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The Solvent Effect In Gas Liquid Chromatography
341
THE SOLVENT EFFECT I N GAS LIQIJID CHROMATOGRAPHY
V PRETORIUS, K LAWSON, E ROHIIER, P APPS UNIVERSITY OF PRETORIA-INST. FOR CHROMATOGRAPHY 1.
-
0002 PRETORIA
SOUTH AFRICA
INTRODUCTION
The s o l v e n t e f f e c t i s a phenomenon which may be employed as one o f t h e many ways o f sample i n t r o d u c t i o n i n g a s - l i q u i d chromotography ( r e f . 1 ) . More p a r t i c u l a r l y i t i s one o f t h e t h r e e modes o f s o l u t e f o c u s i n g . I t i s an e s p e c i a l l y e f f e c t i v e means o f q u a n t i t a t i v e l y and r e p r o d u c i b l y t r a n s f e r r i n g s o l u t e s f r o m d i l u t e s o l u t i o n s t o t h e column ( r e f . 2) w i t h o u t o v e r l o a d i n g under m i l d c o n d i t i o n s . Consequently i t i s o f p a r t i c u l a r v a l u e i n t r a c e a n a l y s i s where t h e r m a l l y l a b i l e s o l u t e s a r e i n v o l v e d . Although t h e t e r m " s o l v e n t e f f e c t " was o n l y c o i n e d l a t e r ( r e f . 3 ) , t h e phenomenon was p r o b a b l y f i r s t observed i n 1969 by Grob ( r e f . 4 ) . U s i n g a d i l u t e s o l u t i o n o f r e l a t i v e l y n o n - v o l a t i l e s t e r o i d s i n b i s ( t r i m e t h y l s i l y l ) acetamide and an excess o f hexane he observed t h a t l a r g e amounts o f sample c o u l d be i n t r o d u c e d i n t o a c a p i l l a r y column w i t h o u t o v e r l o a d i n a " p r o v i d e d t h e columr! temperature i s a t l e a s t 100°C below t h e b o i l i n g p o i n t o f t h e most v o l a t i l e s o l u t e " . Under t h e s e circumstances s t a t i o n a r y phase f o c u s i n g (paragraph 2.1.1.(ii)),
and n o t t h e s o l v e n t e f f e c t . i s dominant.
The f i r s t unequivocal d e s c r i p t i o n and e x p l a n a t i o n o f t h e s o l v e n t e f f e c t , as we understand i t today, was p u b l i s h e d by Deans i n 1971 ( r e f . 5 ) . U n f o r t u n a t e l y he used t h e t i t l e "The sample as i t s own s t a t i o n a r y phase i n gas chromatography'; and r e g r e t t a b l y , b u t u n d e r s t a n d a b l y , t h e i m p o r t o f h i s work remained l a r g e l y unrecognized u n t i l 1982 ( r e f . 5 ) . S i n c e i t s d i s c o v e r y a g r e a t deal has been p u b l i s h e d on t h e s o l v e n t e f f e c t ( r e f s . 7-10). Although t h e p r a c t i c a l u s e f u l n e s s o f t h e e f f e c t became apparent y e a r s ago i t i s o n l y r e c e n t l y t h a t t h e u n d e r l y i n g processes have been u n r a v e l l e d and e x p l a i n e d . I n essence s o l u t e f o c u s i n g u s i n g t h e s o l v e n t e f f e c t i n v o l v e s t h e f o l l o w i n g :
o
a d i l u t e vapour o r l i q u i d sample
o
t h e f o r m a t i o n o f a f i l m o f l i q u i d sample i n t h e i n l e t
348 o
t h e e v a p o r a t i o n o f t h e sample f i l m so t h a t s o l u t e s a r e focused by t h e solvent e f f e c t
o
t h e t r a n s f e r of focused s o l u t e s t o t h e column These i s s u e s may be a r b i t r a r i l y d i v i d e d i n t o fundamental ( p a r a g r a p h 2 ) and
p r a c t i c a l aspects (paragraph 3 ) . The former a r e concerned w i t h t h e s o l v e n t e f f e c t i t s e l f and t h e l a t t e r w i t h i t s p r a c t i c a l o p t i o n s . H i s t o r i c a l l y t h e s o l v e n t e f f e c t has been used f o r l i w i d samples i n open t u b u l a r i n l e t s . T h i s paper w i l l show t h a t b o t h l i q u i d and vapour samples can be handled, t h a t i n l e t geometries o t h e r t h a n open tubes can be used and t h e s o l v e n t e f f e c t can o p e r a t e e i t h e r s t a t i c a l l y o r d y n a m i c a l l y . The f u l l range o f o p t i o n s i s shown i n f i g u r e 1.
F i g . 1. P r a c t i c a l o p t i o n s a v a i l a b l e when.employing t h e s o l v e n t e f f e c t . L i n e s connect c o m p a t i b l e o p e r a t i o n a l parameters.
349
2.
FUNDAMENTAL ASPECTS It i s convenient t o consider t h e a p p l i c a t i o n o f the solvent e f f e c t t o l i q u i d
samples and t o vapour samples s e p a r a t e l y . 2.1
L i q u i d samples ( S t a t i c System) We d i s t i n g u i s h between an
e f f e c t s (paragraph 2 . 1 . 3 ( i ) ) ,
ideal s o l v e n t
e f f e c t (paragraph 2.1.2),
non-ideal
which a r e c l o s e l y connected w i t h t h e s o l v e n t
e f f e c t and a s s o c i a t e d e f f e c t s (paragraph 2.1 . l ) which a r e l e s s c l o s e l y connected. 2.1.1 A s s o c i a t e d e f f e c t s ( i ) Column o v e r l o a d i n g o
Volume o v e r l o a d i n g ( r e f . 2 ) o c c u r s when t h e w i d t h o f t h e vapour band emerging f r o m t h e i n l e t , and a f t e r s t a t i o n a r y phase f o c u s i n g ( p a r a g r a p h 2.1.1
( i i ) ) , i s comparable t o t h e
ncrease i n band w i d t h b r o u g h t about by
n o n - i d e a l chromatographic processes. Volume o v e r l o a d i n g reduces r e s o l u t i o n and r e s u l t s i n chromatographic peaks which a r e w i d e r t h a n t h o s e o b t a i n e d i n non-overloaded s i t u a t i o n s . Focusing t e c h n i q u e s ( e g s o l v e n t e f f e c t , t h e r m a l , s t a t i o n a r y phase) a r e methods o f combattifig volume o v e r l o a d i n g . I t i s i m p o r t a n t t o r e a l i z e , p a r t i c u l a r l y i n t h e p r e s e n t c o n t e x t , t h a t f o c u s i n g t o an e x t e n t much l e s s t h a n t h e column brc?adening i s unnecessary. o
c o n c e n t r a t i o n o v e r l o a d i n g ( r e f . 2 ) occurs when t h e p a r t i t i o n c o e f f i c i e n t s o f t h e s o l u t e s i n t h e f i r s t p o r t i o n o f t h e column change w i t h c o n c e n t r a t i o n . Overloaded peaks can show t a i l i n g o r f r o n t i n g ; t h e l a t t e r i s t h e most common. S o l u t e f o c u s i n g u s i n g t h e s o l v e n t e f f e c t may be p a r t i c u l a r l y e f f i c i e n t and car- r e a d i l y l e a d t o c o n c e n t r a t i o n o v e r l o a d i n g . ( i i ) 5 t a t i o n a r y phase f o c u s i n g ( r e f . 1 1 ) i s always, t o some e x t e n t , a s s o c i a t e d
w i t h t h e s o l v e n t e f f e c t . I t i s caused by d i s s o l u t i o n o f s o l u t e emerging f r o m t h e i n l e t , i n t h e s t a t i o n a r y phase i n t h e f i r s t p o r t i o n o f t h e column. The e x t e n t
of
f o c u s i n g i s l a r g e l y d e t e r m i n e d by t h e p a r t i t i o n r a t i o o f t h e s o l u t e between t h e gas and s t a t i o n a r y phases, and t h u s by t h e t e m p e r a t u r e i n t h e f o c u s i n g r e g i o n . S t a t i o n a r y phase f o c u s i n g i s most p r o f i t a b l y a p p l i e d by d e c r e a s i n g t h e column temperature, o r a t l e a s t t h e t e m p e r a t u r e i n t h e f o c u s i n g r e g i o n d u r i n g t h e f o c u s i n g process and subsequently programming t h e t e m p e r a t u r e .
I n t h e p r e s e n t c o n t e x t two p o i n t s must be emphasized: o
S t a t i o n a r y phase f o c u s i n g can p a r t i a l l y o r c o m p l e t e l y e l i m i n a t e band d i s t o r t i o n a r i s i n g f r o m n o n - i d e a l s o l v e n t e f f e c t s (paragraph 2.1.3
( i ) ) . By
3 50
j u d i c i o u s use o f s t a t i o n a r y phase f o c u s i n g many o f t h e i l l s t o which t h e s o l v e n t e f f e c t i s prone may e f f e c t i v e l y be cured. o
S t a t i o n a r y phase f o c u s i n g masks, t o some degree, event< a t t h e e x i t o f t h e i n l e t . T h i s means t h a t t h e s t u d y o f s o l u t e and s o l v e n t band shapes a t t h e end o f t h e column does n o t n e c e s s a r i l y p r o v i d e a c o r r e c t p i c t u r e o f t h e band shapes i s s u i n g from t h e i n l e t . Most s t u d i e s o f t h e s o l v e n t e f f e c t have r e l i e d on i n f o r m a t i o n o b t a i n e d a t t h e comlumn e x i t .
