Analytical Techniques in Occupational Health ... - ACS Publications

8 Improved Resolution i n H i g h Performance Liquid Chromatography Analysis of Polynuclear Aromatic Hydrocarbons Using Ternary Solvent Systems B A R R Y R. B E L I N K Y

Downloaded by UNIV OF ARIZONA on January 13, 2013 | http://pubs.acs.org Publication Date: April 22, 1980 | doi: 10.1021/bk-1980-0120.ch008

National Institute for Occupational Safety and Health, 4676 Columbia Parkway, Cincinnati, O H 45226

Polynuclear aromatic hydrocarbons (PAH's) a r e produced i n most incomplete combustion processesExamples are internal combustion engines, e f f l u e n t s from c o a l f i r e d e l e c t r i c i t y g e n e r a t i n g p l a n t s , t o b a c c o smoke, and from coking operations i n s t e e l and aluminum r e f i n e r i e s PAH's are also present i n coal tar d e r i v e d and c o a l t a r c o n t a i n i n g p r o d u c t s such as c r e o s o t e and r o o f i n g p i t c h They a r e found i n the water we d r i n k a n d t h e a i r we b r e a t h e , a n d a r e a u b i q u i t o u s component o f o u r e n v i r o n m e n t Concern f o r public h e a l t h and s a f e t y i s stimulated by t h e k n o w l e d g e that many of these compounds a r e potent carcinogensPast a n a l y t i c a l efforts to relate chemical composition to carcinogenicity have used quantitation of the cyclohexane o r benzene s o l u b l e f r a c t i o n ( B S F ) Q ) and

of

benzo(a)pyrene

( B a P ) (2,2,4,5.)

as

indexes o f

carcinogenicityH o w e v e r , t h e BSF i s a n o n s p e c i f i c d e t e r m i n a t i o n , a s many n o n t o x i c compounds a r e f o u n d i n this fractionBaP d e t e r m i n a t i o n s can only give a crude estimate o f c a r c i n o g e n i c i t y because ( a ) i t i s only one o f many carcinogenic PAH's (b) t h e carcinogenic activity o f d i f f e r e n t PAH's vary over a wide range and ( c ) t h e d i s t r i b u t i o n of individual PAH' s can f l u c t u a t e greatly from sample t o sampleThe v a s t c o m p l e x i t y o f P A H s a m p l e s h a s b e e n shown by Lao ( £ ) , who h a s i d e n t i f i e d o v e r 120 P A H ' s i n u r b a n a i r b o r n e p a r t i c u l a t e m a t t e r a n d S e v e r s o n (1), who h a s identified about 900 PAH's i n t o b a c c o smokeI t i s evident, then, that a need exists t o more fully characterize the molecular species found i n PAH mixtures-

This chapter not subject to U.S. copyright. Published 1980 American Chemical Society

In Analytical Techniques in Occupational Health Chemistry; Dollberg, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

OCCUPATIONAL H E A L T H CHEMISTRY

150


Chromatographic Separation

of

PAH's

Both gas chromatography (GC) and high performance l i q u i d chromatography ( H P L C ) have been extensively utilized i n the separation of these complex mixturesGC, e s p e c i a l l y when capillary columns are used, has been shown to have b e t t e r o v e r a l l r e s o l v i n g power t h a n HPLCHPLC c o u p l e d t o a fluorescence d e t e c t o r , on t h e o t h e r h a n d , c a n p r o v i d e s e n s i t i v i t i e s i n the p i c o g r a m range (8) as opposed to the nanogram range f o r GC/KSIn a d d i t i o n , stopped f l o w t e c h n i q u e s a l l o w UV o r f l u o r e s c e n c e s p e c t r a t o be obtained for individual peaks, thus providing c o n f i r m a t i o n of s t r u c t u r e Improvement in resolving power of HPLC thus becomes an o b v i o u s g o a l i n t h e e f f o r t t o i d e n t i f y and q u a n t i t a t e m i x t u r e s o f PAH'sOne way i n which t h i s c a n be a c c o m p l i s h e d i s t h r o u g h t h e use o f s e l e c t i v e m o n i t o r s Selective UV detection has been demonstrated by, among o t h e r s , K r s t u l o v i c and Brown (£) a t t h e U n i v e r s i t y of Rhode IslandWheals (10) u t i l i z e d s e l e c t i v e f l u o r e s c e n c e m o n i t o r i n g to d i f f e r e n t i a t e peaksNeither of these techniques actually improves r e s o l u t i o n ; they merely s i m p l i f y t h e c h r o m a t o g r a m e i t h e r by e l i m i n a t i n g peaks o r by c h a n g i n g t h e r a t i o s o f o v e r l a p p i n g p e a k s s o t h a t t h e y may be d e t e r m i n e d m a t h e m a t i c a l l y The drawback to such a procedure i s that e i t h e r m u l t i p l e d e t e c t o r s must be u s e d o r m u l t i p l e r u n s be made to obtain a complete a n a l y s i s Another method is to optimize those chromatographic parameters which affect resoluticnAlthough this paper i s directed at a pragmatic approach to accomplishing t h i s task, a brief detour i n t o t h e m a t h e m a t i c s i s n e e d e d b e f o r e we p r o c e e d The fundamental equation describing chromatographic resolution, R is g

