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The separation and determination of glycolic and malic acids by ion-exchange chromatography
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"1 i-112 c ; E P A R A T I O N ANI) DE-IIZRMINAIION OF GLYL'OLIC A C I D S BY I O N - L X C H A N G I _ CHI~OMATOGRAPFIY
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of the c,ubox~l~c acids on tlc by thelr ~c:lctmn with 1ocltclc IocI,1tc ,tnd :wnylasc BLIIci~ t,I ill ~' scp~ll,ltcd o l g , l n i c d c l d s f r o m p l a n t tls,,uc o n ,i ,,dlca gel sttlfut ic ; i c l d m 3 p r e g n d t e d c o l u m n S e v e r a l m%'c~ttg,ltors h,lvc stucllcd t h e s c p a r d t l o n o f t h e ,|cid,, w l t h , i n ~ o n - c x c h , l n g e Ic,,m~ l-t,lrlo~ a n d M o r m a n " u~cd x~atcl to c i u t c , ; o m c 50 , I c t d s rl O111 ~.111, i n l o n - c x c i l a r l g c c o l t t m n w l t h n o p r c t l g~.t11111211t S , i i l l u c l s o n d n d T h c d c s u~,cd a n a c c t , l t c f o r m o f rc,,tn w i t h ,i s o d i u m a c e t a t e c l u a n t for t h e n ,~tudy T h r e e d c l d t t t o n a l ,,tud~cs '~ t~ w e r e m a d c ~ tth t h e a c e t a t c ic,,m f o r m . t h e first t w o c m p l o y t n g m a g n c s t u m a c c t , l t c a s c l u d n t ,.ir|d tile lattcr, z i n c , i t e r a t e Da~,le'., et t~/tz StL|dted 9 4 o l g : l n t c ,ic~d,~. u s i n g a n a n t o n - c , ¢ c h : | n g c r c s m m Its f o r m a t c f o r m T h e y c l u t c d c.'lcl'| o f thc,,c , | c l d s w t t h f r o m z e r o to 25 AI f o r m | c a c i d ,ts t h c c l u d n t s T h e a l ' o l c m c n t l o n c d s t u d t c s . ~n s o r n c c,lsc,,, h a d a t w o f o l d p u t po,~c o n c . ,,cpar:|tlon. thc o t h e r d c t c c t t o n T h e p , l p c r - c h r o m a t o g r , | p l ' | l c a n d t i c mcthod~ g e n c r d l l y m c o r p o r , | t c s o m e , I c l d - b a s c i n d i c a t o r i n t o t h e ~ol~,cnt ,,,,stem S o r n c o f t h e m d l c d t o t , ; u~,cd for d e t e c t i o n w m c b r o m o p h c n o l b l u c t 3,~. t h y m o l b l u c t, ,tnd chlolol'~henol red' in s o m e cd,,c,, t h e i n d l c , t t o l c a n b c spr:~ycd o n t o t h e C111 0111'1 [OgrdlT1 in t h e l i q u i d colLiilln rncthod~, v,u m u s m e t h o d , , o f dctcc.t~on w e r e e m p l o y e d B u l c n et td ~' t a n t h e ,~ctcl~ mdt,~lclually, c o l l e c t i n g sm:|ll f r a c t i o n , , a n d t | t r , l t m g c a c h c u t w i t h s t a n d a r d b a s e a n d ,in ,senti I'~,lsc m d t c a t o l I l a t l o w dr|d M o t m , u ~ "~ u s e d , ~ t a n d a r d ba,,c t,t~dtIOn b u t c m p l o ) c d :in , l u t o r n , | t l c s y r i n g e a n d gl:tss c l c c t r o d c s for tile clctcl tllltlLttlOt'! A n u m b e r o f t h c c l c t c r m t n : l t o n s w e r e m a d c b y d s m ~ u l t a r ~ c o u s c a r b , t z o l c a n d c h l o m l c a c i d m e t h o d s ~t A n o t h e r m e t h o d o f d e t c c t t o n w a s t h a t o f S l l n ' n o m u t , t a n d W d l t o n ~ '. w h o u,,cd a d ~ f f c l c n t m l r c f r , t c t o m c t c r t o m o n i t o r t h e c f f l u c n t s o f o r g , m l c acid,, f r o m ,m d n l o n - c x c h a n g c c o l u m n T h i s w:t,, o n c o f t h e first a t t e m p t s to d e t e r m i n e d n c c t l y t h e ;iced c o n c c n t l , t u n n n~ ,~ sy~,tct'n T h e p r c s c n t w o r k d e s c l l b e s the a p p l i c a t i o n o f t h e v a r i o u s m c t h o d s f o r t h e s e p a r a t i o n a n d d e t e c t i o n o f g l y c o l l c ,ind n l a l l c :lcld~
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Paper c hromatoqraph P a p e r c h r o m a t o g r , l p h 5 w,l~ used to m o n i t o r qualltatwel~, effluent fractJon~ from liquid c h r o m a t o g l , l p h l c c o l u m n ~ T h e p a p e r u~ed was W h ~ l t m a n N o [ ;rod was d e v e l o p e d in the f o l l o w i n g s o i x e n t ~y~tem e t h y l form;ice 98",, a q u e o u n f o r m i c acid H ~ O (2 I i. v , v / v ) c o n t a i n i n g 0 15",, ( w , v ) b ~ o m o p h e n o l blue a n d 0 0 5 " , , ( w / v ) s o d i u m f o r m a t e T h e c h r o m a t o g r a m c a n al,,o be d e v e l o p e d with thl~ ~olvenl s y s t e m e x c l u d i n g the m d i c . a t o r T h e i n d i c a t o r c a n t h e n be s p r t l y e d o n t o the p l a t e m an e t h a n o l , c s o l u t i o n a f t e r d r y i n g o f the c h r o m , ~ t o g r a m ~ Ptocvtho'v 1-10 Id o f s a m p l e was ,~potted o n the p a p e r , the s o l v e n t was a l l o w e d to e v a p o r a t e (occ=lslonally t h e p a p e r wa~ h e q t e d ~llghtly to l e m o v e trace~ o f f o r m i c acld a n d w a t e l ) q n d t h e n pluced into a d e v e l o p i n g t a n k T h e cla~omatogr,m~ was r e m o v e d a n d a l l o w e d to a i r - d r y . T h e a p p e a r : r a c e o f a c t d s p o t s req ulred I 5 - 6 0 rain Thm-la) ¢r t'hromatoql aph v M m m t u r e t h m - l a y c r p l a t e s w e r e used T i c w,a~lnveqttg~lted from t w o p o m t s o f v l e w , a s a s e p a r a t i o n techn~qtie a n d a ~ an i d e n t i f i c a t i o n t e c h n i q u e T h e 6-cm x 6-cm plates were c o a t e d wffh ~fllca gel G for t i c a c c o r d i n g to Stud1! (E M e r c k ) . T h e plates were ~pread to a t h i c k n e s s o f I m m a n d acttv:lted m a '~'dCtlUnl ox'el~ for 4 h at 120°C TI,~; v a r i o u s d e v e l o p i n g s o l v e n t ~y~tems a n d visual Merit ifleatton t e c h n i q u e s u s e d were: ( I ) 2 0 " . b u t a n o l m c h l o r o f o r m s p r a y e d w i t h b r o m o p h e n o l b l u e : (2) 10",, b u t a n o l in c h l o r o f o r m ~ p r a y e d w i t h b r o m o p h e n o l b l u e . ( 3 ) e t h a n o l chlcwoforn" a q u e o u s 2 8 " . a m m o n m s o l u t i o n . w a t e r ( 5 3 3 0 3 152 1 5. v v/v x) with identtfie;itlon by s p r a y i n g with b r o m o c r e ~ o l green, b r o m o t h y m o l blue. a n d 2.7-clichlorofluorescem, as well a~ c h a r r i n g ( e a c h I d e n t i f i c a t i o n w a s clone sepal a t e l y ) . (4) a q u e o u s 28"0 a m m o n m s o l u t i o n ~ p r a y e d w i t h b r o m o c r e ~ o l g r e e n . (5) a q t ] e o u s 28",, t l r n m o n n ! s o l u t i o n ' e t h a n o l (l I. v,'v) s p r a y e d with b r o m o c r e ~ o l g r e e n . ( 6 ) a q u e o u s 28". a m m o m a s o l u t i o n ' e t h a n o l ( 1 " 2 . ~ , ~ ) s p r q y e d w i t h b t o m o c r e ~ o l g r e e n : (7) e t h y l f o r m a t e . 