An evaluation of hydrocarbon extraction techniques for tissue

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Dissertations, Theses, and Masters Projects Theses, Dissertations, & Master Projects

1979

An evaluation of hydrocarbon extraction techniques for tissue An evaluation of hydrocarbon extraction techniques for tissue

Colleen Gail Becker College of William and Mary - Virginia Institute of Marine Science

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AN EVALUATION OF HYDROCARBON EXTRACTION

TECHNIQUES FOR TISSUE

A T h e s is

P r e s e n te d to

The F a c u l ty o f th e Schoo l o f M arine S c ie n ce

The C o lleg e o f W il l ia m and Mary i n V i r g i n i a

In P a r t i a l F u l f i l l m e n t

Of th e R equirem ents f o r th e Degree o f

M as te r o f A rts

by

C o lle en B ecker

1979

APPROVAL SHEET

This t h e s i s i s su b m it te d i n p a r t i a l f u l f i l l m e n t o f

th e re q u ire m e n ts f o r th e d e g re e o f

M as te r o f A r ts

Autho

Approved, F eb ru a ry 1971

C ra ig L. s m i th , Ph.D.

ti , M —( V

R udolf H. B i e r i , Ph.D.

4-Uwd (&U---Howard K a to r , Ph.D.

R ober t A. Jo rd a n ,

LR obert E. B la c k , Ph.D.

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS .............................. . . . . . . . . . . . v

LIST OF TABLES. ......................................... v i

ABSTRACT..................................... v i i

INTRODUCTION. . . . . . . ..................... . . .......................................................... 2

EXPERIMENTAL.............................................. 10

MATERIALS. . . . . . . . . . . . . . . . . . ................................ 10

STANDARDS. ....................................................... . . 10

METHODS.............................................. 12

D e te rm in a t io n o f R e p l i c a te Numbers. . . . . . . . . . . 12

P r e p a r a t i o n o f T is su e Homogenate................................ 12

E x t r a c t i o n T e c h n i q u e s ................................ 13

D ig e s t io n i n KOH-methanol..................................... 13

S o x h le t E x t r a c t i o n . . . . . . . . . . . ................... 14

MacLeod's P ro c e d u re . .............................................. 15

E v a p o ra t io n o f Excess S o lv e n t . . . . .......................................15

Column Chromatography . ..................................... 16

Gas Chromatography. .................................................................................16

Method o f Q u a n t i f i c a t i o n ............................................................................ 17

P r e l im in a r y D e te rm in a t io n o f E f f i c i e n c i e s ............................ 18

S t a t i s t i c a l A n a ly s i s ..................................................................................... 19

RESULTS............................................................ 20

DISCUSSION.......................................................................... 29

i i i

TABLE OF CONTENTS (C on tinued )

Page

APPENDIX................................................................. 36

LITERATURE CITED.............................................. 71

VITA. ..................................... 75

iv

ACKNOWLEDGMENTS

The a u th o r w ish es to e x p re s s h e r s i n c e r e a p p r e c i a t i o n to h e r

m a jo r p r o f e s s o r , Dr. C ra ig S m ith , f o r h i s c o n t in u e d s u p p o r t , gu idance

and s e n s e o f humor th ro u g h o u t th e c o u rse o f t h i s s tu d y . Thanks a re

a l s o ex ten d ed to D rs . Howard K a to r , R udolf H. B i e r i and R o b er t E.

B lack f o r c r i t i c a l l y re v ie w in g th e m a n u s c r ip t .

A s p e c i a l d e b t o f th an k s i s owed to Dr. R ober t A. Jo rd a n

f o r h i s p a t i e n c e , encouragem ent, and i n v a lu a b le c r i t i c a l rev iew o f

th e m a n u sc r ip t i n i t s v a r io u s s t a g e s . Thanks a r e a l s o ex ten d ed to

L inda J e n k in s f o r ty p in g th e m a n u s c r ip t .

F i n a l l y , th e a u th o r w ish es to thank h e r husband , James Schwan,

w i th o u t whose p a t i e n c e , s u p p o r t , and " r e a l i s t i c " s e n se o f humor t h i s

p r o j e c t would n o t have been com ple ted .

V

LIST OF TABLES

T ab le Page

1 E x t r a c t i o n T echniques ........................................................ . . . . 9

2 Recovered W eights o f A l i p h a t i c H ydrocarbon F r a c t i o nC a lc u la te d by th e E x t e r n a l S ta n d a rd Method. . . . . . . 21

3 R ecovered W eights o f A rom atic Hydrocarbon F r a c t i o nC a lc u la te d by th e E x t e r n a l S ta n d a rd M ethod.................................. 22

4 N orm alized R ecovered W eights o f H ydrocarbons. .................... 24

5 Student-Newman^-Keul's T e s ts (SNK) ............................. 26

6 S ta n d a rd R ec o v e r ie s as A rc s in e Transform ed P e r c e n tsfrom S p iked B lanks f o r th e Complete T echn iques . . . . . 27

v i

ABSTRACT

The p u rp o se o f t h i s r e s e a r c h was to e v a lu a t e and compare t h r e e methods o f e x t r a c t i o n o f h y d ro ca rb o n s from clam t i s s u e . No s i g n i f i c a n t d i f f e r e n c e be tw een te c h n iq u e s (KOH a l c o h o l i c d i g e s t i o n , S o x h le t e x t r a c t i o n , and MacLeod's p ro c e d u re ) was d e te rm in ed f o r th e f r a c t i o n s q u a n t i t a t e d by th e e x t e r n a l s t a n d a r d method and f o r th e a r c s i n e t ra n s fo rm e d p e r c e n t r e c o v e r i e s o f th e i n t e r n a l s t a n d a r d s . S i g n i f i c a n t d i f f e r e n c e s among te c h n iq u e s w ere d e te rm in ed f o r f r a c t i o n s q u a n t i t a t e d to th e i n t e r n a l s t a n d a r d s e x c e p t hexam ethy lbenzene . I n t e r p r e t a t i o n i s d i f f i c u l t b e c a u se no te c h n iq u e p roved to be cons i s t e n t l y s u p e r i o r . The th r e e te c h n iq u e s must be c o n s id e re d e q u iv a l e n t s i n c e raw r e c o v e r i e s w ere s t a t i s t i c a l l y s i m i l a r and r e c o v e r i e s n o rm a liz e d to th e i n t e r n a l s t a n d a r d s d id n o t behave c o n s i s t e n t l y .

This s tu d y d e m o n s t ra te s t h a t d i f f e r e n t i n t e r p r e t a t i o n s o f d a t a can be made b ased s o l e l y upon th e q u a n t i f i c a t i o n m ethod. N ea r ly a l l th e l o s s e s o f compounds i n th e s p ik e b la n k s were a cco u n ted f o r by t h r e e p r o c e s s e s : c o n c e n t r a t i o n under n i t r o g e n , c o n c e n t r a t i o n by r o t a r ye v a p o r a t io n , and column chrom atography . A com parison o f th e s ta n d a r d r e c o v e r i e s f o r th e clam t i s s u e and th e s p ik e b la n k s i n d i c a t e d a 10-20% lo s s due t o th e p re s e n c e o f th e t i s s u e . Given th e o bserved u n c e r t a i n t i e s w i t h i n th e te c h n iq u e s s t u d ie d h e r e , sm a l l d i f f e r e n c e s be tw een te c h n iq u e s a r e n o t s i g n i f i c a n t . Based upon tim e and equ ipm ent, M acLeod's p ro c e d u re was th e p r e f e r a b l e te c h n iq u e .

AN EVALUATION OF HYDROCARBON EXTRACTION

TECHNIQUES FOR TISSUE

INTRODUCTION

P o t e n t i a l developm ent o f th e U. S . A t l a n t i c c o n t i n e n t a l

s h e l f p e tro le u m r e s e r v e s has in c r e a s e d th e need f o r a c c u r a t e a n a l y t i c a l

te c h n iq u e s to d e te rm in e th e c o n c e n t r a t i o n and co m p o s i t io n o f p e tro le u m

p o l l u t a n t s i n th e m arin e en v iro n m en t. T y p ic a l h y d ro ca rb o n a n a l y t i c a l

scheme c o n s i s t s o f s e v e r a l d i s t i n c t o p e r a t i o n s : c o l l e c t i o n , s t o r a g e ,

e x t r a c t i o n , c o n c e n t r a t i o n , i s o l a t i o n , i d e n t i f i c a t i o n , and q u a n t i f i c a t i o n .

A key s t e p i n most o f th e a n a ly s e s o f b i o l o g i c a l sam ples i s th e

e x t r a c t i o n o f t h e h y d ro c a rb o n s . Many e x t r a c t i o n te c h n iq u e s in v o lv in g

th e s e p a r a t i o n o f th e compounds and i s o l a t i o n from n o n -hydroca rbons

have been d e v e lo p e d . These in v o lv e e x t r a c t i o n w i th a s o l v e n t , s team

d i s t i l l a t i o n and gas d i s t i l l a t i o n . S o x h le t e x t r a c t i o n in v o lv e s an

e x te n s iv e e x t r a c t i o n by an o r g a n ic s o l v e n t fo l lo w ed by p a r t i t i o n i n g

in t o a n o n - p o la r s o l v e n t (C la rk and Blumer, 1967; Blumer e t a l . , 1970;

E h r h a r d t , 1972; C la rk and F in l e y , 1973; F a r r in g to n e t a l . , 1973a;

B i e r i and S tam oud is , 1977). O th e r p ro c e d u re s in c lu d e : a l c o h o l i c

KOH d i g e s t i o n fo l lo w e d by p a r t i t i o n i n g i n t o a n o n - p o la r s o lv e n t

(G r e f f a r d and Meury, 1967; B la y lo c k e t a l . , 1973; Smith e t a l . , 1977);

s team d i s t i l l a t i o n (Ackman and N oble , 1973); h ead sp ace a n a l y s i s o f

v o l a t i l i z e d h y d ro ca rb o n s (H e r tz e t a l . , 1974); g r in d i n g , hom ogenizing ,

o r s o n i c a t i o n w i th an o rg a n ic s o l v e n t (Stegeman and T e a l , 1973; H un te r

e t a l . , 1974; Mackie e t a l . , 1974); and aqueous a l k a l i n e d i g e s t i o n

fo l lo w ed by e t h e r e x t r a c t i o n (MacLeod e t a l . , 1976; W arner, 1976).

2

3

These te c h n iq u e s r e l y on a d i s r u p t i o n o f th e t i s s u e m a t r ix and c e l l s

to g a in a c c e s s to th e h y d ro c a rb o n s , fo l lo w ed by v o l a t i l i z a t i o n o r

o r g a n ic s o l v e n t e x t r a c t i o n .

P r i o r t o t h e a n a l y s i s f o r p e t ro le u m h y d ro c a rb o n s , th e e x t r a c t e d

m a t e r i a l must be i s o l a t e d . A v a r i e t y o f t e c h n iq u e s have been used to

remove l i p i d s and o t h e r n o n -h y d ro ca rb o n s from th e e x t r a c t . T h i n - l a y e r

chrom atography i s a form o f l i q u i d - s o l i d chrom atography em ploying th e

mechanism o f a d s o r p t io n to r e t a i n sam ple m o le c u le s . T h i n - l a y e r chrom ato

graphy i s c a p a b le o f a c c e p t in g d i r t y sam ples and can be used to p ro c e ss

a l a r g e number o f sam ples s im u l ta n e o u s ly . One te c h n iq u e b a sed upon th e

same a d s o r p t io n mechanism i s column chrom atography ( g r a v i t y f l o w ) . T h in -

l a y e r chrom atography and column chrom atography , s i n g l y and i n c o m b in a tio n ,

have been used to i s o l a t e h y d ro ca rb o n f r a c t i o n s (Koons e t a l . , 1965;

Avigan and B lumer, 1968; S ev e r and Haug, 1971; B a r b ie r e t a l . , 1973;

F a r r in g to n and Quinn, 1973; Johnson and C a ld e r , 19 73; Z so ln ay , 19 73).

High p e rfo rm ance l i q u i d chrom atography (HPLC) i s u s e f u l f o r th e

s e p a r a t i o n o f a ro m a t ic h y d ro ca rb o n s b u t i t s u se i n c o n ju n c t io n w i th

s u b se q u e n t gas c h ro m a to g ra p h ic a n a l y s i s has n o t been f u l l y d eve loped .

Amounts o f t o t a l h y d ro ca rb o n s i s o l a t e d by chrom atography

can b e q u a n t i t a t e d g r a v i m e t r i c a l l y i f s u f f i c i e n t m a t e r i a l rem ains

a f t e r th e e v a p o r a t io n o f th e s o l v e n t . One p o s s i b l e d is a d v a n ta g e i s

th e l o s s o f th e more v o l a t i l e h y d ro ca rb o n s ( F a r r in g to n e t a l . , 19 73a) .

A nother d i s a d v a n ta g e , w hich a p p l i e s to a l l b u lk m easurem en ts , i s t h a t

th e q u a n t i t a t i o n in c lu d e s b o th in d ig e n o u s and p e tro le u m hydrocarbons

i n a d d i t i o n to o t h e r o r g a n ic compounds.

As a q u a n t i f i c a t i o n and i d e n t i f i c a t i o n method, i n f r a r e d

s p e c t ro m e try shows l i t t l e p rom ise f o r d e t e c t i n g sm a l l q u a n t i t i e s of

4

p e tro le u m i f b io g e n ic h y d ro c a rb o n s a r e p r e s e n t . Loss o f v o l a t i l e

h y d ro c a rb o n s i s m in im a l, b u t th e a b s o r p t io n f r e q u e n c i e s o f in d ig e n o u s

h y d ro c a rb o n s o v e r la p o r c o in c id e w i th th o se o f p e t ro le u m h y d ro c a rb o n s .

A p o s s i b l e e x c e p t io n to t h i s g e n e r a l i z a t i o n a r e th e a ro m a t ic h y d ro ca rb o n s

a b s o rb in g IR r a d i a t i o n i n th e lo n g w av e len g th r e g io n (Brown e t a l . ,

1973; Z a f i r i o u , 1973) .