«s

-

i

[s^t]

λ

[τϊτ]

f

where α, Ν and k are, r e s p e c t i v e l y , the s e p a r a t i o n factor, theoretical plate number and capacity factor-(24) One can s e e t h r e e r o u t e s t o an i n c r e a s e i n t h e value of r e s o l u t i o n First, one nan increase the plate number Ν which results i n an increase in r e s o l u t i o n p r o p o r t i o n a l t o t h e s q u a r e r o o t o f NThe use of microparticulate packings was a major breakthrough i n t h i s respectI f we a s s u m e , however, t h a t t h e c h r o m a t o g r a p h e r a l r e a d y has a t h i s d i s p o s a l a high e f f i c i e n c y column, then he must go to great


8.

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lengths to obtain any significant increase in resolution because of the proportionality factor. Secondly, one can adjust k , the c a p a c i t y f a c t o r . F i g u r e 1 shows t h e r e l a t i o n s h i p b e t w e e n k ' / d + k ' ) and k . Traditionally t h i s i s a c c o m p l i s h e d by a d j u s t i n g t h e s o l v e n t c o m p o s i t i o n so k' f a l l s b e t w e e n 2 and 10, s i n c e r e s o l u t i o n f a l l s o f f r a p i d l y below a k value of 2, and r e l a t i v e l y little increase i s obtained for values of k g r e a t e r t h a n 10. F i n a l l y t h e v a l u e o f a, the s e l e c t i v i t y f a c t o r can be adjusted. Obviously, one technique f o r changing the selectivity i s to change the s t a t i o n a r y p h a s e . I n d e e d , HPLC a n a l y s i s o f PAH has been done on silica (JUL) » alumina (12), c e l l u l o s e a c e t a t e ( 1 3 , l i i ) and polyamide (13.) c o l u m n s and e v e n s i l i c a c o l u m n s w i t h s p e c i a l i z e d b o n d e d p h a s e s such as 3-(2,4,5,7-Tetranitrofluorenimine)ρropy1diethoxysiloxane (15.)· A c h a n g e i n m o b i l e p h a s e w i l l a l s o a f f e c t a. T h e r e f o r e , i n the present work, the t e r n a r y s o l v e n t s y s t e m a c e t o n i t r i l e / m e t h a n o l / w a t e r was examined over a wide range of concentrations to determine optimum mobile phase c o m p o s i t i o n f o r PAH analysis. A statistical experimental design strategy was u s e d f o r t h e o p t i m i z a t i o n p r o c e s s . f

1

f


f

Development of Optimal

Mobile

Phase

Most o f t h e r e c e n t l i t e r a t u r e (16-20) d e s c r i b i n g PAH a n a l y s i s by HPLC has centered on the use of octadecylsilane bonded s t a t i o n a r y p h a s e s and e i t h e r methanol-water or acetonitrile-water as the mobile phase. 3oth isocratic and g r a d i e n t c o n d i t i o n s h a v e been u t i l i z e d . The g e n e r a l a p p r o a c h t a k e n t o o p t i m i z e chromatographic c o n d i t i o n s u s u a l l y i s as f o l l o w s . One o f t h e two b i n a r y s o l v e n t systems methanol/water or acetonitrile/water i s s e l e c t e d , and an arbitrary i s o c r a t i c c o m p o s i t i o n , s a y 60/n0 o r 7 0 / 3 0 , i s chosen. A standard mixture i s c h r o m a t o g r a p h e d and t h e n t h e organic/water ratio i s adjusted to optimize k'. Depending upon the nature of the sample, a g r a d i e n t may o r may n o t be i m p l e m e n t e d t o r e d u c e a n a l y s i s time while maintaining reasonable separation. If the r e s o l u t i o n i s i n s u f f i c i e n t f o r the a n a l y s i s at hand, the other binary system i s evaluated i n t h e same manner. F i g u r e 2 presents a triangular coordinate system, a l l o w i n g v i s u a l i z a t i o n of a l l combinations of the three solvents. Vertex A represents 100% methanol, vertex β r e p r e s e n t s 100% a c e t o n i t r i l e , and v e r t e x C, 100? w a t e r . P o i n t s X and Y represent the a r b i t r a r y b i n a r y s o l v e n t systems mentioned p r e v i o u s l y .