9 8 " , , f o r m i c a c l d ' w a t e r (2 I I. v'~ v) c o n t a i n i n g 0.015",, ( w / v ) s o d i u m formate Procedure I - 1 0 HI o f acid s a m p l e in 2 M f o r m i c acld w a s placed on the plate; the plate was d r i e d .'it 70 C fol 30 rain a n d d e v e l o p e d After d e v e l o p m e n t the plate wa~ a g a i n d r i e d a n d visualized by e i t h e r ~ p r a y m g el c h a r l m g T h e o n l y e x c e p t i o n to this p r o c e d u r e w~ls a triple d e v e l o p m e n t (de~,elop. dr3'. d e v e l o p , ctc ) w i t h solvent s y s t e m (3) T h e r e t e n t i o n v.'flue,, for the m i g r a t i o n o f maltc a n d g l y c o l l c a c i d s in the v a r i o u s s o l v e n t s y s t e m s are given m T a b l e ! L I q u u i c o h m m c'h; o m a t o g r a p h v C o l u m n ,4 This was an 8-cm long by 0 5-cm i d mlcrocolumn p~cked w i t h I 5 g of 100-200 mesh sdlca gel (Davtdson Chemtcal. BaltH~ore, Md.) Packing
w a s a c c o m p h s h e d by a d d i t i o n o f the a d s o r b e n t w i t h gentle t a p p i n g T h e bed w a s s u p p o r t e d by a glass w o o l plug. a n d the acid s a m p l e w a s placed at the t o p o f the d r y bed T h e s a m p l e w a s e l u t c d w i t h s o l v e n t s y s t e m (3) (see t I c d e v e l o p i n g s o l v e n t s ) Small cuts were t a k e n a n d q u a l i t a t i v e an.'Hysl~ o f the cuts wa,, undert~lken by t.l.c ( w i t h solvent s y s t e m ( 3 ) ) Cohmm B. T h i s w a s a 0 7-cm i d by 50-cm l o n g glass c o l u m n e q u i p p e d w i t h
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a T e f l o n s t o p c o c k a n d a 100-ml s o l v e n t r e s e r v o w T h e c o l u m n w as p a c k e d with 5 0 - 1 0 0 m e s h D o w e x I-X8 a n i o n = e x c h a n g e resin (:t q u a t e r n a r y a m m o m u m salt) T h e c o l u m n wa~ p a c k e d by filhng Hrst w i t h w a t e r a n d t h e n a d d i n g a m i x t u r e o f resin a n d w a t e r to t h e r e s e r v o i r T h e s t o p c o c k w a s o p e n e d a n d t h e resin wa~ a l l o w e d to s ettle in the c o l u m n A glass w o o l p l u g w as used to s u p p o r t the resin bed T h e resin w a s p u t .n t o uts f o r m a t e f o r m by p a s s a g e o f 200 ml o f 2 M s o d m m f o r m a t e o v e r t h e bed. f o l l o w e d b y I0 ml o f 25 M f o r m i c a c i d T h e c o l u m n was t h e n w a s h e d with 200 ml o f w a t e r ,rod put i n t o final f o r m by elutnon with f o r m i c a c i d at the c o n c o n t r a t x o n to be u s e d m the s e p a r a t i o n . M a l i c a n d glycolnc a c i d s a m p l e s w e r e p l a c e d o n the c o l u m n , a n d the c o l u m n w a s e l u t e d w l t h 2 0 0 m l o f 0 2 M f o r m i c aci d F o r t y 5-rPi c u t s w e r e t a k e n a n d a n a l y z e d q u a l i t a t i v e l y b y p a p e r c h r o m a t o g r a p h y T h e result~ i n d i c a t e d a c i d c o m p o n e n t s m cut,, 3 - 1 1 a n d c u t s 2 1 - 2 6 . T h e c o l u m n w as w a s h e d t h o r o u g h l y w i t h w a t e r a n d ,t s i m i l a r s a m p l e w h i c h a l s o i n c l u d e d t e t r a m e t h } l a m m o n i u m b r o m i d e was p l a c e d on t h e c o l u m n Tllm mn×ture was e l u t e d with 50 ml o f w a t e r , f o l l o w e d by 100 ml o f 0.2 M f o r m i c ,acld. a n d finally by 2 0 M f o r m i c a c i d
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C o l m m l D T h i s w a s ,i 2 8 - r a m i d b y 122-¢m long. h o a ~ y - ~ d l l , gl,lg', c o l u m n p a c k e d w i t h 100 2 0 0 m e s h . A m b c ~ l l t c C G - 4 0 0 ; l n l o n - e x c h d n g c i c ' , l n (a q u d t e r n , l r y , I m m o n l u n l ~dlt. R o h m a n d H a a , , C o ) T h c t e s t a w a s p u t ~nto ~t~ f o r m a t e f o r m m a 111;.1111101 s i m i l a r t o th,'ll I.l,,ed for the D o w e l leSln T h e c o l u m n w a s p l a c e d m t h e c h l o m : l t o g l , l l ~ l a l c s y s t e m A typlc,'ll c h t o m a t o g l a m for tile , , e p a t , l t l o n o f g l y c o h c •' l a d m , l h c a c l d ~ t~ g l ~ e u in F i g 3. T i l e g l y c o l l c a n d m a l l c a c i d s h,ld e l u t l o n v o l u m e s o r 8 74 a n d 2 3 . 8 5 m l . r c s p e c t l v e l y . R e p r o d u c i b i l i t y s t u d i e s o n t h e p e a k a r e a r e s p o n s e s ( m 2 m l - t o f e l u a n t ) for v~.lrlOtl,~ g l y c o l l c a c i d c o n c e l l t r a t i o n s w e r e al,,o r u n T i l e r e s u l t s o r t h i s s t u d y ,ire g i v e n In T a b l e il T r i p l i c a t e r u n ~ o r t h e m a l l c a c i d ,tt
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Fig 4 Scann,ng probe clcctron microscope of 50/100 mesh D o w cx I-X8 and I00/200 mesh Ambcrl=tc C G . 4 0 0 ( ~ c a l e 100prn cm - I ) Left Oowcx I-Xg Right A m b e r l , t e C G - 4 0 0
