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Chapter 5 Column Design Selection
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CHAPTER 5
COLUMN DESIGN SELECTION J.C.
KRAAK
Laboratory f o r A n a l y t i c a l Chemistry, U n i v e r s i t y o f Amsterdam,
Nieuwe Achtergracht
166, Amsterdam (The Netherlands)
CONTENTS 1. I n t r o d u c t i o n 75 2. Column system 76 2.1. Column tube m a t e r i a l 76 2.2. Metal tubes 77 2.3. Glass tubes 77 2.4. Tubes o f polymeric m a t e r i a l s 77 2.5. G l a s s - l i n e d tubes 78 3. Column dimensions 78 3.1. Column l e n g t h 78 3.2. I n n e r diameter o f t h e column 78 3.3. Outer diameter and w a l l t h i c k n e s s 79 3.4. I n s i d e s u r f a c e o f columns 8 1 4. Column t u b e c o n f i g u r a t i o n 8 1 5. Column f i t t i n g s 82 5.1. C a p i l l a r y t u b i n g s 82 6. Column end t e r m i n a t o r s 83 6.1. Terminators f o r g l a s s columns 83 6.2. Terminators f o r metal columns 84 7. Porous f i l t e r s 86 8. Connectors f o r coup1 i n g columns 87 9. M u l t i p l e column systems 88 10. Column t h e r m o s t a t i n g devices 88 10.1. C i r c u l a t i n g a i r - b a t h thermostats 90 10.2. Thermostating by means o f heat j a c k e t s 11. Appendix 91 12. References 91
91
1. INTRODUCTION I n high-performance 1 i q u i d chromatography (HPLC), t h e choice and c o n s t r u c t i o n o f t h e column i t s e l f and i n p a r t i c u l a r t h e column f i t t i n g s might be o f d e c i s i v e importance f o r p r e p a r i n g s u c c e s s f u l l y h i g h - e f f i c i e n c y separation columns. A p a r t from r e s i s t a n c e t o h i g h pressures and chemical a c t i o n o f t h e eluent, much a t t e n t i o n has t o be p a i d t o m i n i m i z i n g e x t e r n a l peak broadening caused by column t e r m i n a t o r s and connection
F u r t h e r , t h e p o s s i b i l i t y o f exchanging each separate p a r t ,
s i m p l i c i t y and cheapness o f t h e column design a r e o f importance.
I n t h i s paper,
d i f f e r e n t column systems i n c l u d i n g t h e r m o s t a t i n g devices, commercially a v a i l a b l e o r custom made, w i l l be discussed.
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2. COLUMN SYSTEM 2.1.
Column t u b e m a t e r i a l Before d i s c u s s i n g t h e s u i t a b i l i t y o f t h e d i f f e r e n t column t u b e s t h a t a r e
a v a i l a b l e , i t i s w o r t h w h i l e summarizing t h e p a c k i n g procedures t h a t have been a p p l i e d as t h e s e u s u a l l y r e s t r i c t t h e c h o i c e o f t h e column t u b e m a t e r i a l i n p r a c t i c e . M a i n l y two p a c k i n g procedures have been a p p l i e d up t o now: ( i ) Dry p a c k i n g t e c h n i q u e s : Tamping: small p o r t i o n s o f a l o w - s u r f a c e - a r e a support, c o a t e d o r uncoated, a r e p l a c e d i n t h e column and compressed w i t h a PTFE-tipped p l u n g e r 4 ; Tapping: d r y s o l i d s u p p o r t i s p l a c e d i n t h e column and tapped r a p i d l y u n t i l a dense p a c k i n g i s o b t a i n e d
5
.
( i i ) S o l v e n t f i l t r a t i o n t.echniques: S l u r r y packing: a s l u r r y o f t h e s o l i d s u p p o r t i n t h e e l u e n t o r a n o t h e r s u i t a b l e s o l v e n t i s pumped i n t o t h e column, c l o s e d by a f r i t a t t h e bottom. The column 6 packing i s b u i l t up by f i l t r a t i o n
.
Balanced s l u r r y packing: a s l u r r y o f t h e s o l i d s u p p o r t i n a l i q u i d w i t h a d e n s i t y i d e n t i c a l w i t h t h a t o f t h e s o l i d s u p p o r t i s pumped i n t o t h e column. As a r e s u l t o f t h e v e r y s t a b l e s l u r r y , a homogeneous dense column p a c k i n g i s t h e n 7 b u i l t up by s o l v e n t f i l t r a t i o n
.
Dry p a c k i n g i n v o l v i n g tamping o f t h e s o l i d s u p p o r t w i t h a p l u n g e r i s s u i t a b l e o n l y f o r g l a s s columns. W i t h metal columns, t h e compression o f t h e added l o o s e m a t e r i a l , r e q u i r e d f o r o b t a i n i n g a dense packing, i s s e v e r e l y h i n d e r e d by t h e l a r g e f r i c t i o n a l f o r c e , due t o t h e r e l a t i v e s o f t n e s s o f m e t a l s compared w i t h s o l i d supports. Dry p a c k i n g i n v o l v i n g t a p p i n g can be a p p l i e d w i t h a l l column t u b e m a t e r i a l s . I n p r a c t i c e , however, i t s use i s r e s t r i c t e d t o r e l a t i v e l y l a r g e p a r t i c l e s (e.g.,
> 20 urn). S l u r r y p a c k i n g i s s u i t a b l e f o r a l l t y p e s o f column f o r a l l i n s t a n c e s i n which t h e p r e s s u r e r e s i s t a n c e o f t h e column t u b e i s s u f f i c i e n t . The balanced s l u r r y technique, however, needs such h i g h p r e s s u r e drops i n o r d e r t o o b t a i n r e a s o n a b l e f l u i d v e l o c i t i e s w i t h t h e r a t h e r v i s c o u s s l u r r y t h a t i n most i n s t a n c e s o n l y metal columns can be used. A p a r t f r o m t h e r e s t r i c t i o n i n t h e s e l e c t i o n o f t h e p a c k i n g procedure, o t h e r demands such as r e s i s t a n c e t o chemical a c t i o n by t h e e l u e n t o r i n e r t n e s s o f t h e surface m i g h t p l a y a d e c i s i v e r o l e i n t h e s e l e c t i o n o f t h e column t u b e . Up t o now, g l a s s , metal and t o a l e s s e r e x t e n t p o l y m e r i c m a t e r i a l s have been t e s t e d as column t u b e m a t e r i a l s .
2.2. Metal tubes A l t h o u g h some workers found no s i g n i f i c a n t d i f f e r e n c e s i n column e f f i c i e n c y between d i f f e r e n t k i n d s o f m e t a l tubes f o r dry-packed ( t a p p i n g ) columns o f l o w e f f i c i e n c y * , copper, aluminium and t o a l e s s e r e x t e n t i r o n t u b e s a r e l e s s s u i t a b l e as column t u b e m a t e r i a l s . Owing t o t h e i r r e l a t i v e s o f t n e s s , p i e c e s o f metal a r e e a s i l y s c r a t c h e d f r o m t h e w a l l s o f t h e tubes d u r i n g t h e f i l l i n g
procedure^^'^^.
F u r t h e r t h e s e m e t a l s have a p o o r r e s i s t a n c e t o chemical a c t i o n , have a r a t h e r n o n - i n e r t s u r f a c e which f a v o u r s c a t a l y t i c r e a c t i o n s and a r e l i m i t e d i n p r e s s u r e resistance. S t a i n l e s s - s t e e l ( t y p e 316) columns do n o t have t h e s e disadvantages o f c o p p e r , aluminium and i r o n tubes and a r e v e r y s u i t a b l e . I n most HPLC a p p l i c a t i o n s , 316 s t a i n l e s s - s t e e l column tubes can be used w i t h o u t d i f f i c u l t y . When t h e chemical i n e r t n e s s i s i n s u f f i c i e n t , t h e 316 s t a i n l e s s - s t e e l column can be r e p l a c e d w i t h t h e more expensive b u t more i n e r t t a n t a l u m column t u b e s (Siemens). When v e r y s t r o n g l y a c i d i c m o b i l e phases have t o be used, 316 s t a i n l e s s - s t e e l columns a r e l e s s s u i t a b l e . F o r such i n s t a n c e s , sometimes home-made t i t a n i u m columns, i n c l u d i n g t i t a n i u m t e r m i n a t o r s and i n j e c t i o n systems, have been a p p l i e d suc11 cessfully
.
2.3.
Glass tubes
The t y p e o f g l a s s t h a t i s chosen seems t o be o f m i n o r importance. I n most i n s t a n c e s normal b o r o s i l i c a t e g l a s s seems t o be a good c h o i c e . However, i n o r d e r t o c o n s t r u c t a h i g h - p r e s s u r e r e s i s t a n t g l a s s column, t h e absence o f s t r e s s i n t h e glass tube i s very,important. Stress u s u a l l y occurs a f t e r c u t t i n g o f t h e g l a s s t u b e t o t h e d e s i r e d l e n g t h . Slow h e a t i n g o f t h e g l a s s t u b e t o 600'
and
c o o l i n g s l o w l y u s u a l l y removes t h e s t r e s s and improves c o n s i d e r a b l y t h e p r e s s u r e resistance o f t h e glass tubes. Obviously t h e pressure resistance o f glass tubes i s r e l a t e d t o t h e w a l l thickness. With respect t o possible c a t a l y t i c r e a c t i o n s a t t h e surface, g l a s s columns a r e much more i n e r t t h a n metal columns, as i s known f r o m gas chromatography. 2.4. Tubes o f p o l y m e r i c m a t e r i a l s
A m a j o r r e q u i r e m e n t f o r column t u b e s i n HPLC i s constancy o f t h e co umn the
d i a m e t e r when changing t h e p r e s s u r e d r o p a l o n g them. I f t h i s i s n o t so
s t a b i l i t y o f t h e p a c k i n g i s a f f e c t e d on changing t h e p r e s s u r e drop, wh ch d e s t r o y s t h e column e f f i c i e n c y . A l l a v a i l a b l e p o l y m e r i c tubes, such as PTFE, n y l o n o r p l a s t i c tubes, show a d e f i n i t e p r e s s u r e dependence o f t h e t u b e diameter. A p a r t f r o m t h i s , p o l y m e r i c m a t e r i a l s , e x c e p t PTFE, a r e e a s i l y a t t a c k e d by o r g a n i c s o l v e n t s . F o r t h e s e reasons p o l y m e r i c m a t e r i a l s a r e u n s u i t a b l e as column t u b e m a t e r i a l s i n HPLC. R e c e n t l y , however, t h e a p p l i c a t i o n o f a r a d i a l compressed PTFE column f o r p r e p a r a t i v e LC was r e p o r t e d (Waters Assoc.).
78
2.5.
G l a s s - l i n e d tubes Metal t u b e s i n t e r n a l l y c o a t e d w i t h g l a s s a r e c o m m e r c i a l l y a v a i l a b l e w i t h
diameters up t o 4 mm ( S c i e n t i f i c Glass E n g i n e e r i n g ) . These t u b e s show t h e mechanical s t r e n g t h o f metal columns and t h e i n e r t n e s s and smooth s u r f a c e o f t h e g l a s s columns. However, t h e s e g l a s s - l i n e d t u b e s have seldom been a p p l i e d i n LC. I n c o n c l u s i o n , 316 s t a i n l e s s - s t e e l and g l a s s a r e p r e f e r r e d as column t u b e m a t e r i a l s i n HPLC. Radial compressed PTFE t u b e s m i g h t have advantages i n t h e f u t u r e , as was p o i n t e d o u t by Eon12 r e c e n t l y . A l t h o u g h c o m m e r c i a l l y a v a i l a b l e f o r many y e a r s , g l a s s - l i n e d tubes have found l i t t l e a p p l i c a t i o n i n HPLC so f a r . T e s t i n g o f t h e s e column tubes m i g h t be i n t e r e s t i n g , as t h e advantages o f t h e i n e r t n e s s and hardness o f g l a s s a r e combined w i t h t h e mechanical s t r e n g t h o f metal. 3. COLUMN DIMENSIONS 3.1.
Column l e n q t h
The c h o i c e o f t h e l e n g t h o f t h e column w i l l always be a compromise between t h e d i f f e r e n t demands p u t on t h e d e s i r e d r e s u l t and t h e performance o f t h e 1 i q u i d chromatograph. The l e n g t h of t h e s e p a r a t i o n column i s d e t e r m i n e d p r i m a r i l y by t h e number o f t h e o r e t i c a l p l a t e s r e q u i r e d . F u r t h e r , t h e column l e n g t h i s r e l a t e d t o t h e p a c k i n g t e c h n i q u e a p p l i e d , t h e p r e s s u r e c a p a b i l i t i e s o f t h e d i f f e r e n t LC components and sometimes t h e space a v a i l a b l e i n t h e t h e r m o s t a t . W i t h r e s p e c t t o t h e column p a c k i n g , s h o r t e r columns (e.g., o r g l a s s can be packed more e f f i c i e n t l y t h a n l o n g e r
10-25 cm) o f m e t a l
column^^^-^^.
I n order t o
c o n s t r u c t a l o n g e r column, s h o r t e r columns can be c o u p l e d w i t h o u t a s i g n i f i c a n t decrease i n e f f i c i e n c y 1 6 . W i t h t h e d r y tamping and s l u r r y p a c k i n g techniques, t h e o p t i m a l l e n g t h w i t h r e s p e c t t o column e f f i c i e n c y ranges f r o m 10 t o 25 cm. F o r d r y t a p p i n g t h e l e n g t h i s l e s s c r i t i c a l and m i g h t be up t o 100 cm. I f a f i x e d number o f t h e o r e t i c a l p l a t e s i s needed a t a l i m i t e d p r e s s u r e drop, t h e n H/V versus a P / N ( p r e s s u r e d r o p p e r p l a t e ) p l o t s 1 6 o f d i f f e r e n t p a r t i c l e s i z e s a r e h e l p f u l f o r f i n d i n g t h e o p t i m a l column l e n g t h and p a r t i c l e s i z e . I n g e n e r a l , t h e l e n g t h s o f s e p a r a t i o n columns l i e i n t h e range 10-100 cm. With g l a s s columns, however, no high-speed s e p a r a t i o n s can be e x p e c t e d on l o n g columns because o f t h e i r l i m i t e d p r e s s u r e r e s i s t a n c e . 3.2.
