size-exclusion chromatography on soft packing material under axial compression

8/2/2019 Size-Exclusion Chromatography on Soft Packing Material Under Axial Compression

1/9

E L S E V I E R Journal of Chromatography A, 732 (1996) 17-25

JOURNAL OFCHROMATOGRAPHYA

Size-exclusion chromatography on soft packing material under axialcompress ion

A .V . D a n i l o v , L . G . M u s t a e v a , I .V . V a g e n i n a , M . B . B a r u *Branch o f Shemyakin a nd Ovchinnikov Ins t i tu te o f B ioorganic Chemis try , R uss ian Academy o f Sc iences, Puschino , 142292 M oscow

Region, Russia

Received 4 Octob er 1995; accepted 3 N ovem ber 1995

A b s t r a c t

The influence of soft gel axial compression on the packing structure and the associated chromatographic parameterssuch as dispersion of chromatographic zones, retention volume and peak resolution have been considered for size-exclusionchromatography. Continuous time gel compression is accomplished by the use of a special column with controlled externalpressure applied to the packing. The experimentally obtained improvement in resolution combined with the decrease inseparation time has been proved.K e y w o r d s : Axial compression; Stationary phases, LC; Band broadening

1 . I n t r o d u c t i o n

C o n v e n t i o n a l L C r e m a i n s a c o m m o n m e t h o d o fi so l a t i on a nd pu r i f i c a t i on o f b io log ic a l subs t a nc e s ,e ve n in a pe r iod o f g r e a t p r og r e s s i n a na ly t i c a l a ndp r e p a r a t i v e H P L C a n d a s s o c i a t e d m e t h o d s . F e a t u r e sp e c u l i a r t o b i o l o g i c a l s u b s t a n c e s m a y f a v o u r L C a sl a b i l e , w a te r so lub le b io spe c i e s r e qu i r e e x t r e m e c a r ein ha nd l ing .I t i s w or th m e n t ion ing tha t i n t he c onve n t iona l LCo f b i o m o l e c u l e s m o s t a t t e n t i o n i s g i v e n t o t h ed e v e l o p m e n t o f h i g h l y e f f i c i e n t p a c k i n g m a t e r i a l sw h i c h a r e d e r i v e d m a i n l y f r o m p o l y m e r i c g e l s o rc o m p o s i t e m a t e r i a l s . M a n u f a c t u r e r s h a v e m o d i f i e dm o s t o f t h e p a r a m e t e r s o f s o l i d p h a s e s w h i c h a r ed i r e c t ly r e l a t e d t o pa c k ing se l e c t i v i t y : c he m ic a ls t r uc tu r e o f so r p t ion s i t e s , so r p t ion c a pa c i ty , t he*Corresponding author.

a c c e s s ib i l i t y o f po r e s a nd the f l e x ib i l i t y o f a po lym e r(see the survey in Ref . [1] for de ta i l s ) .

I n pa r a l l e l w i th s e l e c t i v i t y e f f e c t i ve ne s s i nhe r e n tt o a p a c k e d b e d ( r e s p o n s i b l e f o r b a n d b r o a d e n i n g )in f lue nc e s r e so lu t ion in LC . The r e f o r e a u se r f a c e st h e p r o b l e m o f o b t a in i n g a h i g h - q u a l i ty p a c k i n g i n ac o lum n a nd e nsu r ing i t s s t a b i l i t y i n t he c ou r se o f ase pa r a t i on p r oc e s s . D e sp i t e t he g r e a t va r i e ty o fc o l u m n s f o r t h e L C o f b i o p o l y m e r s th a t a re c o m m e r -c i a l l y a va i l a b l e , s l u r r y pa c k ing in a que ous m e d ia bys e d i m e n t a t i o n a n d / o r b y f l o w s t i l l r e m a i n s a s t h em a i n m e t h o d o f p a c k in g . W h i l e h i g h p r e ss u r esse ve r a l t im e s g r e a t e r t ha n the ope r a t i ng p r e s su r e s a r ea pp l i e d du r ing s lu r r y pa c k ing o f r i g id pa r t i c l e s ( suc ha s s i l i c a - ba se d pa r t i c l e s ) , i n t he c a se o f xe r oge l s( s u ch a s d e x t ra n - o r p o l y a c r y l a m i d - b a s e d m a t r i x e s ,e t c . ) m a x i m u m f l o w - r a t e s ( a n d t h e c o r r e s p o n d i n gp r e s s u r e s ) a r e c l o s e t o t h e r e c o m m e n d e d o p e r a t i n gf low - r a t e s ( w h ic h a r e r a the r l ow ) . Th i s r e su l t s i n a

0021-9673/96/$15.00 1996 Elsevier Science BY. All rights reservedS S D I 0021- 9673( 95 ) 01 246 - X


2/9

1 8 A .V . D a n i l o v e t al . / J . C h r o m a t o g r . A 7 3 2 ( 1 9 9 6 ) 1 7 - 2 5

l ong pa c k ing t im e , a nd unde s i r a b l e r e d i s t r i bu t ion o fge l be a ds , a c c o r d ing to s i z e , a long the c o lum n a x i sb e c o m e s m o r e p r o b a b l e . O n t h e o t h e r h a n d , p a c k i n gp r e s su r e c a n be i n su f f i c i e n t t o a c h i e ve a c om ple t e lyh o m o g e n e o u s p a c k e d b e d .