d i f f e r e n c e s m time D o w e × a n d A m b e r h t c resins. T h e results o f a 100-l~m c m - ~ sc:m :ire g i v e n m Fig. 4. A closer c x a m m a t l o n o f a n i n d i v i d u a l resin part=clc o f each resin at 1000-pro c m - ~ a n d 2000-1ram c m - = scan~ is g~ven m F=g 5 RESULTS AND DISCUSS|ON
T h e results o f the p a p e r c h r o m a t o g r a p h i c m e t h o d w e r e i n c o n c l u s i v e G l y c o l l c a n d m a h c as well as h y d r o c h l o r i c qclds h a d Rr v a l u e s o f 100 T h i s m e t h o d seems sufficient for a s p o t test w h e n o n l y o n e k n o w n :tc~d is p r e s e n t m ~ given system. T h e t.I c. m e t h o d s m e r i t m o r e c o n s t d e r a t l o n . T h e s e m e t h o d s are fasr q u a l i t a t i v e tests• In gener-'fl, t.l.c m e t h o d s w h i c h d o n o t use s o m e s t r o n g base o r acid either m t h e d e v e l o p i n g s o l v e n t o r i m p r e g n a t e d r o t e t h e gel result m s e r i o u s tailing. T h e p r o b l e m a s s o c i a t e d wltll i m p r e g n a t i o n o f t h o gel is t h a t o f tdentzficatlon. An a c i d - b a s e s p r a y m d l c a t o r Is g e n e r a l l y used for I d e n t i f i c a t i o n o f time s p o t s O n e c a n r e a d d y see t h a t a c i d i m p r e g n a t i o n w o u l d c h r o m a t e this m e t h o d o f d e t e c t i o n T h e v a r i o u s d e v e l o p i n g s o l v e n t s y s t e m s a r e given m T a b l e I a n d m the E x p e r i m e n t a l part. T h e s y s t e m s c o n t a i n i n g 50')(,, o r m o r e ( b y v o l u m e ) o f a m m o m a s o l u t i o n ( s o l v e n t s y s t e m s (4) a n d (5)) o r 25')o ( b y v o l u m e ) f o r m i c acid ( s o l v e n t s y s t e m (7)) r e s u l t e d m R E values o f 100 for m a l t c as well as glycolic a c i d W h e n time a m m o n m c o n c e n t r a t t o n wqs d e c r e a s e d b y the addH~on o f m o r e e t h a n o l ( s o l v e n t s y s t e m (6)). a slsght s e p a r a t i o n w a s o b s e r v e d b u t t h e r e w a s o v e r l a p p i n g Time s p o t m i g r a t i o n for s o l v e n t system (6) r e s u l t e d m an e l o n g a t e d s p o t w i t h a tr.'Hhng edge (R,) o f 25 a n d a l e a d i n g edge ( R 0 o f 55 for m a l l c a n d c o r r e s p o n d i n g values o f 40 a n d 75 for g l y c o h c (see T a b l e l). W h e n b o t h acid a n d b a s e were r e m o v e d from the d e v e l o p i n g s o l v e n t (solvent s y s t e m s (1) a n d (2)). t a i l i n g fi o m the s p o t p o i n t upwarci was o b s e r v e d • T h e spot for t h e m a h c acid h a d a n Rt v a l u e o f zero a n d :m Ra value o f 40. T h e g l y c o h c r a n g e d f r o m z e r o to 70 for t h e R, a n d R, values. Time o n l y s y s t e m s h o w i n g prom~se for s e p a r a t i o n by t.l c w a s s o l v e n t s y s t e m (3) (53')~o e t h a n o l : 30.3~',, c h l o r o f o r m " 15.2~/A. 28'/o a q u e o u s a m m o n i a s o l u H o n : 1.5~)~,
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F l g 5 S~.~.|nllmLZ p r o b e c l c c l r o n m i c r o s c o p e o f II1¢ ~,lrloB~ d i l l o n c x c h d n g c re~Hl.~ at thu g i v e n m,l~.nd'icdt l o n ~ P h o t o g r d p h ~ U p p e r h:rl l ) o w t : x 11300 l l n l ~ n l - ~ u p p e r rlL.zhl A m b e r l l l o 10(10 p m ~ r n - ~ . l o w e r I~ft. D o w ~ x 20{]0 l : m c m z Iov~c! r~L~lll A m b c r l q c 2 0 0 0 Im~ ~.m
w a t e r , v / v / v / v ) whzch is a c o m b ~ n a u o n or s o m e o f the o t h e r s y s t e m s dlscus~,ed A single d e v e l o p m e n t w~th th~s s o l v e n t s h o w e d a Might s e p a r a t i o n , b u t a triple d e v e l o p m e a t r e s u l t e d in RI values o f 10 for m a h c d a d 60 for g l y c o l l c a c i d . In b o t h cases. there was no evidence of tadmg. T h e i n d i c a t o r s used were b r o m o c r e s o l L~recn. b r o m o p h e n o i blue. a n d d~chlorof l u o r e s c e m T h e b r o m o c r e s o l g r e e n s e e m e d to be the best i n d i c a t o r a n d the dlc h l o r o f i u o r e s c e m the poorest, since the acids s h o w e d no p r o n o u n c e d f l u o r e s c e n c e . C h a r r i n g a l s o p r o v e d to be msufl'ielent, p a r t t c u l a r l y for the g l y c o l l c acid. T h e r e f o r e . t I.e. s e p a r a t i o n s e e m s to bc sufficient, b u t d e t e r m m a t m n a p p e a r s t o be u n h k e l y T h e ion-exchang.c c h r o m a t o ~ r a p h i c c o l u m n s seem to offer t h e best p o s s l b l l | t y for c o m p l e t e s e p a r a t i o n a n d d c t e l m m a t l o n T h e c o l u m n s y s t e m C d e s c r i b e d u n d e r E x p e r i m e n t a l ( p a c k e d with D o w e x I-XS) wa~ e v a l u a t e d first. T h l s c o l u m n y m l d c d p e a k m a x i m a e l u t l o n volume~ o f 13.39 ml a n d 14 97 ml ( T a b l e IV) for g l y c o h c a n d m a h c acids. A n o t h e r a n i o n - e x c h a n g e resin selected ror e v a l u a t i o n w a s Amberl~te C G - 4 0 0 . T h i s c o l u m n D (~ee E x p e r m l e n t a l ) gave e l u t l o n v o l u m e s o r 8.74 a n d 23.85 ml for L;lycohc a n d m a l l c a c i d s ( T a b l e IV) T o c o m p m e the s c p a r a t m ~ p o w e r of each c o l u m n , t h c l e a r e t w o f a c t o r s t h a t c a n be c a l c u l a t e d the t h e o r e t i c a l plate c o u n t ( N ) o1" the c o l u m n a n d the r e s o l u t i o n Factor ( R , ) o f the two p e a k s e l u t e d f r o m the c o l u m n z'~ ~ T h e t h e o r e t i c a l plate c o u n t is c a l c u l a t e d f r o m '