I n n e r d i a m e t e r o f t h e column
A d e c i s i o n about t h e i n n e r d i a m e t e r o f t h e a n a l y t i c a l column depends upon a number o f d i f f e r e n t demands. O b v i o u s l y t h e r e i s a r e l a t i o n s h i p between t h e i n n e r diameter, w a l l t h i c k n e s s and
p r e s s u r e r e s i s t a n c e o f g l a s s o r m e t a l columns. I n
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p r a c t i c e , however, t h i s i s l e s s c r i t i c a l w i t h s t a i n l e s s - s t e e l columns when used a t pressures up t o 400 atm. I n r e c e n t y e a r s f i l l i n g pressures up t o 1000 atm have been a p p l i e d i n o r d e r t o pack more e f f i c i e n t , more r e p r o d u c i b l e and l o n g e r columns16. I n these instances t h e r e l a t i o n s h i p between i n n e r diameter and w a l l t h i c k n e s s becomes c r i t i c a l . Apart from t h e pressure r e s i s t a n c e , o t h e r f a c t o r s might be o f d e c i s i v e importance. I n t r a c e a n a l y s i s , u s u a l l y small samples o f low c o n c e n t r a t i o n a r e a v a i l a b l e and t o o g r e a t a d i l u t i o n o f such samples i n t h e column s e r i o u s l y a f f e c t s t h e i r d e t e c t i o n . The maximal c o n c e n t r a t i o n o f a s o l u t e i n t h e mobile phase a t t h e end o f t h e column ( < cy > i n j e c t e d , Qi,
as a f u n c t i o n o f t h e amount
i s given by t h e f o l l o w i n g equation17:
where E = p o r o s i t y o f t h e mobile phase; A = c r o s s - s e c t i o n a l area o f t h e column; m L = column l e n g t h ; Hi = t h e o r e t i c a l p l a t e h e i g h t o f t h e s o l u t e i.
Eqn. 1 shows c l e a r l y t h a t t h e s m a l l e r t h e c r o s s - s e c t i o n a l area ( p r o p o r t i o n a l t o the square o f t h e i n n e r diameter) t h e h i g h e r i s t h e c o n c e n t r a t i o n o f t h e s o l u t e a t t h e end o f t h e column. Therefore, i n t r a c e a n a l y s i s , h i g h - e f f i c i e n c y ,
s h o r t and
small-diameter columns a r e t o be p r e f e r r e d . The s m a l l e s t i n n e r diameter o f columns t h a t can be packed e f f i c i e n t l y i s about 2 mm ( r e f . 7). Smaller diameters a r e l e s s e f f i c i e n t because o f an i n c r e a s i n g i n f l u e n c e o f t h e w a l l e f f e c t s and t h e r e l a t i v e i n c r e a s e i n t h e e x t e r n a l peak broadening versus t h e d i s p e r s i o n due t o t h e column. I f t h e c o n c e n t r a t i o n and amount o f sample a r e n o t r e s t r i c t e d , columns o f l a r g e r i n n e r diameter a r e advantageous, because of decreasing c o n t r i b u t i o n s o f w a l l e f f e c t s and e x t e r n a l peak broadening t o t h e o v e r a l l column d i s p e r s i o n , as has been shown by several workers18y19. 10 mm I . D .
For a n a l y t i c a l purposes, columns up t o
can be used.
To summarize, column i n n e r diameters between 2 and 10 mm a r e s u i t a b l e f o r a n a l y t i c a l purposes when t a k i n g i n t o account t h e pressure r e s i s t a n c e . Columns w i t h i n n e r diameters o f 3-5 mm seem t o be a good compromise f o r most p r a c t i c a l applications. 3.3. Outer diameter and w a l l t h i c k n e s s As mentioned e a r l i e r , t h e o u t e r diameter i s r e l a t e d t o t h e i n n e r diameter. Up t o u l t i m a t e pressures o f about 400 atm, t h e w a l l t h i c k n e s s o f 316 s t a i n l e s s - s t e e l columns i s n o t so c r i t i c a l , b u t when l a r g e r pressures a r e a p p l i e d , t h e r a t i o o f
80
t h e i n n e r t o t h e o u t e r d i a m e t e r o f t h e t u b e must have a c e r t a i n v a l u e . To ensure a good p r e s s u r e r e s i s t a n c e up t o 1000 atm, t h i s r a t i o should be ca. 0.5. A p a r t f r o m t h i s , an o u t e r d i a m e t e r t h a t can be f i t t e d w i t h c o m m e r c i a l l y a v a i l a b l e end f i t t i n g s such as Swagelok components i s o f t e n v e r y c o n v e n i e n t . F o r g l a s s columns, t h e r a t i o o f i n n e r t o o u t e r d i a m e t e r determines t h e maximal a l l o w a b l e p r e s s u r e drop. I n o r d e r t o r e s i s t p r e s s u r e s up t o 100 atm, t h e w a l l t h i c k n e s s o f g l a s s columns must l i e i n t h e range 3-6 mm. The t h i c k e r t h e g l a s s w a l l , t h e more p r e s s u r e r e s i s t a n t w i l l be t h e g l a s s column, b u t t h e g r e a t e r m i g h t be t h e s t r e s s i n t h e g l a s s . W i t h v e r y t h i c k - w a l l e d g l a s s tubes, extreme c a r e must be t a k e n t o remove s t r e s s . T h i s can be done by s l o w l y h e a t i n g and c o o l i n g , as described before. I n p r a c t i c e thick-walled, 3 mm and O.D. 20 atm
.
s m a l l - d i a m e t e r g l a s s columns (I.D.
12 mm) can be used s a f e l y up t o an u l t i m a t e p r e s s u r e o f about 100
I n an a t t e m p t t o use g l a s s columns a t h i g h e r pressures, an i n g e n i o u s g l a s s w i t h a p r e s s u r e r e s i s t a n c e up t o
column system was developed by S t a h l e t a1."
300 atm. I n t h i s c o n s t r u c t i o n , t h e i n l e t p r e s s u r e i n s i d e and o u t s i d e t h e column
can be k e p t t h e same d u r i n g t h e experiments, as can be seen f r o m F i g . 1.
,glass tube
+
detector F i g . 1. Schematic diagram o f a h i g h - p r e s s u r e r e s i s t a n t g l a s s column system.
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3.4.
I n s i d e s u r f a c e o f columns
Many recommendations have been made about t h e p r e p a r a t i o n o f t h e i n n e r s u r f a c e when p a c k i n g m e t a l columns. It i s o b v i o u s t h a t t h e smoother t h e i n n e r w a l l , t h e b e t t e r t h e ( b a l a n c e d ) s l u r r y f l o a t s i n t o t h e column as t h e f o r c e o f f r i c t i o n a t t h e w a l l d i m i n i s h e s . T h i s improves t h e r e g u l a r i t y o f t h e p a c k i n g s i g n i f i c a n t l y , as was shown by many workers who compared p o l i s h e d o r d r i l l e d metal columns w i t h t h e o r i g i n a l ones 5y9y22y23.O t h e r workers have p r e s c r i b e d a t h o r o u g h washing procedure i n o r d e r t o o b t a i n h i g h l y e f f i c i e n t packed columns. Most o f t h e s e r i t u a l s , however, can b e dispensed w i t h when a p p l y i n g modern h i g h - q u a l i t y seamless 316 stainless-steel
p r e c i s i o n - b o r e tubes. I n most i n s t a n c e s t h e s e tubes can be used
w i t h o u t p r e - t r e a t m e n t o t h e r t h a n washing w i t h an o r g a n i c s o l v e n t i n o r d e r t o remove p o s s i b l e small metal p a r t i c l e s o r d r i l l i n g o i l . The i n n e r s u r f a c e o f g l a s s columns i s u s u a l l y smooth and no f u r t h e r p r e - t r e a t m e n t i s necessary. I n some i n s t a n c e s t h e i n n e r s u r f a c e i s etched; t h i s has no s i g n i f i c a n t e f f e c t on t h e column e f f i c i e n c y i n comparison w i t h t h e smooth-surface t y p e o f g l a s s tubes, b u t i n p r a c t i c e , e t c h e d g l a s s tubes have a s l i g h t l y b e t t e r p r e s s u r e resistance. 4. COLUMN TUBE CONFIGURATION I n HPLC, s t r a i g h t columns a r e u s u a l l y a p p l i e d , i n c o n t r a s t t o gas chromatography where c o i l e d columns a r e p o p u l a r . F o r a number o f reasons, s t r a i g h t columns a r e p r e f e r a b l e i n HPLC; u s u a l l y s e p a r a t i o n s can be o b t a i n e d w i t h s h o r t columns, and c o i l i n g i n o r d e r t o o b t a i n more c o n v e n i e n t t h e r m o s t a t i n g i s o f m i n o r i n t e r e s t . Also, t h e p a c k i n g t e c h n i q u e s , i n p a r t i c u l a r d r y tamping f o r g l a s s columns, need s t r a i g h t columns. On t h e o t h e r hand, t h e s l u r r y p a c k i n g o f c o i l e d m e t a l columns l e a d s t o l e s s homogeneous column packings owing t o s i g n i f i c a n t d i f f e r e n c e s i n a x i a l f l u i d v e l o c i t i e s i n t h e i n s i d e and o u t s i d e t r a c k o f t h e column c r o s s - s e c t i o n . F u r t h e r , c o i l i n g o f s t r a i g h t f i l l e d m e t a l columns s e r i o u s l y a f f e c t s homogeneity of t h e p a c k i n g 5 y 2 4 and decreases t h e column e f f i c i e n c y s i g n i f i c a n t l y . T h i s does n o t o c c u r when columns f i l l e d w i t h l a r g e p a r t i c l e s (e.g.,
porous l a y e r beads)
a r e c o i l e d , because t h e e f f i c i e n c y of such columns i s a l r e a d y l o w even when s t r a i g h t , and c o i l i n g o r t h e use o f o t h e r shapes such as an S or a f i g u r e - o f - e i g h t does n o t i n f l u e n c e t h e poor e f f i c i e n c y . I f one i s a b l e t o pack a c o i l e d column homogeneously,then , a t t h e f l u i d v e l o c i t i e s commonly used, a p o o r e r column e f f i c i e n c y can be expected because o f d i f f e r e n c e s i n a x i a l f l u i d v e l o c i t i e s and t h e absence o f r a d i a l ( t r a n s v e r s e ) m i x i n g . As mentioned b e f o r e , c o u p l i n g o f s h o r t e r columns w i t h U-shaped c o u p l i n g s w i l l be more s u c c e s s f u l . The c o n c l u s i o n s about column dimensions and c o n f i g u r a t i o n can be summarized as f o l l o w s : s t r a i g h t g l a s s and m e t a l columns w i t h a l e n g t h o f 10-25 cm, an i n n e r d i a m e t e r o f 3-5 mm and a w a l l t h i c k n e s s o f h a l f t h e i n n e r d i a m e t e r f o r m e t a l columns and equal t o t h e i n n e r d i a m e t e r f o r g l a s s columns seem t o be a good
82
compromise f o r a n a l y t i c a l purposes. P r e c i s i o n - b o r e columns made o f 316 s t a i n l e s s - s t e e l can u s u a l l y be a p p l i e d w i t h o u t any p r e - t r e a t m e n t . W i t h o t h e r t y p e s o f s t a i n l e s s s t e e l , smoothing o f t h e i n n e r s u r f a c e m i g h t be necessary. When l o n g e r columns a r e d e s i r e d , c o u p l i n g o f s h o r t e r columns i s p r e f e r a b l e because t h e y can be packed more e f f i c i e n t l y than l o n g e r columns.
5. COLUMN FITTINGS 5.1.
C a p i l l a r y tubings I n o r d e r t o connect t h e column by means o f t e r m i n a t o r s t o t h e i n j e c t i o n p o r t
and d e t e c t o r , c a p i l l a r y t u b i n g i s o f t e n used. A t t h e h i g h - p r e s s u r e s i d e u s u a l l y
316 s t a i n l e s s - s t e e l c a p i l l a r y t u b i n g and a t t h e l o w p r e s s u r e ( d e t e c t o r ) s i d e b o t h 316 s t a i n l e s s - s t e e l and PTFE c a p i l l a r y t u b i n g s a r e a p p l i e d . The peak broadening i n c a p i l l a r y t u b i n g , expressed i n terms o f t h e s t a n d a r d d e v i a t i o n ( i n volume u n i t s ) o f t h e e l u t i o n c u r v e as a f u n c t i o n o f t h e l e n g t h and r a d i u s , i s g i v e n by
25,26
2
3 6
(uv)tube = L2a R Dim/ w
+
4 waR L / 24Dim
where L = l e n g t h ; R = r a d i u s ; w = l i q u i d Flow ( m l l s e c ) ; Dm i
= diffusion coefficient
i n t h e eluent. I n p r a c t i c e , t h e f i r s t t e r m i s neg i g i b l e compared w i t h t h e second, and t h e v a r i a n c e (e.g.,
2
u V ) i s g i v e n by
I n o r d e r t o m i n i m i z e e x t r a peak broadening, t h e c a p i l l a r y c o n n e c t i o n tubes s h o u l d be s h o r t and, even more i m p o r t a n t , t h e y s h o u l d have a r a d i u s as s m a l l as p o s s i b l e because o f t h e s q u a r e - r o o t dependence o f (uv)tube on i t . I n p r a c t i c e , however, t h e r a d i u s o f t h e tube cannot be decreased w i t h o u t l i m i t because o f t h e p r e s s u r e drop o v e r t h e c a p i l l a r y , which i s i n v e r s e l y p r o p o r t i o n a l t o t h e f o u r t h power o f t h e r a d i u s . A good compromise w i t h r e s p e c t t o p r e s s u r e d r o p and magnitude o f t h e peak broadening seems t o be an I . D .
o f 0.25 mm and a l e n g t h o f ca. 20-50 mm.
The c h o i c e o f t h e o u t e r d i a m e t e r o f t h e t u b i n g i s n o t c r i t i c a l . I t i s advantageous when c o m m e r c i a l l y a v a i l a b l e Swagelok o r P a r k e r c o n n e c t o r s f i t t h e t u b i n g (O.D.
1/16 i n . ) . Obviously, o n l y PTFE t u b i n g can be used a t t h e l o w - p r e s s u r e s i d e o f t h e column.
a3
6. COLUMN END TERMINATORS I n o r d e r t o o b t a i n a p e r f e c t j u n c t i o n between t h e column end and t h e t e r m i n a t o r , one must ensure t h a t t h e column ends a r e p e r f e c t l y f l a t . 6.1.
