic aeid [RT, 5.12] and selenite [RT, 8.31] separated with

aqueous ammonium carbonate (0.1 % w/v). Mobile phase was delivered, at

0.6 mL/min, to the PL-SAX colllmn.... ... ...... ....... .. ......... 58

Figure 3-7 Isocratie HPLC-AAS ehromatograms of 10 (A) or 20 ng (B) as Se of

selenite [RT, 4.74] and selenate fRT, 6 95] separated on the Pl.-SAX column

with an aqueous mobile phase eontaining 0.1% (w/v) ammonium carbonate

(adjusted to pH 9 with aqueous ammonia). Mobile phase was delivered at to

the column at 0.6 ml/min................... ............. .. ................. ... ....... 59

Figure 3-8 HPLC-AAS chromatograms of 5 (A) or lOng (B) as Se of selenoeystine

[RT, 5.20], selenomethionine rRT, 6.68] and selenoethionine [RT, 8.45]

The an alytes were eluted from the eyanopropyl column with aqueous

acetic aeid (004% v/v) delivered at 0.5 mL/min... .......................... 60

Figure 3-9 HPLC-AAS chromatograms of 10 (A) or 20 (B) ng as Se of selenatc [RT,

2.67], selenite [RT, l07], selenoeystine [RT, 3.94], selenomethionine [RT,

4.36] and selenoethionine [RT, 4.81]. The analytes were eluted form the

cyanopropyl column with 0.015% (v/v) aqueous acetic aeid eontaining 0 1%

(w/v) ammonium aeetate delivered at 0 5 mL/min " 61

Figure 3-10 Regression analysis for the determination of the Iimit of detection for

selenomethionine (peak Area vs Amount, Phase C) . ........ . ., ............ 63

Figure 3-11 Regression analysis for the determination of the Iimit of detection for

selenocystine (Peak Area vs Amount, Phase C) ... ... . ............. .. .. '" .... . .. 64

Figure 3-12 Regression analysis for the determination of the limit of detection for

selenoethionine (Peak Area vs Amount, Phase C) ............... " .................. 65

Figure 3 -13 Regression analysis for the determination of the Iimit of detection for

selenate (Peak Area vs Amount, Phase C) ... "...... . ...... "" ............... " ........... 66

Figure 3-14 Regression analysis for the determination of the limit of detection for

selenite (Peak Area vs Amount, Phase C) " .............. " ......................... "" ... 67

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Figure 3-15 HPLC-AAS chromatograms resu.lting from: A, tap water (20 mL) which

had been spiked with a standard mixture of 4 J.1g each (as Se) oftive Se

eompounds, evaporated to dryness, resolubiUised in hot water The recoveries

of the five Se-an alytes, selenate, selenite, selenocystine, selenomethionine and

selenoethionine (in order of elution, -50 ng as Se/injection) were virtually

quantitative; B, an identical recover.v procedure applied to a control sample

of the tap water ...................................... ., . ..... ....................... . ............. 72

F'igure 3-16 HPLC-AAS chromatograms of phenol extracts of tap water which had becn

spiked with a mixture of selenate, selenite, selenocystine, selenomethionine

and selenoethionine (0.2 Ilg each as Se/mL, in order of elution, -25 ng as

Se/injection) then acidified to pH 3 with (A), acetic acid; (B), hydrochlorie

aeid; or (C), formic acid or basified to pH 10 with (0), aqueous ammonia

prior to extraction. The analytes were duted with aqueous aeetic aeid

(0.04%, v/v) delivered, at 0.5 mUmin, to the eyanopropyl eolumn ............. 74

Figure 3-17 HPLC-AAS ehromatogram of the phenol extraet from tap water which had

been spiked with a mixture of selenite, sl:lenocystine, selenomethionine and

selenoethionine (0 2 Jlg each as Se/ml, in order of elution, -50 ng as

Se/injeetion). The an alytes were eluted with aqueous acetic aeid (0.04% v/v)

delivered, at 0.5 mUmin, to the cyanopropyl colurnn .................................. 75

Figure 3-18 HPLC-AAS ehromatogram of (A) the pht~nol extraet from a ground dried

wheat sampie whieh had been spiked with ~;elenate, selenite, selenocystine,

selenomethionine and selenoethionine (4 J.1g1g eaeh as Se, in order of elution,

-25 ng as SeJinjection) then aeidified to pH 3, prior to extraction.

Chromatogram B resulted from a 0.5 g sample of the same wheat material

which was acidified then extracted in identicaJ fashion. The analytes were

eluted wilh aqueous acetie aeid (0.015% v/v) containing 0.1% (w/v)

ammonium aeetate delivered, at 0.5 mUmin. to the cyanopropyl column .... 76

Figure 3-19 HPLC-AAS ehromatograms of (A), the mixture of selenite [RT, 6.05] and

xii

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selenate [RT, 10 19], (B), the oxidation products ofselenomethionine [RT,

3.42~ 6.07]; or (C), the oxidation products of selenoeystine [RT, 3 52, 601,

10.18]. The analytes were eluted with an aqueous mobile phase containing

o 1 % (w/v) ammonium carbonate (adjusted to pH 8 with aqueous anll11onia)

delivered, at 0 6 mL/min to the PL-SAX column .. , 79

Figure 3-20 HPLC-AAS chromatograms of (A), the mixture ofmethaneselenonic aeid

[RT, 2.42], selenite [RT, 267], methaneseleninic aeid [RT, 4 52],

selenoeystine [RT, 6.00], selenomethionine [RT, 749] and selenoethioninc

[RT, 9.28], (B), the oxidation products ofselenomethionine [RT, 2.39,

12 II]; or (C), the oxidation products ofselenocystine [RT, 240,262,

4.17] The analytes were eluted with aqueous acetic aeid (0.05% v/v)

delivered, at 0.5 mL/min, to the cyanopropyl column .. , . 80

Figure 3-21 HPLC-AAS chromatograms of (A), the mixture of methaneseleninic aeid

[RT, 680] and methaneselenonie aeid [RT, 7 86]; (B) the aqueous solution

of methaneseleninic aeid and methaneselenonic aeid after the APDTC

extraction; or (C), the aqueous solution ofmethaneseleninic aeid and

methaneselenonic aeid after the TBADTC extraction. The an alytes were

eluted with aqueous ammonium carbonate (0 02% w/v) delivered, at 0 6

mL/min, to the PL-SAX column ..... . ... , 82

Figure A-l Simple regression analysis of the observed Peak Areas for selenate with

predicted values from the model . .. 87

Figure A-2 Predicted respo.1se surfaces for peak area for selenate (30 ng as Se) as a

funetion of the tlow rates of oxygen and hydrogen with the flow rate of

mobile phase fixed at (A) 0 5 mL/min; (B) 0.7 mL/min, (C) 0.8 mL/min .. 88

Figure A-3 Regression analysis for the determmation of the Iimit of detcction for

selenomethionine (Peak Area vs Amount, Phase A) ..... 89

Figure A-4 Regression analysis for the determination of the \imit of deteetion for

selenocystine (Peak Area vs Amount) Phase A).. . .. ...... . ......... 90

Figure A-5 Regression analysis for the determination of the Iimit of deteetion for

selenoethionine (Peak Area vs Amount, Phase A) . .......... .. ................... 91

xÎli

• Figure A-6 Regression anaJysis for the determination of the Jimit of detection for

selenite (Peak Area vs Amount, Phase B) ..................... .. .. . . ... .. .............. 92

Figure A-7 Regression analysis for the determination of the li mit of detection for

selenate (Peak A.rea vs Amount, Phase B) ............................................. 93

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Chapter l. Introduc~on

Selenium is a naturaIly-occurring substance that is widely but unevenly distributed

in the earth's crust, having an average abundance of about 0.09 mglkg (Lakin. 1972) and is

commonlY associated with sedimentary rock formations. It was first identified as an

element in 1817 by the Swedish chemist Berzelius. Selenium chemistry is complex and

additional research continul~S to be performed on chemical and biochemical

transformations among valence states and allotropie forms of this element Early interest in

selenium by nutritionists concemed its high concentration in certain range plants and the

consequent toxicosis induced in animaIs lhat grazed on these plants In recent years many

exciting research results have indicated that selenium, depending on its concentration, can

influence mammalian metabolism in a variety of ways; the essentiality of selenium to

homeotherms has become the focus of attention. This element is now known to be an

ultra-trace essential element which is required by laboratory animaIs, food animals and

humans, but the range between dietary rtquirements and toxic levels is relatively narrow.

With the growing importance of selenium chemistry cornes the need for precise methods

of analysis for this element.

1.1 Chemical Aspects of Selenium

Selenium belongs to group VI of the periodic tables. It possesses both metallic and

nonmetallic characteristics and is capable of forming both cationic and anionic salts. Sorne

chemical and physical properties of selenium are listed in Table 1-1. Elemental selenium

can be reduced to -2 (selenide), or oxidized to +4 (selenite) or +6 (selenate) oxidation

states. The stability and insolubility of elemental selenium render it unavailable to plants.

The conversion to this elemental form by natural processes might, thus, be considered as

one mean by which the element is removed from activ~ cycling in the environment.

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Table 1-1. Some chemical and physical properties ofseleniuma

Properties

Relative atomic mass Atomic number Atomic radius Covalent radius Electronegativity (pauling's Scale) Electronic structure Common oxidative states Stable isotopes

Mass Natural abundance (%)

pKa: SeO(OH)2' aqueous Se02(OH)2, aqueous (HSe-), aqueous (H2Se), aqueous

Values

78.96 34 0.14 nm 0.116 nm 2.55 [Ar]3dI04s24p4

-2,0, +4, +6

74 76 77 78 80 82 0.87 9.02 785 23.52 49.82 9.19 2.6 -3 11.0 3.8

a From: Rosenfeld & Beath (1964), Cooper et al. (1974) & Shamberger (1983).

A large number of selenium compounds are known. Many have been identified in

plants, animals and microorganisms. The selenium compounds of greatest interest in

nutrition are presented in Table 1-2. The chemistry of organic selenium compounds has

been reviewed in detail by Klayman and Gunther (1973). Although the chernistty of

selenium is sirnilar to that of sulfur, certain differences result in these elements being

metabolized somewhat differently. First is the difference in the ease of oxidation of Se

(IV) relative to the ease of oxidation of S (IV), the former tending to undergo reduction

and later tending to undergo oxidation. This difference is demonstrated by the foUowing

reaction (Combs and Combs, 1986)'

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The second characteristic relates to differences in the relative acid strengths of H2Se (pKa

3.8) and H2S (PKa 7.0). This difference is reflected in the dissociation behaviors of the

selenohydryl group (-SeH) of selenocysteine (pKa 5.24) relative to the sultbdryl group (

SH) of cysteine (pKa 8.25). Thus, at physiological pH, the sulfhdryl group in cysteine (or

other truols) exists mainly in the protonated form, whereas the selenohydryl group in

selenocysteine (or other selenols) are predominantly dissociated (Shamberger, 1983).

Table 1-2. Organic selenium compounds which have been reported in plantsa

Dimethyl selenide Dimethyl diselenide Selenomethionine Selenomethionine selennxide Selenohomocysteine Selenocystathionine Selenocystine

a From Combs & Combs (1986).

Selenocysteine Selenocysteine-selenic acid Se-methylselenocysteine Se-methylselenomethionine Se-propenylselenocysteine selenoxide Se-containing peptides Seleno-waxes

1.2 Environmental Occurrence of Selenium

Selenium is ubiquitous in the environment, being released by both natural and man

made sources. The primary factor determining the fate of selenium in the environment is

its oxidation state. Selenium is stable in four valence states (-II, 0, +IV and +VI) and

fomlS chemical compounds similar to those of sulfur. The heavy metal selenides (-II) are

insoluble in water, as is elemental selenium. The inorganic selenites and selenate~ are

soluble in water and are therefore more bioavaible, they are readily taken up by plants and

converted to organic compounds such as dimethyl selenide and selenoamino acids .

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1.2.1 Rocks and Soils

Selenium is rarely found in its elemental fonn. It has been found as a major

constitute of 40 minerais and a minor component of 37 others (Cooper et al, 1974)

Typically, it is located in minerai deposits and soil fonnations where a high concentration

of sulfur is found (Painter, 1941). Th~ ~oncentration of selenium in ingenious rocks is low,

usually mu ch less than 1 mg!kg, and similar levels probably occur in metamorphlc rocks.

But sedimentary rocks, such as sandstone, limestone, phosphorite and shales may contain

from < 1 to > 1 00 mglkg (EHC 58, 1987). Canadian ores are considerable richer in

selenium than those of Australia but less rich than sOane of the sedimentary deposits of the

western United States (NRC, 1983). Coal has abundant arnounts of selenium, ranging

from 0.1 to 4 mglkg (Shamberger, 1983) With an unusually high selenium content

(>80,000 mglkg, average 300 mglkg), coal was identmed as the ultimatt environrnental

source of selenium contaminating soils in a seleniferous region of Enshi county in China

(Yang el al., 1983). When the seleniferous co al or oil is bumed, selenium is introduced

into the atmosphere from which selenium redistributed to the earth's surface in rain and

snow.

Seleniferous soils occur in the western USA, Canada, Mexico, China, Colombia

and elsewhere (Rosenfeld and Beath, 1964; Adriano, i 986). Sorne seleniferous soils

contain > 300 mglkg, but the sel~nium concentration of most surface soils varies between

0.1 and 2.0 mglkg, even in those that SUPPl'rt growth of plants toxic to animaJs (NRC,

1983; Girling, 1984). Alkaline and oxidizing conditions favorthe formation and stability of

selenates. which do not form stable adsorption complexes; therefore they are taken up by

plants readily and are easily leached into ground water. Soluble selenites that predominate

in soils of humid and aeid regions are highly toxie but less available to plants. In soils that

are slightly aeid or neutral, selenium is present in organie eompounds which were formed

by plant metabolism by the indigenous vegetation (NRC, 1976, 1983; Adriano, 1986) .

Under reducing conditions, volatile methyldted selenium compounds are easily forrned and

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this represents with leaching a major mechanism for selenium loss from soil (Kabata

Pendias and Pendias, 1984).

1.22 Plants

Rosenfeld and Beath (1964) c.' vided plants into three groups on the basis of their

ability to accumulate selenium when g_ .:>wn on seleniferous soils Most plants contain < 1

mg Selkg when grown on nonseleniferous soils (Girling, 1984). Normal selenium

concentrations in algae and rooted aquatic plants also are < 1 mglkg (Rossi el al., 1976;

Saiki and Lowe, 1987, Hothem and Ohlendorf, 1989). Plant uptake studies have shown

that selenium is readily taken up by plants trom soils rugh in water soluble selenium Table

1-2 presents a list of the organic forms of selenium that hav.;: been reported in plants. The

major chemical forms of selepJum in terrestrial plants are thought to be seleno-analogs of

sulfur-containing amino acids that have been incorporated into proteins (Shibata, el al.,

1992). OIson, et al. (1970) reported that wheat grain grown on selenium-rich land

accumulated selenium mainly in the protein fraction, and that half the selerium in the

fraction was selenomethionine. The major selenium compound in the hydrolysate of

selenium-enriched yeast was also shown to be selenomethionine (Beilstein and Whanger,

1986).

1.2.3 Water

Selenium concentrations in surface water average 2 J.lg/L or less (Robberecht and

Von Grieken, j 982; Nriagu and Wong, 1983; Adriano, 1986; Wiggett and Alfon, 1986).

Drainage from irrigation and soilleaching are the principal sources into water, The highest

natural concentration reported to date is 9000 J.lg/L, almost ail other values falling below

500 JJ.g/L (EHC, 1987). Most water systems contain only a very low concentration of

selenium. High concentrations of selenium have been found in water (400 J.lg/L) in the

vicinity of the Ni-Cu smelter at Sudbury. Ontario (Nriagu and Wong, 1983). Selenium

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intoxication (i.) of domesticated herbivores has also been reported for seleniferous areas

and, more recently, (ii.) of wildlife in marshland areas of central Califomia v.;hich have

been supplemented with agricultural drainage waters that contained elevated levels of total

selenium (Burau, 1985).

1.2.4 Foods and Feeds

The selenium contents of human foods and animal feedstuffs vary widely due to

such factors as the species, the methods of preparation and/or processing, the climatic

conditions during the growing season and the amount of biologically available selenium in

the particular nutrient environment. In sorne areas of the United States, forages contain

sufficiently high selenium concentrations to cause overt sign of selenium intoxication in

;;vestock, in the other regions the levels of selenium in crops and forages are too low to

meet animal requirernents (NRC, 1983) The selenium content of corn and wheat products

from Ontario, Canada., and the midwestem United States have very Jow levels of selenium

(007-0.08 mglkg). This is in direct contrast to the level of selenium in these same foods

grown in western Canada, where levels average 0 56 mglkg (Arthur, 1972). Much of the

variation in the selenium content of foods is due to large scale geographical differences in

environmental selenium Schubert, et al. (1987) reviewed the selenium concentration in

foods that contribute the highest proportion of the daily selenium intake of human

populations in the United States In general, fruits and vegetables were found to oontain

less than 0 01 mg Se/kg whereas grain and dairy products contained higher concentrations

of selenium. Meat and fish contain the highest selenium levels (0.1-2.84 mg Se/kg).