M u c h a t t e n t i o n i s p a i d t o p a c k i n g p r o c e d u r e s a n dp a c k e d b e d s t a b i l i t y i n p r e p a r a t i v e H P L C [ 2 - 4 ] , t h ese pa r a t ion e f f i c i e nc y be ing one o f t he de c i s ivef a c to r s i n t he p r i c e o f a pa r t i c u l a r p r oduc t . I t ha sbe e n show n tha t , be g inn ing w i th a de f in i t e I . D . ,c o lum ns o f t he c onve n t iona l t y pe a r e l e ss s t a b l e tha na na ly t i c a l - s i z e d c o lum ns . Tha t I . D . va lue i s d i c t a t e db y t h e p a c k i n g m a t e r i a l a s w e l l a s t h e m o b i l e p h a s etype , f o r i n s t a nc e a c o lum n w i th I . D . m or e t ha n 22m m a p p e a r s t o b e u n s t a b le w h e n p a c k e d w i t h 1 0 - / z mreversed-phase s i l ica ge l [5] . I t i s be l ieved [6] tha t apa c ke d be d m a y c on ta in l oose , i . e . uns t a b l e , r e g ionsw h e r e m i c r o v o i d s a r e f o r m e d . B e a d s r e a r r a n g eg r a dua l ly w i th t im e a nd the w ho le be d s e t t l e s g iv ingc ha nne l s o r vo id s a t t he c o lum n in l e t . Th i s m a y bec a u s e d b y r e p e a t e d t h e r m a l e x p a n s i o n / c o n t r a c t i o n o fthe pa c k ing be d , pum p pu l sa t ions , e t c . The in f lue nc eo f I . D . va lue on the se e f f e c t s i s c a use d by thede c r e a se i n c o lum n w a l l suppo r t .

S i n c e t h e d i f f e r e n c e b e t w e e n H P L C a n d l o wp r e s s u r e c h r o m a t o g r a p h y a p p e a r s t o b e q u a n t i t a t i v er a the r t ha n qua l i t a t i ve , s im i l a r phe nom e na a l so t a kep la c e w i th t he u se o f so f t pa c k ing m a te r i a l s . O n lyva r i a t i ons i n t he i r va lue s a nd du r a t ion a r e obse r ve d .T h e c a p a c i t y o f x e r o g e l s f o r m e c h a n i c a l d e f o r m a t i o n( f o r i n s t a nc e unde r t he a c t ion o f l i qu id f l ow o r ow ingto va r i a t i ons i n sw e l l i ng in t he g r a d i e n t e lu t i onm ode ) s e e m s to i nc r e a se t he p r oba b i l i t y o f suc hoc c u r r e nc e s ( c om pa r e d to a e r oge l s ) a nd a l so t o c a usea de c r e a se i n t he e f f e c t o f t he c o lum n w a l l s w h ic hs t a b i l i z e s a pa c k ing f o r a na ly t i c a l c o lum ns w i th r i g idge l pa c k ings .

P a c k i n g i m p r o v e m e n t a n d s t a b i li z a ti o n c a n b ea t t a i n e d b y a d a p t i n g t h e c o l u m n v o l u m e t o t h ec h a n g i n g b e d v o l u m e . A x i a l a n d / o r r a d i a l c o m p r e s -s i o n a p p l i e d d u r i n g a c h r o m a t o g r a p h i c p r o c e s s m a k es u c h a d a p t a t i o n p o s s i b l e . H P L C i n s t r u m e n t s b a s e do n b o t h a p p r o a c h e s a r e n o w c o m m e r c i a l l y a v a i l a b l ef r o m a n u m b e r o f m a n u f a c t u r e r s [ 3] . T h e d e s i g n s o fc h o i c e a r e t h o s e u t i l i z i n g t h e d y n a m i c m o d e o fc om pr e s s ion [ 7,8 ]. S um m in g up the re su l t s o f t hes tud ie s on the pe r f o r m a nc e s o f sphe r i c a l a nd a ngu la rs i l i c a - b a s e d p a c k i n g s i n d y n a m i c c o m p r e s s i o n c o l -

um ns [ 6 , 9 - 11 ], t he f o l low ing a dva n ta ge s o f t he l a t te rp a c k i n g s c a n b e m e n t i o n e d : ( i ) p a c k i n g p r o c e d u r ec onve n ie nc e a nd the pos s ib i l i t y t o r e pa c k ; ( i i ) p r e -v e n t i o n o f v o i d f o r m a t i o n a n d l o n g - t e r m s t a b i l i t y ;( i i i ) h igh c o lum n e f f i c i e nc y w i th l ow m a ss t r a ns f e rl im i t a t i on . ( The on ly d i s a dva n ta ge i s t he i r h ighe rpr ice ) .

S im i l a r de s igns a r e a l so u se d in p r e pa r a t ive l ow -p r e s su r e c h r om a tog r a phy . F o r i n s t a nc e , a x i a l pa c k ingte c hno logy w i th a 700 - kP a p r e s su r e r a t i ng i s u se d f o rV a n ta ge TM c o l u m n s f o r b i o c h r o m a t o g r a p h y ( G r a c eI ndus t r i a l , La usa nne , S w i t z e r l a nd ) . Th i s r e su l t s i nh ighe r f l ow - r a t e s , im pr ove d c o lum n be d s t a b i l i t y a ndf a s t er t h r o u g h p u t. " A u t o m a t i c B e d H e i g h t C o l u m n "( e . g . M o d e l A B H C 4 4 0 x 5 0 0 ) m a n u f a c t u r e d b yA m i c o n ( D a n v e r s , M A , U S A ) i s a n e x a m p l e o f th ea p p l i c a t i o n o f c o m p r e s s i o n t o p a c k e d b e d s t h a tc h a n g e t h e i r v o l u m e i n t h e c o u r s e o f c h r o m a t o -g r a ph ic p r oc e du r e s . Th i s c o lum n i s de s igne d top r ov ide a s l i gh t ga s ove r p r e s su r e ( 0 . 5 - 5 p . s . i . ) t o t hes e a l e d s p a c e a b o v e t h e a d j u s t e r a s s e m b l y . T h er e s u l t i n g a d v a n t a g e s m a y b e s u m m a r i z e d a s f o l l o w s :e a s i e r pa c k ing a nd r e ge na t ion , be t t e r r e so lu t ion duet o m i n i m a l d e a d v o l u m e .