( i i i ) S t a t i o n a r y phase s w e l l i n g R e l a t i v e l y l a r g e amounts o f s o l v e n t a r e a s s o c i a t e d w i t h t h e s o l v e n t e f f e c t . I f t h i s s o l v e n t i s n o t s p l i t . ( p a r a g r a p h 3.5) i t passes o n t o t h e column and may cause t h e s t a t i o n a r y phase t o s w e l l . The e x t e n t o f t h e s w e l l i n g , t h e dynamic behaviour o f t h e s w o l l e n f i l m and t h e consequences o f s w e l l i n g a r e discussed elsewhere ( r e f . 1 2 ) . T v p i c a l l y t h e s o l v e n t peak may be d i s t o r t e d by f r o n t i n g and a s o l u t e band immediately b e h i n d t h e s o l v e n t may be sharpened. T h i s process was f i r s t d e s c r i b e d by H a r r i s ( r e f . 13) and was i n v e s t gated by Grob ( r e f . 14) who termed i t 'phase s o a k i n g ' . 2.1.2
The i d e a l s o l v e n t e f f e c t o c c u r s when: n o n - i d e a l chromatographic processes ( d i f f u s i o n e t c i n l e t r e g i o n (paragraph 2.1.3
a r e absent f r o m t h e
(ii))
s o l u t e i n t e r a c t i o n s w i t h t h e surface o f t h e i n l e t a r e n e g l i g i b l e (paragraph 2.1.3
(iv))
s o l u t e c o n c e n t r a t i o n i n t h e gas phase, o u t s i d e t h e f o c u s i n g r e g i o n , i s n e g l i g i b l e (paragraph 2.1.3
(v))
thermal and c o n c e n t r a t i o n e q u i l i b r i a a r e l a t e r a l l y ( i e a t r i g h t angles t o t h e gas f l o w ) mantained (paragraph 2.1.3
(vi))
the solvent f i l m ,
-
once formed, i s s t a t i c
i e does n o t c r e e p
-
and s t a b l e
- i e i t does n o t break up (paragraph 2.1.3 ( v i i ) ) Consider a f i l m o f a d i l u t e s o l u t i o n o f a s i n g l e s o l u t e i n a solvent,spread on t h e s u r f a c e o f t h e i n l e t , which may be an open t u b e o r a packed bed. Consider i n p a r t i c u l a r t h e r e a r edge o f t h e f i l m as i s shown i n F i g u r e 2
351
Fig. 2 . The s t a t i c solvent e f f e c t as applied t o l i q u i d samples. 1 ) Carrier gas flow; 2 ) I n l e t surface; 3 ) Sample f i l m . U$equals t h e 'chromatographic' v e l o c i t y of s o l u t e x and Uf equals the velocity of t h e sample film r e a r edge. Carrier gas passing over t h e f i l m will cause t h e r e a r edge t o evaporate and move forward a x i a l l y . As t h e evaporation proceeds s o l u t e accumulates in t h e rear edge, causing a concomitant increase in the s o l u t e concentration in the gas above the r e a r edge. I f t h e l i n e a r axial v e l o c i t y of t h e r e a r edge of t h e f i l m i s f a s t e r than chromotographic movement of t h e s o l u t e the s o l u t e will accumulate ( i d e a l l y ) in an i n f i n i t e l y narrow band. In o t h e r words t h e s o l u t e i s focused and t h e focusing process i s t h e solvent e f f e c t . Grob ( r e f . 15) r e f e r s t o t h i s process as "solvent trapping".
I t has been shown ( r e f . 6 ) t h a t s o l u t e focusing occurs when, i n the i n i t i a l
-
stages
K;c KO - (3 and i n the f i n a l stages
p > 0
(i) (2)
where k, KO = p a r t i t i o n c o e f f i c i e n t s of t h e s o l u t e (z) and solvent
((1)
between the gas and l i q u i d phases
p
=
phase r a t i o
Solute focusing continues u n t i l t h e r e a r edge of the f i l m reaches t h e f r o n t edge a t which point t h e focused s o l u t e and residual solvent evaporate and pass into t h e column. Equations 1 and 2 suggest t h a t :
352
> KO w i l l
o
all
o
s o l u t e s w i t h Icic< ko can be focused t o an e x t e n t determined by
o
t h e shape o f t h e f i l m i s r e l a t i v e l y u n i m p o r t a n t
o
s o l u t e f o c u s i n g i s independent o f t h e c a r r i e r gas f l o w r a t e
o
f o c u s i n g w i l l occur a t a l l temperatures between t h e m e l t i n g p o i n t and t h e
s o l u t e s f o r which Icic
be focused
fi
b o i l i n g p o i n t o f t h e sample An i d e a l (one w i t h an i n f i n i t e l y f a s t response) d e t e c t o r a t t h e e x i t of t h e i n l e t would produce c o n c e n t r a t i o n - t i m e curves s i m i l a r t o t h o s e shown i n F i g . 3.
I
F i g . 3. The i d e a l s o l v e n t e f f e c t . Peak shapes emerging f r o m an i n l e t w i t h an i d e a l s o l v e n t e f f e c t . a ) S o l v e n t s i g n a l ; b) S o l u t e s i g n a l . I n p r a c t i c e however, t h e s o l v e n t e f f e c t i s n o t i d e a l and t h e s o l u t e bands emerging f r o m t h e i n l e t can be d i s t o r t e d by a number o f phenomena. 2.1.3 Non-ideal e f f e c t s ( i ) Non-focusing Non-focusing occurs when e q u a t i o n 1 i s n o t v a l i d . F i g u r e 4 shows an example.
b
1 260
0
600
400
time
800
(s)
F i g . 4. Non-ideal e t t e c t s : non f o c u s i n g o f s o l u t e . S i g n a l s f r o m a mass spectrometer, i n s e l e c t i v e i o n m o n i t o r i n g , c o u p l e d d i r e c t l y t o a packed bed s o l v e n t e f f e c t i n l e t . a ) S o l u t e : n-decane; b) S o l v e n t :
353 methanol. The sample i s l o p 1 o f 1:lO3 n-decane i n methanol and i n j e c t e d a t 40°C under a f l o w r a t e o f 2 cm3 min-l o f helium. ( i i ) Back d i f f u s i o n Back d i f f u s i o n o f s o l u t e i n t h e gas phase i n t h e f o c u s i n g r e g i o n above t h e r e a r edge o f t h e sample f i l m broadens t h e f o c u s i n g s o l u t e band by an amount, g i v e n i n t i m e u n i t s b y ( r e f . 16) t =
Dg
.
n2 d i 4 E2 16 Vg L
(4)
where Dg
=
d i f f u s i o n c o e f f i c i e n t o f t h e s o l u t e i n t h e gas phase
di
=
i n t e r n a l diameter o f t h e i n l e t
€
=
porosity o f the i n l e t
Vg
=
volume f l o w r a t e i n t h e i n l e t
Back d i f f u s i o n can be s i g n i f i c a n t i n packed bed systems where t h e c a r r i e r gas f l o w r a t e i s s e t t o o p t i m i z e t h e column's chromatographic performance ( F i g . 5 ) .
a
b
F i g . 5. Non-ideal e f f e c t s : back d i f f u s i o n . 5a and 5b show t h e e f f e c t o f d i f f e r e n t c a r r i e r gases on peak w i d t h s emerging from packed bed s o l v e n t e f f e c t i n l e t s . I n b o t h cases t h e sam l e i s 1 5 4 of 1:103 n-octane i n n-hexane i n j e c t e d a t 30°C under 2 cm3 min-y o f c a r r i e r gas flow. The i n l e t b e i n g d i r e c t l y connected t o a mass spectrometer s e l e c t i v e l y m o n i t o r i n g f o r n-octane. I n 5a t h e c a r r i e r gas i s h e l i u m and i n 5b n i t r o g e n . D i f f u s i o n i s known t o be f a s t e r i n h e l i u m t h a n i n n i t r o g e n . Grob ( r e f . 3 3 )
has d i s c u s s e d a s i m i l a r e f f e c t i n an open t u b u l a r i n l e t .
The d e l e t e r i o u s e f f e c t s o f d i f f u s i o n can be b e s t overcome by u s i n g h i g h e r gas v e l o c i t i e s i n an o f f - l i n e system (paragraph 3 . 2 ) . ( i i i ) Chromatographic-type band-broadening processes i n p o r t i o n s o f t h e i n l e t n o t u t i l i z e d f o r t h e s o l v e n t e f f e c t , o r i n c o n n e c t i n g l i n e s between t h e i n l e t and t h e column can i n c r e a s e t h e w i d t h of t h e focused s o l u t e band ( r e f . 1 7 ) .
3 54
These processes should be m i n i m i z e d by procedures s e t o u t i n t h e p r e v i o u s paragraph. An e x p e r i m e n t a l s t u d y o f these processes i s s t i l l l a c k i n g . ( i v ) Solute l a g g a (ref.18) S o l u t e l a g g i n g occurs when
o
A d s o r p t i o n o f s o l u t e on t h e s u r f a c e o f t h e i n l e t i s s i g n i f i c a n t .
o
Slow e v a p o r a t i o n o f t h e focused s o l u t e s occurs i n t h e f i n a l stages o f t h e solvent effect. Both l e a d t o broadened peaks ( F i g u r e 6 ) .