Higham and Tomkins (1993) reported that the selenium concentration of canned tuna fish

ranged from 0.034 to 1 20 mglkg. In summary, selenium is most concentrated in high

protein foods, but the content is greatly influenced by growth condit~ons

1.2.5 Selenium Cycling in Nature

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Like oth(~r elements, selenium is continuously cycled by natural processes Several

diagrammatic schemes have been presented for the environmental cycling of selenium

Major cycle compone'nts have been described in a general model, whlch is IIlustrated in

Figure 1-1. The natural sO\Jrce of selenium for agriculture and most other biological uses

is the soil, from which the element is accumulated, to varying amounts, by plants which

are ultimately consumed by animais The behavior of selenium in the environment Îs

influenced to a large degree by its oxidation state and the consequent differences in the

behavior of its different chemical compounds (CA, 1989) The oxidation state of selenium

in the environment is dependent on ambient conditions, particularly on pH, Eh, and

biological activity (Maier et al., 1988).

Terrestrial Systems

Plants 4 ~ Soils

\ 1 Animais

H Human ActivltJes j+--.

Atmosphere

Volcanism and

Igneous Rocks

Aquatic and Manne Systems

Plants +---+ Sediments

l ')( 1 Arumals+--+ Water

Figure 1-1. The cycling of selenium in nature. (for simplicity, microorganisms are not

included in the scheme)

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1.3 Metabolism or Selenium

Until 1957, the only physiologica1 significance of selenium was thought to be its

toxicity The essentiality of selenium to animal nutrition has been appreciated only since

then Schwarz and Foltz (1957) identified selenium as a part of "Factor 3", which is

important in preventing dietary liver necrosis in rats. InterestingJy, in this same year the

properties ofa newly discovered enzyme, glutathione peroxidase (GSH-Px) were reported

(Mill s, 1957). However, it took a decade and a half to discover that selenium was an

integral component of tbis enzyme (Rotruck, et al., 1972). GSH-Px which is found in

most human and animal tissues, is an enzyme that is involved principally in the destruction

and removal of hydrogen peroxide and lipid hydroperoxides (Rotruck, et al., 1973). The

enzyme thereby protects cellular membranes and lipid-containing organelles trom

peroxidative damage, and together with vitamin E, serves to maintain the integrity of these

membranes (Koller and Exon, 1986, Shambrger, 1986; Zachara, 1992). Amino acid

analyses have identified the form of selenium in GSU-Px as selenocysteine covalently

incorporated into the primary structure of the enzyme (Forstrom, et al., 1978). Another

selenium-containing protein, termed selenoprotein P, was identified in rat plasma and Iiver

(Gomez and Tappel, 1989; Motchnik and Tappel, 1990; Read, et al., 1990) and human

plasma (Deagen, et al., 1991). Other selenium-binding proteins were reported trom

muscle, kidney, and other organs (Reddy and Massaro, 1983; Stadtman, 1983; Haas and

Velten, 1992). Moreover, a significant finding was that sel~nium could counteract the

toxicity of sorne heavy metals such as cadmium and mercury (Ganther, et al., 1972;

Ridlington and Whanger, 1981; Whanger, 199~). This rather unusual feature of one

element, which is highly toxic itself at elevated levels, counteracting the toxicity of heavy

metals has stimulated intensive research into the relationships of selenium with other

elements. The other significant development with selenium has been the demonstration

that it can counteract the deleterious effects of certain chemical carcinogens (Hocman,

1988; Milner, 1985; Vernie, 1984).

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Since food is the primary environmental medium through which man and animais

are exposed to selenium, most data conceming selenium absorption deal with the

gastrointestinal pathway. The intestinal adsorption of soluble selenium compounds by rats

is highly efficient. It has been shown that these animais absorbed 92, 91, and 81 % of doses

of selenite, selenomethionine, and selenocystine, respectively (Thomson and Stewart,

1973~ Thomson et al., 1975). In humans, absorption of sodium selenite or

selenomethionine can exceed 80% for both small and large doses (Griffiths, et al., 1978~

Thomson and Stewart, 1974; Thomson, et al., 1978).

Absorbed selenium is rapidly distributed among the tissues. Under normal

conditions, levels of selenium are higher in the kidney and li ver than in the other major

body tissues. Not ooly is the tissue content of selenium dependent upon the level of total

selenium in the diet, but also upon its chemical fonn In general, selenium is deposit~ in

tissues at higher concentrations when present in diets as organic rather than as inorganic

selenium. Different selenium compound s, however, result in different distribution patterns

in the body. The highest concentrations of selenium trom selenite and selenate is found in

the Iiver and kidney of human and animal following oral administration or injection

(Cavalieri et al., 1966; Jereb, et al., 1975; Thomson and Stewart, 1973). Selenium from

selenomethionine, on the other hand, has been observed to concentrate in the pancreas of

human and rat following intravenous administration and in the pancreas of chick following

oral administration (Ben-Porath and Kaplan, 1969; Cantor, el al., 1975; Lathrop, el QI.,

1972). A rapid decline in blood selenium levels one houT after intravenous administration

of selenite or selenate to human has been reported (Burie, 1974). Several studies indicate

that selenite is chemically altered in the erythrocyte and then transported back into the

plasma where the selenium metabolite binds to plasma proteins (Burie, 1974~ Hirooka and

Lee, el al., 1969).

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In humans and animais, excretion of selenium can occur in the urine, feces, and

expired air. Moreover, the initial rate ofexcretion appears to be dose-dependent (Griffiths,

el al, 1976, Lathrop el al, 1972; McConnell and Roth, 1966; Thomson and Stewart,

1974) Studies with rats have demonstrated that the urinary pathway is the dominant route

for selenium excretion, as long as the dietary selenium exceeds a certain critical threshold

level. The principal urinary selenium metabolite of rats is trimethylselenonium ion (Byard,

1969; Palmer el al., 1969). This form accounts for 20-50% of the urinary selenium,

regardless of the fonn of selenium administered (palmer, et al., 1970). Whether a low or a

high oral dose of selenium, selenomethionine resulted in a less trimethylselenonium in the

urine than when similar levels of selenocystine or selenite were administered (Nahapetian,

et al., 1984). On other hand, two volatile selenium compounds which have been identified

in exhaled air, dimethylselenide and dimethyldiselenide, were only detected in cases of

very high selenium exposure (McConneJJ and Portman, 1952; Jiang, el al., 1983) .

In contrast to sulfur, selenium compounds tend to undergo reductive

transformations in tissues. Ganther (1979) has provided fundamental information on the

mechanism for reduction of selenite to selenide. GSH, anaerobic conditions and NADPH

are ail essential for this reduction. Evidence has been obtained for the formation of

hydrogen selenide trom selenite in tissues (Hsieh and Ganther, 1975).

NADPH NADP NAI>PH NADP

H2SeO, + GSH---+» GSSeSG '-..,:Ir »GSSdI '---" ) H2Se

Challenger (l955) has reviewed the formation of volatile dimethyl selenide by

certain molds fioom inorganic selenium salts. He (1951) has postulated a mechanism which

con~isted of alternate methylation and reduction steps to account for the biosynthesis of

dimethyl selenide from selenite (Figure 1-2):

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ion

anion of Methane seleninic acid

methaneselenonic acid

dimethyl selenonc

dimethyl sclcnide

Figure 1-2. Proposed ofmechanism for the methylation of selenium (Challenger, 1951) .

Dimcthyltelenone Methyl methylJelenite

\ /--(CH,hSe

Dimethylteleniclc

..JIOI

CH'r CH,SeSeCH, DimcthyI dileleniclc

Figure 1-3. Proposed mechanism for the methylation of selenium (Reamer & ZoUer, 1980).

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Subsequently, Reamer and Zoller (1980) modified the scheme to include a concentration

dependent side-reaction which resulted in the production of dimethyldiselenide (Figure 1-

3). This type of mechanism is sirnilar to that of the eorresponding sulfur species.

Moreover, several investigators have used chromatographie identification as evidenee for

the conversion of inorganic selenium into selenoarnino acids by rnicroorganisms. Bottino

et al. (1984) postulated a hypothetieal seheme for the metabolism of selenium by piants

(Figure 1-4). This proposai constitutes a series of methylation and reduction steps that

collectively provides means of elirninating toxic selenium trom the cell.

w CH2-Se-OH

1 /1 seOl- ~ fHNH2 0

COOH Selerute Selenocystelc ICld

W fH2-S~Me CH2SeMe

1 CHNH2

1 COOH

Se-methyl MICI1OC)'IIeine IClenoxicie

~ fHNH2

COOH

MeSe--SeMe Dimethyl diselenidc

" fH2-ile-Me

~ • CHNH10

1 COOH Se-mdhyl selmocysteine sdenone

Selenometluooine

Figure 1-4. Proposed metabolic events that would result in the fonnation of selenoamino

acids and the eventuaJ elimination of selenium from plants.

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Selenium is metabolized by a combination of reduction and methylation processes

Methylated metabolites of selenium include trimethylselenonium ion, the major urinary

metabolite of selenium, and dtmethyl selenide, the volatile selenium metabolite produced

under conditions of selenium intoxication Methylation of selenium has been regarded as a

detoxitication mechanism since these methylated forms are of relatively low aeute toxicity

and are rapidly eliminated Reduction of selenium III VIl'O is probably accompli shed by

reaction with the protein-bound sl. •• bydryl groups, or with low molecular weight thiols,

such as glutathione, to fonn selenotrisulfide (S-Se-S) derivatives. Dietary selenium in

excess of 4 mg/kg is generally considered to be toxic to mammals (Oison, 1986). High

environ.'11ental concentrations of selenium have been documented to adversely affect

reproduction, survival, and development of wild aquatic birds (Ohlendorf, el al, 1986,

1988; Hoffinan, et al, 1988, Heinz and Fitzgerad, 1993). The proximal biochemical roles

of selenium toxicity are not c1ear at the present time, however, it is thought that these

involve the oxidation of and/or binding to eritieal sulfhydryl groups by selenium speeies

present in excessive concentrations Dickson and Tappel (I969) proposed that selenium

toxicity is related to changes in intra-cellular concentrations of reduced GSH, and ex cess

selenium has been shown to interaet with intra-cellular sulfhydryls LeBoeuf et al. (1985)

reported that 3.0-6.0 mglkg selenium as selenite in the diet of rats for 6 weeks caused a

significant dose-dependent increase in hepatic GSH, in oxidized GSSG, and in GSSG to

GSH ratio. Hoffinan et al. (1988) reported that excess dietary selenium, as

selenomethionine, has a more pronounced effect on hepatic glutathione metabolism and

Iipid peroxidation in ducklings than does selenite, which may be related to the pattern of

accumulation.

1.4 Determination and Speciation of Selenium Compounds

The speciation of inorganically and organically bound metals in solution presents a

formidable challenge. Although the technology required to perform rugh-performance

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Iiquid chromatographic (HPLC) separations of metal-containing species is generally

available, conventional HPLC detectors, such as ultraviolet (UV), fluorescence (FL),

electrochemical (EC), Oame Îonization (FI), and infrared (IR), lack the desired degree of

selectivity, sensitivity, and applicability to the se metal bound compounds (Ettre, 1978;

White, 1984). Atomic spectroscopie detectors, on the other hand, are highly specific and

are applicable to variety of sample matrices. The various spectroscopie detectors include

Oame atomic absorption spectroscopy (F AAS), graphite f,Jmace atomic absorption

spectroscopy (GFAAS), and Oame or plasma atomic fluorescence spectroscopy (AFS), as

weil as atomic emission spectroscopy (AES). Several formats of AES (according to the

mode of formation of the plasma) including micfOwave induced plasma (MIP). direct

current plasma (DCP), and inductively coupled plasma (lep) hold great potential as

element-specific detectors for HPLC Many researches agree that plasma emission

spectroscopy will eventually be the method of choice for simultaneous, multielement,

element-specific chromatographic detection. The direct coupling of HPLC to ICP or DCP

detection has been accompli shed in severallaboratories (lrgolic, et al, 1983; McCarthy, et

al., 1983; LaFreniere, et al., 1987; Laborda, et al., 1991) and offered the possibility of

continuously monitoring the selenium in column eluate. However, the higher purchase and

operating costs of these instruments may restrict the availability of these techniques to

certain researchers.

The sensitivity of atomic absorption for selenium is limited by flame absorption in

the region of the resonance line (at 196 nm) and by light scattering resulting from

particulate matter or micro-droplets in the light beam (Henn, 1975). Sensitive techniques

for the dttermination of certain selenium compounds have been developed. Generally,

these methods are based on either the differential rates of generation of selenide or column

chromatographie separations of different selenium species. The most sensitive of these

techniques uses graphite furnace atomic absorption spectroflcopy (GF AAS) for the

14

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determination of selenium in column effluent (Oyamada and Ishizaki, 1986; Laborda, et

al., 1993). GF AAS off ers the advantage of high sensitivity for a single an alyte The

disadvantage of GF AA is that the selenium measurement is discontinuous. Because

GF AAS, as conventionally practised, does not provide a continuous signal, its use requires

that small portions of the column effluent be collected and that each fraction be separately

analyzed. Off-Ime detection provides a histogram of the Se-content which may

compromise quantitation. Direct coupling between HPLC and GFAAS requires (i.) the use

of specially designed fumaces in order to maintain the atomization temperature during the

elution period and that (ii.) the sample be introduced into the fumace in a form whieh

avoid the drying and charring steps (Nygren, et al., 1988) The requirement to dry and ash

a sample prilJf to atomization causes on-Ime HPLC-GF AAS system to be especially

difticult to operate. Recently, Kalbl and co-workers (1993a; 1993b) reported an ion

chromatographie method for the determination of selenite and selenate using liquid

chromatography (LC)-GF AAS provided that the absolute detection Iimits were 1 ng and

0.6 ng as Se, respectively. They also developed a LC-FAAS system that had the absolute

detection Iimit of 8 ng Se for selenite and Il ng Se for selenate.

Many of the speciation techniques currently used for selenium rely on indirect

measurements of one or more of the chemical species. The chemical complexing

techniques, such u~ reagents as 2,3-diaminonaphthalene (Shibata, et al., 1984;

Handelman, et al., 1989; Khuhawar, 1992) and dithiocarbamates (Nakagawa, et al., 1989;

Park and Hardy, 1989; Shofstahl and Hardy, 1990), are sensitive only to Se(IV). The

determination of Se(VI) is accompli shed by observing the difference between total Se

measurements before and after photolytie or chemical reduction. The method has been

developed based on the separation of selenite and selenate from the organic interference(s)

by colunm chromatography on XAD-8 resin followed by hydride generationlGF AAS

(Roden and Tallman, 1982). A relatively new technique, ion chromatography (le), has

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become an accepted method for the direct determination of ionic species in complex

mixtures. The technique has bt~en applied to the simultaneous separation of selenite and

selenate, using either a non-suppressed IC system (Karlson and Frankenberger, 1986a;

1986b) or a suppressed lC system (Zolotov, el al., 1983; Urasa and Ferede, 1987; Goyal,

et al., 1991, McGeehan and Naylor, 1992) with a Na2C03/NaHCO) mixture as the

eluting mobile phase. Although suppressed IC achieves lower detection limits than non

suppressed IC, both techniques lack the sensitivity required for the successful analysis of

many environmental samples. Detection lirnits of 140 and 91 ng of Se for selenite and

selenate, respectively, have been obtained using ICP-AES (McCarthy, et al., 1983), these

values have been reduced to 14, 26 and 54 ng of Se for trimethylselenomum, selenite and

selenate, respectively, using a thermospray nebulizer instead of conventional nebulizers

(Laborda, et al., 1991). The ETAAS method using a pulse mode interface has resulted in

detection Iirnits of 5 ng of selenite and selenate (Chakraborti, 1982). An ion-pairing

(tetrabutylammonium phosphate), reverse-~nase (Whatman Partisil 5 00S-3) separation

of Se(IV) and Se(VI) have been rcported by LaFreniere, et al. (1987) using the HPLC

DIN (direct injection nebulizer)-ICP-AES system. The lirnits of detection (LODs) were

determined to be 42 ng/mL (8 ng) and 71 ng/mL (14 ng) for selenite and selenate,

respectively. These results are superior to the conventional HPLC-ICP-AES LODs for

selenite (7000 ng/mL) and selenate (4550 ng/mL) (McCarthy, et al., 1983). Laborda, et

al. (1993) have reported the limits of detectioll were 1.67, 1.27, 0.76 ng of Se for

trimethylselenonium, selenite and selenate, respectively, by using HPLC-fraction

collection-ET AAS. Tanzer and Heumann (1991) described analytical procedures for the

selective determination of diffèrent selenium species (selenite, selenate and

trimethylselenonium ion) in natural waters using isotope dilution mass spectrometry

(IDMS), which y.elds relatively precise and accurate results even at to low sub-part per

billion concentrations .

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Ion-exchange chromatographic procedures have been reported for the separation

of selenoamino acids trom other ami no acids (Martin and Gerlach, 1969, Benson and

P$\tterson, 1969; Walter, el al., 1969; Martin and Cummins, 1966, McConnel and

Wabnitz, 1964) This technique provides a resolving capability which is appreciably

greater than that offered by paper (Water and Chan, 1967; Barak. and Swanberg, 1967),

thin-layer (Millar, 1966; Spencer, el al., 1966), cJassical column chromatograph'! or gel

filtration. However; the procedures were time consuming (usuaHy more thar. one hour)

and lacked sufficient sensitivity to determine Se-analyte concentrations in most

environmental samples Fortunately, in recent years the development of methods directed

at the selective detection and effective separation of selenoamino acids has drawn

increasing attention. Kraus et al (1985) have described a method for the separation of

methylselenomethionine-selenonium, dimethylselenocysteine-selenonium, and

trimethylselenonium ion in urine by HPLC on a Macherey-Nagel Nuc1eosil 5-SA strong

cation exchanger colurnn with a gradient solvent system containing ammonium phosphate.