H o w e v e r v a r i a t i o n s i n c h r o m a t o g r a p h i c c h a r a c -t e r i s t i c s o f so f t ge l pa c k ing r e su l t i ng f r om i t s c om -p r e s s ion ha ve no t be e n a de qua te ly i nve s t iga t e d ( i nc on t r a s t t o r i g id pa c k ings ) a l t hough the " de a d vo l -u m e " m i n i m i z a t i o n i n e v i t a b l y i n v o l v e s s u c h v a r i a -t i ons . I t shou ld be no te d tha t, i n 1970, E dw a r ds a ndH e f t d e m o n s t r a t e d t h e i m p r o v e d s e p a r a t i o n o f B l u eD e x t r a n 2 0 0 0 a n d N i ( N O 3 ) 2 o n S e p h a d e x G - 5 0u n d e r c o m p r e s s i o n [ 12 ]. W i t h t h e i r m e t h o d o f p a c k -ing , c om pr e s s ion by the a c t ion o f t he hyd r a u l i cp r e s su r e o f t he e lue n t i nvo lve s a n inc r e a se i n t hel ine a r f l ow - r a t e o f t he e lue n t a nd th i s i s a c c om pa n ie dby a r e duc t ion in t he e f f i c i e nc y . Thu s i t is d i f f ic u l t t op r e d i c t t h e p a c k i n g b e h a v i o u r u n d e r f u r t h e r c o m p r e s -s ion be c a use o f t he tw o c oun te r a c t ing p r oc e s se s .

W e ha ve a l r e a dy show n p r e v ious ly [ 13 ] t ha t i on -e x c h a n g e s ep a r at io n o n " s o f t " C M S e p h a d e x C - 2 5i m p r o v e s u n d e r a x i a l c o m p r e s s i o n . C o m p r e s s i o n h a sbe e n a c h ie ve d by the u se o f a spe c i a l r e f ine m e n t i nt h e d e s i g n o f a s t a n d ar d P h a r m a c i a c o l u m n . T h e g e lp a c k i n g r e m a i n s u n d e r d y n a m i c a x i a l c o m p r e s s i o nth r oughou t t he p r oc e s s .

A d e t a i l e d e x a m i n a t i o n o f t h e p a r a m e t e r s o f s o f tp a c k i n g m a t e r i a l s u n d e r d y n a m i c c o m p r e s s i o n i s t h e


3/9

A.V. Danilov et al. / J. Chrom atogr. A 7.32 (1996) 17 -2 5 19o b j e c t i v e o f o u r s t u d y . S i z e - e x c l u s i o n c h r o m a t o g -r a p h y ( S E C ) h a s b e e n c h o s e n f o r th e i n v e s t i g at i o n a sS E C p a c k i n g g e o m e t r i c a l p a r a m e t e r s ( w h i c h a r e f o rt h e m o s t p a r t i n fl u e n c ed b y c o m p r e s s i o n ) a f f e c t p e a kr e t e n t ion a nd d i spe r s ion .

2. Experimental2 . 1 . A p p a r a t u s a n d r e a g e n t s

T h e c h r o m a t o g r a p h i c s y s t e m c o n s i s t ed o f a 2 1 5 0H P L C p u m p ( L K B , S w e d e n ), a s a m p l e i n j e c to r V -7( P ha r m a c ia , S w e de n ) w i th 250 - /x l l oop , a n o r ig ina lc o l u m n o f v a r i a b l e v o l u m e , a n u l t r a v i o l e t p h o t o -m e t r i c d e t e c t o r 2 2 3 8 U v i c o r d S I I a n d a 2 2 1 0 t w o -c h a n n e l r e c o r d e r ( L K B , S w e d e n ) . A l l c h e m i c a l sw e r e o f a n a l y t i c a l g r a d e . S e p h a d e x G - 2 5 S F a n dB l u e D e x t r a n 2 0 0 0 w e r e o b t a i n e d f r o m P h a r m a c i a( S w e d e n ) .

2 .2 . D e s i g n o f th e c o l u m n w i t h a x ia l c o m p r e s s i o nS E C s e p a r a ti o n o f th e B l u e D e x t r a n ( B D ) a n d

p o t a s s i u m d i c h r o m a t e ( P D ) m i x t u r e w a s p e r f o r m e don a c o lum n o f a n o r ig ina l de s ign s im i l a r t o t he onede sc r ibe d in ou r p r e v ious pub l i c a t ions [ 14 , 15 ] . Thec o l u m n w a s a p r e c i s i o n - b o r e g l a s s c y l i n d e r ( 1 0 m mI . D . ) w i th t he h igh inne r su r f a c e f i n i sh . The inne rsu r f a c e w a s c a r e f u l ly s i l a n i z e d to d im in i sh f r i c t i on .T h e k e y u n i t o f t h e c o l u m n w a s a f l o a t i n g p i s t o n( F P ) . The de s ign o f a Te f lon F P se a l m a de i t pos s ib l et o m i n i m i z e f r i c t i o n . T o c o m p r e s s t h e p a c k e d b e dc on t inuous ly , t he F P w a s sub je c t e d to t he a c t ion o fa n e x t e r na l f o r c e i n t he a x i a l d i r e c t ion . To do th i s w eu s e d a s y s t e m o f v a r i a b l e w e i g h t s . F o r c o n v e n i e n c e ,t h e w e i g h t s - F P c o n n e c t i o n w a s m a d e a s a d e t a c h -a b le j o in t .

2 . 3 . P r o c e d u r e sA c o m m e r c i a l d e x t r a n g e l ( S e p h a d e x G - 2 5 S F

gr a de ) w a s u se d . The ge l s lu r r y , p r e v ious ly sw o l l e ni n w a t e r, w a s p o u r e d i n t o t h e c o l u m n . W h e n t h ep a c k i n g h e i g h t b e c a m e c o n s t a n t t h e F P w a s i n s e r t e da n d f u r t h e r b e d c o n s o l i d a t i o n w a s a c c o m p l i s h e d b ya x ia l c om pr e s s ion w i th a n a pp r op r i a t e l oa d , t he

e l u e n t b e i n g c o n t i n u o u s l y s u p p l i e d t o t h e c o l u m n a tthe ope r a t ing f l ow - r a t e .