UV a b s o r p t io n and UV f lu o r e s c e n c e s p e c t ro m e try d e t e c t th e

p re s e n c e o f a ro m a t ic h y d ro ca rb o n s ( Z i tk o and C arson , 1970; Z i tk o and

T ibbo , 1970; E r h a r d t and B lum er, 19 71; Z a f i r i o u , 19 73; Gordon and

K e iz e r , 19 74 ) . The fo rm er method i s b a se d upon a b s o r p t io n o f UV l i g h t ,

th e l a t t e r on e m is s io n . C om ple te ly s a t u r a t e d compounds show no

s e l e c t i v e a b s o r p t io n th ro u g h o u t th e v i s i b l e and u l t r a v i o l e t r e g io n s .

A f t e r m o lecu les have ab so rb ed r a d i a n t energy and b een e x c i t e d to a

h i g h e r e l e c t r o n i c s t a t e , th e y must l o s e t h e i r ex cess energy i n o rd e r

to r e t u r n to th e ground e l e c t r o n i c s t a t e . F lu o re s c e n c e i s th e im m ediate

e m is s io n o f energy as v i s i b l e l i g h t from a m o lecu le a f t e r i t has absorbed

r a d i a t i o n o f a s h o r t e r w a v e le n g th . Both te c h n iq u e s d e t e c t a ro m a t ic

h y d ro ca rb o n s b u t t h e r e i s l i t t l e d i f f e r e n t i a t i o n w i t h i n a m ix tu re o f

a ro m a t ic h y d ro c a rb o n s . However, UV and U V -f lu o rescen ce sc a n n in g o f

ch ro m a to g rap h ic s e p a r a t i o n s a r e u s e f u l i n d e te rm in in g w hich f r a c t i o n s

c o n ta in a ro m a t ic h y d ro c a rb o n s .

The i n c r e a s i n g u t i l i z a t i o n o f gas chrom atography i n d i c a t e s

i t s w ide a c c e p ta n c e as an a n a l y t i c a l te c h n iq u e f o r any sam ple to

which i t may b e a p p l i e d . In r e a l i t y , t h i s means any s t a b l e compound

w hich e x h i b i t s a m e a su re a b le v ap o r p r e s s u r e a t te m p e ra tu re s up to 300°C,

as w e l l as any compound o f w hich a s t a b l e , v o l a t i l e d e r i v a t i v e may

be form ed. C o n d it io n s o f column a d s o r p t io n and d e t e c t o r s e n s i t i v i t y

5

a l s o i n f l u e n c e th e a p p l i c a t i o n o f t h i s a n a l y t i c a l t e c h n iq u e . Gas

chrom atography p ro v id e s a d e g re e o f s e n s i t i v i t y and e f f i c i e n c y which

makes i t i d e a l f o r th e a n a l y s i s o f h y d ro c a rb o n s . I t p ro v id e s an

e x c e l l e n t s e p a r a t i o n and q u a n t i f i c a t i o n method f o r p e t ro le u m m ix tu r e s ,

a lo n g w i th e x t e n s iv e i n f o r m a t io n on th e m o le c u la r w e ig h t ran g e and th e

c o m p le x i ty o f th e m ix tu r e . T e n t a t i v e i d e n t i f i c a t i o n s o f r e s o lv e d and

p a r t i a l l y r e s o lv e d compounds a r e p o s s i b l e i f s t a n d a r d s a r e a v a i l a b l e

f o r s u p e r im p o s i t io n (Koons e t a l . , 1965; Avigan and Blumer, 1968;

S e v e r and Haug, 1971; Youngblood e t a l . , 1971; I l i f f e and C a ld e r , 1974;

F a r r in g to n and M ed e iro s , 19 75; Youngblood and B lumer, 1975; W arner,

19 76; Sm ith e t a l . , 19 77).

Mass s p e c t ro m e try and gas chrom atography-m ass s p e c t ro m e try

(gc-m s) p ro v id e e x c e l l e n t q u a l i t a t i v e a n a ly s e s o f p e t ro le u m h y d ro c a rb o n s .

The b a s i c f u n c t io n o f th e mass s p e c t r o m e te r i s to p roduce io n s from

a sam p le , s e p a r a t i n g them a c c o rd in g to t h e i r m a s s - to - c h a rg e r a t i o s

( m / e ) . C oupling o f mass s p e c t ro m e try w i th gas chrom atography has

p ro v id e d a un iq u e o p p o r tu n i ty to b e g in to i d e n t i f y even complex n a t u r a l l y

o c c u r r in g compounds i n p e t r o le u m s . This te c h n iq u e has been found v e ry

e f f e c t i v e f o r a ro m a t ic h y d ro ca rb o n s (Blumer e t a l . , 1970; S ev e r and

Haug, 1971; Youngblood e t a l . , 1971; H i t e s and Biemann, 1972; B la y lo c k

e t a l . , 1973; Brown e t a l . , 19 73; C la rk and F in l e y , 1973; B i e r i e t a l . ,

1974; I l i f f e and C a ld e r , 1974; Youngblood and B lum er, 1975; W arner,

1976; B i e r i and S tam oud is , 19 77).

P re v io u s e v a lu a t io n s o f h y d ro ca rb o n e x t r a c t i o n te c h n iq u e s

have b een made on sed im en ts (R ohrback and Reed, 1975) and t i s s u e

( F a r r in g to n and M ed e iro s , 1975; MacLeod e t a l . , 1976; G r i t z and Shaw,

19 7 7 ) . The e v a l u a t i o n o f te c h n iq u e s f o r sed im en ts c o n s id e re d t h r e e

a s p e c t s o f th e e x t r a c t i o n : 1) sam ple p r e t r e a tm e n t ; 2) e x t r a c t i o n

t e c h n iq u e s ( S o x h le t , s o n i c a t i o n , h o m o g e n iz a t io n , r e f l u x e x t r a c t i o n ,

and r e c i p r o c a l s h a k e r t a b l e ) ; and 3) c h o ic e o f s o l v e n t s . S o x h le t

e x t r a c t i o n p roved s l i g h t l y more e f f i c i e n t (4%) th an th e o th e r

e x t r a c t i o n t e c h n iq u e s . A c i d - t r e a t e d , w a te r -w ash e d , f r e e z e - d r i e d

s e d im en t e x t r a c t e d w i th to lu e n e -m e th a n o l (3 :7 ) ap p ea red to be th e

b e s t g e n e r a l l y a p p l i c a b l e p ro c e d u re (R ohrback and Reed, 19 75).

F a r r i n g t o n and M edeiros (19 75) e v a lu a te d t h r e e methods o f

e x t r a c t i n g h y d ro ca rb o n s from m arin e o rg an ism s: S o x h le t e x t r a c t i o n ,

h o m o g e n iz a t io n w i th Na2S0^ i n a V i r t i s h o m ogen izer , and KOH-methanol

d i g e s t i o n . The amounts o f e x t r a c t e d h y d ro ca rb o n s from s p ik e d subsam ples

o f clam (M e rc e n a r ia m e rc e n a r ia ) homogenate w ere d e te rm in ed g r a v i m e t r i c a l l y

w i th S o x h le t e x t r a c t i o n s t a t i s t i c a l l y s l i g h t l y more e f f i c i e n t . G r i t z

and Shaw (19 77) u s in g th e s o f t p a r t s o f Saxidomus g ig a n te a ( b u t t e r clam)

compared S o x h le t e x t r a c t i o n fo l lo w ed by 2 h o u r s a p o n i f i c a t i o n to

e i t h e r 2 h o u r o r tw enty-^four h o u r a l k a l i n e e x t r a c t i o n . A f u r t h e r

p a ra m e te r w hich was i n v e s t i g a t e d was th e use o f f u l l y a c t i v a t e d o r

p a r t i a l l y d e a c t i v a t e d a l u m i n a - s i l i c a g e l columns. S im i l a r r e c o v e r i e s

o f added a lk a n e s w ere o b ta in e d by th e t h r e e e x t r a c t i o n m ethods. Twenty-

fo u r h o u r s a p o n i f i c a t i o n and column chrom atography on p a r t i a l l y d e a c t iv a t e d

columns was ju d g ed th e b e s t p ro c e d u re . S o x h le t e x t r a c t i o n fo l lo w ed by

two h o u r s a p o n i f i c a t i o n d id n o t co m p le te ly remove m ethy l e s t e r s and

was th e m ost l a b o r io u s t e c h n iq u e . D i r e c t two h o u r s a p o n i f i c a t i o n was

a l s o q u e s t i o n a b le i n th e rem oval o f e s t e r s and l e d to s e v e re em uls ion

p rob lem s . T h e i r column chrom atography work i l l u s t r a t e d th e need f o r

c a u t io n i n a r b i t r a r i l y a s s ig n in g compounds to d i f f e r e n t f r a c t i o n s .

S e v e r a l a lk e n e s w hich e l u t e d i n th e f i r s t ( a lk a n e ) f r a c t i o n when

p a r t i a l l y d e a c t i v a t e d columns w ere u s e d , a p p ea re d p a r t i a l l y o r t o t a l l y

7

i n th e second (a ro m a t ic ) f r a c t i o n when f u l l y a c t i v e columns w ere employed.

W hile t h i s s i t u a t i o n w i l l cause l i t t l e p roblem i n h e a v i l y p o l l u t e d

sam ples i n w hich a lk e n e s a r e o n ly a s m a l l p e r c e n ta g e o f th e t o t a l hyd ro

c a rb o n s , i n u n p o l lu te d b i o l o g i c a l m a t e r i a l s w here a lk e n e s may be

p ro m in en t , c l a s s i f i c a t i o n o f m o le c u le s as a lk a n e s o r a ro m a t ic s b a se d

o n ly on column ch ro m a to g ra p h ic b e h a v io r m ust be done w i th c a u t i o n . The

r e s u l t s from th e e v a l u a t i o n s tu d y from w hich MacLeodfs p ro c e d u re was

s e l e c t e d a r e c u r r e n t l y b e in g e v a lu a te d (D. Brown, p e r s o n a l com m unication).

An i n t e r c a l i b r a t i o n o f t h r e e a n a l y t i c a l p ro c e d u re s conducted

by F a r r in g to n e t a l . (1 9 7 3 b ) , u s in g a cod l i v e r l i p i d s t a n d a r d c o n ta in in g)

b io g e n ic and p e t ro le u m h y d ro c a rb o n s , i n d i c a t e d s i m i l a r c o n c e n t r a t io n s

o f p r i s t a n e and s q u a le n e and l e v e l s o f c o n ta m in a t io n o f p e tro le u m . The

i n t e r c a l i b r a t i o n c o n c e n t r a t e d on c le a n up and gas chrom atography

p r o c e d u r e s , s i n c e th e l i p i d had a l r e a d y been e x t r a c t e d from th e l i v e r

t i s s u e .

The p u rp o se o f t h i s r e s e a r c h was to e v a lu a t e and compare

t h r e e methods o f e x t r a c t i o n o f h y d ro ca rb o n s from an im al t i s s u e . The

o rgan ism chosen was th e r e a d i l y a v a i l a b l e , common guahog clam M erce n a r ia

m e r c e n a r i a . Three p ro c e d u re s t e s t e d w ere S o x h le t e x t r a c t i o n ( B i e r i

and S tam o u d is , 197 7 ), a l c o h o l i c KOH d i g e s t i o n (Sm ith e t a l . , 1977) and

e t h e r e x t r a c t i o n (MacLeod e t a l . , 1976); th e f i r s t two te c h n iq u e s

have been u sed e x t e n s i v e l y i n t h i s l a b o r a t o r y . A l l th e p ro c e d u re s

in v o lv e d a d i g e s t i o n fo l lo w e d by e x t r a c t i o n w i th a n o n - p o la r s o l v e n t .

They d i f f e r e d by th e method o f e x t r a c t i o n and th e ty p e o f d i g e s t i o n .

To e l i m i n a t e as many o u t s i d e f a c t o r s as p o s s i b l e and to c o n c e n t r a t e

th e e v a l u a t i o n s o l e l y on th e e x t r a c t i o n p ro c e d u re s , i d e n t i c a l column

chrom atography and gas chrom atography p ro c e d u re s w ere used to d e te rm in e

8

h y d ro c a rb o n c o n c e n t r a t i o n s . The th r e e a n a l y t i c a l p ro c e d u re s a re

p r e s e n t e d i n T ab le 1.

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9

EXPERIMENTAL

M a te r i a l s

P e n ta n e , h ex an e , b e n zen e , m e th a n o l , and u n p re se rv e d d i e t h y l

e t h e r , a l l " d i s t i l l e d i n g l a s s " , w ere u t i l i z e d (B u rd ick and J a c k so n ,

Muskegon, M ich ) . D ie th y l e t h e r w i th 2% e th a n o l p r e s e r v a t i v e was n o t

a c c e p ta b le f o r MacLeod's p r o c e d u r e . Up to 0 .5 ml o f e th a n o l rem ains

i n th e e x t r a c t and upon c o n c e n t r a t i o n e th a n o l s e p a r a t e s as a second

p h a se . T h is e t h a n o l i c p h ase may cause bumping and lo s s o f sam ple .

F u r th e rm o re , th e a l c o h o l may d e a c t i v a t e th e s i l i c a g e l column and

n u l l i f y th e s e p a r a t i o n o f s a t u r a t e d and u n s a tu r a t e d h y d ro c a rb o n s .

Sodium c h l o r i d e , anhydrous sodium s u l f a t e and s e a sand w ere S o x h le t

e x t r a c t e d w i th hexane and d r i e d a t 135°C f o r 16 h o u r s . The B io -S i l^ A

s i l i c a g e l , 100-200 mesh, was a c t i v a t e d by h e a t i n g f o r 16 h o u rs a t 210°C.

A l l d e io n iz e d w a te r was e x t r a c t e d w i th hexane p r i o r to u s e . A l l

g la s s w a re was f i r s t washed w i th soapy w a te r , r i n s e d w i th ta p w a te r ,

d r i e d , p la c e d i n d ich ro m a te c le a n in g s o l u t i o n , r i n s e d w i th ta p w a te r ,

d e io n iz e d w a te r and a c e to n e , and baked a t 135°C.