362 FABLI.
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o f t h e p e a k al cas at t h e 50",, c o n f i d e n c e level t n n g c s f l o m -t- I 5 ,tt low c o n c e n t r a t i o n s . u p to 14",, qt h~ghcr c o n c c n t z : t t ~ o n s T h c ave~ngc ~Cldtt~.C c n o r o b t a i n e d f r o m the dnaly,~s o l b o t h a c i d s w a s 2 3 9 " . v,'tth a na, t xm l um c~to~ of 8 6 8 " . ( I n d z v l d t t a l reldu~c error.,, w c t c d c t c ~ m m c d by dsv~dmg the d i f f e r e n c e ~n c o n c c n u a t m n w e i g h e d an d c o n c c n t r , t u o n f o u n d by t h e c o n c e n t r a t i o n w e i g h e d ) A I c a s t - s q u m e s p l o t o f thc,,c d a t a .,,howcd a Imc,t~ ~elat~onshtp 1o exist b e t w e e n the p e a k a l e a a n d p e r c e n t
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( 2 ) m a c r o r e t l c u l a r , a n d ( 3 ) p e l h c u l a r r e s m s t'~. T h e m t c r o r e t l c u l a r r e s i n ]s a s p h e r i c a l r e s i n w~th m l c r o p o r e s t h r o u g h t h e b e a d . T h e m a c r o r e t ~ c u l a r r e s i n s h a v e m ~ c r o p o r e s a s well a s m a c r o p o r e s . T h e m a c r o p o r e s a r e l a r g e e n o u g h t o a l l o w s o l u t e m o l e c u l e s t o p a s s t h r o u g h t h e resin b e d : t h e r e f o r e , t h e m a c r o r e t l c u l a r r e s i n s h a v e a mucla g r e a t e r s u r f a c e a r e a for e x c h a n g e o f i o n s t h a n t h e m l c t o r e t t c u l a r r e s i n s s i n c e t h e y h a v e i n t e r n a l a s well a s e x t e r n a l a c t i v e sites. T h e p e l h c u l a r r e s i n s a r c s o l i d - c o r e b e a d s c o a t e d w~th t o n - e x c h a n g e r e s i n s T h e y a r e u s e d for a n a l y t i c a l r a t h e r t h a n p r e p a r a t i v e w o r k . o w i n g to their low l o a d i n g capabd]ttes. T h e r e s u l t s o f t h e e l e c t r o n m i c r o p r o b e ( F i g 4) s h o w t h a t t h e D o w e x resin m a s p h e r i c a l - t y p e p a r t m l e w h d e the A m b e r l ~ t e r e s i n e o n s x s t s ol c u b i c p a r t i c l e s A c l o s e r e x a m i n a t i o n o f an zndlvldual resin particle o f e a c h resin at the 1000-Hm em " ' a n d 2 0 0 0 - / t i n c m - ~ s c a n s ( F i g . 5) s h o w s a n e v e n g r e a t e r d , f f e r e n c e in t h e s t t r f a c e s t r u c t u r e s . In t h e D o w e x resin, the s u r f a c e ts r a t h e r s m o o t h w i t h n o v i s i b l e p o r e s through the molecules The Amberltte. however, shows numerous pores and ch,tnnels p e n e t r a t t n g the r e s i n m o l e c u l e T h e r e f o r e . t h e A m b e r l t t e a p p e a r s t o be a m a c r o r e t i c u l a r r e s , n w i t h a l a r g e s u r f a c e are,t, intern,al a s well a s e x t e t n a l , w h i l e t h e D o w e x ts e i t h e r a p e l h c u l a r resin w i t h n o m t e r t o r s u r f a c e a e t t v t t y o r a t n l c r o r e t ~ c u l a l t e s l n w i t h p o r e s t o o s m a l l t o a l l o w s o l u t e m o l e c u l e s to p a s s t h r o u g h T h i s s t r u c t u r a l d i f f e r e n c e w o u l d e a s i l y ,'recount for t h e h i g h e r r e s o l u t l o n f a c t o r f o r t h e g l y c o l l c a n d m a l t c a c i d s o n A m b e r l l t e c o m p a r e d to D o w e x r e s t n The author gratefully acknowledges the guld,tnce and able assistance of Dr G e r a l d P e r k i n s , Jr.. M r . R E. L a r a m y . a n d M r D. L. K o h l e r , ,tnd e x p r e s s e s his t h a n k s t o Dr. C. N M c K i n n e y for hts w o r k w i t h t h e e l e c t r o n m i c r o p r o b e SUMMARY A n t o n - e x c h a n g e c h r o m a t o g r a p h i c m e t h o d for t h e s e p , t r a t t o n o f g l y c o l t c a n d mahcactdswasdeveioped It w a s f o u n d t h a t t h e r e s t n s t r u c t u r e p l a y s a n i m p o r t a n t r o l e m t h e r e s o l u t i o n o f t h e t w o a c i d s . T h e best s y s t e m f o r s e p a r a t i n g t h e a c i d s w a s a 2 8 - r a m Ld.. 1 2 2 - c m l o n g A m b e r l l t e C G - 4 0 0 , t a l o n - e x c h a n g e c o l u m n The a c i d s m c o n c e n t r a t i o n s o f 1-10,~, m 2 M fol m m a c i d g a v e h n e a r p e a k a r e a r e s p o n s e s . REFERENCLS
I R D Hartley and (3 J I.dw,,on J Cltrmn,m~tlr. 4 (1960) 410 2 R D Hartlcy and G J Lawson J C/omnttto~lr, I (1962) 69 3 F Stern. G Grumet V Trabal A Mennt,, and H Zm,,.,,ar J Chtonttttotlr 4 A S Ble=wel,,. H C Reeve,, ,llld S J AjI. 4hal Bto, lwm 20(1967) 335 5 L Ch,tfetz ,rod M H Pe,lqcr I ChtomtttoHt 49 (1970) 341) 6 W A Bulen. J E Vorner,md R C Burrcll...ltltll C.hvIH. 24(1952) 187 7 G A H,irlow and D l-I Morman 4hal ('hem 36 (1964)2438 8 0 Samuclson and t Thedu. J Chrtmlaloqr 30 (1967) 556 9 L Bangtsson and O S,=intt~l,,otl, rln,II Clmn At tu. 44 (1969) 217 I0 K S Lc¢ and O
II 12 13 14
Samuul,.on .-lind C l n m
19 ( 1 9 6 5 ) 130
,It/ca 37 (1¢)67)357
U-B Lar,,son. T Isaks~on and O S,tmuel~on..Iota Chem Sttm,I 20(1966) 1965 L D,tvm.,, R D Harrier ,tnd G L Ld~A,,Orl J (htottultoHt 18 (1965) 47 K Shtmomura dnd II F Walton Anal Chvm 37(1965) 1012 C D Scott =n J J Ktrkl,md ted ), Modern Ptcltlltt.~, o/ I..ulmd Chtonttltoqst#/~ht, Wdcy-lnterst.tcn~.c .