152



0

2

4

6

8

10

oo

k'

Figure 1.

Plot of k'/(l + k'j vs. k'

Wate r

C

Acetonitrile Figure 2.

Methanol

Representation of binary solvents X and Y as points in the ternary solvent system ABC



8.

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153

The a r r o w s a l o n g t h e AC and BC axes are the ranges over which the s o l v e n t systems are v a r i e d * Now the question arises: what happens i n t h e center of this triangle where not two but three s o l v e n t s are p r e s e n t ? Are the c a p a c i t y f a c t o r s merely averaged? I f so, α (which i s the ratio of the capacity factors f o r s o l u t e 1 and s o l u t e 2) w i l l n o t be e n h a n c e d * Or do more s u b t l e e f f e c t s o c c u r ? Karger (21), Snyder and Kirkland ( 2 2 ) , B a k a l y a r (2Λ) and others have discussed the expanded solubility parameter c o n c e p t which says t h a t p o l a r i t y as d e f i n e d by t h e H i l d e b r a n d s o l u b i l i t y p a r a m e t e r i s actually a composite of specific intermolecular interactions c o n s i s t i n g o f d i s p e r s i v e , d i p o l e and h y d r o g e n bonding interactionsNo a t t e m p t w i l l be made h e r e t o d e v e l o p t h e s e s u b p a r a m e t e r s on a t h e o r e t i c a l basisSuffice it to say t h a t much i s n o t y e t u n d e r s t o o d r e g a r d i n g the q u a n t i f i c a t i o n o f t h e s e i n t e r a c t i o n s — especially in a q u e o u s m i x t u r e s due t o t h e u n i q u e p r o p e r t i e s o f waterA t any r a t e , we c a n n o t y e t p r e d i c t v a l u e s f o r resolution in a ternary solvent system based on theoretical considerationsTo t r y to determine optimal s o l v e n t r a t i o s i n a t e r n a r y s y s t e m by a t r i a l and e r r o r a p p r o a c h w o u l d i n v o l v e an i n o r d i n a t e number of experiments — f a r more t h a n most a n a l y s t s w o u l d c a r e t o makeNevertheless, a relatively simple way exists to experimentally o p t i m i z e r e s o l u t i o n i n the t e r n a r y systemExperjmgntctl Fluoranthene (MC&B), P y r e n e and B e n z o ( a ) p y r e n e (Eastman Organic Chemicals), Benz(a)anthracene and Chrysene ( A l d r i c h C h e m i c a l Co-) and P e r y l e n e ( P f a u l t z & B a u e r ) were used as received t o make a standard solution with a concentration o f a p p r o x i m a t e l y 0-1 rag/ml o f e a c h PAHMethanol and A c e t o n i t r i l e (UV g r a d e - B u r d i c k & Jackson Laboratories) and water purified with a Millipore S u p e r Q s y s t e m were f i l t e r e d t h r o u g h an 0-5 m i c r o n f i l t e r and d e g a s s e d p r i o r t o useInstrumentation was a Waters A s s o c i a t e s HPLC s y s t e m i n c l u d i n g two M o d e l 6000A pumps, a M o d e l 440 UV detector fixed at 254 nm, a Model 660 solvent p r o g r a m m e r a n d a M o d e l U6K i n j e c t o r A Vydac 201 TP reverse phase c o l u m n ( 1 0 m i c r o n C-18 p a c k i n g , 4-6 mm ID χ 25 cm) was u s e d t h r o u g h o u t -