O p e r a t i n g c o m p r e s s i o n o f t h e g e l p a c k i n g w a s s e tup to 300 kP a . I t i s w or th m e n t ion ing tha t t he r e w e r en o e x p e r i m e n t s w i t h z e r o c o m p r e s s i o n . I t i s i m -poss ib l e t o de t e r m ine the va lue a nd d i r e c t ion o ff r i c t i on be tw e e n the F P a nd the c o lum n w a l l s a t r e s t( w h e n c o m p r e s s i o n i s c o u n t e r a c t e d b y t h e g e l p a c k -i n g ) . T h a t i s w h y t h e e x p e r i m e n t s h a v e b e e n c a r r i e dou t s t a r t i ng f r om 38 kP a c om pr e s s ion in o r de r t or e duc e the unc e r t a in ty . I n i t i a l l y t he c om pr e s s ioninc r e a se c a use s l oc a l de f o r m a t ion o f t he ge l pa r t i c l e sne a r t he F P due to a n a dd i t i ona l l oa d . I n o r de r t op r ov ide un i f o r m d i s t r i bu t ion o f t h i s de f o r m a t ion a l lo v e r t h e g e l b e d , t h e c o l u m n w a s w a s h e d o v e r n i g h t .A f t e r th e p a c k i n g h a d b e e n c o n s o l i d a te d , a s a m p l e o f2 5 0 / z l o f di s ti l le d w a t e r c o n t a i n in g 0 . 2 % ( w / v ) B Da n d 0 . 1 % ( w / v ) P D w a s a p p l i e d t o t h e c o l u m n w i t ha n in j e c t ion va lve . D i s t i l l e d w a te r e lu t ion w a s pe r -f o r m e d a t a f l o w - r a t e o f 0 . 3 m l / m i n a t a m b i e n tt e m p e r a t u r e . T h e s u b s t a n c e s w e r e s p e c t r o p h o t o m e t -r i c a l l y de t e c t e d a t 226 nm .

T h e c o l u m n d e s i g n e n a b l e d v i s u a l i n s p e c t i o n o fv a r i a ti o n s i n t h e b e d h e i g h t a n d t h e i r m e a s u r e m e n t i nthe c ou r se o f c on t r a c t ion .

2 .4 . D a t a a c q u i s i t i o nE x p e r i m e n t a l d a t a a c q u i s i t i o n w a s m a d e w i t h

D O S - b a s e d s o f t w a r e w r i t te n i n B A S I C i n o u r l a b o r a -tory .

T h e r e t e n t i o n v o l u m e o f t h e p e a k s w a s c a l c u l a t e da s t he f i r s t m om e n t , / x1 . B a n d b r o a d e n i n g w a se x p r e s s e d a c c o r d i n g t o t h e s t a n d a r d m e t h o d o fc a l c u l a t i on o f t he pe a k s e c ond c e n t r a l m om e n t , / . ~ .I n t h e c a l c u l a t i o n s , b o t h m o m e n t s h a v e b e e n c o r -r e c t e d f o r t he f i n i t e vo lum e o f t he i n j e c t e d s a m p le .The d i spe r s ion o f t he l e a d ing ( t r 2 ) a nd r e a r ( ~.2 ) pe a ke d g e s r e l a t i v e t o t h e e l u t i o n v o l u m e s o f p e a k m a x -im a , VM , ha s be e n c a l c u l a t e d a c c o r d ing to Eq . 1 a ndEq . 2 , r e spe c t ive ly :2 = ~ ] h j ( V j _ V M ) 2 /~ , h i , at V; VMJ Jw h e r e h j

(2 )a nd V j a r e t he de t e c to r r e sponse a nd the


4/9

2 0 A.V Danilov et al. / J. Chromatogr. A 732 (1996) 17 -2 5

vo lu m e c o r r e spond ing to e f f lue n t f r a c t ion j , r e spe c -t i ve ly .

The e r r o r va lue s e s t im a te d a s t he l e a s t squa r e sd e v i a t i o n f r o m r e g r e s s i o n a n a l y s is d i d n o t e x c e e d 6 %a n d 6 % f o r / . q a n d /. % r e s p e c t i v e l y i n ca s e o f B D a n d9 % a n d 5 % f o r t h e d e s cr i b e d m o m e n t s i n t he c a s e o fP D .

3 . R e s u l t s a n d d i s c u s s io n

3 .1 . M e c h a n i c a l p r o p e r t i e s a n d i n te r n a l g e o m e t r yo f t h e p a c k i n g

T h e e x t e r n a l l y a p p l i e d c o m p r e s s i v e f o r c e c a u s e sin t e r na l r e a c t ive f o r c e s i n a de f o r m e d ge l be d a nd thea s s o c i a t e d v o l u m e c h a n g e s . T h e e l a s t i c b e h a v i o u r o ft h e p a c k e d b e d m u s t d e p e n d o n t h e m e c h a n i c a lp r ope r t i e s o f ge l be a ds a s w e l l a s on the i r a r r a nge -m e n t i n spa c e a nd f r i c t i on on e a c h o the r . F ig . 1s h o w s t h e e x p e r i m e n t a l r e l a t i o n s h i p b e t w e e n t h e g e lb e d h e i g h t L a n d t h e p r e s s u r e a p p l i e d . T h o u g h apa c ke d be d i s no t a so l id , i t s m e c ha n ic a l p r ope r t i e sa ppe a r t o be s im i l a r t o t hose o f e l a s t i c so l id s j udg ingby the c u r ve , soF / S = P -~ - E A L / L o , (3 )w he r e S i s t he c o lum n c r os s - se c t ion a r e a , F a nd P

20 0

1 5 0 ~ B1 0 0 o 1 ~ o 2 ~ o 3 ~ o

p r e s s u r e [kPa]F i g . 1 . B e d h e i g h t v e r s u s a x i a l c o m p r e s s i o n : t h e l i n e c o r r e s p o n d st o l i n e a r r e g r e s s i o n f i t .

a r e t he f o r c e a nd p r e s su r e o f c om pr e s s ion , r e spe c -t i ve ly , a nd A L / L o i s t he r e l a t i ve de f o r m a t ion o f t hege l pa c k ing . C a lc u l a t e d by Eq . 3 , t he m odu lus o fe l a s t ic i t y , E , i s a pp r ox im a te ly e qua l t o 9 . l 05 N m -2( f o r c o m p a r i s o n t h e c o r r e s p o n d i n g v a l u e f o r r u b b e ri s a n o r de r o f m a gn i tude h ighe r ) .

