Process Scale Chromatography - ACS Symposium Series (ACS

5 Process Scale Chromatography The New Frontier in High Performance Liquid Chromatography Downloaded by UNIV LAVAL on October 19, 2015 | http://pubs.acs.org Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch005

A. H .

1

HECKENDORF ,

E. A S H A R E , and C. R A U S C H

Waters Associates, Inc., Milford, M A 01757

In a process scale chromatograph, the concern for continuous utilization of an expensive investment in capital equipment plays a key role in the trade-offs of operation. The scale-up from the laboratory to the industrial scale equipment requires a system engineered to solve a problem greater than the task of purifying larger amounts of compound. The problem is one of operating a system at minimum cost and obtaining high yields of compound at high purity in as short a period of time as possible, and at as high a concentration as possible to minimize cost and solvent removal problems in the recovery process. The use of multiple column segments allows the flexibility of tailoring the column length to the difficulty of varieties of separations and through column sequence and dynamic column length control, many different modes of operation can be performed. High performance liquid chromatography has developed from the analytical scale to the process scale. The evolution of column technology was enhanced by a technique of radial compression, first developed in 1975. This technology was the result of the realization that rather than go to smaller and smaller particle technology to gain resolution in order to enhance the ability of a packed bed structure to resolve compounds, the spaces between particles could be decreased, thus decreasing the amount of dilution that would occur from a given mass transfer rate. This significant step in the evolution of analytical HPLC technology resulted in the ability to change direction away from open column chromatography to the utilization of high-speed, high-resolution, column technology. Current address: NEST Group, Southboro, MA 01772.

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0097-6156/ 85/0271 -0091 $06.00/0 © 1985 American Chemical Society In Purification of Fermentation Products; LeRoith, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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PURIFICATION OF FERMENTATION PRODUCTS

Downloaded by UNIV LAVAL on October 19, 2015 | http://pubs.acs.org Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch005