SEPARATION
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AND
MALIC ACIDS
365
N e w Y o r k . 1971. p 292 15 J C G i c l d m g s D~ncom¢ ~ o f Chronultoglaplav IM.,rcel D e k k c r . N e w Y o r k , 1965. p 369 16 B L K a r g e r ,n J J K i r k l a n d ( E d ) . Modem; P~actl(¢.~ ol LuluU/ C/Iro.lcltoLIrapll~., Wdey-Intersc,encc. N e w Y o r k . 1971. Ch I. p 3 17 I_. R Snyder. J Chromarogr S¢t. 1(1'969)352 18 J Bohemcn and J H Purncll ,n D H Dcsty ( E d ) . Ga~ Chromatograph~ 1938. B u t t e r w o r t h s . London. p 8
"1 i-112 c ; E P A R A T I O N ANI) DE-IIZRMINAIION OF GLYL'OLIC A C I D S BY I O N - L X C H A N G I _ CHI~OMATOGRAPFIY
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'1 ht2 s C p d r d [ I t l n l i n d ~ub,,tJqtlcnl dt~lt~lnlm~.ltlon ol I o x ~ - m o l t ~ C u l d l - ~ C I g h t ol"g d n l c d c l d s I~ h , l l d to d c c o m p l t s h by mo,,t m o d e r n dn,~l.~ttcdl tt~t.hlllcll.l~s 111 ti~c pd',t row oleo,ides. ~ c p a r d t t o n s o f ~,t|lot,s m g a n l c ,tcltls h:l~e I'~¢cn r c p m t c d b y p ; I p c r chromdtogldph~, thm-l,tyel ¢hlomdtoglaph.~ (tic) .tnd i t q u l d ¢ h l o m d t o g r , l p h ) F t a t t l e ) m i d L a w , o n ~ : , , t t , d~ed tile s c p a r . l t l o n o f some 128 m g d n l c a u t d s m s e v e r d l d i f f e r e n t s o l v e n t , , v s t e m s b y i~apel c h t o r r | a t o g l a r d L ~ T h e y Ii,,tcd R. ~ d l u c s f o r t h e s e nunlglOU% ,Icids to1 eLIC]l s o l v e n t %y%tl,~111 Stg111 t,t t~l ~. a l s o u,,ed thl,, t e c h n i q u e t o s t u d y tile sep,ll~ltlotl o r tile urln,tl), o t g , tnlc d¢ld'~ B l e l w e l s t,t t d "~ s t u d i e d t h e s e i ~ a t , t t t o n o f ,,or|it: a c i d s b y t i c w h i l e C h , f f c t z ,tnd P c n n c r ~ s t u d i e d t h e dctec.'tlon
of the c,ubox~l~c acids on tlc by thelr ~c:lctmn with 1ocltclc IocI,1tc ,tnd :wnylasc BLIIci~ t,I ill ~' scp~ll,ltcd o l g , l n i c d c l d s f r o m p l a n t tls,,uc o n ,i ,,dlca gel sttlfut ic ; i c l d m 3 p r e g n d t e d c o l u m n S e v e r a l m%'c~ttg,ltors h,lvc stucllcd t h e s c p a r d t l o n o f t h e ,|cid,, w l t h , i n ~ o n - c x c h , l n g e Ic,,m~ l-t,lrlo~ a n d M o r m a n " u~cd x~atcl to c i u t c , ; o m c 50 , I c t d s rl O111 ~.111, i n l o n - c x c i l a r l g c c o l t t m n w l t h n o p r c t l g~.t11111211t S , i i l l u c l s o n d n d T h c d c s u~,cd a n a c c t , l t c f o r m o f rc,,tn w i t h ,i s o d i u m a c e t a t e c l u a n t for t h e n ,~tudy T h r e e d c l d t t t o n a l ,,tud~cs '~ t~ w e r e m a d c ~ tth t h e a c e t a t c ic,,m f o r m . t h e first t w o c m p l o y t n g m a g n c s t u m a c c t , l t c a s c l u d n t ,.ir|d tile lattcr, z i n c , i t e r a t e Da~,le'., et t~/tz StL|dted 9 4 o l g : l n t c ,ic~d,~. u s i n g a n a n t o n - c , ¢ c h : | n g c r c s m m Its f o r m a t c f o r m T h e y c l u t c d c.'lcl'| o f thc,,c , | c l d s w t t h f r o m z e r o to 25 AI f o r m | c a c i d ,ts t h c c l u d n t s T h e a l ' o l c m c n t l o n c d s t u d t c s . ~n s o r n c c,lsc,,, h a d a t w o f o l d p u t po,~c o n c . ,,cpar:|tlon. thc o t h e r d c t c c t t o n T h e p , l p c r - c h r o m a t o g r , | p l ' | l c a n d t i c mcthod~ g e n c r d l l y m c o r p o r , | t c s o m e , I c l d - b a s c i n d i c a t o r i n t o t h e ~ol~,cnt ,,,,stem S o r n c o f t h e m d l c d t o t , ; u~,cd for d e t e c t i o n w m c b r o m o p h c n o l b l u c t 3,~. t h y m o l b l u c t, ,tnd chlolol'~henol red' in s o m e cd,,c,, t h e i n d l c , t t o l c a n b c spr:~ycd o n t o t h e C111 0111'1 [OgrdlT1 in t h e l i q u i d colLiilln rncthod~, v,u m u s m e t h o d , , o f dctcc.t~on w e r e e m p l o y e d B u l c n et td ~' t a n t h e ,~ctcl~ mdt,~lclually, c o l l e c t i n g sm:|ll f r a c t i o n , , a n d t | t r , l t m g c a c h c u t w i t h s t a n d a r d b a s e a n d ,in ,senti I'~,lsc m d t c a t o l I l a t l o w dr|d M o t m , u ~ "~ u s e d , ~ t a n d a r d ba,,c t,t~dtIOn b u t c m p l o ) c d :in , l u t o r n , | t l c s y r i n g e a n d gl:tss c l c c t r o d c s for tile clctcl tllltlLttlOt'! A n u m b e r o f t h c c l c t c r m t n : l t o n s w e r e m a d c b y d s m ~ u l t a r ~ c o u s c a r b , t z o l c a n d c h l o m l c a c i d m e t h o d s ~t A n o t h e r m e t h o d o f d e t c c t t o n w a s t h a t o f S l l n ' n o m u t , t a n d W d l t o n ~ '. w h o u,,cd a d ~ f f c l c n t m l r c f r , t c t o m c t c r t o m o n i t o r t h e c f f l u c n t s o f o r g , m l c acid,, f r o m ,m d n l o n - c x c h a n g c c o l u m n T h i s w:t,, o n c o f t h e first a t t e m p t s to d e t e r m i n e d n c c t l y t h e ;iced c o n c c n t l , t u n n n~ ,~ sy~,tct'n T h e p r c s c n t w o r k d e s c l l b e s the a p p l i c a t i o n o f t h e v a r i o u s m c t h o d s f o r t h e s e p a r a t i o n a n d d e t e c t i o n o f g l y c o l l c ,ind n l a l l c :lcld~
356
I) I- LINDER
EXPERIMENTAl.
