A Comparison of the Electrophoretic Velocities of Cellophane and Collodion Suspensions With Electroosmotic Velocities Through Membranes of the Same Materials

8/2/2019 A Comparison of the Electrophoretic Velocities of Cellophane and Collodion Suspensions With Electroosmotic Velocities Through Membranes of the Same Mate

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A C O M P A R I S O N O F T H E E L E C T R O P H O R E T I C V E L O C I T I E SO F C E L L O P H A N E A N D C O L L O D I O N S U S P E N S I O N S

W I T H E L E C T R O O S M O T I C V E L O C I T I E S T H R O U G HM E M B R A N E S O F T H E S A M E M A T E R I A L S

BY BETTY MONAGHAN, H. L. WHITE , AND FRAN K URBA N(From the Department of Physiology and the Department of Biologica l Chemistry,Washington U niversity School of Medicine, St. Louis)

(Accepte d for publicat ion, Jun e 29, 1934)Th is pape r is a repo rt of the electropho ret ic veloci ties of cel lophane

and collodion suspensions in ThC14 solut ions of various concen trat ions,and of the e lec t roosmot ic ve loci t ies of the same solut ions throu ghcel lophane and col lodion membranes .

EXPER rM'F.NTALCdlaphane.--For the dect rophores i s de te rm inat ions a col lo ida l suspens ion of

ce l lophane was prepa red by soaking ce l lophane (previous ly washed f ree of g lycer inand dr ied) in a mixture of about equal par t s of ace tone and e ther for severa l days ,put t ing a few cubic cent imeters of th i s mixture in to 100 cc . of water and aera t ingto remove the ace tone and e ther . On two occas ions out of severa l t r i a l s sa t i s -fac tory suspens ions were obta ined; they remained s table for severa l weeks. Weregre t tha t we are unable to spec i fy the fac tors respons ib le for the success orfa i lure of th is procedure . This suspens ion was examined in a Nor thr op-K uni tzce l l of prede termined cross -sec t ion and the cur rent measured . I t var ied f rom1 10 -6 amp. in wa ter to 5 X 10 -8 in 4 X 10 -2 M ThCI4. Th e specific resista nceof each so lu t ion was de termined in the usua l way and the vol t s per cent imeteracross the ce l l ca lcula ted as IR , as sugges ted by Abram son (1929) . Ze ta was

14 micra/sec.calculate d in m~ll~volts as, zeta = The resul ts are shown inv o l t s / c m .Fig. 1, where zeta in m il l~volts is plot ted aga inst the negat iv e logari th m of themolar concent ra t ion of ThCI , . The i sodec t r i c poin t is be tween 3 and 4 10 -6 K.

The e lec t roosmot ic i soe lec t r ic poin t was then de termined on in tac t ce l lophanemembran es. A side arm w as sealed to a tube of 2.5 cm. bore and 6 cm. lengthand a c apil lary of 1 ram. bore f i t ted to the side arm. A cel lophane sheet was t iedover one end of the l a rge tube and sea led t ight wi th collodion . The upper endof the tube was f i t t ed wi th a rub ber s topper and sea led wi th beeswax- ros in cement.This s topper was per fora ted by a t ight ly f i t t ing tube forming an agar -KC1 br idge .

523

The Journal of General Physiology

Published March 20, 1935


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524 C O ~ I P A R I S O N O F ELECTROP HOP ~ETIC V E L O C I T I E ST h e b r i d g e p a s s e d t o a l a r g e c a l o m e l - s a t u r a t e d K C I e l e c tr o d e ; i ts l o w e r e n d j u s ta b o v e t h e m e m b r a n e w a s t u r n e d u p . T h e c e l l w a s f il l e d w i t h t h e s o l u t i o n u n d e ri n v e s t i g a t io n a n d d i p p e d i n t o a b e a k e r c o n t a i n i n g t h e s a m e s o l u t i o n , t h e c i r c u itb e i n g c o m p l e t e d b y a n a g a r b r i d g e i n t h e b e a k e r a n d a n o t h e r c a l o m e l e le c t ro d e .