S ta n d a rd s

The s t a n d a r d s f o r th e s p ik e b la n k s and s p ik e d t i s s u e s a r e

l i s t e d below .

S ta n d a rd A

compound g /1

2-me thy lo c t ade can e 0.0570

2 -m e th y le ic o sa n e 0 .0248

10

compound g /1 -

d o t r i a c o n t a n e n C ^ 0 .0371

hexam ethy lbenzene 0 .0362

b e n z o ( g h i ) p e r y le n e 0 .0356

The s t a n d a r d s f o r th e r e c o v e r i e s o f c o n c e n t r a t i o n u nder

p i t r o g e n , c o n c e n t r a t i o n u n d e r r o t a r y e v a p o r a to r , and column chrom ato

graphy a re l i s t e d be low .

S ta n d a rd B

compound g /1

hex ad ecan e 0.0584

e ic o s a n e nC£Q 0.0242

o c ta c o s a n e nC2 g 0.0236

d o t r i a c o n t a n e n C ^ 0.0260

1 - m e th y l f lu o r e n e 0.0236

p y re n e 0 .0220

b e n z o (a )p y re n e 0 .0238

W arner (19 76) d e te rm in ed t h a t d i f f e r e n t s p ik e c o n c e n t r a t io n s

v a r i e d i n t h e i r r e c o v e r i e s : a s p ik e c o n c e n t r a t i o n o f 10 y g /g had

r e c o v e r i e s i n th e ran g e o f 65-100%; 2 .0 y g /g had a re c o v e ry ran g e o f

70-103%; 0 .4 y g /g had a range o f 53-102%; and 0 .1 y g /g had a range o f

40-90%. W ith th e ran g e o f 3 -8 yg t o t a l e x t r a c t e d hyd rocarbons f o r th e

t i s s u e sam p le s , a n o th e r f a c t o r b e s id e s r e c o v e r i e s sh o u ld be c o n s id e re d

in d e te rm in in g th e s p ik e c o n c e n t r a t i o n . To maximize th e p a ra m e te rs

o f r e c o v e r i e s v e r s u s sam ple m ask ing , th e s p ik e c o n c e n t r a t io n s 0 .1 0 -0 .2 8

Wg/g w ere chosen w hich w ere t h e r e f o r e c lo s e t o th e lo w e s t c o n c e n t r a t io n

(0 .1 0 yg) used by W arner (1976 ) .

12

Methods

D e te rm in a t io n o f R e p l i c a t e Numbers

P r i o r to th e a c t u a l t i s s u e a n a l y s i s , an a t te m p t was made to

d e te rm in e th e number o f r e p l i c a t e s n e c e s s a ry f o r s t a t i s t i c a l l y v a l i d

r e s u l t s . The r e c o v e r i e s f o r th e s p ik e d b la n k s and a 95% c o n f id e n ce

l e v e l w ere u t i l i z e d i n th e c a l c u l a t i o n s . The number o f r e p l i c a t e s

n e c e s s a r y was d ependen t upon th e power o f th e t e s t . An e r r o r o f th e

f i r s t k in d (Type I ) i s made when th e e x p e r im e n te r r e j e c t s th e n u l l

h y p o th e s i s and i t i s t r u e . An e r r o r o f t h e second k in d (Type I I ) i s

made when th e e x p e r im e n te r a c c e p ts th e n u l l h y p o th e s i s and th e a l t e r n a t i v e

i s t r u e . When b o th ty p es o f e r r o r a r e c o n s id e re d i n th e c ase o f a

sam ple o f f i x e d s i z e , i t i s s e e n t h a t a r e d u c t io n i n th e p r o b a b i l i t y

o f a Type I e r r o r (ot) m ust be accompanied by an i n c r e a s e i n th e p r o b a b i l i t y

o f a Type I I e r r o r ( 3 ) . I n an i d e a l s i t u a t i o n , b o th a and 3 a re f ix e d

i n advance; t h i s d e te rm in e s th e r e q u i r e d sam ple s i z e . The power o f a

t e s t i s i t s a b i l i t y to d e t e c t th e a l t e r n a t i v e h y p o th e s i s when i t i s t r u e

( 1 - 3 ) . I t i s o b v io u s ly d e s i r a b l e to have t h i s p r o b a b i l i t y h ig h . In

o r d e r to o p t im iz e th e p a ra m e te r s w i th a a t 0 .0 5 , n in e t e e n r e p l i c a t e s

had a power o f 70% w h i le n in e r e p l i c a t e s had 40%. The n in e r e p l i c a t e s

w ere chosen due to o u t s id e l i m i t i n g f a c t o r s ( l i m i t e d tim e and equ ipm en t) .

However, th e s e c a l c u l a t i o n s p r o v id e some i n d i c a t i o n o f th e l i m i t s o f

any s t a t i s t i c a l h a n d l in g o f th e r e s u l t s . I t cou ld n o t be d e te rm in ed

how v a l i d i t was to u t i l i z e r e s u l t s from s p ik e d b la n k s f o r sp ik e d

t i s s u e .

P r e p a r a t i o n o f t i s s u e homogenate

The t i s s u e and f l u i d s o f e i g h t m edium -sized clams M ercen a r ia

m e rc e n a r ia w ere homogenized a t medium-high speed u s in g a V i r t i s "45"

13

h o m o g en ize r . To d e te rm in e th e hom ogeneity o f th e a l i q u o t s , th e

homogenate was f i r s t f r o z e n i n hexane-w ashed m e ta l i c e cube t r a y s ;

t h r e e f r o z e n cubes w ere w eighed , th e n d r i e d o v e r n ig h t i n a 125°C

oven to a c o n s t a n t w e ig h t . The dry w e ig h t /w e t w e ig h t r a t i o av e rag e

f o r t h e t h r e e t i s s u e cubes was 0.14669 ± 0 .00364 .

E x t r a c t i o n te c h n iq u e s

D ig e s t i o n i n KOH-methanol (Method B)

The te c h n iq u e o f Sm ith e t a l . (1977) was fo l lo w e d . Ten grams

o f hom ogenate (one homogenate cube) w ere w eighed i n t o a t a r e d b o i l i n g

f l a s k . F i f t e e n ml o f 0 .5 N p o ta s s iu m h y d ro x id e i n m ethanol and 50 y l

o f S ta n d a rd A w ere added . The sam ple was r e f lu x e d f o r f o u r h o u r s , th en

a l lo w ed to co o l to room te m p e ra tu re .

The d ig e s t e d m ix tu re was t r a n s f e r r e d to a s e p a r a to r y f u n n e l ;

th e f l a s k was r i n s e d t h r e e t im es w i th m ethanol i n t o th e s e p a r a to r y

fu n n e l . E qual volumes o f s a t u r a t e d s a l t s o l u t i o n and hexane w ere

added; th e volume was e q u i v a l e n t to th e volume o f th e d ig e s t e d m ix tu r e .

The m ix tu re was shaken v ig o r o u s ly , and a f t e r s e p a r a t i o n o f aqueous and

o r g a n ic l a y e r s ( u s u a l ly 0 .5 - 1 h r ) , th e aqueous l a y e r was d ra in e d o f f

apd th e o r g a n ic l a y e r , i n c l u d i n g any em u ls io n , was t r a n s f e r r e d to

c e n t r i f u g e tu b e s and c e n t r i f u g e d a t 2000 rpm f o r f i v e m in u te s . The

s u p e r n a t a n t was t r a n s f e r r e d by P a s t e u r p i p e t to an E rlenm eyer f l a s k

w i th a ground g la s s s to p p e r . The em u ls ion and aqueous l a y e r were

r e tu r n e d to th e s e p a r a t o r y fu n n e l and th e e x t r a c t i o n r e p e a te d th r e e

more t im e s . The combined hexane e x t r a c t s w ere b a c k - e x t r a c t e d fo u r

t im es w i th s a t u r a t e d s a l t s o l u t i o n ; th e aqueous s o l u t i o n s were c e n t r i

fuged when n e c e s s a r y . The s a l t s o l u t i o n was saved and r e - e x t r a c t e d

once w i th h ex an e . A l l hexane e x t r a c t s were combined and d e h y d ra ted

o v e r sodium s u l f a t e o v e r n ig h t .

S o x h le t e x t r a c t i o n (Method C)

The p ro c e d u re o f B i e r i and Stam oudis (1977) was fo l lo w e d .

Ten grams o f homogenate w ere w eighed i n t o a t a r e d 250 ml b o i l i n g f l a s k .

F i f t y y l o f S ta n d a rd A, 5 .5 g KOH p e l l e t s , 60 g m ethano l and 20 ml o f

h e x a n e - e x t r a c t e d d e io n iz e d w a te r w ere added. The m ix tu re was r e f lu x e d

f o r 2 h o u rs a t 83-87°C th e n a l lo w ed to co o l to room te m p e ra tu re . I t

was th e n t r a n s f e r r e d i n t o a c o a r s e - f r i t t e d g l a s s th im b le , w hich was

p la c e d i n a S o x h le t e x t r a c t o r c o n ta in in g 200 ml o f m e th an o l.

E x t r a c t i o n p ro c eed ed f o r 15 h r s a t a speed o f 1 .5 - 2 c y c le s p e r h o u r .

The e x t r a c t was a llo w ed to co o l to room te m p e ra tu re , and th e e x t r a c t

removed and sav e d . F re sh m e thano l was added and th e p ro c e d u re was

r e p e a te d ; th e two m e th an o l e x t r a c t s w ere combined. The t o t a l e x t r a c t

was t r a n s f e r r e d t o a 2000 ml s e p a r a t o r y fu n n e l .

A p prox im a te ly 300 ml o f h e x a n e - e x t r a c te d d e io n iz e d w a te r ,

16 g NaCl and a p p ro x im a te ly 80 ml p e n ta n e were added . The m ix tu re was

shaken th o ro u g h ly and th e l a y e r s a l lo w ed to s e p a r a t e , u s u a l l y 1-2 h r s .

The aqueous l a y e r and any em u ls io n w ere th e n d ra in e d i n t o a b e a k e r ,

and th e c l e a r p e n ta n e l a y e r was t r a n s f e r r e d to an E rlenm eyer f l a s k

equ ipped w i th a ground g la s s s to p p e r . The aqueous p o r t i o n was r e tu r n e d

to th e s e p a r a t o r y fu n n e l and th e p ro c e d u re was r e p e a te d tw ic e . The

p e n ta n e l a y e r and any em uls ion formed in th e t h i r d e x t r a c t i o n were

combined w i th th e o t h e r two e x t r a c t s i n a s e p a r a t o r y fu n n e l and washed

th r e e t im es w i th 500 ml h e x a n e - e x t r a c t e d d e io n iz e d w a te r . I f any

em u ls io n w&s p r e s e n t a f t e r th e t h i r d w ash , th e m ix tu re was shaken w i th

0 . 5 - 1 . 0 g NaCl and l e f t 15-30 min. The w a te r l a y e r was d i s c a r d e d and

15

th e p e n ta n e e x t r a c t was t r a n s f e r r e d to an E rlenm eyer f l a s k ; th e fu n n e l

was r i n s e d w e l l w i th ab o u t 20 ml p e n ta n e w hich was th e n added to th e

e x t r a c t . Ten grams o f anhydrous Na£S0^ w ere added , th e f l a s k s to p p e re d

o v e r n ig h t and th e sam ple a l lo w ed to d e h y d ra te .

MacLeod’s P ro c e d u re (Method A)

Ten grams o f homogenate w ere w eighed i n t o a t a r e d 50 ml,

t e f l o n - l i n e d , s c rew -cap p ed c e n t r i f u g e tu b e . S ix ml o f 4N NaOH and

50 y l o f S ta n d a rd A w ere added and th e m ix tu re shaken v ig o r o u s ly f o r

one m in u te . The tu b e s w ere p la c e d i n a 30°C w a te r b a th f o r 18 h r s .

At th e end o f t h i s p e r i o d , th e sam ples w ere c o o led to room te m p e ra tu re

(MacLeod e t a l . , 1976).

F i f t e e n ml o f p e r o x i d e - f r e e e t h e r w ere added to th e sam p les ,

th e tu b e s recap p ed t i g h t l y and shaken v ig o r o u s ly f o r one m in u te . The

tu b e s w ere th e n c e n t r i f u g e d a t 2000 rpm f o r 10 min. I f th e e t h e r phase

was c l e a r , i t was t r a n s f e r r e d w i th a P a s t e u r p i p e t to a 50 ml g l a s s

s to p p e r e d c e n t r i f u g e tu b e . A pprox im ate ly 1 g o f anhydrous Na2S0^ was

added , w i th o u t a g i t a t i o n . The e x t r a c t i o n was r e p e a te d w i th 10 ml o f

e t h e r . The e x t r a c t s w ere combined and 3 g o f anhydrous Na2S0^ were

added . The sam ples w ere l e f t s to p p e r e d o v e r n ig h t (MacLeod e t a l . ,

1976).

E v a p o ra t io n o f e x ce ss s o lv e n t

Sample volumes f o r KOH a l c o h o l i c d i g e s t i o n and S o x h le t

e x t r a c t i o n w ere re d u ced to ab o u t 6 ml i n a warm w a te r b a th u s in g a

r o f a r y e v a p o r a to r co n n ec ted to a w a te r a s p i r a t o r f o r rem oval o f v a p o rs .

The c o n c e n t r a t e d sam ple was th e n t r a n s f e r r e d by P a s t e u r p i p e t i n t o 15 ml

tu b es and f u r t h e r c o n c e n t r a t e d , i n a w arm -w ater b a th 30°C f o r e t h e r

sam ples and 6 0 °C f o r a l l o t h e r s ) u nder a g e n t l e ^ s t re a m , to about

1 .0 m l.

