Purification of Fermentation Products - American Chemical Society

dampener (Fluid Metering Company) or with a Beckman 112 HPLC pump equipped with a ... (2.5 X 25 cm) equipped with a steel plunger. When solubility...
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Preparative Reversed Phase High Performance Liquid Chromatography A Recovery and Purification Process for Nonextractable Polar Antibiotics R. D. SITRIN, G. CHAN, P. DEPHILLIPS, J. DINGERDISSEN, J. VALENTA, and K. SNADER Smith Kline & French Laboratories, Philadelphia, PA 19101 Reversed phase high performance liquid chromatography (RPHPLC) has found wide use as an analytical tool in monitoring antibiotic fermentation production, isolation, and purification schemes. The recent introduction of suitable instrumentation and affordable preparative reversed phase packing materials allows the traditional advantages of RPHPLC - speed and resolution - to be applied to preparative work. Classically, isolation and purification processes for polar, charged, non-solvent extractable antibiotics such as peptides or glycopeptides have required multistep, medium to low resolution procedures such as charcoal, cellulose, ion-exchange or size exclusion techniques often with low recovery. However, by the nature of its mechanism, reversed phase HPLC is particularly suited to solve these separation problems because undesirable very polar highly colored contaminants elute at the solvent front ahead of the desired material. Thus, it is possible to introduce such a step very early in a purification scheme on a relatively crude isolate. We w i l l demonstrate examples of such one step purifications on very crude fermentation isolates to yield highly purified homogeneous products from milligram to gram and potentially kilogram scales. One problem frequently encountered in the recovery and purification of fermentation products such as antibiotics and peptides is the necessity for multistep procedures to obtain pure materials. For substances which cannot be extracted into ethyl acetate or methylene chloride and which typically cannot be chromatographed on s i l i c a , purification often requires repetitive chromatographic 0097-6156/ 85/ 0271 -0071 $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.

PURIFICATION OF FERMENTATION PRODUCTS

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s t e p s on a d s o r p t i o n media such as polyamide, Sephadex, B i o g e l , c h a r c o a l and i o n exchange o r XAD r e s i n s . Such m u l t i s t e p schemes o f t e n y i e l d p r o d u c t s i n low r e c o v e r y and, b e i n g l i m i t e d i n c a p a c i t y , are d i f f i c u l t t o s c a l e - u p t o p r o c e s s l e v e l . However, r e v e r s e d phase h i g h performance l i q u i d chromatography (RPHPLC), w i d e l y used as an a n a l y t i c a l procedure (1,2), has g r e a t u t i l i t y as w e l l i n the p r e p a r a t i v e mode f o r i s o l a t i o n and p u r i f i c a t i o n , o f t e n i n one s t e p . A r e c e n t Chemical A b s t r a c t s s e a r c h o f t h e l i t e r a t u r e (19801983) i n d i c a t e d t h a t , out o f 7,000 p o s t i n g s f o r HPLC papers, o n l y 100 d i s c u s s e d p r e p a r a t i v e work. Furthermore, the b u l k o f t h e p r e p a r a t i v e papers were e i t h e r t h e o r e t i c a l d i s c u s s i o n s ( 3 - 8 ) , d e s c r i p t i o n s o f l a r g e s c a l e (multigram) s e p a r a t i o n s on s i l i c a o r p r e p a r a t i v e r e v e r s e d phase work on a n a l y t i c a l columns and i n s t r u ments (100 mg s c a l e ) ( 9 - 1 4 ) . The most r e c e n t development i n t h i s a r e a i s d i s p l a c e m e n t chromatography (15-18), b u t so f a r t h i s has been a p p l i e d o n l y i n l i m i t e d cases on a n a l y t i c a l s c a l e i n s t r u m e n t s . A l t h o u g h s e v e r a l e x c e l l e n t r e v i e w s on p r e p a r a t i v e HPLC have been p u b l i s h e d (19-21), few papers d e s c r i b i n g gram o r l a r g e r p r e p a r a t i v e r e v e r s e d phase chromatography were e v i d e n t (22-23). Several s u c c e s s f u l examples o f such s e p a r a t i o n s are d e s c r i b e d h e r e , r a n g ing from 100 mg t o gram s c a l e . Furthermore, these s e p a r a t i o n s a l s o demonstrate e f f i c i e n t one-step procedures f o r p r e p a r i n g pure p r o d u c t s s t a r t i n g from crude f e r m e n t a t i o n i s o l a t e s . The r e c e n t i n t r o d u c t i o n o f i n d u s t r i a l s c a l e equipment and p a c k i n g s from Waters, Whatman and E l f - A q u i t a i n e a l l o w s these procedures t o be s c a l e d up t o p r o c e s s l e v e l . Background RPHPLC i s performed on columns packed w i t h s i l i c a g e l t o which a hydrocarbon, u s u a l l y w i t h 8 o r 18 c a r b o n s , has been c h e m i c a l l y a t t a c h e d (I). P a r t i t i o n i n g o f a compound o c c u r s between t h e hydrophobic s t a t i o n a r y phase and a p o l a r aqueous m o b i l e phase. S o l v e n t systems u s u a l l y c o n s i s t o f m i x t u r e s o f methanol o r a c e t o n i t r i l e w i t h water o r b u f f e r . E l u t i n g strength increases w i t h decreases i n p o l a r i t y and, i n g e n e r a l , compounds o f s i m i l a r s t r u c t u r e e l u t e i n o r d e r o f d e c r e a s i n g p o l a r i t y as c a n be seen f o r a s t a n d a r d m i x t u r e ( F i g u r e l a ) o f m e t h y l , e t h y l , p r o p y l and b u t y l parabens and the dye t a r t r a z i n e ( 2 4 ) . The f o u r parabens have r e l a t i v e l y l a r g e a l p h a - v a l u e s , where

k« = ( t - t ) / t Q

0

where t i s t h e r e t e n t i o n time o f a peak o f i n t e r e s t and t is the r e t e n t i o n time o f an u n r e t a i n e d s u b s t a n c e . Such s e l e c t i v i t y f o r homologs i s a c h a r a c t e r i s t i c o f RPHPLC and has g r e a t p o t e n t i a l i n the n a t u r a l p r o d u c t s a r e a where m i x t u r e s o f homologs a r e f r e q u e n t l y encountered. The h i g h l y charged t a r t r a z i n e m o l e c u l e , which would s t i c k i r r e v e r s i b l y t o a s i l i c a column, e l u t e s a t t h e f r o n t . T h i s demonstrates one o f the b e n e f i c i a l a s p e c t s o f RPHPLC, Q

