Ultrahigh Resolution Chromatography - ACS Publications - American

7 Advances in Liquid Chromatographic Selectivity DAVID H. FREEMAN Department of Chemistry, University of Maryland, College Park, M D 20742

Downloaded by UNIV LAVAL on April 8, 2016 | http://pubs.acs.org Publication Date: April 26, 1984 | doi: 10.1021/bk-1984-0250.ch007

Historical Development of Rapid HPLC Separations In the past twelve years, HPLC, or high performance liquid chromatography, has been simplified. It has become easier to learn, and easier to apply to chemical separation problems. HPLC signifies the rapid achievement of high resolution separations. The technique requires usually no more than a number of minutes for each compound separated. This aspect is shown in Figure 1 where the time for separating hemoglobin variants has been reduced from fourteen hours to 7 minutes. This trend, expressed in terms of the time needed to separate multi-component mixtures, has decreased steadily over the past forty years. A sampling of the separation literature illustrates this in Figure 2. The number of components separated per unit time when plotted as a logarithm is, approximately, a straight line function of the year reported. The separation capabilities of current HPLC technology, as implied by Figure 2, did not arrive by a sudden jump into the "HPLC era". HPLC today is a cumulative advance, a result of progress made by a cast of many thousands who over the years have worked to expand the scope and depth of the field, and who have made their results known to others. This growth has mushroomed into a cooperative social effect in the form of an informal network for information exchange. I estimate the availability of 100,000 HPLC instruments, at least the same number of liquid chromatographers, and perhaps several times that number of LC columns. Centers that spread information effectively and provide practical guidance include application laboratories, discussion groups, short courses and meeting symposia. 0097-6156/ 84/ 0250-0077506.00/ 0 © 1984 American Chemical Society

Ahuja; Ultrahigh Resolution Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

ULTRAHIGH RESOLUTION CHROMATOGRAPHY


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F i g u r e 1. The s e p a r a t i o n of hemoglobin v a r i a n t s by c l a s s i c a l L C ( 1 ) and by modern HPLC ( 2 ) techniques.


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Advances in Liquid Chromatographic Selectivity


The number of t r a i n e d chromatographers i s expand i n g r a p i d l y . Modern i n s t r u m e n t a t i o n i s becoming more automated and e a s i e r to use. The g a i n i n g of p r a c t i c a l e x p e r i e n c e i s a c c e l e r a t e d by c o n t a c t w i t h the a v a i l a b l e p o o l of o t h e r u s e r s . A p o r t i o n of t h i s i s apparent i n the p u b l i s h e d l i t e r a t u r e . To see the v a l u e of h a v i n g a c q u i r e d HPLC s e p a r a t i o n s k i l l s , one may examine the " h e l p wanted advert i s e m e n t s i n Chemical and E n g i n e e r i n g News. The commercial p o p u l a r i z a t i o n of HPLC was begun i n the e a r l y 1970's a t the same time t h a t the Nobel l a u r e a t e , the l a t e R.B. Woodward, was p u r s u i n g the s e p a r a t i o n , and l a t e r the proof of p u r i t y , of the t h e r m a l l y l a b i l e c o b e s t e r i n t e r m e d i a t e s formed i n the s y n t h e s i s of v i t a m i n B12. One of Woodward's p o s t d o c t o r a l r e s e a r c h a s s o c i a t e s , Helmut Hamburger, devoted a y e a r ' s e f f o r t to t r i a l and e r r o r a p p l i c a t i o n s of the use of s i l i c a g e l a d s o r b e n t . The Harvard-MIT community knew of the d i f f i c u l t i e s b e i n g e x p e r i e n c e d . Professor M e r r i l l of the MIT c h e m i c a l e n g i n e e r i n g department, a l s o on the board of d i r e c t o r s of Waters A s s o c i a t e s , communicated these d i f f i c u l t i e s to James Waters. H i s company, Waters A s s o c i a t e s , had s t a r t e d an a p p l i c a t i o n s l a b o r a t o r y i n 1967. Woodward's c h e m i c a l i n s i g h t s were needed, as were Waters' t e c h n i c a l i n s i g h t s , i n o r d e r to a c h i e v e a proper h a r n e s s i n g of the speed and r e s o l u t i o n of the new HPLC t e c h n o l o g y . I t was d e c i d e d t h a t the problem was to be s o l v e d i n Woodward's l a b o r a t o r y u s i n g the technology t h a t Waters was eager to s h a r e . The success of t h i s v e n t u r e i s shown by the chromatogram i n F i g u r e 3. Subsequent work moved f a s t e r as HPLC was r e c o g n i z e d as a more p o w e r f u l b a s i s f o r p r o v i n g the p u r i t y of the i s o l a t e d products. A s i g n i f i c a n t p r e c e d e n t had been put to work: the w i l l i n g n e s s of i n d u s t r y to undertake the s o l v i n g of d i f f i c u l t s e p a r a t i o n problems as a b a s i s f o r subsequent s a l e s of i n s t r u m e n t a t i o n and r e l a t e d supplies. The r a t e of s o l v i n g such s e p a r a t i o n problems i s a less frequently discussed subject. An e m p i r i c a l a p p r o a c h i s a t r a d i t i o n t h a t even today i s dominant i n s o l v i n g s e p a r a t i o n problems. Choosing or f i n d i n g the r i g h t c o m b i n a t i o n of l i q u i d and s t a t i o n a r y phases to o b t a i n the needed c h e m i c a l s e l e c t i v i t y i s u s u a l l y an " e d u c a t e d " t r i a l and e r r o r p r o c e s s . I f analogous m i x t u r e s have been s e p a r a t e d , t h a t can p r o v i d e a time-saving boost. Ahuja; Ultrahigh Resolution Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