1
a
I
5
10
15 MWS
5
m
15 YWS
F i g . 6. The e f f e c t o f r a p i d i n l e t h e a t i n g on s o l u t e peak shapes emerging f r o m packed bed solvenr; e f f e c t i n l e t s . 6a shows a chromatogram o f 1)n-octane; 2 ) n-nonane; 3 ) 2,4 d i m e t h y l hepan-3-one; 4) n-decane; 5 ) p - c r e s o l ; 6 ) n-undecane; 7 ) 2,4 d i m e t h y l a n i l i n e ; 8 ) n-dodecane; 9) n-decanol; 10) n - t r i d e c a n e ; 11) n - t e t r a d e c a n e ; 12) n-pentadecane. 20 1 o f a 1:lO7 s o l u t i o n i n n-hexane was i n j e c t e d o n t o a packed bed i n l e t connected t o a 19m, 0.3mm i . d . column c o a t e d w i t h c r o s s - l i n k e d SE-30. The sample was i n j e c t e d a t 50°C, on c o m p l e t i o n o f t h e s o l v e n t e f f e c t t h e i n l e t and column were temperat u r e programmed a t 10°C min-1. The c a r r i e r gas was hydrogen a t 50 cm sec-1. 6b was an i d e n t i c a l experiment except t h a t t h e i n l e t was b a l l i s t i c a l l y heated on c o m p l e t i o n o f t h e s o l v e n t e f f e c t . A s p e c i a l k i n d o f s o l u t e l a g g i n g a l s o o c c u r s when t h e i n l e t i s c o a t e d w i t h s t a t i o n a r y phase ( r e f . 1 9 ) . I n t h i s case s o l u t e s w i t h h i g h p a r t i t i o n r a t i o s between t h e s t a t i o n a r y phase and t h e gas phase chromatograph t o o s l o w l y t o remain i n t h e advancing back edge o f t h e s o l v e n t f i l m . F o c u s i n g , t h e r e f o r e ,
does
n o t occur. Grob ( r e f . 20) demonstrated t h i s phenomenon and c o i n e d t h e t e r m "band broadening i n space" t o d e s c r i b e i t . He termed t h e removal o f s t a t i o n a r y phase Prom t h e i n l e t r e g i o n t o be " c r e a t i n g a r e t e n t i o n gap". Both terms, i n
355
o u r view, a r e unnecessary. ( v ) S o l u t e escape S o l u t e escape ( r e f . 21) r e f e r s t o s o l u t e which i s t r a n s p o r t e d o u t of t h e i n l e t during t h e focusing period. The f r a c t i o n a l escape i s g i v e n by Mxe -
MXS
b+P
(5)
where Mxe 'M ,
=
mass o f s o l u t e t o escape
=
mass o f o r i g i n a l sample
S o l u t e escape i s c o n t r o l l e d s o l e l y by t h e vapour p r e s s u r e o f t h e s o l u t e o v e r t h e sample. The g r e a t e r t h e vapour p r e s s u r e t h e g r e a t e r t h e degree o f escape.
A
l a r g e p o l a r i t y d i f f e r e n c e between s o l v e n t and s o l u t e w i l l i n c r e a s e
t h e degree o f escape and s h o u l d be avoided i f p o s s i b l e . Grob J r ( r e f s . 20, 22,
23) has c o i n e d t h e terms "band broadening i n t i m e " , " p a r t i a l s o l v e n t t r a p p i n g " and "two s t e p chromatography" (among o t h e r s ) . Except f o r s o l u t e s which do n o t obey e q u a t i o n 1 s o l u t e escape may be prevented by p l a c i n g a f i l m o f pure s o l v e n t ahead o f t h e sample f i l m ( r e f . 21) I n p r a c t i c e t h i s i s t r i c k y t o manage and achieves l i t t l e . The degree o f escape f a l l s r a p i d l y w i t h p a r t i t i o n r a t i o ( e q u a t i o n 5 ) and i s n e g l i g i b l e f o r say n-decane d i s s o l v e d i n n-hexane. Escaped s o l u t e can o f t e n be r e f o c u s e d by phase s w e l l i n g ( r e f . 10). ( v i ) Thermal e f f e c t s o
E v a p o r a t i v e c o o l i n g o c c u r s on t h e r e a r edge o f t h e sample f i l m when t h e h e a t needed t o e v a p o r a t e t h e sample i s n o t s u p p l i e d q u i c k l y enough ( r e f . 2 4 ) . Temperature drops o f up t o 20°C have been measured i n packed bed systems made o f g l a s s . C o o l i n g i s p r o b a b l y i n s i g n i f i c a n t i n open t u b u l a r systems.
As f a r as we can a s c e r t a i n e v a p o r a t i v e c o o l i n g does n o t , i n p r a c t i c e , detrimentally a f f e c t the solvent e f f e c t .
o
L a t e r a l thermal g r a d i e n t s may a l s o o c c u r i n packed bed systems where t h e packing i s p o o r l y conducting. I n such s i t u a t i o n s t h e r e a r edge o f a sample f i l m w i l l move a t d i f f e r e n t
a x i a l v e l o c i t i e s across t h e t u b e and may broaden s o l u t e bands as a r e s u l t . D i r e c t e x p e r i m e n t a l e v i d e n c e o f such g r a d i e n t s i s s t i l l l a c k i n g .
356
( v i i ) Film i n s t a b i l i t y
o
F i l m creep occurs when, once t h e sample has been formed, t h e f i l m as a whole i s moved a x i a l l y f o r w a r d by shear f o r c e s e x e r t e d by t h e c a r r i e r gas. Creep i s g r e a t e s t i n smooth w a l l e d open t u b u l a r systems and l e a s t i n porous packing packed beds ( r e f . 2 5 ) . F i l m c r e e p does n o t a f f e c t t h e s o l v e n t f o c u s i n g b u t i n c r e a s e s t h e l e n g h t o f i n l e t r e q u i r e d f o r a p a r t i c u l a r s i z e o f sample.
o
F i l m break up may be a t t r i b u t e d t o f i l m i n s t a b i l i t y and shear f o r c e s . The former i s e v i d e n t when t h e s u r f a c e t e n s i o n o f t h e sample i s g r e a t e r t h a n t h e excess s u r f a c e f r e e - e n e r g y o f t h e i n n e r s u r f a c e s o f t h e i n l e t and i s most p r e v a l e n t w i t h p o l a r s o l v e n t s ( r e f . 2 6 ) . Shear f o r c e s f r o m t h e c a r r i e r gas can cause t h e w e t t e d f i l m on a smooth w a l l e d open t u b u l a r i n l e t t o break up f o r m i n g l e n s e s . Rough-wall open t u b e and packed bed i n l e t s a r e l e s s
2.2
prone t o s h e a r - f o r c e break up.
Vapour samples ( S t a t i c Systems) ( r e f . 27) F o r s o l v e n t e f f e c t f o c u s i n g t h e sample s h o u l d c o n s i s t o f vapours i n a non-
i n t e r a c t i n g gas. The sample may e x i s t as such, eg o r g a n i c p o l l u t a n t s i n a i r , o r may be formed by p a s s i n g c a r r i e r gas o v e r t h e sample (head-space s a m p l i n g ) .
A f i l m o f s o l v e n t i s l a i d down i n t h e i n l e t and sample vapour i s passed o v e r t h i s f i l m . Focusing o c c u r s by t h e mechanism o u t l i n e d i n paragraph 2.1.2 and t h e c o n d i t i o n f o r f o c u s i n g i s a l s o g i v e n by e q u a t i o n s 1 and 2. The sample f e e d i s c o n t i n u e d u n t i l t h e e v a p o r a t i n g r e a r edge approaches t h e f r o n t edge. Sample f e e d i s t e r m i n a t e d and p u r e c a r r i e r gas i s i n t r o d u c e d . Subsequent events p a r a l l e l t h o s e p r e v i o u s l y o u t l i n e d f o r l i q u i d samples. S o l u t e escape does n o t o c c u r i n t h i s sampling mode, except f o r non-focusinp s o l u t e s ; t h i s can be a m a j o r advantage. Vapour samples c o n t a i n i n g l a r g e amoun s o f water can he e a s i l y handled u s i n g t h i s t e c h n i q u e . Water i s a n o n - f o c u s i n g s o l u t e on a n o n - p o l a r s o l v e n t such as hexane and i s t h e r e f o r e n o t accumulated 2.3
Dynamic Systems ( L i q u i d samples) Dynamic systems a r e a r e l a t i v e l y new i n n o v a t i o n i n v o l v i n g a moving
(dynamic) sample " f i l m " and c o u n t e r c u r r e n t c a r r i e r gas f l o w ( r e f . 2 6 ) . They have n o t y e t been s t u d i e d i n as g r e a t d e t a i l as have s t a t i c systems and t h e t h e o r e t i c a l t r e a t m e n t and d i s c u s s i o n presented h e r e i s c o n s e q u e n t l y s k e t c h y . Consider a tube, c o n t a i n i n g an a x i a l porous l a y e r and a gas channel w i t h
357
one end dipped i n t o a l i q u i d sample and t h e o t h e r end connected t o a c a r r i e r gas s u p p l y ( F i g u r e 7 ) . The l i q u i d sample r i s e s by c a p i l l a r y a c t i o n i n t h e porous l a y e r w h i l e t h e c a r r i e r gas evaporates sample a t t h e upper edge, c a u s i n g i t t o move downwards. E q u i 1 i b r i u m . o c c u r s when t h e volume r a t e o f c a p i l l a r y
r i s e equals t h e volume r a t e o f e v a p o r a t i o n . S o l u t e i s focused i n t h e e v a p o r a t i o n zone by processes s i m i l a r t o t h o s e i n t h e s t a t i c systems p r e v i o u s l y d e s c r i b e d (paragraph 2 ) .