I t i s w or th m e n t ion ing tha t t he ge l be d e xpe r i e nc e sde f o r m a t ion g r a dua l ly . I t ha s t a ke n a l ong t im e( a bou t s e ve r a l hou r s ) f o r t he ge l be d to s t a b i l i z e a f t e rthe c om pr e s s ion inc r e a se i n t he c ou r se o f t he de -s c r ib e d e x p e r i m e n t . S i m i l a r p h e n o m e n a t a k e p l a c e i nr ig id pa c k ing m a te r i a l s [ 8 ] . Thus da m pe d s t a b i l i z a -t i o n o f a n a n g u l a r 1 0 - / x m s i l i c a p a c k e d b e d w a so b s e r v e d o v e r 1 0 0 h . T h e r e a r r a n g e m e n t o f t h e b e du n d e r c o m p r e s s i o n d u e t o t h e c o l la p s e o f t h e u n -s t a b l e r e g ions c r e a t e d du r ing the pa c k ing s t e p p r o -v ide s a n e xp la na t ion f o r suc h da m pe d s t a b i l i z a t i on .D e f o r m a t i o n o f r i g i d s i l i c a - b a s e d p a c k i n g s u p o na x ia l c om pr e s s ion s e e m s to be i r r e ve r s ib l e ; i nd i r e c te v i d e n c e c a n b e f o u n d i n t h e d a t a o f W u a n d L o h s e[ 10 ] . The y r a n a n e xpe r im e n t t o e luc ida t e t he i n -f lu e n c e o f s u c c e s s i v e l y d e c r e a s e d c o m p r e s s i o n o n t h ec o l u m n p e r f o r m a n c e o f s i l i c a p a c k i n g . N o e f f e c t o nt h e c o l u m n e f f i c i e n c y a n d b a c k - p r e s s u r e w a s o b -s e r v e d w i t h a f o u r - f o ld d e c re a s e i n c o m p r e s s i o n f r o mi ts m a x i m u m v a l u e , i n d i c at i n g t h a t th e b e d w a s v e r ys table .I n c on t r a s t , a n i nc r e a se i n t he be d he igh t w a so b s e r v e d u p o n i n s t a n t a n e o u s S e p h a d e x p a c k i n g d e -c o m p r e s s i o n ( f r o m 2 8 0 k P a t o z e r o ) , t h o u g h t h ep r o c e s s w a s s l o w . T h e r e s i d u a l d e f o r m a t i o n a f t e rc o m p l e t e r el a x a t io n w a s a p p r o x i m a t e l y 5 0 % o f t h ein i t i a l va lue ( t ha t o f de f o r m a t ion a t 280 kP a ) .The r e f o r e pa r t i a l r e ve r s ib i l i t y o f t he c om pr e s s ione f f e c t on so f t pa c k ing m a te r i a l s suc h a s de x t r a n -b a s e d o n e s h a s b e e n o b s e r v e d . M i c r o s c o p i c s t u d i e so f s a m p l e d g e l b e a d s d i d n o t r e v e a l a n y s u b s t a n t i a lc ha nge s i n t he i r sphe r i c a l f o r m a nd the qua n t i t y o fd e s t r o y e d b e a d s d i d n o t e x c e e d 1 - 2 % o f th e i r t o ta lqua n t i t y .

I n v i e w o f B D t o t a l e x c l u s i o n f r o m t h e g e lpa r t i c l e s o f S e pha de x G - 25 , i t s r e t e n t ion vo lum e(/x ) m u s t c o r r e s p o n d t o t h e m o v i n g m o b i l e p h a s ec o l u m n v o l u m e , V . T h e r e te n t i o n v o l u m e o f t o t a ll ypene t ra t ing PD ( /z t l ) i s equa l to the to ta l l iquid phasec o lum n vo lum e , V t , a c c o r d ing ly . Thus t he i nne rv o l u m e o f g e l p a c k i n g V i s e q u a l t o / J , t1_ p o . T y p i c a lc u r v e s c o r r e s p o n d i n g t o V , V a n d V u n d e r p a c k i n g


5/9

A.V. Danilov et al. / J. Chromatogr. A 732 (1996) 17-25 212 0 -

1 5 -

E 1 0 -o

5 -

B

C

i i i1O 0 2 0 0 3 0 0p re s s u re [k Pa ]

Fig. 2. Effect of axial compression on SEC retention volumes (A)V,,, (B) V~, (C) V=V,-Vo.

for maximum packing density of incompressiblespheres of equal size (rhombohedral and face-cen-tered cubic array).

Sephadex compression may be supposed to resultnot only in the rearrangement of the packed bed intoa tighter array but also in the deformation of theparticles themselves. The latter phenomenon is sup-ported by the decrease in the V value (at pressuresabove 200 kPa) and reversibility of bed deformation.Slow and incomplete restoration of the bed heightcan be attributed to the generation of a new packingstructure more compact than the initial one and alsoto a residual deformation of beads that persists underthe action of inter-particle friction. Unfortunately, theprecise determination of the prevailing process upona compression of particular value is not possible withthe data obtained.

compression are shown in Fig. 2. While the decreasein V is almost linear with the rise in P, the decreasein V starts at more than 200 kPa compression. It isrepresented by the sigmoid shape of the V /Vo curve(Fig. 3), V~/V being the ratio of pore volume to voidvolume.

The second curve in Fig. 3 also illustrates thevariation of another dimensionless parameter Vo/V ,that is almost equal to the inter-particle porosity ofgel packing. The V /V~ value varies from 0.40 to 0.25with the increase in compression from 38 to 280 kPa.The resulting porosity value corresponds to the value

3.2. Band broadening upon packi ng compress ion

The elution profiles in Fig. 4 corresponding to theinitial and maximum values of Sephadex packingcompression indicate a decrease in retention volumesin parallel with the reduction in the width of peaks.The influence of the packing compression on bandbroadening is depicted in Fig. 5.Analysis of the data presented in Fig. 2 and Fig. 5suggests different results for compression in the case

0 . 4 '

0 .2 "

Vo / V t

V / V 0

0.0 00 1 ; 0 2 ; 0 3 0 0pressure [kPa]

Fig. 3. Effect of axial compression on packing parameters~/VoandVo/V~.

