Ultrahigh Resolution Chromatography - American Chemical Society

1 Overview: Multiple Pathways to Ultrahigh Resolution Chromatography SATINDER AHUJA

Downloaded by 80.82.77.83 on December 30, 2017 | http://pubs.acs.org Publication Date: April 26, 1984 | doi: 10.1021/bk-1984-0250.ch001

Development Department, Pharmaceuticals Division, CIBA-GEIGY Corporation, Suffern, NY 10901

Today virtually every chromatographer understands what is implied by the term high resolution chromatography even though there is no hard and fast definition for it. Generally, high resolution gas chromatography entails gas-liquid chromatography with capillary columns and high resolution liquid chromatography involves the use of bonded phase packed columns with less than 30μ particle size. Webster's dictionary defines ultra as beyond what is ordinary. Hence we expect to cover in this book beyond what is ordinarily considered high resolution chromatography viz. ultrahigh resolution chromatography. Even though there are no fixed rules to determine whether a given technique provides high resolution or not, the limits of resolution of any technique are well known to the practitioners. Hence, there is a constant thrust to improve resolution, i.e. separation between two or more components. Therefore, what constitutes high resolution is constantly changing. Since selec tivity offered by each technique can be truly unique it would be desirable to arrive at some meaningful definitions in terms of Ν, α or k' values for high resolution in each technique. After this is accomplished, it would be easier to determine whether a given improvement leads to ultrahigh resolution. Future symposia w i l l address t h i s problem. In the i n t e r i m i t i s p a t e n t l y c l e a r t h a t , i n d i v i d u a l l y , none of the terms (Ν, α and k ) i n the r e s o l u t i o n equation i s s u f f i c i e n t to describe r e s o l u t i o n (jL) : f

Resolution = R

1

g

= 1 ^ 4T

[^] [^r]

Where: Ν = number of t h e o r e t i c a l p l a t e s α = separation f a c t o r k peak c a p a c i t y , =

k

1

I t i s erroneous to d e f i n e r e s o l u t i o n only i n terms of Ν or as i s f r e q u e n t l y done by many chromatographers. An approach 0097-6156/ 84/0250-0001 $06.00/0 © 1984 American Chemical Society

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


2

ULTRAHIGH RESOLUTION CHROMATOGRAPHY

that seems to have some merit i s d e f i n i n g r e s o l u t i o n i n terms of the number of components that can be separated i n a given u n i t of time. The time s c a l e i s a b e t t e r measure of r e s o l u t i o n than the commonly used t h e o r e t i c a l p l a t e s s c a l e . Of course, t h e o r e t i c a l plates/second, attempts to address t h i s question but misses the mark s i n c e the number of t h e o r e t i c a l plates/second does not n e c e s s a r i l y assure s e p a r a t i o n of a v a r i e t y of components. The frequency of component overlap i s s i g n i f i c a n t i n chromatograms f o r which r e l a t i v e component spacing i s governed by random f a c t o r s . Giddings ej: a l (2) c a l c u l a t e only 36% recovery f o r 100 components from a system with a peak c a p a c i t y of 200. A 90% recovery would r e q u i r e a peak c a p a c i t y of 1900 or 20 m i l l i o n t h e o r e t i c a l p l a t e s . In those circumstances where a s i n g l e component can be i s o l a t e d as a single-component peak with an 80% p r o b a b i l i t y , two components can be simultaneously i s o l a t e d with a p r o b a b i l i t y of only (0.80) or 0.64. Thus the p r o b a b i l i t y of i s o l a t i n g ten components simultaneously i s q u i t e small ( 0 . 8 0 ) = 0.107). Consequently, with systems of enormous r e s o l v i n g power, i t i s d i f f i c u l t to r e s o l v e a small number of components s i m u l t a neously. This can have s i g n i f i c a n t i m p l i c a t i o n s with respect to the way we handle data and design o p t i m i z a t i o n procedures. There are m u l t i p l e pathways to achieve u l t r a h i g h r e s o l u t i o n because a v a r i e t y of modes of s e p a r a t i o n are a v a i l a b l e . I t i s important to choose the best mode of s e p a r a t i o n f o r a given problem and optimize i t r a t h e r than optimize a f a v o r i t e mode of s e p a r a t i o n . Some of the modes of s e p a r a t i o n used today are: high performance t h i n - l a y e r chromatography (HPTLC), c a p i l l a r y GLC, s e v e r a l modes of high performance l i q u i d chromatography (HPLC), s u p e r c r i t i c a l f l u i d chromatography, f i e l d flow f r a c t i o n a t i o n , e l e c t r o p h o r e s i s , electroosmosis and i s o e l e c t r i c f o c u s i n g . Of these, c a p i l l a r y GLC and HPLC are p r o v i d i n g the main t h r u s t i n u l t r a h i g h r e s o l u t i o n chromatography. S u p e r c r i t i c a l f l u i d chromatography i s opening some new avenues and, h o p e f u l l y , w i l l provide r e s o l u t i o n that i s not p o s s i b l e now with GLC or HPLC. Hence, mainly these subjects have been covered i n t h i s book. Whereas, some improvements i n r e s o l u t i o n have been obtained with HPTLC, f u r t h e r improvements are p o s s i b l e i n the area of d e t e c t i o n and q u a n t i f i c a t i o n . U l t r a h i g h r e s o l u t i o n with the other modes of s e p a r a t i o n s t i l l remains w i t h i n the domain of a handful of experts. Some information i s included on these under M i s c e l l a neous techniques, however, a d e t a i l e d d i s c u s s i o n of these techniques i s d e f e r r e d to a f u t u r e symposium. 2