Column chrom atography

A 10 x 300 mm ch ro m a to g ra p h ic column w i th a s e a l e d - i n c o a r se

p o r o s i t y f r i t t e d d i s c i n th e b o tto m was u sed . The column was packed

by p o u r in g a hexane s l u r r y o f a c t i v a t e d B i o - S i l s i l i c a g e l i n t o th e

tu b e . The s i l i c a was s e t t l e d by v i b r a t i o n to a h e i g h t o f 1 7 .5 cm

(a b o u t 7 g ) . About 0 .5 cm o f S o x h l e t - e x t r a c t e d sand was added . The

column was washed w i th ab o u t 40 ml h ex an e . When th e s o lv e n t l e v e l

re a c h e d th e top o f th e a d s o r b e n t , th e sam ple was added u s in g a P a s t e u r

p i p e t . A f t e r th e l e v e l o f th e sam ple had reach ed th e top o f th e

a d s o r b e n t , th e tu b e was r i n s e d w i th abou t 1 ml h exane . C o l l e c t i o n o f

th e f r a c t i o n s began im m ed ia te ly a f t e r th e sam ple was added. F i r s t

th e column was e l u t e d w i th h e x an e . The f i r s t 5 ml w ere d i s c a r d e d ;

th e n e x t 15 ml w ere sav ed and l a b e l e d as th e a l i p h a t i c f r a c t i o n . Then

th e column was e l u t e d w i th 40 /60 (v /v ) b en zen e -h ex an e m ix tu re ; th e

n e x t 30 ml w ere c o l l e c t e d and l a b e l e d as th e a ro m a t ic f r a c t i o n .

F r a c t i o n s w ere c o n c e n t r a te d to a b o u t 0 .2 ml as d e s c r ib e d above (Sm ith

e t a l . , 19 77; B i e r i and S tam oud is , 1977).

Gas chrom atography (gc)

The gas chrom atograph was a V a r ia n 2740 w i th d u a l colum ns,

d i r e c t d a t a o u tp u ts to s t r i p c h a r t r e c o r d e r s i n a H e w le t t -P a c k a rd 3352B

l a b o r a t o r y d a t a sy s tem . G lass c a p i l l a r y columns, c o a te d by th e method

o f Grob e t a l . (1 9 7 7 ) , w ere 20 m x 0 .2 8 mm w i th 0.20% SE52, a s i l i c o n

m e th y l p h e n y l l i q u i d p h a se , and w ere programmed from 5 0 -240°C a t 6°C/

m in u te ( th e n h e ld u n t i l n C ^ d e t e c t e d ) . The He c a r r i e r gas had a

17

p r e s s u r e o f a round 12 p s i w i th a t o t a l flow r a t e o f 30 m l/m in u te . With

th e s p l i t t e r s open, th e He f low i n t o th e columns was 6 -8 m l/m in u te . The

flam e i o n i z a t i o n d e t e c t o r had a H2 f low r a t e o f 30 m l/m in u te and an

a i r f low o f 300 m l/m in u te a t 265°C. S e n s i t i v i t y was 1 x 10” H amp/mv.

Response f a c t o r s o f a l i p h a t i c and a ro m a t ic s t a n d a r d s ( l i s t e d

i n T ab le s A24 and A25 Appendix) w ere used f o r th e q u a n t i f i c a t i o n o f th e

sam ple f r a c t i o n s . Two f r a c t i o n s w ere i n j e c t e d s im u l ta n e o u s ly as a

tim e s a v in g s t e p . The columns w ere in d e p e n d e n t ly c a l i b r a t e d and th e

two f r a c t i o n s n e v e r compared one to a n o th e r . W ith th e s p l i t t e r s c lo se d

and a 50°C oven, sam ples w ere i n j e c t e d ; t im ed th r e e seconds th en th e

s y r in g e s w ere removed. T em pera tu re programming was s t a r t e d one m in u te

a f t e r th e rem oval o f th e s y r i n g e s . S p l i t t e r s w ere opened f o r each

ch an n e l when th e s o l v e n t peak a p p e a re d .

Method o f q u a n t i f i c a t i o n

S ta n d a rd s (T ab le s A24 and A25 Appendix) w ere f i r s t i n j e c t e d

i n t o each column; one column was u sed e x c lu s iv e ly f o r a ro m a t ic com

pounds and th e o t h e r f o r a l i p h a t i c compounds. Once each peak was

i d e n t i f i e d and a s s ig n e d a r e t e n t i o n in d e x and a r e a , th e F f a c t o r was

c a l c u l a t e d f o r each peak as fo l lo w s :

p _ c o n c e n t r a t i o n (m g /y l) x i n j e c t i o n volume ( y l ) x 1000a r e a ( m i c r o v o l t - s e c )

w here th e c o n c e n t r a t i o n was th e known s t a n d a r d c o n c e n t r a t i o n and th e

a r e a o f th e peak u n d e r s tu d y .

Then f o r each s t a n d a r d a g raph was made f o r F f a c t o r v e rsu s

r e t e n t i o n i n d i c e s ( R . I . ) .

Peaks i n th e sam ple chromatogram were th en i d e n t i f i e d and

a s s ig n e d r e t e n t i o n i n d i c e s and a r e a s . The a r e a f o r each peak was

18

m u l t i p l i e d by th e F f a c t o r ( t a k e n from th e s t a n d a r d g raph f o r t h a t

p a r t i c u l a r c h an n e l and r e t e n t i o n in d ex ) to g iv e th e ygrams i n j e c t e d .

This number was th e n m u l t i p l i e d by th e K f a c t o r as f o l lo w s :

K _ ______ t o t a l volume (ml)_____ _____________1000 x d ry w e ig h t (g) x i n j e c t i o n volume ( y l )

where d ry w e ig h t = d ry w e ig h t /w e t w e ig h t r a t i o x t o t a l sam ple w et w e ig h t

to g iv e th e yg/g d ry w e ig h t f o r t h a t p eak . The h y d ro ca rb o n w e ig h t was

t o t a l e d f o r a l l th e peaks to g e t th e t o t a l e x t r a c t e d h y d ro ca rb o n s f o r

e i t h e r th e a l i p h a t i c o r a ro m a t ic f r a c t i o n .

This c a l c u l a t i o n p ro c e s s was c l a s s i f i e d as th e e x t e r n a l

s t a n d a r d method w hich assumes 100% re c o v e ry f o r t h e e x t r a c t e d h y d ro c a rb o n s .

The i n f e r n a l s t a n d a r d method c a l c u l a t e s th e t h e o r e t i c a l r e c o v e ry f o r

e x t r a c t e d h y d ro c a rb o n s b a se d upon th e known p e r c e n t r e c o v e r i e s o f added

s t a n d a r d s .

P r e l im in a r y d e te r m in a t io n o f e f f i c i e n c i e s

Loss o f h y d ro ca rb o n s i s a p o s s i b l e p roblem a t a number o f

s t e p s c r i t i c a l to th e a n a ly s e s . To e s t i m a te th e o c c u r re n c e and th e

m agn itude o f l o s s , e x t r a c t i o n e f f i c i e n c i e s w ere d e te rm in ed f o r each

te c h n iq u e u s in g s t a n d a r d s i n p l a c e o f sam ples . E x t r a c t i o n e f f i c i e n c i e s

w ere d e te rm in e d f o r each o f th e t h r e e methods by p l a c i n g 50 y l of

S ta n d a rd A s o l u t i o n i n th e a p p r o p r i a t e v e s s e l ( s e e e x t r a c t i o n p ro c e d u re s

a b o v e ) , and t r e a t i n g i t as sam ple . Loss d u r in g s o l v e n t r e d u c t io n

was e s t im a te d by p l a c i n g a 100 y l a l i q u o t S ta n d a rd B, d i l u t e d to 100 ml,

i n a b o i l i n g f l a s k , and t r e a t i n g i t as a sam ple . Loss d u r in g column

chrom atography was m easured by f r a c t i o n a t i o n o f a 100 y l a l i q u o t o f

S ta n d a rd B t h a t had been d i l u t e d to 1 .0 ml w i th h exane . Loss d u r in g

e v a p o r a t io n u n d e r ^ was e s t im a te d by r e d u c t io n o f a 100 y l a l i q u o t

19

o f S ta n d a rd B ( d i l u t e d to 1 .0 ml i n hexane) to ab o u t 0 .2 ml i n a 15 ml

g l a s s s to p p e r e d c e n t r i f u g e tu b e . S ta n d a rd c o n c e n t r a t i o n s w ere m easured

by GC and t h e i r e f f i c i e n c i e s d e te rm in e d .

S t a t i s t i c a l A n a ly s is

P a r a m e t r i c te c h n iq u e s w ere chosen f o r th e s t a t i s t i c a l a n a ly s i s

o f th e d a t a , and th e d a t a w ere examined to c o n f irm t h a t th ey s a t i s f i e d

th e u n d e r ly in g a ssu m p tio n s f o r w hich th e te c h n iq u e s a r e v a l i d :

hom ogeneity o f v a r i a n c e ; no rm al d i s t r i b u t i o n ; and random sam p les .

B a r t l e t t ^ t e s t o f hom ogeneity o f v a r i a n c e (S nedeco r , 1956) was computed

f o r th e e x t r a c t e d re c o v e re d w e ig h ts and f o r th e a r c s i n e tra n s fo rm e d

p e r c e n t r e c o v e r i e s f o r th e i n t e r n a l s t a n d a r d s . In th e h a n d l in g o f th e

p e r c e n ta g e s , i f t h e form o f th e p a r e n t p o p u la t io n i s known to be

r e a s o n a b ly c lo s e to a d i s t r i b u t i o n f o r w hich t h e r e i s a s t a n d a r d

( p a r a m e t r i c ) th e o r y , o r i f th e d a t a can be t ra n s fo rm e d so t h a t such i s

th e case* th e n p a r a m e t r i c p ro c e d u re s e x t r a c t more in f o r m a t io n from th e

d a t a th a n do n o n p a ra m e tr ic p ro c e d u re s ( S t e e l and T o r r i e , 1960). A rc s in e

t r a n s f o r m a t io n f u l f i l l s t h i s r e q u i r e m e n t . A no ther p ro c e d u re to check

th e norm al d i s t r i b u t i o n o f d a ta i s a g ra p h ic method d e s c r i b i n g th e

r e l a t i o n s h i p s e x p re s s e d be th e norm al d i s t r i b u t i o n a v a i l a b l e i n terms

o f p r o b a b i l i t y p a p e r . D ata which fo l lo w th e norm al p r o b a b i l i t y curve

w i l l p l o t as a s t r a i g h t l i n e on t h i s g r i d , t h u s , t h e r e i s a v a i l a b l e a

q u ic k method o f t e s t i n g th e n o r m a l i ty o f a s e r i e s o f o b s e r v a t io n s . The

p a r a m e t r i c te c h n iq u e s o f ANOVA and S tudent-N ew m an-K eul 's T e s t were

a p p l i e d to th e e v a l u a t i o n d a t a .

RESULTS

N ine r e p l i c a t e s w ere u sed f o r th e S o x h le t e x t r a c t i o n and

KOH a l c o h o l i c d i g e s t i o n ; t h i r t e e n r e p l i c a t e s w ere used f o r MacLeod's

p r o c e d u re . B a r t l e t t ' s T e s ts and t h e p r o b a b i l i t y g raphs i n d i c a t e d t h a t

th e e x t r a c t e d re c o v e re d w e ig h ts and p e r c e n t s t a n d a r d r e c o v e r i e s w ere

n o rm a lly d i s t r i b u t e d i f t h e Y/9 KOH a l c o h o l i c d i g e s t i o n r e p l i c a t e f o r

e x t r a c t e d re c o v e re d w e ig h ts i s n o t in c lu d e d . The a ro m a tic and a l i p h a t i c

h y d ro c a rb o n f r a c t i o n s o f clam t i s s u e e x t r a c t s as d e te rm in ed by gas

chrom atography a r e g iv e n i n T ab les 2 and 3 , r e s p e c t i v e l y . These w e ig h ts

w ere c a l c u l a t e d u s in g th e e x t e r n a l s t a n d a r d method. Mean (x) r e c o v e r i e s

f o r th e a l i p h a t i c f r a c t i o n w ere 7 .04 y g /g d ry w e ig h t f o r MacLeod's

p ro c e d u re , 5 ,2 4 Ug/g dry w e ig h t f o r th e S o x h le t e x t r a c t i o n and 7 .96

y g /g d ry w e ig h t f o r KOH a l c o h o l i c d i g e s t i o n . For th e a ro m a t ic f r a c t i o n ,

th e meay (x) r e c o v e r i e s w ere 4 .27 y g /g dry w e ig h t , 3 .73 y g /g dry w e ig h t

and 3 .95 y g /g dry w e ig h t r e s p e c t i v e l y . An a n a l y s i s o f v a r i a n c e (ANOVA)

was p e rfo rm ed on th e means f o r each te c h n iq u e (T ab le A26 Appendix) and

showed no s i g n i f i c a n t d i f f e r e n c e be tw een te c h n iq u e s (P < 0 .0 5 ) . An

e x a m in a t io n o f th e gas chromatograms i n d i c a t e d t h a t a l l t h r e e te c h n iq u e s

w ere e x t r a c t i n g th e same o r s i m i l a r compounds. The a l i p h a t i c f r a c t i o n

was c h a r a c t e r i z e d by th e n - a l k a n e s , t i ro u 8^1 n ^ 3 i* a ro m a t ic

f r a c t i o n had two d i s t i n c t g roups o f p eak s : one w i th r e t e n t i o n i n d i c e s

be tw een 1700 and 2300; th e o t h e r w i th r e t e n t i o n i n d i c e s be tw een 2800 and

3100. The l a r g e s t a n d a r d d e v i a t i o n d e te rm in e d f o r KOH a l c o h o l i c d i g e s t i o n

20

TABLE 2

RECOVERED WEIGHTS OF ALIPHATIC HYDROCARBON FRACTION

CALCULATED BY THE EXTERNAL STANDARD METHOD

G-lG/G DRY WEIGHT TISSUE)

R e p l i c a te s T echniques

MacLeod * s P ro ce d u re

S o x h le tE x t r a c t i o n

KOHA lc o h o l icD ig e s t io n

n 8 .93 2 .20 4 .8 1

2 7 .45 4 .3 2 9.59

3 8 .60 5 .03 7 .87

4 4 .63 7 .14 6 .12

5 4 .92 6 .92 4.19

6 3 .07 6 .5 8 5 .81

7 5.72 3 .9 8 8.73

8 5 .8 7 3 .0 1 4.99

9 7 .30 7 .9 8 19.55

10 10.00

11 9 .6 4

12 8 .33

13 7 .03

x + s 7 .04 + 2 .09 5 .24 + 2 .0 1 7 .96 + 4 .72

( s p ik e s n o t in c lu d e d i n r e c o v e re d w e ig h ts )

21

TABLE 3

RECOVERED WEIGHTS OF AROMATIC HYDROCARBON FRACTION

CALCULATED BY THE EXTERNAL STANDARD METHOD

(yG/G DRY WEIGHT TISSUE)

R e p l i c a te s Techniques

MacLeod1s P ro c e d u re



#1 4 .2 3 2.56 2 .36

2 5 .0 3 3.67 2 .47

3 5.19 4 .0 1 5 .96

4 4 .7 3 3 .92 2 .84

5 3 .03 4 .37 2 .95

6 2 .8 8 3 .36 2 .83

7 4 .12 4 .1 1 4 .3 8

8 3 .67 3.17 2 .78

9 5 .87 4 .44 8 .97

10 2 .96

11 4 .9 3

12 4 .8 4

13 4 .05

x + s 4 .2 7 + 0 .9 4 3 .73 ± 0 .6 1 3 .95 + 2 .21

( s p ik e s n o t in c lu d e d in re c o v e re d w e ig h ts )

22

23

was due to r e p l i c a t e #9. W i t h . t h i s r e p l i c a t e e x c lu d e d , th e s t a n d a r d

d e v i a t i o n f o r th e KOH method was s i m i l a r to th o se f o r th e o t h e r two

te c h n iq u e s and s t i l l no d i f f e r e n c e s betw een te c h n iq u e s w ere d e t e c t e d by

ANOVA.

