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Electro-osmosis on glass fiber filter paper
416
SHORT COMMUNICATIONS
VOL. 20 (I956)
Electro-osmosis on glass fiber filter paper I n a n a t t e m p t to find a stabilizing m e d i u m o t h e r t h a n cellulose p a p e r for t h e s e p a r a t i o n of s e r u m glycoproteins b y i o n o g r a p h y , glass fiber filter p a p e r w a s e m p l o y e d . I t was o b s e r v e d t h a t t h e o r d e r of m a g n i t u d e of electroosmosis w a s m u c h g r e a t e r on this glass p a p e r t h a n t h a t w h i c h h a s been o b s e r v e d on cellulose p a p e r 1. A few e x p e r i m e n t s were set u p to d e t e r m i n e t h e e x t e n t of t h e electroosmosis. T h e P r e c i s i o n I o n o g r a p h * was u s e d for all separations. All r u n s were m a d e u s i n g a veronal buffer at p H 8.6, ionic s t r e n g t h 0.05, a t e m p e r a t u r e of 4 ° C a n d a p o t e n t i a l g r a d i e n t of five volts per c e n t i m e t e r . T h e c u r r e n t carried b y t h e glass p a p e r w a s o b s e r v e d to be twice t h a t carried b y • -. . . . . . . . . . i i¸,1¸¸! i~',¸ ~ J t h e cellulose p a p e r u n d e r otherwise identical ex.i 1 . p e r i m e n t a l conditions. T h e cellulose p a p e r u s e d was W h a t m a n No. I (o. 5 inch width) a n d t h e i-glass p a p e r w a s No. X - 9 3 4 - A H ( m a d e b y .,m / I : ! H u r l b ufiber t P a p e r Co. a n d d i s t r i b u t e d b y H. R e e v e Angel a n d Co., Inc. N e w Y o r k 7, N.Y.). T h e l a t t e r sheets, i2 x x5 inches in size, were c u t into strips o. 5 × 15 inches. I n t h e first set of experim e n t s (i in Fig. x) 5 /~1 of a o.oi % solution of 2 A b r o m p h e n o l blue w a s u s e d as t h e m i g r a n t . In all cases t h e m i g r a n t was a d d e d as a t h i n s t r e a k across t h e full w i d t h of t h e strips m i d w a y . A b e t w e e n t h e ends. Six strips were r u n s i m u l t a n e ously, t h r e e of cellulose p a p e r a n d t h r e e of glass. T h e m o b i l i t y of t h e b r o m p h e n o l blue w a s 4- o.77 • • microns/second/volt/cm (the 4- sign i n d i c a t i n g A • m i g r a t i o n to t h e positive electrode) on t h e cellulose paper, a n d - - o . 4 4 on t h e glass paper. I n t h e n e x t e x p e r i m e n t , (2 in Fig. x) five/~l of a 1 % solution ~. A • of c r y s t a l l i n e b o v i n e p l a s m a a l b u m i n w a s applied to each of t h e six strips. T h e m o b i l i t y of t h e b o v i n e Fig. I. A = bovine p l a s m a a l b u m i n ; B p l a s m a a l b u m i n w a s 4- 0.26 on t h e cellulose p a p e r b r o m p h e n o l blue; G = bovine p l a s m a ?a n d - - o . 6 4 on t h e glass paper. T h e location of globulin; vertical b r o k e n line = p o i n t of t h e p r o t e i n b a n d s in each case was d e t e r m i n e d a p p l i c a t i o n of m i g r a n t to strips. T h e second b y use of t h e b r o m p h e n o l blue t e c h n i q u e I. s t r i p in each set is t h e glass filter paper. I n A n a t t e m p t was t h e n m a d e to s e p a r a t e a t h e glass s t r i p in 3, t h e lightly s h a d e d m i x t u r e on glass paper. T h e m i g r a n t in t h e n e x t p o r t i o n i n d i c a t e s d i s t r i b u t i o n of globulin series of e x p e r i m e n t s (3 in Fig. i) was a solution all t h e w a y f r o m t h e origin. c o n t a i n i n g i % crystalline b o v i n e p l a s m a a l b u m i n a n d x % crystalline bovine p l a s m a 7-globulin (Fraction II). I n all cases t h e p r o t e i n s a m p l e s were o b t a i n e d f r o m t h e R e s e a r c h Division, A r m o u r a n d C o m p a n y , Chicago• T h e mobilities of t h e bovine p l a s m a a l b u m i n a n d bovine p l a s m a ?