Gradient Thin-Layer Chromatogr

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Gradient Thin-Layer Chromatography of P lant Extracts

G . M a t y s i k / E . Soczewi~ski*Depar tme nt o f I no r gan i c and Ana l y t i ca l Chem is t r y , Med i ca l Academy, S tasz ica 6 , 20- 81 Lub l i n , Po land

Ke y W o rd sT h i n - l a y e r c h r o m a t o g ra p h yStepw ise g r ad ien t e l u t i onChamber f o r g r ad ien t TLCDens i t omet r y o f i soc r a ti c and g r ad ien t ch r omatogr ams

S u m m a r yA mo di f ied Niederwieser cham ber for s tepwise gradiente lut ion, consist ing of a PTFE capi l lary to store the ser iesof e luents and a hor izo nta l g lass sandwich cham ber wi tha g l ass d i s t r i bu to r was used f o r t he c hr omatogr aph i cseparat ion of complex p lant ext racts (Seboren, Hemor i -gen and Pectosol) used in therapy. Densi tograms de-mon st rate ma rked ly improv ed separations o f the ex-t racts in compar ison to isocrat ic e lut ion.

I n t ro d u c t i o nGr ad ien t e l u t i on i n t h i n - l ayer ch r omatog r aphy can beobtaine d in several ways [1 ] :1 . Mu l t i zona l deve lopmen t : t he use o f mu l t i comp one nt

eluents which are pa r t ia l l y separated dur ing deve lop-ment ( f r on ta l ch r omatogr aphy) , f o r m ing an e l uen tst rength gradient a long the layer .

2. Dev elopm ent w i th a st rong solvent (e.g. , acetone) ofan ads orbent layer exposed to vapou rs of less polarsolvents (e.g. , benzene) in Var io-KS , De Zeeuw orKaiser type chambers.

3. T he use of mix ed layers of vary ing surface area, ac t iv i tyetc.

4. D el ive ry of an e luent whose co mp osi t ion is var ied in acon t i nuous o r s t epw ise manner [ 1 - 7 ] .

In recent papers [8, 9] a s imple chamber was used forg r ad ien t TLC, r epr esen t i ng a mod i f i ca t i on o f t he N ieder -wieser -Honegger device [2, 3] . The more conv enient newvers ion [9 ] consists of a hor izo nta l g lass cham ber wi th aglass d is t r ibuto r , a long PTFE tub ing to store the e luent

*) Produced by Polish Reagents, Melgiewska 18, 20 --234 Lublin,Poland

f ract ion s and a sy r inge at tached to the base plate. Th~plunger o f the syr inge can be dr iven in two di rect ions by~screw in a nu t at tached to the b ase plate. The dif feren~is in the d ist r ib uto r geo me t ry [9] : in our chamber th~eluen t i s del ivered f ro m the ed ge of the th in layer ~:tha t zonal app l icat ion of the sample is possib le. Anigradient program, two - or m ul t i co mp on ent , i s producecby int roduc ing smal l vo lum es of e luent f ract ions (e40.2 ml) int o a ser ies of sma l l test tubes and the n suckin!t hem i n to t he PTFE cap i l l a r y .

Exper imenta lSandwich chamber f o r g r ad ien t TLC * ) was used w i th !3-m PTF E co il (1. D. = 1.2 mm ) a ttach ed t o a glass syrin;edr iven by manual ly rotated screw [9] . The precoated plate55 0X 100 mm (Si l i ca 60, E. Me rck, D armstad t , FR G) with~1-cm wide margin cleaned of adsorbent, were pre-wettecwi th one d ist r ib uto r v olum e (0.1 ml ) of the f i rs t eluer,:f rac t ion (10 % ethyl acetate in chloro form ) and the sample!were spotted on the star t l ine. Three medicinal preparat ions(Herba pol , Poland) were used as com plex samples.a) Seboren (Fru ctus Past inacae, Ra dix Bardanae, Radlx

Ur t icae , R h i zoma Ca lami ) ,b) Hem or igen (Er igeron Canadensis) ,c) Pectosol (R ad ix Inula e, Lichen Islandicus , Radix Sap0na

r iae, Herba Hy ssopi , Herba Thym i )oSam ple Preparation:a) Seboren: 250 g was evaporated, the oi ly residue treatee

w ith 20 ml of eth yl acetate, f i l te red an d evaporatecagain. 0.59 5 g of the d ry residue was dissolved in 3 rnl0"eth yl acetate.