Selenite, sel~noethionine, selenomethionine, selenocystine, and trimethylselenonium ion

were studied by Blotcky and Hansen (1985). They reported several separation procedures

inc1uding ion exchange chromatography and automated liquid chromatography which can

be coupled with neutron activation for the determination of these compounds in urine.

Selenomethionine, selenocystine and trimethyJselenonium have been recently determined

on a silica gel sintered TLC plate, which provided a detection limit of 0.4 ng as selenium

(Hasunuma, el al., 1991).

Recently, a novel thermochemical hydride generating (THG) interface has been

described for the determination of arsenic (Blais, el al., 1990) and selenium (Blais, el al.,

1991) containing species in HPLC eluate. In the an quartz interface assembly, eluate was

pyrolyzed ir\ an oxygen supported flame, reacted with excess hydrogen to fonn the

corresponding hydrides, and atomized in a second cool diftùsion tlame maintained jUil

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below the optical beam of the spectrometer. Subsequently, surface response

methodologies were used to optimize the separationldetection of selenocholine and

trimethylselenonium by HPLC-THG-AAS (Huyghue3-Despointes, 1991). Detection limits

of 5 and 7 ng (as selenium) respectively, for these two compounds were achieved when

they were eluted from a cyanopropyl bonded phase column using a mixture of

tricthylammonium and trimethylsulfonium ions in a methanol-l % acetic acid as the mobile

phase (Huyghues-Despointes, et al., 1991). The THG interface provided a sensitive and

selective method for determination of selenium analytes. However, the principal limitation

of the THG device was the requirement for a predominantly methanotic mobile phase (>

60% CH30H) to support the combustion process. This limitation somewhat restricts the

separatory modes which can be used to resolve ionic analytes in the automated

chromatography system. In contrast to e'quivalent detector responses to As-analytes

[As(V), As(III) and As(-nI)], the THG-AAS response to selenate was only 20% of the

response to selenite or trimethylselenonium (Blais, 1990). Fortunately, tbis limitations

have been overcome by rnodifying the CUITent interface design. The low cost of this quartz

interface coupled with its relatively bigh sfnsitivity and selectivity make it an attractive

approach for routine analyses of selenium compounds in biological system.

1.5 Research Objectives

The objectives of these studies were to deveJop methods for the detection and

separation of selenium compounds by HPLC with on-fine detection by AAS. The

operation of a novel HPLC-AAS interface design was to he optimized for selenium

detection and then evaluated for the detectionlquantitation of selenium analytes in HPLC

column eluate. The optimized operation of this interface was then to be used as the basis

of an analytical technique to speciate the readily extractable fraction of the total selenium

burden in natural waters and plants .

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Cbapter 2. Determination of Selenoamino Acids by "PLe-THG-AAS

2.1 Synopsis

Selcnoamino acids have attracted much attention, because Se-compounds such as

selenomethionine, selenocystine and selenocysteine are considered to be the main selenium

sources in naturally occurring food and feedstuffs. These compounds have been also

hypothesized to be as metabolites in the biological pathways of selenium incorporation and

excretion in living systems. Selenomethionine is used as selenium supplements for man and

animais and selenocysteine is a part of the active site of the enzyme glutathione

peroxidease. The identification and determination of the se sele'1ium compounds in

environmental samples is, therefore, of great importance The aim of tbis study is to

develop a convenient and sensitive method for detennining traces of selenomethionine,

selenocystine and selenocysteinl~ by HPLC-THG-AAS. After tagging the free selenol

group of selenocysteine with phenylmercuric cation, a procedure was optimized for

determination of these three selenoarnino acids based on formation of the 2,4-

dinitrophenyl (DNP) derivative. Then, the product N-DNP-selenoamino acids were

separated on the HPLC column with on-line detection by AAS.

2.2 Materials and Methods

2.2.1 Reagents and Chemicals

AlI solvents were "distilled in glass" or "HPLC" grade (BDH, Inc., Montreal,

Que.). Acetic acid was certified ACS reagent grade. Triethylamine was purified "gold

label" grade (Aldrich Chemicals Co, Milwaukee, Wis). Water was double-distilled and

deionized. Aqueous anunonia, tetramethylammonium hydrmdde :md tetrabutylammonium

hydroxide were analytîcal grade (Aldrich Chemicals Co, Milwaukee, Wis). Ail other

chemicals were ACS reagent grade or better (Aldrich Chemicals Co., Milwaukee, Wis).

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Selenocystine and selenomethionine were purchased from Sigma Chemical Co. (St. Louis,

MO, U S.A.) and were used without further purification.

Tetramethylammonium nitrate and tetrabutylammonium nitrate were prepared by

the dropwise addition of 0 5 M nitric acid to 0 5 M tetramethylammonium hydroxide and

0.5 M tetrabutylammonium hydroxide, respectively

2.2.2 Synthesis ofOrganoselenium Compound

Following the general method of Carty, et al. (1983), phenylmercury

selenocysteine (Ph-Hg-selenocysteine) was prepared by the reaction of selenocysteine with

phenylmercury acetate. Selenocystine (50 mg, 0.15 nunol) was added to water (15 mL).

Upon addition of 1 equivalent of sodium hydroxide, the diselenide dissolved. Excess

sodium borohydride (50 mg) in watler was added slowly to the above solution under a

nitrogen atmosphere. During 20 min for stirring, the solution graduaUy became colorless.

Sufficient hydrochloric acid, 10% (v/v), was ther. added to destroy excess sodium

borohydride and to lower the pH to 4 An equivalent of phenylmercury acetate (100 mg,

0.30 rnrnol) in ethanol (25 mL) was added to the reaction mixture which was stirred for a

further 3 hours Filtration to remove metallic seleniumoy evaporation of the filtrate almost

to dryness and reconstitution of the residue in 20 mL ethanol containing a few drops of

water aWorded crystals which were recovered by filtration. The crystals were washed

sparingly with ethanol The combined ethanolic mother liquors were concentrated to

produce a second crop of crystals. The product migrated as a single spot on silica gel thin

layer chromatography (TLC) plates with nvo different solvent systems [Rf = 0.5 with

butanoVacetic acid/water (4.1 1, v/v/v), Rf = 03 with methanoVbenzene (7:3 v/v)] and

tested positive when visualized with two chromogenic spray reagents [02% ninhydin in

ethanol or 3% aqueous hydrogen peroxide followed by 1 % diphenylcarbazide in 95%

ethanol foHowed by exposure of the treated TLC plate to an ammonia atmosphere]. The

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purple eolour suggested the presence of mercury The presence of both selenium and

mercury was corroborated by HPLC-AAS using either a selenium or a mereury hollow

cathode lamp

2 2.3 Instruments

The instruments for this study consisted of an HPLC system [(Beckman Model

100 A pump and Model 401 program module), and an autosampler (LKB, model 2157)]

interfaeed with an atomic absorption speCirometer (Phillips, PU9100 set at 1964 nm)

which was equipped with a high energy selenium hollow cathode lamp (photron super

lamp system, Australia) and a deuterium background correction system. Optimization

experiments were performed in the flow injection mode (no column) with deuterium

background correction Since the use of background correction increased the noise of the

detector appreciably, the correction system was not used for chromatographie analyses.

Narrow-bore stainless-steel tubing (0007 cm. id) was used post-in je ct or. The 50 J..lm i.d

silica transfer line was eonnected to the HPLC tubing via a capillary reducing union (0 16

--0.08 cm, Chromatographie Specialties, Brockville Ont.) Chromatograms were

recorded with a recording integrator (Hewlett Packard model 3390A).

2.2.4 HPLC-Thermochemical Hydride Generation-ASS Interface

The HPLC-Thermochemical Hydride Generation-AAS (HPLC-THG-AAS)

interface has been described previously (Blais el al, 1990). A diagram of this

thermochemical hydride generator (THG) is presented in Figure 2-1. The ail quartz

assembly (LaSalle Scientific, Inc, Ont.) consisted of an optical tube (a, 9 mm i.d. x Il

mm o.d. x 12 cm) which was positioned in the AAS optimal beam, an analytical tlame tube

(b, 4 mm i.d. x 6 mm 0 d. x 8 cm) and a side arrn which met the analytical tlame tube at an

angle of 45°. This side arm contained a combustion chamber (c, 7 mm i d. x 9 mm 0 d x 4

cm) and was titted with gas inlets for oxygen and hydrogen (e, f; 2 mm i.d. x 3.2 mm o.d.

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a.

c. b .

h. g. ~

HPLC eJuate

Figure 1·1. HPLC-mG-AAS interface consisting of (a) optical tube (9 mm i.d. x Il mm

o.d. x 12 cm); (h) analyticaJ fiame tube (4 mm i.d. x 6 mm o.d. " 8 cm); (c) combustion chamber (7 mm i.d. x 9 mm o.d. x 4 cm); (d) thermospray tube (4

mm i.d. x 6 mm o.d. x 8 cm); (e), (t) oxygen and hydrogen inlets (2 mm i.d. x 3.2 mm o.d. x S cm, 2.5 cm apart); (g) deactivated capillary sillca transfer line (50 ~m i.d. x 20 cm); (h) quartz guide tube (2 mm i.d x 3.2 mm o.d. x 10 cm);

(j) analytical oxygen quartz tube inlet (2 mm i.d. x 3.2 mm o.d x 1 S cm) .

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x 5 cm, 2.5 cm apart). The thermospray assembly consisted of an outer tube (d, 4 mm i d

x 6 mm o.d. x 8 cm) and a deactivated capillary silica transfer line (g, 50 Ilm i d x 20 cm)

connected to the HPLC column outlet and centered within the thermospray tube by a

quartz guide tube (h, 2 mm i d x 3 2 mm 0 d. x 10 cm, with outlet bore constricted to 1

mm i.d) The thermaspray tube was heated with a coil of resistance wire (40 cm, 22-

guage Chromel 875 alloy, Hoskins Alloys, Toronto, Ont) which was insulated with

refractive wool (FiberfTax, The Carborundum Co., Niagara Falls, N Y) and surrounded by

a shaped firebrick casing held in place by a hose clamp The heating element was powered

with a current of 4 to 5 amps (2 amps on standby) supplied by an AC variable transformer

and monitored with a standard ammeter Two stainless steel modified Swagelok

assemblies (Forsyth and Marshall, 1985) were used to position the silica guide tube (h)

within the therrnospray tube (d) and the analytical oxygen quartz tube inlet (j, 2 mm i.d. x

3.2 mm o.d x 15 cm) within the analytical tube (b). The tip of the analytical oxygen inlet

(j) was positioned approximately 0 5 cm from the optical tube intersection to maintain the

analytical fiame slightly removed from the AAS beam

In operation, HPLC column eluate was thermosprayed into the pyrolysis chamber

and combusted in an oxygen-rich atmosphere Downstream, the combustion products

were mixed with hydrogen to convert them to their hydrides, the product hydrides were

subsequently atomized in the cool micro diffusion fiame. The ope;ating conditions of the

assembly were as follows' fiow rates of oxygen and hydrogen to the pyrolysis chamber,

650 and 1700 mL/min; tlow rate of oxygen to the analytical flame, 170 mUmin

2.2.5 Preparation of N-DNP-Selenoamino Acids

To 1 ml (20 llg/mL as Se) of selenomethionine, selenocystine or Ph-Hg-

selenocysteine, contained in 5 mL phosphate buffer (pH, 9, J, 0.2 M) was added 1 ml of

10% (w/v) 2.4-dinitrofiuorobenzene (DNFB) (freshly pl'epared in methanol). The reaction

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mixture was stirred under nitrogen for 2 hours room temperature in the dark. The reaction

mixture was extracted three times with 5 mL benzene to remove unreacted DNFB,

acidifed to pH 2 with 1 M hydrochloride acid, and further extracted three times with 5 ml

of diethyl ether The combined ether extracts were dried with anhydrous sodium sulfate,

filtered, transferred to a dry graduated tube then evaporated to dryness under a gentle

stream of dry nitrogen at 35°C. The residue was redissolved in 3 mL of methanol. The

final solution was storee! in the dark at 4°C to await until HPLC-THG-AAS analysis.

The yield of N-DNP-selenomethionine was assessed by comparing the HPLC

THG-AAS response (peak area) of N-DNP-selenomethionine with the background

corrected peak area of standard selenomethionine.

2.2.6 HPLC Conditions

N-DNP-selenoamino acids were separated on a Nucleosil5-N02 colurnn (0.46 cm

i.d. x 15 cm, 5 ~ particle size, CSC, Ltd, Montreal, Qc) with a methanolic mobile phase

containing varying amounts of acetic acid and triethylamine. Several mobile phase

compositions were tested. A Nucleosil CI8 (0.46 cm i.d. x 15 cm, 5 ~ particle size, CSC,

Ltd, Montreal, Qc) colurnn was also evaluated during tbis study.

%.3 Results and Discussion

2.3.1 Optirnization of the Preparation ofN-DNP-Selenoamino Acids

The use of DNFB used to derivatize amino acids has been first developed by

Sanger (Sanger, 1945; Proter and Sanger, 1948). Since his c1assic work on insu lin, DNFB

has been used routinely for decades to form relatively non-polar derivatives of amino acids

that are useful in analysis. The dinitrophenylation procedures commonly in use have two

distinct disadvantages: long reaction time and low yields. The Sanger procedure as

modified by Rao and Sober (1954) required stirring for 2-5 hours with sodium bicarbonate

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and excess DNFB in ethanol-water at room temperature. In an attempt to improve the

yields, Schroeder and LeGette (1953) developed a procedure which produced yields of

76-100%. However, this method involved Il extractions and three washings, thus

requiring a lengthy work-up period. In an effort to improve the yield and reaction time, a

systematic search (Vinson and Pepper, 1972) was made for a reaction solvent system that

was ditTerent from the ethanol-water medium.

The dinitrophenylation procedure has been applied to the identification and

determination of selenium compounds. Ganther and Kraus (1984) reported a method for

trapping and identitying hydrogen selenide (H2Se) and methylselenol (CH3SeH). These

analytes were reacted with DNFB to form stable dinitrophenyl selenoethers which were

detected by thin layer chromatography (TLC), high-perfomance Iiquid chromatography

(HPLC), and mass spectrometry (MS). Ganther, el al., (1984) and Kraus, et al., (1983)

identified selenocysteine in selenoprotein based on the formation of a 2,4-dinitrophenyl

(DNP) derivative. During the curent study, the 2,4-dinitrophenylation procedure was

optimized for selenoamino acids.

2.3.1.1 The pH of the Buffer Solution

In the traditionat 2,4-dinitrophenylation procedure, sodium carbonate or sodium

biocarbonate has been used as a component of the reaction system. Some attention has

been given to the influence of variation in the pH of the reaction medium (Bunnett and

Randall, 1958; Bunnett and Garst, 1965). Bunnett and Hermann (1970) investigated the

kinetics of the reactions of DNFB with glycine, proline and N-phenylglycine, and reported

that the pH dependence of the rates of DNFB reaction with amino acids is entireJy

accounted for by the effect of pH on the degree of ionization of the amino acids. There was

no evidence for base catalysis of the substitution reaction. It was of interest to determine

whether reactions of other amine acids with DNFB respond to catalysis by bases~ variation

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100

90

l 80 ,-.

~ '-'

"0 ())

-:;:. 70

60

50 L-____ -J _______ -L ______ ~ ______ ~ _____ ~

7 8 9 10 1 1 12

pH of Buffer

Filun 2-2. The influence of the buffer pH on the yield ofN-DNP-selenumethionine

in pH might enable the selective dinitrophenylation of certain aminoacyl moieties. Buffer

solutions composed of mixtures of sodium dihydrogen phosphate and disodium hydrogen

phosphate (pH, 7-12; J, 0.2 M) were used as media for the 2,4-dinitrophenyl substitution

reaction during tbis study. The effeet of the pH of the reaction medium on the yield ofN

DNP-selenomethionine is presented as Figure 2-2. The optimum pH value was at 9. The

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yielrl of N-DNP-selenomethionine (quantity and repeatability) was measured by three

replicate derivative assays at a each pH value The method used for the derivatization of

selenomethionine was Apl-'roach 1 as outlined in Table 2-1.

2.3.1.2 Solvents of the Extraction

There are two extraction steps during the 2,4-DNP derivatization procedure. The

tirst extraction was perfbrmed to remove unreacted DNFB reagent and the second

extraction was performed to recover the 2,4-dinitrophenyl derivative of amino acids ioto

an organic phase. The eifeet of the extracting solvent was investigated by comparing the

recoveries of 2,4-DNP selenomethionine with diethyl ether, toluene and benzene The

results are recorded in Table 2-1. ft can be seen that the recovery of 2,4-0NP

selenomethionine with the benzene/ether combination was greater than the recovery with

either etherlbenzene or ether/toluene or the benzeneJbenzene combinations. The

etherlbenzene, ether/toluene, or benzenelbenzene combinations were found to give similar

results.

Under optimum conditions for N-DNP-selenomethionine, the derivatization

procedure resulted in virtually quantitative recoveries ofN,N-di-DNP-selenocystine (Table

2-2). In summary this procedure for the deterrnination of nanomolar levels of

selenomethionine and selenocystine proved to be convenient, effective and quantitative.

2.3.2 The Effect(s) ofPotentiai Interferences on the Dinitrophenylation Procedure

The influence of other amino acids on the efficiency of recovery of 2,4-DNP

selenomethionine was investigated. The following amino acids, in admixture with

selenomethionine, were subjected to the derivatization procedure' a-alanine, cysteine,

cystine, lysine, glutarnic acid, histidine, methionine, phenylalanine, proline, and tyTosine.