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

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namely, t h a t v e r y p o l a r contaminants a r e n o t r e t a i n e d and t h e r e f o r e do not i n t e r f e r e w i t h t h e chromatographic p r o c e s s . The f a c t t h a t aqueous s o l v e n t s c a n be used i s advantageous i n p r o c e s s i n g f e r m e n t a t i o n p r o d u c t s where p o l a r charged w a t e r - s o l u b l e m a t e r i a l s such as p e p t i d e s a r e f r e q u e n t l y e n c o u n t e r e d . Other advantages o f RPHPLC a l r e a d y w e l l known i n a n a l y t i c a l a p p l i c a t i o n s a l s o c a r r y over t o p r e p a r a t i v e work. They i n c l u d e : f a s t e r e q u i l i b r a t i o n times on s o l v e n t changes, i n t r i n s i c a l l y h i g h e r r e s o l u t i o n and c a p a c i t i e s than found on s i l i c a g e l a l o n e , and the a b i l i t y t o h a n d l e a wide d i v e r s i t y o f compounds through the use o f c o n t i n u o u s o r step g r a d i e n t s . As w i l l be d e s c r i b e d l a t e r , t h e use o f such g r a d i e n t s a l l o w s f o r l i t e r a l l y u n l i m i t e d i n j e c t i o n volumes thus a v o i d i n g time-consuming l y o p h i l i z a t i o n o r concentration steps. S m a l l e r s c a l e p r e p a r a t i v e RPHPLC. P r e p a r a t i v e chromatography c a n of course be r u n on many s c a l e s . When c o n f r o n t e d w i t h a f e r m e n t a t i o n b r o t h c o n t a i n i n g an unknown a n t i b i o t i c , a f i r s t o b j e c t i v e i s to i s o l a t e enough m a t e r i a l t o determine n o v e l t y . W i t h today's i n s t r u m e n t a t i o n [mass spectrometry (MS), i n f r a r e d , u l t r a v i o l e t (UV), and n u c l e a r magnetic resonance (NMR) s p e c t r o s c o p y ] , t h i s c a n o f t e n be done w i t h 1-2 mg o f m a t e r i a l , r e a d i l y prepared on a n a l y t i c a l o r s e m i - p r e p a r a t i v e columns (4.6 o r 10 X 250 mm) w i t h 10 m i c r o n p a c k i n g . However, f o r more d e t a i l e d s p e c t r o s c o p i c and b i o l o g i c a l s t u d i e s , l a r g e r amounts ( s e v e r a l hundred mg) a r e f r e q u e n t l y r e q u i r e d . F o r t h i s s c a l e we employ g l a s s columns 2.5 X 50 cm (100 P S I l i m i t ) w i t h l a r g e r p a r t i c l e s u p p o r t s . Because o f p r e s s u r e l i m i t a t i o n s , f l o w r a t e s a r e o f t e n l i m i t e d t o 15 ml/min. R e c e n t l y , s e v e r a l p a c k i n g s (Merck L i C h r o p r e p RP-18 25-40 m i c r o n , Whatman Prep 40 ODS-3 37-60 m i c r o n o r Baker Bonded PhaseO c t a d e c y l (C13) 40 m i c r o n ) have become a v a i l a b l e f o r such columns. However, when gram q u a n t i t i e s o f p r o d u c t s a r e r e q u i r e d , these g l a s s columns a r e i n s u f f i c i e n t and l a r g e r i n s t r u m e n t s a r e n e c e s s a r y as d e s c r i b e d below. M a t e r i a l s and Methods A n a l y t i c a l HPLC was r u n on a Beckman Model 345 Chromatograph equipped w i t h a Beckman 165 d e t e c t o r operated a t 220 o r 254 nm, a s indicated. I n t e r m e d i a t e s c a l e chromatography was r u n on Whatman Prep 40 ODS-3 (37-60 m i c r o n ) d r y packed, o r Merck L i C h r o p r e p RP-18 (25-40 m i c r o n ) s l u r r y packed (60% methanol-water) i n t o g l a s s columns (2.54 X 50 cm, Altex-Beckman). Columns were equipped w i t h a p r e s s u r e gauge (Ace G l a s s ) and p r e s s u r e r e l e a s e v a l v e s (Nupro) s e t a t 90 PSI. E l u t i o n was performed w i t h an FMI m e t e r i n g pump (maximum f l o w 15 ml/min a t 100 PSI) equipped w i t h an FMI p u l s e dampener ( F l u i d M e t e r i n g Company) o r w i t h a Beckman 112 HPLC pump equipped w i t h a p r e p a r a t i v e head (maximum f l o w 30 ml/min a t 2000 PSI). E f f l u e n t s were m o n i t o r e d w i t h an ISCO UA-5 (Instrument S p e c i a l t i e s Company), o r a Gow Mac Model 80-850 v a r i a b l e wavelength d e t e c t o r (Gow Mac I n s t r u m e n t s ) a t 210 o r 254 nm. Samples were i n t r o d u c e d v i a a Rheodyne T e f l o n V a l v e ( R a i n i n I n s t r u m e n t s ) , o r by p r e a d s o r p t i o n o f a s o l u t i o n o f a n t i b i o t i c onto

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

PURIFICATION OF FERMENTATION PRODUCTS

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r e v e r s e d phase p a c k i n g o r c e l i t e by c o n c e n t r a t i o n on a r o t a r y e v a p o r a t o r and p a c k i n g onto a s m a l l A l t e x Beckman g l a s s pre-column (2.5 X 25 cm) equipped w i t h a s t e e l p l u n g e r . When s o l u b i l i t y p e r m i t t e d , l a r g e r volumes were i n j e c t e d through t h e pump i n a s o l v e n t c o n t a i n i n g lower amounts o f o r g a n i c m o d i f i e r than r e q u i r e d for elution. L a r g e r s c a l e chromatography was r u n on a J.Y. Chromatospac Prep 100 ( J . Y . I n s t r u m e n t s ) o r a Waters Prep-500A equipped w i t h a Whatman Magnum 40 (4.8 X 50 cm) column d r y packed w i t h Whatman P a r t i s i l Prep 40 ODS-3 (37-60 um) and mounted i n p l a c e o f t h e r a d i a l compression chambers. I n j e c t i o n s were c a r r i e d out by pumping on d i l u t e s o l u t i o n s o f samples i n a s o l v e n t o f lower e l u t i n g power. D e t e c t i o n was by the Gow Mac model 80-850 U.V. d e t e c t o r a t 210 o r 254 nm. B u r d i c k and J a c k s o n a c e t o n i t r i l e (UV grade) was used f o r a n a l y t i c a l and g l a s s column work. Baker a c e t o n i t r i l e (HPLC) was used f o r l a r g e r s c a l e work. Water f o r p r e p a r a t i v e work was d e i o n i z e d and g l a s s d i s t i l l e d . P l a t e counts were determined by the formula Ν = 5.54 ( t / w

e 5

)