I n t r o d u c t i o n t o t h e Technology o f Chromatography I f one compares t h e e f f i c i e n c y o r r e s o l v i n g power o f s m a l l e r p a r t i c l e m a t e r i a l s i n a packed bed s t r u c t u r e t o t h e l o a d r e q u i r e m e n t s o f p r e p a r a t i v e chromatography ( F i g u r e 1 ) , one c a n see t h a t , i n f a c t , r e s o l u t i o n d e c r e a s e s w i t h l o a d . Hence, t h e d r i v e toward s m a l l e r p a r t i c l e s i s a g o a l f o r t h e a n a l y t i c a l chromatographer, b u t i t i s n o t a m u t u a l l y shared g o a l f o r t h e p r e p a r a t i v e chromatographer. Because prep i s t h e r e l a t i v e enrichment o f one compound v s . a n o t h e r , i t i s t h e a b i l i t y t o i s o l a t e compounds from a packed bed s t r u c t u r e i n h i g h y i e l d and h i g h mass i n a s h o r t p e r i o d o f t i m e . T h i s R a d i a l Compression t e c h n o l o g y has been packaged i n a v a r i e t y o f forms, t h e s m a l l e s t o f which i s t h e SEP-PAK c a r t r i d g e f o r sample p r e p a r a t i o n f o r t h e a n a l y t i c a l chromatographer. A SEP-PAK c a r t r i d g e i l l u s t r a t e s how column t e c h n o l o g y c a n f u n c t i o n much l i k e a l i q u i d - l i q u i d e x t r a c t o r , w h i l e u t i l i z i n g an i m m o b i l i z e d bed. The SEP-PAK C-18 c a r t r i d g e i s f i l l e d w i t h s i l i c a g e l w h i c h i s c o v a l e n t l y bonded w i t h an o c t a d e c y l s i l a n e e t h e r . T h i s C-18 ( o c t a d e c y l ) c o a t i n g f u n c t i o n s l i k e t h e non-polar s o l v e n t i n a l i q u i d / l i q u i d e x t r a c t o r , and i f one passes a p o l a r s o l v e n t a c r o s s t h i s column, a p a r t i t i o n phenomenon o c c u r s . I f one needs t o s e p a r a t e t h e components o f a m i x t u r e , one c a n s e l e c t s o l v e n t c o n d i t i o n s which w i l l o p t i m i z e t h e p a r t i t i o n i n g a b i l i t y o f t h e n o n - p o l a r i m m o b i l i z e d phase. I f one uses w a t e r , t h e f u r t h e s t i n p o l a r i t y away from t h e n o n - p o l a r C-18 group, n e u t r a l p o l a r and n o n - p o l a r sample components a r e p a r t i t i o n e d i n t o t h e s u r f a c e c o a t i n g . I f t h e amount o f m i s c i b l e n o n - p o l a r s o l v e n t i n t h e m o b i l e phase i s s e l e c t i v e l y i n c r e a s e d , one c a n s e l e c t i v e l y e l u t e o r p a r t i t i o n away from t h a t bonded phase t h e v a r i o u s components. I f one i n c r e a s e s even f u r t h e r t h e non-polar component o f t h e m o b i l e phase, t h e a d d i t i o n a l components b e g i n t o e l u t e . Thus, r a t h e r q u i c k l y we c a n f r a c t i o n a t e a complex m i x t u r e i n t o s e v e r a l components, o r f r a c t i o n a t e a s i m p l e m i x t u r e i n t o i t s i n d i v i d u a l components. Column Technology Design The t e c h n o l o g y o f f e r e d t o t h e p r o c e s s i n d u s t r y i s a c o m b i n a t i o n o f column t e c h n o l o g y , column c h e m i s t r y and system a r c h i t e c t u r e o p t i m i z e d around a p a r t i c u l a r t a s k . The k e y element t o most c h r o m a t o g r a p h i c systems i s based on column d e s i g n , and t h e parameters f o r column performance a r e o p t i m i z e d . I f one were concerned about t h e e f f e c t i v e n e s s o f t h e packed bed s t r u c t u r e , and i n k e e p i n g i t i n p l a c e f o r l o n g p e r i o d s o f t i m e , and one wanted t o m i n i m i z e t h e use o f p r e s s u r e because t h e t r e n d i s towards h i g h e r p r e s s u r e s t o g e t b e t t e r r e s o l u t i o n , one needs an a l t e r n a t e approach t o s c a l i n g up column c a p a c i t y . One c a n g e t t h a t b e t t e r r e s o l u t i o n f o r a p a r t i c u l a r p a r t i c l e s i z e w i t h o u t t h e i n c r e a s e d p r e s s u r e t h r o u g h r a d i a l compression technology. The need t o improve r e s o l u t i o n as i n t h e a n a l y t i c a l w o r l d i s t h e r e , b u t i n t h e p r e p a r a t i v e w o r l d t h e r e i s a l s o t h e need t o c o n t r o l t h e bed s t r u c t u r e over l o n g p e r i o d s o f t i m e . The use o f massive o v e r l o a d and massive overworking o f t h e column t o serve p r o d u c t i o n p u r p o s e s , demands a more r i g i d c o n t r o l over t h e

In Purification of Fermentation Products; LeRoith, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

HECKENDORF ET AL.

Process Scale Chromatography


104

w

103h

10um 37jum 75um

102

1

10

_1_ 1 02

1 03

1 04

ug SAMPLE / gm PACKING F i g u r e 1. Load vs.

particle

size.