The influence of these potential interferents on the yield 1 recovery of N-DNP-

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Table 2-1. The influence of extracting solvent on the yield ofN-DNP-selenomethionine

Approach 1 st Extractiona 2nd Extractionb

1 Ether Benzene

II Ether Toluene

III Benzene Benzene

IV Benzene Ether

a three successive 5 ml washes of the crude reaction mixture. b three successive 5 ml washes after pH adjustment to 2. c standard deviation based on three replicate trials .

Table 2-2. The yields ofN-DNP-selenoamino acids

Analyte yield (%)

Selenomethionine 99.8 ± 1.3*

Selenocystine 97.9 ± 0.44

* standard deviation based on three replicate trials .

28

Yield (%)

87.0 ± 1.5c

85.3 ± 2.7

86.7 ± 0.25

99.8 ± 1.3

•

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selenomethionine are presented in Table 2-3. The recovery of N-DNP-selenomethionine

(0.25 ~mols) remained relatively unaffected by a 20-fold ex cess of other N-DNP-amino

acids (0.5 J.1mols of each of the amino acids). However, the yield of N-DNP-

selenomethionine was decreased somewhat by a 100-fold excess of N-DNP-amino acids

(2.5 J..lmols of each above amino acids). No AAS response to selenium was observed upon

injection of the mixture of N-DNP-amino acids using the same HPLC operating

conditions. The method used for the derivatization of N-DNP-selenomethionine was

Approach IV (Table 2-1).

Table 2-3. The effect ofpotential interference on yield ofN-DNP-selenomethionine

Selenomethionine Mixture of 10 amino acids + Yield (%) (Ilmols) (J.1mols of each AA)

0.25 0 99.8 ± 1.3*

0.25 0.5 99.3 ± 1.1

0.25 2.5 86.4 ± 1.3

0 2.5 0

* standard deviation based on three replicate trials. + mix. of Met, Cys, Cys-Cys, Pro, Trp, Glu, His, Phe, Aja, Lys.

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2.3.3 Chromatographie Separation of N-DNP-Selenoamino Acids

The separation of2,4-DNP-amino acids has been reponed using a native eeUulose

eolumn (Fukuhara and Yuasa, 1987) and a C18 reverse phase column (Kozuku, et al.,

1982). Fariss and Reed (1987) and Reed, et al (1980) have deseribed a method for

determining sulfur-eontaining amino acids or their derivatives. This procedure is based

upora the initiai formation of S-earboxyrnethyl derivatives of free tlûols followed by the

conversion of free amino group to 2,4-dinitrophenyl derivatives. Chromatography of this

reaction mixture was perforrned on a 3-aminopropylsilane derivatived siliea column eluted

with a sodium or ammonium aeetate gradient in a water/methanoVacetic acid solvent at

pH 4.5, but the column is not commerciaIly available.

Di-DNP-monoselenide, Di-DN-P-diselenide and DNP-methylselenide have been

separated by TLC with heptaneJchloroformlpyridine (5:5:1 v/v/v) and bezenelpyridine (8:2

v/v) solvent systems, and by HPLC with a heptanelcbJorofonn (80:20 v/v) mobile phase

on Nuc1eosil NH2-bonded column (Ganther and Kraus, 1984). Se-DNP-selenocysteine

was separated from other water-soluble DNP-amino acids by using 10 J.lm C18 Bondapak

column with methanoVwater (30:70 v/v) eluate solvent system (Ganther, et al., 1984).

Since the detector response of the HPLC-THG-AAS system was slightly

dependent 011 the composition orthe mobile phase, it was deemed prudent to develop and

optimize an isocratic separation. A reversed-phase column appeared to be the approach

method of choice for separating N-DNP-amino acid derivatives, however, this approach

was incompatible with the THG interface because of the high proportion of water required

in the mobile phase. The separation of N-DNP-selenoamino acids was etfected on the

Nucleosil S-N02 column. Several methanolic mobile phase compositions were

investigated. N-DNP-selenomethionine and N,N-di-DNP-selenocystine were separated

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with a methanolic mobile phase containing 0.8 ~mL triethylamine and 0.05 ~mL

acetic acid, delived at 0.65 ml/min. The chromatogram is presented as Figure 2-3.

Figure 2-3. HPLC-THG-AAS chromatogram of N-ONP-selenomethionine (a> and N,N

di-DNP-selenocystine (b) separated on a Nucleosil 5 N02 column with using

a methanolic mobile phase containing acetic acid and triethylamine (O.OS and

0.8 f.1IJmL, respectively) delivered at 0.65 mUmin.

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In contrast to the formation of N-DNP-selenomethionine and N,N-di-DNP-

selenocystine, selenocysteine reacts with DNFB to form Se-DNP-selenocysteine, which is

freely soluble in water. The Se-DNP derivative of selenocysteine is stable when stored

under acidic conditions and is hydrolyzed relatively slowly under strictly anoxic

conditions. Under mildly basic conditions, however, decomposition by the intramolecular

Se -+ N rearrangement of the DNP group takes place (Smiles rearrangement). Kraus, et

B CH)I DNP-SC-CH2-CH-COO- --... rSe-CH2-C&-CO(Y)- •

1 N2 l "J2

NH2 NH1-DNP

al. (1983) and Ganther, et al. (1984) obtained chromatographie evidence that this

rearrangement occurred with Se-DNP-selenocysteine in the presence of methyl iodide and

sodium barbital under anaerobic conditions te form Se-methyl-N-DNP-selenocysteine,

which was used for the identification of selenocysteine ns a constituent of gluthathione

peroxidase by mass speetrometry. However, it is difficult to control tbis reaction, because

of contaminating substances which cao Interfere with the intrarnolecular rearrangement via

competing ineramolecular reactions. Consequently, it is necessary to purify the Se-DNP

selenocysteine to a high degree before performing the Smiles rearrangement. Moreover, il

is necessary to identifY a method for blocking the Iiberated selenol to fonn a stable product

which i8 amenable to chromatographie determination. Previous investigators have used

conventional aIiphatic agents such as iodoacetic acid to derivatize the selenium in various

selenoproteins. Our starting point was the idea that if selenium could be coupJed to an

aromatie reagent, the resulting derivative would be more stable and have desirable

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solubility properties. A possible approach for blocking the labile selenol functionaJ group

(-SeH) involved the reaction of selenocysteine with phenylmercury acetate to fonn

phenylmercury selenocysteine. This approach is also interesting because the mercury

present in aquatic organisms might be present (at least in part) as methylmercury

selenocysteine or phenylmercury selenocysteine. Carty, el al. (1983) and Arnold, et al.

(1982) suggested that Hg-Se binding is stronger than Hg-S binding. There is no

information about the synthesis of phenylmercury selenocysteine (ph-Hg-selenocysteine)

• c

5

b

Figure 2-4. HPLC-THG-AAS chromatograms ofN-DNP-selenomethionine (a), N,N-di

DNP-selenocystine (b) and N-DNP-Ph-Hg-selenocysteine (c) obtained on

Nucleosil 5-N02 column with a methanolic mobile phase containing 6 J.lUmL

tetrabutyJammonium nitrate and 0.9 J.1UmL oftriethylamine. delivered at 0.65

mUmin.

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in the Iiterature. However, the method for synthesis of methylmercury selenocysteinate

monohydrate complex is avaiJable (C~rty, el al, 1983). A phenylmercuric complex of

selenocysteine was formed to block the selenol functional group, then trus complex was

reacted with DNFB to fonn N-DNP-Ph-Hg-selenocysteine, which can be extracted into an

organic solvent simultaneously with N-DNP-selenomethionine and N,N-di-DNP

selenocystine. Figure 2-4 presents the chromatograms of N-DNP-selenometruonine, N,N

di-DNP-selenocystine and N-DNP-Ph-Hg-selenocysteine, developed with methanolic

mobile pha.se containing triethylamine and tetrabutylammonium nitrate. It can be seen that

c .. 1 ... N~ ~

b ... ... ""

Figure 1-5. HPLC-THG-AAS chromatogram of N-DNP-selenomethionine (a), N,N-di

DNP-selenocystine (b) and N-DNP-Ph-Hg-selellocysteine (c) separated 011 a

Nucleosil 5-N02 column with a methanolic mobile phase containing acetic

acid and triethylamine (0.05 and 0.8 J.1llmL respectively) delivered al 0.65

mUmin.

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the three eompounds ean be separated to baseline with this chromatographie condition.

But tetrabutylammonium nitrate as ion-pair rcagent was not a suitable additive for the

THG interface system Apparently, this reagent causee! an accelerated degradation of the

silica surface (\f the interface Moreover, N-DNP-seJenomethionine, N,N-di-DNP

selenocystine and N-DNP-Ph-Hg-selenocysteine were separated with methanolic mobile

phase containing acetic aci~ and triethylamine, as shown as Figure 2-5

2.4 Conclusion

A convenient procedure was developed for simultaneously determining

selenomethionine, selenocystine and selenocysteine by HPLC-THG-AAS, based on the

derivatization of these analytes with Sanger's reagent After blocking the free selenol

group (for selenocysteine) with phenylmercuric cation, the selenoamino acids were reacted

with 2,4-dinitrofluorobenzene in phosphate buffer (pH 9) The product N-DNP

derivatives were recovered into diethyl ether, concentrated and resolubilized in rr.ethanol

N-DNP-selenomethiorùne, N,N-di-DNP-selenocystine and N-DNP-Ph-Hg-selenocysteine

were separated on a Nucleosil 5-N02 column with methanolic mobile phase containing

acetic acid and triethylamine. It is believed that trus procedure has the potential to become

a routine method for the determination of selenoamino acids in samples .

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Cbapter 3. Approacbes to tbe Speciation of Se(-II), Se(IV) and

Se(VI) Compounds in Natural Water and Wheat by

HrLC-AAS

3.1 Syn,'p.i.

~elenium occurs in water, soil and plant systems in four oxidation states: -II, 0,

+IV and +VI. ft is important to know the selenium species distribution because each has a

different geochemical and biochemical behavior. A number of methods for the

determination of the total selenium content are available, however, methods that are

capable of distinguishing the differcnt chemical forms (different Se compounds) are searce.

None of methods for speciating hoth organic and inorganic selenium provides a complete

yet sensitive method for determining these compounds in environmental samples. One of

the diffieulties of exploring the various speeies of selenium in the environrnent is the very

low total concentration of this element in most matrices A fundamental requirement of

an} selenium speciation method is that it be very sensitive.

The use of atomic spectrometry as a detection system entails the coupling of this

virtually element-specifie detector with a flowing stream of liquid column eluate. The

interest of this combination of techniques foeuses on the separation and quantification of

different chemicaJ species of an element in a sample. The prototype HPLC-THG-AAS

interface has been used for the detectiol1 of arsonium, selenonium analytes and

selenoamino acids in HPLC column eluate. The principal limitation of this prototype

design remains the requirement for an organic rich (> 6(010) mobile phase which aets as a

fuel to support the combustion proeess. A new prototype HPLC-AAS interface was under

aetive development in our laboratory when these studies were initiated. Preliminary studies

had demonstrated that it eould he operated successfully for the detection of traces of

arsenic compounds in either aqueous or methanolic HPLC mobile phases It was

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postulated that the new interlàce design could be e.xploited advantageously for the

determination of selenium analytes in environmental samples The purpose of this work

was to modify and optimize this new HPLC-AAS interface for detection of selenium

analytes The optimized operation of the interface-detection system was to be applied to

the determination of Se(-II), Se(IV) and Se(VI) compounds Different isolation and pre

concentration procedures and different chromatographie approaches were also to be

explored in an effort to provide unambiguous estimates of the levels of these analytes in

selected natural water and wheat samples.

3.2 Materials and Methods

3.2.1 Reagents and Chemicals

Ali solvents were "distilled in glass" or "HPLC" grade (BOu, Inc. Montreal,

Que.). Acetic acid and formic acid were certified ACS reagent grade. Water was distilled

and deionized (Milli-Q system, Millipore Corp, Montreal, Qc). Ammonium hydroxide,

ammonium carbonate and ammonium acetate were analytical grade (Aldrich Chernieals

Co., Milwaukee, Wis). Ali other chemicals were reagent grade or better (Aldrich

Chemicals Co., Milwaukee, Wis) Selenocystine, selenomethionine and selenoethionine

were purchased from Sigma Chemical Co. (St Louis, MO, U.S A) Sodium selenite and

sodium selenate were obtained from Aldrich (Aldrich Chemical Co. Milwaukee, Wis).

Selenium pellets, 99.995%, was purchased from Ftuka (Ftuka Chemicals Co., Milwaukee,

Wis.).

3.22 Syntheses ofOrganoselenium Compounds

Some of organoselenium compounds of interest were not available commercially,

which necessitated their synthesis for this study. However most of the procedures required

for the preparation of these compounds were avaitable in the literature .

Meth.neseleninic .cid (CBl Se02HJ

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Methaneseleninic acid was prepared by the reaction of dimethylselenide with

hydrogen peroxide (Bird and Challenger, 1942) A mixture of sodium

formaldehydesulphoxylate (2 5 g), sodium hydroxide (1.6 g), and powdered selenium (3.2

g) in 25 ml of water \Vas stirred for 30 min (until the selenium had dissolved completely)

then .llmended with the dropwise addition of 2. J mL dimethyl sulfate. The reaction mixture

was refluxed at 40-50°C for 2 hours. A red oil (dimethyl selenide) separated on the

addition of water (10 mL) to the crude product mixture. After removing the aqueous

phase, 6.5 mL of 30% hydrogen peroxide was added to the residual red oil and the

mixture was refluxed for a further 30 min. The reaction solution was concentrated to a

smatl volume and the erude product was set aside to crystallize.

Methaneselenonic acid, potassium salt (CHlSeOlK]

Potassium methaneselenonate was obtained by the reaction of methaneseleninic

acid with potassium permanganate (Bird and Challenger, 1942). Methaneseleninic acid (1

g) and potassium hydroxide (0.15 g) were dissolved in 10 ml water, and slowly treated

with aqueous potassium permanganate (0 84 g in 10 ml H20) for 10 min. The mixture

became wann and was finally exactly neutralized Wl~h potusium hydroxide and filtered.

The solution was evaporated to dryness (an essential precaution) and the residue, when

crystallised from ethanol, atTored white crystals.

Triphenylphosphine selenide [(C.JI!5)"PSe]

According the method of the formation of triphenylphosphine sulfide (Hannestad,

et al., 1989), triphenylphosphine selenide (TPPSe) was prepared by the reaction of

triphenylphosphine with elemental selenium. A mixture oftriphenylphosphine (1.66 g) and

powered selenium (0.5 g) in 20 mL of diethyl ether was stirred for overnight. The white

crystals of the selenide separated on the surface of the flask. Theo, toluene (30 mL) was

added to the reaction mixture to dissolve the selenide. Filtration removed metallic

selenium and the filtrate was evaporated almost to dryness. The residue, dissolved in 30

ml ethanol and 10 ml toluene, was refrigerated to form white crystals. The selenide when

38

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recrystallised from ethanol airorded a mass of perfectly white long needle crystals. The

melting point of the crystals is 185-186°C [184-186°C for TPPSe in Iiterature

(Kosolapoff, 1950)].

Reactions of selenomethionine and selenocystine with hydrogen perodde

Aqueous selenomethionine or selenocystine (5 mL, 20 J.lglmL as Se) was mixed

with 0.1 ml 30% hydrogen peroxide and reacted overnight. The reaction solutions were

evaporated aImost to dryness and the residues were dissolved in 5 ml water. Aliquots

were injected into the HPLC-AAS.

3.2.3 HPLC-ASS Interface

The instruments for tbis part of the studies have been described in section 2.2 3 of

this thesis. A description of the HPLC-AAS interface has been reported previously

(Momplaisir, et al., 1994). A diagram ofthis interface is presented in Figure 3-1. The main

body (all quartz, LaSalle Scientific, Inc, Ont.) consisted of an opticaJ tube (a, 7 mm i.d. x

9 mm o.d. x 12 cm) which was positioned in the AAS optimal ~ a pyrolysis chamber

(h, 9 mm i.d. x Il mm o.d. x 4 cm), a thermospray tube (c, 4 mm i.d. x 6 mm o.d. x 6 cm),

and two side arms (d; e, 4 mm i d. x 6 mm o.d. x 7 cm), which met the thermospray tube

at an angle of 45°, and served as inlets for oxygen and hydrogen, respectively. The

thermospray asspmbly consisted of a deactivated capillary silica transfer tine (f, 50 J1I1l i.d.

x 20 cm) connected to the HPLC column outlet and centered within the thermospray tube

(c) by a quartz ~ide tube (g, 2 mm i.d x 3.2 mm o.d. x 8 cm, with outlet bore constricted

to 1 mm i.d.). Both the pyrolysis chamber and the thermospray tube were surrounded by

heating coils of resistance wire (22-guage Chromel 875 a1loy, Hoskins Alloys, Toronto,

Ont.) which were insulated with refractive wool (Fiberfrax, The Carborundum Co.,

Niagara Falls, N. Y) and surrounded by shaped tirebrick casing helds in place with a hose

clamp. The heating elements were separately energized with the AC variable transformers,

to produce a current of 5 and 6 amps respectively as monitored with a standard ammeter.

39

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•

8.

d.