a
b 9
-2
F i g . 7a) Schematic diagram o f operation o f solvent e f f e c t i n t h e dynami c mode. 1 ) Pure gas ( f o r l i q u i d samples) o r head space sample; 2) Glass tube; 3) Pure s o l v e n t ( f o r head space samples) o r d i l u t e l i q u i d sample; 4) Region o f porous bed w e t t e d by l i q u i d ; 5 ) Solute/head space component m o l e c u l e c a r r i e d up by c a p i l l a r y r i s e o f l i q u i d ; 6) C a p i l l a r y r i s e o f l i q u i d ; 7 ) Chromatographic t r a n s p o r t o f s o l u t e s ; 8 ) S o l u t e s a c c u m u l a t i n g i n e v a p o r a t i o n zone when 5 and 6 a r e f a s t e r t h a n 7; 9) Dry r e g i o n o f porous bed. 7b) P l a n view. 3. 3.1
PRACTICAL ASPECTS S t a t i c vs dynamic systems
o
s t a t i c systems may be o n - l i n e o r o f f - l i n e ;
o
s t a t i c systems a r e p r e s e n t l y more w e l l - d e v e l o p e d t h a n a r e dynamic systems
o
dynamic systems can h a n d l e l a r g e r samples t h a n can s t a t i c systems
o
dynamic systems a r e more c o m p l i c a t e d t h a n s t a t i c systems
3.2 o
dynamic systems o n l y o f f - l i n e
O n - l i n e vs o f f - l i n e systems ' o f f - l i n e systems can employ h i g h c a r r i e r gas f l o w s t h e r e b y s h o r t e n i n g t h e
358
t i m e needed t o complete t h e s o l u t e f o c u s i n g
o
off-line
sampling can be done i n p a r a l l e l w i t h t h e chromatography; t h i s
s h o r t e n s a n a l y s i s t i m e i n r o u t i n e work o
o f f - l i n e systems a v o i d i n t r o d u c i n g n o n - v o l a t i l e r e s i d u e s i n t o t h e column
o
most o f t h e s o l v e n t i s removed d u r i n g o f f - l i n e o p e r a t i o n and t h i s reduces t h e amount o f s o l v e n t i n t r o d u c e d on t h e column
o 3.3
o
o n - l i n e systems a r e s i m p l e r t h a n o f f - l i n e systems Open vs packed tube i n l e t s open tubes, p a r t i c u l a r l y t h o s e w i t h smooth w a l l a r e prone t o f i l m creep and i n s t a b i l i t y . T h i s problem i s f a r l e s s i n packed tubes
o
packed tubes can handle l a r g e r samples p e r u n i t l e n g h t t h a n can open tubes
o
packed t u b e i n l e t s must be independenc'ently heated t o a v o i d l a g g i n g ; when open tubes a r e used t h e chromatograph oven i s s u f f i c i e n t
3.4
o
D i r e c t vs s p l i t l e s s i n j e c t i o n s p l i t l e s s i n j e c t i o n e n t a i l s v a p o r i s a t i o n and can l e a d t o decomposition o f thermally l a b i l e solutes
o
d i r e c t i n j e c t i o n does n o t r e q u i r e v a p o r i s a t i o n . I n p r o p e r l y d e a c t i v a t e d i n l e t s s o l u t e s can be t r a n s f e r r e d t o t h e column a t l o w temperatures
3.5
Solvent s p l i t t i n g r e f e r s t o t h e removal o f s o l v e n t d u r i n g f o c u s i n g b e f o r e t h e focused s o l u t e s a r e t r a n s f e r r e d t o t h e column s o l v e n t s p l i t t i n g occurs a u t o m a t i c a l l y i n o f f - l i n e systems and can be invoked i n o n - l i n e systems by u s i n g a Deans s w i t c h ( r e f . 2 9 ) . i f t h e s o l v e n t i s n o t s p l i t t h e l a r g e amounts o f s o l v e n t p a s s i n g i n t o t h e column can cause s t a t i o n a r y phase s w e l l i n g ( r e f . 14) and t a i l i n g o f solvent peaks i n a p r o p e r l y designed system n o n - s p l i t t i n g o f s o l v e n t does n o t cause problems ( F i g . 8 )
359 F i g . 8. A p r o t o t y p e i n l e t f o r t h e s t a t i c solvent e f f e c t 1 ) C a r r i e r gas i n l e t 2 ) Septum n u t 3 ) Septum purge 4 ) Gas chromatograph oven l i d 5 ) Packing 6 ) Fused s i l i c a l i n e 7) Connection 8 ) Heater c o i l s
3.6
tiardware A l a r g E number o f i n l e t s designed f o r s o l u t e f o c u s i n g u s i n g t h e s o l v e n t
e f f e c t have been d e s c r i b e d ( r e f s . 30, 31, 3 2 ) ; t h e f i n a l word has y e t t o be written. F i g u r e s 8 and 9 a r e schematic r e p r e s e n t a t i o n s o f s e v e r a l p r o t o t y p e i n l e t s b e i n g developed i n o u r l a b o r a t o r i e s .
4. PRACTICAL EXAMPLES F i g u r e 10 shows a chromatogram o f a headspace sample generated by a dynamic sol vent e f f e c t i n l e t . F i g u r e 6b shows a chromatogram o f a l i q u i d sample generated by a s t a t i c solvent e f f e c t operating on-line.
360
F i g . 9. Diagrammatic v e r t i c a l s e c t i o n o f system used t o t r a n s f e r sample f r o m an o f f - l i n e , dynamic, s o l v e n t e f f e c t sampler t o a c a p i l l a r y GLC column
1) 2) 3) 4) 5) 6) 7) 8) 9)
9
10)
-7
9
C a r r i e r gas i n l e t Removable cap Ground g l a s s j o i n t O f f - l i n e , dynamic, s o l v e n t e f f e c t sampler Heater c o i l s Purge gas e x i t Porous packed bed To column E l e c t r i c a l power s u p p l y . S o l u t e s and s o l v e n t a r e evaporated f r o m t h e o f f - l i n e , dynamic, s o l v e n t e f f e c t sampler by e l e c t r i c a l h e a t = i n g and condensed i n t o t h e porous bed where on-1 i n e , s t a t i c , nonsolvent s p l i t t i n g solvent e f f e c t focusing i s carried out. Seat
361
F i g . 10. A chromatogram o f v o l a t i l e s f r o m 10 m l o f Simonsig Colombar wine sampled by s t r i p p i n g w i t h 15 rnl m i n - I o f p u r e N2 f o r 30 min and t r a p p i n g by o f f - l i n e , dynamic s o l v e n t e f f e c t f o c u s i n g on p u r e n-hexane a t 30°C. Chromatographic c o n d i t i o n s as i n F i g u r e 6. REFERENCES
1 V. P r e t o r i u s and W. B e r t s c h , J . HRC & CC, 6 (1983) 64. 2 V. P r e t o r i u s , K. Lawson and W. B e r t s c h , J. HRC & CC, 6 (1983 185. 3 K. Grob and K. Grob Jr, J. Chrom, 94 (1974) 53. 4 K. Grob and G. Grob, J. Chrom. S c i . , 7 (1969) 584. 5 D.R. Deans, Anal. Chem., 43 (1971) 2026. 6 V. P r e t o r i u s , C.S.G. P h i l l i p s and W. B e r t s c h , 3 . HRC & CC, 6 ( 1 983) 232. 7 F.J. Yang, A.C. Brown and S.P. Cram, 3 . Chrom., 158 (1978) 91 8 W.G. Jennings, R.R. Freeman and T.A. Rooney, J. HRC & CC, 1 1978) 275. 9 K. Knauss, J . Fulleman and M.P. T u r n e r . J . HRC & CC. 4 (1981 641. 10 R.G. J e n k i n s , i n "Proceedings o f t h e F o u r t h I n t e r n a t i o n a l Symposium on C a p i l l a r y Chromatography, Hindelang, 1981", R.E. K a i s e r ed., H e u t h i g , H e i d e l b e r g , 1981, 563 pp. 11 V. P r e t o r i u s , E.R. Rohwer and K.H. Lawson, S . A f r . J. Chem, 37(2) (1984) 65. 12 V. P r e t o r i u s e t a l . I n p r e p a r a t i o n . 13 W.E. H a r r i s , J. Chrom. S c i . , 11 (1973) 184. 14 K. Grob J r and B. S c h i l l i n g , Chromatographia, 17 (1983) 361. 15 K. Grob J r , J . Chrom., 264 (1983) 7. 16 V. P r e t o r i u s e t a l . I n p r e p a r a t i o n . 17 V. P r e t o r i u s e t a l . I n p r e p a r a t i o n . 18 V. P r e t o r i u s , C.S.G. P h i l l i p s and W . B e r t s c h , J. HRC & CC, 6 (1983) 321. 19 V. P r e t o r i u s , K.H. Lawson, E.R. Rohwer and W. B e r t s c h , J . HRC & CC, 7 (1984) 92. 20 K. Grob Jr, J. Chrom., 213 (1981) 3. 21 V. P r e t o r i u s , K. Lawson and W . B e r t s c h , J . HRC & CC, 6 (1983) 419. 22 K. Grob J r , Chromatographia, 17 (1983) 357. 23 K. Grob J r , J. Chrom., 253 (1982) 17. 24 V. P r e t o r i u s , P. Apps, E.R. Rohwer and K.H. Lawson, J. HRC & CC, 7 (1984) 210. 25 V. P r e t o r i u s , K.H. Lawson, P. Apps and W. B e r t s c h , J . Chrom., 279 (1983) 233. 26 P. Sandra, I . Temmerman and M. Verstappe, J. HRC & CC, 6 (1983) 501. 27 V. P r e t o r i u s e t a l . I n p r e p a r a t i o n .
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V. P r e t o r i u s , P. Apps, E.R. Rohwer and K.H. Lawson, J. HRC & CC, 7 (1984) 212. 29 D.R. Deans, Chromatographia, 1/2 (1968) 18. 30 E. Geeraert, D. de Schepper and P. Sandra, J. HRC & CC, 6 (1983) 386. 31 K. Grob and K. Grob Jr, J . Chrom., 151 (1978) 311. 31 G. Schomburg, H. Behlau, R. Dielmann, F. Weeke and H. Husmann, J . Chrom., 142 (1977) 87. 33 K. Grob J r , J . HRC & CC, 8 (1984) 461. 28
THE SOLVENT EFFECT I N GAS LIQIJID CHROMATOGRAPHY
V PRETORIUS, K LAWSON, E ROHIIER, P APPS UNIVERSITY OF PRETORIA-INST. FOR CHROMATOGRAPHY 1.