154


The g o a l o f t h i s i n v e s t i g a t i o n was t o d e t e r m i n e optimum m o b i l e phase composition for PAH analysis using the ternary solvent system acetonitrile/ methanol/waterA model PAH standard solution was p r e p a r e d c o n s i s t i n g o f t h r e e p a i r s o f compounds l i s t e d in order of increasing retention: FluoranthenePyrene, Benz(a)anthracene-Chrysene, and PeryleneBenzo(a)pyreneThe b a s i c o p t i m i z a t i o n s t r a t e g y was as follows: 1) an i s o c r a t i c t e r n a r y s o l v e n t s y s t e m was found which gave maximum r e s o l u t i o n of the fluoranthene-pyrene peaks at a k = 5; 2) two additional isocratic ternary solvent systems were found t o o p t i m i z e the r e s o l u t i o n of each o f the o t h e r two PAH p a i r s ; 3) a s o l v e n t g r a d i e n t was constructed which incorporated a l l three i s o c r a t i c compositionsA l l three optimal i s o c r a t i c systems were determined simultaneously by means of a Simplex statistical design strategyThe statisical design employed f o r the study r e q u i r e s t h a t a b o l d a p p r o a c h be u s e d : that i s , both the upper and lower bounds of the region being investigated should be chosen such that they completely enclose the r e g i o n of i n t e r e s t Figure 3 shows t h i s regionIn the case of acetonitrile/ methanol/water the two v e r t i c e s where t h e e l u e n t i s strongest for the solutes of interest are pure m e t h a n o l and p u r e a c e t o n i t r i l e The w e a k e s t e l u e n t i s pure waterH o w e v e r , w i t h p u r e w a t e r t h e PAH's would never be eluted f r o m t h e c o l u m n , so t h a t p a r t i c u l a r choice is impracticalTherefore, a 65/35 methanol/water composition was c h o s e n as t h e w e a k e s t mobile phase along the AC axis, and a 55/45 acetonitrile/water 'composition as t h e l o w e r bound o f t h e BC a x i s These points are labeled C* and D respectivelyA S i m p l e x d e s i g n f o r a t h r e e component mixture r e q u i r e s a t r i a n g u l a r c o o r d i n a t e system which obviously has only three verticesEecause of the c o n s t r a i n t we h a v e p u t on t h e u p p e r bound for water content, not three but four vertices existBy connecting points Β and C, two roughly equal triangular areas a r e g e n e r a t e d , e a c h o f w h i c h c a n be thought of as representing an independent ternary s o l v e n t systemT h e s e two t r i a n g u l a r r e g i o n s ABC and BC'D' c a n be r e p r e s e n t e d on an isometric orthogonal plot a s s e e n i n F i g u r e 4Vertex D i s technically a pseudocomponent r a t h e r t h a n a component o f the system because i t i s a c t u a l l y a m i x t u r e of the components Β ( a c e t o n i t r i l e ) and C (water)Likewise, pseudocom-


f

f

f

1

f


8.

Analysis

BELiNKY

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Aromatic

155

Hydrocarbons

1

ponent C i s a methanol/water mixtureWhen r e a d i n g the o r t h o g o n a l graph, the line AB r e p r e s e n t s zero content of C L i n e s p a r a l l e l t o AB m o v i n g c l o s e r t o vertex C represent successively higher C concen trationAcetonitrile i s 100% at point Β and d e c r e a s e s i n a l i n e a r f a s h i o n t o z e r o a l o n g t h e BA a n d BC l i n e s , a n d t o 55% a c e t o n i t r i l e a l o n g t h e BD lineOur o b j e c t i v e i s t o generate response s u r f a c e s which w i l l a l l o w t h e p r e d i c t i o n o f r e s o l u t i o n a t any p o i n t w i t h i n t h e r e g i o n s ABC and B C D ' The r e s p o n s e surface i s described mathematically by t h e s p e c i a l c u b i c m o d e l shown i n e q u a t i o n 2f