HPLC ~ eluate

Figure 3-1. HPLC-AAS interface consisting of (a) optical tube (7 mm i.d. x 9 mm o.d. x

12 cm); (b) pyrolysis chamber (9 mm i.d. x Il mm o.d. x 4 cm); (c)

thennospray tube (4 mm i.d. x 6 mm o.d. x 6 cm); (d), (e) quartz guide tubes

for gas inlets (4 mm i.d. x 6 mm o.d. x 7 cm); (f) a deactivated capiUary sillca transfer line (SO J,lm i.d. x 20 cm); (g) quartz guide tube (2 mm i.d x 3.2 mm

o.d. x 8 cm); Ü) oxygen quartz tube inlet (2 mm i.d. x 3.2 mm o.d x 10 cm);

(h) hydrogen quartz tube inlet (2 mm i.d. x 3.2 mm o.d x 10 cm).

40

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•

Three standard Swagelok fittings were separately used to position the silica guide tube (g)

within the thermospray tube (c), the oxygen quartz inlet tube (j, 2 mm i.d x. 3.2 mm o.d x.

10 cm) within the tube (d), and the hydrogen quartz intet tube (h, 2 mm i d. x 32 mm 0 d

x. 10 cm) within the tube (e). The outtet of the silica guide tube (g) was positioned

approximately 0.2 cm behind the tip of the captllary transfer line (f). The unheated optical

tube was supported within an aluminum casing by firebrick disks and refractive wool at

both extremes only leaving most of the optical tube ~x.posed. Oxygen and hydrogen were

controlled with flowmeters (Matheson, Toronto, Ont) and transferred to the gas inlets of

the interface using Teflon tubing (2.48 mm i.d. x. 4 mm i.d., Cole-Panner Co., Chicago,

Il.) which had been heat shrunk on to the quartz tube gas inlets (j, h) to provide a gas-tight

seal.

A diffused flame within I-IPLC-AAS interface was smoothJy ignited as follows: (1)

current to both heating elements was increased to 5 A; (2) the hydrogen flow rate was

adjusted to 1.95 Llmin, then ignited with an open flame held sequentially to both exits of

the optical tube; (3) the oxygen flow rate was gradually increased to 60 mL/min or more

until auto ignition produced a visible Bame at the tip of the oxygen iolet (j); (4) the

hydrogen flow rate was first decreased until the Bames at each exit of the optical tube

were extinguished then renrmed to its original value (no visible flame should be present at

the ends of the optical tube); (5) the capillary transfer tine (f) was introduced into the silica

guide tube (g) and the tip was positioned just dOWDstream from the hot region, (6) the

HPLC eluate was rapidly increased to 0.5 mUrnin and (as soon as possible) the tip of the

capïllary transfer line was advanced towards the junction of the pyrolysis chamber (b) and

the thermospray tube (c); (7) the position of the capillary tip was subsequently adjusted to

produce a maximum thennospray effect which is accompanied by a characteristic "spray"

sound. Shut-down of the interface was performed smoothly by following this procedure in

the reverse order.

41

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•

3.2.4 Optimization Procedure

The optimization of the interface operating parameters was performed using a

response surface methodology incorporating a statisticaJ experimental design (Debaun's

cuboctahedron design), with the 110w rate of eluate (FE, 0.5--0.8 mUrnin), oxygen flow

rate to the pyrolysis chamber (02, 2>-85 ml/min) and hydrogen flow rate to the

pyrolysis chamber (H2, 1.~2.45 Llmin) as the three independent variables. These

ranges were identified by preliminary experiments, which had determined the optimal flow

rates corresponding to a maximum response (the center point). The detector response to

eqi-molar amounts of selenomethionine and sodium selenate (30 ng as Se, 25 f.J.1 injection)

were recorded in triplicate for each of 15 experimental points For these experiments, the

HPLC column was replaced with a wide bore stainless steel tubing (l mm i.d. x 20 cm)

and water was used as the sample carrier. No AAS response was observed upon injection

ofwater in these conditions. The AAS respons: to equivaJent molar amounts of ditTerent

selenium standards (selenomethionine vs seleno,;ystine vs selenoethionine vs selenate vs

selenite vs trimethylselenonium iodide, 20 ng as Se/injection) were recorded under optimal

operating conditions.

3.2.5 HPLC Conditions

Selenomethionine, selenocystine and selenoethionine were separated on a silica

based cyanopropyl column (5 ~m silica support, 0.46 mm i.d. x IS cm, LC-CN, Supelco,

Ine., Bellefonte, PA) with 0.5 mL/min of aqueous aeetic aeid (0.04% v/v) as the mobile

phase.

Selenate and selenite were separated on a PL-SAX strong anion exchange column

(8 ~m, 15 x 4.6 mm, Poiyrner Laboratories Inc., Amherst, MA. USA) with 0.6 mUmin of

aqueous ammonia (pH = 9) mobile phase containing ammonium carbonate (0.\% w/v) .

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Methaneseleninic aeid and methaneselenonie acid were separated on the PL-SAX column

with aqueous ammonium carbonate (0 02% w/v), flow rate' 0 6 ml/min

Mixtures of selenate, selenite, selenomethionine, selenocystine and selenoethionine

were separated on the cyailopropyl column with aqueous aeetic acid (0.015% v/v)

containing ammonium aeetate (0.1 % w/v), flow rate: 0 5 mL/min These chromatographie

conditions wcre a1so used for the extracts rrom sarnples.

Several other chromatographie approaches were investigated. Methods for ion

pairing, reversed phase and cation exchange were tested. The stationary phases included

Nucleosil Cl8 (0.46 cm i.d. x 15 cm, 5 J1 particle size, CSC, Ltd, Montreal, Qc) and

Nucleosil SA (0.46 cm i.d. x 15 cm, 5 j.1 particle size, CSC, Ltd, Montreal, Qc). Ion

pairing agents whieh were 'l.,sayed included tetramethylammonium hydroxide and

tetrabutylammonium hydroxide with acetic acid or formic acid.

3.2.6 Calibration

The calibration curves for the five selenium analytes were obtained by analysis of

sequential dilutions of a fresh standard solution containing these analytes (25 J1L

injection), under several HPLC conditions. The limits of detectior. (LODs) were detennine

from the calibration curves using a first-order error proragation model with base-Une

noise normally distributed (Foleyand Dorsey, 1984).

3.2.7 Recovery,'Stability Trials

Five selenium compounds were assayed for their stabilities under severa)

treatments. A standard mixture (2 mL, 2 J1g/mL as Se for each compound) of selenate,

selenite, selenomethionine, selenocystine and selenoethionine was added to 10 mL of

distilled and deionized water, to 10 mL of tap water and to 10 ml of 80% ethanol. The

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resulting solutions were heated to 80°C for 30 min, then evaporated to dryness. The

residue was dissolved in 2 mL ofwater, and analyzed by HPLC-AAS.

Tap water (20 ml) which had been spiked with 0 or 2 ml of standard solution

containing five selenium compounds (2 Jlg/mL as Se for each analyte) were separately

evaporated to dryness. The residue was triturated three times 5 ml hot (80°C) distilled

deionized water. The combined aqueous extracts were concentrated to 2 mL and 25 J.1L

aliquots analyzed directly by HPLC-AAS.

Plant sampi es (mixed feedstuffconsisting mainly ofwheat) were kindly supplied by

Prof. E. Chavez, Dept. of Animal Science, Macdonald Campus of McGill University. They

had been ground to pass a 50 mesh screen. Ground sample (0.5 g) wa~ blended three times

with 8 ml. of hot (80°C) 80% ethanol or hot water (80°C) for 5 min. The combined

supematant fractions were evaporated to dryness and the residues, resuspended in 10 ml.

water, was defatted by extraction with 10 ml diethyl ether. The aqueous phase was

concentrated to 2 ml, centrifuged and the supematant fraction was filtered through 0.45 J.1

m membrane filter paper. Aliquots, 25 J.1L or 50 J.1L, were injected into the HPLC-AAS.

Sample (0.5 g) in a 2S-mL Pyrex centrifuge tube was vortexed (1 min) with 1 ml.

ofwater (80°C) and then centrifuged at 2000 rpm. The supematant was recovered and the

insoluble materials were again blended WÎth hot water (1 mL). The procedure was

repeated a third time. The combined extracts was quantitatively transferred to a separatory

funnel, mixed with 1.5 ml Beetic acid and diluted with water to result in a final volume of

20 mL. The aqueous solution was extracted three times with Iiquefied phenol (1 x 10 ml

and 2 x 5 ml). The phenol extracts were combined, diluted with 10 ml of diethyl ether

and back extracted three times with 5 mL water. The aqueous extracts were combined,

washed three times with 5 mL of diethyl ether and evaporated to dryness under reduced

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pressure at 37 oC. The residue was resolubilised in 2 ml water, filtered through 0.45 ~m

membrane tilter paper and 25 or 50 J.1L of a1iquots of the filtrate were injected into the

HPLC-AAS.

3.3 Results and Discussion

3.3.1 Optimization of the HPLC-AAS Interface

An improved interface design for the on-fine AAS detection of selenium and

arsenic compounds in HPLC colurnn eluate has been described (Momplaisir, el al., 1994).

This interface design (Figure 3 -1) represents a configurational modification and a

simplification of the previous THG interface (Figure 2-1) The pyrolysis chamber in the

current prototype has been moved from a separate side arm to become an extension of the

lower sample introduction tube. Furthermore, two si de arms, which enter the base of the

pyro1ysis charnber at an angle of 45°, served as inlets for oxygen and hydrogen.

Additionally, the hydride generationlatomiz&tion processes are combined with the

pyrolysis process in the current design. By contrast, in the previous THG interface, the

atomÎzation process was etfected by passing the combustion products through a smal1

oxygen supported analytical flame maintained just upstream from the optic:a1 tube.

In operation, Iiquid HPLC column eluate is nebulized by thermospray effect into

the pyrolysis chamber and combusted in an hydrogenloxygen atmosphere. The combustion

products are thought to be converted to their hydrides in the hydrogen-rich atmosphere of

the pyrolysis chamber. The optimum flow rate of hydrogen was 30-fold greater than the

flow rate of oxygen which corresponded to a IS-fold stoichiometrlc excess. The product

hydrides are subsequently atomized and entrained into an unheated optical tube by the

expanding pyro1ysis product gases.

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The determination of the optimal operating parameters of the interface for

selenium detection was performed in a flow injection mode using water as the sample

carrier. Energy inputs to the thermospray and pyrolysis chamber coits were maintamed

constant. The interface operatÎng parameters were optimized using a response surface

methodology (Debaun's cuboctahedron design) using peak area as the dependent variable,

and with the tlow rate of liquid carrier (FE), the flow rates of oxygen (02) and hydrogen .. (H2) to the pyrolysis chamber as-the three independent variables. preliminary experiments

were perfonned to seaf'ch out a maximum response which served as the center point of the

experimental design The experiments were carried-out by recording the instrumental

response to selenomethionine afid selenate under different combinations of interface

operating parameters. The results were modeled by using the RSREG procedure (Ieast

squares multiple regression) of the SAS statistical software (SAS Institute, Cary, NI,

USA). The multiple regression analysis procedure calculated a "best fit" mathematical

expression which related analyte peak area to FE, 02 and H2 Table 3-1 and Table A-I

present the obselved and predicted peak areas for selenomethionine and selenate,

respectively (NOTE: Tables and Figures with numbers starting preceded by an A, will be

found in appendices). The SAS statistic,a1 results of the experiments are given in Table 3-2

and Table A-2. This combination resulted in two models, which were characterized bya

signification lack nf fit at the 95% level of confidence (p < 0.05). However, variations

between observed and predicted responses were generally lower than 1 ()O/., except one

point for selenomethionine (15.74%). The predicted values which were calœlated trom

the regression equation for each model are plotted against the observed values in Figure 3-

2 and Figure A-). According to the regression of observed values on predicted values, the

model fitted the experimental data reasonably weU (r = 0.977139, slope = 1.00002 for

selenomethionine; r -= 0.989444, slope = 1.00001 for selenate). Furthermore, plots of the

distribution of the residuals about the regression tine indicated that these residual errors

were randomly distributed.

46

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Table 3 .. 1. Observed and predicted Peak Area for selenomethionine as a function orthe flow rates of mobile phase, oxygen and hydrogen.

Point Factors FEa 02b

(rnIlmin) (mUmin)

1 0.5 29 2 0.5 49 3 0.5 78 4 0.5 49 5 0.7 29 6 0.7 29 7 0.7 78 8 0.7 78 9 0.7 49 10 0.7 49 11 0.7 49 i2 0.8 29 13 0.8 49 14 0.8 49 15 0.8 18

a flow rate of the mobile phase

b flow rate of oxygen

C flow rate ofhydrogen

Response

H2c Observed Predicted

(Urnin)

1.95 57828 61800.29 1.25 67569 62948.26 1.95 75716 76693.64 2.45 89862 89523.23 1.25 60138 63369.55 2.45 77571 71207.82 1.25 28146 32575.58 2.45 82331 81023.56 1.95 94056 94797.91 1.95 94667 94797.91 1.95 95678 94797.91 1.95 75983 75132.76 1.25 67750 64103.51 2.45 80040 88036.53 1.95 61138 57028.63

d deviation of observed trom predicted values (lobs. -pred.1) / obs. x 100

41

Deviation

(%"1

6.87 6.84 1.29 0.38 5.37 8.20 15.74 1.59 0.79 0.14 0.92 1.12 4.50 9.99 6.12

•

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Table 3-2. The statistial results of analysis of variance and regression estimates for selenomethionine as a function of the flow rates of mobile phase, oxygen and hydrogen.

Response Mean Root MSE R-Square Coef. of Variation

Regression Of Linear 3 Quadratic 3 Crossproduct 3 Total Resress. 9

73898 6366.188124

0.9548 8.6148

TYPEI SS R-Square 1735642625 0.32'/6 1802996970 0.4026 737115350 0.1646

4275754944 0.9548

F-Ratio 14.275 14.829 6.063

11.722

_R_e_si_du_al _____ D_f __ Sum of Squares Lack of fit 3 201299647 Pure error 2 1342109 Total error 5 202641756

Mean Square F-Ratio 67099882 100.0

671054 40528351

Parameters Of Estimate Std. Error T-Ratio Intercept 1 -296836 97177 -3.055 FEa 1 572071 231326 2.473

°2b 1 3799.398 947.230 4.011

Ht: 1 94213 43198 2.181

FE*FE 1 -342286 169865 -2.015 °2*FE 1 -2244.726 836.777 -2.683

°2·°2 1 -34.765 5.758 -6.037

H2*FE 1 -9005.431 34219 -0.263

H2*02 1 690.641 213.450 3.236

H2*H2 1 -27407 9530.194 -2.876

a flow rate of the mobile phase (0.5--0.8 mL/min)

b flow rate of oxygen (29-78 mUmin)

C flow rate ofhydrogen (1.25-2.45 Llmin)

48

Prob. 0.0069 0.0064 0.0404 0.0072

Prob. 0.0099

Peob. 0.0283 0.0563 0.0102

0.0810

0.1000 0.0437

0.0018

0.8029

0.0231

0.0348

•

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•

Regression· Lirar modcI: Y :a 1 + bX, for scleDOIIIdhionine Variable Intercept Siope

Parame. Estimate Standard Error T Value Prob. Level 0.811887 45768280534 00002 0.9999 1.000021 006038038 16562 0.0001

Analysis of Vananœ Source OF Sum of Squares Mean Square F Vruue Model 1 4.27S8E9 4 2758E9 274.301 Error 13 2 0264E8 1.S588E7 Total (corr.) 14 4.4'784E9 Correlation Coefficient = 0.977139, R-squared = 9S 48%

Regression of Obsenred on Predicted 12~--------------------·----·----------~--------~ .. ' ,-'

10 r- -S-i = ... 8 f----

,,~

È 6 r----1 .c o

4 r----

4 6

o o

~4_ - -

8 Predicted (1: 10000)

o

c __

1

10

Residuals for the Regression of Observed on Predict .. j

12

8.---------------·----------------------------~

6 of ___ _

o

o

• ----- -~----o o.

S' 4 --~ - ---- ----- • .. -.-8 0 .. 0 :

... 2 r---------4-------+------ -.-t:----- ---- -1---~ .. .: i Or-------~------~------~,---=·-,~·~----~

:1 • : :s! (2) rJ ~ (4) • - --_.---- -~- -- - -.-- -

(6) f----

(8) 2 4 6 8

Predided (1 10000)

Figure 3-2. Simple regression analysis of the observed Peak Area.s for selenomethionine with predicted values trom the model.

49

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The resulting regression equation was lhen further simplified by fixing one variable

FE (the flow rate of eluate) of the predicted equation, therefore responses (peak area)

only varied with the other two variables 02 (the flow rate of oxygen) and H2 (the flow

rate of hydrogen) (Table 3-3 and Table A-3) Effects and interactions were studied

visuaJly by using the predicted response surtàce plots (SYST AT, Evanston, IL, USA)

Three dimensional response surfaces describing the predicted effects on peak area of

selenium were obtained from the resulting regression equation. The selenium response

surface plots are presented in Figure 3·3 and Figure A-2. As indicated by these figures, a

minimum response was observed at low H2 and high 02 Increasing H2 resulted in higher

responses up to a maximum response Over the ranges of operating parameters studied,

the difference between low and high responses was generally less than 50% variation.

Thus, the net nebulizationlatomizationldetection process was only moderately affected by

appreciable variations in these interface operating parameters .

The predicted optimal operating parameters of the interface were as follows' f)ow

rate of eluate, 0.7 mUmin, oxygen, 60 mUmin; hydrogen, 1 95 Llmin. The detector

responses to different selenium conlpounds were not affected by the magnitude of the

current supplied to the heating element of the thermospray tube or the pyrolysis chamber

provided that sufficient heat was supplied (cuITent of 5 amps or more) to gener'lte a stable

the thennospray etrect and combustion process. The same quartz interface device has been

operated (virtually daily) for up to six months without appreciable loss in response.