b) Hemor igen : 150g was evaporated and the oi ly residuet reated wi th 15 ml of eth yl acetate, f i l tered and eva.pora ted again. 0.18 g o f th e dr y residue was dissolved :3 ml of ethyl acetate.

c) Pectosoh 30g was evaporated and the oi ly residuet reated wi th 9 ml of ethy l acetate, f i l tered and eva.porated. 0.0 4g of the d ry residue was d issolved i :1 ml of e thy l acetate.

The samples conta ined non volat i le , m odera te ly p01a:com pone nts of the preparat ions; 4-/~1 samples of the~

178 Chrom atographia, Vo l. 26 (198-

0009-5893/88 0178-03 ~ 03.00/0 9 198 8 Friedr. Viewe g & S ohn Verlagsgesellschaftmb H


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s olu tio ns w e r e s p o t t e d . T h e c h r o m a t o g r a m s w e r e d e v e l o p e din t he i s o cr a ti c m o d e ( 1 : 1 e t h y l a c e t a t e c h l o r o f o r m ) a n dw ith a l in e a r g r a d e i n t ( 1 0 - 2 0 - 3 0 - 4 0 - 5 0 - 7 0 % e t h y l a c e t a t e i nc h lo r o fo r m , i n t r o d u c e d i n t o t h e c o i l i n t h e o p p o s i t e o r d e r ) .

F o r d e v e l o p m e n t o n a 9 -c m d i s t a n c e , s i x 0 . 0 8 - m l f r a c t io n so f t h e e l u e n t w e r e n e e d e d .T h e d e n s i to g r a m s w e r e o b t a i n e d u s i n g a S h i m a d z u C S - 9 3 0d e n s i t o m e t e r a t 2 5 4 n m .

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Fig, 1D e n s i to g r a ms ( 2 5 4 n m ) o f Se b o r e n : F ig . l a : i s o c ra t ic e lu t io n ,1 : 1 e t h y l a ce t a te + c h lo r o f o r m ; F ig . lb : s t e p w is e g r a d ie n t e lu t io n .( 1 0 -2 0 - 30 - 4 0 -5 0 - 70 % e t h y l a c e t a te in c h lo r o f o r m ) .

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1 0 . 0 ~ [ 1 . 0 I b , 3 . 0 : i ; . " , ' :.Fig. 2D e n s i to g r a ms ( 2 5 4 n m ) o f H e m o r ig e n . F ig . 2 a : i s o c ra t ic e lu t io n ,1 : 1 e t h y l a c e t at e + c h lo r o f o r m; F ig . 2 b : s t e p w is e g r a d ie n t e lu t io n .( 1 0 - 2 0 - 3 0 - 4 0 - 5 0 - 7 0 % e t h y l a c e ta te i n c h l o r o f o r m ) .

Chromatographia,V o l . 2 6 ( 1 9 8 8 ) 179


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F i g . 3Densi tograms (254 nm) o f Pectoso l . Fig . 3a: isocrat ic e lu t ion,1 : 1 e thy l acetate + ch loro form ; Fig . 3 b : stepwise grad ien t e lu t io n.( 10-20-30-40-50-70 % eth yl acetate in ch loro form ).