94

PURIFICATION OF FERMENTATION PRODUCTS p o t e n t i a l c h a n n e l i n g and v o i d i n g o f t h a t bed s t r u c t u r e through s o l v e n t changes, s o l v e n t s t r i p p i n g , and g e n e r a l abuse t h a t o c c u r s i n the p r o c e s s environment t o c o n t r o l c o s t s . I f one can keep p r e s s u r e s low, one a l s o c a n i n c r e a s e t h e speed w i t h which one c a n f l o w t h r o u g h t h a t column and, t h u s , the s e p a r a t i o n time i s g o i n g t o be a t a minimum. One wants t o get the b e s t e f f i c i e n c y out o f t h a t bed s t r u c t u r e t h a t one can, r e g a r d l e s s o f t h a t f a c t t h a t one w i l l l o s e a good p o r t i o n o f t h a t e f f i c i e n c y through l o a d a b i l i t y . And, one i s g o i n g t o want t o have the b e s t c a p a c i t y f o r s e p a r a t i n g compounds t h a t one can w i t h a f i x e d bed l e n g t h .


R = 1/4

vHï

a-1 α

f

k k» +1

The r e s o l u t i o n e q u a t i o n i s composed o f t h r e e p a r t s : the e f f i c i e n c y parameter, N, the c h e m i s t r y o r s e l e c t i v i t y parameter, a l p h a , and the c a p a c i t y o r l o a d a b i l i t y parameter, k'· I n the r e s o l u t i o n e q u a t i o n f o r the p r e p a r a t i v e environment, t h i s e q u a t i o n i s a l r e a d y f i x e d by the c h e m i s t r y p a r a m e t e r s . F o r example, t h e c a p a c i t y f a c t o r i s s e t f o r the optimum l o a d from the a n a l y t i c a l d a t a ; t h e c h e m i s t r y o r s e l e c t i v i t y parameter i s s e t by t h e a n a l y t i c a l work-up where one h a s t e s t e d a l l the p o s s i b l e c o m b i n a t i o n s o f c h e m i s t r y r e q u i r e d t o get the s e p a r a t i o n ; and the e f f i c i e n c y parameter i s f i x e d by the amount o f l o a d t h a t i s g o i n g t o be p u t on t h a t bed structure. The e f f i c i e n c y , N, i s a f u n c t i o n o f the l e n g t h and the equivalent to a t h e o r e t i c a l plate.

height

Ν = L/H

The h e i g h t e q u i v a l e n t t o a t h e o r e t i c a l p l a t e i s composed o f t h r e e components o f the e q u a t i o n :

H = A + B/u + Cu

The A term (Eddy d i f f u s i o n ) i s the term m i n i m i z e d by r a d i a l c o m p r e s s i o n t e c h n o l o g y ; the Β term i s p o s i t i v e l y i n f l u e n c e d by l i n e a r v e l o c i t y i n c r e a s e s . I f one wants t o go t o h i g h e r speeds, the l o n g i t u d i n a l d i f f u s i o n ( t h e Β term) g e t s s m a l l e r . T h i s i s good because one i s g o i n g t o want t o u s e the system a t a s h i g h a r a t e as p o s s i b l e t o get the maximum throughput o u t o f t h i s i n v e s t m e n t . However, the C term i s n e g a t i v e l y a f f e c t e d by t h i s i n c r e a s e i n l i n e a r v e l o c i t y , u , So one needs some f l u i d v e l o c i t y c o n t r o l t o be a b l e t o o p t i m a l l y get the s e p a r a t i o n over the w i d e s t p o s s i b l e o p e r a t i n g range. Thus, the g o a l s a r e t o d r i v e the Η term down ( t h e h e i g h t e q u i v a l e n t t o a t h e o r e t i c a l p l a t e down), get the optimum u s e of p r e s s u r e , and t o m a i n t a i n Η over a wide dynamic f l o w range, so t h a t one can do a number o f d i f f e r e n t s e p a r a t i o n s on the same system a t d i f f e r e n t l i n e a r v e l o c i t i e s .


5.

HECKENDORF ET AL.