10

High Pressure L i q u i d Chromatography A great deal of improvement i s being made i n general equipment to minimize band broadening. I t i s important to e l i m i n a t e or minimize dead volumes. S p e c i a l i n j e c t o r s with minimum dead volume are being experimented with to optimize s e p a r a t i o n s . S i m i l a r l y , detectors with small c e l l volumes are being developed


1. AHUJA

3

Overview

to minimize post-column band broadening. As shown by the f o l l o w i n g equation, band-broadening can be minimized by g i v i n g consider a t i o n to each component:


W

T

=

W

+

W

+

W

+

W

i f d c i n j e c t o r f i t t i n g s detector column Major improvements are needed i n conventional columns (4.6 mm i . d . ) used f o r HPLC. Even though columns are a v a i l a b l e that can provide more than 10,000 plates/column or more than 100,000 plates/meter, l a r g e column to column v a r i a t i o n s can be found between manufacturers and from one batch to the next (3). Further improvements r e q u i r e b e t t e r understanding of column v a r i a b i l i t i e s and the s e p a r a t i o n processes o c c u r r i n g i n the column. An approach f o r improvement i n t h i s area has provided a new set of columns of smaller diameter, i . e . microbore columns. These columns can provide u l t r a h i g h r e s o l u t i o n when s e v e r a l columns are combined i n a row. Due to the f a c t that a l i n e a r r e l a t i o n s h i p can be obtained between e f f i c i e n c y and column length, columns of a m i l l i o n t h e o r e t i c a l p l a t e s or more can be packed (4)· K r e j c i e t a l (5) have described an open tubular column that i s capable of p r o v i d i n g up to 1,250,000 t h e o r e t i c a l p l a t e s with an e f f e c t i v e n e s s of 50 t h e o r e t i c a l p l a t e s / s e c . The column (21 m χ 60 ym I.D.) used l , 2 , 3 - t r i s ( 2 - c y a n o e t h o x y ) propane as s t a t i o n a r y phase and hexane saturated with s t a t i o n a r y phase as mobile phase, with a l i n e a r v e l o c i t y of 0.18 cm/sec. The above-mentioned p l a t e count i s was obtained with a n a l y s i s time of 6 hours 54 minutes. A l t e r n a t i v e l y , a s i n g l e column (330 ym diameter) packed with 3 ym C^g reverse phase packing can y i e l d up to 110,000 p l a t e s f o r 1 meter length. Separation of p r i o r i t y p o l l u t a n t mixture of 15 PAH components was demonstrated by Yang on one such column (6). Ion-pair chromatography has provided numerous a p p l i c a t i o n s . U l t r a h i g h r e s o l u t i o n can be obtained when a mixed mode i s used i . e . one of the components i s separated on the b a s i s of i o n - p a i r formation and the other i s not (7). Separation of Isophenindamine and phenindamine deserves s p e c i a l mention s i n c e an argentated HPLC mobile phase can b r i n g about t h i s d i f f i c u l t separation based on the p o s i t i o n of double bond ( 8 ) .