T e r m i n a t o r s f o r g l a s s columns F o r g l a s s columns, two p r i n c i p l e s a r e commonly a p p l i e d f o r o b t a i n i n g a
p r e s s u r e - r e s i s t a n t seal i n g between t h e g ass column and t h e e n d - f i t t i n g : connectors a r e l u t e d on b o t h column ends
( i ) metal
and ( i i) PTFE-coated metal c o n n e c t o r s
a r e pressed on t h e f l a t column ends.
A
6
C
F i g . 2. Column t e r m i n a t o r systems f o r g l a s s columns. F i g . 2a shows s c h e m a t i c a l l y t h e p r i n c i p l e o f a l u t e d e n d - f i t t i n g .
The l u t e d
column e n d - f i t t i n g s have a number o f disadvantages i n p r a c t i c e , such as: (1) t h e s y n t h e t i c g l u e s do n o t w i t h s t a n d a number o f o r g a n i c s o l v e n t s ; ( 2 ) a t t h o s e p l a c e s were t h e c o n n e c t o r s a r e l u t e d on t o t h e g l a s s tube, s t r e s s u s u a l l y o c c u r s , which l o w e r s t h e mechanical s t r e n g t h and p r e s s u r e r e s i s t a n c e c o n s i d e r a b l y , and ( 3 ) t h e l u t i n g techniques r e q u i r e a great experience i n handling glues i n order
t o a p p l y them s u c c e s s f u l l y . Two examples o f t h e o t h e r s e a l i n g p r i n c i p l e a r e shown i n F i g s . 2b and 2c. The f i r s t t y p e ( F i g . 2 b ) , which i s e s p e c i a l l y s u i t a b l e f o r t h i c k - w a l l e d s t r a i g h t g l a s s columns, has been used f o r many y e a r s w i t h g r e a t success27. The g l a s s tube, w i t h p e r f e c t l y f l a t ends, i s p l a c e d i n a metal t u b e w i t h a s c r e w - t h r e a d a t b o t h
84 ends. Then PTFE-coated metal end f i t t i n g s a r e p l a c e d a t b o t h ends o f t h e g l a s s t u b e and t h e s e PTFE-coated c o n n e c t o r s a r e t i g h t l y pressed on t h e f l a t column ends o f the glass tube w i t h nuts. This type o f s e a l i n g i s r e s i s t a n t against very h i g h pressures, f a r above t h e b r e a k i n g - p o i n t o f t h i c k - w a l l e d g l a s s tubes, which ranges f r o m 100-150 atm. The p e r f e c t c o n n e c t i o n w i t h t h e p a c k i n g i s a n o t h e r advantage
o f t h i s column system. An analogous s e a l i n g system i s shown.in F i g . 2c. A t about 5 w f r o m t h e column ends a c a v i t y i s c u t . Two h a l f - c y l i n d r i c a l p i e c e s of PTFE a r e p l a c e d i n these c a v i t i e s and PTFE-coated c o n n e c t o r s w i t h screw-threads a r e p l a c e d a t b o t h ends
o f t h e g l a s s tube. With a n u t , which i s f i x e d by t h e p i e c e s o f PTFE, t h e c o n n e c t o r
i s p u l l e d on t o t h e f l a t column end. Both o f t h e systems i n F i g s . 2b and 2c a r e c o m m e r c i a l l y a v a i l a b 1 e (Siemens; Packard-Becker).
6.2.
T e r m i n a t o r s f o r metal columns A l t h o u g h t h e c o n s t r u c t i o n o f h i g h - p r e s s u r e r e s i s t a n t end f i t t i n g s f o r m e t a l
columns i s easy, many f a i l u r e s i n t h e a p p l i c a t i o n of HPLC m i g h t be a t t r i b u t e d t o u n s a t i s f a c t o r y i n s t a l l a t i o n o f t h e end f i t t i n g s . A p a r t f r o m custom-made t e r m i n a t o r s , m o d i f i e d s t a i n l e s s - s t e e l r e d u c i n g unions o f t h e Swagelok t y p e a r e u s u a l l y a p p l i e d . The m o d i f i c a t i o n o f Swagelok r e d u c i n g u n i o n s i s necessary i n o r d e r t o m i n i m i z e peak broadening, as shown i n F i g . 3. A l l o r i g i n a l Swagelok r e d u c i n g u n i o n s ( F i g . 3a) have a b o r e i n t h e c e n t r e o f about 6 mm l e n g t h and a m i n i m a l I . D .
o f 1.2 mm.
F u r t h e r , r e d u c i n g unions have a l a r g e b o r e a t t h e n o n - r e d u c i n g s i d e . F i t t i n g columns w i t h t h e s e u n m o d i f i e d t e r m i n a t o r s w i l l l e a d t o a l a r g e c o n t r i b u t i o n o f
,ferrules
\
I
A
).
dead volume
t
-reducing
t
original F i g . 3. M o d i f i c a t i o n o f Swagelok unions.
t
bored
6
unic
85 e x t e r n a l peak b r o a d e n i n g due t o these l a r g e dead volumes (e.g.,
m i x i n g chambers).
I n o r d e r t o m i n i m i z e t h e dead volumes, t h e r e d u c i n g u n i o n s have t o be bored t h r o u g h and d r i l l e d o u t , as shown i n F i g . 3b. A p a r t f r o m t h i s m o d i f i c a t i o n , a n o t h e r problem m i g h t a r i s e w i t h t h e j u n c t i o n o f t h e column end and t h e u n i o n s e a t . The h i g h - p r e s s u r e r e s i s t a n t s e a l i n g o f t h e Swagelok u n i o n s and column t u b e t a k e s p l a c e a t t h e c o n i c a l f e r r u l e s pressed on t o t h e tube. The d i s t a n c e f r o m t h e f e r r u l e s t o t h e column end i s c r i t i c a l w i t h r e s p e c t t o t h e p e r f e c t j u n c t i o n o f column end and u n i o n s e a t . An i m p e r f e c t j u n c t i o n r e s u l t s i n s i g n i f i c a n t e x t r a peak broadening, e s p e c i a l l y when exchanging d i f f e r e n t column tubes and t e r m i n a t o r s . I n o r d e r t o a v o i d t h e s i t u a t i o n where each column t u b e would f i t o n l y one p a i r of expensive t e r m i n a t o r s , an a l t e r n a t i v e m o d i f i c a t i o n o f Swagelok r e d u c i n g u n i o n s i s more s u i t a b l e 2 * ( F i g . 4 ) . I n t h i s c o n s t r u c t i o n , t h e s e a l i n g no l o n g e r appears 0
Rulon
pillary
l.D=030mrn
grooves :50-100
top view
Pm
r
metal filter $ 2 m m . 3 rn I
‘2.9rnrn
F i g . 4. Schematic diagram o f a mod f i e d Swagelok r e d u c i n g u n i o n used as column t e r m i n a t o r . From r e f . 2 8 . a t t h e c o n i c a l f e r r u l e s b u t on t h e f l a t column ends by means o f a P T F E - f i l l e d Swagelok t e r m i n a t o r . The i n s t a l l e d f e r r u l e s s e r v e o n l y t o o b t a i n a g r i p on t h e t u b e i n o r d e r t o f i x t h e n u t s f o r p u l l i n g t h e P T F E - f i l l e d c o n n e c t o r on t o t h e column-end.
T h i s i m p l i e s t h a t t h i s t y p e o f c o n n e c t o r can be exchanged w i t h e v e r y
column t u b e o f t h e same i n n e r and o u t e r d i a m e t e r . An analogous method o f s e a l i n g a t t h e column ends can b e o b t a i n e d when u s i n g c o m m e r c i a l l y a v a i l a b l e porous f r i t s i n a Kel-F r i n g ( A l t e x S c i e n t i f i c ) . These f r i t s f i t i n t h e d r i l l e d - o u t Swagelok r e d u c i n g unions. When s e a l i n g a t t h e column ends, w e l l e d f l a n g e s can b e used i n s t e a d o f f e r r u l e s . A p a r t f r o m these Swagelok c o u p l i n g s , s p e c i a l t e r m i n a t o r c o n n e c t i o n systems w i t h w e l l e d f l a n g e s o r screw-threads, custom made or c o m n e r c i a l l y 29,30
a v a i l a b l e ( H e w l e t t - P a c k a r d ) , have been developed d u r i n g t h e l a s t few y e a r s The s o - c a l l e d s p l i t - s t r e a m arrangement c o n n e c t o r s 14y31’32
i n combination w i t h
.
86
b l
injection (Yalve
11 N
w
t
'tpurnp
needle valve
glass beads teflon fei,rules -
iporous
filter
F i g . 5. Schematic r e p r e s e n t a t i o n o f a s p l i t - s t r e a m column i n l e t t e r m i n a t o r . i n j e c t i o n v a l v e s ( F i g . 5 ) a r e noteworthy. These i n l e t systems were found t o improve t h e peak shapes s i g n i f i c a n t l y on l a r g e - d i a m e t e r columns. The g r e a t v a r i e t y o f commercially a p p l i e d t e r m i n a t o r s makes t h e exchange o f d i f f e r e n t p a r t s c o m p l i c a t e d and more e f f o r t s h o u l d be made t o a c h i e v e s t a n d a r d i z a t i o n o f column t e r m i n a t o r s i n t h e n e a r f u t u r e . I n t h i s r e s p e c t , one f i r m (Chrompack) d e l i v e r s zero-dead-volume a d a p t o r s f o r a l l t y p e s o f d i f f e r e n t c o m m e r c i a l l y a v a i l a b l e column designs. 7. POROUS FILTERS S p e c i a l c a r e must be t a k e n t o a v o i d p r e s s i n g p a r t i c l e s i n t o t h e c a p i l l a r y t u b i n g s and d e t e c t o r . F o r t h i s purpose, coverage o f t h e column p a c k i n g on b o t h s i d e s w i t h PTFE- o r glass-wool, o r i n s t a l l a t i o n o f porous metal o r PTFE f r i t s i n t h e column t e r m i n a t o r s , i s a p p l i e d . Glass-wool and t o a l e s s e r e x t e n t PTFE-wool a r e l e s s s u i t a b l e when u s i n g small p a r t i c l e s and h i g h p r e s s u r e drops. Porous s t a i n l e s s - s t e e l f i l t e r s a r e t o be p r e f e r r e d f o r keeping t h e p a c k i n g i n t h e column
a t h i g h p r e s s u r e s (e.g.,
porous PTFE f i l t e r s deform a t about 70 atm). Three main
demands a r e made upon porous f i l t e r s : (i)
t h e f i l t e r must r e t a i n p a r t i c l e s o f t h e packing, i n c l u d i n g t h e s m a l l e r ones;
(ii)
t h e p e r m e a b i l i t y o f t h e f i l t e r must be l a r g e ;
( i i i ) t h e peak broadening i n t h e porous f i l t e r must be s m a l l compared w i t h t h a t o f t h e column.
I n o r d e r t o r e t a i n t h e p a r t i c l e s , f i l t e r s w i t h a p o r e s i z e o f h a l f t h e mean p a r t i c l e s i z e a r e a g o o d t c h o i c e . The p e r m e a b i l i t y o f porous metal f r i t s depends
on t h e p o r e s i z e and t h i c k n e s s o f t h e frit, b u t i n p r a c t i c e i t i s u s u a l l y much l a r g e r i n comparison w i t h t h e column. However, i f v e r y small p a r t i c l e s ( e . g . , d u s t ) a r e p r e s e n t , b l o c k i n g o f t h e f i l t e r m i g h t occur, which decreases t h e p e r m e a b i l i t y s i g n i f i c a n t l y . T h e r e f o r e , i t i s p r e f e r a b l e t o remove t h e f i n e s , u s u a l l y p r e s e n t i n t h e s o l i d s u p p o r t s , by s e d i m e n t a t i o n b e f o r e p a c k i n g t h e column. The peak broadening due t o metal f r i t s i s u s u a l l y v e r y small when t h e y a r e i n s t a l l e d p r o p e r l y (i.e.,
w i t h a p e r f e c t j u n c t i o n w i t h t h e p a c k i n g and c a p i l l a r y
i n l e t and o u t l e t t u b i n g ) . When a p p l y i n g c o m m e r c i a l l y a v a i l a b l e column t e r m i n a t o r s f i t t e d w i t h porous metal f r i t s , one has t o check c a r e f u l l y whether " m i x i n g chambers" a r e p r e s e n t owing t o a bad j u n c t i o n a t t h e r e d u c i n g s i d e o f t h e union. F o r i n s t a n c e , when u s i n g a porous f r i t i n a Kel-F r i n g i n c o m b i n a t i o n w i t h a Swagelok r e d u c i n g union, one must be s u r e t h a t t h e u n i o n i s bored t h r o u g h and d r i l l e d o u t . PTFE f i l t e r s a r e l e s s p r e s s u r e r e s i s t a n t ( < 70 atm) and a r e b e t t e r a p p l i e d a t t h e l o w - p r e s s u r e ( d e t e c t o r ) s i d e o f t h e column. However, porous 316 s t a i n l e s s - s t e e l o r nickel f i l t e r s are oreferable.
8. CONNECTORS FOR COUPLING COLUMNS Several t y p e s o f c o n n e c t o r s f o r c o u p l i n g columns have been developed
5,16
(Siemens). The c o n n e c t o r s must be designed i n such a way t h a t no e x t r a peak broadening occurs. I n t h i s r e s p e c t , when t h e column t e r m i n a t o r s a r e f i t t e d w i t h 1/16-in.
c a p i l l a r y t u b i n g , a s i m p l e low-dead-volume c o u p l i n g i s o b t a i n e d when
a p p l y i n g c o m m e r c i a l l y a v a i l a b l e zero-dead-volume Swagelok unions (Swagelok E a s t e r n ) . These unions a r e e s p e c i a l l y designed f o r gas and l i q u i d chromatography. When a p p l y i n g t h e much cheaper normal 1 / 1 6 - i n . through a t a bore o f 1/16-in.
Swagelok unions, one has t o b o r e
i n o r d e r t o ensure a p e r f e c t j u n c t i o n o f t h e
c a p i l l a r i e s (see F i g . 3 ) . A p a r t f r o m t h i s s i m p l e c o u p l i n g p o s s i b i l i t y , s p e c i a l c o u p l i n g c o n n e c t o r s c o n s i s t i n g o f two column end t e r m i n a t o r s ( i . e . ,
t w i n connectors)
a r e c o m m e r c i a l l y a v a i l a b l e . F o r g l a s s columns ( t y p e as shown i n F i g . 2b) and metal columns w i t h w e l l e d screw-threads,
s p e c i a l l y shaped p i e c e s o f PTFE as shown i n
F i g . 6 have been used s u c c e s s f u l l y .