0 . 0 0 S.O0 30.00

B A

1 0 . 0 0 1 5 . 0 0 2 0 . 0 0 2 5 . 0 0e l u t i o n v o l u m e [r o l l

Fig. 4. Elution profiles obtained upon 38 kPa (A) and 280 kPa (B)compression pressure.


6/9

2 2 A.~ Danilov et al. / J. Chromatogr. A 732 (1996) l 7- 25

0.50-

0,25'

0.000

3

2

B Di / 11 0 0 2 0 0 3 0 0

p r e s s u r e [ k P a ]F i g . 5 . P e a k v a r i a n c e v e r s u s a x i a l c o m p r e s s i o n .

under consideration (SEC on soft packing) and inchromatographic separations on silica-based pack-ings. As SEC separation is caused by the distinctionin distribution of substances between mobile andstagnant liquid phases, volume variations of the latterupon compression influence the efficiency of themethod significantly. That was the case in ourexperiment, as /z 1 and P-2 variations were comparableto their initial values in order of magnitude (61% and75% of their initial values for BD and 38% and 23%for PD respectively). The retention variation as suchupon axial or radial compression of rigid silica-basedparticles is not reported in the literature. Thus thenegligibility of this effect can be indirectly deducedfor the latter case (see Ref. [10] for example).

As already discussed (see Fig. 2), the drop in the/z 1 value results largely from the decrease in Vrather than in V~. To a first approximation, observednarrowing of peaks can be related to the reduction intheir retention (which is rather significant for thecase under discussion). The difference in the dy-namics of the /22 decrease for BD and DP isdetermined by a variation in the packed bed ef-ficiency, i.e. the number of theoretical plates, N, orthe height equivalent to the theoretical plate, H, asp2=#~/N or I.t.2=Ix~H/L.The corresponding varia-tions in N and H values for both substances areshown in Fig. 6 and Fig. 7, respectively. A differentpattern of variation of these parameters for BD andPD is readily apparent. An essentially uniformdecrease in N is observed for BD in the range of

100-

E =

5 0 "o .

P D

a~0'1 0 0 2p r e s s u r e [ k P a ]

F i g . 6 . N u m b e r o f t h e o r e t i c a l p l a t e s v e r s u s c o m p r e s s i o n p r e s s u r e :t h e l in e s c o r r e s p o n d t o s e c o n d ( B D ) a n d t h i r d ( P D ) p o w e rp o l y n o m i a l r e g r e s s i o n f i t s .

compression pressure from 38 to 200 kPa, and thisdecrease becomes slower on further pressure eleva-tion. In contrast, the slope of the N curve for PDincreases at P>200 kPa. It should be noted that thevariation pattern of the H value also changes whencompression pressure is elevated above 200 kPa.

In order to consider the observed differences in theplots of H versus P for BD and PD the well knownGiddings expression [16] can be used. For SEC, theexpression for H will be (thus sorption is supposedto be absent):

4 -

3"EE

,1- :1D

BD

p r e s s u r e [ k P a]F i g . 7 . H E T P v e r s u s c o m p r e s s i o n p r e s s u r e .


7/9

A.V. Dan ilov et al . / J . Chrornatogr. A 732 (1996 ) 1 7- 25 23H = H L + H sM + HM =

D M d p u d ~= b u + CSM 1+ 1 / a d . + DM/CMI.,td~ ( 4 )w h e r e H L , HSM , a n d H M a r e t h e p l a t e h e i g h t c o n t r i -b u t i o n s d u e t o l o n g i t u d i n a l - d i f f u s i o n , s t a t i o n a r y - m o -b i l e - p h a s e , a n d i n t r a - p a r t i c l e m o b i l e p h a s e m a s st r a n s f e r p r o c e s se s , r e sp e c t i v e l y ; u i s t h e l i n e a r f l o w -r a t e ; d p i s th e d i a m e t e r o f t h e p a c k i n g p a r t i c le s , D Ma n d D S M a r e so l u t e - d i f f u s i o n c o e f f i c i e n t s c o r r e -s p o n d i n g t o e x tr a - p a r ti c l e a n d s t a g n a n t m o b i l e p h a s e ,r e sp e c t i v e l y ; a , b , C SM a r e t h e c o e f f i c i e n t s o f t h er e s p e c t i v e d i s p e r s i o n t e r m s , t h e i r m a g n i t u d e s b e i n gg e n e r a l l y a f u n c t i o n o f t h e n a tu r e a n d t h e g e o m e t r yo f p a c k i n g a n d i ts p o r e s t ru c t u re .O w i n g t o a sm a l l D M , H L c o n t r i b u t i o n t o t h eo v e r a l l p l a t e h e i g h t i s n e g l i g i b l e f o r B D . B e s i d e s , t h el i n ea r f l o w - r a t e o f th e m o b i l e p h a s e i n c r e a se s w i t ht h e r e d u c t i o n i n p a c k i n g p o r o s i t y w h e n c o m p r e s s i o ni s e l e v a t e d , t h i s r e su l t s i n a d e c r e a se i n t h e H L v a l u e .A s d e x t r a n d o e s n o t p e n e t r a t e i n t o t h e p o r e s o fp a c k e d b e a d s , t h e c o r r e s p o n d i n g H S M v a l u e i s e q u a lt o z e r o , a n d , i n e s s e n c e , i t s p e a k b r o a d e n i n g d e p e n d so n l y o n i ts d i s p e r s i o n c a u s e d b y i n t er - p a rt i cl e m o b i l ep h a s e m a s s t r a n s f e r p r o c e s s e s , d e s c r i b e d b y t h e H Mt e r m o f E q . 4 .H M i s a f u n c t i o n o f t h e l i n e a r f l o w - r a t e , u , a n d o ft h e p a r t i c l e m e a n d i a m e t e r , a l p. I n th e c o n t e x t o f th er a n d o m w a l k t h e o r y , t h e d p v a l u e i n t h e H M t e r m i su se d t o r e p r e se n t t h e c h a r a c t e r i s t i c d i s t a n c e s c o v e r e db y v a r i o u s e x c h a n g e p r o c e s s e s b e t w e e n v e l o c i t ye x t r e m e s i n t h e m o b i l e p h a s e . T h e m a g n i t u d e o ft h e se d i s t a n c e s i s d e t e r m i n e d b y t h e p a r t i c l e s i z e a sw e l l a s b y a v e r a g e d d i s t a n c e s b e t w e e n t h e i r s u r f a c e sa n d / o r c e n t r e s . B e d c o m p r e s s i o n r e s u l t s i n a t i g h t e rp a c k i n g t h a t i s e q u i v a l e n t t o t h e r e d u c t i o n i n t h ea p p a r e n t d p v a l u e i n t h e H M t e r m o r t h e c o r r e -s p o n d i n g v a r i a t io n i n t h e v a l u e s o f a a n d c Mc o e f f i c i e n t s ( p r o v i d e d t h e p a r t i c l e d i a m e t e r i s c o n -s t a n t ) . I t i s v e r y d i f f i c u l t t o e s t i m a t e t h i s e f f e c tq u a n t i t a ti v e l y b e c a u s e o f th e c o m p l e x i t y o f t h ed e sc r i p t i o n c o n s i s t e n t w i t h v a r i a t i o n s i n t h e b e ds t r u c t u r e . I t i s w o r t h m e n t i o n i n g t h a t i t h a s t o r e su l ti n t h e d e c r e a se i n t h e H M c o n t r i b u t i o n t o t h e o v e r a l lp l a t e h e i g h t w i t h u h e l d c o n s t a n t . A l t e r n a t i v e l y , t h ef l o w - v e l o c i t y i n c r e a s e c o r r e s p o n d i n g t o t h e o p e r a t i n gc o n d i t i o n s u n d e r d i s c u s s i o n i s k n o w n t o c a u s e t h e