2

where t i s r e t e n t i o n time and w 5 i s w i d t h a t h a l f h e i g h t . V a l u e s f o r p l a t e counts a r e g i v e n as p l a t e s p e r column and n o t n o r m a l i z e d t o p l a t e s p e r meter. The p o l y o x i n m i x t u r e was a water e x t r a c t o f the a g r i c u l t u r a l product o b t a i n e d from Kaken Kagaku Company. The o t h e r a n t i b i o t i c s were o r i g i n a l l y unknown i s o l a t e s from t h e SK&F f e r m e n t a t i o n s c r e e n . #

Results Low p r e s s u r e g l a s s column s e p a r a t i o n s . P l a t e counts a r e n o r m a l l y used i n a n a l y t i c a l columns as a measure o f e f f i c i e n c y and s e p a r a t i o n p o t e n t i a l . We have found t h e d e t e r m i n a t i o n o f p l a t e c o u n t s t o be d e s i r a b l e f o r t h e p r e p a r a t i v e columns i n o r d e r t o m o n i t o r p a c k i n g procedures and changes i n column performance w i t h time. Using t h e parabens (Figure l b ) a t a n a l y t i c a l l o a d i n g l e v e l s on a g l a s s column packed w i t h 37-60 m i c r o n p a r t i c l e s , 500 t o 800 p l a t e s c a n be o b t a i n e d . T h i s compares w i t h s e v e r a l thousand p l a t e s f o r a n a l y t i c a l columns w i t h s m a l l e r p a r t i c l e s (5 m i c r o n s ) , as seen i n Figure l a . These numbers should be used o n l y f o r c o m p a r a t i v e purposes, s i n c e p l a t e counts tend t o drop on i n c r e a s e d l o a d i n g , (4-6,19) and t h e parabens tend t o g i v e h i g h e r p l a t e counts than complex a n t i b i o t i c s . Although seemingly low, 500 p l a t e s i s s u f f i c i e n t t o o b t a i n adequate r e s o l u t i o n when a l p h a v a l u e s a r e 1.5 t o 2.0. Very t i g h t s e p a r a t i o n s ( a l p h a v a l u e s under 1.2) r e q u i r e h i g h e r e f f i c i e n c y , and t h e r e f o r e c a n o n l y be r u n w i t h d i f f i c u l t y on such l a r g e p a r t i c l e systems. When c o n f r o n t e d w i t h low a l p h a v a l u e s , expensive p r e p a r a t i v e 10 m i c r o n columns ( f o r example t h e Whatman Magnum 20, 10,000 p l a t e s ) r u n under non-overload c o n d i ­ t i o n s a r e r e q u i r e d . Because such columns a r e s m a l l and operate w i t h h i g h back p r e s s u r e s , they a r e l i m i t e d both i n l o a d i n g

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

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Inj.

1

Figure l a . Separations on a p r e p a r a t i v e column 150 mm. M o b i l e phase: D e t e c t i o n : 254 nm (0.5

2

3

4

5

Minutes

of parabens and t a r t r a z i n e (0.1 μg each) (Beckman U l t r a s p h e r e ODS, 5 μπι, 4.6 χ 60% methanol. Flow: 1 ml/min, 1700 p s i . AUFS).

F i g u r e l b . S e p a r a t i o n s o f parabens and t a r t r a z i n e (0.5-1 mg each) on an a n a l y t i c a l column (Whatman P a r t i s i l Prep 40 0DS-3, 37-60 μπι, 2.5 χ 50 cm). M o b i l e phase: 60% methanol. Flow: 20 ml/min. D e t e c t i o n : 254 nm (1 AUFS).

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

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c a p a c i t y and maximum a l l o w a b l e flow r a t e . F u r t h e r m o r e , these h i g h r e s o l u t i o n , s m a l l p a r t i c l e columns s u f f e r v e r y s e v e r e p l a t e count d r o p s on e x c e s s i v e l o a d i n g . Thus, the p l a t e count of 10,000 does not c a r r y o v e r t o l a r g e s c a l e p r e p a r a t i v e work. In o r d e r to get h i g h t h r o u g h p u t , the l a r g e r p a r t i c l e , lower r e s o l u t i o n systems a r e necessary. Thus, h i g h e r a l p h a - v a l u e s need to be o b t a i n e d e i t h e r by v a r y i n g s o l v e n t , pH or p a c k i n g c h e m i s t r y , or by a d j u s t i n g f e r m e n t a t i o n parameters to remove o f f e n d i n g m a t e r i a l s . I f none of t h e s e can be done, s c a l e - u p w i t h r e a s o n a b l e throughput w i l l be very d i f f i c u l t . Examples. U s i n g the g l a s s systems on p a r t i a l l y p u r i f i e d p r e p a r a t i o n s , we have s e p a r a t e d m i x t u r e s of p o l y o x i n s L, B, A and K, [ ( 2 5 ) , F i g u r e 2] and g i l v o c a r c i n s V and M [ ( 2 6 ) , F i g u r e 3 ] . The p o l y o x i n s are p o l a r n u c l e o s i d e s and o r i g i n a l l y r e q u i r e d e x t e n s i v e chromatography on c e l l u l o s e to y i e l d p r o d u c t s f o r c h e m i c a l c h a r a c t e r i z a t i o n and c o u l d not be chromatographed on s i l i c a ( 2 5 ) . U s i n g p a i r e d i o n chromatography w i t h h e p t a - f l u o r o b u t y r i c a c i d (HFBA), the f o u r major components of the complex which d i f f e r i n amino a c i d c o n t e n t were r e a d i l y s e p a r a t e d i n 100 mg q u a n t i t i e s (Figure 4). S t r u c t u r e s of these a n t i b i o t i c s were a s s i g n e d by f a s t atom bombardment mass s p e c t r o m e t r y and NMR (27). In a second example of p r e p a r a t i v e RPHPLC, an e t h y l a c e t a t e e x t r a c t of an unknown a n t i b i o t i c which had been chromatographed on s i l i c a was f u r t h e r f r a c t i o n a t e d i n t o homologs ( F i g u r e 5 ) . A n a l y s i s by UV, MS and NMR i n d i c a t e d t h a t these m a t e r i a l s were g i l v o c a r c i n s M and V, d i f f e r i n g o n l y i n the presence of a m e t h y l or v i n y l side c h a i n (26). Separation