To summarize one can s t a t e t h a t t h e d e s i g n and i n s t a l l a t i o n o f column t e r m i n a t o r s i s o f paramount importance i n HPLC. For metal columns, m o d i f i e d Swagelok r e d u c i n g u n i o n s ( s e e F i g . 3 ) seem t o f u l f i l t h e demands o f s m a l l e x t r a peak broadening, p r e s s u r e r e s i s t a n c e and ease o f i n s t a l l a t i o n . S p e c i a l c a r e must be t a k e n when i n s t a l l i n g porous metal f r i t s . F o r g l a s s columns, a t e r m i n a t o r design as shown i n F i g . 2b seems t o be t h e most s u i t a b l e . I n a l l designs, one s h o u l d m i n i m i z e t h e d i a m e t e r and l e n g t h o f c a p i l l a r y t u b i n g s as much as p o s s i b l e . I n t h i s r e s p e c t , t h e c o n n e c t i o n between column t e r m i n a t o r s and d e t e c t o r and i n j e c t i o n p o r t s h o u l d a l s o be minimized. F o r t u n a t e l y , most d e t e c t i o n and i n j e c t i o n systems a r e f i t t e d w i t h
88
teflon
capill
Fig. 6. Some coupling connectors for glass columns and metal columns with screwthreads. 1/16-in. capillary tubing. By applying zero-dead-volume Swagelok unions, a perfect low-dead-volume connection between terminator and detector or injector is obtained. 9. MULTIPLE COLUMN SYSTEMS Mu1 tiple column chromatography is a chromatographic t e c h n i q ~ e ~in ~ -which ~~' a number of different columns are used in order to minimize the separation time of a sample containing compounds with very large differences in capacity ratio. For this purpose, a switching valve has to be used for directing groups of compounds that are not separated on the first column to other columns with favourable separation conditions for this particular group of compounds. In process gas chromatography, on-1 ine multiple column systems are frequently appl ied36. In HPLC, however, the high demands put on the switching valve, with respect to pressure resistance and extra peak broadening, have delayed the use of multiple column systems so far. In this respect, a switching valve, particularly designed for multiple column operation in HPLC meeting these demands, is now commercially available (Siemens) (Fig. 7). Apart from this special switching valve, high-pressur sampling valves can be successfully applied in multi-column operation. Fig. 8 shows schematically how multiple column systems with such high-pressure injection valves are operated. 10. COLUMN THERMOSTATING DEVICES The adjustment of the column temperature has proved to be extremely important in ion-exchange ~hromatography~~. In other forms of 1 iquid chromatography, however, considerable effects of temperature on the degree and order of retention are also found27y38. In liquid-1 iquid chromatography, temperature adjustment is essential in order to obtain stable phase systems (i.e. minimal stripping of the stationary phase). In order to obtain stable columns and reproducible 1 iquid chromatographic
89
F i g . 7. Schematic diagram o f a commercia l y a v a i l a b l e s w i t c h i n g v a l v e . 1, I n l e t ; 2, o u t l e t s ; 3, a i r s u p p l y . From r e f . 35. data, column t e m p e r a t u r e c o n t r o l t o w i t h n r 0.1'
seems t o be a good compromise
i n practice. For t h i s purpose, m a i n l y two t h e r m o s t a t i n g d e v i c e s a r e a p p l i e d : a i r - b a t h t h e r m o s t a t s and l i q u i d t h e r m o s t a t i n g u s i n g j a c k e t e d columns. I n a l l systems, t h e temperature s t a b i l i t y , a d j u s t a b l e t e m p e r a t u r e range and h e a t c a p a c i t y a r e i m p o r t a n t c o n s i d e r a t i o n s . F o r a i r - b a t h t h e r m o s t a t s , t h e shape and c a p a c i t y o f t h e oven, t h e ease o f column i n s t a l l a t i o n and s a f e t y a s p e c t s a r e a d d i t i o n a l f a c t o r s .
column 1
inj. port detector F i g . 8. M u l t i - c o l u m n o p e r a t i o n u s i n g an i n j e c t i o n v a l v e .
90
10.1. C i r c u l a t i n g a i r - b a t h t h e r m o s t a t s Two h e a t i n g p r i n c i p l e s a r e a p p l i e d i n c i r c u l a t i n g a i r - b a t h t h e r m o s t a t s : ( i ) e l e c t r i c a l h e a t i n g i n c o m b i n a t i o n w i t h a h i g h v e l o c i t y a i r blower, arid ( i i ) by c i r c u l a t i o n o f l i q u i d f r o m a w a t e r - b a t h t h e r m o s t a t t h r o u g h t h e oven w a l l s , combined w i t h a h i g h v e l o c i t y a i r blower. The s t a b i l i t y o f t h e e l e c t r i c a l l y heated t y p e i s u s u a l l y b e t t e r t h a n
? .'1
For
t h e l i q u i d f e e d types t h e s t a b i l i t y depends on t h e t e m p e r a t u r e s t a b i l i t y o f t h e water-bath t h e r m o s t a t used f o r l i q u i d c i r c u l a t i o n . I n p r a c t i c e , t e m p e r a t u r e s t a b i l i t i e s o f i- 0.1'
can be o b t a i n e d when u s i n g a w a t e r - b a t h w i t h a temperature
s t a b i l i t y o f i- O.0lo, which i s common f o r most w a t e r - b a t h t h e r m o s t a t s . The l o w e r l i m i t o f t h e t e m p e r a t u r e range o f t h e e l e c t r i c a l l y heated a i r - b a t h t h e r m o s t a t s , i f no c o o l i n g tubes a r e p r e s e n t , can be as much as 20'
because o f
t h e h e a t produced by t h e a i r blower. I n c o m m e r c i a l l y a v a i l a b l e l i q u i d chromatographs, t h e ovens can be s i t u a t e d i n a v e r t i c a l o r h o r i z o n t a l p o s i t i o n . With r e s p e c t t o t h e main r e q u i r e m e n t s o f temperature s t a b i l i t y , no d i f f e r e n c e s can be expected f r o m t h e g e o m e t r i c a l p o s i t i o n o f t h e oven. The o n l y d i f f e r e n c e between t h e s e c o n s t r u c t i o n s i s r e f l e c t e d i n t h e ease o f i n s t a l l i n g columns and c o n n e c t i n g t h e d e t e c t i o n and i n j e c t i o n systems t o t h e column w i t h o u t u s i n g l o n g c a p i l l a r y t u b i n g s . I n p r a c t i c e , a h o r i z o n t a l l y p o s i t i o n e d oven proved t o be more f l e x i b l e when changing d e t e c t o r s . The dimensions o f t h e oven must be l a r g e enough t o accommodate a number o f columns a t l e a s t 50 cm i n l e n g t h . A i r - b a t h t h e r m o s t a t s must have t h e p o s s i b i l i t y o f p u r g i n g t h e oven w i t h n i t r o g e n i n o r d e r t o m i n i m i z e t h e r i s k o f e x p l o s i o n when u s i n g flammable s o l v e n t s .
1 / 1 6 t O 114"
termlnotor
t o Injector
Column
I
Water jacket
I
t o detector
7=% Nut
Metal
washer
S
F i g . 9. Schematic diagram o f a t h e r m o s t a t e d column u s i n g a w a t e r - j a c k e t . r e f . 39.
From
91 10.2. T h e r m o s t a t i n g by means o f h e a t j a c k e t s If no a i r - b a t h t h e r m o s t a t i s a v a i l a b l e , e x c e l l e n t column t h e r m o s t a t i n g can be o b t a i n e d by i n s t a l l a t i o n o f a m e t a l , g l a s s o r perspex j a c k e t around t h e column, f e d by a c i r c u l a t i n g w a t e r - b a t h ( V a r i a t ~ ) ~ ' .A l e a k - f r e e i n s t a l l a t i o n o f h e a t j a c k e t s around t h e column can b e achieved w i t h O - r i n g s e a l s o r bored r u b b e r stoppers. Depending on t h e t e m p e r a t u r e s t a b i l i t y o f t h e c i r c u l a t i n g w a t e r - b a t h ( u s u a l l y ca. O . 0 l o ) , t e m p e r a t u r e c o n t r o l o f b e t t e r t h a n
f
0.1'
can be o b t a i n e d . A d i s -
advantage o f many h e a t j a c k e t s i s t h e poor t e m p e r a t u r e c o n t r o l a t t h e t o p and bottom o f t h e column. I n t h i s r e s p e c t , h e a t j a c k e t s t h a t i n c l u d e t h e r m o s t a t i n g o f t h e column t e r m i n a t o r s a r e p r e f e r a b l e 3 '
(see Fig. 9 ) . Despite t h e e x c e l l e n t
temperature c o n t r o l , h e a t - j a c k e t e d columns a r e l e s s c o n v e n i e n t i n p r a c t i c e w i t h r e s p e c t t o l o c a l i z a t i o n o f column leakage and column exchange.
11. APPENDIX L i s t o f suppliers A l t e x S c i e n t i f i c I n c . , 1780 F o u r t h S t r e e t , Berkeley, C a l i f . 94710, U. S. A. Chrompack B.V., Kuipersweg 6 , Middelburg, The N e t h e r l a n d s . Hewlett-Packard and Co., Avondale D i v i s i o n , R t . 41 and S t a r r Rd., Avondale, Pa. 19311, U. S. A. Packard-Becker B.V., Vulcanusweg 259, P.O. Box 519, D e l f t , The Netherlands. S c i e n t i f i c Glass E n g i n e e r i n g , Pty. Ltd., 111 Arden S t r e e t , N o r t h Melbourne, A u s t r a l i a 3051. Siemens AG, E 634 R h e i n b r k k e n S t r a s s e , P o s t f a c h 211080, 7500 K a r l s r u h e 21, G. F. R. "Swagelok", Crawford F i t t i n g Co., 884 East 1 4 0 t h S t r e e t , Cleveland, Ohio, U. S. A. Varian A s s o c i a t e s , 2700 M i t c h e l l D r i v e , Walnut Creek, C a l i f . 94598, U. S. A. Waters A s s o c i a t e s Inc., Maple S t r e e t , M i l f o r d , Mass. 01757, U. S. A.
12. REFERENCES 1 J.F.K. Huber and J.A.R.J. Hulsman, Anal. Chim. Acta, 38(1967)305. 2 J.F.K. Huber, J.A.R.J. Hulsman and C.A.M. M e i j e r s , J. Chromatogr. , 62(1971)79. 3 J.J. K i r k l a n d , W.W. Jan, H.J. S t o k l o s a and C.H. D i l k s , Jr., J. Chromatogr. S c i . , 15( 1977)303. 4 J.F.K. Huber, J.C. Kraak and H. Veening, Anal. Chem., 44(1972)1554. 5 J.J. K i r k l a n d , J. Chromatogr. S c i . , 7(1969)7. 6 J.J. K i r k l a n d , J. Chromatogr. Sci., 9(1971)206. 7 R.E. Majors, Anal. Chem., 44(1972)1722. 8 B.L. Karger and H. B a r t h , Anal. L e t t . , 4(1971)595. 9 B.L. Karger, K. Conroe and H. E n g e l h a r d t , J. Chromatogr. Sci., 8(1970)242. 10 I. Halasz and P. Walking, J . Chromatogr. S c i . , 7(1969)129. 11 K.M. Jonker, H. Poppe and J.F.K. Huber, Chromatographia, i n press. 12 C.H. Eon, J . Chromatogr., 149(1978)29. 13 R.E. Majors, J. Chromatogr. Sci., 11(1973)88. 14 T.J.N. Webber and E.H. M c K e r r e l l , J. Chromatogr., 122(1976)243. 15 H.R. L i n d n e r , H.P. K e l l e r and R.W. F r e i , J. Chromatogr. S c i . , 14(1976)234. 16 J.C. Kraak, H. Poppe and F. Smedes, J. Chromatogr., 122(1976)147. 17 C.A.M. M e i j e r s , J.A.R.J. Hulsman and J.F.K. Huber, Z. Anal. Chem., 261(1972)347. 18 J.H. Knox and J.F. Parcher, Anal. Chem., 41(1969)1599. 19 J.H. Knox, G.R. L a i r d and P.A. Raven, J. Chromatogr., 122(1976)129. 20 J.F.K. Huber, J. Chromatogr. S c i . , 7(1969)85. 21 K.W. S t a h l , E. Schuppe and H. P o t t h a s t , GIT Fachz. Lab., 17(1973)563. 22 B. V e r s i n o and H. S c h l i t t , Chromatographia, 5(1972)332.
92
23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
J. Asshauer and I . Halasz, J. Chromatogr. Sci., 12(1974)139. R.P.W. Scott, J. Gas Chromatogr., 5(1967)183. R.Aris, Proc. Roy. SOC., Ser. A, 235(1956)67. R.P.W. S c o t t and P. Kucera, J. Chromatogr. Sci., 9(1971)641. C.P. Terweij-Groen and J.C. Kraak, J. Chromatogr. , 138(1977)245. J.C. Kraak and P. B i j s t e r , J . Chromatogr., 143(1977)499. J.H. Knox, Chem. Ind., (1975)29. I . Halasz, H.O. Gerlach, A. Kroneisen and P. Walkling, Z. Anal. Chem., 234 (1968)97. J.J. K i r k l a n d , W.W. Jan, H.J. Stoklosa and C.H. D i l k s , Jr., J. Chromatogr. Sci., 15 (1977)303. A.W.J. de Jong, H. Poppe and J.C. Kraak, J. Chromatogr., 148(1978)127. L.R. Snyder, J. Chromatogr. Sci., 8(1970)692. C.D. S c o t t , D.D. C h i l c o t e and N.E. Lee, Anal. Chem., 45(1972)85. J.F.K. Huber, R. van d e r Linden, E. Ecker and M. Oreans, J. Chromatogr., 83 (1973)267. H. Oster, Prozess Chromatographie, Akad. Verlagsges., F r a n k f u r t , 1973. R. Dybczjmski, J. Chromatogr. , 31(1967)155. J.C. Kraak and J.F.K. Huber, J. Chromatogr., 102(1974)333. Gy. Vigh, J. Chromatogr., 117(1976)424.