Results and DiscussionT h e c h r o m a t o g r a m s v i e w e d u n d e r U V l i g h t de m on st ra te da m u c h b e t t e r s e p a r a t i o n f o r t h e g r a d i e n t e l u t i o n mode( l a r g e r n u m b e r o f b e t t e r d e f i n e d z o n e s ) . T h e e st im a tio nw a s c o n f i r m e d b y c o m p a r i s o n o f d e n s i t o g ra m s o f is~c r a t i c ( F i g . l a - 3 a ) a n d g r a d i e n t c h r o m a t o g r a m s ( F igl b - 3 b ) . T h e n u m b e r s o f p e a k s d i s ti n g u i s h e d b y th e in t~g r a t o r w e r e ( f o r i s o c r a t i c a n d g r a d i e n t e l u t i o n , r e sp ec tiv el y ) : f o r S e b o r e n , 7 a n d 1 7 ; f o r H e m o r i g e n , 1 1 an d 12( h o w e v e r , t h e p e a k s f o r g r a d i e n t e l u t i o n , F i g . 2 b , a re m uchh i g h e r a n d s h a r p e r ) a n d f o r P e c t o s o l , 7 a n d 1 1 . D u e t ot hec o m p l e x c o m p o s i t i o n o f t h e e x t r a c t s , s e p a r a ti o n wa:o b v i o u s l y i n c o m p l e t e ; h o w e v e r , t h e s h a r p ne s s o f t h e zonesu n d e r U V l i g h t a n d t h e m u c h b e t t e r d e n s i t o g r a m s in dic ate dt h a t i n t h e c a se o f g r a d i e n t e l u t i o n t h e a n a l y s i s o f t h e mainc o m p o n e n t s i s m a r k e d l y b e t t e r ; a l s o , i s o l a t i o n o f t h e zonesf r o m c h r o m a t o g r a m s l b - 3 b w o u l d r e s u l t i n s im p l e frac.t i o n s .

ConclusionsS t e p w i s e g r a d i e n t e l u t i o n c a n b e r e a l i z e d i n re la ti ve lys i m p l e a n d i n e x p e n s i v e s a n d w i c h c h a m b e r s w i t h glassd i s t r i b u t o r a n d a P T F E c o i l t o s t o r e t h e s e ri es o f el ue nt sT h e s e p a r a t i o n e f f i c i e n c y is m u c h b e t t e r t h a n i n is oc ra ticd e v e l o p m e n t d u e t o t h e e l i m i n a t i o n o f t h e " g e n e r a l e l u t i 0 ;p r o b l e m " , c o m p r e s s i n g e f f e c t o f t h e g r a d i e n t a n d en ha nc edm u t u a l d i s p l a c e m e n t o f t h e s o l u te s , e s p e c i a l l y e f f e c ti v e f0~m o d e r a t e ( ~ 1 ) k ' v a l u e s .T h e f i n a l R F v a l u e s f o r a g iv e n g r a d i e n t p r o g r a m c an bec a l c u l a t e d a n d t h e m i g r a t i o n p a t h s o f th e i n d i v i d u a l com .p o n e n t s s i m u l a t e d w i t h a p e r s o n a l c o m p u t e r , w h i c h faciF i.t a t e s th e o p t i m i z a t i o n o f t h e g r a d i e n t p r o g r a m f o r d iff ic u lts a m p l e s [ 6 ] .

Acknow l edgemen t sT h a n k s a r e d u e t o D r . W . H o l s k a , t h e h e a d o f t h e D ep a~ .m e n t o f A n a l y t i c a l C h e m i s t r y o f T a r c h o m i n P ha rrn ~.c e u t i c a l P l a n t s " P o l f a " a n d t o M r s . A . K u l i 6 s k a , f o r th eirh e l p i n t h e d e n s i t o m e t r i c a n a ly s is .

References[ 1 ] F . Ge iss , Fundamenta ls o f Th in Layer Chromatography,H0th ig , Heide lberg 1987; pp. 346 and 395.[2 ] A. Niederwieser, C. G. Honegger, Adv. Chromatogr. (N.Y.) ,2 , 123 (1969).[3 ] A. N iede rw iese r , Chromatographia 2 , 362 (1969).[4 ] G. Matys ik, E. Soczewi~ski , J. Chromatogr. 369, 19 (19861.[5 J K. Glowniak, G. Matysik, M. Bieganowska, E. Soczewi~sk~,Ch romatographia 22, 307 (198 6).16 ] W. Mar kows ki , E. Soczew i4ski , G. Matysik, J. Liq.Chromatogr. 10, 1261 (1987).17 ] J. Pick, J. L iq . Chromatogr. 10, 182 (1987).[8 ] G. Matysik, E.Soczewit }sk i , J. Chromatogr. 446, 275 (1988).191 E. Soczewi6skL G. Maty sik, J. Planar Chrom atogr. in press.

Received: Nov. 1, 1988Acc epte d: No v. 10, 1986A

180 Chromatographia, Vol. 26 (1988

Gradient Thin-Layer Chromatogr

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