The p e r c e n t r e c o v e r i e s o f th e s t a n d a r d compounds a f t e r a r c s i n e

t r a n s f o r m a t i o n from th e s p ik e d clam t i s s u e w ere :

S ta n d a rd P e r c e n t Recovery (_x - s )

2 -m e th y lo c ta d e c a n e 54 .10 + 12 .53

2 -m e th y le ic o sa n e 50 .84 + 12 .64

d o t r i a c o n t a n e n C ^ 41 .57 + 9 .6 0

h exam ethy lbenzene 36 .90 + 7 .04

b e n z o ( g h i ) p e r y le n e 65 .54 + 11.59

(T ab le s A1-A5 Appendix)

A s t a t i s t i c a l e x am in a t io n o f th e s t a n d a r d and in d ig e n e o u s

compounds showed no d i f f e r e n c e i n th e r e c o v e r i e s f o r th e t h r e e te c h n iq u e s

The s t a n d a r d compounds d id , how ever, show (by ANOVA, Table A27 Appendix)

a s i g n i f i c a n t v a r i a t i o n i n t h e i r p e r c e n t r e c o v e r i e s . The S tu d e n t -

Newman-Keul’s T e s t (SNK T ab le A6 Appendix) i l l u s t r a t e s th e d i f f e r e n c e s .

The d a t a l i s t e d i n T ab le s 2 and 3 w ere n o rm a liz e d to 100%

e f f i c i e n c y u s in g th e i n t e r n a l s t a n d a r d method. In t h i s m ethod, th e

a r e a o f th e s e l e c t e d s t a n d a r d was assumed to i n d i c a t e 100% re c o v e ry and

th e a r e a s f o r th e o th e r peaks were n o rm a l iz e d i n r e l a t i o n to t h i s

s t a n d a r d . The means f o r th e n o rm a l iz e d d a ta a r e l i s t e d i n T ab le 4.

ANOVA (T a b le s A28 and A29 Appendix) f o r th e n o rm a liz e d d a ta r e v e a le d

s i g n i f i c a n t d i f f e r e n c e s be tw een methods f o r 2 -m e th y lo c ta d e c a n e , 2-

m e th y le ic o s a n e , n C32* and b e n z o ( g h i ) p e r y le n e ; t h e r e was no s i g n i f i c a n t

TABLE 4

NORMALIZED RECOVERED WEIGHTS OF HYDROCARBONS (yG/G DRY WEIGHT TISSUE)

(x + s)

I n t e r n a l S ta n d a rd

A l i p h a t i c F r a c t i o n

T echniques

MacLeod’s P ro ce d u re



2 -m e th y lo c ta d ec a n e*

2-'rmethy l e i cos ane*

nC32*

1 3 .0 8 ± 3 .73 9 .0 8 ± 2 .13

13 .82 + 3 .9 1 9 .5 7 + 2 .6 8

15 .11 + 3 .6 8 9 .4 6 + 5 .72

15 .27 + 6 .05

17 .28 ± 10.30

10 .96 ± 5 .88

hexam ethy lbenzene

b e n z o ( g h i ) p e r y le n e *

A rom atic F r a c t io n

1 2 . 6 9 + 4 . 2 6 9 . 1 2 + 1 . 2 9 1 1 . 8 4 + 7 .78

7 .2 1 + 1 .22 5 .94 + 1 .0 8 5 .05 + 2 .17

T ab les A7-A11 Appendix

*AN0VA i n d i c a t e d s i g n i f i c a n t d i f f e r e n c e be tw een te c h n iq u e s a t th e 95% c o n f id e n c e l e v e l .

24

25

d i f f e r e n c e i n e x t r a c t i o n r e c o v e r i e s f o r h ex am ethy lbenzene . The S tu d e n t -

Newman-Keul1s T e s ts f o r t h e fo u r s i g n i f i c a n t f r a c t i o n s i l l u s t r a t e th e

d i f f e r e n c e s i n T ab le 5.

S p ik ed b la n k s w ere ru n f o r each te c h n iq u e to p ro v id e an

i n d i c a t i o n o f th e l o s s e s due s o l e l y to th e p re s e n c e o f t i s s u e . The

means (x) and s t a n d a r d d e v i a t i o n s ( s ) a r e i l l u s t r a t e d i n T ab le 6.

ANOVA (T ab le 30 Appendix) i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e

be tw een th e e x t r a c t i o n r e c o v e r i e s o f th e t h r e e te c h n iq u e s a t th e 95%

c o n f id e n c e l e v e l . ANOVA (T ab le A30 Appendix) i n d i c a t e d no d i f f e r e n c e

be tw een compounds i n e x t r a c t i o n r e c o v e r i e s f o r KOH a l c o h o l i c d i g e s t i o n

and S o x h le t e x t r a c t i o n ; th e SNK T e s t f o r MacLeod's p ro c e d u re i s i l l u s t r a t e d

i n T ab le A19 (A p p en d ix ) .

C o n c e n t r a t io n u n d e r c o n c e n t r a t io n u n d e r r o t a r y e v a p o r a to r

and column chrom atography w ere e v a lu a te d u t i l i z i n g a s p ik e w i th s ta n d a rd s

s i m i l a r to th e ones usee} f o r th e s p ik e d t i s s u e and b la n k s . The r e c o v e r i e s

f o r th e s t a n d a r d s a t v a r io u s s t e p s i n th e te c h n iq u e s w ere:

P ro c e d u re P e rc e n t Recovery (x _ s

p ro c e d u re b la n k 70 .4 + 1 5 .0

column chrom atography andc o n c e n t r a t i o n u n d e r N2 8 0 .8 + 6 .4

c o n t e n t r a t i o n by r o t a r ye v a p o r a to r and N2 9 1 .8 + 4 .0

c o n c e n t r a t i o n u n d e r N2 9 4 .1 + 3 .0

(T ab le s A16^A18; A20-A22 Appendix)

ANOVA (T ab le A31 Appendix) i n d i c a t e d no d i f f e r e n c e betw een

compounds f o r th e two c o n c e n t r a t i o n s t e p s . The SNK f o r th e column

chrom atography (T ab le A23 Appendix) showed a s i g n i f i c a n t d i f f e r e n c e

TABLE 5

STUDENT-NEWMAN-KEUL1s TESTS (SNK)

A l i p h a t i c F r a c t i o n N orm alized to 2 -m e th y lo c ta d ec a n e I n t e r n a l S ta n d a rd

S o x h le t MacLeod’s KOHE x t r a c t i o n P ro c e d u re A lc o h o l ic d i g e s t i o n

9 .0 8 1 3 .08 15 .27

A l i p h a t i c F r a c t i o n N orm alized to 2 -m e th y le ic o sa n e I n t e r n a l S ta n d a rd

S o x h le t MacLeod's KOHE x t r a c t i o n P ro c e d u re A lc o h o l ic d i g e s t i o n

9 .5 7 13 .82 17 .28

A l i p h a t i c F r a c t i o n N orm alized to n C ^ I n t e r n a l S ta n d a rd

S o x h le t KOH MacLeod'sE x t r a c t i o n A lc o h o l ic d i g e s t i o n P ro ced u re

9 .4 6 10 .96 15 .11

A rom atic F r a c t i o n N orm alized to b e n z o ( g h i ) p e r y le n e I n t e r n a l S ta n d a rd

KOH S o x h le t MacLeod'sA lc o h o l ic d i g e s t i o n E x t r a c t i o n P ro ced u re

5 .0 5 5 .9 4 7 .21

T ab les A12-A15 Appendix

Those compounds s h a r i n g a common u n d e r l i n e w ere n o t s i g n i f i c a n t l y d i f f e r e n t . R e s u l t s a r e i n term s o f y g /g d ry w e ig h t t i s s u e .

26

TABLE 6

STANDARD RECOVERIES AS ARCSINE TRANSFORMED PERCENTS FROM

SPIKED BLANKS FOR THE COMPLETE TECHNIQUES (x + s ’)

I n t e r n a l S ta n d a rd T echniques

MacLeodVs P ro ce d u re



2-me thy l o c t ade cane

2 -m e th y le i cos ane

nC32

h ex ame th y lb e n z e n e

b e n z o ( g h i ) p e r y le n e

7 4 .0 + 8 .2

7 6 .0 + 11 .1

5 1 .6 + 11 .1

5 4 .0 ± 9 .2

78.4 + 9 .9

7 7 .3 ± 3 .3

79 .6 + 11 .3

9 1 .1 + 4 5 .0

5 4 .8 + 6 .9

8 1 . 7 + 5 .2

74 .5 ± 5 .2

76 .7 + 10 .4

5 5 .6 + 26 .2

5 3 .3 + 8 .3

77.4 + 9 .7

(T ab le s A16-A18 Appendix)

27

28

(P<0.05) be tw een 311 t ie two compounds nC2 g and b e n z o (a )p y re n e .

The r e p r o d u c i b i l i t y o f gas ch ro m ato g rap h ic i n j e c t i o n s f o r

a l i p h a t i c and a ro m a t ic f r a c t i o n s i s i l l u s t r a t e d i n T ab les A24 and A25.

DISCUSSION

T his i n v e s t i g a t i o n was conducted to e v a l u a t e t h r e e commonly

u sed p ro c e d u re s f o r th e e x t r a c t i o n o f h y d ro ca rb o n s from clam t i s s u e .

The t o t a l r e c o v e re d h y d ro ca rb o n s from MacLeod's p ro c e d u re (1 1 .3 1 y g /g

dry w e ig h t)* KOH a l c o h o l i c d i g e s t i o n (1 1 .9 1 y g /g d ry w e ig h t ) , and

S o x h le t e x t r a c t i o n (8 .9 7 y g /g dry w e ig h t) i n d i c a t e d t h a t MacLeod's

p ro c e d u re and KOH a l c o h o l i c d i g e s t i o n ap p ea red m a r g in a l ly more e f f i c i e n t

th a n S o x h le t e x t r a c t i o n b u t t h e s e d i f f e r e n c e s w ere n o t s t a t i s t i c a l l y

s i g n i f i c a n t . N o rm a l iz a t io n o f th e re c o v e re d h y d ro ca rb o n f r a c t i o n s

to i n t e r n a l s t a n d a r d s p roduced anomalous r e s u l t s (T ab le s 4 and 5 ) .

S i g n i f i c a n t d i f f e r e n c e s among te c h n iq u e s w ere d e te rm in ed f o r f r a c t i o n s

n o rm a l iz e d to a l l s t a n d a r d s e x c e p t h ex am ethy lbenzene . I n t e r p r e t a t i o n

i s d i f f i c u l t b e ca u se no te c h n iq u e proved to be c o n s i s t e n t l y s u p e r i o r .

The o n ly c o n s i s t e n t o b s e r v a t io n i n t h i s e v a l u a t i o n was t h a t S o x h le t

e x t r a c t i o n was l e a s t e f f i c i e n t f o r e x t r a c t i o n o f a l i p h a t i c h y d ro c a rb o n s .

However, th e t h r e e te c h n iq u e s must be c o n s id e re d e q u iv a l e n t s i n c e raw

r e c o v e r i e s w ere s t a t i s t i c a l l y s i m i l a r and r e c o v e r i e s n o rm a liz e d to

th e i n t e r n a l s t a n d a r d s d id n o t behave c o n s i s t e n t ly *

Commonly u sed te c h n iq u e s f o r th e q u a n t i f i c a t i o n o f h y d ro

ca rb o n s from e n v iro n m e n ta l sam ples p r e s e n t l y in c lu d e g r a v im e t r i c

and gas c h ro m a to g ra p h ic d e te r m in a t io n u s in g e i t h e r th e e x t e r n a l s t a n d a r d

method o r th e i n t e r n a l s t a n d a r d method. G ra v im e tr ic d e te r m in a t io n

p r o v id e s a b u lk m easurement o f th e e x t r a c t e d h y d ro ca rb o n s w i th v e ry

29

l i t t l e q u a l i t a t i v e d a ta and y i e l d s no i n d i c a t i o n o f th e e f f i c i e n c y o f

t h e e x t r a c t i o n o f h y d ro c a rb o n s . G ra v im e tr ic methods a r e most u s e f u l

w here r e l a t i v e l y l a r g e amounts (10“ ^ g) o f each h y d ro c a rb o n f r a c t i o n

e x i s t (C la rk , 1973). Gas chrom atography p r o v id e s b o th a s e p a r a t i o n and

a q u a n t i t a t i v e e s t i m a t e o f s p e c i f i c compounds. Q u a n t i f i c a t i o n by th e

e x t e r n a l s t a n d a r d method p ro v id e s no i n d i c a t i o n o f th e e f f i c i e n c y o f

h y d ro ca rb o n e x t r a c t i o n . The i n t e r n a l s t a n d a r d method p ro v id e s some

i n d i c a t i o n o f e x t r a c t i o n e f f i c i e n c i e s , how ever, v a r i a t i o n s i n r e c o v e r i e s

o f th e i n t e r n a l s t a n d a r d s th em se lv es in d u ce u n c e r t a i n t i e s i n r e s u l t s .