CHAPTER 5
COLUMN DESIGN SELECTION J.C.
KRAAK
Laboratory f o r A n a l y t i c a l Chemistry, U n i v e r s i t y o f Amsterdam,
Nieuwe Achtergracht
166, Amsterdam (The Netherlands)
CONTENTS 1. I n t r o d u c t i o n 75 2. Column system 76 2.1. Column tube m a t e r i a l 76 2.2. Metal tubes 77 2.3. Glass tubes 77 2.4. Tubes o f polymeric m a t e r i a l s 77 2.5. G l a s s - l i n e d tubes 78 3. Column dimensions 78 3.1. Column l e n g t h 78 3.2. I n n e r diameter o f t h e column 78 3.3. Outer diameter and w a l l t h i c k n e s s 79 3.4. I n s i d e s u r f a c e o f columns 8 1 4. Column t u b e c o n f i g u r a t i o n 8 1 5. Column f i t t i n g s 82 5.1. C a p i l l a r y t u b i n g s 82 6. Column end t e r m i n a t o r s 83 6.1. Terminators f o r g l a s s columns 83 6.2. Terminators f o r metal columns 84 7. Porous f i l t e r s 86 8. Connectors f o r coup1 i n g columns 87 9. M u l t i p l e column systems 88 10. Column t h e r m o s t a t i n g devices 88 10.1. C i r c u l a t i n g a i r - b a t h thermostats 90 10.2. Thermostating by means o f heat j a c k e t s 11. Appendix 91 12. References 91
91
1. INTRODUCTION I n high-performance 1 i q u i d chromatography (HPLC), t h e choice and c o n s t r u c t i o n o f t h e column i t s e l f and i n p a r t i c u l a r t h e column f i t t i n g s might be o f d e c i s i v e importance f o r p r e p a r i n g s u c c e s s f u l l y h i g h - e f f i c i e n c y separation columns. A p a r t from r e s i s t a n c e t o h i g h pressures and chemical a c t i o n o f t h e eluent, much a t t e n t i o n has t o be p a i d t o m i n i m i z i n g e x t e r n a l peak broadening caused by column t e r m i n a t o r s and connection
F u r t h e r , t h e p o s s i b i l i t y o f exchanging each separate p a r t ,
s i m p l i c i t y and cheapness o f t h e column design a r e o f importance.
I n t h i s paper,
d i f f e r e n t column systems i n c l u d i n g t h e r m o s t a t i n g devices, commercially a v a i l a b l e o r custom made, w i l l be discussed.
76
2. COLUMN SYSTEM 2.1.
Column t u b e m a t e r i a l Before d i s c u s s i n g t h e s u i t a b i l i t y o f t h e d i f f e r e n t column t u b e s t h a t a r e
a v a i l a b l e , i t i s w o r t h w h i l e summarizing t h e p a c k i n g procedures t h a t have been a p p l i e d as t h e s e u s u a l l y r e s t r i c t t h e c h o i c e o f t h e column t u b e m a t e r i a l i n p r a c t i c e . M a i n l y two p a c k i n g procedures have been a p p l i e d up t o now: ( i ) Dry p a c k i n g t e c h n i q u e s : Tamping: small p o r t i o n s o f a l o w - s u r f a c e - a r e a support, c o a t e d o r uncoated, a r e p l a c e d i n t h e column and compressed w i t h a PTFE-tipped p l u n g e r 4 ; Tapping: d r y s o l i d s u p p o r t i s p l a c e d i n t h e column and tapped r a p i d l y u n t i l a dense p a c k i n g i s o b t a i n e d
5
.
( i i ) S o l v e n t f i l t r a t i o n t.echniques: S l u r r y packing: a s l u r r y o f t h e s o l i d s u p p o r t i n t h e e l u e n t o r a n o t h e r s u i t a b l e s o l v e n t i s pumped i n t o t h e column, c l o s e d by a f r i t a t t h e bottom. The column 6 packing i s b u i l t up by f i l t r a t i o n
.
Balanced s l u r r y packing: a s l u r r y o f t h e s o l i d s u p p o r t i n a l i q u i d w i t h a d e n s i t y i d e n t i c a l w i t h t h a t o f t h e s o l i d s u p p o r t i s pumped i n t o t h e column. As a r e s u l t o f t h e v e r y s t a b l e s l u r r y , a homogeneous dense column p a c k i n g i s t h e n 7 b u i l t up by s o l v e n t f i l t r a t i o n
.
Dry p a c k i n g i n v o l v i n g tamping o f t h e s o l i d s u p p o r t w i t h a p l u n g e r i s s u i t a b l e o n l y f o r g l a s s columns. W i t h metal columns, t h e compression o f t h e added l o o s e m a t e r i a l , r e q u i r e d f o r o b t a i n i n g a dense packing, i s s e v e r e l y h i n d e r e d by t h e l a r g e f r i c t i o n a l f o r c e , due t o t h e r e l a t i v e s o f t n e s s o f m e t a l s compared w i t h s o l i d supports. Dry p a c k i n g i n v o l v i n g t a p p i n g can be a p p l i e d w i t h a l l column t u b e m a t e r i a l s . I n p r a c t i c e , however, i t s use i s r e s t r i c t e d t o r e l a t i v e l y l a r g e p a r t i c l e s (e.g.,
> 20 urn). S l u r r y p a c k i n g i s s u i t a b l e f o r a l l t y p e s o f column f o r a l l i n s t a n c e s i n which t h e p r e s s u r e r e s i s t a n c e o f t h e column t u b e i s s u f f i c i e n t . The balanced s l u r r y technique, however, needs such h i g h p r e s s u r e drops i n o r d e r t o o b t a i n r e a s o n a b l e f l u i d v e l o c i t i e s w i t h t h e r a t h e r v i s c o u s s l u r r y t h a t i n most i n s t a n c e s o n l y metal columns can be used. A p a r t f r o m t h e r e s t r i c t i o n i n t h e s e l e c t i o n o f t h e p a c k i n g procedure, o t h e r demands such as r e s i s t a n c e t o chemical a c t i o n by t h e e l u e n t o r i n e r t n e s s o f t h e surface m i g h t p l a y a d e c i s i v e r o l e i n t h e s e l e c t i o n o f t h e column t u b e . Up t o now, g l a s s , metal and t o a l e s s e r e x t e n t p o l y m e r i c m a t e r i a l s have been t e s t e d as column t u b e m a t e r i a l s .
2.2. Metal tubes A l t h o u g h some workers found no s i g n i f i c a n t d i f f e r e n c e s i n column e f f i c i e n c y between d i f f e r e n t k i n d s o f m e t a l tubes f o r dry-packed ( t a p p i n g ) columns o f l o w e f f i c i e n c y * , copper, aluminium and t o a l e s s e r e x t e n t i r o n t u b e s a r e l e s s s u i t a b l e as column t u b e m a t e r i a l s . Owing t o t h e i r r e l a t i v e s o f t n e s s , p i e c e s o f metal a r e e a s i l y s c r a t c h e d f r o m t h e w a l l s o f t h e tubes d u r i n g t h e f i l l i n g
procedure^^'^^.
F u r t h e r t h e s e m e t a l s have a p o o r r e s i s t a n c e t o chemical a c t i o n , have a r a t h e r n o n - i n e r t s u r f a c e which f a v o u r s c a t a l y t i c r e a c t i o n s and a r e l i m i t e d i n p r e s s u r e resistance. S t a i n l e s s - s t e e l ( t y p e 316) columns do n o t have t h e s e disadvantages o f c o p p e r , aluminium and i r o n tubes and a r e v e r y s u i t a b l e . I n most HPLC a p p l i c a t i o n s , 316 s t a i n l e s s - s t e e l column tubes can be used w i t h o u t d i f f i c u l t y . When t h e chemical i n e r t n e s s i s i n s u f f i c i e n t , t h e 316 s t a i n l e s s - s t e e l column can be r e p l a c e d w i t h t h e more expensive b u t more i n e r t t a n t a l u m column t u b e s (Siemens). When v e r y s t r o n g l y a c i d i c m o b i l e phases have t o be used, 316 s t a i n l e s s - s t e e l columns a r e l e s s s u i t a b l e . F o r such i n s t a n c e s , sometimes home-made t i t a n i u m columns, i n c l u d i n g t i t a n i u m t e r m i n a t o r s and i n j e c t i o n systems, have been a p p l i e d suc11 cessfully
.
2.3.
Glass tubes
The t y p e o f g l a s s t h a t i s chosen seems t o be o f m i n o r importance. I n most i n s t a n c e s normal b o r o s i l i c a t e g l a s s seems t o be a good c h o i c e . However, i n o r d e r t o c o n s t r u c t a h i g h - p r e s s u r e r e s i s t a n t g l a s s column, t h e absence o f s t r e s s i n t h e glass tube i s very,important. Stress u s u a l l y occurs a f t e r c u t t i n g o f t h e g l a s s t u b e t o t h e d e s i r e d l e n g t h . Slow h e a t i n g o f t h e g l a s s t u b e t o 600'
and
c o o l i n g s l o w l y u s u a l l y removes t h e s t r e s s and improves c o n s i d e r a b l y t h e p r e s s u r e resistance o f t h e glass tubes. Obviously t h e pressure resistance o f glass tubes i s r e l a t e d t o t h e w a l l thickness. With respect t o possible c a t a l y t i c r e a c t i o n s a t t h e surface, g l a s s columns a r e much more i n e r t t h a n metal columns, as i s known f r o m gas chromatography. 2.4. Tubes o f p o l y m e r i c m a t e r i a l s
A m a j o r r e q u i r e m e n t f o r column t u b e s i n HPLC i s constancy o f t h e co umn the
d i a m e t e r when changing t h e p r e s s u r e d r o p a l o n g them. I f t h i s i s n o t so
s t a b i l i t y o f t h e p a c k i n g i s a f f e c t e d on changing t h e p r e s s u r e drop, wh ch d e s t r o y s t h e column e f f i c i e n c y . A l l a v a i l a b l e p o l y m e r i c tubes, such as PTFE, n y l o n o r p l a s t i c tubes, show a d e f i n i t e p r e s s u r e dependence o f t h e t u b e diameter. A p a r t f r o m t h i s , p o l y m e r i c m a t e r i a l s , e x c e p t PTFE, a r e e a s i l y a t t a c k e d by o r g a n i c s o l v e n t s . F o r t h e s e reasons p o l y m e r i c m a t e r i a l s a r e u n s u i t a b l e as column t u b e m a t e r i a l s i n HPLC. R e c e n t l y , however, t h e a p p l i c a t i o n o f a r a d i a l compressed PTFE column f o r p r e p a r a t i v e LC was r e p o r t e d (Waters Assoc.).
78
2.5.
G l a s s - l i n e d tubes Metal t u b e s i n t e r n a l l y c o a t e d w i t h g l a s s a r e c o m m e r c i a l l y a v a i l a b l e w i t h
diameters up t o 4 mm ( S c i e n t i f i c Glass E n g i n e e r i n g ) . These t u b e s show t h e mechanical s t r e n g t h o f metal columns and t h e i n e r t n e s s and smooth s u r f a c e o f t h e g l a s s columns. However, t h e s e g l a s s - l i n e d t u b e s have seldom been a p p l i e d i n LC. I n c o n c l u s i o n , 316 s t a i n l e s s - s t e e l and g l a s s a r e p r e f e r r e d as column t u b e m a t e r i a l s i n HPLC. Radial compressed PTFE t u b e s m i g h t have advantages i n t h e f u t u r e , as was p o i n t e d o u t by Eon12 r e c e n t l y . A l t h o u g h c o m m e r c i a l l y a v a i l a b l e f o r many y e a r s , g l a s s - l i n e d tubes have found l i t t l e a p p l i c a t i o n i n HPLC so f a r . T e s t i n g o f t h e s e column tubes m i g h t be i n t e r e s t i n g , as t h e advantages o f t h e i n e r t n e s s and hardness o f g l a s s a r e combined w i t h t h e mechanical s t r e n g t h o f metal. 3. COLUMN DIMENSIONS 3.1.
Column l e n q t h
The c h o i c e o f t h e l e n g t h o f t h e column w i l l always be a compromise between t h e d i f f e r e n t demands p u t on t h e d e s i r e d r e s u l t and t h e performance o f t h e 1 i q u i d chromatograph. The l e n g t h of t h e s e p a r a t i o n column i s d e t e r m i n e d p r i m a r i l y by t h e number o f t h e o r e t i c a l p l a t e s r e q u i r e d . F u r t h e r , t h e column l e n g t h i s r e l a t e d t o t h e p a c k i n g t e c h n i q u e a p p l i e d , t h e p r e s s u r e c a p a b i l i t i e s o f t h e d i f f e r e n t LC components and sometimes t h e space a v a i l a b l e i n t h e t h e r m o s t a t . W i t h r e s p e c t t o t h e column p a c k i n g , s h o r t e r columns (e.g., o r g l a s s can be packed more e f f i c i e n t l y t h a n l o n g e r
10-25 cm) o f m e t a l
column^^^-^^.
I n order t o
c o n s t r u c t a l o n g e r column, s h o r t e r columns can be c o u p l e d w i t h o u t a s i g n i f i c a n t decrease i n e f f i c i e n c y 1 6 . W i t h t h e d r y tamping and s l u r r y p a c k i n g techniques, t h e o p t i m a l l e n g t h w i t h r e s p e c t t o column e f f i c i e n c y ranges f r o m 10 t o 25 cm. F o r d r y t a p p i n g t h e l e n g t h i s l e s s c r i t i c a l and m i g h t be up t o 100 cm. I f a f i x e d number o f t h e o r e t i c a l p l a t e s i s needed a t a l i m i t e d p r e s s u r e drop, t h e n H/V versus a P / N ( p r e s s u r e d r o p p e r p l a t e ) p l o t s 1 6 o f d i f f e r e n t p a r t i c l e s i z e s a r e h e l p f u l f o r f i n d i n g t h e o p t i m a l column l e n g t h and p a r t i c l e s i z e . I n g e n e r a l , t h e l e n g t h s o f s e p a r a t i o n columns l i e i n t h e range 10-100 cm. With g l a s s columns, however, no high-speed s e p a r a t i o n s can be e x p e c t e d on l o n g columns because o f t h e i r l i m i t e d p r e s s u r e r e s i s t a n c e . 3.2.