-
0002 PRETORIA
SOUTH AFRICA
INTRODUCTION
The s o l v e n t e f f e c t i s a phenomenon which may be employed as one o f t h e many ways o f sample i n t r o d u c t i o n i n g a s - l i q u i d chromotography ( r e f . 1 ) . More p a r t i c u l a r l y i t i s one o f t h e t h r e e modes o f s o l u t e f o c u s i n g . I t i s an e s p e c i a l l y e f f e c t i v e means o f q u a n t i t a t i v e l y and r e p r o d u c i b l y t r a n s f e r r i n g s o l u t e s f r o m d i l u t e s o l u t i o n s t o t h e column ( r e f . 2) w i t h o u t o v e r l o a d i n g under m i l d c o n d i t i o n s . Consequently i t i s o f p a r t i c u l a r v a l u e i n t r a c e a n a l y s i s where t h e r m a l l y l a b i l e s o l u t e s a r e i n v o l v e d . Although t h e t e r m " s o l v e n t e f f e c t " was o n l y c o i n e d l a t e r ( r e f . 3 ) , t h e phenomenon was p r o b a b l y f i r s t observed i n 1969 by Grob ( r e f . 4 ) . U s i n g a d i l u t e s o l u t i o n o f r e l a t i v e l y n o n - v o l a t i l e s t e r o i d s i n b i s ( t r i m e t h y l s i l y l ) acetamide and an excess o f hexane he observed t h a t l a r g e amounts o f sample c o u l d be i n t r o d u c e d i n t o a c a p i l l a r y column w i t h o u t o v e r l o a d i n a " p r o v i d e d t h e columr! temperature i s a t l e a s t 100°C below t h e b o i l i n g p o i n t o f t h e most v o l a t i l e s o l u t e " . Under t h e s e circumstances s t a t i o n a r y phase f o c u s i n g (paragraph 2.1.1.(ii)),
and n o t t h e s o l v e n t e f f e c t . i s dominant.
The f i r s t unequivocal d e s c r i p t i o n and e x p l a n a t i o n o f t h e s o l v e n t e f f e c t , as we understand i t today, was p u b l i s h e d by Deans i n 1971 ( r e f . 5 ) . U n f o r t u n a t e l y he used t h e t i t l e "The sample as i t s own s t a t i o n a r y phase i n gas chromatography'; and r e g r e t t a b l y , b u t u n d e r s t a n d a b l y , t h e i m p o r t o f h i s work remained l a r g e l y unrecognized u n t i l 1982 ( r e f . 5 ) . S i n c e i t s d i s c o v e r y a g r e a t deal has been p u b l i s h e d on t h e s o l v e n t e f f e c t ( r e f s . 7-10). Although t h e p r a c t i c a l u s e f u l n e s s o f t h e e f f e c t became apparent y e a r s ago i t i s o n l y r e c e n t l y t h a t t h e u n d e r l y i n g processes have been u n r a v e l l e d and e x p l a i n e d . I n essence s o l u t e f o c u s i n g u s i n g t h e s o l v e n t e f f e c t i n v o l v e s t h e f o l l o w i n g :
o
a d i l u t e vapour o r l i q u i d sample
o
t h e f o r m a t i o n o f a f i l m o f l i q u i d sample i n t h e i n l e t
348 o
t h e e v a p o r a t i o n o f t h e sample f i l m so t h a t s o l u t e s a r e focused by t h e solvent e f f e c t
o
t h e t r a n s f e r of focused s o l u t e s t o t h e column These i s s u e s may be a r b i t r a r i l y d i v i d e d i n t o fundamental ( p a r a g r a p h 2 ) and
p r a c t i c a l aspects (paragraph 3 ) . The former a r e concerned w i t h t h e s o l v e n t e f f e c t i t s e l f and t h e l a t t e r w i t h i t s p r a c t i c a l o p t i o n s . H i s t o r i c a l l y t h e s o l v e n t e f f e c t has been used f o r l i w i d samples i n open t u b u l a r i n l e t s . T h i s paper w i l l show t h a t b o t h l i q u i d and vapour samples can be handled, t h a t i n l e t geometries o t h e r t h a n open tubes can be used and t h e s o l v e n t e f f e c t can o p e r a t e e i t h e r s t a t i c a l l y o r d y n a m i c a l l y . The f u l l range o f o p t i o n s i s shown i n f i g u r e 1.
F i g . 1. P r a c t i c a l o p t i o n s a v a i l a b l e when.employing t h e s o l v e n t e f f e c t . L i n e s connect c o m p a t i b l e o p e r a t i o n a l parameters.
349
2.
FUNDAMENTAL ASPECTS It i s convenient t o consider t h e a p p l i c a t i o n o f the solvent e f f e c t t o l i q u i d
samples and t o vapour samples s e p a r a t e l y . 2.1
L i q u i d samples ( S t a t i c System) We d i s t i n g u i s h between an
e f f e c t s (paragraph 2 . 1 . 3 ( i ) ) ,
ideal s o l v e n t
e f f e c t (paragraph 2.1.2),
non-ideal
which a r e c l o s e l y connected w i t h t h e s o l v e n t
e f f e c t and a s s o c i a t e d e f f e c t s (paragraph 2.1 . l ) which a r e l e s s c l o s e l y connected. 2.1.1 A s s o c i a t e d e f f e c t s ( i ) Column o v e r l o a d i n g o
Volume o v e r l o a d i n g ( r e f . 2 ) o c c u r s when t h e w i d t h o f t h e vapour band emerging f r o m t h e i n l e t , and a f t e r s t a t i o n a r y phase f o c u s i n g ( p a r a g r a p h 2.1.1
( i i ) ) , i s comparable t o t h e
ncrease i n band w i d t h b r o u g h t about by
n o n - i d e a l chromatographic processes. Volume o v e r l o a d i n g reduces r e s o l u t i o n and r e s u l t s i n chromatographic peaks which a r e w i d e r t h a n t h o s e o b t a i n e d i n non-overloaded s i t u a t i o n s . Focusing t e c h n i q u e s ( e g s o l v e n t e f f e c t , t h e r m a l , s t a t i o n a r y phase) a r e methods o f combattifig volume o v e r l o a d i n g . I t i s i m p o r t a n t t o r e a l i z e , p a r t i c u l a r l y i n t h e p r e s e n t c o n t e x t , t h a t f o c u s i n g t o an e x t e n t much l e s s t h a n t h e column brc?adening i s unnecessary. o
c o n c e n t r a t i o n o v e r l o a d i n g ( r e f . 2 ) occurs when t h e p a r t i t i o n c o e f f i c i e n t s o f t h e s o l u t e s i n t h e f i r s t p o r t i o n o f t h e column change w i t h c o n c e n t r a t i o n . Overloaded peaks can show t a i l i n g o r f r o n t i n g ; t h e l a t t e r i s t h e most common. S o l u t e f o c u s i n g u s i n g t h e s o l v e n t e f f e c t may be p a r t i c u l a r l y e f f i c i e n t and car- r e a d i l y l e a d t o c o n c e n t r a t i o n o v e r l o a d i n g . ( i i ) 5 t a t i o n a r y phase f o c u s i n g ( r e f . 1 1 ) i s always, t o some e x t e n t , a s s o c i a t e d
w i t h t h e s o l v e n t e f f e c t . I t i s caused by d i s s o l u t i o n o f s o l u t e emerging f r o m t h e i n l e t , i n t h e s t a t i o n a r y phase i n t h e f i r s t p o r t i o n o f t h e column. The e x t e n t
of
f o c u s i n g i s l a r g e l y d e t e r m i n e d by t h e p a r t i t i o n r a t i o o f t h e s o l u t e between t h e gas and s t a t i o n a r y phases, and t h u s by t h e t e m p e r a t u r e i n t h e f o c u s i n g r e g i o n . S t a t i o n a r y phase f o c u s i n g i s most p r o f i t a b l y a p p l i e d by d e c r e a s i n g t h e column temperature, o r a t l e a s t t h e t e m p e r a t u r e i n t h e f o c u s i n g r e g i o n d u r i n g t h e f o c u s i n g process and subsequently programming t h e t e m p e r a t u r e .
I n t h e p r e s e n t c o n t e x t two p o i n t s must be emphasized: o
S t a t i o n a r y phase f o c u s i n g can p a r t i a l l y o r c o m p l e t e l y e l i m i n a t e band d i s t o r t i o n a r i s i n g f r o m n o n - i d e a l s o l v e n t e f f e c t s (paragraph 2.1.3
( i ) ) . By
3 50
j u d i c i o u s use o f s t a t i o n a r y phase f o c u s i n g many o f t h e i l l s t o which t h e s o l v e n t e f f e c t i s prone may e f f e c t i v e l y be cured. o
S t a t i o n a r y phase f o c u s i n g masks, t o some degree, event< a t t h e e x i t o f t h e i n l e t . T h i s means t h a t t h e s t u d y o f s o l u t e and s o l v e n t band shapes a t t h e end o f t h e column does n o t n e c e s s a r i l y p r o v i d e a c o r r e c t p i c t u r e o f t h e band shapes i s s u i n g from t h e i n l e t . Most s t u d i e s o f t h e s o l v e n t e f f e c t have r e l i e d on i n f o r m a t i o n o b t a i n e d a t t h e comlumn e x i t .