Phenindamine

Isophenindamine



4

Several c h o i e s t r i e l i q u i d c r y s t a l s have been evaluated as s t a t i o n a r y phases i n HPLC (9). C h o l e s t e r i c e s t e r s , f o r the most part, are l i q u i d c r y s t a l s i n the range 20 to 1 0 0 ° C C h o i e s t e r y l 2-ethylhexanoate was coated on C o r a s i l I I , and a mixture of these androgens was chromatographed between 0 and 8 0 ° C The capacity f a c t o r increased 559% and 489% f o r androstenedione and Δ* androstdienedione while the change f o r testosterone was -100%. Using a c h i r a l r e c o g n i t i o n r a t i o n a l e , P i r k l e , et_ a l (10) designed a c h i r a l f l u o r o a l c o h o l i c bonded s t a t i o n a r y phase which separates the enantiomers of s u l f o x i d e s , lactones, and d e r i v a t i v e s of a l c o h o l s , amines, amino a c i d s , hydroxy a c i d s , and mercaptans. A c r o s s - l i n k e d polystyrene r e s i n with f i x e d ligands of the type (R)-N , N -dibenzyl-l,2-propanediamine i n the form of a copper (II) complex d i s p l a y s high e n a n t i o s e l e c t i v i t y i n l i g a n d exchange chromatography of amino acids (11). A microbore column (100 mm χ 1 mm i.d.) packed w i t h p a r t i c l e s of dp = 5-10 ym generated up to 3500 t h e o r e t i c a l p l a t e s , enabling a complete r e s o l u t i o n of a mixture of three racemic amino a c i d s i n t o s i x components under i s o c r a t i c c o n d i t i o n s . The combination of various chromatographic mechanisms (e.g. by the use of column switching) can expand the 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 that obtained with a s i n g l e s e l e c t i v i t y mechanism. Freeman (12) c a l c u l a t e s a t r i l l i o n compounds could be separated with the a v a i l a b l e column switching technology. Column switching can be viewed as a s e r i e s of group separations where the i n d i v i d u a l compound separation i s the l a s t step i . e . f i n a l s u b - c l a s s i f i c a t i o n i n t o the sub-group. Detectors such as l a s e r fluorometry, FT-IR, mass spectro meter and flame-based detectors can be used to obtain d e s i r e d s e l e c t i v i t y and r e s o l u t i o n . A phosphorus-selective detector f o r HPLC can provide u l t r a h i g h r e s o l u t i o n . In the phosphorus-selec t i v e chromatogram, the analyte peak i s w e l l r e s o l v e d from neighboring peaks and the s i g n a l - t o - n o i s e r a t i o i s a l s o much higher than f o r the RI detector (13).


1

f

C a p i l l a r y Gas

f

Chromatography

High r e s o l u t i o n chromatography evolved with the advent of c a p i l l a r y gas chromatography; however, t h i s f i e l d remained dormant f o r a long p e r i o d of time because of d i f f i c u l t i e s involved i n the preparation of s u i t a b l e c a p i l l a r y columns. Most of these problems have now been resolved p r o v i d i n g an impetus f o r u l t r a high r e s o l u t i o n chromatography. One method of o b t a i n i n g a very large number of t h e o r e t i c a l p l a t e s by the e x p l o i t a t i o n of increased column length, avoiding the l i m i t a t i o n s imposed by the increased pressure drop, i s that of r e c y c l e chromatography. Jennings e£ a l (14) were able to generate i n excess of 2,000,000 t h e o r e t i c a l p l a t e s . I t can be best employed where those components that can be separated by



1.