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FREEMAN


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An important development i n HPLC has been the r e c e n t p r o g r e s s i n s y s t e m a t i z i n g column and s o l v e n t selectivity. The c o l l e c t i o n and improved a c c e s s i b i l i t y of l a r g e a r r a y s o f e m p i r i c a l s e p a r a t i o n r e s u l t s a r e p r o v i d i n g the major b a s i s f o r f a s t e r problem s o l v i n g . The f a s t s e p a r a t i o n r a t e s shown i n F i g u r e s 2a and 3 do not convey the speed of o b t a i n i n g the s e l e c t i v i t y i n the f i r s t p l a c e . I f one were to add the time needed to f i n d t h i s s e l e c t i v i t y (about one y e a r ) to the time a x i s shown i n F i g u r e 3, t h a t would c r e a t e a very d i f f e r e n t i m p r e s s i o n o f whether t h i s was an example of a " h i g h speed" separation. Hence, to be r e a l i s t i c , the s e p a r a t i o n shown i n F i g u r e 3 r e q u i r e d a time investment of a year p l u s h a l f an hour. S i n c e then, v a s t improvements have been made i n the speed o f f i n d i n g c h e m i c a l s e l e c t i v i t y and these w i l l be the theme i n t h i s d i s c u s s i o n . In i t s e a r l y b e g i n n i n g s , the t r i a l and e r r o r a p p r o a c h i n v o l v e d numerous s o r b e n t s , and even more numerous c o m b i n a t i o n s o f l i q u i d s and a d d i t i v e s . I t f o l l o w s t h a t the c o m p l e x i t y o f LC problem s o l v i n g has been a t l e a s t i n p a r t r e l a t e d to the number o f e q u a l l y p l a u s i b l y d e c i s i o n s needed i n c h o s i n g an e f f e c t i v e c o m b i n a t i o n o f l i q u i d and s o r b e n t . The l i k e l i h o o d o f h i t t i n g the proper c o m b i n a t i o n o f l i q u i d and sorbent by g u e s s i n g was, and s t i l l i s , quite small. In a d d i t i o n , the o l d e r a d s o r b e n t s were not d e s i g n e d to f u n c t i o n as such. I n s t e a d they were chosen because o f t h e i r a v a i l a b i l i t y , t h e i r p a s t a p p l i c a t i o n s h i s t o r y , and t h e i r e s t i m a t e d s u i t a b i l i t y based on c o n s i d e r a t i o n s o f t h e i r c h e m i c a l structure. The o c c a s i o n a l l a c k o f r e p r o d u c i b i l i t y , low l o a d c a p a c i t y or sample s i z e l i m i t a t i o n , peak t a i l i n g , c h e m i c a l r e a c t i v i t y , and u n c e r t a i n s o l v e n t i n t e r a c t i o n s slowed the problem s o l v i n g p r o c e s s considerably. Before 1970, s e p a r a t i o n s k i l l s i n LC were r e s e r v e d f o r the r e l a t i v e l y few p r a c t i t i o n e r s who had g a i n e d e x p e r t e s e from r e l a t i v e l y tedious experimentation. Sorbent