I n n e r d i a m e t e r o f t h e column
A d e c i s i o n about t h e i n n e r d i a m e t e r o f t h e a n a l y t i c a l column depends upon a number o f d i f f e r e n t demands. O b v i o u s l y t h e r e i s a r e l a t i o n s h i p between t h e i n n e r diameter, w a l l t h i c k n e s s and
p r e s s u r e r e s i s t a n c e o f g l a s s o r m e t a l columns. I n
79
p r a c t i c e , however, t h i s i s l e s s c r i t i c a l w i t h s t a i n l e s s - s t e e l columns when used a t pressures up t o 400 atm. I n r e c e n t y e a r s f i l l i n g pressures up t o 1000 atm have been a p p l i e d i n o r d e r t o pack more e f f i c i e n t , more r e p r o d u c i b l e and l o n g e r columns16. I n these instances t h e r e l a t i o n s h i p between i n n e r diameter and w a l l t h i c k n e s s becomes c r i t i c a l . Apart from t h e pressure r e s i s t a n c e , o t h e r f a c t o r s might be o f d e c i s i v e importance. I n t r a c e a n a l y s i s , u s u a l l y small samples o f low c o n c e n t r a t i o n a r e a v a i l a b l e and t o o g r e a t a d i l u t i o n o f such samples i n t h e column s e r i o u s l y a f f e c t s t h e i r d e t e c t i o n . The maximal c o n c e n t r a t i o n o f a s o l u t e i n t h e mobile phase a t t h e end o f t h e column ( < cy > i n j e c t e d , Qi,
as a f u n c t i o n o f t h e amount
i s given by t h e f o l l o w i n g equation17:
where E = p o r o s i t y o f t h e mobile phase; A = c r o s s - s e c t i o n a l area o f t h e column; m L = column l e n g t h ; Hi = t h e o r e t i c a l p l a t e h e i g h t o f t h e s o l u t e i.
Eqn. 1 shows c l e a r l y t h a t t h e s m a l l e r t h e c r o s s - s e c t i o n a l area ( p r o p o r t i o n a l t o the square o f t h e i n n e r diameter) t h e h i g h e r i s t h e c o n c e n t r a t i o n o f t h e s o l u t e a t t h e end o f t h e column. Therefore, i n t r a c e a n a l y s i s , h i g h - e f f i c i e n c y ,
s h o r t and
small-diameter columns a r e t o be p r e f e r r e d . The s m a l l e s t i n n e r diameter o f columns t h a t can be packed e f f i c i e n t l y i s about 2 mm ( r e f . 7). Smaller diameters a r e l e s s e f f i c i e n t because o f an i n c r e a s i n g i n f l u e n c e o f t h e w a l l e f f e c t s and t h e r e l a t i v e i n c r e a s e i n t h e e x t e r n a l peak broadening versus t h e d i s p e r s i o n due t o t h e column. I f t h e c o n c e n t r a t i o n and amount o f sample a r e n o t r e s t r i c t e d , columns o f l a r g e r i n n e r diameter a r e advantageous, because of decreasing c o n t r i b u t i o n s o f w a l l e f f e c t s and e x t e r n a l peak broadening t o t h e o v e r a l l column d i s p e r s i o n , as has been shown by several workers18y19. 10 mm I . D .
For a n a l y t i c a l purposes, columns up t o
can be used.
To summarize, column i n n e r diameters between 2 and 10 mm a r e s u i t a b l e f o r a n a l y t i c a l purposes when t a k i n g i n t o account t h e pressure r e s i s t a n c e . Columns w i t h i n n e r diameters o f 3-5 mm seem t o be a good compromise f o r most p r a c t i c a l applications. 3.3. Outer diameter and w a l l t h i c k n e s s As mentioned e a r l i e r , t h e o u t e r diameter i s r e l a t e d t o t h e i n n e r diameter. Up t o u l t i m a t e pressures o f about 400 atm, t h e w a l l t h i c k n e s s o f 316 s t a i n l e s s - s t e e l columns i s n o t so c r i t i c a l , b u t when l a r g e r pressures a r e a p p l i e d , t h e r a t i o o f
80
t h e i n n e r t o t h e o u t e r d i a m e t e r o f t h e t u b e must have a c e r t a i n v a l u e . To ensure a good p r e s s u r e r e s i s t a n c e up t o 1000 atm, t h i s r a t i o should be ca. 0.5. A p a r t f r o m t h i s , an o u t e r d i a m e t e r t h a t can be f i t t e d w i t h c o m m e r c i a l l y a v a i l a b l e end f i t t i n g s such as Swagelok components i s o f t e n v e r y c o n v e n i e n t . F o r g l a s s columns, t h e r a t i o o f i n n e r t o o u t e r d i a m e t e r determines t h e maximal a l l o w a b l e p r e s s u r e drop. I n o r d e r t o r e s i s t p r e s s u r e s up t o 100 atm, t h e w a l l t h i c k n e s s o f g l a s s columns must l i e i n t h e range 3-6 mm. The t h i c k e r t h e g l a s s w a l l , t h e more p r e s s u r e r e s i s t a n t w i l l be t h e g l a s s column, b u t t h e g r e a t e r m i g h t be t h e s t r e s s i n t h e g l a s s . W i t h v e r y t h i c k - w a l l e d g l a s s tubes, extreme c a r e must be t a k e n t o remove s t r e s s . T h i s can be done by s l o w l y h e a t i n g and c o o l i n g , as described before. I n p r a c t i c e thick-walled, 3 mm and O.D. 20 atm
.
s m a l l - d i a m e t e r g l a s s columns (I.D.
12 mm) can be used s a f e l y up t o an u l t i m a t e p r e s s u r e o f about 100
I n an a t t e m p t t o use g l a s s columns a t h i g h e r pressures, an i n g e n i o u s g l a s s w i t h a p r e s s u r e r e s i s t a n c e up t o
column system was developed by S t a h l e t a1."
300 atm. I n t h i s c o n s t r u c t i o n , t h e i n l e t p r e s s u r e i n s i d e and o u t s i d e t h e column
can be k e p t t h e same d u r i n g t h e experiments, as can be seen f r o m F i g . 1.
,glass tube
+
detector F i g . 1. Schematic diagram o f a h i g h - p r e s s u r e r e s i s t a n t g l a s s column system.
81
3.4.
I n s i d e s u r f a c e o f columns
Many recommendations have been made about t h e p r e p a r a t i o n o f t h e i n n e r s u r f a c e when p a c k i n g m e t a l columns. It i s o b v i o u s t h a t t h e smoother t h e i n n e r w a l l , t h e b e t t e r t h e ( b a l a n c e d ) s l u r r y f l o a t s i n t o t h e column as t h e f o r c e o f f r i c t i o n a t t h e w a l l d i m i n i s h e s . T h i s improves t h e r e g u l a r i t y o f t h e p a c k i n g s i g n i f i c a n t l y , as was shown by many workers who compared p o l i s h e d o r d r i l l e d metal columns w i t h t h e o r i g i n a l ones 5y9y22y23.O t h e r workers have p r e s c r i b e d a t h o r o u g h washing procedure i n o r d e r t o o b t a i n h i g h l y e f f i c i e n t packed columns. Most o f t h e s e r i t u a l s , however, can b e dispensed w i t h when a p p l y i n g modern h i g h - q u a l i t y seamless 316 stainless-steel
p r e c i s i o n - b o r e tubes. I n most i n s t a n c e s t h e s e tubes can be used
w i t h o u t p r e - t r e a t m e n t o t h e r t h a n washing w i t h an o r g a n i c s o l v e n t i n o r d e r t o remove p o s s i b l e small metal p a r t i c l e s o r d r i l l i n g o i l . The i n n e r s u r f a c e o f g l a s s columns i s u s u a l l y smooth and no f u r t h e r p r e - t r e a t m e n t i s necessary. I n some i n s t a n c e s t h e i n n e r s u r f a c e i s etched; t h i s has no s i g n i f i c a n t e f f e c t on t h e column e f f i c i e n c y i n comparison w i t h t h e smooth-surface t y p e o f g l a s s tubes, b u t i n p r a c t i c e , e t c h e d g l a s s tubes have a s l i g h t l y b e t t e r p r e s s u r e resistance. 4. COLUMN TUBE CONFIGURATION I n HPLC, s t r a i g h t columns a r e u s u a l l y a p p l i e d , i n c o n t r a s t t o gas chromatography where c o i l e d columns a r e p o p u l a r . F o r a number o f reasons, s t r a i g h t columns a r e p r e f e r a b l e i n HPLC; u s u a l l y s e p a r a t i o n s can be o b t a i n e d w i t h s h o r t columns, and c o i l i n g i n o r d e r t o o b t a i n more c o n v e n i e n t t h e r m o s t a t i n g i s o f m i n o r i n t e r e s t . Also, t h e p a c k i n g t e c h n i q u e s , i n p a r t i c u l a r d r y tamping f o r g l a s s columns, need s t r a i g h t columns. On t h e o t h e r hand, t h e s l u r r y p a c k i n g o f c o i l e d m e t a l columns l e a d s t o l e s s homogeneous column packings owing t o s i g n i f i c a n t d i f f e r e n c e s i n a x i a l f l u i d v e l o c i t i e s i n t h e i n s i d e and o u t s i d e t r a c k o f t h e column c r o s s - s e c t i o n . F u r t h e r , c o i l i n g o f s t r a i g h t f i l l e d m e t a l columns s e r i o u s l y a f f e c t s homogeneity of t h e p a c k i n g 5 y 2 4 and decreases t h e column e f f i c i e n c y s i g n i f i c a n t l y . T h i s does n o t o c c u r when columns f i l l e d w i t h l a r g e p a r t i c l e s (e.g.,
porous l a y e r beads)
a r e c o i l e d , because t h e e f f i c i e n c y of such columns i s a l r e a d y l o w even when s t r a i g h t , and c o i l i n g o r t h e use o f o t h e r shapes such as an S or a f i g u r e - o f - e i g h t does n o t i n f l u e n c e t h e poor e f f i c i e n c y . I f one i s a b l e t o pack a c o i l e d column homogeneously,then , a t t h e f l u i d v e l o c i t i e s commonly used, a p o o r e r column e f f i c i e n c y can be expected because o f d i f f e r e n c e s i n a x i a l f l u i d v e l o c i t i e s and t h e absence o f r a d i a l ( t r a n s v e r s e ) m i x i n g . As mentioned b e f o r e , c o u p l i n g o f s h o r t e r columns w i t h U-shaped c o u p l i n g s w i l l be more s u c c e s s f u l . The c o n c l u s i o n s about column dimensions and c o n f i g u r a t i o n can be summarized as f o l l o w s : s t r a i g h t g l a s s and m e t a l columns w i t h a l e n g t h o f 10-25 cm, an i n n e r d i a m e t e r o f 3-5 mm and a w a l l t h i c k n e s s o f h a l f t h e i n n e r d i a m e t e r f o r m e t a l columns and equal t o t h e i n n e r d i a m e t e r f o r g l a s s columns seem t o be a good
82
compromise f o r a n a l y t i c a l purposes. P r e c i s i o n - b o r e columns made o f 316 s t a i n l e s s - s t e e l can u s u a l l y be a p p l i e d w i t h o u t any p r e - t r e a t m e n t . W i t h o t h e r t y p e s o f s t a i n l e s s s t e e l , smoothing o f t h e i n n e r s u r f a c e m i g h t be necessary. When l o n g e r columns a r e d e s i r e d , c o u p l i n g o f s h o r t e r columns i s p r e f e r a b l e because t h e y can be packed more e f f i c i e n t l y than l o n g e r columns.
5. COLUMN FITTINGS 5.1.
C a p i l l a r y tubings I n o r d e r t o connect t h e column by means o f t e r m i n a t o r s t o t h e i n j e c t i o n p o r t
and d e t e c t o r , c a p i l l a r y t u b i n g i s o f t e n used. A t t h e h i g h - p r e s s u r e s i d e u s u a l l y
316 s t a i n l e s s - s t e e l c a p i l l a r y t u b i n g and a t t h e l o w p r e s s u r e ( d e t e c t o r ) s i d e b o t h 316 s t a i n l e s s - s t e e l and PTFE c a p i l l a r y t u b i n g s a r e a p p l i e d . The peak broadening i n c a p i l l a r y t u b i n g , expressed i n terms o f t h e s t a n d a r d d e v i a t i o n ( i n volume u n i t s ) o f t h e e l u t i o n c u r v e as a f u n c t i o n o f t h e l e n g t h and r a d i u s , i s g i v e n by
25,26
2
3 6
(uv)tube = L2a R Dim/ w
+
4 waR L / 24Dim
where L = l e n g t h ; R = r a d i u s ; w = l i q u i d Flow ( m l l s e c ) ; Dm i
= diffusion coefficient
i n t h e eluent. I n p r a c t i c e , t h e f i r s t t e r m i s neg i g i b l e compared w i t h t h e second, and t h e v a r i a n c e (e.g.,
2
u V ) i s g i v e n by
I n o r d e r t o m i n i m i z e e x t r a peak broadening, t h e c a p i l l a r y c o n n e c t i o n tubes s h o u l d be s h o r t and, even more i m p o r t a n t , t h e y s h o u l d have a r a d i u s as s m a l l as p o s s i b l e because o f t h e s q u a r e - r o o t dependence o f (uv)tube on i t . I n p r a c t i c e , however, t h e r a d i u s o f t h e tube cannot be decreased w i t h o u t l i m i t because o f t h e p r e s s u r e drop o v e r t h e c a p i l l a r y , which i s i n v e r s e l y p r o p o r t i o n a l t o t h e f o u r t h power o f t h e r a d i u s . A good compromise w i t h r e s p e c t t o p r e s s u r e d r o p and magnitude o f t h e peak broadening seems t o be an I . D .
o f 0.25 mm and a l e n g t h o f ca. 20-50 mm.
The c h o i c e o f t h e o u t e r d i a m e t e r o f t h e t u b i n g i s n o t c r i t i c a l . I t i s advantageous when c o m m e r c i a l l y a v a i l a b l e Swagelok o r P a r k e r c o n n e c t o r s f i t t h e t u b i n g (O.D.
1/16 i n . ) . Obviously, o n l y PTFE t u b i n g can be used a t t h e l o w - p r e s s u r e s i d e o f t h e column.
a3
6. COLUMN END TERMINATORS I n o r d e r t o o b t a i n a p e r f e c t j u n c t i o n between t h e column end and t h e t e r m i n a t o r , one must ensure t h a t t h e column ends a r e p e r f e c t l y f l a t . 6.1.