f

1

f

1


1

Y = biXi + b

2 3

+

Y)2X2

X2X3 +

+

t>3X3

+ t>i2XlX2

+

^ 1 3 X 1 X 3

Eq. 2

12123X1X2X3

A m e a s u r e d v a l u e Y, w h i c h c o u l d be t h e c a p a c i t y f a c t o r o r r e s o l u t i o n , i s shown a s t h e sum o f t h e individual contributions of each component o r pseudocomponent i n t h e systemΧι, X and X rep resent t h e f r a c t i o n o f each pseudocomponent p r e s e n t The b coefficients a r e determined experimentallyNote that t h e r e a r e no t e r m s s u c h a s b i 1 X 1 X 1 p r e s e n t because a s o l v e n t s i n t e r a c t i o n with itself h a s no physical meaning i n such a systemThe r e s p o n s e s (Y v a l u e s ) a r e measured a t each of the concentrations d e p i c t e d g r a p h i c a l l y i n F i g u r e 5, w h i c h r e p r e s e n t s o n e o f t h e two t e r n a r y s y s t e m s p r e v i o u s l y d e s c r i b e d The c o n c e n t r a t i o n o f each o f t h e pseudocomponents i s g i v e n around the peripheryThe s e v e n closed circles are the experimentally determined responses a t each v e r t e x , a t t h e m i d p o i n t o f each b i n a r y system, and a t the c e n t r o i d o f t h e t r i a n g l e These seven p o i n t s w i l l be u s e d t o g e n e r a t e t h e b c o e f f i c i e n t s i n E q u a t i o n 2The three open circles are also experimentally d e t e r m i n e d v a l u e s , a n d w i l l be c o m p a r e d with values calculated from t h e s p e c i a l c u b i c model t o determine the degree o f f i t A l l t e n values are determined in duplicate so t h a t an e s t i m a t e o f p r e c i s i o n c a n be madeThe c a l c u l a t i o n o f t h e " b " v a l u e s i s shown i n Equations 3 through 9, T a b l e I - S u b s t i t u t i n g t h e b values i n Equation 2 w i l l allow the prediction of Y for any g i v e n s o l v e n t c o m p o s i t i o n w i t h i n t h e c o n f i n e s of t h e model assuming t h e model i s an accurate r e p r e s e n t a t i o n o f the response s u r f a c e 2

3


156


Figure 3.


Ternary sohent systems A B C and B C D '

Figure 4. Orthogonal representation of ternary solvent systems A B C and B C D '

Figure 5. Ternary solvent system de sign; check points for determination of hck-of-fit indicated by open circles.

c·ι


8. BELiNKY

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Table I


Calculation

o fCoefficients

f o rSpecial

Cubic

Model

t>! = Y

A

Eq< 3

b

2

= Y

B

Eq,4

b

3

= Y

c

Eq.5 2 ( Y

Y

1'

A

B

) -

bis

= ΜΥ

Α

(

0

- 2(Y + Y )

Eq.7

b

= MY

B C

)

- 2(Y + Y )

Eq.8

A

B

}

= Mï

2 3

A

+

bi2

Ε (

c

B

c

Y

AC

+

Y

6

BC^

Y

a

n

d

Y

i n

The t h r e e c h e c k p o i n t s Y ABC> ABBC ABCC F i g u r e 5 a r e used t o v e r i f y the v a l i d i t y o f the modelThis i s accomplished by examining the d i f f e r e n c e between t h e observed and p r e d i c t e d v a l u e s o f Y a t each checkpointThe l a c k o f f i t i s g i v e n by the expression i n Equation 1 0 A

S

LF

=

fJ

X

(Yoi " Y!)'

Eq. 10

where Y i i s t h e o b s e r v e ^ average response a t t h c h e c k p o i n t , and Y. i s the p r e d i c t e d response a t t h e i t h c h e c k p o i n t (from equation 2 ) Q

D u p l i c a t e v a l u e s o f Y f o r a l l t e n p o i n t s a r e used t o determine t h e pooled e r r o r v a r i a n c e ( E q u a t i o n 1 1 ) and the r e p l i c a t i o n e r r o r v a r i a n c e ( E q u a t i o n 1 2 ) -


OCCUPATIONAL H E A L T H

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CHEMISTRY

where Y j ! a n d Y^ a r e t h e d u p l i c a t e r e s p o n s e determinations a t each of the ten solvent compositions * 2


And finally t h e l a c k o f f i t v a r i a n c e i s compared t o the r e p l i c a t i o n e r r o r v a r i a n c e u s i n g an F - t e s t ( E q * 13)i which i n t h i s case b e i n g t h r e e components w i t h t e n d e g r e e s o f f r e e d o m , i s 3-71I f this ratio i s less t h a n 3 - 7 1 , t n e n t h e f i t i s good a n d t h e e q u a t i o n f o r t h e r e s p o n s e s u r f a c e c a n be c o n s i d e r e d v a l i d * (F-test)