Selectivity

A c r u c i a l a s p e c t o f chromatography i s the importance o f m i n i m i z i n g the time needed f o r the s o l u t e to t r a n s i t back and f o r t h between the sorbent and l i q u i d s t r u c t u r e s . T h i s i s n e c e s s a r y i n o r d e r to o b t a i n sharp peaks, as demonstrated by M a r t i n and Synge i n 1941 ( 8 ) . T h i s e x p e r i m e n t a l p r i n c i p l e was a d a p t e d to the i o n exchange s e p a r a t i o n o f amino a c i d s Ahuja; Ultrahigh Resolution Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

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(15 min per component) i n 1963 by Hamilton ( 9 ) . H o r v a t h (10) used impermeable beads as a base f o r an i o n exchange r e s i n f i l m to s e p a r a t e n u c l e o t i d e s and n u c l e o s i d e s a t 5 min per component.


These major s t r i d e s i n s t a t i o n a r y phase d e s i g n — m a x i m i z i n g the r a t e of mass t r a n s f e r — a r e now added to a b o o s t e d sample l o a d c a p a c i t y . An o p t i m a l a r c h i t e c t u r e , s u i t e d f o r purposes of speed and l o a d c a p a c i t y , i s o b t a i n e d when the s o r p t i v e f u n c t i o n a l groups are bonded to a h i g h l y porous s u b s t r a t e . The s o r b e n t s most f r e q u e n t l y chosen f o r c u r r e n t use are the porous s i l i c a d e r i v a t i v e s . Most p o p u l a r a r e the h y d r o p h o b i c or " r e v e r s e phase" s o r b e n t s w i t h bonded a l k y l groups. These are i d e a l l y s u i t e d f o r the p a r t l y aqueous c a r r i e r phases that a r e s u i t a b l e f o r s e p a r a t i o n s r e l a t e d to the l i f e s c i e n c e s , an a r e a that a c c o u n t s f o r most of the c u r r e n t use of HPLC. Many hundreds of c h e m i c a l d e r i v a t i v e s of porous s i l i c a have been p r e p a r e d and t e s t e d ( 1 1 ) . The a b i l i t y to f a b r i c a t e r e p r o d u c i b l y a h i g h performance LC column has c o n s i d e r a b l e commercial v a l u e . As a r e s u l t , column m a n u f a c t u r e r s are apt to be r e l u c t a n t to convey the c h e m i s t r y used to p r e p a r e the bonded f u n c t i o n a l group s t r u c t u r e s , but they o f t e n a l l o w u s e r s to make educated guesses as to what those s t r u c t u r e s might be. Porous s i l i c a m i c r o p a r t i c l e s l e s s than 10 micrometers i n s i z e a r e used, among o t h e r purposes, to s e p a r a t e the l e a s t water s o l u b l e as w e l l as the l e a s t i n t e r a c t i v e compounds. The use of b a s i c f u n c t i o n a l groups ( i . e . amine) on porous s i l i c a can be an a p p r o p r i a t e c h o i c e f o r s e p a r a t i o n s of a t l e a s t s l i g h t l y p o l a r compounds that are hexane s o l u b l e , and even f o r aqueous s e p a r a t i o n s of h i g h l y p o l a r compounds, such as sugars i n aqueous media. Ion exchange d e r i v a t i v e s of s i l i c a compete w i t h c a r e f u l l y p r e p a r e d i o n exchange r e s i n (12), a l t h o u g h the l a t t e r has one advantage i n terms of i t s higher load c a p a c i t y . The o n l y p r e d i c t a b l e s e l e c t i v i t y i n HPLC i s t h a t t h a t p r o v i d e d by the n o n - i n t e r a c t i v e c o n d i t i o n s t h a t may be a c h i e v e d w i t h the porous a d s o r b e n t s used i n nonaqueous s i z e e x c l u s i o n chromatography. Predictab i l i t y i s based on a knowledge of the r e l a t i v e pore and s o l u t e s i z e s . The o p t i m a l s i z e r a t i o f o r s o l u t e s e p a r a t i o n i s f o u r to one ( 1 3 ) .