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F I G . 1. E l e c t r o p h o r e t i c z e t a p o t e n t i a l s i n m i l U v o l t s o f a c e l l o p h a n e s u s p e n s i o ne , a n d e l e c tr o o s m o t i c v e l o c i t y i n a r b i t r a r y u n i t s o f t h r e e c e ll o p h a n e m e m b r a n e sO , a s a f u n c t i o n o f t h e T h C 1 4 c o n c e n t r a t io n .T h e m o v e m e n t o f t h e m e n i s c u s i n t h e n e a r l y h o r i z o n t a l c a p i ll a r y s i d e a r m w a so b s e r v e d w i t h a m i c r o s c o p e . 1 0 0 v o l t s w e r e a p p l i e d a t t h e e l e c t r o d e s . T h ec u r r e n t t h r o u g h t h e c e l l w a s a l w a y s m e a s u r e d . P l a t i n u m e l e c t ro d e s w e r e u s e da t f i r s t b u t w i t h t h e s e t h e c u r r e n t w a s n o t t h e s a m e o n r e v e r s a l o f p o l a r i t y ; w i t h



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B. M ONAGHAN~ H. L . WHITE~ AND ]? . URBAN 5 2 5c a l o m e l e l e c t r o d e s i t w a s u n c h a n g e d a l t h o u g h i t r o s e s l o w l y w i t h t i m e , d u e , p r e -s u m a b l y , t o t h e d i f fu s i o n o f K C 1 f r o m t h e a g a r b r i d g e s . H i g h e r c o n c e n t r a t i o n st h a n 4 1 0 3 T h C 1 4 c o u l d n o t b e i n v e s t i g a t e d b e c a u s e o f t h e h e a t i n g e f f e c to f t h e l a r g e r c u r r e n ts . T h e a v e r a g e o f s e v e ra l r e a d i n g s w i t h e a c h d i r e c t i o n o fc u r r e n t w a s t a k e n . T h e r e s u l t s o n t h r e e m e m b r a n e s , w i t h r a t e o f e l e c t r o o sm o t i ct r a n s p o r t e x p r e s se d i n a r b i t r a r y u n i t s , a r e g i v e n i n F i g . 1 .

A b s o l u t e v a l u e s o f z e t a b y t h e e l e c t r o o s m o t i c m e t h o d c a n b e g i v e n o n l y i f t h eE . ~ . F . a c r o s s t h e m e m b r a n e i s k n o w n . T h i s m u s t b e o n l y a s m a l l f r a c t i o n , ino u r e x p e r i m e n t s , o f th e 1 0 0 v o l t s a t t h e e le c t ro d e s . W e h a v e a t t e m p t e d t o d e t e r -m i n e t h i s b y d e t e r m i n i n g t h e r e s i s ta n c e o f c el l o p h an e m e m b r a n e s i n a n a p p a r a t u so f t h e t y p e d e s c r i b ed b y G r e e n , W e e c h , a n d M i c h a e ll s (1 92 9) a n d m u l t i p l y i n g b yt h e c u r r e n t . W e h a v e n o t y e t s u c c ee d e d , h o w e v e r , i n m e a s u r i n g t h e s e r e s is t a n c esw i t h c o n s i s t e n t e n o u g h r e s u l t s t o j u s t i f y a s t a t e m e n t a s t o t h e E . ~ . F . a c r o s s t h em e m b r a n e s . I t a p p e a r s p r o b a b l e t h a t t h e m e m b r a n e r e s i s ta n c e s a r e s o l o w a sn o t t o b e m e a s u r a b l e w i t h m u c h a c c u r a c y . I t i s im p r o b a b l e t h a t t h e E . ~ .~ .a c r o s s t h e m e m b r a n e r e m a i n s a c o n s t a n t f r a c t i o n o f t h e a p p l i e d E .M . ~ . i n t h ev a r i o u s s o l u ti o n s , s i n ce t h e r a t i o o f s u r f a c e t o b u l k c o n d u c t i v i t y m u s t b e h i g ha n d s i n c e t h e d i f fu s i o n o f K C 1 i n t r o d u c e s a n i n c o n s t a n t e r r o r . T h e r e f o r e , t h ez e t a - c o n c e n t r a t i o n c u r v e m a y n o t b e o f t h e s a m e s h a p e a s t h e d e c t r o o s m o t i ct r a n s p o r t - c o n c e n t r a t i o n c u r v e. N e v e r t h e l es s , s in c e t h e p e r c e n t i l e c h a n g e s u n d e r -g o n e b y z e t a a r e b e y o n d q u e s t i o n m u c h g r e a t e r t h a n t h o s e o f ~ . ~ . ~ . a c r o s s t h em e m b r a n e , t h e t r a n s p o r t - c o n c e n t r a t i o n c u r v e p r o b a b l y d o e s n o t g r e a t l y d i f f e rq u a l i t a t i v e l y f r o m t h e z e t a - c o n c e n t r a t i o n c u r v e . I n a n y e v e n t , t h e i s o e le c t r i ep o i n t t o d e c t r o o s m o s i s i s a c c u r a t e l y l o c a t e d a t 4 1 0 ~ ~ T h C h , a c o n c e n t r a t i o na b o u t 1 0 t i m e s a s g r e a t a s th e i s o d e c t r i c p o i n t t o e l e c tr o p h o re s i s. T h e a d v a n -t a g e o f c o m p a r i n g t w o p r o c e s s e s , a s e l e c t r o o s m o s i s a n d e l e c t r o p h o r e s i s , b y a c o m -p a r i s o n o f t h e i r is o e l ec t r ic p o i n t s r a t h e r t h a n b y a n e v a l u a t i o n o f z e t a a t v a l u e so t h e r t h a n z e ro , is t h a t c o m m o n t o a l l n u n p o i n t m e t h o d s .