T e r m i n a t o r s f o r g l a s s columns F o r g l a s s columns, two p r i n c i p l e s a r e commonly a p p l i e d f o r o b t a i n i n g a
p r e s s u r e - r e s i s t a n t seal i n g between t h e g ass column and t h e e n d - f i t t i n g : connectors a r e l u t e d on b o t h column ends
( i ) metal
and ( i i) PTFE-coated metal c o n n e c t o r s
a r e pressed on t h e f l a t column ends.
A
6
C
F i g . 2. Column t e r m i n a t o r systems f o r g l a s s columns. F i g . 2a shows s c h e m a t i c a l l y t h e p r i n c i p l e o f a l u t e d e n d - f i t t i n g .
The l u t e d
column e n d - f i t t i n g s have a number o f disadvantages i n p r a c t i c e , such as: (1) t h e s y n t h e t i c g l u e s do n o t w i t h s t a n d a number o f o r g a n i c s o l v e n t s ; ( 2 ) a t t h o s e p l a c e s were t h e c o n n e c t o r s a r e l u t e d on t o t h e g l a s s tube, s t r e s s u s u a l l y o c c u r s , which l o w e r s t h e mechanical s t r e n g t h and p r e s s u r e r e s i s t a n c e c o n s i d e r a b l y , and ( 3 ) t h e l u t i n g techniques r e q u i r e a great experience i n handling glues i n order
t o a p p l y them s u c c e s s f u l l y . Two examples o f t h e o t h e r s e a l i n g p r i n c i p l e a r e shown i n F i g s . 2b and 2c. The f i r s t t y p e ( F i g . 2 b ) , which i s e s p e c i a l l y s u i t a b l e f o r t h i c k - w a l l e d s t r a i g h t g l a s s columns, has been used f o r many y e a r s w i t h g r e a t success27. The g l a s s tube, w i t h p e r f e c t l y f l a t ends, i s p l a c e d i n a metal t u b e w i t h a s c r e w - t h r e a d a t b o t h
84 ends. Then PTFE-coated metal end f i t t i n g s a r e p l a c e d a t b o t h ends o f t h e g l a s s t u b e and t h e s e PTFE-coated c o n n e c t o r s a r e t i g h t l y pressed on t h e f l a t column ends o f the glass tube w i t h nuts. This type o f s e a l i n g i s r e s i s t a n t against very h i g h pressures, f a r above t h e b r e a k i n g - p o i n t o f t h i c k - w a l l e d g l a s s tubes, which ranges f r o m 100-150 atm. The p e r f e c t c o n n e c t i o n w i t h t h e p a c k i n g i s a n o t h e r advantage
o f t h i s column system. An analogous s e a l i n g system i s shown.in F i g . 2c. A t about 5 w f r o m t h e column ends a c a v i t y i s c u t . Two h a l f - c y l i n d r i c a l p i e c e s of PTFE a r e p l a c e d i n these c a v i t i e s and PTFE-coated c o n n e c t o r s w i t h screw-threads a r e p l a c e d a t b o t h ends
o f t h e g l a s s tube. With a n u t , which i s f i x e d by t h e p i e c e s o f PTFE, t h e c o n n e c t o r
i s p u l l e d on t o t h e f l a t column end. Both o f t h e systems i n F i g s . 2b and 2c a r e c o m m e r c i a l l y a v a i l a b 1 e (Siemens; Packard-Becker).
6.2.
T e r m i n a t o r s f o r metal columns A l t h o u g h t h e c o n s t r u c t i o n o f h i g h - p r e s s u r e r e s i s t a n t end f i t t i n g s f o r m e t a l
columns i s easy, many f a i l u r e s i n t h e a p p l i c a t i o n of HPLC m i g h t be a t t r i b u t e d t o u n s a t i s f a c t o r y i n s t a l l a t i o n o f t h e end f i t t i n g s . A p a r t f r o m custom-made t e r m i n a t o r s , m o d i f i e d s t a i n l e s s - s t e e l r e d u c i n g unions o f t h e Swagelok t y p e a r e u s u a l l y a p p l i e d . The m o d i f i c a t i o n o f Swagelok r e d u c i n g u n i o n s i s necessary i n o r d e r t o m i n i m i z e peak broadening, as shown i n F i g . 3. A l l o r i g i n a l Swagelok r e d u c i n g u n i o n s ( F i g . 3a) have a b o r e i n t h e c e n t r e o f about 6 mm l e n g t h and a m i n i m a l I . D .
o f 1.2 mm.
F u r t h e r , r e d u c i n g unions have a l a r g e b o r e a t t h e n o n - r e d u c i n g s i d e . F i t t i n g columns w i t h t h e s e u n m o d i f i e d t e r m i n a t o r s w i l l l e a d t o a l a r g e c o n t r i b u t i o n o f
,ferrules
\
I
A
).
dead volume
t
-reducing
t
original F i g . 3. M o d i f i c a t i o n o f Swagelok unions.
t
bored
6
unic
85 e x t e r n a l peak b r o a d e n i n g due t o these l a r g e dead volumes (e.g.,
m i x i n g chambers).
I n o r d e r t o m i n i m i z e t h e dead volumes, t h e r e d u c i n g u n i o n s have t o be bored t h r o u g h and d r i l l e d o u t , as shown i n F i g . 3b. A p a r t f r o m t h i s m o d i f i c a t i o n , a n o t h e r problem m i g h t a r i s e w i t h t h e j u n c t i o n o f t h e column end and t h e u n i o n s e a t . The h i g h - p r e s s u r e r e s i s t a n t s e a l i n g o f t h e Swagelok u n i o n s and column t u b e t a k e s p l a c e a t t h e c o n i c a l f e r r u l e s pressed on t o t h e tube. The d i s t a n c e f r o m t h e f e r r u l e s t o t h e column end i s c r i t i c a l w i t h r e s p e c t t o t h e p e r f e c t j u n c t i o n o f column end and u n i o n s e a t . An i m p e r f e c t j u n c t i o n r e s u l t s i n s i g n i f i c a n t e x t r a peak broadening, e s p e c i a l l y when exchanging d i f f e r e n t column tubes and t e r m i n a t o r s . I n o r d e r t o a v o i d t h e s i t u a t i o n where each column t u b e would f i t o n l y one p a i r of expensive t e r m i n a t o r s , an a l t e r n a t i v e m o d i f i c a t i o n o f Swagelok r e d u c i n g u n i o n s i s more s u i t a b l e 2 * ( F i g . 4 ) . I n t h i s c o n s t r u c t i o n , t h e s e a l i n g no l o n g e r appears 0
Rulon
pillary
l.D=030mrn
grooves :50-100
top view
Pm
r
metal filter $ 2 m m . 3 rn I
‘2.9rnrn
F i g . 4. Schematic diagram o f a mod f i e d Swagelok r e d u c i n g u n i o n used as column t e r m i n a t o r . From r e f . 2 8 . a t t h e c o n i c a l f e r r u l e s b u t on t h e f l a t column ends by means o f a P T F E - f i l l e d Swagelok t e r m i n a t o r . The i n s t a l l e d f e r r u l e s s e r v e o n l y t o o b t a i n a g r i p on t h e t u b e i n o r d e r t o f i x t h e n u t s f o r p u l l i n g t h e P T F E - f i l l e d c o n n e c t o r on t o t h e column-end.
T h i s i m p l i e s t h a t t h i s t y p e o f c o n n e c t o r can be exchanged w i t h e v e r y
column t u b e o f t h e same i n n e r and o u t e r d i a m e t e r . An analogous method o f s e a l i n g a t t h e column ends can b e o b t a i n e d when u s i n g c o m m e r c i a l l y a v a i l a b l e porous f r i t s i n a Kel-F r i n g ( A l t e x S c i e n t i f i c ) . These f r i t s f i t i n t h e d r i l l e d - o u t Swagelok r e d u c i n g unions. When s e a l i n g a t t h e column ends, w e l l e d f l a n g e s can b e used i n s t e a d o f f e r r u l e s . A p a r t f r o m these Swagelok c o u p l i n g s , s p e c i a l t e r m i n a t o r c o n n e c t i o n systems w i t h w e l l e d f l a n g e s o r screw-threads, custom made or c o m n e r c i a l l y 29,30
a v a i l a b l e ( H e w l e t t - P a c k a r d ) , have been developed d u r i n g t h e l a s t few y e a r s The s o - c a l l e d s p l i t - s t r e a m arrangement c o n n e c t o r s 14y31’32
i n combination w i t h
.
86
b l
injection (Yalve
11 N
w
t
'tpurnp
needle valve
glass beads teflon fei,rules -
iporous
filter
F i g . 5. Schematic r e p r e s e n t a t i o n o f a s p l i t - s t r e a m column i n l e t t e r m i n a t o r . i n j e c t i o n v a l v e s ( F i g . 5 ) a r e noteworthy. These i n l e t systems were found t o improve t h e peak shapes s i g n i f i c a n t l y on l a r g e - d i a m e t e r columns. The g r e a t v a r i e t y o f commercially a p p l i e d t e r m i n a t o r s makes t h e exchange o f d i f f e r e n t p a r t s c o m p l i c a t e d and more e f f o r t s h o u l d be made t o a c h i e v e s t a n d a r d i z a t i o n o f column t e r m i n a t o r s i n t h e n e a r f u t u r e . I n t h i s r e s p e c t , one f i r m (Chrompack) d e l i v e r s zero-dead-volume a d a p t o r s f o r a l l t y p e s o f d i f f e r e n t c o m m e r c i a l l y a v a i l a b l e column designs. 7. POROUS FILTERS S p e c i a l c a r e must be t a k e n t o a v o i d p r e s s i n g p a r t i c l e s i n t o t h e c a p i l l a r y t u b i n g s and d e t e c t o r . F o r t h i s purpose, coverage o f t h e column p a c k i n g on b o t h s i d e s w i t h PTFE- o r glass-wool, o r i n s t a l l a t i o n o f porous metal o r PTFE f r i t s i n t h e column t e r m i n a t o r s , i s a p p l i e d . Glass-wool and t o a l e s s e r e x t e n t PTFE-wool a r e l e s s s u i t a b l e when u s i n g small p a r t i c l e s and h i g h p r e s s u r e drops. Porous s t a i n l e s s - s t e e l f i l t e r s a r e t o be p r e f e r r e d f o r keeping t h e p a c k i n g i n t h e column
a t h i g h p r e s s u r e s (e.g.,
porous PTFE f i l t e r s deform a t about 70 atm). Three main
demands a r e made upon porous f i l t e r s : (i)
t h e f i l t e r must r e t a i n p a r t i c l e s o f t h e packing, i n c l u d i n g t h e s m a l l e r ones;
(ii)
t h e p e r m e a b i l i t y o f t h e f i l t e r must be l a r g e ;
( i i i ) t h e peak broadening i n t h e porous f i l t e r must be s m a l l compared w i t h t h a t o f t h e column.
I n o r d e r t o r e t a i n t h e p a r t i c l e s , f i l t e r s w i t h a p o r e s i z e o f h a l f t h e mean p a r t i c l e s i z e a r e a g o o d t c h o i c e . The p e r m e a b i l i t y o f porous metal f r i t s depends
on t h e p o r e s i z e and t h i c k n e s s o f t h e frit, b u t i n p r a c t i c e i t i s u s u a l l y much l a r g e r i n comparison w i t h t h e column. However, i f v e r y small p a r t i c l e s ( e . g . , d u s t ) a r e p r e s e n t , b l o c k i n g o f t h e f i l t e r m i g h t occur, which decreases t h e p e r m e a b i l i t y s i g n i f i c a n t l y . T h e r e f o r e , i t i s p r e f e r a b l e t o remove t h e f i n e s , u s u a l l y p r e s e n t i n t h e s o l i d s u p p o r t s , by s e d i m e n t a t i o n b e f o r e p a c k i n g t h e column. The peak broadening due t o metal f r i t s i s u s u a l l y v e r y small when t h e y a r e i n s t a l l e d p r o p e r l y (i.e.,
w i t h a p e r f e c t j u n c t i o n w i t h t h e p a c k i n g and c a p i l l a r y
i n l e t and o u t l e t t u b i n g ) . When a p p l y i n g c o m m e r c i a l l y a v a i l a b l e column t e r m i n a t o r s f i t t e d w i t h porous metal f r i t s , one has t o check c a r e f u l l y whether " m i x i n g chambers" a r e p r e s e n t owing t o a bad j u n c t i o n a t t h e r e d u c i n g s i d e o f t h e union. F o r i n s t a n c e , when u s i n g a porous f r i t i n a Kel-F r i n g i n c o m b i n a t i o n w i t h a Swagelok r e d u c i n g union, one must be s u r e t h a t t h e u n i o n i s bored t h r o u g h and d r i l l e d o u t . PTFE f i l t e r s a r e l e s s p r e s s u r e r e s i s t a n t ( < 70 atm) and a r e b e t t e r a p p l i e d a t t h e l o w - p r e s s u r e ( d e t e c t o r ) s i d e o f t h e column. However, porous 316 s t a i n l e s s - s t e e l o r nickel f i l t e r s are oreferable.
8. CONNECTORS FOR COUPLING COLUMNS Several t y p e s o f c o n n e c t o r s f o r c o u p l i n g columns have been developed
5,16
(Siemens). The c o n n e c t o r s must be designed i n such a way t h a t no e x t r a peak broadening occurs. I n t h i s r e s p e c t , when t h e column t e r m i n a t o r s a r e f i t t e d w i t h 1/16-in.
c a p i l l a r y t u b i n g , a s i m p l e low-dead-volume c o u p l i n g i s o b t a i n e d when
a p p l y i n g c o m m e r c i a l l y a v a i l a b l e zero-dead-volume Swagelok unions (Swagelok E a s t e r n ) . These unions a r e e s p e c i a l l y designed f o r gas and l i q u i d chromatography. When a p p l y i n g t h e much cheaper normal 1 / 1 6 - i n . through a t a bore o f 1/16-in.
Swagelok unions, one has t o b o r e
i n o r d e r t o ensure a p e r f e c t j u n c t i o n o f t h e
c a p i l l a r i e s (see F i g . 3 ) . A p a r t f r o m t h i s s i m p l e c o u p l i n g p o s s i b i l i t y , s p e c i a l c o u p l i n g c o n n e c t o r s c o n s i s t i n g o f two column end t e r m i n a t o r s ( i . e . ,
t w i n connectors)
a r e c o m m e r c i a l l y a v a i l a b l e . F o r g l a s s columns ( t y p e as shown i n F i g . 2b) and metal columns w i t h w e l l e d screw-threads,
s p e c i a l l y shaped p i e c e s o f PTFE as shown i n
F i g . 6 have been used s u c c e s s f u l l y .