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The most i m p o r t a n t s t a t i o n a r y phase s e l e c t i o n s a r e u s u a l l y aimed a t the f o u r major chromatographic c a t e g o r i e s o f p a r t i t i o n (normal phase on bonded amine or r e v e r s e d phase w i t h bonded a l k y l ) , a d s o r p t i o n ( i . e . , s i l i c a ) , i o n exchange and s i z e e x c l u s i o n . There i s a wide d i v e r s i t y of a v a i l a b l e t r a d e names, s y n t h e t i c nuances, or g e o m e t r i c ones such as column s i z e and shape. However, over t h r e e - f o u r t h s of HPLC i s done w i t h c h r o m a t o g r a p h i c columns p r e p a r e d as porous s i l i c a d e r i v a t i v e s w i t h bonded a l k y l f u n c t i o n a l groups. Other bonded groups t h a t a r e noteworthy i n c l u d e d i o l f u n c t i o n a l i t y f o r aqueous s i z e e x c l u s i o n and bonded c o u n t e r i o n s f o r ion-exchange. The r e s u l t i s an i n c r e a s i n g l y w e l l f o c u s s e d i n t e r e s t i n r e l a t i v e l y few, but w i d e l y used, column t y p e s . I t i s known t h a t n o m i n a l l y i d e n t i c a l column types do not e x h i b i t i d e n t i c a l separation behavior i n p r a c t i c e . T h i s nonu n i f o r m i t y has provoked u s e r i n t e r e s t i n the q u e s t i o n of the e x t e n t to which s o r p t i v e r e p r o d u c i b i l i t y o c c u r s from column to column and, s i m i l a r l y , among d i f f e r e n t m a n u f a c t u r e r s . R e s e a r c h e r s , as w e l l as a p p l i c a t i o n s s p e c i a l i s t s , must have w e l l d e f i n e d s o r p t i v e s t r u c t u r e s i f t h e i r r e s u l t s are to be t r u l y i n t e r c o r a p a r a b l e . One b a s i s f o r i r r e p r o d u c i b l e column performance among the bonded phases comes from the d i f f i c u l t y i n removing the s i l a n o l groups from the s i l i c a substrate. In some a p p l i c a t i o n s the groups p r o v i d e needed a d d i t i o n a l i n t e r a c t i o n s w h i l e i n o t h e r c a s e s t h i s i s unwanted. C u r r e n t l y i t i s d i f f i c u l t to measure the s i l a n o l c o n c e n t r a t i o n d e f i n i t i v e l y , and i t remains a s y n t h e t i c c h a l l e n g e to c o m p l e t e l y r e move the s i l a n o l groups through end-capping r e a c tions. SOLVENT SELECTIVITY Improvements i n s t a t i o n a r y phase d e s i g n have advanced to a more c o h e r e n t t e c h n o l o g y f o r a c h i e v i n g , a t l e a s t i n p r a c t i c a l terms, w e l l defined sorptive effects. E q u a l l y important, s i m i l a r p r o g r e s s has been made toward improved p r a c t i c a l u n d e r s t a n d i n g of l i q u i d phase c o m p o s i t i o n s needed to a c h i e v e c h e m i c a l s e l e c t i v i t y . P r e l i m i n a r y s o l v e n t s e l e c t i o n has been reduced to the use of s o l v e n t t r i a d s , one f o r aqueous and another f o r non-aqueous systems ( 1 4 ) . Thus, aqueous m i x t u r e s f o r r e v e r s e d phase HPLC, or RPLC, a r e p r e p a r e d w i t h methanol, a c e t o n i t r i l e , and/or t e t r a h y d r o f u r a n as


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l i q u i d phase m o d i f i e r s . I n c r e a s i n g the water c o n t e n t i n RPLC i n t e n s i f i e s the l i p o p h i l i c i n t e r a c t i o n s between sample and s t a t i o n a r y phase.