Paper c hromatoqraph P a p e r c h r o m a t o g r , l p h 5 w,l~ used to m o n i t o r qualltatwel~, effluent fractJon~ from liquid c h r o m a t o g l , l p h l c c o l u m n ~ T h e p a p e r u~ed was W h ~ l t m a n N o [ ;rod was d e v e l o p e d in the f o l l o w i n g s o i x e n t ~y~tem e t h y l form;ice 98",, a q u e o u n f o r m i c acid H ~ O (2 I i. v , v / v ) c o n t a i n i n g 0 15",, ( w , v ) b ~ o m o p h e n o l blue a n d 0 0 5 " , , ( w / v ) s o d i u m f o r m a t e T h e c h r o m a t o g r a m c a n al,,o be d e v e l o p e d with thl~ ~olvenl s y s t e m e x c l u d i n g the m d i c . a t o r T h e i n d i c a t o r c a n t h e n be s p r t l y e d o n t o the p l a t e m an e t h a n o l , c s o l u t i o n a f t e r d r y i n g o f the c h r o m , ~ t o g r a m ~ Ptocvtho'v 1-10 Id o f s a m p l e was ,~potted o n the p a p e r , the s o l v e n t was a l l o w e d to e v a p o r a t e (occ=lslonally t h e p a p e r wa~ h e q t e d ~llghtly to l e m o v e trace~ o f f o r m i c acld a n d w a t e l ) q n d t h e n pluced into a d e v e l o p i n g t a n k T h e cla~omatogr,m~ was r e m o v e d a n d a l l o w e d to a i r - d r y . T h e a p p e a r : r a c e o f a c t d s p o t s req ulred I 5 - 6 0 rain Thm-la) ¢r t'hromatoql aph v M m m t u r e t h m - l a y c r p l a t e s w e r e used T i c w,a~lnveqttg~lted from t w o p o m t s o f v l e w , a s a s e p a r a t i o n techn~qtie a n d a ~ an i d e n t i f i c a t i o n t e c h n i q u e T h e 6-cm x 6-cm plates were c o a t e d wffh ~fllca gel G for t i c a c c o r d i n g to Stud1! (E M e r c k ) . T h e plates were ~pread to a t h i c k n e s s o f I m m a n d acttv:lted m a '~'dCtlUnl ox'el~ for 4 h at 120°C TI,~; v a r i o u s d e v e l o p i n g s o l v e n t ~y~tems a n d visual Merit ifleatton t e c h n i q u e s u s e d were: ( I ) 2 0 " . b u t a n o l m c h l o r o f o r m s p r a y e d w i t h b r o m o p h e n o l b l u e : (2) 10",, b u t a n o l in c h l o r o f o r m ~ p r a y e d w i t h b r o m o p h e n o l b l u e . ( 3 ) e t h a n o l chlcwoforn" a q u e o u s 2 8 " . a m m o n m s o l u t i o n . w a t e r ( 5 3 3 0 3 152 1 5. v v/v x) with identtfie;itlon by s p r a y i n g with b r o m o c r e ~ o l green, b r o m o t h y m o l blue. a n d 2.7-clichlorofluorescem, as well a~ c h a r r i n g ( e a c h I d e n t i f i c a t i o n w a s clone sepal a t e l y ) . (4) a q u e o u s 28"0 a m m o n m s o l u t i o n ~ p r a y e d w i t h b r o m o c r e ~ o l g r e e n . (5) a q t ] e o u s 28",, t l r n m o n n ! s o l u t i o n ' e t h a n o l (l I. v,'v) s p r a y e d with b r o m o c r e ~ o l g r e e n . ( 6 ) a q u e o u s 28". a m m o m a s o l u t i o n ' e t h a n o l ( 1 " 2 . ~ , ~ ) s p r q y e d w i t h b t o m o c r e ~ o l g r e e n : (7) e t h y l f o r m a t e . 9 8 " , , f o r m i c a c l d ' w a t e r (2 I I. v'~ v) c o n t a i n i n g 0.015",, ( w / v ) s o d i u m formate Procedure I - 1 0 HI o f acid s a m p l e in 2 M f o r m i c acld w a s placed on the plate; the plate was d r i e d .'it 70 C fol 30 rain a n d d e v e l o p e d After d e v e l o p m e n t the plate wa~ a g a i n d r i e d a n d visualized by e i t h e r ~ p r a y m g el c h a r l m g T h e o n l y e x c e p t i o n to this p r o c e d u r e w~ls a triple d e v e l o p m e n t (de~,elop. dr3'. d e v e l o p , ctc ) w i t h solvent s y s t e m (3) T h e r e t e n t i o n v.'flue,, for the m i g r a t i o n o f maltc a n d g l y c o l l c a c i d s in the v a r i o u s s o l v e n t s y s t e m s are given m T a b l e ! L I q u u i c o h m m c'h; o m a t o g r a p h v C o l u m n ,4 This was an 8-cm long by 0 5-cm i d mlcrocolumn p~cked w i t h I 5 g of 100-200 mesh sdlca gel (Davtdson Chemtcal. BaltH~ore, Md.) Packing
w a s a c c o m p h s h e d by a d d i t i o n o f the a d s o r b e n t w i t h gentle t a p p i n g T h e bed w a s s u p p o r t e d by a glass w o o l plug. a n d the acid s a m p l e w a s placed at the t o p o f the d r y bed T h e s a m p l e w a s e l u t c d w i t h s o l v e n t s y s t e m (3) (see t I c d e v e l o p i n g s o l v e n t s ) Small cuts were t a k e n a n d q u a l i t a t i v e an.'Hysl~ o f the cuts wa,, undert~lken by t.l.c ( w i t h solvent s y s t e m ( 3 ) ) Cohmm B. T h i s w a s a 0 7-cm i d by 50-cm l o n g glass c o l u m n e q u i p p e d w i t h
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a T e f l o n s t o p c o c k a n d a 100-ml s o l v e n t r e s e r v o w T h e c o l u m n w as p a c k e d with 5 0 - 1 0 0 m e s h D o w e x I-X8 a n i o n = e x c h a n g e resin (:t q u a t e r n a r y a m m o m u m salt) T h e c o l u m n wa~ p a c k e d by filhng Hrst w i t h w a t e r a n d t h e n a d d i n g a m i x t u r e o f resin a n d w a t e r to t h e r e s e r v o i r T h e s t o p c o c k w a s o p e n e d a n d t h e resin wa~ a l l o w e d to s ettle in the c o l u m n A glass w o o l p l u g w as used to s u p p o r t the resin bed T h e resin w a s p u t .n t o uts f o r m a t e f o r m by p a s s a g e o f 200 ml o f 2 M s o d m m f o r m a t e o v e r t h e bed. f o l l o w e d b y I0 ml o f 25 M f o r m i c a c i d T h e c o l u m n was t h e n w a s h e d with 200 ml o f w a t e r ,rod put i n t o final f o r m by elutnon with f o r m i c a c i d at the c o n c o n t r a t x o n to be u s e d m the s e p a r a t i o n . M a l i c a n d glycolnc a c i d s a m p l e s w e r e p l a c e d o n the c o l u m n , a n d the c o l u m n w a s e l u t e d w l t h 2 0 0 m l o f 0 2 M f o r m i c aci d F o r t y 5-rPi c u t s w e r e t a k e n a n d a n a l y z e d q u a l i t a t i v e l y b y p a p e r c h r o m a t o g r a p h y T h e result~ i n d i c a t e d a c i d c o m p o n e n t s m cut,, 3 - 1 1 a n d c u t s 2 1 - 2 6 . T h e c o l u m n w as w a s h e d t h o r o u g h l y w i t h w a t e r a n d ,t s i m i l a r s a m p l e w h i c h a l s o i n c l u d e d t e t r a m e t h } l a m m o n i u m b r o m i d e was p l a c e d on t h e c o l u m n Tllm mn×ture was e l u t e d with 50 ml o f w a t e r , f o l l o w e d by 100 ml o f 0.2 M f o r m i c ,acld. a n d finally by 2 0 M f o r m i c a c i d
358