95


I f one c o n s i d e r s t h e e f f e c t s o f p a c k i n g m a t e r i a l p a r t i c l e s i z e on l i n e a r v e l o c i t y , i t has been determined t h a t i f one d e c r e a s e s p a r t i c l e s i z e by a f a c t o r o f two, the p r e s s u r e i n c r e a s e s as i t s square. Thus, the p r o d u c t i v i t y i n c r e a s e i s p o t e n t i a l l y j e o p a r d i z e d f o r a n e s t h e t i c i n c r e a s e i n r e s o l u t i o n . One needs t o t e s t the a c t u a l r e s o l u t i o n a c h i e v e d i n chromatograms of " u n r e s o l v e d peaks" by t a k i n g f r a c t i o n s t h r o u g h the r e g i o n o f i n t e r e s t and analyzing t h e i r composition. A n a l o g o u s l y , the c h o i c e o f pore s i z e on p r o d u c t i v i t y must be e v a l u a t e d t o o . The r e c e n t i n t e r e s t i n l a r g e pore ( g r e a t e r t h a n 300 angstrom) p a c k i n g m a t e r i a l s f o r chromatography o f macromolecules such a s p e p t i d e s and p r o t e i n s was i n i t i a t e d t o a l l o w h i g h e r r e c o v e r i e s from each c h r o m a t o g r a p h i c r u n , a s w e l l a s t o e l i m i n a t e "memory" e f f e c t s from entrapped m o l e c u l e s on subsequent r u n s . However, the l a r g e r the pore s i z e , the s m a l l e r the s u r f a c e area becomes. T h i s r e s u l t s i n lower c a p a c i t y f a c t o r s , k , a s w e l l a s lower t o t a l c a p a c i t y from a chromatographic system. Thus, t e s t i n g of r e l a t i v e l o a d a b i l i t y and c a p a c i t y from the p a c k i n g m a t e r i a l s i s n e c e s s a r y t o o p t i m i z e the p r o d u c t i v i t i e s o f any system. A s i g n i f i c a n t c o n s i d e r a t i o n i n the p r o d u c t i o n environment i s the c o n c e n t r a t i o n of the p r o d u c t i n the e l u e n t due t o the c o s t s a s s o c i a t e d w i t h sample r e c o v e r y systems and compound s t a b i l i t y during recovery. C o n c e n t r a t i o n i s a f u n c t i o n of the l e n g t h and d i a m e t e r o f t h e column. The w i d e r the column and the l o n g e r i t i s , the more d i l u t e the p r o d u c t stream i s going t o be. Thus, the more energy and t i m e r e q u i r e d t o r e c o v e r the components of i n t e r e s t , and the h i g h e r the p o t e n t i a l f o r p r o d u c t y i e l d l o s s e s due to degradat i o n of p u r i f i e d p r o d u c t . Thus, c o n s i d e r a t i o n of techniques t o prevent product d i l u t i o n i n a chromatographic system are i m p o r t a n t . The amount o f time i n v o l v e d w i t h t h e s e p a r a t i o n i s g o i n g t o be a f u n c t i o n of the l e n g t h of the column, the l i n e a r v e l o c i t y , and the c a p a c i t y term we d i s c u s s e d above. I f one wants to get the most f l e x i b l e column system f o r a v a r i e t y of s e p a r a t i o n problems, what one i s g o i n g t o want t o do i s o p t i m i z e each o f t h e s e terms ( f l u i d v e l o c i t y , c o n c e n t r a t i o n and s e p a r a t i o n t i m e ) . What one i s f o r c e d t o c o n s i d e r i s the a b i l i t y t o have a dynamic column l e n g t h c o n t r o l b u i l t i n t o the system a r c h i t e c t u r e , w h i c h w i l l m i n i m i z e the amount o f d i l u t i o n , s h o r t e n the amount o f time t o get components o u t , keep the p r e s s u r e a t i t s l o w e s t p o s s i b l e l e v e l f o r maximum o p e r a t i n g c a p a b i l i t y , and not s a c r i f i c e the a b i l i t y t o s e p a r a t e compounds. I f one i n c o r p o r a t e s r a d i a l c o m p r e s s i o n technology i n t o the c h r o m a t o g r a p h i c system, i t o f f e r s one the a b i l i t y t o segment the column i n t o s h o r t segments w i t h o u t s a c r i f i c i n g f l u i d d i s t r i b u t i o n o r i n c r e a s i n g p r o d u c t d i l u t i o n t h r o u g h t h i s s e g m e n t a t i o n o f the bed; w h i l e , a t the same t i m e , i t o f f e r s the a b i l i t y t o get the maximum e f f i c i e n c y of our sample f e e d r a t e w i t h r e s p e c t t o the column l e n g t h . I n F i g u r e 2, one can see t h a t the r a t i o of the column l e n g t h a v a i l a b l e over the minimum l e n g t h n e c e s s a r y t o get a s e p a r a t i o n , p l o t t e d a g a i n s t the e f f i c i e n c y of the sample feed r a t e , goes t h r o u g h a n optimum. And, depending on l o a d , t h i s optimum can s h i f t , so t h a t the need f o r h a v i n g a v a r i a b l e l e n g t h column w i t h i n a s i n g l e system i s a p p a r e n t , e s p e c i a l l y when one compares t h a t same L over L min. r a t i o a g a i n s t the e f f i c i e n c y of s o l v e n t u t i l i z a t i o n .