In normal phase HPLC, or NPLC, hexane i s a w i d e l y used d i l u e n t and the common m o d i f i e r s a r e one or a c o m b i n a t i o n of dichlororaethane, chloroform and e t h e r . Remember, bonded amine d e r i v a t i v e s of s i l i c a are a l s o used f o r normal phase s e p a r a t i o n o f sugars and o t h e r h i g h l y p o l a r compounds. For simple m i x t u r e s s e l e c t i v i t y can o f t e n be found q u i c k l y . I f the sample m i x t u r e i s a t l e a s t s p a r i n g l y water s o l u b l e , RPLC may be the f i r s t choice. I f the sample i s water i n s o l u b l e , then NPLC is preferred. Once the s t a t i o n a r y phase i s chosen, t e s t i n g w i t h i n the s o l v e n t m o d i f i e r t r i a d i s then u s u a l l y done by t r i a l and e r r o r . Such experiments can be c a r r i e d out by automated i n s t r u m e n t a t i o n programmed f o r r e p e t i t i v e s c o u t i n g e x p e r i m e n t s . Those who d e a l r o u t i n e l y w i t h the w i d e l y v a r i e d s e p a r a t i o n problems t h a t show up i n a p p l i c a t i o n s l a b o r a t o r i e s share the o p i n i o n t h a t the easy problems i n HPLC have a l r e a d y been s o l v e d . A p p l i c a t i o n chemists estimate i n f o r m a l l y that simple s o l v e n t b l e n d i n g w i t h r e l a t i v e l y few a d d i t i v e s w i l l p r o v i d e the s e l e c t i v i t y needed to s e p a r a t e 90% o f the r o u t i n e l y e n c o u n t e r e d m i x t u r e s . User support i s a key m a r k e t i n g a c t i v i t y t h a t i n c l u d e s the p r o v i s i o n of e s t i m a t e d separation c o n d i t i o n s or enhancements, recommended columns, s o l v e n t s , a d d i t i v e s and o t h e r t i p s or recommendations. T h i s may be p r o v i d e d , f o r example, by an a p p l i c a t i o n s l a b o r a t o r y i n response to a telephone inquiry. S e p a r a t i o n problems become s u b s t a n t i a l l y more d i f f i c u l t as the number of components i n c r e a s e s much above t e n . Such c o m p l e x i t y i s o f t e n c h a r a c t e r i s t i c of e n v i r o n m e n t a l and many b i o l o g i c a l samples. Ionic i n t e r a c t i o n s and i o n exchange, f o r example, may o f f e r p o t e n t i a l l y u n l i m i t e d s e l e c t i v i t y but the c o n d i t i o n s f o r o p t i m a l s e l e c t i v i t y are c o r r e s p o n d i n g l y more d i f f i c u l t to f i n d . ROLE OF

ADDITIVES

The growing s e p a r a t i o n c a p a b i l i t i e s of HPLC i n c l u d e an expanding c a t a l o g of l i q u i d phase a d d i tives. Such compounds are p l a c e d i n the c a r r i e r i n o r d e r to s t a b i l i z e or augment the c h e m i c a l