of a G l y c o p e p t i d e

Antibiotic

Mixture

The i s o l a t i o n and s e p a r a t i o n of t h r e e g l y c o p e p t i d e a n t i b i o t i c s (28) from a crude XAD-7 f e r m e n t a t i o n b r o t h e x t r a c t f u r t h e r e x e m p l i f i e s the advantages of p r e p a r a t i v e RPHPLC. F i g u r e 6a g i v e s an a n a l y t i c a l chromatogram of a crude i s o l a t e of a m i x t u r e of a n t i b i o t i c s of i n t e r e s t . The a n t i b i o t i c was not s o l v e n t e x t r a c t a b l e and c o u l d not be chromatographed on s i l i c a . In e x p l o r a t o r y work w i t h t h i s complex, samples were p r o c e s s e d through s e v e r a l p u r i f i c a t i o n s t e p s (XAD, i o n exchange, Sephadex, LH-20) and f i n a l l y on p r e p a r a t i v e RPHPLC u s i n g a n a l y t i c a l and g l a s s p r e p a r a t i v e columns. I t was shown t h a t the peaks l a b e l e d I, I I and I I I were the a n t i b i o t i c components of i n t e r e s t . The m u l t i s t e p sequence was n e c e s s a r y to remove the p o l a r c o n t a m i n a n t s which e l u t e at the f r o n t of the a n a l y t i c a l chromatogram and which t a i l i n t o the d e s i r e d p e a k s . However, gram q u a n t i t i e s of each a n t i b i o t i c were needed and c o u l d not be o b t a i n e d by t h a t p r o c e s s because of the c o m p l e x i t i e s of the c l e a n - u p p r o c e d u r e and the l i m i t e d c a p a c i t y of the g l a s s columns. D e s i g n of the s e p a r a t i o n . I t i s w e l l known t h a t g r a d i e n t s c a n improve apparent r e s o l u t i o n i n chromatography ( I ) . The use of a g r a d i e n t i n a s e p a r a t i o n such as t h a t shown i n F i g u r e 6a would be i d e a l i n t h a t adequate c l e a r a n c e of the p o l a r c o n t a m i n a n t s c o u l d

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

4.

SITRIN ET AL.

Preparative Reversed Phase HPLC

11

R

Polyoxln L

II

B

CH OH

HO

OH

A

CH OH

COOH

OH

2

2

>N

HN

COOH R OC

O ^ N

2

1

OH

IV >N

I

0 = CHNCH . O .



ι H,NCH HÇR

3

Ν

OH

3

OH

ι

III

Downloaded by UNIV OF SYDNEY on April 3, 2013 | http://pubs.acs.org Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch004

R

2

HO

H

OH

HOCH

I CHoOCNH, 2

II ο F i g u r e 2. S t r u c t u r e s of p o l y o x i n s L, B, A, and K.

OH

OMe

V

R = CH = CH

M

R = CH

3

F i g u r e 3. S t r u c t u r e s of g i l v o c a r c i n s V and M.

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

2

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PURIFICATION OF FERMENTATION PRODUCTS

20

40

60

80

10of

120

140

160

180

200 Minutes

10% Acetonitrile F i g u r e 4. S e p a r a t i o n o f a p o l y o x i n m i x t u r e . Sample: p o l y o x i n e x t r a c t (100 mg). Column: L i C h r o p r e p RP-18, 25-40 μπι, 2.5 χ 50 cm. M o b i l e phase: 0.015 M HFBA. Flow: 14 ml/min, 80 p s i . D e t e c t i o n : 254 nm.

Gilvocarcin V

Gilvocarcin M

Inj.

1

2

/

\

3

Hours

F i g u r e 5. S e p a r a t i o n of a g i l v o c a r c i n m i x t u r e . Sample: gilvo­ c a r c i n (40 mg). Column: L i C h r o p r e p RP-18, 25-40 μπι, 2.5 χ 50 cm. M o b i l e phase: 3 5 % a c e t o n i t r i l e . Flow: 8 ml/min, 80 p s i . D e t e c t i o n : 254 nm.

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

4.

SITRIN ET AL.