-globulin on cellulose p a p e r were 4-0.26 a n d 4- 0.02 respectively. O n glass p a p e r t h e mobilities were - - 0 . 6 4 a n d - - o . 9 x respectively. T h e m o b i l i t y of d e x t r a n , w h i c h h a s o f t e n been u s e d as a electroosmotic i n d i c a t o r in ionographic research, w a s f o u n d to be - - 14. T h e location of t h e d e x t r a n zone on t h e glass filter p a p e r strip w a s d e t e r m i n e d b y i m m e r s i n g t h e strip in w a r m c o n c e n t r a t e d sulfuric acid which resulted in a b r o w n c h a r r e d zone. Since t h e glass paper, as c o n t r a s t e d w i t h cellulose paper, does n o t yield a colored b a c k g r o u n d w i t h c a r b o h y d r a t e - d e t e c t i n g agents, t h e l a t i t u d e of choice of developing a g e n t is a wide one. U p o n s c a n n i n g t h e s t a i n e d i o n o g r a m s w i t h a n a u t o m a t i c s c a n n i n g device l, u s e d to d e t e r m i n e t h e a m o u n t of m a t e r i a l in a g i v e n b a n d , it w a s f o u n d t h a t t h e glass p a p e r a b s o r b s a l m o s t t h r e e t i m e s as m u c h light as W h a t m a n No. i p a p e r (L 4 optical d e n s i t y u n i t s a n d 0.93 optical d e n s i t y u n i t s respectively). T h e b r o m p h e n o l blue g a v e as s h a r p a b a n d on t h e glass p a p e r as it did on t h e cellulose paper, b u t t h e a l b u m i n b a n d was slightly m o r e diffuse on t h e glass p a p e r t h a n on t h e cellulose paper. T h e ?-globulin p a t t e r n s were q u i t e diffuse on t h e glass p a p e r strips, w i t h t h e m a t e r i a l being s p r e a d o u t in t h e fashion of a c o n t i n u o u s s p e c t r u m all t h e w a y f r o m t h e p o i n t of origin. If o n f u r t h e r e x a m i n a t i o n t h e different p o r t i o n s of t h e broad zone on t h e i o n o g r a m s are f o u n d to r e p r e s e n t real differences in t h e c h a r a c t e r of t h e ?-globulin, this_electrosmotic f r a c t i o n a t i o n m a y p r o v e to be of v a l u e in t h e s t u d y of globulin heterogeneity. A t first it m i g h t a p p e a r t h a t s u b s t a n c e s
1~
* Precision Scientific C o m p a n y , Chicago 47, Illinois.
VOL. 2 0
(z956)
SNORT COMMUNICATIONS
4Z7
with a lower net negative charge yield diffuse zones on the ionograms, b u t dextran, which exhibits only a very low electrophoretic mobility in free solution, gave a s h a r p band.
Department of Biochemistry, The Graduate School and Stritch School o] Medicine, Loyola University, Chicago, IlL (U.S.A.)
EDWARD W. BERMES, Jr. HUGH J. McDONALD
1 H. J. McDoNALD, R. J. LAPPE, E. P. MARBACH, R. H. SPITZER AND M. C. URBIN, Ionography; Electrophoresis in Stabilized Media, Year Book Publishers, Chicago, Ill., 1955. Received F e b r u a r y 2r, st, i956
Elution apparatus for quantitative paper chromato9raphy I n the course of recent studies on p l a s m a corticosteroids it was necessary q u a n t i t a t i v e l y to elute large n u m b e r s of p a p e r strips 7 × 1.5 cm in a minimal volume of solvent. The a p p a r a t u s illustrated greatly facilitated the procedure. I n place of micropipettes, 1.5 m m bore glass capillary t u b i n g was used to collect t h e eluate, one end being b e n t up at a right angle and flared to receive the end of the p a p e r ; each t u b e was calibrated at o.i and 0.2 ml. The t a n k and inner t r o u g h are constructed in stainless steel'. The t r o u g h is m o u n t e d on a screw at each end, b y m e a n s of which its height is adjusted to a c c o m m o d a t e strips of different lengths. The slope of the tubes m a y be varied; t h e y are attached by clips to s h o r t sections of steel t u b i n g m o u n t e d on a 5 m m rod, and these m a y be rotated on the rod by slackening a fixing screw.