7. F R E E M A N


selectivity. C o n s i d e r the f o l l o w i n g examples: ( a ) Reverse phase p a r t i t i o n i n g of c a r b o x y l i c a c i d s o r amine compounds i s more d e f i n i t i v e when the compounds a r e kept i n t h e i r l e s s p o l a r s t a t e ; i . e . , when the c a r b o x y l groups a r e p r o t o n a t e d ( n o n - i o n i c and t h e r e f o r e l e s s p o l a r ) by the p r e s e n c e o f a c i d . S i m i l a r e f f e c t s a r e o b t a i n e d a t h i g h e r pH w i t h the use of b a s i c media f o r c o n v e r t i n g ammonium to amine. Such c o n d i t i o n s , known as i o n s u p p r e s s i o n , a r e g e n e r a t e d i n the c a r r i e r phase. (One l i m i t a t i o n i s the i n c r e a s i n g s o l u b i l i t y of the s i l i c a s u b s t r a t e above pH 8.) ( b ) E x c e s s i v e e l e c t r o s t a t i c i n t e r a c t i o n s o f metal i o n s a r e s u p p r e s s e d by c o m p l e x i n g a n i o n s , such as the use o f gamma-hydroxybuterate f o r the c a t i o n exchange of r a r e e a r t h s ( 1 5 ) . ( c ) Unwanted c h e l a t i o n e f f e c t s take p l a c e between c e r t a i n a n t i b i o t i c s and the s t a i n l e s s s t e e l i n LC systems. T h i s e f f e c t can be swamped out by a d d i n g EDTA to the c a r r i e r . CARRIER INDUCED SELECTIVITY Experiments show t h a t s e l e c t i v i t y can be i n t r o d u c e d , o r i n d u c e d , on the b a s i s of the c a r r i e r composition alone. To i l l u s t r a t e , i o n exchange has been regarded as the main s e p a r a t i o n p r o c e s s t h a t r e s u l t s when e l e c t r o l y t e s a r e chromatographed on a s t a t i o n a r y phase h a v i n g bound c o u n t e r i o n s . It i s now r e c o g n i z e d t h a t the same i o n exchange mechanism can be a c h i e v e d i n the absence of f i x e d charge sites. T h i s i s a c h i e v e d , f o r example, by a d d i t i o n to the c a r r i e r l i q u i d of h y d r o p h o b i c i o n s under r e v e r s e phase c o n d i t i o n s . The e x p l a n a t i o n i s t h a t the h y d r o p h o b i c ends of the i o n s a r e sorbed by the l i p o p h i l i c sorbent. As a p r a c t i c a l example, the c o u n t e r i o n s needed f o r c a t i o n exchange can be o b t a i n e d from p e n t y l s u l f o n a t e s a l t added to the carrier. S i m i l a r l y , a n i o n exchange i s o b t a i n e d i n the presence of c a r r i e r w i t h o c t y l ammonium s a l t . Such e f f e c t s a r e turned o f f by s t o p p i n g the a d d i t i v e f e e d to the c a r r i e r ( 1 6 ) . The use o f the p r e c e d i n g c a r r i e r i n d u c e d i o n exchange e f f e c t i s n o t a b l y c o m p l i c a t e d because the u n d e r l y i n g r e v e r s e phase p a r t i t i o n i n g e f f e c t i s s t i l l o p e r a t i o n a l . T h i s o c c u r s w i t h i o n exchange r e s i n s as w e l l . The e x i s t e n c e i n a s i n g l e s t a t i o n a r y phase o f m u l t i p l e c h r o m a t o g r a p h i c mechanisms has the e f f e c t of expanding the b a s i s f o r s e l e c t i v i t y , but i t a l s o adds to the d i f f i c u l t y of c h r o m a t o g r a p h i c o p t i m i z a t i o n .



86


When u t i l i z i n g the i o n exchange mechanism the degree of s o l u t e i o n i z a t i o n i s c o n t r o l l e d by pH, and the p a r t i t i o n i n g e f f e c t i s r e s p o n s i v e to c a r r i e r solvent blending. These a r e independent v a r i a b l e s . I t i s i m p o r t a n t to know t h a t HPLC o f f e r s o p t i o n s to c o n t r o l s e l e c t i v i t y though the mobile phase i n o r d e r to i n d u c e i o n exchange, to a l t e r the i o n i c c h a r g e s t a t e of the sample or of the s t a t i o n a r y phase, or to remove e i t h e r or b o t h of these charge e f f e c t s by c o n t r o l l i n g the c a r r i e r f e e d c o m p o s i t i o n . An i m p o r t a n t s p i n - o f f from these advances i s they a r e l e a d i n g toward improved c h e m i c a l u n d e r s t a n d i n g of the b e h a v i o r and p r o p e r t i e s of the l i q u i d phase. An i m p r e s s i v e s e p a r a t i o n of o p t i c a l l y a c t i v e amino a c i d s has been demonstrated through the use of c h i r a l r e a g e n t s that induce the l i g a n d exchange mechanism Q17)· The r e s u l t i s i l l u s t r a t e d i n F i g u r e 4. The D,L e l u t i o n o r d e r of the compounds depends upon whether a D o r L c h i r a l r e a g e n t i s used, and the c h i r a l s e p a r a t i o n t e r m i n a t e s i f a r a c e m i c r e a g e n t i s used. While o n l y a few i l l u s t r a t i o n s of c a r r i e r phase c o n t r o l of HPLC s e l e c t i v i t y have been g i v e n , i t s h o u l d be r e c o g n i z e d the h a r n e s s i n g of l i q u i d phase c o m p o s i t i o n to c o n t r o l HPLC s e l e c t i v i t y i s p r o v i d i n g a major c o r r i d o r f o r a c h i e v i n g s e p a r a t i o n s i n an i n c r e a s i n g l y s y s t e m a t i c manner. ULTRA-RESOLUTION A s y s t e m a t i c b a s i s f o r the combining of independent s e l e c t i v i t y mechanisms can p r o v i d e a major b o o s t to the o v e r a l l s e l e c t i v i t y . The o v e r a l l e f f e c t i s m u l t i p l i c a t i v e based on the s e p a r a t i n g power, or peak c a p a c i t y , of each of the steps. E i t h e r i m p l i c i t l y or e x p l i c i t l y , t h i s i s the w i d e l y used b a s i s f o r m u l t i - s t e p s e p a r a t i o n schemes. The s e r i a l i m p l e m e n t a t i o n of m u l t i p l e o r i g i n s of s e l e c t i v i t y i s the most p r a c t i c a l approach a t present. I t has been adapted f o r columnar LC u s i n g a " h e a r t c u t t i n g " t e c h n i q u e (18) i n t r o d u c e d i n gas chroma tography. The use of a number, N, o f d i f f e r e n t chromatog r a p h i c mechanisms i n sequence i s known as column switching. Assume each mechanism has the same e f f e c t i v e peak c a p a c i t y . In i t s s i m p l e s t form, the use of such a c h r o m a t o g r a p h i c sequence expands the