I t w a s t h o u g h t t h a t t h i s d i f fe r en c e i n t h e e l e c t r o p h o re t i c a n d e l e c t ro o s m o t i ci s o e le c t r ic p o i n t s o n c e l l o p h a n e m i g h t b e d u e t o i n a b i l i t y o f t h e t h o r i u m i o n t op e n e t r a t e i n t o t h e s m a l l p o re s o f t h e c e l lo p h a n e m e m b r a n e s i n a r e a s o n a b l e ti m e .T h a t t h i s i s n o t t h e e x p l a n a t i o n w a s s h ow n b y t h e e x p e r i m e n t a l fi n d in g s t h a t ( 1)m e m b r a n e s a l l o w e d t o s o a k f o r m a n y d a y s i n a c o n c e n t r a t i o n o f 1 X 1 0 - 5 ~ T h C 1 4w e r e s t i l l c h a r g e d a s i n w a t e r , ( 2 ) a c t i v e l y f i l t e r in g t h i s s o l u t i o n t h r o u g h a m e m -b r a n e f o r s e v e r a l h o u r s u n d e r p r e s s u r e d i d n o t r e v e r s e t h e s i g n o f c h a r g e o n t h em e m b r a n e , a n d ( 3 ) e v e n m o r e s t r i k i n g , t h r e e m e m b r a n e s w h o s e s i g n o f c h a r g eh a d b e e n r e v e r s e d w i t h a s t r o n g t h o r i u m s o l u t i o n (1 1 0 3 M ) a n d w h o s e p o r e sm u s t t h e r e f o r e h a v e c o n t a i n e d s u f f i c i e n t t h o r i u m t o b r i n g a b o u t r e v e r s a l , v e r yq u i c k l y s h o w e d a n e g a t i v e z e t a p o t e n t i a l w h e n p l a c e d i n 1 X 1 0 5 ~ T h C 14 .

T h e p o s s i b i l i ty w a s t h e n c o n s i d e re d t h a t t h e c e l l o p h a n e s us p e n s io n u s e d i n t h ee l e c tr o p h o r e si s s t u d y w a s s i m p l y a m o r e s o l u b le f r a c t io n o f t h e c e l l op h a n e m e m -b r a n e , w i t h s o m e w h a t d i f f er e n t ch e m i c a l p r o p e r t i e s f r o m t h o s e o f t h e u n t r e a t e dm e m b r a n e . T h e e x p e r i m e n t s w e r e t h e re f o r e r e p e a t e d o n c o ll o d i o n s i n ce w i t h t h i s



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526 COMPARISON 0~' ELECTROPHOKETIC VELOCITIESmaterial suspensions could be obtained which were certainly of the same corn-position as the membrane.

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M O L A R CONCENTRATION ThCl4Fro. 2. Electrophoretic zeta poten tials in miUivolts of a collodion suspension

as a function of ThCh concentration. A-B represents the isoelectric zone forelectroosmosis in five collodion membranes.Collodion.--Collodion membranes of varying pore size were prepared by the

metho d described by Bjerr um and Manegold (1927). The electroosmotic experi-ments on the collodion membranes were carried out exactly as with cellophane.