T h is s tu d y d e m o n s t ra te s t h a t dtH eaent tntesip/ietattons ofi data can be

made based &o£e£y upon the qu,ayit£££cation method. i n p re v io u s s t u d i e s ,

G r i t z and Shaw (1 9 7 7 ) , W arner (1 9 7 6 ) , and B la y lo c k e t a l . (1973) used

th e i n t e r n a l s t a n d a r d method; F a r r in g to n and M edeiros (1975) used th e

g r a v im e t r i c method and MacLeod e t a l . (1976) used a g r a v im e t r i c method

and a gas c h ro m a to g rap h ic method u t i l i z i n g a s t a n d a r d added a f t e r

e x t r a c t i o n and c o n c e n t r a t i o n p ro c e d u re s . I t i s im p o r ta n t t h a t s e v e r a l

methods o f q u a n t i f i c a t i o n y i e l d th e same r e s u l t s b e f o r e c o n c lu d in g

t h a t one e x t r a c t i o n te c h n iq u e i s more e f f i c i e n t th a n a n o th e r .

A l l o f t h e added h y d ro ca rb o n s t a n d a r d s w ere re c o v e re d in

l e s s th an 100% y i e l d . These s p ik e r e c o v e r i e s a p p ea r to be s i m i l a r to

r e c o v e r i e s c i t e d i n o t h e r r e f e r e n c e s (B la y lo c k e t a l . , 1973; W arner,

1976; G r i t z and Shaw, 1977). D i r e c t com parison i s n o t a p p r o p r i a t e

s i n c e d i f f e r e n t o rg a n ism s , s p ik e compounds and c o n c e n t r a t i o n s were

u sed . The s l i g h t l y low er l e v e l s re c o v e re d i n t h i s s tu d y may be due

to th e low c o n c e n t r a t i o n o f s p ik e d s t a n d a r d s ( 0 .1 0 - 0 .2 8 y g / g ) .

P re v io u s works (W arner, 1976; G r i t z and Shaw, 1977) used c o n c e n t r a t i o n

31

l e v e l s two to t e n t im es th o s e u t i l i z e d i n t h i s s tu d y . W arner (1976)

d e m o n s tra ted t h a t r e c o v e ry y i e l d d e c r e a s e s w i th d e c r e a s in g s t a n d a r d

c o n c e n t r a t i o n . P r e l im in a r y work i n t h i s l a b o r a to r y i n d i c a t e d t h a t

t h e s e l e c t e d s t a n d a r d c o n c e n t r a t i o n s gave a d eq u a te re c o v e ry and showed

l i t t l e i n t e r f e r e n c e w i th th e in d ig e n o u s h y d ro c a rb o n s . The c o n c e n t r a t i o n

o f th e s p ik e was chosen to b e s i m i l a r i n m agn itude to i n d i v i d u a l

compounds i n th e sam ple so t h a t d e t e c t o r n o n l i n e a r i t y w ould n o t be

t ro u b le so m e .

Nearly a l t the loAAes ofi compounds In the A pike btanks were

accounted fior by three procesAes: concentration under nitrogen,

concentration by rotary evaporation, and column chromatograpky.

H exam ethylbenzene and n C ^ had th e lo w e s t re c o v e ry y i e l d s . Hexa

m e th y lb en zen e i s a c o m p a ra t iv e ly low b o i l i n g h y d ro ca rb o n w hich m ight

be s u s c e p t i b l e to v o l a t i l i z a t i o n , how ever, p re v io u s work (B lay lo ck

e t a l . , 1973) has i n d i c a t e d h i g h e r r e c o v e r i e s f o r compounds w i th lower

b o i l i n g p o i n t s th a n h ex am eth y lb en zen e . S in c e hexam ethy lbenzene was

n o t u sed i n th e e v a l u a t i o n o f th e s t e p s w i t h i n th e p ro c e d u re s (con

c e n t r a t i o n u n d e r n i t r o g e n , r o t a r y e v a p o r a t io n , and column chrom ato

graphy) , t h e r e i s no yay o f d e te r m in in g w here th e l o s s e s o c c u r re d .

A comparison o& the Atandard recjoverles fior tk e clam tiSAue and the

A pike btankA Indicated a 10-20% Zoaa due to the presence o£ the tiSAue

uilth ke^omethythenzene having one o& the higher Zoaacs. T h is i n d i c a t e d

a p o s s i b l e i n t e r f e r e n c e w i th e x t r a c t i o n by th e t i s s u e m atrix* Low

n ^32 y*-e ld s were d e te rm in e d f o r th e s p ik e d b la n k s and s p ik e d t i s s u e

sam ples w hich underw ent column ch rom atography , b u t n o t f o r th e sam ples

c o n c e n t r a t e d on ly u n d e r r o t a r y e v a p o r a t io n and n i t r o g e n . T h is would

s u g g e s t t h a t s i l i c a g e l i n th e column chrom atography p ro c e d u re

r e t a i n s th e h i g h e r m o le c u la r w e ig h t compounds, how ever, p re v io u s work

( G r i t z and Shaw, 19 77) d id n o t r e p o r t low r e c o v e r i e s o f n C ^*

A p o s s i b l e f a c t o r i n th e d e te r m in a t io n o f l o s s e s may be th e

low c o n c e n t r a t i o n s o f s t a n d a r d s u sed i n t h i s s tu d y . For exam ple,

a g iv e n p ro c e d u re may r e t a i n a c o n s t a n t amount o f compound r e g a r d l e s s

o f th e o r i g i n a l c o n c e n t r a t i o n . T h is lo s s would be m inim al w here th e

o r i g i n a l c o n c e n t r a t i o n i s l a r g e w i th r e s p e c t to t h a t l o s s b u t h ig h ly

s i g n i f i c a n t when m agn itudes a re s i m i l a r .

The v a r i a b i l i t y be tw een r e p l i c a t e s may be p a r t i a l l y due

to th e gas chrom atography p ro c e d u re . For th e n - p a r a f f i n s l a r g e r th an

nC2 5 > th e r e p r o d u c i b i l i t y o f gas c h ro m a to g rap h ic i n j e c t i o n s may v a ry

as much as 12-22%. T h is cou ld be due to a change i n s e n s i t i v i t y w i th

i n c r e a s i n g m o le c u la r w e ig h t o r a change i n th e shape and s i z e o f

peaks a t th e u p p e r end o f th e chromatogram (C la rk , 1973). However,

t h e a ro m a t ic s t a n d a r d s (T ab le A25, Appendix) v a r i e d from on ly 1.5-9% ,

e x c lu d in g b e n z o ( g h i ) p e r y le n e Which v a r i e d more th a n 50%. B enzo(gh i)

p e r y le n e i s one o f th e h ig h e r m o le c u la r w e ig h t compounds examined.

The e x p la n a t io n f o r t h i s v a r i a b i l i t y i s p ro b a b ly th e same as f o r th e

l a r g e r n - p a r a f f i n s . MacLeod e t a l . (1976) checked th e r e p r o d u c i b i l i t y

o f r e p l i c a t e gas c h ro m a to g rap h ic sam ple i n j e c t i o n s and found v a r i a t i o n s

o f 10-17% f o r nC£ 4 to n 29 n ^30 p r y i n g 51% and nC3 ^ , 38%. Grob

and Grob (1978) have r e p o r t e d l y e l im in a te d t h i s problem w i th on-column

i n j e c t i o n s o n to th e g l a s s c a p i l l a r y columns. Gas ch ro m ato g rap h ic

i n j e c t i o n s i n t h i s s tu d y w ere h ig h ly r e p r o d u c ib l e f o r most compounds.

The v a r i a b i l i t y w i t h i n th e r e c o v e re d h y d ro ca rb o n w e ig h ts ranged from

17-^0%. B la y lo c k e t a l . (19 73) found t h a t v a r i a t i o n i n re c o v e ry o f

th e i n d i v i d u a l n - a lk a n e s was two to e i g h t t im es low er f o r th e h ig h

c o n c e n t r a t i o n l e v e l s th a n f o r th e minimum and i n t e r m e d i a t e c o n c e n t r a t i o n

l e v e l s . A gain , t h i s i n d i c a t e s th e im p o r tan ce o f th e e f f e c t o f concen

t r a t i o n l e v e l s on th e r e s u l t s . Given the observed uncertainties within

the techniques Atudlcd here, AmaZZ dlfiierences between techniques are

not Algnljjlcant. Choice o f one te c h n iq u e o v e r a n o th e r b ased on th e s e

s m a l l d i f f e r e n c e s ( i . e . Rohrback and Reed*s (1975) recom m endation o f

one te c h n iq u e o v e r a n o th e r b ased on a 4% d i f f e r e n c e ) i s n o t v a l i d .

T h is e v a l u a t i o n a t te m p te d to c o n c e n t r a t e on j u s t a few

te c h n iq u e s and f a c t o r s i n f l u e n c i n g th e e f f i c i e n t e x t r a c t i o n o f hyd ro

carbons from o rg a n ism s . The s e l e c t i o n o f th e t h r e e te c h n iq u e s (S o x h le t

e x t r a c t i o n j MacLeodfs p ro c e d u re , and KOH a l c o h o l i c d ig e s t i o n ) was

b a sed upon s e v e r a l f a c t o r s : p re v io u s u t i l i z a t i o n i n th e l a b o r a to r y

( B i e r i and S tam o u d is , 1977; Smith e t a l . , 1977); u t i l i z a t i o n in

p re v io u s e v a l u a t i o n s t u d i e s (B la y lo c k e t a l . , 1973; F a r r in g to n and

M ed e iro s , 1975; G r i t z and Shaw, 1977); and p re v io u s d e te r m in a t io n o f

h ig h r e c o v e r i e s and s i m p l i f i c a t i o n o f te c h n iq u e s (W arner, 1976;

MacLeod e t a l . , 1976). S im i l a r y i e l d s w ere ex p ec te d due to th e

b a s i c s i m i l a r i t i e s betw een te c h n iq u e s (an a l k a l i n e d i g e s t i o n fo llow ed

by an o rg a n ic s o l v e n t e x t r a c t i o n ) and th e r e s u l t s o f p re v io u s e v a lu a

t i o n s ( F a r r in g to n and M ed e iro s , 1975; G r i t z and Shaw, 1977). A lk a l in e

d i g e s t i o n p r i o r to S o x h le t e x t r a c t i o n was pe rfo rm ed to t o t a l l y d i s r u p t

th e c e l l f o r e a s i e r a c c e s s to hyd ro ca rb o n s c o n ta in e d w i t h i n th e c e l l .

I t was f e l t t h a t S o x h le t e x t r a c t i o n would n o t g iv e com ple te re c o v e ry

as h y d ro ca rb o n s a s s o c i a t e d w i th th e c e l l l i p i d s may n o t be a d e q u a te ly

s e p a r a t e d from th e t i s s u e s (B la y lo c k e t a l . , 1973). S e l e c t i o n o f

clam t i s s u e was i n d i c a t e d by th e p re v io u s u t i l i z a t i o n o f s h e l l f i s h

34

f o r h y d ro c a rb o n e x t r a c t i o n s t u d i e s (B la y lo c k e t a l . , 1973; F a r r in g to n

and M ed e iro s , 1975; G r i t z and Shaw, 1977). T h is s tu d y encompassed

p ro c e d u re s o f p a s t e v a lu a t io n s by in c lu d in g a l k a l i n e d i g e s t i o n and

S o x h le t e x t r a c t i o n ( F a r r in g to n and M ed e iro s , 1975; G r i t z and Shaw,

1977).

Th is s tu d y has t r i e d to improve on p re v io u s e v a l u a t i o n s by

d e m o n s t ra t in g th e need to e v a lu a t e te c h n iq u e s u s in g s e v e r a l q u a n t i

t a t i v e m ethods, by th e u se o f a l a r g e r number o f r e p l i c a t e s , and by

th e a p p l i c a t i o n o f s t a t i s t i c a l t e s t s f o r s i g n i f i c a n c e o f r e s u l t s .

An i n f r e q u e n t l y m en tioned d i f f i c u l t y i s th e fo rm a t io n o f

p e r s i s t e n t em u ls io n s d u r in g th e e x t r a c t i o n p ro c e d u re . In t h i s s tu d y ,

r e c o v e r i e s f o r S o x h le t e x t r a c t i o n and KOH a l c o h o l i c d i g e s t i o n w ere

n o t low ered w here an em uls ion was p r e s e n t compared to when i t was

a b s e n t . At th e p r e s e n t t im e , th e m ost s a t i s f a c t o r y t r e a tm e n t con

s i s t s o f e x te n s iv e tim e consum ing, s o l v e n t - s o l v e n t p a r t i t i o n i n g .

Em ulsions a p p ea r to be due to a number o f f a c t o r s . B la y lo c k e t a l .