( i i i ) S t a t i o n a r y phase s w e l l i n g R e l a t i v e l y l a r g e amounts o f s o l v e n t a r e a s s o c i a t e d w i t h t h e s o l v e n t e f f e c t . I f t h i s s o l v e n t i s n o t s p l i t . ( p a r a g r a p h 3.5) i t passes o n t o t h e column and may cause t h e s t a t i o n a r y phase t o s w e l l . The e x t e n t o f t h e s w e l l i n g , t h e dynamic behaviour o f t h e s w o l l e n f i l m and t h e consequences o f s w e l l i n g a r e discussed elsewhere ( r e f . 1 2 ) . T v p i c a l l y t h e s o l v e n t peak may be d i s t o r t e d by f r o n t i n g and a s o l u t e band immediately b e h i n d t h e s o l v e n t may be sharpened. T h i s process was f i r s t d e s c r i b e d by H a r r i s ( r e f . 13) and was i n v e s t gated by Grob ( r e f . 14) who termed i t 'phase s o a k i n g ' . 2.1.2
The i d e a l s o l v e n t e f f e c t o c c u r s when: n o n - i d e a l chromatographic processes ( d i f f u s i o n e t c i n l e t r e g i o n (paragraph 2.1.3
a r e absent f r o m t h e
(ii))
s o l u t e i n t e r a c t i o n s w i t h t h e surface o f t h e i n l e t a r e n e g l i g i b l e (paragraph 2.1.3
(iv))
s o l u t e c o n c e n t r a t i o n i n t h e gas phase, o u t s i d e t h e f o c u s i n g r e g i o n , i s n e g l i g i b l e (paragraph 2.1.3
(v))
thermal and c o n c e n t r a t i o n e q u i l i b r i a a r e l a t e r a l l y ( i e a t r i g h t angles t o t h e gas f l o w ) mantained (paragraph 2.1.3
(vi))
the solvent f i l m ,
-
once formed, i s s t a t i c
i e does n o t c r e e p
-
and s t a b l e
- i e i t does n o t break up (paragraph 2.1.3 ( v i i ) ) Consider a f i l m o f a d i l u t e s o l u t i o n o f a s i n g l e s o l u t e i n a solvent,spread on t h e s u r f a c e o f t h e i n l e t , which may be an open t u b e o r a packed bed. Consider i n p a r t i c u l a r t h e r e a r edge o f t h e f i l m as i s shown i n F i g u r e 2
351
Fig. 2 . The s t a t i c solvent e f f e c t as applied t o l i q u i d samples. 1 ) Carrier gas flow; 2 ) I n l e t surface; 3 ) Sample f i l m . U$equals t h e 'chromatographic' v e l o c i t y of s o l u t e x and Uf equals the velocity of t h e sample film r e a r edge. Carrier gas passing over t h e f i l m will cause t h e r e a r edge t o evaporate and move forward a x i a l l y . As t h e evaporation proceeds s o l u t e accumulates in t h e rear edge, causing a concomitant increase in the s o l u t e concentration in the gas above the r e a r edge. I f t h e l i n e a r axial v e l o c i t y of t h e r e a r edge of t h e f i l m i s f a s t e r than chromotographic movement of t h e s o l u t e the s o l u t e will accumulate ( i d e a l l y ) in an i n f i n i t e l y narrow band. In o t h e r words t h e s o l u t e i s focused and t h e focusing process i s t h e solvent e f f e c t . Grob ( r e f . 15) r e f e r s t o t h i s process as "solvent trapping".
I t has been shown ( r e f . 6 ) t h a t s o l u t e focusing occurs when, i n the i n i t i a l
-
stages
K;c KO - (3 and i n the f i n a l stages
p > 0
(i) (2)
where k, KO = p a r t i t i o n c o e f f i c i e n t s of t h e s o l u t e (z) and solvent
((1)
between the gas and l i q u i d phases
p
=
phase r a t i o
Solute focusing continues u n t i l t h e r e a r edge of the f i l m reaches t h e f r o n t edge a t which point t h e focused s o l u t e and residual solvent evaporate and pass into t h e column. Equations 1 and 2 suggest t h a t :
352
> KO w i l l
o
all
o
s o l u t e s w i t h Icic< ko can be focused t o an e x t e n t determined by
o
t h e shape o f t h e f i l m i s r e l a t i v e l y u n i m p o r t a n t
o
s o l u t e f o c u s i n g i s independent o f t h e c a r r i e r gas f l o w r a t e
o
f o c u s i n g w i l l occur a t a l l temperatures between t h e m e l t i n g p o i n t and t h e
s o l u t e s f o r which Icic
be focused
fi
b o i l i n g p o i n t o f t h e sample An i d e a l (one w i t h an i n f i n i t e l y f a s t response) d e t e c t o r a t t h e e x i t of t h e i n l e t would produce c o n c e n t r a t i o n - t i m e curves s i m i l a r t o t h o s e shown i n F i g . 3.
I
F i g . 3. The i d e a l s o l v e n t e f f e c t . Peak shapes emerging f r o m an i n l e t w i t h an i d e a l s o l v e n t e f f e c t . a ) S o l v e n t s i g n a l ; b) S o l u t e s i g n a l . I n p r a c t i c e however, t h e s o l v e n t e f f e c t i s n o t i d e a l and t h e s o l u t e bands emerging f r o m t h e i n l e t can be d i s t o r t e d by a number o f phenomena. 2.1.3 Non-ideal e f f e c t s ( i ) Non-focusing Non-focusing occurs when e q u a t i o n 1 i s n o t v a l i d . F i g u r e 4 shows an example.
b
1 260
0
600
400
time
800
(s)
F i g . 4. Non-ideal e t t e c t s : non f o c u s i n g o f s o l u t e . S i g n a l s f r o m a mass spectrometer, i n s e l e c t i v e i o n m o n i t o r i n g , c o u p l e d d i r e c t l y t o a packed bed s o l v e n t e f f e c t i n l e t . a ) S o l u t e : n-decane; b) S o l v e n t :
353 methanol. The sample i s l o p 1 o f 1:lO3 n-decane i n methanol and i n j e c t e d a t 40°C under a f l o w r a t e o f 2 cm3 min-l o f helium. ( i i ) Back d i f f u s i o n Back d i f f u s i o n o f s o l u t e i n t h e gas phase i n t h e f o c u s i n g r e g i o n above t h e r e a r edge o f t h e sample f i l m broadens t h e f o c u s i n g s o l u t e band by an amount, g i v e n i n t i m e u n i t s b y ( r e f . 16) t =
Dg
.
n2 d i 4 E2 16 Vg L
(4)
where Dg
=
d i f f u s i o n c o e f f i c i e n t o f t h e s o l u t e i n t h e gas phase
di
=
i n t e r n a l diameter o f t h e i n l e t
€
=
porosity o f the i n l e t
Vg
=
volume f l o w r a t e i n t h e i n l e t
Back d i f f u s i o n can be s i g n i f i c a n t i n packed bed systems where t h e c a r r i e r gas f l o w r a t e i s s e t t o o p t i m i z e t h e column's chromatographic performance ( F i g . 5 ) .
a
b
F i g . 5. Non-ideal e f f e c t s : back d i f f u s i o n . 5a and 5b show t h e e f f e c t o f d i f f e r e n t c a r r i e r gases on peak w i d t h s emerging from packed bed s o l v e n t e f f e c t i n l e t s . I n b o t h cases t h e sam l e i s 1 5 4 of 1:103 n-octane i n n-hexane i n j e c t e d a t 30°C under 2 cm3 min-y o f c a r r i e r gas flow. The i n l e t b e i n g d i r e c t l y connected t o a mass spectrometer s e l e c t i v e l y m o n i t o r i n g f o r n-octane. I n 5a t h e c a r r i e r gas i s h e l i u m and i n 5b n i t r o g e n . D i f f u s i o n i s known t o be f a s t e r i n h e l i u m t h a n i n n i t r o g e n . Grob ( r e f . 3 3 )
has d i s c u s s e d a s i m i l a r e f f e c t i n an open t u b u l a r i n l e t .
The d e l e t e r i o u s e f f e c t s o f d i f f u s i o n can be b e s t overcome by u s i n g h i g h e r gas v e l o c i t i e s i n an o f f - l i n e system (paragraph 3 . 2 ) . ( i i i ) Chromatographic-type band-broadening processes i n p o r t i o n s o f t h e i n l e t n o t u t i l i z e d f o r t h e s o l v e n t e f f e c t , o r i n c o n n e c t i n g l i n e s between t h e i n l e t and t h e column can i n c r e a s e t h e w i d t h of t h e focused s o l u t e band ( r e f . 1 7 ) .
3 54
These processes should be m i n i m i z e d by procedures s e t o u t i n t h e p r e v i o u s paragraph. An e x p e r i m e n t a l s t u d y o f these processes i s s t i l l l a c k i n g . ( i v ) Solute l a g g a (ref.18) S o l u t e l a g g i n g occurs when
o
A d s o r p t i o n o f s o l u t e on t h e s u r f a c e o f t h e i n l e t i s s i g n i f i c a n t .
o
Slow e v a p o r a t i o n o f t h e focused s o l u t e s occurs i n t h e f i n a l stages o f t h e solvent effect. Both l e a d t o broadened peaks ( F i g u r e 6 ) .