8

2

L¥

τ S|. < 3 . 7 1

Eq. 1 3

A total o f seventeen different m o b i l e phase compositions are required t o generate the response s u r f a c e f o r t h e two S i m p l e x d e s i g n s a s shown i n F i g u r e 6* E a c h r u n i s done i n duplicate, and t h e e n t i r e design i s randomized t o reduce experimental bias* Runs 12 t h r o u g h 17 a r e t h e c h e c k p o i n t s u s e d to determine v a r i a n c e and goodness o f f i t * The o v e r a l l d e s i g n i s shown i n T a b l e I I * Capacity f a c t o r s , column e f f i c i e n c y i n terms o f Ν, and r e s o l u t i o n w e r e c a l c u l a t e d f o r e a c h o f t h e s i x peaks i n t h e chromatograms* Initial attempts to g e n e r a t e r e s p o n s e s u r f a c e s u s i n g e i t h e r k o r R^ were failures as i n d i c a t e d by t h e F - t e s t i n e q u a t i o n 13New s e t s o f c o e f f i c i e n t s f o r t h e s p e c i a l cubic model were generated, using log k and l o g R as the measured r e s p o n s e s , which gave acceptable F-tests* Orthogonal plots were t h e n g e n e r a t e d f o r each o f t h e t h r e e p a i r s o f t e s t compounds* Although the response s u r f a c e s were g e n e r a t e d u s i n g t h e l o g o f t h e r e s p o n s e , the i s o p l e t h s , f o r c l a r i t y , are i d e n t i f i e d by t h e actual response, r a t h e r than the l o g o f the response* F i g u r e 7 shows t h e r e s p o n s e s u r f a c e f o r t h e c a p a c i t y factor f o r f l u o r a n t h e n e as a f u n c t i o n of solvent composition* The r e s p o n s e s u r f a c e f o r r e s o l u t i o n of fluoranthene/pyrene is shown in Figure 6* Superimposed i s the i s o p l e t h f o r a c a p a c i t y f a c t o r of 5The s o l v e n t c o m p o s i t i o n c o r r e s p o n d i n g t o a k' o f 5 at which resolution i s g r e a t e s t i s shown by t h e circle* Response s u r f a c e s f o r t h e o t h e r two s o l u t e p a i r s were g e n e r a t e d s i m i l a r l y * F i g u r e 9 shows that resolution f o r b e n z ( a ) a n t h r a c e n e and c h r y s e n e i s a t a maximum w i t h i n t h e l o w e r t r i a n g u l a r r e g i o n * Again the p o i n t o f maximum r e s o l u t i o n f o r a c a p a c i t y f a c t o r o f 5 is indicated* The p e r y l e n e / B a P response surface i s shown i n Figure 10* T h i s Figure demonstrates the f

f

s



Β

0 1 0 0 1/2 0 1/2 1/2 C 1/3 1/3 1/6 2/3 1/6 2/3 1/6 1/6

A

1 0 0 0 1/2 1/2 0 0 0 1/3 0 2/3 1/6 1/6 0 0 0

Run

1 2 3 14 5 6 7 ε 9 10 1 1 12 13 14 15 16 17

Design

0 0 1 0 C 1/2 1/2 0 1/2 1/3 1/3 1/6 1/6 2/3 1/6 2/3 1/6

C*

Component

Simplex

0 0 0 1 0 0 0 1/2 1/2 0 1/3 0 0 0 1/6 1/6 2/3

D* 0 100 0 55 50 0 50 77-5 27-5 33-3 51-7 16,7 66.7 16,7 75-8 25-8 53-3

100 0 65 0 50 82,5 32-5 0 32,5 55 21 ,7 77-5 27,5 60 10,8 43-3 10,5

Methanol

(%)

Optimization

Composition

Solvent

Acetonitrile

f o r Ternary

Table I I


0 0 35 45 0 17-5 17.5 22,5 40 11 -7 26,7 5.8 5.8 23.3 13.3 30.8 35.8

Water

AP

u

I

Y

D C D D

"ABC yBC 0 AAAEC ABBC yABCC Υ ΒBC D yBCCD

YCD

YBD

vBC

yAC

Y Y

Response


Figure 7. Response surface for capacity factor, k', of fluoranthene


Analysis of Polynuclear

Aromatic

Hydrocarbons


8. BELiNKY


162



A

Figure 10. Response surface for resolution of perylene and benzo(a)pyrene; ( ) is the isopleth for k' (perylene)

A

Figure 11. Optimized solvent gradient for acetonitrile/methanol/water ternary system



8. BELiNKY

Analysis of Polynuclear

36

24

36

24

Aromatic

'

12

12

Hydrocarbons

'

'

163

0

0

Figure 12. Comparison of binary and ternary solvent systems for "soft" coal tar pitch: (A) acetonitrile/water, 55:45 to acetonitrile/water, 72:28; linear, SO min; (B) acetonitrile/methanol/water, 48:8:44 to acetonitrile/methanol/water, 43:37:20; linear, 30 min.