(1973) found t h a t th e a d d i t i o n o f e x c e s s iv e w a te r ten d ed to red u ce

th e e x t r a c t i o n e f f i c i e n c y b e c a u se o f em u ls ion fo rm a t io n d u r in g th e

s o l v e n t e x t r a c t i o n p ro c e d u re . G r i t z and Shaw (1977) and p re v io u s

work i n t h i s l a b o r a t o r y i n d i c a t e d t h a t norm al a lk a n e s o lv e n t s such as

hexane o r p e n ta n e caused em u ls io n p ro b lem s . The use o f d ic e d r a t h e r

th a n homogenized t i s s u e re d u ced th e fo rm a tio n o f an em uls ion w i th o u t

a f f e c t i n g h y d ro ca rb o n re c o v e ry ( G r i t z and Shaw, 1977). No em uls ion

was formed i n t h i s s tu d y w i th th e u se o f an aqueous r a t h e r th an an

a l c o h o l i c d i g e s t i o n m ix tu r e . One prob lem w hich may o ccu r w i th

M acLeod's p ro c e d u re and KOH a l c o h o l i c d i g e s t i o n i s th e fo rm a t io n o f

a g e l upon s o l v e n t e v a p o r a t io n . This h ig h ly v is c o u s m a t e r i a l can

35

p lu g an a d s o r p t io n chrom atography column, p r e v e n t in g s o lv e n t f low .

However, th e sam ple g e l can be d i s s o lv e d i n m eth y len e c h lo r id e and

f i l t e r e d th ro u g h a bed o f ch ro m a to g rap h ic s i l i c a g e l prew ashed w i th

m e th y len e c h l o r i d e . The m ethy lene c h lo r id e s o l v e n t i s th e n d i s p l a c e d

by hexane f o r a d s o r p t io n chrom atography (MacLeod e t a l . , 1976).

Drawing o f c o n c lu s io n s from th e r e s u l t s i n any e v a l u a t i o n o f

h y d ro c a rb o n e x t r a c t i o n te c h n iq u e s sh o u ld always be done w i th th e c o r r e c t

p e r s p e c t i v e . The r e s u l t s o f t h i s s tu d y and p re v io u s work i l l u s t r a t e

t h a t th e d e te r m in a t io n o f th e m ost e f f i c i e n t p ro c e d u re may be i n

f lu e n c e d by th e s e l e c t i o n o f a g iv en s p i k e , c o n c e n t r a t io n o r q u a n t i

f i c a t i o n m ethod. Any c o n c lu s io n drawn sh o u ld be c o n d i t i o n a l upon th e

f a c t o r s t h a t in fb u e n c e th e r e s u l t s o f th e e v a l u a t i o n .

O th e r f a c t o r s become p e r t i n e n t i n i n f l u e n c in g a d e c i s io n when

a l l t h r e e te c h n iq u e s e x t r a c t th e same r e l a t i v e am ounts. When tim e and

equipm ent a r e c o n s id e r e d , i t becomes r e a d i l y a p p a re n t t h a t MacLeod’s

p ro c e d u re i s th e p r e f e r r e d te c h n iq u e . By u s in g aqueous c a u s t i c f o r

d i g e s t i o n , t h e r e was no need f o r e x t r a c t i o n s t e p s to remove a lc o h o l

p r i o r to t h e s i l i c a g e l chrom atography . The e n t i r e d i g e s t i o n and

e x t r a c t i o n s t e p s w ere c o n v e n ie n t ly c a r r i e d o u t i n a s i n g l e sc rew -

cap c e n t r i f u g e tu b e . S o x h le t e x t r a c t i o n and KOH a l c o h o l i c d i g e s t i o n

r e q u i r e d e i t h e r e x t e n s iv e tim e o r equipm ent o r b o th .

A d d i t io n a l work i s needed to g e n e r a l i z e th e e v a lu a t io n o f

th e s e t e c h n iq u e s . The e f f e c t o f d i f f e r e n t t i s s u e ty p e s and s t a n d a r d

c o n c e n t r a t i o n s a r e w orthy o f s tu d y . The fo rm a t io n o f em uls ions o r

g e l s p r e s e n t s a d i f f i c u l t y w i t h i n t h e s e te c h n iq u e s which a l s o d e se rv e s

f u r t h e r s tu d y .

A P P E N D I X

36

37

F ig u re 1A, Gas chromatogram o f th e (A) a ro m a t ic f r a c t i o n and (B)

a l i p h a t i c f r a c t i o n o f qlani t i s s u e e x t r a c t e d by MacLeod's

p ro c e d u re , w i th th e s ta n d a rd p p e a k s 1) hex am eth y lb en zen e ,

2) b e n z o ( q h i ) p e r y le n e , 3) 2 -m e th y lo c ta d e c a n e , 4) 2- m e th y le ic o s a n e , and 5) n C ^ *

TABLE Al

ARCSINE TRANSFORMED PERCENT RECOVERY FOR 2-METHYLOCTADECANE

FROM SPIKED CLAM TISSUE

R e p l i c a t e s Techniques

MacLeod* s P ro c e d u re



#1 61 .30 42 .70 47.98

2 62.37 41.. 39 53 .23

3 57 .57 51 .23 49 .17

4 40 .9 8 63 .02 41.35

5 23 .73 71 .05 40 .61

6 42 .98 67.15 45 .23

7 51. 30 47 .25 53 .78

8 73.09 49 .26 50 .30

9 43 .20 73.21 66.70

10 66 .12

11 61 .60

12 81.19

13 57 .11

X 4- s 54 .10 + 12. 53

38

TABLE A2

ARCSINE TRANSFORMED PERCENT RECOVERY FOR 2-METHYLEICOSANE


Rep l i c a t es T echn iques

MacLeod's P ro c e d u re



n23

4

5

6

7

8

8

9

10

11

12

13

55 .38

67 .41

61.29

38 .79

20 .94

31 .63

37 .63

69 .83

£9. 83

4 2 .9 8

71.46

61 .20

76 .90

57 .87

41 .25

46 .7 8

48 .72

62 .75

53. 37

67 .17

45 .21

4 3 .31

43 .31

59 .28

48 .63

49.56

50 .23

38.47

37.26

43 .17

52 .01

50 .64

50 .64

45 .10

x + s 5 0 .8 4 + 12 .64

39

TABLE A3

ARCSINE TRANSFORMED PERCENT RECOVERY FOR nC


32

R e p l ic a te s T echn iques

MacLeod1s P ro ced u re



#1

2

3

4

5

6

7

8

9

10

11

12

13

4 6 .74

4 2 .03

62 .63

39 .62

36 .62

3 6 .7 8

28.92

60 .55

38 .22

64 .59

54 .95

57 .68

4 1 .5 4

2 7 .38

38 .43

39 .01

41 .25

45 .83

39 .38

40 .17

36 .04

4 7 .21

37 .91

38 .92

41 .03

34 .71

29 .18

3 2 .78

42 .62

30 .48

35 .60

x + s 41 .5 7 + 9 .6 0

40

TABLE A4

ARCSINE TRANSFORMED PERCENT RECOVERY FOR HEXAMETHYLBENZENE


R e p l i c a t e s T echn iques' ' •' " ' ’ ' • - - , _

MacLeod's S o x h le t KOHP ro ce d u re E x t r a c t i o n A lc o h o l ic

D ig e s t io n

#1 5 0 .1 1 36 .60 41 .43

2 4 5 .7 3 3 2 .68 30 .48

3 3 4 .87 45 .63 32 .01

4 28 .54 4 5 .03 41 .94

5 2 6 .20 51 .19 34 .25

6 34 .21 38 .52 32.35

7 35.12 48 .76 35 .68

8 36 .81 3 1 .00 36 .10

9 36 .85 4 2 .38 30 .30

10 39 .07

11 36 .51

12 20 .80

13 33 .04

x + s 36 .9 0 + 7 .0 1

41

TABLE A5

ARCSINE TRANSFORMED PERCENT RECOVERY FOR BENZO(GHI) PERYLENE


R e p l i c a t e s Techniques

MacLeod’s S o x h le t KOHP ro c e d u re E x t r a c t i o n A lc o h o l ic

D ig e s t io n

//I 57 .4 8 74.16 90 .13

2 57 .25 66.45 78 .18

3 69 .50 57.09 83.30

4 60.39 71.13 70.25

5 42 .91 67.29 69.79

6 57. 63 48 .12 63. 29

7 69 .30 63 .78 71.92

8 61 .83 53 .34 66.70

9 67 .60 72.83 93 .94

10 47 .90

11 5 4 .40

12 69 .90

13 54 .06

x + s 65 .54 + 11.59

42

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TABLE A7

ALIPHATIC HYDROCARBON FRACTION (pG/G DRY WEIGHT TISSUE)

NORMALIZED TO 2-METHYL0 CTADECANE INTERNAL STANDARD


MacLeodrs P ro ce d u re



#1

2

3

4

5

6

7

8

9

10

11

12

13

14 .57

11 .94

14.94

11. 30

20. 73

7* 13

11 .15

8 .03

16 .90

15 .12

15 .64

10 .25

12 .31

5 .15

10 .43

9 .82

11 .33

9 .74

9 .8 0

8 .42

6.12

10.90

10 .0 2

18 .02

16 .00

14 .80

10.32

12.84

16 .23

9 .92

29.31

x 13 .08 x 9 .0 8 15.27

s i g n i f i c a n t

44

TABLE A8

ALIPHATIC HYDROCARBON FRACTION (yG/G DRY WEIGHT TISSUE)

NORMALIZED TO 2-METHYLEICOSANE INTERNAL STANDARD


MacLeod’ s S o x h le t KOHP ro c e d u re E x t r a c t i o n A lc o h o l ic

D ig e s t io n

til 16 .12 5.33 9 .89

2 1 1 .05 9 .2 3 19 .35

3 14 .03 8 .21 15.67

4 11 .94 11 .38 15 .91

5 23 .49 12 .97 11.25

6 9. 70 9 .79 13.46

7 15 .20 8 .80 16 .78

8 8 .41 6 .95 9 .85

9 1 6 .98 13.46 43 .35

10 1 3 .9 9

11 15.75

12 1 0 .8 3

13 12 .15

x 13 .82 9 .57 17 .28


45

TABLE A9

ALIPHATIC HYDROCARBON FRACTION (yG/G DRY WEIGHT TISSUE)

R e p l ic a te s

NORMALIZED TO nC32 INTERNAL STANDARD

T echn iques

MacLeod1s P ro c e d u re



n2

3

4

5

6

7

8

9

10

11

12

13

19 .10

1 7 .7 3

13 .73

11 .69

1 3 .4 1

8 .35

1 9 .8 1

9 .69

1 9 .10

1 5 .4 8

17 .54

14 .44

16 .92

9 .35

9 .57

10 .28

9 .5 0

9 .5 3

8 .53

10 .23

8. 79

9 .4 0

6 .23

6 .35

14.52

8 .18

10.118 .63

10 .27

9 .12

25 .20

x 15 .11 9 .46 10.96


46

TABLE A10

AROMATIC HYDROCARBON FRACTION (yG/G DRY WEIGHT TISSUE)

NORMALIZED TO HEXAMETHYLBENZENE INTERNAL STANDARD

R e p l i c a t e s T echniques

MacLeod*s S o x h le t KOHP ro ce d u re E x t r a c t i o n A lc o h o l ic

D ig e s t io n

//I 8 ,44

2 10.99

3 14 .88

4 16 .56

5 11 ,56

6 8 .39

7 11.738 9 .97

9 15 .92

10 7 .58

11 13 .50

12 23 .26

13 12 .26

x 12 .69

6 ,99 5 .10

11 .23 8 .11

8 .79 18.62

8 .7 1 6 .77

8 .54 8 .61

8 .72 8 .74

8 .43 12.75

10 .23 7 .7 0

1 0 .48 29 .60

9 .1 2 11 .84

n o t s i g n i f i c a n t

47

TABLE A ll

AROMATIC HYDROCARBON FRACTION C|iG/G DRY WEIGHT TISSUE)

NORMALIZED TO BENZO(GHI)PERYLENE INTERNAL STANDARD


MacLeod’s S o x h le t KOHP ro ce d u re E x t r a c t i o n A lc o h o l ic

D ig e s t io n

#1 7 .36 3 .45 2.62

2 8 .79 5 .52 3 .16

3 7.47 7.02 7.15

4 7 .83 5 .5 1 4 .04

5 7.05 6.49 4 .23

6 5 . 00 6 .98 4 .47

7 5 .95 6 .4 4 6.09

8 5 .94 5 . 94 4 .17

9 8 .68 6 .10 9 .54

10 6 .18

11 9 .0 6

12 6 .92

13 7 .49

x 7 .21 5 .94 5 .05


49

TABLE A12

STUDENT-NEWMAN-KEULS ’ ?EST FOR THE ALIPHATIC HYDROCARBON FRACTION

NORMALIZED TO 2-METHYLOCTADECANE INTERNAL STANDARD

e r r o r

Ms

17.7322257

d f

28 S- = -v| MS e r r o r -J = 1 .2911X V V 2n1 ,n 2

S- = -^ 1 7 .7 3 2 2 2 5 7 /9 = 1.40365

t re a tm e n t

x


9 .0 8

Wp = P x S-A

MacLeod'sP ro ced u re

13 .08

KOH A lc o h o l ic D ig e s t io n

15 .27

P 2

q a (P ,2 8 ) 2 .90

Wp 4 .07

Wp 3 .74

15 .27 - 9 .0 8 = 6 .19*

13 .08 - 9 .0 8 = 4 .0 *

3

3 .50

4 .9 1

* s i g n i f i c a n t

Those compounds s h a r in g a common u n d e r l in e a re n o t s i g n i f i c a n t l yd i f f e r e n t .

49

TABLE A13

student- newman- k e u ls ' TEST FOR THE ALIPHATIC HYDROCARBON FRACTION

NORMALIZED TO 2-METHYLEICOSANE INTERNAL STANDARD

Ms df

e r r o r 38.94647 28

t r e a tm e n t


l j 3 8 -9 4 6 4 7 ^ 2§ i » =

" \ P -

9135396

94647/9 = 2.08023

Wp = P x S-


9 .5 7

MacLeod1s P ro ce d u re

13 .82

P 2 3

q a ( P ,28) 2 .90 3 .50

Wp 6 .0 3 7 .28

Wp 5 .54

17 .2 8 - 9 .5 7 = 7 .71*

KOH A lc o h o l ic Diges t i o n

17 .28

Those compounds s h a r in g a common u n d e r l i n e a r e n o t s i g n i f i c a n t l y d i f f e r e n t .