1




EFFICIENCY OF SAMPLE FEED RATE

LOAD

COLUMN LENGTH / LENGTH (MIN)

SOLVENT UTILIZATION

LOAD

COLUMN LENGTH / LENGTH (MIN) F i g u r e 2.

E f f e c t s o f dynamic column l e n g t h c o n t r o l .


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HECKENDORF ET AL.


97

I f one c a n f i x t h e column l e n g t h a t a n optimum f o r t h e sample feed r a t e , t h e n one c a n f i x t h e amount o f s o l v e n t r e q u i r e d t o g e t t h a t separation.


F l u i d Velocity Control One must l o o k a t t h e f a c t o r s a f f e c t i n g sample d i s t r i b u t i o n t o see how one c a n c o n t r o l t h i s f l u i d v e l o c i t y parameter, t o a l l o w s e g m e n t a t i o n o f t h e column. S c a l i n g parameters f o r sample d i s t r i b u t i o n i n v o l v e f l u i d d i s t r i b u t i o n , c o n t r o l o v e r temperature and, a s has been d i s c u s s e d , a p r e s s u r e c o n s i d e r a t i o n a s w e l l as a m i x i n g volume c o n c e r n . I f one does not c o n t r o l m i x i n g volumes a d e q u a t e l y , t h e r e w i l l be an a u t o m a t i c i n c r e a s e i n t h e volume o f t h e p r o d u c t . I f one u t i l i z e s a l a r g e volume t a p e r a t t h e end o f t h e column t o c o n t r o l f l u i d v e l o c i t y , a s has been done i n t h e l a b o r a t o r y column t e c h n o l o g i e s , one c a n g e t a smooth a d d i t i o n o f sample onto t h e column a t l o w l i n e a r v e l o c i t i e s . However, i n a p r o d u c t i o n environment where one i s going t o t r y t o o p t i m a l l y pump t h a t bed s t r u c t u r e , one c a n see from t h e Van Deemeter p l o t comparison t o t h e f l u i d v e l o c i t y p r o f i l e s w i t h i n t h i s schematic o f a column ( F i g u r e 3) t h a t one w i l l be o p e r a t i n g a t d i f f e r e n t l i n e a r v e l o c i t i e s w i t h i n t h a t d i s t r i b u t i o n o r i f a c e . T h i s adds volume t o t h e p r o d u c t , and c o n s e q u e n t l y , t h e r e ' s a l o s s o f r e s o l v i n g power w i t h i n t h e system. C o n s i d e r i n g t h e e f f e c t o f momentum on t h e volume o f t h e peak t h r o u g h t h i s schematic h e r e , one had a p o i n t s o u r c e o f sample and s o l v e n t a t h i g h l i n e a r v e l o c i t i e s , t h e c e n t e r o f t h e peak would be d r i v e n downwards w i t h i n t h e column