Moreover, greater than 95% recovery of the initial detector response to selenium was

achieved by simply washing the inner surfaces of the interface with 60% hydrofluoric acid

Under interface operating conditions which provided a maximum response for

selenomethionine, the detector provided equivaIent responses to compounds containing

selenium in different formaI oxidation states Relative AAS responses observed for

50

•

•

•

Table 3-3. Simplified polynomial expression for the predicted peak area for

selenomethionine (30 ng as Se) as a function of the flow rates of oxygen and hydrogen to

the pyrolysis chamber The flow rate of mobile phase fixed at (a) 0 5 mVmin; (b) 0 7

mUmin, or (c) 0 8 roUmin.

(a) FEQ = 0.5 mL/min [Figure 3-3 (A)]

Peak Area = - 96372 + 267702639 x oi+ 89710.28438 x H2 c_ 34.764777

x 02 x 02 + 690.641327 x H2 x 02 - 27407 x H2 x H2

(b) FE = 0.7 mL/min [Figure 3-3 (B)] Peak Area = - 64106.44 + 2228.08118 x 02 + 87909 1981 x H2 - 34 764777

x 02 x 02 + 690 641327 x H2 x 02 - 27407 x H2 x H2

(c) FE = 0.8 mL/min [Figure 3-3 (C)] Peak Area = - 58242 24 + 2003.60857 x 02 + 87008.655 x H2 - 34.764777

x 02 x 02 + 690.641327 x H2 x 02.27407 x H2 x H2

a flow rate of the mobile phase b flow rate of oxygen (29 - 78 mUmin)

C flow rate ofhydrogen (1.25 - 2.45 Urnin)

51

•

•

•

B •

Filure 3-3. Prcdicted response surfaces for ptak area for selenomethionine (30 ns as Se) as a function of the flow rates of oxygen and hydrogen with the flow rate of mobile phase fixed at (A) O.S mUmin; (B) 0.7 mUmin; (C) 0.8 mUmin.

52

•

•

•

different representative oxidation states of selenium compounds are presented in Table 3-

4. By contrast, it had been observed previously with the THG interface, that selenate was

less efficiently detecte:l (18 %) when compared with selenomethionine (Blais, et al.,

1991). An appreciable improvement in the detection efficiency for Se (VI) was evidenced

by equivalent mol al' responses obtained for Se (-II) and Se (IV) with this new interface.

3.32 Chromatographie Separation of Selenium Compounds

Much of the effort in the area of selenium analytical chemistry has been directed

toward accu rate and precise determinations of selenium in inorganic, organic, and

biological matrices. Few methods have been developed which permit the simultaneous

detennination of different selenium compounds. A method for the simultaneous

detennination of selenite, selenate and sorne of the comman selenoamino acids was not

available. The aim of tbis research was to develop a method to simultaneously determine

of Se (-II), Se (IV) and Se (VI) using an automated ehromatography separation eoupled

with on-fine detection by AAS. This method might then Sfrve as a rapid screening

technique for extractible Se-residues in biologieal samples.

Selenomethionine, selenoeystine and selenoethionine were separated to baseline on

a Nueleosil S-SA column, with a aqueous acetie acid (0.1% v/v) mobile phase containing

0.05% (v/v) triethylamine or aqueous ammonium acetate (0.12% w/v) delivercd al 0.5

mL/min. These same analytes were also separated on a Nucleosil C 18 column with

aqueous aeetie acid (005% v/v) containing 0.2% (v/v) tetramethylammonium hydroxide

or aqueous: ammonium acetate (0.06% w/v) delivered at 0.6 mUmin. The chromatograms

of Figure 3-4 and Figure 3 .. 5 demonstrate the selectivities that can be achieved using

different HPLC stationary and mobile phases (the "spikes" in the ehromatograms result

directly from the recording integrator). It ean be secn that the differences in selenium

response resulted from different the mobile phase compositions, therefore, the mobile

S3

• Table 3-4. Relative AAS responses of selenium compounds

Compound Relative Response (%)

Selenomethionine loo.0±0.7 •

Selenocystine 103.1 ± 0.3

Selenoethionine 97.3 ± 0.5

Selenate 98.0±0.5

• Selenite 100.8 ±0.3

Trimethylselenonium iodide 99.5 ±0.4

• Standard deviation based on three repli~ate analyses

• 54

•

•

• b

phase affects the detector response somewhat in trus case. With these chromatograp~jc

conditions, however, selenite and selenate were not separated from each other. due

principally to a lack of retention on either of the se co!umns.

With the PL-SAX column (a strong anion exchange mùterial), mathaneselenillk

acid and methaneselenonic acid were separated with aqueous ammonium carbonate

(0.02% w/v). Moreover, selenite can be separated from these two compound! by

increasing the amount of ammonium carbonate (0 1 % w/v) in the mobile phase, but

selenate was totally retained on the column under these conditions. The resulting

chromatograms are shown as Figure 3-6 It is suggested that this problem might be solved

by using a solvent program. Due to the limitations of the isocratic pump, gradient systems

were not investigated during this study. Selenite and selenate were weil separated with

aqueous ammonia (pH =- 9) containing 0.1% (w/v) ammonium carbonate. Figure 3-7

presents the separation of selenite and selenate recorded under opttl'JlÏzed chromatographie

conditions.

A normal-phase HPLC approach (using a cyanopropyl bonded stationary phase)

proved to be a better alternative for separating selenomethionine, selenocystine and

selenoethionine with aqueous acetic acid (0.04% v/v) as a mobile phase (Figure 3-8).

Moreover, the five selenium compounds (selenate, selenite, selenocystine,

selenomethionine and selenoethionine, the arder of elutian) could be separated ta baseline

on this column using a mobile phase consisting 0.015% (v/v) aqueous acetic acid

containing 0.1% (w/v) ammonium acetate resulting low ng Iimits of detection of these

compound s, as indicated by the chromatograms of Figure 3-9.

•

•

•

A .

• • 1a ~~~_~I ___ ~f ____ ! ____ ~

(MIN) B .

~ 3 • • 1 •

1 1 1 1 .. (MIN)

Figure 3-4. HPLC-AAS chromatograms of seJenocystine [RT, 4.52 min (A); 4.86 (B)], selenomethionine[RT, 5.32 (A); 5.85 (B)] and selenoethionine [RT, 5.96 (A); 6.93 (8)] (0.125 J,Lg as Se for each compound) separated on the Nueleosil SSA eolumn with O.S mUmin of mobile phase consisting of (A) aqueous aeetic acid (0.1% v/v) eontaining 0.05% (v/v) triethylamine or (B) aqueous ammonium acetate (0.12% w/v).

S6 ft

•

•

•

A.

1 tl

• (Mlhl

B.

1 • • tl .,

(MIN)

Figure 3-5. HPLC-AAS chromatograms of selenocystine [RT, 3.00 (A); 3.0S (B)], selenomethionine [RT, 4.47 (A); 4.41 (B)] and selenoethionine [RT, 7.93 (A); 7.97 (D)] (0.3 J1g as Se for each compound) separated on the Nucleosil e18 colurnn with 0.6 mUmin of mobile phase consisting of (A) aqueous acetic acid (0.05% v/v) containing 0.2% (v/v) tetramethylammonium hydroxide or (B) aqueous ammonium acetate (0.06% w/v).

57

•

•

•

A.

B.

a ~~I--~,--~--~----.

(MIN)

• • Il

1 1

1 1

• .. (MIN)

Filure 3-6. HPLC-AAS chromatograms of (A) methaneseleninic acid [RT, 6.80] and methaneselenonic acid [RT, 7.86] separated with aqueous ammonium carbonate (0.02% w/v) and (B) methaneseleninic acid [RT, 4.54], methaneselenonic acid [RT, 5.12] and selenite [RT, 8.31] separated with aqueous ammonium carbonate (0.1% w/v). Mobile phase was delivered, at 0.6 mL/min, to the PL-SAX column.

58

•

•

•

A .

B •

3 1

3 , • 1

• 1

• 1

• 1

12 1

, . 1

.. (MIN)

.. (MIN)

Figure 3-7. Isocratic HPLe-AAS chromatograms of 10 (A) or 20 ns (B) as Se of selenite [RT, 4.74] and selenate [RT, 6.95) separated on the PL-SAX colurnn with an aqueous mobile phase containins 0.1% (w/v) ammonium carbonate (adjusted to pH 9 with aqueous ammonia). Mobile phase was delivered at to the column at 0.6 mUmin.

59

•

•

•

A.

" ... -.

B .

3 1

3 L • 1

1 1

• 1

• 1 12

1

12 1

(MIN)

.. (MIN)

Figure 3-8. HPLC-AAS chromatograms of 5 (A) or lOng (B) as Se of selenocystine [RT, 5.20], selenomethionine [RT, 6.68] and selenoethionine [RT, 8.45]. The analytes were eJuted from the cyanopropyl column with aqueous acetic acid (0.04% v/v) delivered at 0.5 m1lmin.

60

•

•

•

A.

B •

1

1 1 • 1

• 1

• ,

• ,

11 1

11 ,

• (MIN)

• (MIN)

Figurt 3-9. HPLC-AAS chromatograms of 10 (A) or 20 (B) ng as Se of selenate [RT, 2.67), selenite [RT, 3.07], selenocystine [RT, 3.94], selenomethionine [RT, 4.36] and selenoethionine [RT, 4.81]. The analytes were eluted fonn the cyanopropyl column with 0.015% (v/v) aqueous acetic &cid containing 0.1% (w/v) ammonium acetate delivered at 0.5 rnUmin.

61

•

•

•

3.3.3 Calibration and !.imit of Detection (LODs)

Chromatographie limits of detection (LODs) for the selenium analytes under

conditions of optimal chromatography were estimated by analyzing seriaI dilutions of a

stock solution of standards and fiuang the observed peak areas to a Iinear regression model

with background noise assumed to be normally distributed. The LODs were calculated

from their calibration curves by using a first-order error propagation model (Foley and

Dorsey, 1984):

where, S, i, Ss and si are the si ope, intereept, and their respective standard deviation of ,

the calibration curve obtained via linear regression~ and sB is the standard deviation of the

spectroscopie blank signal. The factor 3 in the numerator of equation (l} gives a practical

confidence level (between 90 and 99.7 %) which is proportional to the goodness of fit on

the probability distribution of the blank signal and the accuracy of sB. The standard

deviation of the baseline noise was given by:

SB == Np-p / r (2)

where Np_p is the peak-to-peak noise (integrated on a 30 min blank run) and r is a

parameter dependent on the type of noise. In all cases, the baseline noise was normally

distribution (Gaussian) and r was assumed to be 5.

The regression plots and analyses of variance for the five selenium analytes are

presented in Figure 3-10,3-11,3-12, 3-l3, 3-14, A-3, A-4, A-S, A-6, and A-7.

The linear calibration models were highly correlated (0.9971 < r < 0.9986) in the

concentration range studied (5 ng to 50 ng as Se). The calculated Iimits of detection are

reported in Table 3-5. From trus table it is evident that equivalent limits of detection (1-2

ng as Se) were obtained for an alytes contairùng selenium in ditrerent formai oxidation

states under several HPLC conditions. The Iimits of detection for these selenium analytes

62

------------""------------------------

•

•

•

Regression - Linear madel: Y = a + bX, for selenometruonme (5-40 Ilg Se, phase C) Variable Parame. Estimate Standard Error T Value Prob. Level Intercept Slope

3253.217 746.0863 4.360 00008 2331.948 303327 76879 0.0001

___ Analysis of VaJiance Source OF Sum of Squares Mean Square FValue Model 1 l.3377ElO 1 3377ElO 25910 377 Error 13 2.9424E7 2.2634E6 Total (COR.) 14 ~~_1;..-..3~4;.;..07.;.;;;E;.;;1.O~ ~ _______________ _ Correlation Coefficient = O.9;~)o2; R-squared = 99.78%

! ~

E.

Regression of Peak Area on Amount 140,000,--------------

120,000

100,000 --

80,000

60,000

40,000

20,000

a 0

1 - - --

, , 1 ~ - -

10

-- -- 1

~ - -- , ,

20 30 AmouDt (og as Se)

40

Residuals Regression of Peak Area on Amount

50

4,000 ~---~--------------------.

3,000 - ~ - -- , - -- -. , ,

2,000 1---- ---- - ---, :

i 1,000 ----~- -~----: 1 - ---- -_. -; -1 --- -- ----- 1 ,

::t • :; 0 • • • • ~ (1,000) -_ ...... - , -- -- -- ----. • , (2,000) ............ - · - ---- . . ,

(3,000) -- -- - - 1 _ ... --, , , ,

(4,000) ; ; : :

0 10 20 30 40 50 AmouDt (Dg as Se)

Figure 3-10. Regression analysis for the determination of the limit of detection for selenomethionine (peak Area vs Amount, Phase C)

63

•

•

•

Regression - LlOear model: Y "" a + bX, for selenocystinc (,..w f.&I Sc, phase C) Variable Parame Estimate Standard Error T Value Prob. Level Inlerccpt 2934 514 7625796 3848 0.0020 ~ 2157659 31.0033 69594 00001

Analysis of Variance Sour~ OF Sum of Squares Mean Square F Value Model 1 1 14S2EIO 1.1452E10 4843.384 Error 13 3.0739E7 2.3646E6 Total (corr.) 14 1.1 48JE 10 Correlallon Coefficient = 0998661. R-squared = 99 73%

Regression of Peak Area on Amount 140,000 r---------------------,

120,000

100,000

! 80,000 < ~ l 60,000

40,000 r---"..-." .

- -- ...,.-.: " ..•.. "

.' ----~.,..~,. -- -.. . " ." .. , .. , .r'

_____ of ___ :/ .. , .. , ... ~ ___ .. . , .. ,." - .... ~~ .. .. .;~ .. ".

" " "

20,000 --- -.>""" - -,.. ....

o .. "".' .. .&-I ---~--_.__'_ _____ ~ __ __._J

o lf) 20 30 40 SO Amouot (Dg as Se)

Residuals Regression of Peak Area on Amount 4.000 ,...---------------------...,

3,000

2,000 -- ------ - --- - ----• i 1.000 1 --------;- -------,----- ---;--_._-

• ! ~ O~-----------~--------------~ 1 Ir: (1,000) --- ---.----

(2.000) r--- -.- --

(3,000) -.--

(4,000) 0 10

---- -- -- ---- - ----, - -- ----

1

20 30 40 50 Amount (ag as Se)

Figure 3-11. Regression analysis for the determination of the Iimit of detection for selenacystine (peak Area vs Amount, Phase C)

64

•

•

•

Regression. Linear model: Y = a + bX. tor sclenoethiorune (5-40 l1g Se. phase C) Variable Parame Esumate Standard 'Enor T Value Prob Le\'~l Intercept Siope

2675.819 7625796 2612 00215 2200.145 416431 52833 00001

Analysls ofVanance Source OF Sum of S!].uares Mean Square F Value Model 1 1 1908E JO 1.l908E JO 2791.358 Error 13 5 545~,E7 4. 2660E6 Total (con.) 14 1 l%'3EIO Correlation Coefficient = 0 997679; R-squared == 99.54%

Regression of Peak Area on Amount 140,000 ~----_._--------------.......

"' .. -< ..li!(

120,000 r----

100,000 r----

BO,OOO f----

1. 60,000 f----

40,000 f----

10 1 1

20 30 40 Amount (Dg as Se)


50

4,000 .-----------------------.

3,000 f-- -

2.000 f----

i 1,000 f-----

Il :si! Or-----1: • ~ (1,000) f---- .----

(2,000) f----- --

(3,000) f- - --

• (4,000) 0

• f. ___ _ •

1

10

__ O •

• - -- - '1' - - - -. - -- - - \ ,

• • ---- - . ----- - - - - . ----

1

20

•

30 Amount (Dg as Se)

- . • 40 50

Figure l-ll. Regression analysis for the determination of the li mit of detection for selenoethionine (peak Area vs Amount, Phase C)

65

•

•

•

RegressIon - Linear model Y = a + bX, for selenate (5-40 118 Sc, phase C)

Vanable Parame. Esumate Standard Error T Value Prob. Level Inlercept Siope

68.5772 886 118~ 0.077 0.9395 2128.246 360258 59076 0.0001

Analysis of Vanance Source DF Sum of Squares Mean Square F Value Model 1 1 1142EIO ...;.1.....;.1....;..14-2":'El .... 0~---'3'-4-8=9.=91;...8---Error 13 4.1S06E7 3. 1 927E6 TotaI (corr.) 14 1. Il 84E 10 Correlation Coefficient = 0.998143; R-squared = 99 63%

Regression of Peak Area on ADlount 140,000 ,...--------------------------,

120,000

100,000

! 80,000

i 60,000 f- -l. 40,000 t- -

10

~ - - r

20 30 50 Amount (Dg as Se)

Residuals Regression of Peak Area on Amount 4,000,..---------------------,

3,000 1-- --

2,000 1-----

"Ï 1,000 f---

D

-- . ---, -------t-----

1 0 • ~ (1,000) --- • ---

(2,000) t- -

(3,000) t- - --

(4,000)0--

, '" .. -..

, , 1

10

- . , , , - -~ --- ----. ,

1 : 1

------ ~ --------.l- ---------i-- ------ ----, l ,

•

1

20

,

• - ____ o. , , 1

30 Amount (Dg as Sf)

•

, - --.