H M r i se . T h e t o t a l e f f e c t o f t h e se o p p o s i t e t e n d e n c i e sw h i c h a r e d e p e n d e n t o n s e v e r a l p a r a m e t e r s c a n n o t b ep r e d i c t e d e a s i l y . A s s h o w n i n F i g . 7 , t h e s e t e n d e n -c i e s c o m p e n s a t e f o r e a c h o t h e r i n th e c a s e o f B D ,a n d H r e m a i n s p r a c t i c a l ly c o n s t a n t u p o n c o m p r e s -s i on . T h e o b s e r v e d H v a r i a ti o n s a r e c o m p a r a b l e t ot h e e r r o r v a l u e .

I n t h e c a s e o f P D , H E a p p e a r s t o c o n t r i b u t e m o r em a r k e d l y t o t h e o v e r a l l p l a t e h e i g h t t h a n i n t h e c a seo f B D , t h e d i f f u s i o n c o e f f i c i e n t o f P D b e i n g s e v e r a lo r d e r s o f m a g n i t u d e g r e a t e r t h a n t h e d e x t r a n D M . F o rt h e s a m e r e a s o n t h e p a t t e r n o f H M v a r i a ti o n f o r P Du p o n b e d c o m p r e s s i o n c a n d i ff e r f r o m t h a t o f B D .B e s i d e s , d i s p e r s i o n d u e t o t h e s t a t i o n a r y - m o b i l e -p h a s e m a s s t r a n s f er p r o c e s s ( H s M t e rm ) c o n t r i b u te se s s e n t ia l l y t o H i n th e c a s e o f P D . T h e e x a c te x p r e s s i o n f o r HSM a c c o r d i n g t o t h e n o n e q u i l i b r i u mt h e o r y [ 1 7 ] m a y b e u s e d f o r th e d e s c r i p t i o n o f t h eS E C s t a t i o n a r y - p h a s e d i s p e r s i o n i n t e r m s o fg e o m e t r i c a l p a r a m e t e r s o f t h e g e l p a c k i n g

ud~ KsEcVi/V ttd2pHSM = CSM DSM -- 3 0 ( 1 + KsEcWi/V0)2 DSM (5)w h e r e K sE c i s t h e S E C d i s t r i b u t i o n c o e f f i c i e n t . F o rPD, KSEc = 1 , sO i f u an d dp a re con s tan t , t he HSMvalu e i s p r op or t io na l to the (V~/Vo) / ( I +V~/V0) 2 ra t ioa n d m u s t r e d u c e w i t h t h e i n c r e a se ( s e e F i g . 3 ) o f t h ep a r a m e t e r V i / V . I n o r d e r t o t a k e i n t o a c c o u n t t h ei n f l u e n c e o f th e f l o w - v e l o c i t y i n c r e a se o n H SM it isc o n v e n i e n t t o r e la t e t h e f o r m e r v a l u e t o f l o w - r a t e a n db e d p o r o s i t y . S i n c e V o / Q = L / u , w h e r e Q i s t h e f l o w -ra t e , and Vco~= L S , where V~o~ i s t h e c o l u m n v o l u m e ,t h e n u i s e q u a l t o QVco1/SV . Or, w i th Vco~V~, l i nea rf l o w v e l o c i t y m a y b e t a k e n t o b e a p p r o x i m a t e l yp r o p o r t i o n a l t o V~/V o r ( 1 + V ~/ Vo ) w i t h a n a c c u r a c yo f Q / S c o n s t a n t . T h u s i f Q a n d d p a r e k e p t c o n s t a n t ,t h e H S M v a l u e i s p r o p o r t i o n a l t o t h e r a t i o (Vi /Vo) /( 1 + V ~/ Vo ) a n d m u s t i n c r e a se w i t h a V i / V r i se . F ig . 8d e p i c t s t h e c u r v e s i l l u s t r a t i n g v a r i a t i o n s i n ( V ~ / V o ) /( l + V i / V 0 ) 2 a n d ( V i / V o ) / ( l + V i / V o ) r a t i o s u n d e rc h a n g e s o f t h e V~ /V p a r a m e t e r w h e r e t h e p o i n tsm a r k t h e i r v a l u e s c o r r e s p o n d i n g t o t h e e x p e r i m e n t a ld a t a o n V i / V . T h e c o n d i t i o n o n d p c o n s t a n c y i s v a l i di n t h e p r e s s u r e r a n g e 0 - 2 0 0 k P a w i t h V a p p r o x i -m a t e l y e q u a l t o a c o n s t a n t . A s c o m p r e s s i o n i n c r e a s e sf u r t h e r , V~ d i m i n i sh e s i n a w a y t h a t m a y b e c o r r e l a t e dw i t h t h e d e f o r m a t i o n o f p ar t ic l e s a n d t h u s w i t h