FREEMAN


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Chiral eluant l-Proline (8 χ 10~ M)


3

Chiral eluant: D-Proline (8 * 10" M) 3

Ractmic eluant: Di-Proline (8 χ 1(Γ Μ) 3

J L Time (minutes)

Figure 4. Separation by HPLC of the D,L amino a c i d enantiomers using a c h i r a l reagent. The D,L order i s obtained when L - P r o l i n e i s added to the c a r r i e r i n the presence of c u p r i c s u l f a t e . The enantiomer r e s o l u t i o n i s reversed using D-Proline. No o p t i c a l r e s o l u t i o n i s obtained when the c a r r i e r chiràlty i s removed, i . e . with the use of the racemic D,L eluant (17).


88



o v e r a l l s e l e c t i v i t y of the LC system by the Nth power of t h a t o b t a i n e d from a s i n g l e s e l e c t i v i t y mechanism. To i l l u s t r a t e , the s e r i a l combination of three d i f f e r e n t column mechanisms, each h a v i n g a v a l u e of ten as i t s e f f e c t i v e peak c a p a c i t y , w i l l be expected to be a b l e to s e p a r a t e a thousand components. E x p e r i m e n t a l l y , two column s w i t c h i n g can be demonstrated u s i n g a p p a r a t u s no more e l a b o r a t e than a conventional six-port valve. Column s w i t c h i n g can c r e a t e tremendous s e p a r a t i n g power, but i t i s a requirement t h a t each one i n the sequence of s e l e c t i v i t y mechanisms not be redundant. On t h i s b a s i s i t was c a l c u l a t e d t h a t a t r i l l i o n compounds c o u l d be s e p a r a t e d w i t h a v a i l a b l e column s w i t c h i n g t e c h nology (19). The i s o l a t i o n of s p e c i f i c compounds u s i n g u l t r a - s e l e c t i v e column s w i t c h i n g HPLC i s d i f f e r e n t i n concept from the s e p a r a t i o n of a l l compounds i n a sample mixture i n a s i n g l e chromatogram. The l a t t e r i s a t y p i c a l g o a l i n c a p i l l a r y gas chromatography. Column s w i t c h i n g s h o u l d be viewed i n s t e a d as a s e r i e s of group s e p a r a t i o n s where the r e a l i z a t i o n of i n d i v i d u a l compound s e p a r a t i o n , i . e . the f i n a l s u b - c l a s s i f i c a t i o n or sub-group, would be the l a s t s t e p i n the chromatography. The l i m i t a t i o n s of the s i n g l e chromatogram approach a r e d i s c u s s e d by G i d d i n g s elsewhere i n t h i s symposium (20). I f the number of independent c o n t r i b u t i o n s to s e l e c t i v i t y from the c a r r i e r i s c o n s i d e r e d i n c o m b i n a t i o n w i t h the f o u r c i t e d s t a t i o n a r y phase c a t e g o r i e s , the peak c a p a c i t y of an HPLC system, r e f e r r i n g to the maximum number of s e p a r a b l e compounds, becomes e x c e e d i n g l y l a r g e , and i t may w e l l be u n l i m i t e d . T h i s r e a s o n i n g p o i n t s to the advantage of combining s h o r t columns i n an u l t r a h i g h s e l e c t i v i t y scheme, as opposed to the use of a s i n g l e u l t r a - h i g h r e s o l u t i o n column. Recent b r e a k t h r o u g h s over the p a s t two or t h r e e y e a r s i n p r o t e i n s e p a r a t i o n s by HPLC can be summarized on an approximate n u m e r i c a l b a s i s u s i n g the f o r e g o i n g c o n s i d e r a t i o n s based on peak c a p a c i t y . A rough e s t i m a t e of p r e s e n t l y a v a i l a b l e peak c a p a c i t y f o r p r o t e i n s can be e s t i m a t e d from the a v a i l a b l e peak c a p a c i t i e s : from aqueous s i z e e x c l u s i o n ( a p p r o x i m a t e l y 5 ) , aqueous p a r t i t i o n (100), and i o n exchange ( 2 0 0 ) . The e s t i m a t e of 200 f o r the