because o f momentum. I f one u s e s a d i s t r i b u t i o n p l a t e t o i n t e r r u p t t h a t momentum and t o a d e q u a t e l y d i s t r i b u t e t h e sample a c r o s s t h e bed s t r u c t u r e , l e s s o f a momentum parameter w i l l be i n t r o d u c e d t o t h a t s e p a r a t i o n . This l a t t e r s i t u a t i o n c a n be a c h i e v e d most e f f e c t i v e l y i f one has e l i m i n a t e d the w a l l e f f e c t through r a d i a l compression technology, and has o b t a i n e d a u n i f o r m d e n s i t y bed t h r o u g h t h e same technology. S c h e m a t i c a l l y , t h i s f l u i d d i s t r i b u t i o n system has been a c h i e v e d on o u r t e c h n o l o g y t h r o u g h t h e use o f a s e t o f t h i n , l o w volume d i s k s , supported by t h e f o r c e o f t h e a x i a l b r i d g i n g o f t h e p a c k i n g under r a d i a l c o m p r e s s i o n , so t h a t t h e sample i s p u t on i n a u n i f o r m manner, r e g a r d l e s s o f f l o w r a t e through t h e bed. S c h e m a t i c a l l y , one c a n g e t a v a r i e t y o f band p r o f i l e s from column d e s i g n and bed s t r u c t u r e parameters. On t h e t o p l e f t o f F i g u r e 4 i s one p r o f i l e from a h i g h l i n e a r v e l o c i t y f l o w o f t h e column. The c e n t e r p r o f i l e shows t h e e f f e c t o f w a l l e f f e c t and p o i n t source d i s t r i b u t i o n o f sample and s o l v e n t a t h i g h l i n e a r v e l o c i t i e s . I f one e l i m i n a t e s t h e w a l l e f f e c t by u t i l i z i n g v e r y wide d i a m e t e r columns and s m a l l e r p a r t i c l e p a c k i n g , b u t s t i l l has a p o i n t source d i s t r i b u t i o n o f sample and s o l v e n t , one w i l l g e t band d i s t o r t i o n i n t h e c e n t e r o f t h e band a t h i g h e r f l u i d v e l o c i t i e s . The n e t r e s u l t i n a p r o c e s s environment would be a broad d i l u t e p r o d u c t peak. I f one c a n combine w a l l e f f e c t , bed d e n s i t y and f l u i d d i s t r i b u t i o n c o n t r o l , one c a n narrow t h i s band and i n c r e a s e the c o n c e n t r a t i o n o f p r o d u c t , a s i l l u s t r a t e d on t h e bottom o f F i g u r e 4.



98


FAVORED FLOW PATTERNS

UNIFORM S T R U C T U R E ELIMINATION O F F A V O R E D F L O W

F i g u r e 4.

Band s p r e a d i n g .


5.

HECKENDORF ET AL.