AHUJA

5

Overview

conventional high r e s o l u t i o n GC are r e t a i n e d i n that system, and an unresolved f r a c t i o n i s d i r e c t e d i n t o the r e c y c l e u n i t to achieve s e p a r a t i o n . A route to " o p t i m i z a t i o n " i s based on maximizing the r e l a t i v e r e t e n t i o n s of a l l the s o l u t e s i n that sample (15, 16). This concept was employed to p r e d i c t what lengths of d i s s i m i l a r c a p i l l a r y columns should be coupled to achieve a s p e c i f i c binary l i q u i d phase mixture column (17). Takeoka et a l (18) suggested a method f o r the o p t i m i z a t i o n of the s e p a r a t i o n of a model system, which could not be r e s o l v e d on e i t h e r of two 30 m columns coated with d i s s i m i l a r l i q u i d phases. One column r e q u i r e d 25, and the other 57 minutes f o r the a n a l y s i s ; n e i t h e r d e l i v e r e d s e p a r a t i o n of a l l components. Coupling c a l c u l a t e d short lengths of the two columns permitted the complete s e p a r a t i o n of the mixture i n l e s s than 3 minutes. Open-tubular columns u t i l i z e d i n gas chromatography have, with few exceptions, been of 0.2 mm diameter or l a r g e r and have t h e r e f o r e provided at most only a few thousand e f f e c t i v e t h e o r e t i c a l p l a t e s per meter l e n g t h . Nevertheless, the t h e o r e t i c a l background f o r design of column e f f i c i e n c i e s of 1 0 or more e f f e c t i v e p l a t e s per meter was presented as e a r l y as 1958. Laub et a l (19) have explored the p r a c t i c a l l i m i t s of the e a r l y t h e o r e t i c a l work and i n p a r t i c u l a r , the f a b r i c a t i o n and pro p e r t i e s of c a p i l l a r y columns of inner diameter ranging from 0.3 to 0.035 mm. The l a t t e r e x h i b i t s on the order of 2 χ 10 Ν /m f o r k of 17. Much higher e f f i c i e n c i e s could be r e a l i z e d wStn the mass spectrometric d e t e c t o r . A comparison of e f f i c i e n c y of HPLC and GC columns was made by Widmer et_ a l (20). For example, 3 ym packed 10 cm column gives 15,000 p l a t e s or 150,000 plates/meter. This compares with 11,880 p l a t e s obtained with 0.1 mm i . d . w a l l coated c a p i l l a r y GC columns. These data suggest that greater e f f i c i e n c y i n terms of number of plates/meter i s p o s s i b l e with HPLC. 5

h

ff

f

Super C r i t i c a l F l u i d Chromatography

(SFC)

Since high s o l u t e d i f f u s i v i t y , lower v i s c o s i t y and e x c e l l e n t s o l v a t i n g p r o p e r t i e s can be obtained with s u p e r c r i t i c a l f l u i d s , higher chromatographic e f f i c i e n c i e s and f a s t e r a n a l y s i s time than l i q u i d chromatography can be obtained with SFC (21). I t i s a l s o p o s s i b l e to separate n o n - v o l a t i l e high molecular weight compounds at r e l a t i v e l y low temperatures. While extremely l a r g e numbers of t h e o r e t i c a l p l a t e s are p o s s i b l e with l a r g e r diameter columns (22, 23), c a l c u l a t i o n s from chromatographic theory of the i n t e r n a l diameters and column lengths necessary to achieve r e l a t i v e l y high e f f i c i e n c i e s i n reasonable a n a l y s i s times i n d i c a t e that column diameters of 50 to 100 ym i . d . are necessary f o r h i g h - r e s o l u t i o n SFC (23). For example, more than 10 e f f e c t i v e t h e o r e t i c a l p l a t e s are p o s s i b l e i n l e s s than two hours on 30-m long columns of 50 ym i . d . 5


6



UV and fluorescence detectors can be used f o r SFC. Carbon dioxide and n i t r o u s oxide have made p o s s i b l e the use of convent i o n a l GC flame detectors (24). Based on studies of a l i p h a t i c f r a c t i o n of a solvent r e f i n e d c o a l product with s u p e r c r i t i c a l CO2 at 40°C and conventional FID d e t e c t i o n , i t was concluded (21) that the e f f i c i e n c i e s of SFC are approaching those of GC (plate heights of 0.30 mm have been obtained i n c a p i l l a r y SFC, which compare favorably with the 0.20-0.25 mm p l a t e heights normally obtained i n c a p i l l a r y GC). Optimization scheme s i m i l a r to that used f o r HPLC can be used f o r choosing a m o d i f i e r f o r carbon dioxide based SFC (25). Miscellaneous A multichannel detector f o r i n - s i t u a n a l y s i s of f l u o r e s c e n t m a t e r i a l s on TLC p l a t e s was i n v e s t i g a t e d (26). The o p t i c a l system was designed to obtain a fluorescence spectrum from each p o s i t i o n along the e l u t i o n a x i s of a one-dimensional p l a t e without the mechanical scanning. By use of tetraphenylporphine and octaethylporphine i t can be shown how overlapping spots can be resolved i n t o t h e i r components. Isomeric s o l u t e s that have c l o s e l y r e l a t e d s t r u c t u r e w i t h s l i g h t d i f f e r e n c e i n v.p. and degree of i n t e r a c t i o n with conv e n t i o n a l s t a t i o n a r y phases provide d i f f i c u l t separation problems. L i q u i d c r y s t a l , N,N -bis(p-methoxybenzylidene)-a,a -bi-pt o l u i d i n e , was used f o r separation of s e v e r a l PAH, i n general, and benzo (a) pyrane and benzo (e) pyrene i n p a r t i c u l a r (27). This s t a t i o n a r y phase i s mostly u s e f u l f o r isomers that are r i g i d to s e m i r i g i d and d i f f e r s l i g h t l y i n t h e i r length-tobreadth r a t i o . The r e l a t e d e l e c t r o k i n e t i c e f f e c t s of e l e c t r o p h o r e s i s and electroosmosis may be used to achieve u l t r a h i g h r e s o l u t i o n of charged substances (28). The e l e c t r o p h o r e s i s i s c a r r i e d out i n 75 ym i . d . g l a s s c a p i l l a r i e s . Reversed phase chromatography i s performed by electroosmotic pumping of an a c e t o n i t r i l e mobile phase. The values of HETP f o r 9-methylanthracene and perylene are 19 ym and 25 ym r e s p e c t i v e l y . The low reduced p l a t e height values suggest column packing i r r e g u l a r i t i e s are l e s s important when electroosmotic flow i s used i n s t e a d of flow generated by pressure. Two dimensional e l e c t r o p h o r e s i s provide u l t r a h i g h r e s o l u t i o n of v a r i o u s p r o t e i n s (29). For example, by combining i s o e l e c t r i c focusing (IEF) with perpendicular e l e c t r o p h o r e s i s i n the presence of the detergent sodium dodecyl s u l f a t e (SDS), the r e s u l t i n g r e s o l u t i o n of 10,000 p r o t e i n s could be t h e o r e t i c a l l y obtained. Samples of human c e l l s showed 100-2000 p r o t e i n spots. f