Preparative Reversed Phase HPLC

79

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take p l a c e b e f o r e e l u t i o n o f Peak I . F i g u r e 6b shows an a n a l y t i c a l chromatogram o f the same crude m a t e r i a l e l u t e d w i t h a gradient of a c e t o n i t r i l e . I n t h i s case t h e f r o n t m a t e r i a l i s c o m p l e t e l y r e s o l v e d from t h e d e s i r e d peaks. When 2 g o f crude complex was chromatographed on a g l a s s column u s i n g a step g r a d i e n t a s u b s t a n t i a l c l e a n - u p was o b s e r v e d , b u t r e s o l u t i o n and throughput were s t i l l l i m i t e d by column s i z e and f l o w r a t e l i m i t a t i o n s (See F i g u r e 7 ) . T h i s s e p a r a t i o n took o v e r 6 hours and y i e l d e d m i n i m a l amounts o f p r o d u c t s . S c a l i n g up t o l a r g e r i n s t r u m e n t s was n e c e s s a r y . Scale-up instrumentaiton. Two c o m m e r c i a l l y a v a i l a b l e i n s t r u m e n t s were used f o r t h e s c a l e - u p work, a J.Y. Chromatospac 100 and a Waters Prep-500A equipped w i t h a Whatman Magnum 40 column, as d e s c r i b e d i n M a t e r i a l s and Methods. The JY u n i t uses an a x i a l compression system t o o b t a i n t i g h t p a c k i n g . I n o u r hands,"both systems d i s p l a y e d e q u i v a l e n t r e s o l u t i o n . The Waters u n i t w i t h t h e Whatman column had s l i g h t l y lower c a p a c i t y because i t s column was s m a l l e r , but s o l v e n t changes and sample i n j e c t i o n s were e a s i e r t o c a r r y out w i t h i t s r e c i p r o c a t i n g pump. I n both c a s e s , a Gow Mac v a r i a b l e w a v e l e n g t h UV d e t e c t o r was used a t 210 o r 254 nm. T h i s d e t e c t o r i s very d e s i r a b l e as i t shows a l i n e a r response t o very c o n c e n t r a t e d s o l u t i o n s o f compounds a t t h e i r o p t i m a l absorbances. D e t e c t i o n o f the d e s i r e d m a t e r i a l i s enhanced s i n c e t h i s d e t e c t o r can be used a t the same wavelength as t h a t used i n c o r r e s p o n d i n g a n a l y t i c a l work. I n our hands, the use o f a normal v a r i a b l e w a v e l e n g t h d e t e c t o r r u n a t a w a v e l e n g t h somewhat removed from t h e maximum, as o f t e n recommended, has f r e q u e n t l y g i v e n m i s l e a d i n g r e s u l t s due t o the numerous UV-absorbing contaminants i n fermen­ t a t i o n products. The c e l l o f the Gow Mac d e t e c t o r has a 0.1 mm p a t h l e n g t h making i t 100 times l e s s s e n s i t i v e t h a n an a n a l y t i c a l UV d e t e c t o r w i t h a 10 mm p a t h l e n g t h . The c e l l d e s i g n can a l s o h a n d l e 500 ml/min, the maximum f l o w r a t e o f both l a r g e s c a l e chromatographs. Both l a r g e s c a l e systems d i s p l a y e d lower r e s o l u t i o n (200-300 p l a t e s ) than t h e g l a s s columns when e v a l u a t e d w i t h t h e parabens, but they s t i l l had s u f f i c i e n t r e s o l v i n g power t o s e p a r a t e t h e paraben m i x t u r e ( F i g u r e 8 ) . I n t h e o r y , e f f i c i e n c y i s s t i l l s u f f i c i e n t f o r r e s o l v i n g the t h r e e major components i n F i g u r e 4 ( a l p h a ' s o f 1.6 and 1.9). U s i n g a l p h a = 1.6, Ν = 300, and k'= 10 ( t y p i c a l v a l u e s from F i g u r e 4 and c o n d i t i o n s used f o r p r e p a r a t i v e work) and t h e well-known ( 1 ) r e s o l u t i o n e q u a t i o n : R

s

= 1/4 ( a - l ) V ^ T k ' / ( k ' + l )

where R ( r e s o l u t i o n ) i s t h e r a t i o o f the d i s t a n c e between two peaks d i v i d e d by t h e i r average band w i d t h , a c a l c u l a t e d r e s o l u t i o n of 2.3 i s a c h i e v e d . Such a v a l u e i m p l i e s b a s e l i n e r e s o l u t i o n (_1) and i s w e l l i n excess o f t h a t needed t o r e s o l v e peaks I and I I ( F i g u r e 6a). (The minor peaks have r e l a t i v e a l p h a ' s o f o n l y 1.25 i m p l y i n g an R = 0.96, which i s s t i l l s u f f i c i e n t i f c e n t e r c u t s a r e taken.) These c a l c u l a t i o n s use p l a t e counts determined f o r i d e a l substances under n o n - o v e r l o a d c o n d i t i o n s . F o r c o n d i t i o n s s

s

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

80

PURIFICATION OF FERMENTATION PRODUCTS

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Peak I

Inj.

1 5

L. 9 Minutes

F i g u r e 6a. A n a l y t i c a l s e p a r a t i o n s of a crude g l y c o p e p t i d e complex (isocratic). Sample: crude a n t i b i o t i c e x t r a c t . Column: Beckman U l t r a s p h e r e ODS, 5 μπι, 4.6 χ 150 mm. M o b i l e phase: 35% a c e t o ­ n i t r i l e i n 0.1 M KH2PO4PH 3.2. Flow: 1.5 ml/min. D e t e c t i o n : 220 nm.

F i g u r e 6b. A n a l y t i c a l s e p a r a t i o n s o f a crude g l y c o p e p t i d e complex ( g r a d i e n t ) . Sample: crude a n t i b i o t i c e x t r a c t . Column: Beckman U l t r a s p h e r e ODS, 5 μπι, 4.6 χ 150 inm. M o b i l e phase: 27 t o 37% a c e t o n i t r i l e i n 0.1 M KH2PO4PH 3.2. Flow: 1.5 ml/min. D e t e c t i o n : 220 nm.

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

4.

81

Preparative Reversed Phase HPLC

SITRIN ET AL.

ι

Downloaded by UNIV OF SYDNEY on April 3, 2013 | http://pubs.acs.org Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch004

(50 mg)

Inj.

1

2

3

4

5

6

Hours

F i g u r e 7. S m a l l - s c a l e p r e p a r a t i v e s e p a r a t i o n of g l y c o p e p t i d e complex. Sample: crude a n t i b i o t i c e x t r a c t (2 g ) . Column: Merck L i C h r o p r e p RP-18, 25-40 μπι, 2.5 χ 50 cm. M o b i l e phase: to 30% a c e t o n i t r i l e i n 0.1 M KH P04pH 3.2. Flow: 14 ml/min, 90 p s i . D e t e c t i o n : 210 nm (Gow Mac). 2

I

'

>-

Inj.

30

60

Minutes

F i g u r e 8. S e p a r a t i o n o f parabens on Magnum 40 column (4.8 χ 50 cm). Sample: parabens and t a r t r a z i n e (60-80 mg each). Column: Whatman P a r t i s i l Prep 40 0DS-3 (37-60 μπι). M o b i l e phase: 6 0 % methanol. Flow: 100 ml/min. D e t e c t i o n : 254 nm.

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

82

PURIFICATION OF FERMENTATION PRODUCTS run a t h i g h l o a d i n g (>10 mg/g) lower p l a t e counts a r e observed but o f t e n o t h e r b e n e f i c i a l e f f e c t s such as those observed i n d i s p l a c e ment chromatography become e v i d e n t ( 1 8 ) . An example o f a p r e p a r a t i v e s e p a r a t i o n o f a p u r i f i e d f e r m e n t a t i o n e x t r a c t i n order t o o b t a i n s u i t a b l e e l u t i o n c o n d i t i o n s using the Magnum column, i s shown i n F i g u r e 9a. The sample (2 g ) , was i n j e c t e d through t h e pump i n a s o l v e n t system lower i n a c e t o n i t r i l e content than needed t o e l u t e Peak I . E l u t i o n w i t h a s t e p g r a d i e n t y i e l d e d ( a f t e r d e s a l t i n g ) each o f the pure components w i t h b e t t e r than 90% r e c o v e r y and p u r i t y (see F i g u r e 9b). Loading i n t h i s case was a p p r o x i m a t e l y 1 mg o f Component I p e r gram o f adsorbant.