40 50

Figure 3-13. Regression analysis for the determination of the limit of detection for selenate (peak Area vs Amount, Phase C)

66

•

•

•

Regression - Linear model: Y = a + bX. for selenite (5-40 el Se. phase C) _V~an=·a;;;.;bl~e ___ ...;.Panu~e. Estimate Standard Error T Value Prob Level

Intercept 1645.831304 608.51095923 2.70~ 0.0180 Slope 1471.954065 24.73948854 59498 00001

Analysis of Variance Source DF Sum of Squares Mean Square F Value Model 1 5 3299E9 5 3299E9 3540.031 Enor 13 1.9573E7 1.50S6E6 Total (corr.) 14 5.349SE9 Correlation Coefficient = 0.998169; R·squared '"' 99.63%

Regression of Peak Area on Amount 140,000 ,-------------------------,

120,000 r----

100,000 r-- -

- \

~ 80,000 ... --.., . .,-~ .. l.

!Ill

il :t ." .•

60,000

40,000 , ---

20,000

- -1 •••••••••••••

- - - 1 - - -•••• - ••••• ~~ ........... -•••••• ;.~- r .' .... , ... ,.. ...... .~ ........ ~.; ...... _ ....... : ----, --

.. ~ ......• O~----~------~------~------~------~ o 10 20 30 40 50

Amount (Dg as Se)

Residuals Regression of Peak Area on Amount 4,000 ,--------:------------------,

3,000 r- -- , -- - . - . , . , . : : • 2,000 - - -- ,----, -------!. ------- ----~ ------ ,

1,000 --- . ------f ---r .. _-1 • • • ... ... 0 · ":' • ~ • ~ (1,000) • r- --- .---- ---

• (2,000)

, ... ---- -- -- , -- -- · ! . · 1 ,

(3,000)

(4,000) 0 10 20 30 40 50

AlDount (Dg as Se)

Figure 3-14. Regression analysis for the determination of the limit of detection for selenite (peak Area vs Amount, Phase C)

67

•

•

•

Table 3-5. Estimates of the chromatographie limits of detection· (ng of Se) for selenium analytes in HPLC eluate.

Mobile Phase Composition

Analytes Phase AQ PhaseBb Phase Cc

Selenomethionine 0.99 0.98

Selenocystine 1.16 1.08

Selenoethionine 1.07 1.42

Selenite 2.10 1.28

Selenate 1.92 1.27

a 0.04% (v/v) aqueous acetic acid was delivered to the cyanopropyl boned phase

column at O. S mVmin.

b 1.1 mM aqueous ammonia contairüng 0.1 % (w/v) ammomum carbonate was delivered on PL-SAX column at 0.6 ml/min.

e 0.01 % (v/v) aqueous acetic acid containing 0.05% (w/v) ammonium acetate was

delivered to the cyanopropyl boned phase column at 0.5 ml/min. • LOD = 3 [sB2 + Si2 + (i/S)2 sS:Z ]~ 1 S

68

.

•

•

•

were improved approximately ten-fold relative to selenonium analytes using same HPLC

column by the HPLC-THG-AAS system (Huyghues-Despointes, et al., 1991). The current

studies were undertaken to demonstrate the applicability of this interface to the

determination of selenium analytes in biologieal matrices

3.3.4 Sample Extraction and Analysis

Selenite and selenate have been determined in environmental water samples by

extraction with dithizone (Oyamada and Ishizaki, 1986) and ammonium pyrrolidine

dithiocarbamate (Atsuya, el al., 1991), in feeds and plants by extraction with

diethylammonium N,N-diethyldithioearbamate (Hoequellel and Candillier, 1991) and other

thiocarbamates (Chambers and MeClellan, 1976; Kumpulainen, el al., 1983; Kôlbl, el al.,

1993c). In this approaeh, Se (IV) was extracted complexometrically with dithizone

solution from acidie solutions (pH < 5), and subsequently Se (VI) was extracted with

dithizone solution after the reduetion to Se (IV) with hydroxylamine hydrochloride or

hydrochloride.

Several methods for extraction of free selenoamino acids have been described

including the use of trichloroacetic acid (TCA solution) (peterson and Butler, 1971), hot

ethanol (80%) (Bottino, et al., 1984), hot acidic ethanol (HCI:EtOH = 2:8) (Aono, et al.,

1990), and hot water (OIson, et al., 1970).

Given the feasibility of separating anionie selenium compounds and selenoamino

acids in the same chromatogram, it was of interest to develop/optimize an extraction

procedure for these analytes from aqueous media and plant extracts

Wiedmeyer and May (1993) investigated the st orage characteristics of selenate,

selenite, and selenomethionine in water. This study led to questions concerning the

69

•

•

•

stabilities of the mixture selenium analytes during extraction process The stabilities of

selenate, selenite, selenomethionine, selenocystine and selenoethionine during hot water or

hot ethanol treatments were investigated during this study A standard solution containing

these five selenium analytes was spiked in to water or ethanol The resulting solutions

were heated and th en evaporated to dryness. The residues were dissolved in water, and

analyzed by HPLC-AAS. The results are presented in Table 3-6 It can be see that selenite

remained relatively unaffected by different treatments. The recoveries of selenocystine,

selenometiëonine and selenoethionine were slightly lower with method B; presumably a

portion of ,the se selenoamino acids were partitioned into the ether phase. However, the

recovery of selenocystinc was decreased appreciably with method C and D. Good

reeoveries of ail five selenium compounds were observed with method A, whieh was then

further applied for sample extractions .

The tap water was spiked or unspiked with a mixture standard solution containing

five selenium eompounds, evaporated to dryness and the residues were washed three times

with 5 mL hot water. The water washes were combined, coneentrated to volume and

analyzed. The recoveries of the five selenium analytes were unaffected by other

components of the tap water sample, the evaporation of the water or the resolubilization

of the residues in hot water. The recovery was virtually quantitative for each analyte

(Figure 3-1SA). For the control tap water sample (no added selenium standards) there was

no discemible detector response (Figure 3-15B) above background at any point in the

chromatogram.

Several procedures were evaluated for the recovery of selenium analytes from the

feed samples. These procedures included the extraction of the sample with hot (80 OC)

water or 80% (v/v) ethanol However, the chromatographie behavior of the mixture of the

five selenium analytes in the presence of plant co-extracts was different from the

70

• Table 3-6. The Recoveries of selenium compounds trom water extract

Extraction Procedure

Compounds A B C 0

Selenate 95.11 ± 1.86* 97.92±0 73 70.77 ± 0.70 103.26± 080

Selenite 94.89± 2.30 95.08±2.62 98.52 ± 2.06 9869 ± 0.81

• Selenocystine 89.05 ± 2.04 80.41 ± 1.31 69.14 ± 0.71 65.22 ± 1.03

Selenomethionine 94.86 ± 2.15 80.75 ± 1.53 90.73 ± 1.64 9003 ± 0.58 -

Selenoethionine 93.25 ± 2.11 77.34 ± 3.37 91.46 ± 1.33 9085 ±O.27

Method A: treated with 0.0. H20 (80°C) for 30 min.

Method B: treated with 0.0. H20 (80°C) for 30 min, th.en extracted with 3xS ml Ether.

Method C: treated with Tap H20 (80°C) for 30 min.

Method D: treated with SOOIo EtOH (SOOe) for 30 min.

* Standard deviation based on three replicate trials .

• 71

•

•

•

A.

B.

3 1

•

•

•

• 1

tl

12

1

.. (MIN)

.. (MIN)

Figure 3-15. HPLC-AAS chromatograms resulting from: A, tap water (20 mL) which had been spiked with a standard mixture of 4 ~g each (as Se) of five Secompounds, evaporated to dryness, resolubillised in hot water. The recoveries of the five Se-anaJytes, selenate, selenite, selenocystine • selenomelhionine and selenoethionine (in order of elution, -50 ng as Selinjection) were virtuaJIy quantitative; B. al' identical recovery procedure applied to a control sample of the tap water.

72

•

•

•

chromatograprue behavior of the standards alone. Moreover, the detector response to the

standards was changed appreciably by the eo-extracts frona the sample. Because of these

interferences, it was considered necessary to devise procedures is to clean up the erude

extracts prior to chromatographie analysis

A rnethod involving the sequential partitioning of selenium or arsenic analytes from

aqueous solution into liquefied phenol and subsequent recovery back into water after

diluting the combined phenol with diethyl ether had been previously described for

selenonium compounds (Huyghues-Oespointes, 1991) and arsenic compounds

(Momplaisir et al., 1991, 1994). A mixture of selenate, selenite, selenocystine,

selenomethionine and selenoethionine standards (4 J.l.g each as Se) was partitioned from 20

mL water (pH, 3) into liquefied phenol (one 10 mL followed by two 5 mL extractions).

The combined phenolic extracts were diluted with 70 mL of diethyl ether, and selenium

eompounds were subsequently repartitioned baek into water (3 x 5 mL) The aqueous

extracts were concentrated virtually to dryness, and the residue was resolubilized in water

and analyzed by HPLC-AAS. Initial trials consisted of varying the pH of the initial

solution. The recoveries of selenite, selenocystine, selenomethionine and selenoethionine

were improved appreciable by using acetic acid (Figure 3-16A) rather than hydrochloric

acid (Figure 3-16B) or formic acid (Figure 3-16C) to adjust the pH of the initial aqueous

sample solution to 3. Dy contrast, adjustment of the initial pH to 10 with aqueous

ammonia resulted in partial recovery of selenomethionine and selenoethionine only (Figure

3-160). Selenate was recovered to the extent of 10% with the acetic acid treatment but

not recovered in any of the other trials Approximately 90% of the added selenate wu ,

detected in the initial aqueous fraction in each of the trials The recovery of the three

selenoamino acids and selenite from 20 ml of water using acidification with aeetie acid

(1.5 mL120 mL sample) resulted in an efficient recovery (>85%) of each an alyte (Figure 3-

17, Table 3-7). This technique was also attempted on the plant material. Figure 3-18A

73

•

•

•

A.

c.

1 1

1 ,

• ,

• ,

• ,

• 1

t • 1

,:1 ,

.. (MIN)

.. (MIN)

B.

D.

1 1

S 1

• ,

• !

• ,

• ,

, . ,

,. , .. (MIN)

• (MIN)

Figure 3-16. HPLC-AAS chromatograms ofphenol extracts oftap water which had been __ .. ,. spiked with a mixture of selenate, selenite, selenocystine. selenomethionine and selenoethionine (0.2 Jlg each as Se/ml, in order of elution, -25 ng as Selinjection) then acidified to pH 3 with (A), aeetic acid~ ('B), hydroehlorie aeid; or (C). fonnie acid or basified to pH 10 with (0). aqueous ammonia prior to extraction. The analytes were eluted with aqueous acetic aeid (0.04%, v/v) delivered, at 0.5 mUmin, to the cyanopropyl column.

74

•

•

•

3 1 • 1 • 1

12 1

(MIN)

Figure 3-17. HPLC-AAS chrornatogram oCthe phenol extrad fi'om tap water which had becn spiked with a mixture oC selenite, selenocystine, selenomethionine and selenoethionine (0.2 J.l.8 cach as Se/mL, in order of elution, -50 na as Se/injection). The analytes were eluted with aqueous acetic acid (0.04% v/v) delivered, at 0.5 mUrnin, to the cyanopropyl column.

75

•

•

•

A.

3 • • 11 ~ __ ~I __ ~i~ __ ~I~ __ .I __ ~

(MIN)

B.

1 3 • • 11 r---~I--~I~--~I----.I----••

(MIN)

Figure 3-18. HPLC-AAS ehromatogram of (A) the phenol extract from a ground dried wheat sample which had been spiked with selenate, selenite, selenocystine, selenomethionine and selenoethionine (4 J.1g/g each as Se, in order of elution, -25 ng as Se/injection) then acidified to pH 3, prior t.o extrac.1ion. Chromatogram B resulted from a 0.5 g sample of the same wheat material which was acidified then extracted in identieaI fashion. The analytes were eluted with aqueous aeetie acid (0.015% v/v) containing 0.1% (w/v) ammonium aeetate delivered, at 0.5 mIlmin, to the cyanopropyl column.

76

•

•

•

Table 3-7. The Recoveries of selenium compounds from phenol extract for water and wheat samples

Compounds Water sample Wheat sample

Selenate 35.3%

Selenite 89.5% 72.9%

Selenocystine 88.0% 57.5%

SeJenomethionine 96.7% 60.0%

Selenoethionine 95.3% 73.2%

77

•

•

•

presents the chromatogram which resulted from a group wheat sample which had been

spiked with 4 Jlglg (as Se) of each of the five standard selenium compounds then

subjected to the phenol partitioning procedure Although less than quantitative. the

reeoveries of the five selenium ânalytes (Table 3-7) were eonsidered sufficient to permit

the phenol extraction technique to form the basis of a preliminary screening procedure to

detect readily extractable selenium compoun<''\ in ground plant samples. No readily

extractable selenium eompounds were deteeted in this particular set of ground wheat

sample despite the faet that the level of total selenium was in excess of 2 J.lglg (Figure 3-

18B).

Other selenium compounds which might have been present include the oxidation

products of selenoamino acids such as selenocysteic acid, selenomethionine selenoxide cr

selenomethionine selenone. The reactions of selenomethionine and selenocystine with

hydrogen peroxide have been investigated during trus study On the basis of the

chromatographic dates (Figure 3-19; Figure 3-20), selenomethionine and selenocystine

were converted completely to their oxidation products. Selenite was round in the reaction

solutions of both eompounds, and selenate was observed in the reaetion solution of

selenocystine. The unknown compound (RT, 3.42 in Figure 3-19-8, RT, 12.11 in Figure

3-20-B) is, presumably, either selenomethionine selenoxide or selenomethionine selenone.

In addition, there is other unknown compound which appeared in the reaction solution of

selenocystine. This study was undertaken to explore the reaction of selenoamino acids

with hydrogen peroxide because of their r('!levance to the mechanisms of selenium

functions in biochemistry. Sorne of the oxidation products of selenoamino acids have been

determined in plant samples. Moreover, elemental selenium lTtight also be presented in

plants. It can be determined by HPLC-AAS, based on the formation oftriphenylphosphine

selenide wruch has been synthesized during this study.

78

•

•

•

A .

B.

c.

J , • ,

a 1 1 1

a 1 ! ,

• , ta ,

• tl

• (MIN)

1 _

(MIN)

• tl , , .. (MIN)

Figure 3-19. HPLC-AAS chromatograms of (A), the mixture of selenite [RT, 6.05] and selenate [RT, 10.19]; (8), the oxidation products of selenomethionine [RT, 3.42; 6.07]; or (C), the oxidation products ofselenocystine [RT, 3.52; 6.01; 10.18]. The analytes were eluted with an aqueous mobile phase containing 0.1% (wlv) ammonium carbonate (adjusted to pH 8 with aqueous ammonia) delivered, at 0.6 mUmin to the PL-SAX colurnn.

79

•

•

•

A.

B.

1

3 1 • 1 • 1

t • 1

i • 1 1

.. (MIN)

• tl 1 1 ..

lUIN\

C.

i r • 1 • 1

t. 1 ..

(MIN)

Figure 3-20. HPLC-AAS chromatograms of (A), the mixture of methaneselenonic acid [RT, 2.42], selenite [RT, 2.67], methaneseleninic acid [RT, 4 52], selenocystine [RT, 6.00], selenomethionine [RT, 7.49] and selenoethionine [RT, 9.28]; (B), the oxidation products of selenomethionine [RT, 2.39; 12.11]; or (C), the oxidation products of selenocystine [RT, 2.40; 2.62; 4.17]. The analytes were eluted with aqueous acetic acid (0.05% v/v) delivered, at 0.5 ml/min, to the cyanopropyl column.

80

•

•

•

On the other hand, a method for the extraction of methaneseleninic aeid and

methaneselenonic acid usmg ammonium pyrrolidinedithiocarbamate (APDTC),

diethyldithiocarbamate (DEDTC), and tetrabutylammonium dipthyldithiocarbamate

(TBADTC) was also tested during this study The aqueous mixture of methaneseleninic

aeid and methaneselenonic aeid was mixed with 1 % APDTC (in 4 mL water), 1 % DEDTC

(in 4 mL water), or 1% TBADTC (in 4 mL chloroform) a;1d extracted three times with 5

mL chloroform, respectively The aqueous phase was evaporated to dryness, the residue

dissolved in water and analyzed by HPLC-AAS. Similar results were obtained from the

APDTC and DEDTe extraction procedures, both selenium compounds were extracted

into an organic phase. In contrast, both selenium compounds were only partially extracted

into organic phase with TBADTC reagent, as indicated as Figure 3-21. This may have

been caused by the low solubility ofTBADTC in water

3.3.5 Suggested Future Experiments

The phenol extraction procedure was considered sufficient for the routine

extraction of selenium-anions and selenoamino aeids in water and plant samples. However,

the recovery of selenate from the phenol extraction is low. Thus, it is suggested that

further research on this compound should be directed to increasing the effieiency of the

extraction It Îs anticipated that selenium containing proteolytic fragments could be

released from plant samples by enzymatic hydrolysis (such as the use of pronase or

trypsin-pepsin) and that same chromatographie procedures cou Id also be apptied to the

hydrolysate

3.4 Conclusion

The new HPLC-AAS interface design has been demonstrated to provide equivalent

molar response for analyses containing Se (-II), Se (+IV) and Se (+VI). Several

approaches for the separation of selenium-anions and selenoamino acids have been

81

•

•

•

A.

B.

c.