8/9

24 A .V . D a n i l o v e t a l . / J. C h r o m a t o g r . A 7 3 2 ( 1 9 9 6 ) 1 7 - 2 5

1.0

0.5

38kPa 280kPa

B

f

0 ,0 , I J I . . ,2 3V i / V o

Fig. 8. Curv es i llus tra ting vari ation in (A) (Vi/V~0/(1 +VJVo) 2 and(B) ( V ~ / V o ) / ( I + V f l V o ) The points mark their values corre-sponding to the experimental data on V,/V .

va r i a t i ons i n d v a nd p r oba b ly i n D S M . The c om bine de f f e c t o f t he va r i a t i ons o f a l l t he se pa r a m e te r s onH S M c a nno t be c ons ide r e d p r ope r ly w i th in t he c on -t e x t o f t he a pp r ox im a t io n invo lve d . A s a r e su l t o f t heo p p o s i t e l y d i r e c t e d c h a n g e s o f s e p a r a t e c o m p o n e n t sthe ove r a l l H c u r ve f o r P D a ppe a r s t o be o f a r a the rc om ple x sha pe ( s e e F ig . 7 ) .T h e a b o v e c o n s i d e r a t io n o f t h e i n fl u e n ce o f c o m -p r e s s ion on the p l a t e he igh t sugge s t s t ha t r e so lu t ionc a n b e f u r t h e r i m p r o v e d b y h o l d i n g t h e f l o w v e l -oc i ty , u , c ons t a n t ( a t l e a s t f o r subs t a nc e s w i th ane g l ig ib l e c on t r ibu t ion to H ETP due to l ong i tud ina ld i f f u s ion ) . B u t t he s e pa r a t ion t im e w i l l i nc r e a se i nth i s c a se , so t he u se r ha s t o c hoose be tw e e n r e s -o lu t ion a nd se pa r a t ion spe e d .

3.3. Peak asymmetry and resolutionA s show n in . F ig . 4 , s i gn i fi c a n t a sym m e t r y i s

obse r ve d f o r bo th subs t a nc e s . I t s va lue , e xp r e s se d inte rm s of the t r/~ - ra t io , dec rease d su bs tan t ia l ly (1 .84t i m e s ) f o r B D a n d r e m a i n e d c o n s t a n t w i t h i n t h ee x p e r i m e n t a l e r r o r f o r P D w i t h i n c r e a s i n g p r e s s u r eon the pa c k ing ( s e e F ig . 9 ) . I t i s im poss ib l e t oe xp la in t h i s e f f e c t i n t he c on te x t o f t he s im p l i f i e dr a n d o m w a l k t h e o r y . P e a k t a i l i n g m a y b e a t t r i b u t e d

to d i s c on t inu i t i e s i n t he f l ow pa t t e r n , w h ic h a r er e d u c e d t o a m i n i m u m d u e t o b e d c o n s o l i d at i o n u p o nc o m p r e s s i o n . T h i s c o n c l u s i o n i s s u p p o r t e d b y t h ef a c t th a t t he a s y m m e t r y d e c r e a s e i s o b s e r v e d o n l y f o rsubs t a nc e s e xc lude d f r om the i n t r a - pa r t i c l e vo lum e .T h e e x p r e s s i o n f o r r e s o l u t i o n c o r r e c t e d f o r p e a ka s y m m e t r y ( R [ ) h a s b e e n u s e d f o r th e e s t i m a t io n o ft h e s e p a r a t i o n q u a l i t y o f B D a n d P D :

t 0V M - - V M (6 )R s - ~ + % '

w h e r e V a n d V ~ a r e th e e l u ti o n v o l u m e s o f th e B Da n d P D p e a k m a x i m a , r e s p e c t i v e l y .

The in f lue nc e o f c on t r a c t ion o f t he so f t ge lpa c k ing on pe a k se pa r a t ion i s de p ic t e d in F ig . 10 .T h e c u r v e h a s a m a x i m u m i n t h e 2 0 0 - k P a r e g i o nw he r e t he r e so lu t ion e xc e e ds i t s i n i t i a l va lue 1 . 25t i m e s . T h e o b s e r v e d v a r i a t io n i n t h e g i v e n p a r a m e t e rc a n b e e a s i l y e x p l a in e d . D i s p e r s io n o f th e c h r o m a t o -g r a ph ic z one s de c r e a se s ( s e e F ig . 5 ) w h i l e t her e te n t i o n d i f f e r e n ce r e m a i n s a p p r o x i m a t e l y c o n s t a n t

T r O t 0 ~ ra s V M - - V M ~ - ' [ t K I - I , Z l m V i .I f t he d i s t r ibu t ion c oe f f i c i e n t doe s no t a l t e r s i g -n i f i c a n t ly unde r c on t r a c t ion o f t he ge l be d ( i n t her a n g e w h e r e V ~ c o n s t a n t ) t h en , w i t h th e c o n s tr a i n t o fp e a k n a r r o w i n g , s i m i l a r p h e n o m e n a m a y b e s u p -p o s e d t o h o l d a l s o f o r s u b s t a n c e s w i t h 0 < K s z c < ls inc e i n t h is c a se t he / z ] + l - / x ~ d i f f e r e nc e e qua l t o V~( k . j + l j ~ - j, , S EC -- .,S EC , a l so r e m a in s a pp r o x im a te ly c ons t a n t .

PDi0 100 200 300

p r e s s u r e [kPa]Fig. 9. Effect of axial compression on peak asymmetry.


9/9

size-exclusion chromatography on soft packing material under axial compression

Documents