99


System A r c h i t e c t u r e H a v i n g d e s i g n e d an optimum column f o r s e p a r a t i o n , we can now i n c o r p o r a t e t h i s t e c h n o l o g y i n t o t h e optimum system a r c h i t e c t u r e . T h i s system a r c h i t e c t u r e s h o u l d i n c o r p o r a t e t h e segmented column design that i s necessary f o r a maximally f l e x i b l e u n i t separation p r o c e s s i n a p r o d u c t i o n environment. Segmented column d e s i g n a l l o w s column sequence c o n t r o l w i t h i n t h e system. Thus, one can get a more c o n t i n u o u s r e c o v e r y o f p r o d u c t o r a h i g h e r o p e r a t i n g e f f i c i e n c y out o f the system. And, because one i s segmenting t h a t column, one can use more o f t h e column bed f o r an o p e r a t i o n a t any s i n g l e time t h a n would be a b l e t o be done w i t h a s i n g l e l a r g e column. S c h e m a t i c a l l y ( F i g u r e 5 ) , one can compare a s e p a r a t i o n o f two components—one on a l o n g column and one on a s h o r t o n e — a n d one can see t h a t , w i t h a segmented d e s i g n one can p u l l out i n t o the p r o d u c t stream a m a t e r i a l t h a t i s p a r t i a l l y p u r i f i e d o r m a t e r i a l t h a t i s f u l l y p u r i f i e d , and the p a r t i a l l y p u r i f i e d m a t e r i a l i s t h e n sequenced onto a n o t h e r column segment f o r f u r t h e r p u r i f i c a t i o n . T h i s m i n i m i z e s t h e amount o f dead time between sample stream a d d i t i o n s and a l s o a l l o w s one t o v i e w what's g o i n g on w i t h i n t h e column a t a h i g h e r f r e q u e n c y r a t e t h a n one i s a b l e t o do on a l a r g e r column. Through p a r a l l e l column segment o p e r a t i o n ( F i g u r e 6 ) , one can c o n t i n u a l l y l o a d p r o d u c t onto t h e columns a t s h o r t e r c y c l e s , t h u s g e t t i n g a h i g h e r product stream o u t p u t . I f one i s g o i n g t o c o n t r o l column sequence, t h i s a l l o w s one to u t i l i z e complex s o l v e n t sequences a s w e l l ( F i g u r e 7 ) . F o r example, f o r c e r t a i n samples one w i l l have t o put on a sample; a f t e r t h a t s e p a r a t i o n i s c o m p l e t e , one w i l l have t o f l u s h t h a t bed s t r u c t u r e and t h e n one w i l l have t o r e - e q u i l i b r a t e t h a t bed s t r u c t u r e b e f o r e the next sample can be added. I f one uses a segmented column system, one c a n o p e r a t e each column i n d e p e n d e n t l y i n terms o f t h e o p e r a t i o n sequence, and o p t i m a l l y g e t t h e downstream s i d e o f t h e system f u n c t i o n i n g a t i t s maximum because, at each s t e p , t h e r e w i l l be a product coming out o f t h e bed. I n a t h r e e column system, on Column One the column o p e r a t i o n a l sequence w i l l be sample, f l u s h , e q u i l i b r a t e , and t h e n on Column Two one w i l l have t o e q u i l i b r a t e , sample, f l u s h ; and t o set up Column Three i n sequence, one i s g o i n g t o e q u i l i b r a t e t h r o u g h t h e f i r s t two s t e p s and t h e n l o a d sample and t h e n f l u s h . Note t h a t t h e p r o d u c t i s c o n t i n u o u s l y coming out o f t h e system a f t e r each sequence. T h i s i s d i f f e r e n t t h a n i f one had a s i n g l e column and one had t o w a i t f o r l o n g e r p e r i o d s o f t i m e between e v e n t s . I t a l l o w s optimum u t i l i z a t i o n o f downstream r e c o v e r y equipment w i t h l e s s b a t c h o p e r a t i o n . Dynamic column l e n g t h c o n t r o l and column sequence c o n t r o l a l s o o f f e r t h e a b i l i t y t o f r a c t i o n a t e complex m i x t u r e s by t h e use of v a r i o u s column segments t o p e r f o r m t h e s e p a r a t i o n t a s k s d i f f e r e n t l y . F o r example: I n the c o n f i g u r a t i o n i n F i g u r e 8, one can f l u s h t h e e a r l y e l u t e r s from a complex m i x t u r e , and t h e n shunt the p a r t i a l l y p u r i f i e d p r o d u c t onto t h e s e p a r a t i o n segments o f t h e system. And s i m u l t a n e o u s l y , f l u s h t h e l o n g e l u t e r s o f f t h a t f i r s t column segment a s w e l l . T h i s g i v e s one some f l e x i b i l i t y i n o p e r a t i o n by a l l o w i n g t h e system t o h a n d l e b o t h complex and s i m p l e s e p a r a t i o n p r o c e s s e s on the same equipment.