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B . M O N A G H A N , H . L . W H I T E , A N D F . U R B A N 5 2 7Since the mem branes d i ffered in permea bi l i ty (es t imated average po re d iame terof most permeable membrane between 2 and 3 t imes tha t o f leas t permeable) theelectroosmotic velocit ies varie d greatly at conce ntrations other than isoelectric,but the curves for all five membranes crossed the isoelectric point at concentra-t ions between 4 and 6 X 10 5 M ThC h.Microscopic collodion suspensions for electrophoretic measurements were pre-pared f rom the same s tock col lod ion so lu t ion used for the p reparat ion of the mem -branes. One pa rt of this solution was diluted with ten parts of the solvent (alco-hol and ethe r). Distil led w ater was then added slowly, with shaking, until ami lky suspension was ob tained . Th e ether and mo st o f the alcohol were thenrem oved by ae ration. 1 cc. of this stock suspension was adde d to 250 cc. of thevarious thorium solutions unde~ investigation. It should be me ntioned that th isproced ure is not alway s successful in producing suspensions of the desired pa rticles ize (1 to 5#) ; o f ten the par tic les c lumped rather rap id ly . Bu t in about a dozentrials, two suitable suspensions were obtained; these were kept in the ice boxand used as a stock suspension for all futur e electrop horetic determ inations.Th e electrophore tic zeta-pote ntial curve and the electroosmotic isoelectric zoneare shown in Fig. 2. He re again the isoeleetric conc entration (between 1 and2 X 10 6 ~ ThCh) fo r the par t ic les i s very much less than that found for themembranes (4 to 6 X 10 6 ~ Th Ch) .

DISCUSSIONT h a t t h e d i f f e r e n c e i n t h e i s o e l e c tr i c p o i n t o f c e l l o p h a n e a n d c o l-

l o d io n p a rt i c l e s as c o m p a r e d w i t h m e m b r a n e s o f t h e s a m e m a t e r i a li s d u e t o t h e s m a l l s i ze o f t h e m e m b r a n e p o r e s is i n d i c a t e d b y t h ef a c t t h a t i n v e r y la r g e c a p i l l a r ie s ( 3 0 0 / ~ ra d i u s ) o f p y r e x g la s s , o r o n af l a t g l a s s s u r f a c e , t h e s a m e c o n c e n t r a t i o n o f T h C 1 4 ( a s w e l l a s A 1 C l aa n d F e C 13 ) w h i c h i s i s o e l e c t r i c f o r e l e c t r o o s m o s i s is a l s o i s o e l e c t r i cf o r e l e c t r o p ho r e s i s w i t h p y r e x p a r ti c l e s ( M o n a g h a n , W h i t e , a n d U r b a n( 1 9 3 5 ) ) .

T h e b e h a v i o r of t h e m e m b r a n e s i s p r o b a b l y t o b e a t t r i b u t e d t o t h ei n f lu e n c e o f t h e s m a l l p o r e s i n p r e v e n t i n g c o m p l e t e d e v e l o p m e n t o f t h ee l e c tr i c a l d o u b l e l a y e r s. A c c o r d i n g t o M c B a i n a n d K i s t l e r ( 1 9 28 ) ,t h e l a r g e s t p o r e s in c e l l o p h a n e 60 0 m e m b r a n e s a r e o f t h e o r d e r o f m a g -n i t u d e o f 2 - 3 1 0 - ~ c m . i n r a d i u s . L e t u s n o w c o n s i d e r t h e p r o b -a b l e t h i c k n e s s o f t h e d i f f u s e d o u b l e l a y e r ( f o r a r e c e n t d i s c u s s i o n s e eM t i l l e r, 1 9 33 ) . T h e t h i c k n e s s o f t h e d o u b l e l a y e r d e c r e a s e s w i t h i n -c r e a s in g c o n c e n t r a t i o n , t h e d e c r e a s e b e in g f a s t e r t h e h i g h e r t h e v a l e n c y

4 . 3 2 1 0 - ~ c m . w h e r eo f t h e i o n s , a c c o r d i n g t o t h e e x p r e s s i o n ~ .=



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B . M O N A G H A N , H . L . W H I T E , A N D 1 . U R B A N 529

o f 5 .6 X 1 0 - 6 c m . a t t h e e l e c t r o p h o r e t i c t h o r i u m i s o e l e c tr i c c o n c e n -t r a t i o n ( 3 X 1 0 - 6 ~ T h C 1 , ) . T h i s f ig u r e m a y b e s e v e r a l h u n d r e dp e r c e n t f r o m t h e t r u e v a l u e ; n e v e r t h e l e s s it s e em s c e r t a i n t h a t t h ed o u b l e l ay e r t h i c k n e s s a t t h i s c o n c e n t r a t i o n i s m u c h g r e a t er t h a n t h er a d i u s of t h e m e m b r a n e p o r e s a n d c o n s e q u e n t l y t h a t t h e d o u b l e la y e rw i l l b e v e r y m u c h c o m p r e s s e d i n t h e m e m b r a n e p o re s . I t f o ll o w s