Fig. z. Papers are held between a single thin glass sheet below and small rectangles of 7 tuna glass plate above. The t a n k is covered by a glass lid which rests on a s p o n g e - r u b b e r rim. The b o t t o m of the t a n k is covered with the eluting solvent, and after inserting the papers the t r o u g h is half filled with solvent; it is unnecessary to await equilibration. Using this procedure 2 big cortisol was eluted into 0.05 ml absolute ethanol w i t h o u t detectable loss in ten recovery experiments.
Department o/Physiology, University o[ Cape Town (South Alrica)
BARRY LEWlS Received F e b r u a r y -,ist, t956
" The stainless steel a p p a r a t u s was constructed by the Marks Engineering Works Association, Capetown.
SHORT COMMUNICATIONS
VOL. 20 (I956)
Electro-osmosis on glass fiber filter paper I n a n a t t e m p t to find a stabilizing m e d i u m o t h e r t h a n cellulose p a p e r for t h e s e p a r a t i o n of s e r u m glycoproteins b y i o n o g r a p h y , glass fiber filter p a p e r w a s e m p l o y e d . I t was o b s e r v e d t h a t t h e o r d e r of m a g n i t u d e of electroosmosis w a s m u c h g r e a t e r on this glass p a p e r t h a n t h a t w h i c h h a s been o b s e r v e d on cellulose p a p e r 1. A few e x p e r i m e n t s were set u p to d e t e r m i n e t h e e x t e n t of t h e electroosmosis. T h e P r e c i s i o n I o n o g r a p h * was u s e d for all separations. All r u n s were m a d e u s i n g a veronal buffer at p H 8.6, ionic s t r e n g t h 0.05, a t e m p e r a t u r e of 4 ° C a n d a p o t e n t i a l g r a d i e n t of five volts per c e n t i m e t e r . T h e c u r r e n t carried b y t h e glass p a p e r w a s o b s e r v e d to be twice t h a t carried b y • -. . . . . . . . . . i i¸,1¸¸! i~',¸ ~ J t h e cellulose p a p e r u n d e r otherwise identical ex.i 1 . p e r i m e n t a l conditions. T h e cellulose p a p e r u s e d was W h a t m a n No. I (o. 5 inch width) a n d t h e i-glass p a p e r w a s No. X - 9 3 4 - A H ( m a d e b y .,m / I : ! H u r l b ufiber t P a p e r Co. a n d d i s t r i b u t e d b y H. R e e v e Angel a n d Co., Inc. N e w Y o r k 7, N.Y.). T h e l a t t e r sheets, i2 x x5 inches in size, were c u t into strips o. 5 × 15 inches. I n t h e first set of experim e n t s (i in Fig. x) 5 /~1 of a o.oi % solution of 2 A b r o m p h e n o l blue w a s u s e d as t h e m i g r a n t . In all cases t h e m i g r a n t was a d d e d as a t h i n s t r e a k across t h e full w i d t h of t h e strips m i d w a y . A b e t w e e n t h e ends. Six strips were r u n s i m u l t a n e ously, t h r e e of cellulose p a p e r a n d t h r e e of glass. T h e m o b i l i t y of t h e b r o m p h e n o l blue w a s 4- o.77 • • microns/second/volt/cm (the 4- sign i n d i c a t i n g A • m i g r a t i o n to t h e positive electrode) on t h e cellulose paper, a n d - - o . 4 4 on t h e glass paper. I n t h e n e x t e x p e r i m e n t , (2 in Fig. x) five/~l of a 1 % solution ~. A • of c r y s t a l l i n e b o v i n e p l a s m a a l b u m i n w a s applied to each of t h e six strips. T h e m o b i l i t y of t h e b o v i n e Fig. I. A = bovine p l a s m a a l b u m i n ; B p l a s m a a l b u m i n w a s 4- 0.26 on t h e cellulose p a p e r b r o m p h e n o l blue; G = bovine p l a s m a ?a n d - - o . 