164

Figure 13. Comparison of binary and ternary solvent systems for "hard" coal tar pitch: same solvent ratios as Figure 13; 40-min linear gradient.


8. BELiNKY

Analysis

of Polynuclear

Aromatic

165

Hydrocarbons

f l e x i b i l i t y i n choosing optimized solvent composition* The k = 5 i s o p l e t h i s roughly p a r a l l e l to the R = 4*5 i s o p l e t h o v e r t h e c o m p o s i t i o n r a n g e i n d i c a t e d by coordinates A = 0*1, Β = 0*39, C = 0*51 t o Β = 0*62, D = 0*38In p r a c t i c e then, the choice of which optimal t e r n a r y s o l v e n t s h o u l d be u s e d may t a k e o t h e r considerations (such as viscosity or ease of generating the f i n a l gradient) into account* In F i g u r e 11 a g r a d i e n t i s d r a w n t h r o u g h t h e t h r e e p o i n t s c h o s e n a s h a v i n g maximum r e s o l u t i o n a t k' = 5 f o r e a c h of the pairs studied* The initial mobile phase c o m p o s i t i o n o f t h i s s y s t e m i s 4 8 * 4 % a c e t o n i t r i l e , 7-8% m e t h a n o l and 43-8% w a t e r * The final composition is 43% a c e t o n i t r i l e , 3 7 % m e t h a n o l a n d 2 0 % w a t e r * The resolution i n a c t u a l samples f o r the o p t i m i z e d t e r n a r y m o b i l e p h a s e s was c o m p a r e d to that obtained with the optimized binary mobile phase ( a c e t o n i t r i l e / w a t e r ) w h i c h had been used previously* F i g u r e 12 shows a c o m p a r i s o n o f c h r o m a t o g r a m s o b t a i n e d u s i n g t h e b i n a r y and t e r n a r y solvent systems* The s a m p l e was a s o f t c o a l t a r p i t c h w h i c h c o n t a i n s a h i g h p r o p o r t i o n o f low m o l e c u l a r weight PAH's* The s i x test compounds are i d e n t i f i e d i n the f i g u r e , along with t h e two m a j o r components, phenanthrene and anthracene* Note t h e near b a s e l i n e r e s o l u t i o n o f t h e fluoranthene/pyrene p a i r i n the ternary system, and the resolution of t h e two large early-eluting compounds, p h e n a n t h r e n e and a n t h r a c e n e * Figure 13 shows a s i m i l a r p a i r o f c h r o m a t o g r a m s of a b i n a r y sample o f a hard c o a l t a r p i t c h w i t h many high moleular weight PAH's present* I n the upper chromatogram, t h e p y r e n e peak has a s h o u l d e r , w h i c h i s nearly completely resolved i n the ternary system chromatogram* The two peaks between Pyrene and Benz(a)anthracene above a r e seen as f o u r peaks i n t h e lower chromatogram* f

s

1


f

Summary The u s e o f an a c e t o n i t r i l e / m e t h a n o l / w a t e r e l u e n t has been shown to improve resolution of PAH c h r o m a t o g r a p h e d on a V y d a c r e v e r s e p h a s e c o l u m n * This improved r e s o l u t i o n should r e s u l t i n g r e a t e r p r e c i s i o n and accuracy i n t h e q u a n t i t a t i o n o f i n d i v i d u a l PAH* Determination of optimal solvent concentration i s simplified through a p p l i c a t i o n of s t a t i s t i c a l design techniques* T h e s e t e c h n i q u e s c a n be f u r t h e r utilized to investigate various ternary mobile phases i n combination w i t h d i f f e r e n t s t a t i o n a r y phases*


166

OCCUPATIONAL

HEALTH

CHEMISTRY

Disclaimer Mention of company names o r p r o d u c t s d o e s n o t c o n s t i t u t e e n d o r s e m e n t by t h e N a t i o n a l Institute for O c c u p a t i o n a l S a f e t y and H e a l t h * A c k n o w l e d gement


The a u t h o r i s i n d e b t e d t o Mr- D e n n i s H i l l o f t h e CDC Parklawn Computer Center who developed the programs t o g e n e r a t e t h e r e s p o n s e s u r f a c e c u r v e s , and t o Dr* A l e x a n d e r W* T e a s s f o r v a l u a b l e d i s c u s s i o n s .