1
a
I
5
10
15 MWS
5
m
15 YWS
F i g . 6. The e f f e c t o f r a p i d i n l e t h e a t i n g on s o l u t e peak shapes emerging f r o m packed bed solvenr; e f f e c t i n l e t s . 6a shows a chromatogram o f 1)n-octane; 2 ) n-nonane; 3 ) 2,4 d i m e t h y l hepan-3-one; 4) n-decane; 5 ) p - c r e s o l ; 6 ) n-undecane; 7 ) 2,4 d i m e t h y l a n i l i n e ; 8 ) n-dodecane; 9) n-decanol; 10) n - t r i d e c a n e ; 11) n - t e t r a d e c a n e ; 12) n-pentadecane. 20 1 o f a 1:lO7 s o l u t i o n i n n-hexane was i n j e c t e d o n t o a packed bed i n l e t connected t o a 19m, 0.3mm i . d . column c o a t e d w i t h c r o s s - l i n k e d SE-30. The sample was i n j e c t e d a t 50°C, on c o m p l e t i o n o f t h e s o l v e n t e f f e c t t h e i n l e t and column were temperat u r e programmed a t 10°C min-1. The c a r r i e r gas was hydrogen a t 50 cm sec-1. 6b was an i d e n t i c a l experiment except t h a t t h e i n l e t was b a l l i s t i c a l l y heated on c o m p l e t i o n o f t h e s o l v e n t e f f e c t . A s p e c i a l k i n d o f s o l u t e l a g g i n g a l s o o c c u r s when t h e i n l e t i s c o a t e d w i t h s t a t i o n a r y phase ( r e f . 1 9 ) . I n t h i s case s o l u t e s w i t h h i g h p a r t i t i o n r a t i o s between t h e s t a t i o n a r y phase and t h e gas phase chromatograph t o o s l o w l y t o remain i n t h e advancing back edge o f t h e s o l v e n t f i l m . F o c u s i n g , t h e r e f o r e ,
does
n o t occur. Grob ( r e f . 20) demonstrated t h i s phenomenon and c o i n e d t h e t e r m "band broadening i n space" t o d e s c r i b e i t . He termed t h e removal o f s t a t i o n a r y phase Prom t h e i n l e t r e g i o n t o be " c r e a t i n g a r e t e n t i o n gap". Both terms, i n
355
o u r view, a r e unnecessary. ( v ) S o l u t e escape S o l u t e escape ( r e f . 21) r e f e r s t o s o l u t e which i s t r a n s p o r t e d o u t of t h e i n l e t during t h e focusing period. The f r a c t i o n a l escape i s g i v e n by Mxe -
MXS
b+P
(5)
where Mxe 'M ,
=
mass o f s o l u t e t o escape
=
mass o f o r i g i n a l sample
S o l u t e escape i s c o n t r o l l e d s o l e l y by t h e vapour p r e s s u r e o f t h e s o l u t e o v e r t h e sample. The g r e a t e r t h e vapour p r e s s u r e t h e g r e a t e r t h e degree o f escape.
A
l a r g e p o l a r i t y d i f f e r e n c e between s o l v e n t and s o l u t e w i l l i n c r e a s e
t h e degree o f escape and s h o u l d be avoided i f p o s s i b l e . Grob J r ( r e f s . 20, 22,
23) has c o i n e d t h e terms "band broadening i n t i m e " , " p a r t i a l s o l v e n t t r a p p i n g " and "two s t e p chromatography" (among o t h e r s ) . Except f o r s o l u t e s which do n o t obey e q u a t i o n 1 s o l u t e escape may be prevented by p l a c i n g a f i l m o f pure s o l v e n t ahead o f t h e sample f i l m ( r e f . 21) I n p r a c t i c e t h i s i s t r i c k y t o manage and achieves l i t t l e . The degree o f escape f a l l s r a p i d l y w i t h p a r t i t i o n r a t i o ( e q u a t i o n 5 ) and i s n e g l i g i b l e f o r say n-decane d i s s o l v e d i n n-hexane. Escaped s o l u t e can o f t e n be r e f o c u s e d by phase s w e l l i n g ( r e f . 10). ( v i ) Thermal e f f e c t s o
E v a p o r a t i v e c o o l i n g o c c u r s on t h e r e a r edge o f t h e sample f i l m when t h e h e a t needed t o e v a p o r a t e t h e sample i s n o t s u p p l i e d q u i c k l y enough ( r e f . 2 4 ) . Temperature drops o f up t o 20°C have been measured i n packed bed systems made o f g l a s s . C o o l i n g i s p r o b a b l y i n s i g n i f i c a n t i n open t u b u l a r systems.
As f a r as we can a s c e r t a i n e v a p o r a t i v e c o o l i n g does n o t , i n p r a c t i c e , detrimentally a f f e c t the solvent e f f e c t .
o
L a t e r a l thermal g r a d i e n t s may a l s o o c c u r i n packed bed systems where t h e packing i s p o o r l y conducting. I n such s i t u a t i o n s t h e r e a r edge o f a sample f i l m w i l l move a t d i f f e r e n t
a x i a l v e l o c i t i e s across t h e t u b e and may broaden s o l u t e bands as a r e s u l t . D i r e c t e x p e r i m e n t a l e v i d e n c e o f such g r a d i e n t s i s s t i l l l a c k i n g .
356
( v i i ) Film i n s t a b i l i t y
o
F i l m creep occurs when, once t h e sample has been formed, t h e f i l m as a whole i s moved a x i a l l y f o r w a r d by shear f o r c e s e x e r t e d by t h e c a r r i e r gas. Creep i s g r e a t e s t i n smooth w a l l e d open t u b u l a r systems and l e a s t i n porous packing packed beds ( r e f . 2 5 ) . F i l m c r e e p does n o t a f f e c t t h e s o l v e n t f o c u s i n g b u t i n c r e a s e s t h e l e n g h t o f i n l e t r e q u i r e d f o r a p a r t i c u l a r s i z e o f sample.
o
F i l m break up may be a t t r i b u t e d t o f i l m i n s t a b i l i t y and shear f o r c e s . The former i s e v i d e n t when t h e s u r f a c e t e n s i o n o f t h e sample i s g r e a t e r t h a n t h e excess s u r f a c e f r e e - e n e r g y o f t h e i n n e r s u r f a c e s o f t h e i n l e t and i s most p r e v a l e n t w i t h p o l a r s o l v e n t s ( r e f . 2 6 ) . Shear f o r c e s f r o m t h e c a r r i e r gas can cause t h e w e t t e d f i l m on a smooth w a l l e d open t u b u l a r i n l e t t o break up f o r m i n g l e n s e s . Rough-wall open t u b e and packed bed i n l e t s a r e l e s s
2.2
prone t o s h e a r - f o r c e break up.
Vapour samples ( S t a t i c Systems) ( r e f . 27) F o r s o l v e n t e f f e c t f o c u s i n g t h e sample s h o u l d c o n s i s t o f vapours i n a non-
i n t e r a c t i n g gas. The sample may e x i s t as such, eg o r g a n i c p o l l u t a n t s i n a i r , o r may be formed by p a s s i n g c a r r i e r gas o v e r t h e sample (head-space s a m p l i n g ) .
A f i l m o f s o l v e n t i s l a i d down i n t h e i n l e t and sample vapour i s passed o v e r t h i s f i l m . Focusing o c c u r s by t h e mechanism o u t l i n e d i n paragraph 2.1.2 and t h e c o n d i t i o n f o r f o c u s i n g i s a l s o g i v e n by e q u a t i o n s 1 and 2. The sample f e e d i s c o n t i n u e d u n t i l t h e e v a p o r a t i n g r e a r edge approaches t h e f r o n t edge. Sample f e e d i s t e r m i n a t e d and p u r e c a r r i e r gas i s i n t r o d u c e d . Subsequent events p a r a l l e l t h o s e p r e v i o u s l y o u t l i n e d f o r l i q u i d samples. S o l u t e escape does n o t o c c u r i n t h i s sampling mode, except f o r non-focusinp s o l u t e s ; t h i s can be a m a j o r advantage. Vapour samples c o n t a i n i n g l a r g e amoun s o f water can he e a s i l y handled u s i n g t h i s t e c h n i q u e . Water i s a n o n - f o c u s i n g s o l u t e on a n o n - p o l a r s o l v e n t such as hexane and i s t h e r e f o r e n o t accumulated 2.3
Dynamic Systems ( L i q u i d samples) Dynamic systems a r e a r e l a t i v e l y new i n n o v a t i o n i n v o l v i n g a moving
(dynamic) sample " f i l m " and c o u n t e r c u r r e n t c a r r i e r gas f l o w ( r e f . 2 6 ) . They have n o t y e t been s t u d i e d i n as g r e a t d e t a i l as have s t a t i c systems and t h e t h e o r e t i c a l t r e a t m e n t and d i s c u s s i o n presented h e r e i s c o n s e q u e n t l y s k e t c h y . Consider a tube, c o n t a i n i n g an a x i a l porous l a y e r and a gas channel w i t h
357
one end dipped i n t o a l i q u i d sample and t h e o t h e r end connected t o a c a r r i e r gas s u p p l y ( F i g u r e 7 ) . The l i q u i d sample r i s e s by c a p i l l a r y a c t i o n i n t h e porous l a y e r w h i l e t h e c a r r i e r gas evaporates sample a t t h e upper edge, c a u s i n g i t t o move downwards. E q u i 1 i b r i u m . o c c u r s when t h e volume r a t e o f c a p i l l a r y
r i s e equals t h e volume r a t e o f e v a p o r a t i o n . S o l u t e i s focused i n t h e e v a p o r a t i o n zone by processes s i m i l a r t o t h o s e i n t h e s t a t i c systems p r e v i o u s l y d e s c r i b e d (paragraph 2 ) .