" ~

:1 ,

S 1

:1 ,

• ,

• ,

• ,

• ,

• ,

• ,

tl ,

11 1

, . , •

(MIN)

.. (MIN)

• (MIN)

Figure 3-21. HPLC-AAS chromatograms of (A), the mixture ofmethaneseleninic acid [RT, 6.80] and methaneselenonic acid [RT, 7.86]; (8) the aqueous solution of methaneseleninic acid and methaneselenonic acid after the APDTC extraction; or (C), the aqueous solution of methaneseleninic acid and methaneselenonic acid after the TBADTC extraction The an alytes were eluted with aqueous ammonium carbonate (0 02% w/v) delivered, at 0.6 ml/min, to the PL-SAX column

82

•

•

•

evaluated on a variety of different stationary phases and different mobile pnases. A method

for the simultaneous determination of selenate, selenite, selenocystine, selenomethionine

and selenoethionine was developed by using the HPLC with 011 fme detection by AAS,

and a mobile phase containing of aqueous acetic acid/ammonium acetate delivered to the

cyanopropyl bonded phase column The equivak\~nt low ng limits of detection (1-2 ng as

Se) for different oxidation states of selenium analytes were obtained using a variety of

different HPLC conditions This represents approximately a ten-fold improvement relative

to selenonium analytes using the same HPLC column by HPLC-THG-AAS system. It is

possible ~o recover selenium-anions and selenoaminÛ' acids from water and plants with the

same extraction procedure, then to separate these ana\\)ltes to baseline using a cyanopropyl

bonded phase column. A phenol extraction procedure \for selenate, selenite, "Ielenocystine,

selen lmethionine and selenoethionine in water and plr·ots was investigated for the tirst

time This procedure serves to (i ) separate the analyte,~ from interfering co-extractives

while, at the same time, (ii.) concentrating the analytes. T\ie HPLC-AAS system provides

an inexpensive alternative to conventional techniques for the detena1!!1ation of selenium

analytes in samples .

83

•

•

•

Appendices

Table A-I. Observed and predicted Peak Area for selenate as a function of the flow rates of mobile phase, oxygen and hydrogen.

Point Factors FEa 02h

(mL/min) (mL/min)

1 0.5 29 2 0.5 49 3 0.5 78 4 0.5 49 5 0.1 29 6 0.1 29 7 0.7 78 8 0.7 78 9 0.1 49 10 0.7 49 11 0.7 49 12 0.8 29 13 0.8 49 14 0.8 49 15 0.8 18

a flow rate of the mobile phase h flow rate of oxygen C flow rate ofhydrogen

Response

H2c Observed Predicted

(L/min)

1.95 74676 76814.55 1.25 82793 83991.89 1 95 88426 86166.61 2.45 101122 100037.1 1.25 84220 19414.40 2.45 59888 58060.73 1.25 26992 28344.74 2.45 75045 80317.89 1.95 100710 101062.2 1.95 100976 1010622 1.95 101506 101062.2 1.95 69121 73608.07 1.25 74265 76516.50 2.45 83730 81357.39 1.95 60862 5648851

d deviation of observed from predicted values (lobs.-pred.1) / obs. x 100

84

Deviation (%y1

2.86 1.44 2.56 1.01 5.71 3.05 5.01 7.03 0.35 0.09 0.44 6.49 3.03 2.83 7.19

•

•

•

Table A-2. The statistial results ofanalysis of variance and regression estimates for selenate as a function of the flow rates of mobile phase, oxygen and hydrogen.

Response Mean Root MSE R-Squarc Coef of Variation

Regression Of Linear 3 Quadratic 3 Crossproduct 3 Total Regress. 9

78955 '871277916

0.9790 6.1697

TYPE 1 SS R-Sguare 1207060304 0.2134 2707093717 04786 1623640229 0.2870 5537794249 0.9790

F-Ratio Prob. }{,.956 0.0047 38027 0.0007 22.808 0.0024 25.930 0.0011

--------------------~~--~~--~----~~~----~-------Residual Df Sum of Squares Mean Square F-Ratio Prob. Lack offit 3 118318319 39439440 2402 0.0041 Pure error 2 Total error 5

Parameter Df Intercept 1 FEa 1

°2b 1

H2c 1

FE*FE 1

°2*FE 1

°i"02 1

H2*FE 1

H2*02 1

H2*H2 1

328424 118646743

164212 23729349

Estimate Std. Error -198740 74358 405534 177006

3290.92931 742.801260

98755 33054

-233315 129977 -1800.7902 640.285075

-43.284414 4.406193

-31123 26183

1247.05459 163.327289

-35386 7292.31116

a flow rate of the mobile phase (0.5-0.8 mUmin)

b flow rate of oxygen (29-78 mUmin)

C flow rate ofhydrogen (1.2>-2.45 Umin)

85

T-Ratio Prob. -2.673 0.0442 2.291 0.0706 4.540 0.Ov02

2.988 0.0305

-1.795 0.1126 -2.812 0.0347

-9.824 0.0002

-1.189 0.2880

7.635 0.0006

-4.8,2 0.0047

•

•

•

Ta:'Je A-3. Simplified polynomial expression for the predicted peak area for selenate as a

function of the flow rates of oxygen and hydrogen with the flow rate of mobile phase

fixed at (a) 0.5 rnVrnin, (b) 0.7 mL/min; or (c) 0.8 mUmin.

(a) FEQ = 0.5 mL/min [Figure A-2 (A)]

Peak Area = -54301.75 + 2390.5342 x 02b+ 83193.5 x H2 c_ 43.284414

x 02 x 02 + 1247.054585 x H2 x 02 .. 35386 x H2 x H2

(b) FE = 0.7 mL/min [Figure A-2 (8)] Peak Area = -2919055 + 2030.376158 x 02 + 76968 9 x H2 - 43.284414

x 02 x 02 + 1247.054585 x H2 x 02 - 35386 x H2 x H2

(c) FF ::: 0.8 mL/min [Figure A-2 (C)] Peak Area = - 23634.4 + 1850.297137 x 02 + 73856 6 x H2 - 43.284414

x 02 x 02 + 1247.054585 x H2 x 02 - 35386 x H2 x H2

a flow rate of the mobile phase b flow rate of oxygen (29 - 78 mUmin)

C flow rate ofhydrogen (1.25 - 2.45 Umin)

86

•

•

•

______ ~--R--eg"":r~es~s-lo-n _-L_i~near, mode!. y:. a + bX, for selenate Vanable Parame ESÙmate Standard Error T Value Prob Level (ntcreeril 1.035324 32988159657 00003 09998

..,.:S::,:I.=Jope::.=.-____ -=-I-=.OOOO.::..;;...;:..::..lO'--__ 0.04,.:..,05:;.;9,.;;;,6.;;.;;82:.-_---.:2:;.;4....;;6.::..33:.-___ --'-0 _00,,-0~1 ___ _

Analysis of Vanance Source OF Sum of Squares Mean Square Model 1 55378E9 55378E9 Error 13 1 1865E8 9.1267E6 Total (corr.) 14 56564ElO CorrelatJon Cccfficient::: 0989444; R-squared = 97.90%

Regression of Observed on Predicted

i 10 l-

= .... 8 ~

4

1

6

, , -1 ~ __

1

8 Predided (x 10000)

,

1

10

F Value 606.770

Residuals for the Regression of Observed on Predicted

12

8r---------------------------------------------------·~

6 -'

g 4 , • , , 1 o 1 1 1. .... 2 - ---. --- !" ---. ------.-~----- --+---. __ . - -+ -------------~ !. i Or-------·------------------~-------•• ·------~ Il • • :; (2) 1---- .--- .------ -

, , ac (4) r'" Iw __ .... ---- ~--- --------

(6)

(8) 2

i

4

• , ,

•

6 8 Predided (x 10000)

1

10 12

Figure A-t. Simple regression analysis of the observed Peak Areas for selenate with predicted values from the model.

87

•

•

•

B •

c .

Figure A-2. Predicted response surfaces for peak area for selenate (30 ng as Sc) as a function of the tlow rates of oxygen and hydrogen with the tlow ratc of mobile phase fi."(ed at (A) 0.5 mUrnin; (B) 0.7 mUmin; (C) 0.8 mUmin.

88

•

•

•

RegressIon - Lmear model' Y = a + bX, for selenomethlOrune (5-30 Jlg Se, phase A)

Variable Intercept Slope

Parame Eshmate Standard Error T Value Prob Level S227627 11517356 4539 00011 1549315 610204 58166 00001

Analvsls of Vanance Source OF Sum of Squares Mean Square T Value Model 1 3916EIO 1.3936ElO 3383284 Error 10 4119Œ7 4.119lE6 Total (corr.) II 1 3977EIO Correlation CoefficIent = 0.998525, R-squared = 99 71%

Regression of Peak Area on Amount 140,000 ,--------------------.,------,

120,000

100,000

~ 80,000 r<

~

i = -,::s

'ii

60,000 r-

40,000

20,000

Amount (ng as Se)

Residuals Regression of Peak Area on Amount 4,000.-----------------------, • 3,000

2,000 r- -- • - . - - --,

1,000 r---- ---,.. -_. _.-. -.-.- ~---- - - . -+------ i -----f-----I 1 1 1 1 1 •

O~-----.------------~------------------~----~

c.: (1,000) • (2,000) _._-~

, • 4 ___ -- ---- , .. --- - ---• , 1 • 1

---- . - ---- ----- i ----: , ---, 1

5 10 15 20 25 30 35

(3.000) [--

(4.000) o'----'-------"'---·--L-----l----L---'------I

Amount (ng as Se)

Figure A-J. Regression analysis for the detennination of the limit of detection for selenomethionine (peak Area vs Arnount, Phase A)

89

•

•

•

-- ----------------

RegreSSIOn - Lmear model Y = a + b'" for selenOC)'stmc (5-30 Ilg Sc. phase A) Vanaùle Intercept Slope

Parame Estimate Standard Error T Value Prob Level 1646893 1508012 0 109 09152 3912696 7989642 48972 00001

AnalYSIS of Vanancc Source DF Sum of Squares Mean Square Model 1 6936E 10 1 6936E 10 Error 10 7 0617E7 7 0617E6 Total (corr,) 11 17006E1O CorrelatIon CoeffiCient = 0.997912, R-squared = 9958%

Regression of Peak Area on Amount 140,000

120,000 1-

100,000 ~ ~

80,000 .. < ~ ~ 60,000 &:

40,000

20,000

0 ".," 1 1

0 5 IO 15 20 25 Amouot (Dg as Se)

T Value 2398267

30


35

6,000 r-----------------------,

4,000

• 2,000 --* -,- . .;

, ~ ,

• ,

= • • ~ 0 .• ~ :

(2,000) • • (4,000) •

(6,000) . 1

0 5 IO 15 20 25 30 35 Amount (og as Se)

Figure A-4. Regression analysis for the detennination of the limit of detection for selenocystine (peak Area vs Arnount, Phase A)

90

•

•

•

Regression - Lmear model' Y = a + bX, for selenoethionine (S .. 30 eg Se. phase A) Variable Parame. Estimate Standard Error T Value Prob. Level Intercept Slope

2078.(1452 1257.756 1 652 0.1295 3542.125 66.637S2 53 155 0.0001 ----

Analysis of Variance Source DF Sum of Squares Mean Square FValue Model 1 1.3880ElO 1.3880EIO 2825.467 Error 10 4.9124E7 4.9124E6 Total (corr ) 11 1 3929EI0 Correlation Coefficient = 0.99823S; R-squared = 99.65%

Regression of Peak Area on AmouDt 140,000 ,--------------------......

120,000

100,000 -

! 80,000 --~

~ 60,000

40,000

20,000

0 0

- -"1-

5 10

t- ...... ,

15 20 AmouDt (Dg as Se)

, , ~ - -.. r j ~ ......

-----t 1 - ---

1 1

25 30 35

Residuals Regression of Peak Area on Amount 5.000 ,..---------------------,

1

4.000 -- .... - .. --- .. - , - ------~1 .. ---i , , 1

1 .... _______ 1 ___ , 1

3,000 , ,

- _ ... -- .... --- ..... +-o~--, 1 1

1

1 !

2,000 , , , _ L ___ .... -... -- -- . ---------, - --- -----r---- _ .. _-

oS • 1 1 ~ 1,000 --- - ---_ .......... .... -----~ .. --- - . -_.-

! • • 0 1

1 • (1,000) • , ___ Mt ~ -r--- -- -_ .. of ----- -- 1-"" .. -- ._-, , 1

(2,000) , 1 1 ,

_ .. -t- ........ t ...... ---- -1---- - ... --..... ---- ----'--1 -t--- - _._-,

• 1

(3,000) 1

.... -.- --- .. - .... r----- --~-- ------1 r"-" , , 1 !

(4,000) ; 1 i 1 i

0 5 10 15 20 25 30 35 AmouDt (Dg as Se)

Figure A-S. Regression analysis for the determination of the limit of detection for selenoethionine (peak Area vs Arnaunt, Phase A)

91

•

•

•

Variable Intercept Slope

Regression - Linear model: Y = a + bX. for selenite (10-50 eS Se. phase B) Parame Estimate Standard Error T Value Prob. Level 12850 998.7294 12.867 0.0001 1631.063 30 1128 54.165 0.0001 ,----

Analysis of Vanance Source OF Sum of Squares Mean Square F Value Model 1 7.981lE9 7.981lE9 2933855 Error 13 3536SE7 2.7203E6 Total (corr.) 14 80164E9 Correlation Coefficient = 0.997792; R-squared = 99.56%

Regression of Peak Area on Amount 140,000 r----------------------,

120,000 ----

100,000 ,..---

e 80,000 --~

i 60,000 e----

-- 1 ---

, ~ ~ - - t

, - - - 1 -- !

o ~ ____ ~ ____ ~~ ___ ~ ____ ~ ______ ~l ______ ~

o 10 20 30 40 50 60 Amount (Dg 15 Se)

Residuals Regression of Peak Area on Amount 4,000 ,----..... -.------------,-------,

3,000 1----

2,000 1----

- - - .. , , ----- .. , ,

, , i 1,000 r-----------i------ - -t---t-----a • :se 0 '

, , Il : Q: (1,000) 1-'---- - ----- '0----- -:---- ----j'

(2,000) r---

(3,000) r----

____ • _,_ 0 __ -

1

, -- -- -1 -- ---

- --- . - --,

- - - - - t - -

• • --1----- -------, ----

--,--, ,

(4,000) 0~--1L-.0--........ 20----"30---4-L0----'-50------J60

Amount (Dg 15 Se)

Figure A-6. Regression anaIysis for the determination of the Iimit of detection for selenite (peak Area vs Amount, Phase D)

92

•

•

•

Regression - Linear model: Y = a + bX, Co..r selenate (lO-SO J.lg Se, phase B) Variable Parame. Estirnatc Standard Error T Value Prob Levet Intercept Siope

1679.S00 13IS.118 1277 0.2239 1892.677 39.6S23 47.732 0.0001

Analysis oC Vanance Source OF Sum of Squares Mean Square F Value Model 1 1.0747EIO l.0747EIO 2278.328 Error 13 6.1320E7 4.71670E6 Total (corr.) 14 1.0808E9 Correlation CoeffiCient = 0.9971S9; R-squared = 99.43%

Regression of Peak Area on Amount 140,000 .----~-------------.------..,

120,000 -

100,000

! 80,000 ~ -~ • 60,000 ~-~

40,000 r---

,

-- ,

, , - --1

10 1

20 30 40 50 Amount (ng as Se)


60

6,000,-----:------------------, ,

4,000 r--- ! - - - - 1

1 • --1---

, - - - -- t , -t-- - --

1

.; 2,000 r--- ----:. ---- - -j---------- t ,

-- .... - -- ---- -

.. • 1 ~ O~-----T------~-----~-------r------~·------~ ! ! •

(2,000) r---

(4,000) --

(6,000) 0

, ---- ..

........ i

1 !

i JO

, ,

........ .. • + ---- ...... ---_ .. ---- , ~----20 30

Amount (ng as Se) 40 50 60

Figure A-7. Regression analysis for the detennination of the limit of detection for selenate ~.>eak Area vs ArnouRt, Phase B)

93

•

•

•

REFERENCES

Adriano, D.C., (1986). Trace elements in the terrestrial environment. Springer-Verlag, New York.

Aono, T., Nakaguchi, Y., Hiraki, K., and Nagai, T., (1990). Determination of seleno-amino acid in natural water samples. Geochem. J., 24: 255-261.

Arnold, A.P., Canty, A.J, Skelton, B.W, and White, A.H., (1982). Mercury (II) selenolates. Crystal structures of polymerie Hg(SeMeh and the tetrameric pyridinates [{HgCI(py)(SeEt)}4J and [{HgCI(py)0.5(SeBul)}4]. J. Chem. Soc. Dation, 607-613.

Arthur, D., (1972). Selenium content of canadian foods. Cano lmt. Food Sci. Technol. J., 5:165-169.

Atsuya, 1., Itoh, K., and Ariu, K., (1991). Preconcentration by copreclpitation of lead and selenium with Nilpyrrolidine dithiocarrnate complex and their simultaneous detennination by internai standard atomic absorption spectrometry with the solid sampling te~nique. Pure & Appl. Chem., 63: 1221-1226.

Barak, A.J. and Swanberg, S.C., (1967). Determination of selenomethionine in biological sources by chromatography and neutron activation anaIysis. J. Chromatogr., 31:282-284.

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Selenium Speciation by High Performance Liquid ...digitool.library.mcgill.ca/thesisfile55507.pdf · Selenium has been shown to have multiple biochemical effects rangmg from nutrient