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Factors P o t e n t i a l l y Limiting

Scale-up

W i t h s u i t a b l e s o l v e n t systems worked o u t , the q u e s t i o n arose as t o what e x t e n t t h i s p r o c e s s c o u l d be s c a l e d up t o produce gram quant i t i e s o f each component. Three p o t e n t i a l l i m i t a t i o n s e x i s t e d : 1) S u p p l i e s o f p u r i f i e d i n t e r m e d i a t e , 2) l o a d i n g c a p a c i t y o f t h e column, 3) s i z e o f column and c o s t o f p a c k i n g . B y p a s s i n g complex i s o l a t i o n scheme. The m u l t i s t e p procedure used to prepare t h e s t a r t i n g m a t e r i a l f o r t h i s chromatographic s e p a r a t i o n was found t o be the primary b o t t l e n e c k . T h e r e f o r e , i n t h e next s c a l e - u p experiment, 25 g o f a crude XAD i s o l a t e ( c o n t a i n i n g 2.2 g o f Component I ) i n 4 l i t e r s o f 17% a c e t o n i t r i l e i n b u f f e r was pumped onto t h e column a t 250 ml/min. S e q u e n t i a l step e l u t i o n s (20, 22, 24 and 26% a c e t o n i t r i l e a t 250 ml/min) r e s u l t e d i n n e a r l y b a s e l i n e r e s o l u t i o n o f t h e t h r e e components ( F i g u r e s 10a and 10b) a g a i n w i t h h i g h r e c o v e r y and p u r i t y (>85%), i n l e s s than 3 hours. S i n c e s e p a r a t e experiments had i n d i c a t e d t h a t a c c e p t a b l e r e s o l u t i o n c o u l d be o b t a i n e d a t h i g h e r f l o w r a t e s , a l l s c a l e - u p work was performed a t 250 ml/min. Thus, t h e p r e p a r a t i v e r e v e r s e d phase column not o n l y y i e l d e d c h r o m a t o g r a p h i c a l l y pure p r o d u c t s but c o u l d a c h i e v e t h e p r e l i m i n a r y p u r i f i c a t i o n w i t h h i g h r e c o v e r y i n hours, a process which o t h e r w i s e took s e v e r a l days t o do. When 50 g ( c o n t a i n i n g a p p r o x i m a t e l y 3 g o f each component) was i n j e c t e d i n 6 l i t e r s o f b u f f e r a l o n e , no breakthrough was observed, and on g r a d i e n t e l u t i o n , t h e chromatogram shown i n F i g u r e 11a was o b t a i n e d . A l t h o u g h r e s o l u t i o n between the t h r e e components had d e t e r i o r a t e d , the complex had been e f f i c i e n t l y separated from t h e contaminants i n l e s s than 2 hours. T h i s f i r s t chromatography step g i v e s a "window" c u t which i s r e l a t i v e l y f r e e o f contaminants o f h i g h e r and lower p o l a r i t y and thereby more amenable t o rechromatography. Indeed, on rechromatography, a s u p e r i o r s e p a r a t i o n o f t h e complex i n t o i t s components o c c u r r e d . The p o o r l y r e s o l v e d m i x t u r e of Components 1,11 and I I I from F i g u r e 11a was p o o l e d , d i l u t e d w i t h b u f f e r and rechromatographed t o y i e l d , a f t e r d e s a l t i n g , 3 g each o f the pure components ( F i g u r e l i b ) . U s i n g t h e above twos t e p procedure ( i . e . s e v e r a l s e p a r a t i o n s on 50 g o r more o f crude e x t r a c t f o l l o w e d by rechromatography o f t h e pooled p u r i f i e d complex) over 10 grams o f the major component was o b t a i n e d , w i t h the same h i g h r e c o v e r y and p u r i t y as shown i n F i g u r e 9b.

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

4.

SITRIN ET AL.

83

Preparative Reversed Phase HPLC

I

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(550 mg)

Inj.

1

2

3

4

Hours

F i g u r e 9a. S m a l l - s c a l e p r e p a r a t i v e s e p a r a t i o n of g l y c o p e p t i d e complex on Magnum 40 column (4.5 χ 50 cm). Sample: purified a n t i b i o t i c (2 g ) . Column: Whatman P a r t i s i l Prep 40 ODS-3 ( 3 7 60 μπι). M o b i l e phase: 20 t o 26% a c e t o n i t r i l e i n 0.1 M KH2PO4PH 6.0. Flow: 100 ml/min. D e t e c t i o n : 210 nm.

Antibiotic Component

Initial Content

Isolated Weight

HPLC Purity

I II III

600 my 350 mg 250 mg

550 mg 250 mg 200 m g

>95% >90% >95%

Inj. F i g u r e 9b. Recovery data f o r s m a l l - s c a l e s e p a r a t i o n o f g l y c o ­ p e p t i d e complex on Magnum 40 column. Sample: p u r i f i e d component I . Column: Beckman U l t r a s p h e r e ODS, 5 μπι, 4.6 χ 150 mm). M o b i l e phase: 27 t o 37% a c e t o n i t r i l e i n 0.1 M KH P04pH 3.2. Flow: 1.5 ml/min. D e t e c t i o n : 220 nm. 2

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

84

PURIFICATION OF FERMENTATION PRODUCTS

% Acetonitrile 30 26 - 24 - 22 20

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- 17.5

30

60

90

120

150 Minutes

Preparative Separation

F i g u r e 10a. Scale-up p r e p a r a t i v e s e p a r a t i o n of g l y c o p e p t i d e com­ p l e x (25 g r u n ) . Sample: crude a n t i b i o t i c complex (25 g ) . Column: Whatman P a r t i s i l Prep 40 ODS-3 (37-60 μπι) , Whatman Magnum 40 (4.8 χ 50 cm). M o b i l e phase: 17.5 t o 26% a c e t o n i t r i l e i n 0.1 M KH2PO4PH 6.0. Flow: 250 ml/min. D e t e c t i o n : 210 nm. m 1.3g

I

2.ôg

π 17g

Minutes

F i g u r e 10b. A n a l y s i s o f f r a c t i o n s f o r s c a l e - u p s e p a r a t i o n of g l y c o p e p t i d e complex (25 g r u n ) . Sample: p o o l s from 25 g r u n . Column: Beckman U l t r a s p h e r e ODS (5 μπι), 4.6 χ 150 mm. Mobile phase: 27 t o 37% a c e t o n i t r i l e i n 0.1 M KH2PO4PH 3.2. Flow: 1.5 ml/min. D e t e c t i o n : 220 nm.