f



1.

AHUJA

Overview

7

Literature Cited 1. S. Ahuja, Recent Developments in High Performance Liquid Chromatography, Metrochem '80, October 3, 1980 2. J. C. Giddings, J. M. Davis and M. R. Schure, This Symposium 3. S. Ahuja, Presented at New York Chromatography Society Meeting, June 10, 1981 4. R. P. W. Scott, J. Chromatog. Sci., 18, 49 (1980) 5. M. Krejci, K. Tesarik and J. Pajurek, J. Chromatog., 191, 17 (1980) 6. F. J. Yang, This Symposium 7. S. Ahuja, P. Liu and J. Smith, Personal Communication, August 7, 1979 8. R. J. Tscherne and H. Umagat, J. Pharm. Sci., 69, 342 (1980) 9. P. Taylor and P. L. Sherman, J. Liq. Chromatog., 3, 21 (1980) 10. W. H. Pirkle, D. W. House and J. M. Finn, J. Chromatog., 192, 143 (1980) 11. V. A. Davankov and A. A. Kurganov, Chromatographia, 13, 339 (1980) 12. D. Freeman, This Symposium 13. T. L. Chester, Anal. Chem., 52, 1621 (1980) 14. W. Jennings, This Symposium 15. H. J. Maier and O. C. Karpathy, J. Chromatog., 8, 308 (1962) 16. R. J. Laub and J. H. Purnell, Anal. Chem., 48, 799 (1976) 17. D. F. Ingraham, C. F. Shoemaker and W. J. Jennings, J. Chromatog., 239, 39 (1982) 18. G. Takeoka, H. M. Richard, M. Mehran, and W. Jennings, Personal Commuication 19. B. J . Lambert, R. J. Laub, W. L. Roberts and C. A. Smith, This Symposium 20. H. M. Widmer and K. Grolimund, This Symposium 21. W. P. Jackson, Β. E. Richter, J. C. Fjeldsted, R. C. Kong and M. L. Lee, This Symposium 22. S. R. Springston and M. Novotny, Chromatographia, 14, 679 (1981) 23. P. A. Peaden and M. L. Lee, J. Chromatog., 259, 1 (1983) 24. J . C. Fjeldsted, R. C. Kong and M. L. Lee, Personal Communi cation 25. L. G. Randall, See This Book 26. M. L. Gianelli, J. B. Callis, Ν. H. Anderson and G. D. Christian, Anal. Chem, 53, 1357 (1981) 27. W. L. Zielinski, Jr., Indus. Res./Develop., p. 178, Feb. 1980 28. J. W. Jorgenson and K. D. Lukacs, J. Chromatog., 218, 209 (1981) 29.

N. L. Anderson, Trends Anal. Chem., 1, 131 (1982)

RECEIVED

November 10, 1983


Ultrahigh Resolution Chromatography - American Chemical Society

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