a
b 9
-2
F i g . 7a) Schematic diagram o f operation o f solvent e f f e c t i n t h e dynami c mode. 1 ) Pure gas ( f o r l i q u i d samples) o r head space sample; 2) Glass tube; 3) Pure s o l v e n t ( f o r head space samples) o r d i l u t e l i q u i d sample; 4) Region o f porous bed w e t t e d by l i q u i d ; 5 ) Solute/head space component m o l e c u l e c a r r i e d up by c a p i l l a r y r i s e o f l i q u i d ; 6) C a p i l l a r y r i s e o f l i q u i d ; 7 ) Chromatographic t r a n s p o r t o f s o l u t e s ; 8 ) S o l u t e s a c c u m u l a t i n g i n e v a p o r a t i o n zone when 5 and 6 a r e f a s t e r t h a n 7; 9) Dry r e g i o n o f porous bed. 7b) P l a n view. 3. 3.1
PRACTICAL ASPECTS S t a t i c vs dynamic systems
o
s t a t i c systems may be o n - l i n e o r o f f - l i n e ;
o
s t a t i c systems a r e p r e s e n t l y more w e l l - d e v e l o p e d t h a n a r e dynamic systems
o
dynamic systems can h a n d l e l a r g e r samples t h a n can s t a t i c systems
o
dynamic systems a r e more c o m p l i c a t e d t h a n s t a t i c systems
3.2 o
dynamic systems o n l y o f f - l i n e
O n - l i n e vs o f f - l i n e systems ' o f f - l i n e systems can employ h i g h c a r r i e r gas f l o w s t h e r e b y s h o r t e n i n g t h e
358
t i m e needed t o complete t h e s o l u t e f o c u s i n g
o
off-line
sampling can be done i n p a r a l l e l w i t h t h e chromatography; t h i s
s h o r t e n s a n a l y s i s t i m e i n r o u t i n e work o
o f f - l i n e systems a v o i d i n t r o d u c i n g n o n - v o l a t i l e r e s i d u e s i n t o t h e column
o
most o f t h e s o l v e n t i s removed d u r i n g o f f - l i n e o p e r a t i o n and t h i s reduces t h e amount o f s o l v e n t i n t r o d u c e d on t h e column
o 3.3
o
o n - l i n e systems a r e s i m p l e r t h a n o f f - l i n e systems Open vs packed tube i n l e t s open tubes, p a r t i c u l a r l y t h o s e w i t h smooth w a l l a r e prone t o f i l m creep and i n s t a b i l i t y . T h i s problem i s f a r l e s s i n packed tubes
o
packed tubes can handle l a r g e r samples p e r u n i t l e n g h t t h a n can open tubes
o
packed t u b e i n l e t s must be independenc'ently heated t o a v o i d l a g g i n g ; when open tubes a r e used t h e chromatograph oven i s s u f f i c i e n t
3.4
o
D i r e c t vs s p l i t l e s s i n j e c t i o n s p l i t l e s s i n j e c t i o n e n t a i l s v a p o r i s a t i o n and can l e a d t o decomposition o f thermally l a b i l e solutes
o
d i r e c t i n j e c t i o n does n o t r e q u i r e v a p o r i s a t i o n . I n p r o p e r l y d e a c t i v a t e d i n l e t s s o l u t e s can be t r a n s f e r r e d t o t h e column a t l o w temperatures
3.5
Solvent s p l i t t i n g r e f e r s t o t h e removal o f s o l v e n t d u r i n g f o c u s i n g b e f o r e t h e focused s o l u t e s a r e t r a n s f e r r e d t o t h e column s o l v e n t s p l i t t i n g occurs a u t o m a t i c a l l y i n o f f - l i n e systems and can be invoked i n o n - l i n e systems by u s i n g a Deans s w i t c h ( r e f . 2 9 ) . i f t h e s o l v e n t i s n o t s p l i t t h e l a r g e amounts o f s o l v e n t p a s s i n g i n t o t h e column can cause s t a t i o n a r y phase s w e l l i n g ( r e f . 14) and t a i l i n g o f solvent peaks i n a p r o p e r l y designed system n o n - s p l i t t i n g o f s o l v e n t does n o t cause problems ( F i g . 8 )
359 F i g . 8. A p r o t o t y p e i n l e t f o r t h e s t a t i c solvent e f f e c t 1 ) C a r r i e r gas i n l e t 2 ) Septum n u t 3 ) Septum purge 4 ) Gas chromatograph oven l i d 5 ) Packing 6 ) Fused s i l i c a l i n e 7) Connection 8 ) Heater c o i l s
3.6
tiardware A l a r g E number o f i n l e t s designed f o r s o l u t e f o c u s i n g u s i n g t h e s o l v e n t
e f f e c t have been d e s c r i b e d ( r e f s . 30, 31, 3 2 ) ; t h e f i n a l word has y e t t o be written. F i g u r e s 8 and 9 a r e schematic r e p r e s e n t a t i o n s o f s e v e r a l p r o t o t y p e i n l e t s b e i n g developed i n o u r l a b o r a t o r i e s .
4. PRACTICAL EXAMPLES F i g u r e 10 shows a chromatogram o f a headspace sample generated by a dynamic sol vent e f f e c t i n l e t . F i g u r e 6b shows a chromatogram o f a l i q u i d sample generated by a s t a t i c solvent e f f e c t operating on-line.
360
F i g . 9. Diagrammatic v e r t i c a l s e c t i o n o f system used t o t r a n s f e r sample f r o m an o f f - l i n e , dynamic, s o l v e n t e f f e c t sampler t o a c a p i l l a r y GLC column
1) 2) 3) 4) 5) 6) 7) 8) 9)
9
10)
-7
9
C a r r i e r gas i n l e t Removable cap Ground g l a s s j o i n t O f f - l i n e , dynamic, s o l v e n t e f f e c t sampler Heater c o i l s Purge gas e x i t Porous packed bed To column E l e c t r i c a l power s u p p l y . S o l u t e s and s o l v e n t a r e evaporated f r o m t h e o f f - l i n e , dynamic, s o l v e n t e f f e c t sampler by e l e c t r i c a l h e a t = i n g and condensed i n t o t h e porous bed where on-1 i n e , s t a t i c , nonsolvent s p l i t t i n g solvent e f f e c t focusing i s carried out. Seat
361
F i g . 10. A chromatogram o f v o l a t i l e s f r o m 10 m l o f Simonsig Colombar wine sampled by s t r i p p i n g w i t h 15 rnl m i n - I o f p u r e N2 f o r 30 min and t r a p p i n g by o f f - l i n e , dynamic s o l v e n t e f f e c t f o c u s i n g on p u r e n-hexane a t 30°C. Chromatographic c o n d i t i o n s as i n F i g u r e 6. REFERENCES
1 V. P r e t o r i u s and W. B e r t s c h , J . HRC & CC, 6 (1983) 64. 2 V. P r e t o r i u s , K. Lawson and W. B e r t s c h , J. HRC & CC, 6 (1983 185. 3 K. Grob and K. Grob Jr, J. Chrom, 94 (1974) 53. 4 K. Grob and G. Grob, J. Chrom. S c i . , 7 (1969) 584. 5 D.R. Deans, Anal. Chem., 43 (1971) 2026. 6 V. P r e t o r i u s , C.S.G. P h i l l i p s and W. B e r t s c h , 3 . HRC & CC, 6 ( 1 983) 232. 7 F.J. Yang, A.C. Brown and S.P. Cram, 3 . Chrom., 158 (1978) 91 8 W.G. Jennings, R.R. Freeman and T.A. Rooney, J. HRC & CC, 1 1978) 275. 9 K. Knauss, J . Fulleman and M.P. T u r n e r . J . HRC & CC. 4 (1981 641. 10 R.G. J e n k i n s , i n "Proceedings o f t h e F o u r t h I n t e r n a t i o n a l Symposium on C a p i l l a r y Chromatography, Hindelang, 1981", R.E. K a i s e r ed., H e u t h i g , H e i d e l b e r g , 1981, 563 pp. 11 V. P r e t o r i u s , E.R. Rohwer and K.H. Lawson, S . A f r . J. Chem, 37(2) (1984) 65. 12 V. P r e t o r i u s e t a l . I n p r e p a r a t i o n . 13 W.E. H a r r i s , J. Chrom. S c i . , 11 (1973) 184. 14 K. Grob J r and B. S c h i l l i n g , Chromatographia, 17 (1983) 361. 15 K. Grob J r , J . Chrom., 264 (1983) 7. 16 V. P r e t o r i u s e t a l . I n p r e p a r a t i o n . 17 V. P r e t o r i u s e t a l . I n p r e p a r a t i o n . 18 V. P r e t o r i u s , C.S.G. P h i l l i p s and W . B e r t s c h , J. HRC & CC, 6 (1983) 321. 19 V. P r e t o r i u s , K.H. Lawson, E.R. Rohwer and W. B e r t s c h , J . HRC & CC, 7 (1984) 92. 20 K. Grob Jr, J. Chrom., 213 (1981) 3. 21 V. P r e t o r i u s , K. Lawson and W . B e r t s c h , J . HRC & CC, 6 (1983) 419. 22 K. Grob J r , Chromatographia, 17 (1983) 357. 23 K. Grob J r , J. Chrom., 253 (1982) 17. 24 V. P r e t o r i u s , P. Apps, E.R. Rohwer and K.H. Lawson, J. HRC & CC, 7 (1984) 210. 25 V. P r e t o r i u s , K.H. Lawson, P. Apps and W. B e r t s c h , J . Chrom., 279 (1983) 233. 26 P. Sandra, I . Temmerman and M. Verstappe, J. HRC & CC, 6 (1983) 501. 27 V. P r e t o r i u s e t a l . I n p r e p a r a t i o n .
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V. P r e t o r i u s , P. Apps, E.R. Rohwer and K.H. Lawson, J. HRC & CC, 7 (1984) 212. 29 D.R. Deans, Chromatographia, 1/2 (1968) 18. 30 E. Geeraert, D. de Schepper and P. Sandra, J. HRC & CC, 6 (1983) 386. 31 K. Grob and K. Grob Jr, J . Chrom., 151 (1978) 311. 31 G. Schomburg, H. Behlau, R. Dielmann, F. Weeke and H. Husmann, J . Chrom., 142 (1977) 87. 33 K. Grob J r , J . HRC & CC, 8 (1984) 461. 28