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C u t , , x~,ere ;.ig;.llrl t.'ll, ell ( 5 - r n l ) a n d a n a l ~ . / e d b) i'~,lpel ~ . h l o m , l t o g r a p h y Spot,, a p p e d r c d in c u t s I a n d 2 ( t c t l a m e t h y l . ' l m n a o n l u m b i o m i d e ) . 15 23 ( g l y c o l i ¢ a c i d ) a n d 34 3 7 ( m a l l c a c i d ) Cohtnl/I C 1 11i~.%';Is ,I ( ) 9 5 - c m o d bx 2 8 - n l i l l I t l I')% 1 2 2 - c m l o n g h c , l x ) - w a l l g l , l s ~ c ( ~ l u m n p d c k e d w i t h 5 0 I(10 m e q l l)ox~ex I - X S , l ~ d c ~ c l l b e d a b o x c e x c e p t t h a t ~talnle,,~ ,,tecl lilting,, w e r e u s e d ;.it e l t h e l e n d fol b e d , , u p p o l t T h e filtlng~ wel e filled x~tth gla,~,, b e a d , , t o e l i m ~ n a l e d e , l d ~ o l u n l e dn(I b , l c k - m l x l n g T h e ¢oit.mt~ ~ : ~ p l a c e d in d c l o s e d ,,y,,tem. a s , , h e w n in [ i g I. S,inlple~ ~ c r e i n l e c t c d d n c c t l y oil tile c o l u m n , a n d t h e ~tle,lm e f f l u e n t wd~ n ~ o n l l o r e d d l r e c t l 3 b y ,in R-401 ¢llffelcntt,iI r e r r d c t o m c t c r ( W , i t e i ~ As~ocldtc,,. F i , i m n ~ g h , l m . M d ~ ) A t),p~cal t l , l c e Io~ g l ) ' c o l t c a n d m a l l c ac~cls I,; g i v e n m r i g 2 (.,~)
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Fig 4 Scann,ng probe clcctron microscope of 50/100 mesh D o w cx I-X8 and I00/200 mesh Ambcrl=tc C G . 4 0 0 ( ~ c a l e 100prn cm - I ) Left Oowcx I-Xg Right A m b e r l , t e C G - 4 0 0
d i f f e r e n c e s m time D o w e × a n d A m b e r h t c resins. T h e results o f a 100-l~m c m - ~ sc:m :ire g i v e n m Fig. 4. A closer c x a m m a t l o n o f a n i n d i v i d u a l resin part=clc o f each resin at 1000-pro c m - ~ a n d 2000-1ram c m - = scan~ is g~ven m F=g 5 RESULTS AND DISCUSS|ON
T h e results o f the p a p e r c h r o m a t o g r a p h i c m e t h o d w e r e i n c o n c l u s i v e G l y c o l l c a n d m a h c as well as h y d r o c h l o r i c qclds h a d Rr v a l u e s o f 100 T h i s m e t h o d seems sufficient for a s p o t test w h e n o n l y o n e k n o w n :tc~d is p r e s e n t m ~ given system. T h e t.I c. m e t h o d s m e r i t m o r e c o n s t d e r a t l o n . T h e s e m e t h o d s are fasr q u a l i t a t i v e tests• In gener-'fl, t.l.c m e t h o d s w h i c h d o n o t use s o m e s t r o n g base o r acid either m t h e d e v e l o p i n g s o l v e n t o r i m p r e g n a t e d r o t e t h e gel result m s e r i o u s tailing. T h e p r o b l e m a s s o c i a t e d wltll i m p r e g n a t i o n o f t h o gel is t h a t o f tdentzficatlon. An a c i d - b a s e s p r a y m d l c a t o r Is g e n e r a l l y used for I d e n t i f i c a t i o n o f time s p o t s O n e c a n r e a d d y see t h a t a c i d i m p r e g n a t i o n w o u l d c h r o m a t e this m e t h o d o f d e t e c t i o n T h e v a r i o u s d e v e l o p i n g s o l v e n t s y s t e m s a r e given m T a b l e I a n d m the E x p e r i m e n t a l part. T h e s y s t e m s c o n t a i n i n g 50')(,, o r m o r e ( b y v o l u m e ) o f a m m o m a s o l u t i o n ( s o l v e n t s y s t e m s (4) a n d (5)) o r 25')o ( b y v o l u m e ) f o r m i c acid ( s o l v e n t s y s t e m (7)) r e s u l t e d m R E values o f 100 for m a l t c as well as glycolic a c i d W h e n time a m m o n m c o n c e n t r a t t o n wqs d e c r e a s e d b y the addH~on o f m o r e e t h a n o l ( s o l v e n t s y s t e m (6)). a slsght s e p a r a t i o n w a s o b s e r v e d b u t t h e r e w a s o v e r l a p p i n g Time s p o t m i g r a t i o n for s o l v e n t system (6) r e s u l t e d m an e l o n g a t e d s p o t w i t h a tr.'Hhng edge (R,) o f 25 a n d a l e a d i n g edge ( R 0 o f 55 for m a l l c a n d c o r r e s p o n d i n g values o f 40 a n d 75 for g l y c o h c (see T a b l e l). W h e n b o t h acid a n d b a s e were r e m o v e d from the d e v e l o p i n g s o l v e n t (solvent s y s t e m s (1) a n d (2)). t a i l i n g fi o m the s p o t p o i n t upwarci was o b s e r v e d • T h e spot for t h e m a h c acid h a d a n Rt v a l u e o f zero a n d :m Ra value o f 40. T h e g l y c o h c r a n g e d f r o m z e r o to 70 for t h e R, a n d R, values. Time o n l y s y s t e m s h o w i n g prom~se for s e p a r a t i o n by t.l c w a s s o l v e n t s y s t e m (3) (53')~o e t h a n o l : 30.3~',, c h l o r o f o r m " 15.2~/A. 28'/o a q u e o u s a m m o n i a s o l u H o n : 1.5~)~,
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F l g 5 S~.~.|nllmLZ p r o b e c l c c l r o n m i c r o s c o p e o f II1¢ ~,lrloB~ d i l l o n c x c h d n g c re~Hl.~ at thu g i v e n m,l~.nd'icdt l o n ~ P h o t o g r d p h ~ U p p e r h:rl l ) o w t : x 11300 l l n l ~ n l - ~ u p p e r rlL.zhl A m b e r l l l o 10(10 p m ~ r n - ~ . l o w e r I~ft. D o w ~ x 20{]0 l : m c m z Iov~c! r~L~lll A m b c r l q c 2 0 0 0 Im~ ~.m
w a t e r , v / v / v / v ) whzch is a c o m b ~ n a u o n or s o m e o f the o t h e r s y s t e m s dlscus~,ed A single d e v e l o p m e n t w~th th~s s o l v e n t s h o w e d a Might s e p a r a t i o n , b u t a triple d e v e l o p m e a t r e s u l t e d in RI values o f 10 for m a h c d a d 60 for g l y c o l l c a c i d . In b o t h cases. there was no evidence of tadmg. T h e i n d i c a t o r s used were b r o m o c r e s o l L~recn. b r o m o p h e n o i blue. a n d d~chlorof l u o r e s c e m T h e b r o m o c r e s o l g r e e n s e e m e d to be the best i n d i c a t o r a n d the dlc h l o r o f i u o r e s c e m the poorest, since the acids s h o w e d no p r o n o u n c e d f l u o r e s c e n c e . C h a r r i n g a l s o p r o v e d to be msufl'ielent, p a r t t c u l a r l y for the g l y c o l l c acid. T h e r e f o r e . t I.e. s e p a r a t i o n s e e m s to bc sufficient, b u t d e t e r m m a t m n a p p e a r s t o be u n h k e l y T h e ion-exchang.c c h r o m a t o ~ r a p h i c c o l u m n s seem to offer t h e best p o s s l b l l | t y for c o m p l e t e s e p a r a t i o n a n d d c t e l m m a t l o n T h e c o l u m n s y s t e m C d e s c r i b e d u n d e r E x p e r i m e n t a l ( p a c k e d with D o w e x I-XS) wa~ e v a l u a t e d first. T h l s c o l u m n y m l d c d p e a k m a x i m a e l u t l o n volume~ o f 13.39 ml a n d 14 97 ml ( T a b l e IV) for g l y c o h c a n d m a h c acids. A n o t h e r a n i o n - e x c h a n g e resin selected ror e v a l u a t i o n w a s Amberl~te C G - 4 0 0 . T h i s c o l u m n D (~ee E x p e r m l e n t a l ) gave e l u t l o n v o l u m e s o r 8.74 a n d 23.85 ml for L;lycohc a n d m a l l c a c i d s ( T a b l e IV) T o c o m p m e the s c p a r a t m ~ p o w e r of each c o l u m n , t h c l e a r e t w o f a c t o r s t h a t c a n be c a l c u l a t e d the t h e o r e t i c a l plate c o u n t ( N ) o1" the c o l u m n a n d the r e s o l u t i o n Factor ( R , ) o f the two p e a k s e l u t e d f r o m the c o l u m n z'~ ~ T h e t h e o r e t i c a l plate c o u n t is c a l c u l a t e d f r o m '