7. FREEMAN


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l a t t e r includes estimated s e l e c t i v i t y c o n t r i b u t i o n s due to pH, s a l t , and o r g a n i c m o d i f i e r . The cumula t i v e number ( t h e p r o d u c t o f i n d i v i d u a l peak c a p a c i t i e s ) o f HPLC s e p a r a b l e p r o t e i n s i s thus e s t i m a t e d a t over 100,000. HPLC has n o t completed i t s growth n o r i s i t near a p p r o a c h i n g i t s l i m i t . To be s u r e , simple s e p a r a t i o n problems a r e now g r e e t e d by an e x c e s s of s e p a r a t i o n c a p a b i l i t y . The s e p a r a t i o n o f complex m i x t u r e s and the s t r e a m l i n i n g o f m u l t i p l e s e l e c t i v i t y a p p l i c a t i o n s i s o f growing i n t e r e s t . Only p a r t l y d e v e l o p e d i s the need f o r s e n s i t i v e and selective detectors. T h i s a r e a i s f e l t by many t o be the weak l i n k i n HPLC where t h e r e i s s t i l l much room and need f o r advancement.

Literature Cited 1.

Prins, H.K. and Huisman, T.H.J., Nature 175, 903 (1950).

2.

Chang, S.H., Gooding, K.M. and Regnier, F . E . , J . Chromatogr., 42, 355 (1976).

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Strain, Η., "Chromatographic Adsorption Analysis," Revised. Interscience Pub., Inc. New York, 1945.

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Lederer, E. and Lederer, Μ., "Chromato graphy," Elsevier, Amsterdam, 1957.

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Snyder, L.R. and Kirkland. J.J., "Introduction to Liquid Chromatography, Wiley-Interscience, New York, 1979.

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Martin, A.J.P. and Synge, R.L.M., Biochem. J. 35, 1358 (1941).

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Unger, K.K., "Porous Silica," Elsevier, Amster dam, 1979, Chap. 3 (249 references).

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Elchuk, S. and Cassidy, R.M., Anal. Chem. 51, 1434 (1979).

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Freeman, D.H. and Poinescu, I . C . , Anal. Chem. 49, 1183-1188 (1977).

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Snyder, L.R., "Gradient Elution" in "High Performance Liquid Chromatography," C. Horvath, Ed. Academic Press, New York, 1980.

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Blasius, Ε., Augustin, H., Hausler, H., Janzen, Κ.-P. and Wagner, Η., in Heftman, E., "Chromatography, a Handbook of Chromatographic and Electrophoretic Methods," 3rd Ed., Van Nostrand, Reinhold, New York, 1975, pp. 847-853 and 860.

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Skelly, N.E., Anal. Chem. 54, 712 (1982).

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Hare, P.E. and Gil-Av, Ε . , Science 204, 12261228 (1979).

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Deans, D.R., Chromatographia 1, 12-22 (1968).

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Freeman, D.H., Anal. Chem. 53, 2-5 (1981).

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Giddings, J . C . , Davis, J.M., Schure, M.R.; Chapter 2 in this book. RECEIVED March 5, 1984


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