To summarize one can s t a t e t h a t t h e d e s i g n and i n s t a l l a t i o n o f column t e r m i n a t o r s i s o f paramount importance i n HPLC. For metal columns, m o d i f i e d Swagelok r e d u c i n g u n i o n s ( s e e F i g . 3 ) seem t o f u l f i l t h e demands o f s m a l l e x t r a peak broadening, p r e s s u r e r e s i s t a n c e and ease o f i n s t a l l a t i o n . S p e c i a l c a r e must be t a k e n when i n s t a l l i n g porous metal f r i t s . F o r g l a s s columns, a t e r m i n a t o r design as shown i n F i g . 2b seems t o be t h e most s u i t a b l e . I n a l l designs, one s h o u l d m i n i m i z e t h e d i a m e t e r and l e n g t h o f c a p i l l a r y t u b i n g s as much as p o s s i b l e . I n t h i s r e s p e c t , t h e c o n n e c t i o n between column t e r m i n a t o r s and d e t e c t o r and i n j e c t i o n p o r t s h o u l d a l s o be minimized. F o r t u n a t e l y , most d e t e c t i o n and i n j e c t i o n systems a r e f i t t e d w i t h
88
teflon
capill
Fig. 6. Some coupling connectors for glass columns and metal columns with screwthreads. 1/16-in. capillary tubing. By applying zero-dead-volume Swagelok unions, a perfect low-dead-volume connection between terminator and detector or injector is obtained. 9. MULTIPLE COLUMN SYSTEMS Mu1 tiple column chromatography is a chromatographic t e c h n i q ~ e ~in ~ -which ~~' a number of different columns are used in order to minimize the separation time of a sample containing compounds with very large differences in capacity ratio. For this purpose, a switching valve has to be used for directing groups of compounds that are not separated on the first column to other columns with favourable separation conditions for this particular group of compounds. In process gas chromatography, on-1 ine multiple column systems are frequently appl ied36. In HPLC, however, the high demands put on the switching valve, with respect to pressure resistance and extra peak broadening, have delayed the use of multiple column systems so far. In this respect, a switching valve, particularly designed for multiple column operation in HPLC meeting these demands, is now commercially available (Siemens) (Fig. 7). Apart from this special switching valve, high-pressur sampling valves can be successfully applied in multi-column operation. Fig. 8 shows schematically how multiple column systems with such high-pressure injection valves are operated. 10. COLUMN THERMOSTATING DEVICES The adjustment of the column temperature has proved to be extremely important in ion-exchange ~hromatography~~. In other forms of 1 iquid chromatography, however, considerable effects of temperature on the degree and order of retention are also found27y38. In liquid-1 iquid chromatography, temperature adjustment is essential in order to obtain stable phase systems (i.e. minimal stripping of the stationary phase). In order to obtain stable columns and reproducible 1 iquid chromatographic
89
F i g . 7. Schematic diagram o f a commercia l y a v a i l a b l e s w i t c h i n g v a l v e . 1, I n l e t ; 2, o u t l e t s ; 3, a i r s u p p l y . From r e f . 35. data, column t e m p e r a t u r e c o n t r o l t o w i t h n r 0.1'
seems t o be a good compromise
i n practice. For t h i s purpose, m a i n l y two t h e r m o s t a t i n g d e v i c e s a r e a p p l i e d : a i r - b a t h t h e r m o s t a t s and l i q u i d t h e r m o s t a t i n g u s i n g j a c k e t e d columns. I n a l l systems, t h e temperature s t a b i l i t y , a d j u s t a b l e t e m p e r a t u r e range and h e a t c a p a c i t y a r e i m p o r t a n t c o n s i d e r a t i o n s . F o r a i r - b a t h t h e r m o s t a t s , t h e shape and c a p a c i t y o f t h e oven, t h e ease o f column i n s t a l l a t i o n and s a f e t y a s p e c t s a r e a d d i t i o n a l f a c t o r s .
column 1
inj. port detector F i g . 8. M u l t i - c o l u m n o p e r a t i o n u s i n g an i n j e c t i o n v a l v e .
90
10.1. C i r c u l a t i n g a i r - b a t h t h e r m o s t a t s Two h e a t i n g p r i n c i p l e s a r e a p p l i e d i n c i r c u l a t i n g a i r - b a t h t h e r m o s t a t s : ( i ) e l e c t r i c a l h e a t i n g i n c o m b i n a t i o n w i t h a h i g h v e l o c i t y a i r blower, arid ( i i ) by c i r c u l a t i o n o f l i q u i d f r o m a w a t e r - b a t h t h e r m o s t a t t h r o u g h t h e oven w a l l s , combined w i t h a h i g h v e l o c i t y a i r blower. The s t a b i l i t y o f t h e e l e c t r i c a l l y heated t y p e i s u s u a l l y b e t t e r t h a n
? .'1
For
t h e l i q u i d f e e d types t h e s t a b i l i t y depends on t h e t e m p e r a t u r e s t a b i l i t y o f t h e water-bath t h e r m o s t a t used f o r l i q u i d c i r c u l a t i o n . I n p r a c t i c e , t e m p e r a t u r e s t a b i l i t i e s o f i- 0.1'
can be o b t a i n e d when u s i n g a w a t e r - b a t h w i t h a temperature
s t a b i l i t y o f i- O.0lo, which i s common f o r most w a t e r - b a t h t h e r m o s t a t s . The l o w e r l i m i t o f t h e t e m p e r a t u r e range o f t h e e l e c t r i c a l l y heated a i r - b a t h t h e r m o s t a t s , i f no c o o l i n g tubes a r e p r e s e n t , can be as much as 20'
because o f
t h e h e a t produced by t h e a i r blower. I n c o m m e r c i a l l y a v a i l a b l e l i q u i d chromatographs, t h e ovens can be s i t u a t e d i n a v e r t i c a l o r h o r i z o n t a l p o s i t i o n . With r e s p e c t t o t h e main r e q u i r e m e n t s o f temperature s t a b i l i t y , no d i f f e r e n c e s can be expected f r o m t h e g e o m e t r i c a l p o s i t i o n o f t h e oven. The o n l y d i f f e r e n c e between t h e s e c o n s t r u c t i o n s i s r e f l e c t e d i n t h e ease o f i n s t a l l i n g columns and c o n n e c t i n g t h e d e t e c t i o n and i n j e c t i o n systems t o t h e column w i t h o u t u s i n g l o n g c a p i l l a r y t u b i n g s . I n p r a c t i c e , a h o r i z o n t a l l y p o s i t i o n e d oven proved t o be more f l e x i b l e when changing d e t e c t o r s . The dimensions o f t h e oven must be l a r g e enough t o accommodate a number o f columns a t l e a s t 50 cm i n l e n g t h . A i r - b a t h t h e r m o s t a t s must have t h e p o s s i b i l i t y o f p u r g i n g t h e oven w i t h n i t r o g e n i n o r d e r t o m i n i m i z e t h e r i s k o f e x p l o s i o n when u s i n g flammable s o l v e n t s .
1 / 1 6 t O 114"
termlnotor
t o Injector
Column
I
Water jacket
I
t o detector
7=% Nut
Metal
washer
S
F i g . 9. Schematic diagram o f a t h e r m o s t a t e d column u s i n g a w a t e r - j a c k e t . r e f . 39.
From
91 10.2. T h e r m o s t a t i n g by means o f h e a t j a c k e t s If no a i r - b a t h t h e r m o s t a t i s a v a i l a b l e , e x c e l l e n t column t h e r m o s t a t i n g can be o b t a i n e d by i n s t a l l a t i o n o f a m e t a l , g l a s s o r perspex j a c k e t around t h e column, f e d by a c i r c u l a t i n g w a t e r - b a t h ( V a r i a t ~ ) ~ ' .A l e a k - f r e e i n s t a l l a t i o n o f h e a t j a c k e t s around t h e column can b e achieved w i t h O - r i n g s e a l s o r bored r u b b e r stoppers. Depending on t h e t e m p e r a t u r e s t a b i l i t y o f t h e c i r c u l a t i n g w a t e r - b a t h ( u s u a l l y ca. O . 0 l o ) , t e m p e r a t u r e c o n t r o l o f b e t t e r t h a n
f
0.1'
can be o b t a i n e d . A d i s -
advantage o f many h e a t j a c k e t s i s t h e poor t e m p e r a t u r e c o n t r o l a t t h e t o p and bottom o f t h e column. I n t h i s r e s p e c t , h e a t j a c k e t s t h a t i n c l u d e t h e r m o s t a t i n g o f t h e column t e r m i n a t o r s a r e p r e f e r a b l e 3 '
(see Fig. 9 ) . Despite t h e e x c e l l e n t
temperature c o n t r o l , h e a t - j a c k e t e d columns a r e l e s s c o n v e n i e n t i n p r a c t i c e w i t h r e s p e c t t o l o c a l i z a t i o n o f column leakage and column exchange.
11. APPENDIX L i s t o f suppliers A l t e x S c i e n t i f i c I n c . , 1780 F o u r t h S t r e e t , Berkeley, C a l i f . 94710, U. S. A. Chrompack B.V., Kuipersweg 6 , Middelburg, The N e t h e r l a n d s . Hewlett-Packard and Co., Avondale D i v i s i o n , R t . 41 and S t a r r Rd., Avondale, Pa. 19311, U. S. A. Packard-Becker B.V., Vulcanusweg 259, P.O. Box 519, D e l f t , The Netherlands. S c i e n t i f i c Glass E n g i n e e r i n g , Pty. Ltd., 111 Arden S t r e e t , N o r t h Melbourne, A u s t r a l i a 3051. Siemens AG, E 634 R h e i n b r k k e n S t r a s s e , P o s t f a c h 211080, 7500 K a r l s r u h e 21, G. F. R. "Swagelok", Crawford F i t t i n g Co., 884 East 1 4 0 t h S t r e e t , Cleveland, Ohio, U. S. A. Varian A s s o c i a t e s , 2700 M i t c h e l l D r i v e , Walnut Creek, C a l i f . 94598, U. S. A. Waters A s s o c i a t e s Inc., Maple S t r e e t , M i l f o r d , Mass. 01757, U. S. A.
12. REFERENCES 1 J.F.K. Huber and J.A.R.J. Hulsman, Anal. Chim. Acta, 38(1967)305. 2 J.F.K. Huber, J.A.R.J. Hulsman and C.A.M. M e i j e r s , J. Chromatogr. , 62(1971)79. 3 J.J. K i r k l a n d , W.W. Jan, H.J. S t o k l o s a and C.H. D i l k s , Jr., J. Chromatogr. S c i . , 15( 1977)303. 4 J.F.K. Huber, J.C. Kraak and H. Veening, Anal. Chem., 44(1972)1554. 5 J.J. K i r k l a n d , J. Chromatogr. S c i . , 7(1969)7. 6 J.J. K i r k l a n d , J. Chromatogr. Sci., 9(1971)206. 7 R.E. Majors, Anal. Chem., 44(1972)1722. 8 B.L. Karger and H. B a r t h , Anal. L e t t . , 4(1971)595. 9 B.L. Karger, K. Conroe and H. E n g e l h a r d t , J. Chromatogr. Sci., 8(1970)242. 10 I. Halasz and P. Walking, J . Chromatogr. S c i . , 7(1969)129. 11 K.M. Jonker, H. Poppe and J.F.K. Huber, Chromatographia, i n press. 12 C.H. Eon, J . Chromatogr., 149(1978)29. 13 R.E. Majors, J. Chromatogr. Sci., 11(1973)88. 14 T.J.N. Webber and E.H. M c K e r r e l l , J. Chromatogr., 122(1976)243. 15 H.R. L i n d n e r , H.P. K e l l e r and R.W. F r e i , J. Chromatogr. S c i . , 14(1976)234. 16 J.C. Kraak, H. Poppe and F. Smedes, J. Chromatogr., 122(1976)147. 17 C.A.M. M e i j e r s , J.A.R.J. Hulsman and J.F.K. Huber, Z. Anal. Chem., 261(1972)347. 18 J.H. Knox and J.F. Parcher, Anal. Chem., 41(1969)1599. 19 J.H. Knox, G.R. L a i r d and P.A. Raven, J. Chromatogr., 122(1976)129. 20 J.F.K. Huber, J. Chromatogr. S c i . , 7(1969)85. 21 K.W. S t a h l , E. Schuppe and H. P o t t h a s t , GIT Fachz. Lab., 17(1973)563. 22 B. V e r s i n o and H. S c h l i t t , Chromatographia, 5(1972)332.
92
23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
J. Asshauer and I . Halasz, J. Chromatogr. Sci., 12(1974)139. R.P.W. Scott, J. Gas Chromatogr., 5(1967)183. R.Aris, Proc. Roy. SOC., Ser. A, 235(1956)67. R.P.W. S c o t t and P. Kucera, J. Chromatogr. Sci., 9(1971)641. C.P. Terweij-Groen and J.C. Kraak, J. Chromatogr. , 138(1977)245. J.C. Kraak and P. B i j s t e r , J . Chromatogr., 143(1977)499. J.H. Knox, Chem. Ind., (1975)29. I . Halasz, H.O. Gerlach, A. Kroneisen and P. Walkling, Z. Anal. Chem., 234 (1968)97. J.J. K i r k l a n d , W.W. Jan, H.J. Stoklosa and C.H. D i l k s , Jr., J. Chromatogr. Sci., 15 (1977)303. A.W.J. de Jong, H. Poppe and J.C. Kraak, J. Chromatogr., 148(1978)127. L.R. Snyder, J. Chromatogr. Sci., 8(1970)692. C.D. S c o t t , D.D. C h i l c o t e and N.E. Lee, Anal. Chem., 45(1972)85. J.F.K. Huber, R. van d e r Linden, E. Ecker and M. Oreans, J. Chromatogr., 83 (1973)267. H. Oster, Prozess Chromatographie, Akad. Verlagsges., F r a n k f u r t , 1973. R. Dybczjmski, J. Chromatogr. , 31(1967)155. J.C. Kraak and J.F.K. Huber, J. Chromatogr., 102(1974)333. Gy. Vigh, J. Chromatogr., 117(1976)424.