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o f t h e p e a k al cas at t h e 50",, c o n f i d e n c e level t n n g c s f l o m -t- I 5 ,tt low c o n c e n t r a t i o n s . u p to 14",, qt h~ghcr c o n c c n t z : t t ~ o n s T h c ave~ngc ~Cldtt~.C c n o r o b t a i n e d f r o m the dnaly,~s o l b o t h a c i d s w a s 2 3 9 " . v,'tth a na, t xm l um c~to~ of 8 6 8 " . ( I n d z v l d t t a l reldu~c error.,, w c t c d c t c ~ m m c d by dsv~dmg the d i f f e r e n c e ~n c o n c c n u a t m n w e i g h e d an d c o n c c n t r , t u o n f o u n d by t h e c o n c e n t r a t i o n w e i g h e d ) A I c a s t - s q u m e s p l o t o f thc,,c d a t a .,,howcd a Imc,t~ ~elat~onshtp 1o exist b e t w e e n the p e a k a l e a a n d p e r c e n t
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( 2 ) m a c r o r e t l c u l a r , a n d ( 3 ) p e l h c u l a r r e s m s t'~. T h e m t c r o r e t l c u l a r r e s i n ]s a s p h e r i c a l r e s i n w~th m l c r o p o r e s t h r o u g h t h e b e a d . T h e m a c r o r e t ~ c u l a r r e s i n s h a v e m ~ c r o p o r e s a s well a s m a c r o p o r e s . T h e m a c r o p o r e s a r e l a r g e e n o u g h t o a l l o w s o l u t e m o l e c u l e s t o p a s s t h r o u g h t h e resin b e d : t h e r e f o r e , t h e m a c r o r e t l c u l a r r e s i n s h a v e a mucla g r e a t e r s u r f a c e a r e a for e x c h a n g e o f i o n s t h a n t h e m l c t o r e t t c u l a r r e s i n s s i n c e t h e y h a v e i n t e r n a l a s well a s e x t e r n a l a c t i v e sites. T h e p e l h c u l a r r e s i n s a r c s o l i d - c o r e b e a d s c o a t e d w~th t o n - e x c h a n g e r e s i n s T h e y a r e u s e d for a n a l y t i c a l r a t h e r t h a n p r e p a r a t i v e w o r k . o w i n g to their low l o a d i n g capabd]ttes. T h e r e s u l t s o f t h e e l e c t r o n m i c r o p r o b e ( F i g 4) s h o w t h a t t h e D o w e x resin m a s p h e r i c a l - t y p e p a r t m l e w h d e the A m b e r l ~ t e r e s i n e o n s x s t s ol c u b i c p a r t i c l e s A c l o s e r e x a m i n a t i o n o f an zndlvldual resin particle o f e a c h resin at the 1000-Hm em " ' a n d 2 0 0 0 - / t i n c m - ~ s c a n s ( F i g . 5) s h o w s a n e v e n g r e a t e r d , f f e r e n c e in t h e s t t r f a c e s t r u c t u r e s . In t h e D o w e x resin, the s u r f a c e ts r a t h e r s m o o t h w i t h n o v i s i b l e p o r e s through the molecules The Amberltte. however, shows numerous pores and ch,tnnels p e n e t r a t t n g the r e s i n m o l e c u l e T h e r e f o r e . t h e A m b e r l t t e a p p e a r s t o be a m a c r o r e t i c u l a r r e s , n w i t h a l a r g e s u r f a c e are,t, intern,al a s well a s e x t e t n a l , w h i l e t h e D o w e x ts e i t h e r a p e l h c u l a r resin w i t h n o m t e r t o r s u r f a c e a e t t v t t y o r a t n l c r o r e t ~ c u l a l t e s l n w i t h p o r e s t o o s m a l l t o a l l o w s o l u t e m o l e c u l e s to p a s s t h r o u g h T h i s s t r u c t u r a l d i f f e r e n c e w o u l d e a s i l y ,'recount for t h e h i g h e r r e s o l u t l o n f a c t o r f o r t h e g l y c o l l c a n d m a l t c a c i d s o n A m b e r l l t e c o m p a r e d to D o w e x r e s t n The author gratefully acknowledges the guld,tnce and able assistance of Dr G e r a l d P e r k i n s , Jr.. M r . R E. L a r a m y . a n d M r D. L. K o h l e r , ,tnd e x p r e s s e s his t h a n k s t o Dr. C. N M c K i n n e y for hts w o r k w i t h t h e e l e c t r o n m i c r o p r o b e SUMMARY A n t o n - e x c h a n g e c h r o m a t o g r a p h i c m e t h o d for t h e s e p , t r a t t o n o f g l y c o l t c a n d mahcactdswasdeveioped It w a s f o u n d t h a t t h e r e s t n s t r u c t u r e p l a y s a n i m p o r t a n t r o l e m t h e r e s o l u t i o n o f t h e t w o a c i d s . T h e best s y s t e m f o r s e p a r a t i n g t h e a c i d s w a s a 2 8 - r a m Ld.. 1 2 2 - c m l o n g A m b e r l l t e C G - 4 0 0 , t a l o n - e x c h a n g e c o l u m n The a c i d s m c o n c e n t r a t i o n s o f 1-10,~, m 2 M fol m m a c i d g a v e h n e a r p e a k a r e a r e s p o n s e s . REFERENCLS
I R D Hartley and (3 J I.dw,,on J Cltrmn,m~tlr. 4 (1960) 410 2 R D Hartlcy and G J Lawson J C/omnttto~lr, I (1962) 69 3 F Stern. G Grumet V Trabal A Mennt,, and H Zm,,.,,ar J Chtonttttotlr 4 A S Ble=wel,,. H C Reeve,, ,llld S J AjI. 4hal Bto, lwm 20(1967) 335 5 L Ch,tfetz ,rod M H Pe,lqcr I ChtomtttoHt 49 (1970) 341) 6 W A Bulen. J E Vorner,md R C Burrcll...ltltll C.hvIH. 24(1952) 187 7 G A H,irlow and D l-I Morman 4hal ('hem 36 (1964)2438 8 0 Samuclson and t Thedu. J Chrtmlaloqr 30 (1967) 556 9 L Bangtsson and O S,=intt~l,,otl, rln,II Clmn At tu. 44 (1969) 217 I0 K S Lc¢ and O
II 12 13 14
Samuul,.on .-lind C l n m
19 ( 1 9 6 5 ) 130
,It/ca 37 (1¢)67)357
U-B Lar,,son. T Isaks~on and O S,tmuel~on..Iota Chem Sttm,I 20(1966) 1965 L D,tvm.,, R D Harrier ,tnd G L Ld~A,,Orl J (htottultoHt 18 (1965) 47 K Shtmomura dnd II F Walton Anal Chvm 37(1965) 1012 C D Scott =n J J Ktrkl,md ted ), Modern Ptcltlltt.~, o/ I..ulmd Chtonttltoqst#/~ht, Wdcy-lnterst.tcn~.c .
SEPARATION
OF GLYCOLIC
AND
MALIC ACIDS
365
N e w Y o r k . 1971. p 292 15 J C G i c l d m g s D~ncom¢ ~ o f Chronultoglaplav IM.,rcel D e k k c r . N e w Y o r k , 1965. p 369 16 B L K a r g e r ,n J J K i r k l a n d ( E d ) . Modem; P~actl(¢.~ ol LuluU/ C/Iro.lcltoLIrapll~., Wdey-Intersc,encc. N e w Y o r k . 1971. Ch I. p 3 17 I_. R Snyder. J Chromarogr S¢t. 1(1'969)352 18 J Bohemcn and J H Purncll ,n D H Dcsty ( E d ) . Ga~ Chromatograph~ 1938. B u t t e r w o r t h s . London. p 8