4 7r ~ ,f r o m t h e c o n d e n s e r e q u a t i o n ~ - D t h a t , c h a r g e d e n s i t y , ~ ,r e m a i n i n g t h e s a m e , ~ i s d i r e c t ly p r o p o r t i o n a l t o t h e d i s t a n c e b e t w e e nt h e p la t e s . A s c h e m a t i c r e p r e s e n t a t i o n o f t h e p r o b a b l e c o u r s e o f t h ep o t e n t i a l - d is t a n c e c u r v e s a s t h e t h o r i u m c o n c e n t r a t i o n i s i n c re a s e d isg i v e n i n F ig . 3 ( se e a ls o M o n a g h a n , W h i t e , a n d U r b a n ( 1 9 3 5 )) . I n w a t e r( C u r v e 1 ) w h e r e t h e d o u b l e l a y e r i s n o r m a l l y q u i t e d i ff u s e , t h e z e t ap o t e n t i a l i n t h e S m a l l - p o re d m e m b r a n e w i l l b e g r e a t l y r e d u c e d f r o mt h e n o r m a l v a l u e b e c a u s e o f t h e n e c e s s a r i l y c o m p r e s s e d s t a t e o f t h ed i f f u s e l a y e r . I n 3 X 1 0 - 6 x~ T h C 1 4, w h i c h i s i s o e l e c t r i c f o r t h e p a r -t i cl e s , t h e m e m b r a n e ( e le c t ro o s m o t i c) z e t a p o t e n t i a l h a s t h e s a m e s i g na s i n w a t e r ( C u r v e 2 ). I n 4 X 1 0 - s ~ T h C I ~ (C u r v e 3 ) t h e m e m b r a n ei s i s o e le c t ri c , w h i l e t h e s i g n o f e l e c t r o p h o r e t i c z e t a i s r e v e r s e d . I ns t r o n g e r s o l u t io n s , 4 X 1 0 - s ~ T h C1 4, w h e r e t h e d o u b l e l a y e r t h i c k n e s sa p p r o a c h e s t h e p o r e r a d i u s , t h e p o t e n t i a l o f t h e m e m b r a n e w i l l a p -p r o a c h t h a t o f t h e p a r ti c le s , b o t h b e i n g o f r e v e r s ed s i g n ( C u r v e 4 ) .

S U G A R YI t i s d e m o n s t r a t e d t h a t t h e i s o e le c tr i c c o n c e n t r a t i o n o f T h C I ~ i s

m u c h g r e a t e r f o r e l e ct r o o s m o s i s i n s m a l l -p o r e d m e m b r a n e s ( ce ll o-p h a n e , c o l l o d i o n ) t h a n f o r e l e c tr o p h o r e s i s o f p a r t i c l e s o f t h e s a m em a t e r i a l . A n e x p l a n a t i o n f o r t h e d i ff e re n c e i s a d v a n c e d , b a s e d o n t h ei n f lu e n c e o f t h e s m a l l p o r e s i n p r e v e n t i n g c o m p l e t e d e v e l o p m e n t o f t h ee l e c t r i c a l d o u b l e l a y e r .

B I B L I O G R A P H YAbramson, H. A. , 1929,J . Gen. Phys iol. , 12, 469.Bjerrum, N. , an d Manegold , E. , 1927, Kolloid-Z. , 42, 97 .

t Particularly since this va lue of X designates mea n double layer thickness an dn o t the d is tance f rom the w al l to t h e o u t e r l imi t of the layer .



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5 3 0 CO MP A RI SO N O F E L E CT RO P H O RE T I C V E L O CI T IE SGreen, A . A. , W eech, A. A. , and M ichaelis, L. , 192 9, I . Gen. Physiol ., 12, 473.McBain , J . W. , and Kisf le r , S . S . , 1928, J. Gen. Physiol . , 12, 187.Mo n a g h a n , B . R . , Wh i t e , H . L . , a n d U r b a n , F . , 1 9 3 5 , Co l lo i d s y mp o s i u m mo n o -graph, Bal t imore , T he W il liams & Wilk ins Co. , 11 , in press.Mi i l le r , H. , 1933, in Cold Spr ing H arb or sym posia on quan t i ta t ive b io logy, ColdSpr ing H arbo r , Lon g Is land Biologica l Associa tion , 1 , I .


A Comparison of the Electrophoretic Velocities of Cellophane and Collodion Suspensions With Electroosmotic Velocities Through Membranes of the Same Materials

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