A

>

A B A B A B


A B

< F i g u r e 5.

A B PRODUCT STREAM

Column segmentation f o r optimum column

SAMPLE

FLUSH

utilization.

EQUILIBRATE

" PRODUCT STREAM FLUSH

EQUILIBRATE

SAMPLE

"9 Q EQUILIBRATE

SAMPLE

PRODUCT STREAM FLUSH

pu. 3 1

gure 6.

^ PRODUCT STREAM

Column sequence c o n t r o l ( f o r complex s o l v e n t sequences.)



5.

HECKENDORF ET AL.

SAMPLE FLUSH EQUILIBRATE SAMPLE

101


EQUILIBRATE SAMPLE FLUSH EQUILIBRATE

EQUILIBRATE EQUILIBRATE SAMPLE FLUSH

PRODUCT PRODUCT PRODUCT PRODUCT

F i g u r e 7. Column sequence c o n t r o l ( f o r complex s o l v e n t sequences.)

INLET FEED

FLUSH LONG ELUTERS

PRODUCT STREAM ""FLUSH EARLY ELUTERS

PRODUCT STREAM ' INLET FEED

F i g u r e 8. Dynamic column l e n g t h c o n t r o l and column sequence c o n t r o l ( f o r complex m i x t u r e s i m p l i f i c a t i o n . )



F i g u r e 9.

Segment 1

Segment

2

PrepLC 5Û0A

P2I

Begin Injection

Load: 15 gm Flow Rale: 500 ml/min Column: 57 mm χ 30 cm silica

-ι 4 3 2 1 0 Time (min.)

KILOPRFP Flow Schematic (Dynamic Column Length Control "Recycle")

Sample

segment-2

Begin Injection segmenM

150 gm 4 Umin (15 cm χ 60 cm) χ 2 (14 χ PrepPAK Cartridge) χ 2

Unit Separation Process System

Comparison o f column performance a t e q u i v a l e n t l o a d i n g .

t

Time (min.) Mobile Phase

KILOPREP Load: Flow Rate: Column:



5.

H E C K E N D O R F ET AL.


103

T h i s t e c h n o l o g y has been b u i l t i n t o the Waters p i l o t p l a n t u n i t c a l l e d t h e KILOPREP P r o c e s s Chromatography System. I t ' s the f i r s t o f a s e r i e s of i n c r e a s i n g l y l a r g e r chromatographs i n c o r p o r a t i n g r a d i a l compression technology i n t o the system. The performance o f t h i s l a r g e r system can be p r e d i c t e d from the d a t a g e n e r a t e d i n an e q u i v a l e n t l a b o r a t o r y d e v i c e as i n F i g u r e 9. T h i s i s i m p o r t a n t t o p r o c e s s development programs because i t m i n i m i z e s the amount of product and s o l v e n t needed to work out a development program. The t e c h n o l o g y of h i g h performance l i q u i d chromatography has been s u c c e s s f u l l y extended from the a n a l y t i c a l s c a l e t o the p r o c e s s s c a l e . The a b i l i t y t o c o n t r o l the v a r i o u s o p e r a t i o n parameters t o s c a l e up d i r e c t l y from t h e l a b o r a t o r y t o the p i l o t p l a n t and beyond to the p r o d u c t i o n environment has been developed. T h i s t e c h n o l o g y can be combined w i t h o t h e r s e p a r a t i o n s t e c h n o l o g i e s , such a s membrane s e p a r a t i o n s , t o p r o v i d e p a r t i c l e - f r e e s o l v e n t s , u l t r a p u r e p r o d u c t s , and c o n c e n t r a t e d product streams. T h i s w i l l g i v e the o p p o r t u n i t y t o d e a l w i t h f u t u r e s e p a r a t i o n s problems of the chemical process i n d u s t r y . R E C E I V E D October 4, 1984


Process Scale Chromatography - ACS Symposium Series (ACS

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