Abstract The analysis of polynuclear aromatic hydrocarbons (PAH's) presents one of the most challenging tasks to the occupational health chemist. The problems arise from the needs to separate large numbers of structurally similar compounds and simultaneously detect them at extremely low levels. High performance liquid chromatography (HPLC) is a frequently used tool for such analyses. This paper applies statistical experimental design techniques to the improvement of HPLC resolution through optimization of solvent selectivity effects in a ternary solvent system. Literature Cited 1.

Wallcave, L., Garcia, H., Feldman, R., L i j i n s k y , W . , and Shubik, P. T o x i c o l . Appl. Pharmacol., (1971) 18, 41-52.

2.

" S c i e n t i f i c and Technical Assessment Report on Particulate Polycyclic Organic Matter". U. S. Environmental Protection Agency (1975) No. PB 241 799.

3.

Sawicki, Ε., Stanley, T. W., Elbert, W. C., Meeker, J. and McPherson, S. Atmos. Env., (1967) 1, 131.

4.

Sawicki, E., Elbert, W., Stanley, T. W., Hauser, T. R. and Fox, F. Τ. Int. J. Air P o l l . , (1960) 2, 273.

5.

Jackson, J. O., Warner, P.O. and Mooney, T. F. Jr. A. I. Η. A. J., (174) 35, 276.


8.

6.

BELiNKY

Analysis

of Polynuclear

Aromatic

Hydrocarbons

Lao, R. C., Thomas, R. S., Oja, H. and Dubois,


L. Anal. Chem., (1973) 45, 908.

7.

Severson, R. F., Snook, M. E., Akin, F. J . , and Chortyk, O. T. In "Carcinogenesis, V o l . 3: Polynuclear Aromatic Hydrocarbons"; Jones, Ρ. W. and Freudenthal, R. I . , Ed.; Raven Press: New York, 1978.

8.

Christenson, R. G. and May, W. Ε. J. Liq. Chrom. (1978) 1 (3)85.

9.

K r s t u l o v i c , A. M., Rosie, D. M. and Brown, Ρ. R. Anal. Chem., (1976) 48, 1383.

10.

Wheals, Β. Β., Vaughan, C. G. and Whitehouse, M. J. J. Chromatogr., (1975) 106, 109.

11.

Boden, H. J. Chr. Sci., (1976) 14 (8), 391.

12.

Golden, C. and Sawicki, Ε. Anal. Let., (1976) 9 (10), 957.

13.

K l i m i s c h , H. J. J. Chromatogr. (1973) 83, 11.

14.

K l i m i s c h , H. J. Anal. Chem. (1973) 45 (11), 960.

15.

Lochmuller, Chromatogr.

16.

Eisenberg, W. C. J. Chr. Sci., (1975) 16 ( 4 ) , 145.

17.

Dong, M., Locke, D. C. and Ferrand, E. Anal. Chem., (1976 48 (2), 368.

18.

May, W. E., Chesler, S. Ν., Cram, S. Ρ., Gump, B. Η., Hertz, Η. S., Enagonio, D. Ρ. and Dyszel, S. M. J. Chr. Sci., (1975) 13 (11), 535.

19.

Oyler, A. R., Bodenner, D. L., Welch, Κ. J., Liukkonen, R. J . , Carlson, R. M., Kopperman, H. L. and Caple, R. Anal. Chem., (1978) 50 ( 7 ) , 837.

20.

Fox, M. A. and Staley, S. W. Anal. Chem. (1976) 48 ( 7 ) , 992.

21.

Karger, B. L. J. Chromatogr. (1976) 125, 71.

C. H. and (1975) 108, 85.

Amoss, C. W. J.



168


22.

Snyder, L. R. and Kirkland, J. J. "Introduction t o Modern Liquid Chromatography" pp 2 1 5 f f , John W i l e y & Sons, I n c . , New York (1974).

23.

B a k a l y a r , S. R., M c I l w r i c k , R. and Roggendorf, E. J. Chromatogr. (1977) 142, 353.

24.

S n y d e r , L. R. and Kirkland, J. J. "Introduction t o Modern Liquid Chromatography" pp 35-38, John W i l e y & Sons, I n c . , New York (1974).

25.

"Strategy of Experimentation," Revised Edition, Ε. I. du Pont de Nemours & Co., Wilmington, DE, October, 1975, p. 51.

RECEIVED October 30, 1979.


Analytical Techniques in Occupational Health ... - ACS Publications

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