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

4.

SITRIN ET AL.

85

Preparative Reversed Phase HPLC

%

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Acetonitrile π 30

F i g u r e l i a . Scale-up p r e p a r a t i v e s e p a r a t i o n of g l y c o p e p t i d e com­ p l e x (50 g r u n ) . Sample: crude a n t i b i o t i c complex (50 g) i n 6 L b u f f e r . Column: Whatman P a r t i s i l Prep 40 ODS-3 (37-60 μπι), What­ man Magnum 40 (4.8 χ 50 cm). M o b i l e phase: 0 t o 287 a c e t o n i t r i l e i n 0.1 M KH P04pH 6.0. Flow: 250 ml/min. D e t e c t i o n : 210 nm. 0

2

F i g u r e l i b . Rechromatography of pooled f r a c t i o n s f o r s c a l e - u p s e p a r a t i o n of g l y c o p e p t i d e complex (50 g r u n ) . Column: What­ man P a r t i s i l Prep 40 0DS-3 (37-60 μπι) , Whatman Magnum 40 (4.8 χ 50 cm). M o b i l e phase: 10 t o 28% a c e t o n i t r i l e i n 0.1 M Kl^PO^pH 6.0. Flow: 250 ml/min. D e t e c t i o n : 210 nm.

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

PURIFICATION OF FERMENTATION PRODUCTS

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The use o f the column as a c o n c e n t r a t o r i s n o t a b l e . P o o l s c o n t a i n i n g r e l a t i v e l y l a r g e volumes o f f r a c t i o n s (10-20 l i t e r s o r l a r g e r ) c a n be mixed w i t h e q u a l volumes o f b u f f e r t o d i l u t e out the a c e t o n i t r i l e and pumped onto the column w i t h complete r e t e n t i o n . Normal e l u t i o n c a n g i v e the d e s i r e d chromatographic s e p a r a t i o n o r , u s i n g a very s t r o n g s o l v e n t , the e n t i r e product c a n be c o n c e n t r a t e d i n t o two t o t h r e e l i t e r s , a volume s u i t a b l e f o r d e s a l t i n g , l y o p h i l i z a t i o n , e t c . Such a s t e p takes l e s s time than l y o p h i l i z a t i o n o r e v a p o r a t i o n o f the e n t i r e volume. Loading c a p a c i t y . The second f a c t o r which l i m i t s throughput, i n d i c a t e d e a r l i e r , i s t h e l o a d i n g c a p a c i t y o f the column. Table I shows the r e s u l t s o f a l o a d i n g study w i t h homogeneous Compound I w h i c h i n d i c a t e s t h a t " o v e r l o a d " (as d e f i n e d (I) by a 10% l o s s i n k ) o c c u r s a t l e v e l s o f 1-2 mg o f pure m a t e r i a l per g a d s o r b e n t . The k v a l u e s i n Table I are apparent k's s i n c e t h e chromtographic procedure uses a step g r a d i e n t i n o r d e r t o i n j e c t l a r g e volumes. A c t u a l k's a r e somewhat lower. A l s o , l o s s o f r e s o l u t i o n ( d e f i n e d by N) o c c u r s as l o a d i n g i n c r e a s e s beyond t h a t l e v e l . On r e c a l c u l a t i o n o f r e s o l u t i o n observed a t the h i g h e s t l e v e l t e s t e d (13 mg/g) u s i n g Ν = 53, a l p h a = 1.6 and k'= 16, a m i n i m a l l y a c c e p t a b l e v a l u e o f 1.0 i s o b t a i n e d . I n t h e o r y , t h i s p r o b a b l y r e p r e s e n t s the maximum l o a d i n g f o r s e p a r a t i o n o f Component I from Component I I . A t p r e s e n t , q u a n t i t i e s are n o t a v a i l a b l e t o c o n f i r m t h i s . Although s u b s t a n t i a l l y h i g h e r l o a d i n g s are p o s s i b l e by u s i n g displacement chromatography, t h e o v e r a l l throughput would s t i l l depend on the c a p a c i t y o f the column t o p u r i f y the l a r g e r amounts o f crude e x t r a c t s needed t o prepare the p r e c u r s o r s f o r t h e d i s p l a c e m e n t chromatography s t e p . The maximum l o a d i n g of crude e x t r a c t i s y e t t o be determined because o f supply c o n s i d e r a t i o n s . 1

1

Cost l i m i t a t i o n s . The t h i r d l i m i t a t i o n t o s c a l e up i s the s i z e o f column used and c o s t o f p a c k i n g . I n c a r r y i n g out l a r g e s c a l e work on crude e x t r a c t s , the c o s t o f p a c k i n g i s dependent on column life. We have found t h a t the p a c k i n g t u r n s dark i n c o l o r and l o s e s r e s o l u t i o n a f t e r s e v e r a l 50 g r u n s . However, i t can be c o m p l e t e l y r e g e n e r a t e d by e x t r u s i o n , s o a k i n g i n DMSO and r e p a c k ­ ing. Because the p a c k i n g c a n be r e g e n e r a t e d and reused, i t s modest c o s t ($700/kg) i s not a s e r i o u s o b s t a c l e t o i t s use on crude m a t e r i a l . Should f u r t h e r s c a l e - u p be r e q u i r e d , l a r g e r columns w i t h 4 and 20 times t h e c a p a c i t y as the Prep-40 column are a v a i l a b l e from Whatman, as a r e the i n d u s t r i a l u n i t s from Whatman and Waters. Conclusions In summary, we have demonstrated the p o t e n t i a l u t i l i t y o f p r e p a r a t i v e r e v e r s e d phase HPLC as a t o o l t o p u r i f y f e r m e n t a t i o n p r o d u c t s i n m u l t i g r a m s c a l e on l a b o r a t o r y i n s t r u m e n t s . Secondly, due t o i t s i n t r i n s i c s e l e c t i v i t y and c o m p a t i b i l i t y w i t h the use o f h i g h f l o w r a t e s , RPHLC i s a u s e f u l t o o l f o r r a p i d l y p u r i f y i n g crude f e r m e n t a t i o n e x t r a c t s t o a l e v e l o f p u r i t y e q u i v a l e n t t o t h a t o b t a i n e d by m u l t i s t e p time consuming p r o c e d u r e s . We