50

TABLE A14

STUDENT-NEWMAN-KEULS 1 TEST FOR THE ALIPHATIC HYDROCARBON FRACTION

NORMALIZED TO nC32 INTERNAL STANDARD

M$ d f

e r r o r 15.686578 28 686578/9 = 1.32017

Sx = -^| 1 5 .6 8 6 5 7 8 -^1 5 .686578-\/ ■ = 1 .21442(9x13)

Wp = P x S-x

t r e a tm e n t S o x h le tE x t r a c t i o n

KOH A lc o h o l ic D ig e s t io n

MacLeod’sP ro ced u re

x 9 .46 10.96 15.11

q a ( P ,28) 2 .90 3 .50

Wp 3 .5 2 4 .25

Wp 3 .8 3 4 .62

15.11 - 9 .46 = 5 .65*

15 .11 - 10 .96 = 4 .15*

10 .96 - 9 .4 6 = 1 .5



51

TABLE A15

STUDENT-NBWMAN-KEULS 1 TEST FOR THE AROMATIC HYDROCARBON FRACTION

NORMALIZED TO BENZQ(GHI)PERYLENE INTERNAL STANDARD

Ms d f

e r r o r 2.328833456 28 2 .3 2 8 8 3 3 4 5 6 -^ 13+92(13x9)

S- =-^ | 2 .328833456/9 = .50868399

Wp = P x S^

= .46792109

t r e a tm e n t KOH A lc o h o l ic Diges t i o n

5 .05

S o x h le tE x t r a c t io n

5 .94

MacLeod1s P ro ce d u re

7 .21

qa(P ,28) 2 .90 3 .50

Wp 1 .47 1 .7 8

Wp 1 .35 1 .64

7 .21 - 5 .05 = 2 .16*

7.21 - 5 .94 = 1.27

5 .9 4 - 5 .05

* s i g n i f l e a n t


52

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TABLE A24

RESPONSE FACTORS (F FACTORS) FOR ALIPHATIC HYDROCARBON STANDARDS

(yG/microvolt- s econds)

Compounds R e p l i c a te s

#1 #2 #3 #4 #5 x + s

nG13 1 .64 1 .4 1 1 .41 1 .41 1 .42 1 .45 + 0 . 1 0

nC14 1.32 1 .3 8 1 .40 1.39 1 .37 1 .37 + 0 .03

nC15 0.99 1 .3 0 1 .3 4 1 .40 1 .38 1 .2 8 + 0 .1 6

nC16 1.67 1 .55 1 .5 3 1.59 1 .55 1 .5 7 + 0 .05

nC17 1 .45 1 .7 4 1 .65 1 .64 1 .67 1 .6 3 + 0 .11

nC18 1.59 1 .57 1 .54 1 .54 1 .5 6 1 .5 6 + 0 .0 2

nC19 1. 78 1 .66 1.69 1 .61 1 .67 1 .6 8 + 0 .0 6

nC20 1 .6 8 1 .75 1 .68 1 .67 1 .70 1 .69 + 0 .03

nC21 1 .7 1 1 .7 0 1 .7 3 1 .7 0 1 .72 1 .7 1 + 0 .0 1

nC22 1 .55 1 .59 1.59 1 .4 9 1 .57 1 .5 6 ± 0 .0 4

nC23 1 .9 1 1 .9 1 1 .8 8 1 .8 8 1 .88 1.89 + 0 .02

nC24 1 .38 1.39 1 .59 1.39 1 .4 0 1 .4 3 + 0 .09

nC25 2 .27 1 .9 1 2 .67 2 .1 5 2 .07 2 .2 1 + 0 .2 8

nC26 1 .93 2 .0 0 2 .50 1 .8 1 2.09 2 .06 + 0 .26

nC28 2 .54 2 .86 3 .09 2 .05 2.57 2 .62 + 0 .3 9

nC29 1. 66 2 .63 2 .77 1 .8 9 2 .00 2 .19 + 0 .4 8

nC30 3 .0 1 3.06 3 .02 3 .1 8 3 .0 1 3 .03 + 0 .09

nG31 2 .8 4 3 .16 4 .14 2 .82 2.84 3 .1 6 + 0 .56

nC32 2 .01 3 .75 3 .67 2 .34 3 .47 3 .22 + 0 .59

61

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62

TABLE A26

ANOVA OF RECOVERED WIEGHTS OF ALIPHATIC HYDROCARBON

FRACTION EXTRACTED BY MACLEODfS PROCEDURE,

SOXHLET EXTRACTION AND KOH ALCOHOLIC DIGESTION

■........ . ...7— —-- ' ... T— '

S purees o f V a r i a t i o n d f Sums o f Squares Mean S quares F

Among T echn iques 2

W ith in T echn iques 28

34.7859

263.5803

17.3929

9.4135

1.847

T o ta l 30 298.3663

ANOVA OF RECOVERED WEIGHTS OF AROMATIC HYDROCARBON

FRACTION EXTRACTED BY MACLEOD'S PROCEDURE,

SOXHLET EXTRACTION AND KOH ALCOHOLIC DIGESTION

S ources o f V a r i a t i o n d f Sums o f S quares Mean Squares F

Among T echn iques 2

Within, T echniques 28

1 .6018

52.5690

0.8009

1.8774

0.4265

T p t a l 30 54 .1708

63

TABLE A27

ANOVA OF THE ARCSINE TRANSFORMED PERCENT RECOVERIES OF THE ADDED

STANDARDS EXTRACTED BY MACLEOD*S PROCEDURE, SOXHLET

EXTRACTION AND KOH ALCOHOLIC DIGESTION

S ources o f V a r i a t i o n d f Sums o f S quares Mean Squares F

Among T echn iques

W ith in T echn iques

2

154

76.0657

35883.3835

38.0328

233.0089

0.6322

T o ta l 156 35959.4492

ANOVA OF THE ARCSINE TRANSFORMED PERCENT RECOVERIES OF THE

STANDARDS: 2-METHYLOCTADECANE, 2-METHYLEICOSANE, nC32,

HEXAMETHYLBENZENE, AND BENZ0 ( GHI) PERYLENE

ADDED

S ources o f V a r i a t i o n df Sums o f Squares Mean Squares F

Among Compounds

W ith in Compounds

4

150

15532.5147

17811.4194

3883.128

118.742

32 .70*

T o t a l 154 33343.9342

^ s i g n i f i c a n t a t th e 95% c o n f id e n c e l e v e l

TABLE A28

ANOVA OF THE ALIPHATIC FRACTION RECOVERED WEIGHTS NORMALIZED TO

2-METHYLOCTADECANE EXTRACTED BY MACLEOD'S PROCEDURE, SOXHLET


S ources o f V a r i a t i o n d f Sums o f Squares Mean Squares F

Among Techniques 2 178.8058 89.4029 5.04*

W ith in Techniques 28 496.5023 17.7322

T o ta l 30 675.3081


2-METHYLEICOSANE EXTRACTED BY MACLEOD'S PROCEDURE, SOXHLET


Sources o f V a r i a t i o n d f Sums o f Squares Mean Squares F

Among T echniques 2 268.6736 134.3368 3.44*

W ith in Techniques 28 1090.5013 38.9464

T o ta l 30 1359.1749

65

TABLE A28 (C o n tin u e d ) .


nC32 EXTRACTED BY MACLEOD'S PROCEDURE, SOXHLET



Among T echn iques 2 181.9136

W ith in T echn iques 28 417.6588

T o ta l 30 599.5724

* s i g n i f i c a n t a t th e 95% c o n f id e n ce l e v e l

90 .9568 5 .87*

15.4688

66

TABLE A29

ANOVA OF THE AROMATIC FRACTION RECOVERED WEIGHTS NORMALIZED TO

HEXAMETHYLBENZENE EXTRACTED BY MACLEOD'S PROCEDURE, SOXHLET


Sources o f V a r i a t i o n df Sums o f Squares Mean Squares F

Among T echniques

W ith in T echn iques

2

28

69.7002

715.6501

34.8501

25.5589

1 .36

T o ta l 30 785.3503

ANOVA OF THE AROMATIC FRACTION RECOVERED WEIGHTS NORMALIZED TO

BENZ0 ( GHI) PERYLENE EXTRACTED BY MACLEOD'S PROCEDURE, SOXHLET



Among T echn iques

W ith in T echniques

2

28

25.7050

65.2073

12.8525

2.3288

5 .51*

T o ta l 30 90.9123

^ s i g n i f i c a n t a t th e 95% c o n f id e n c e l e v e l

67

TABLE A30

ANOVA OF THE ARCSINE TRANSFORMED PERCENT RECOVERIES OF THE SPIKED BLANKS

(2-METHYLOCTADECANE, 2-METHYLEICOSANE, nC32 , HEXAMETHYLBENZENE AND

BENZO(GHI)PERYLENE) EXTRACTED BY MACLEOD'S PROCEDURE, SOXHLET


Sources o f V a r i a t i o n d f Spms o f S quares Mean Squares F

Among T echniques 2 1581.31546 790 f657 2.29

W ith in T echniques 72 24796.3944 344.394

T o ta l 74 26377.709

ANOVA OF THE ARCSINE TRANSFORMED PERCENT RECOVERIES OF THE SPIKED BLA1

( 2-METHYLOCTADECANE, 2-METHYLEICOSANE, nC32>

HEXAMETHYLBENZENE AND BENZO(GHI)PERYLENE)

Sources o f V a r i a t i o n df Sums o f Squares Mean Squares F

Among compounds 4 6574.8445 1643.7111 5.81*

W ith in compounds 70 19802.8653 282.8980

T o ta l 74 26377.709

* s i g n i f l e a n t a t th e 95% c o n f id e n c e l e v e l

TABLE A31

ANOVA OF THE ARGSINE TRANSFORMED PERCENT RECOVERIES OF ADDED

SPIKES AFTER CONCENTRATION UNDER NITROGEN

Source o f V a r i a t i o n d f Sums o f Squares Mean Squares F

Among Compounds 6 288.6017 48.1002 1 .2 8

W ith in Compounds 28 1046.9280 37.3902

T o ta l 34 1335.5297

ANOVA OF THE ARCSINE TRANSFORMED PERCENT RECOVERIES OF ADDED

SPIKES AFTER CONCENTRATION BY ROTARY EVAPORATION


Among Compounds 6 245.6788 40.9464 0.954

W ith in Compounds 28 1200.7880 42.8852

T o t a l 34 1446.4668

69

TABLE A31 (C o n tin u e d ) .

ANOVA OF THE ARCSINE TRANSFORMED PERCENT RECOVERIES OF ADDED

SPIKES AFTER COLUMN CHROMATOGRAPHY


Among Compounds 6 621.9788 103.6631 3.273*

Withii} Compounds 28 886.8080 31.6717

T o ta l 34 1508.786

* s i g n i f i c a n t a t t h e 95% c o n f id e n c e l e v e l

70

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A vigan , J

B a r b i e r ,

B i e r i , R.

B i e r i , R.

B la y lo c k ,

B lpmer, M

Brown, R.

C la rk , R.

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H. and V. C. S tam oud is . 1977. The f a t e o f p e tro le u m h y d ro ca rb o n s from a No. 2 f u e l o i l s p i l l i n a s e m in a tu r a l e s t u a r i n e en v iro n m en t. Pages 332-344 i n F a te and E f f e c t s o f P e tro leu m H ydrocarbons i n M arine Organisms and E cosystem s. Douglas A. Wolfe ( e d . ) . Pergamon P r e s s , I n c . New York.

H . , A. L. W alker, B. W. Lew is, G. L oser and R. J . H u g g e t t . 1974. I d e n t i f i c a t i o n o f h y d ro ca rb o n s i n an e x t r a c t from e s t u a r i n e w a te r accom odated No. 2 f u e l o i l . Pages 149-153. i n NBS Spec. Pub-1. 409. M arine P o l l u t i o n M o n ito r in g ( P e t r o le u m ) . P ro c e e d in g s o f a Symposium and Workshop a t NBS. G a i th e r s b u r g , M ary land . May 13-17 .

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A ., T. D. S e a r l and J . J . E l l i o t t . 1973. D i s t r i b u t i o n o f heavy h y d ro ca rb o n s i n some A t l a n t i c Ocean w a te r s . Pages 505-520 i n P r o c e e d in g s o f 19 73 J o i n t C onference on P r e v e n t io n and C o n tro l o f O i l S p i l l s . API/EPA/USCG. W ash ing ton , D. C. March 13 -15 .

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C la rk , R. C ., J r . and M. Blumer. 1967. D i s t r i b u t i o n o f n - p a r a f f i n si n m arin e o rgan ism s and s e d im e n ts . Limnology and Oceanography 12: 79 -87 .

C la rk , R. C ., J r . and J . S. F in l e y . 1973. T echniques f o r a n a l y s i s o f p a r a f f i n h y d ro ca rb o n s and f o r i n t e r p r e t a t i o n o f d a ta to a s s e s s q i l s p i l l e f f e c t s i n a q u a t i c o rg a n ism s . Pages 161-172 i n P ro ce e d in g s o f 1973 J o i n t C onference on P r e v e n t io n and C o n tro l o f O i l S p i l l s . API/EPA/USCG. W ashing ton , D. C. March 1 3 -1 5 .

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Grob, K.

H e r tz ,

H u n te r ,

I l i f f e ,

Johnson ,

Koons, C

M ackie,

MacLeod,

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VITA

C o lle e n G a i l Becker

Born i n New Haven, C o n n e c t ic u t , F eb ru a ry 12, 1952. G raduated

from Lyman H a l l High S c h o o l , W a l l in g f o r d , C o n n e c t ic u t i n June 1970.

R ece ived a B a c h e lo r o f A r ts d eg ree from Q u in n ip ia c C o l le g e , Hamden,

C o n n e c t ic u t i n May 19 74, w i th a m ajor i n b io lo g y .

In Septem ber 19 74, th e a u th o r e n te r e d th e School o f M arine

S c ie n c e , th e C o lleg e o f W ill iam and Mary as a g ra d u a te s t u d e n t .

75

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