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

4. SITRIN ET AL.

Preparative Reversed Phase

87

HPLC

TABLE I . L o a d i n g Study - Component

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Injection 5

Weight (g)

Volume* ) (ml)

Loading (mg/g)

c

k' >

Ν

R d) s

0.2

80

0.3

19.2

147

1.7

0.2

2000

0.3

19.3

142

1.7

0.6

220

1.0

17.8

122

1.6

1.2

440

2.0

17.7

121

1.6

2.4

880

4.0

17.2

95

1.4

5.0

1900

8.3

16.2

61

1.1

7.7

1000

12.8

17.4

51

1.0

a) E l u a n t :

24% CH3CN i n 0.1 M phosphate b u f f e r pH 6.0 a t 200 ml/min. Whatman Prep 40 ODS-3 P a c k i n g (37-60 m i c r o n ) i n Magnum 40 Column 50 X 4.8 cm; UV d e t e c t i o n a t 210 urn.

b) Sample d i s s o l v e d i n b u f f e r , no a c e t o n i t r i l e . p r e - e q u i l i b r a t e d w i t h same b u f f e r . c ) Apparent k', not c o r r e c t e d

Column was

f o r e q u i l i b r a t i o n time.

d) C a l c u l a t e d u s i n g a l p h a = 1.6.

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

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recommend a two-step procedure t o get maximum throughput o f m a t e r i a l : 1) S e v e r a l runs, loaded as h e a v i l y as p o s s i b l e w i t h crude e x t r a c t s t o get a "window" c o n t a i n i n g the d e s i r e d m a t e r i a l ( s ) , 2) rechromatography o f pooled c u t s t o produce t h e pure product. F i n a l l y , the o v e r a l l l o s s i n p l a t e count i n g o i n g from a 5 m i c r o n a n a l y t i c a l column run a t low l o a d i n g w i t h i d e a l s u b s t a n c e s t o a l a r g e r p a r t i c l e p r e p a r a t i v e column (37-60 m i c r o n s ) run a t h i g h l o a d i n g w i t h complex s u b s t r a t e s i s indeed s t r i k i n g . N e v e r t h e l e s s , f o r p r e p a r a t i v e purposes such r e s o l u t i o n i s s t i l l s u f f i c i e n t t o separate compounds c l e a n l y w i t h h i g h r e c o v e r y p r o v i d e d t h a t a l p h a - v a l u e s are l a r g e enough (>1.5). I t would appear t h a t e x c e s s i v e c o n c e r n w i t h p l a t e counts i n such systems i s unwarranted u n l e s s very c l o s e s e p a r a t i o n s are b e i n g r u n . Acknowledgment We g r a t e f u l l y acknowledge the e x c e l l e n t t e c h n i c a l a s s i s t a n c e o f Mr. George Udowenko and the support o f o t h e r members o f the SK&F Department o f N a t u r a l P r o d u c t s Pharmacology f o r s u p p l i e s o f f e r m e n t a t i o n p r o d u c t s . We thank Dr. James Chan f o r d a t a on the s t r u c t u r e o f the p u r i f i e d p o l y o x i n s .

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Snyder, L.; Kirkland, J. "Introduction to Modern Liquid Chromatography", Second Edition, John Wiley, New York, 1979. Kabra, P.; Maron, L. "Liquid Chromatography in Clinical Analysis", Humana Press, Clifton, NJ, 1981. Poppe, H.; Kraak, J. J. Chromatog. , 1983, 295, 395. De Jong, Α.; Poppe, H.; Kraak, J. J. Chromatog. 1981, 209, 432. De Jong, Α.; Poppe, H.; Kraak, J. J. Chromatog. 1978, 148, 127. De Jong, Α.; Kraak, J.; Poppe, H.; Nooitgedacht, F. J. Chromatog. 1980, 193, 181. Hupe, K.; Lauer, H. J. Chromatog. 1981, 203, 41. Coq, B.; Cretier, G.; Rocca, J. Anal. Chem. 1982, 54, 2271. Sugnaux, F.; Djerassi, C. J. Chromatog. 1982, 248, 373. Rabel, F. Am. Lab. 1980, 12, 126. Hupe, K.; Lauer, H.; Zech, K. Chromatographia 1980, 13, 413. Verzele, M.; Dewaele, C.; Van Dijck, J.; Van Haver, D. J. Chromatog. 1982, 249, 231. Kagan, M.; Kraevskaya, M.; Vasilieva, V.; Zinkevich, Ε. J. Chromatog. 1981, 219, 183. Fiedler, H. J. Chromatog. 1981, 209, 103. Horvath, C.; Nahum, Α.; Frenz, J. J. Chromatog. 1981, 218, 365. Kalasz, H.; Horvath, C. J. Chromatog. 1981, 215, 295. Kalasz, H.; Horvath, C. J. Chromatog. 1982, 239, 423. Horvath, C.; Frenz, J.; Rassi, Z. J. Chromatog. 1983, 255, 273.

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

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19. Snyder, L. and Kirkland, J., ob. cit., Chapter 15, "Preparative Liquid Chromatography". 20. Nettleton, D. J. Liq. Chromatog. 1981, 4 (Supp. 1), 141. 21. Haywood, P.; Munro, G. In "Developments in Chromatog­ raphy - 2"; C.F.H. Knapman, Ed.; Applied Science Publishers, London 1981, p.33. 22. Eisenbeiss, F.; Henke, H. J. of High Res. Chromatog. and Chromatog. Comm. 1980, 2, 733. 23. Gasparrini, F.; Cacchi, S.; Cagliotti, L.; Misiti, D.; Giovannoli, M. J. Chromatog. 1980, 194, 239. 24. Merck Index, 10th Edition, M. Windholz, Ed., Rayway, NJ. 1983, pl303. 25. Isono, K.; Asahi, K.; Suzuki, S. J. Am. Chem. Soc. 1969, 91, 7490. 26. a) Nakano, H. et al. J. Antibiotics 1981, 34 266; b) Takahashi, K. et al. Ibid 1981, 34, 271. 27. Chan, J.; Yeung, Ε.; Roberts, G.; Sitrin, R. unpublished data. 28. Sitrin, R.; Chan, G.; DeBrosse, C.; Dingerdissen, J.; Hoover, J.; Jeffs, P.; Roberts, G.; Rottschaeffer, S.; Valenta, J.; Snader, K.; 24th Interscience Conference on Antimicrobial Agents and Chemotherapy 1984, Abs. No. 1137. RECEIVED September 7, 1984

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