6 4 on t h e glass paper. T h e location of globulin; vertical b r o k e n line = p o i n t of t h e p r o t e i n b a n d s in each case was d e t e r m i n e d a p p l i c a t i o n of m i g r a n t to strips. T h e second b y use of t h e b r o m p h e n o l blue t e c h n i q u e I. s t r i p in each set is t h e glass filter paper. I n A n a t t e m p t was t h e n m a d e to s e p a r a t e a t h e glass s t r i p in 3, t h e lightly s h a d e d m i x t u r e on glass paper. T h e m i g r a n t in t h e n e x t p o r t i o n i n d i c a t e s d i s t r i b u t i o n of globulin series of e x p e r i m e n t s (3 in Fig. i) was a solution all t h e w a y f r o m t h e origin. c o n t a i n i n g i % crystalline b o v i n e p l a s m a a l b u m i n a n d x % crystalline bovine p l a s m a 7-globulin (Fraction II). I n all cases t h e p r o t e i n s a m p l e s were o b t a i n e d f r o m t h e R e s e a r c h Division, A r m o u r a n d C o m p a n y , Chicago• T h e mobilities of t h e bovine p l a s m a a l b u m i n a n d bovine p l a s m a ?-globulin on cellulose p a p e r were 4-0.26 a n d 4- 0.02 respectively. O n glass p a p e r t h e mobilities were - - 0 . 6 4 a n d - - o . 9 x respectively. T h e m o b i l i t y of d e x t r a n , w h i c h h a s o f t e n been u s e d as a electroosmotic i n d i c a t o r in ionographic research, w a s f o u n d to be - - 14. T h e location of t h e d e x t r a n zone on t h e glass filter p a p e r strip w a s d e t e r m i n e d b y i m m e r s i n g t h e strip in w a r m c o n c e n t r a t e d sulfuric acid which resulted in a b r o w n c h a r r e d zone. Since t h e glass paper, as c o n t r a s t e d w i t h cellulose paper, does n o t yield a colored b a c k g r o u n d w i t h c a r b o h y d r a t e - d e t e c t i n g agents, t h e l a t i t u d e of choice of developing a g e n t is a wide one. U p o n s c a n n i n g t h e s t a i n e d i o n o g r a m s w i t h a n a u t o m a t i c s c a n n i n g device l, u s e d to d e t e r m i n e t h e a m o u n t of m a t e r i a l in a g i v e n b a n d , it w a s f o u n d t h a t t h e glass p a p e r a b s o r b s a l m o s t t h r e e t i m e s as m u c h light as W h a t m a n No. i p a p e r (L 4 optical d e n s i t y u n i t s a n d 0.93 optical d e n s i t y u n i t s respectively). T h e b r o m p h e n o l blue g a v e as s h a r p a b a n d on t h e glass p a p e r as it did on t h e cellulose paper, b u t t h e a l b u m i n b a n d was slightly m o r e diffuse on t h e glass p a p e r t h a n on t h e cellulose paper. T h e ?-globulin p a t t e r n s were q u i t e diffuse on t h e glass p a p e r strips, w i t h t h e m a t e r i a l being s p r e a d o u t in t h e fashion of a c o n t i n u o u s s p e c t r u m all t h e w a y f r o m t h e p o i n t of origin. If o n f u r t h e r e x a m i n a t i o n t h e different p o r t i o n s of t h e broad zone on t h e i o n o g r a m s are f o u n d to r e p r e s e n t real differences in t h e c h a r a c t e r of t h e ?-globulin, this_electrosmotic f r a c t i o n a t i o n m a y p r o v e to be of v a l u e in t h e s t u d y of globulin heterogeneity. A t first it m i g h t a p p e a r t h a t s u b s t a n c e s
1~
* Precision Scientific C o m p a n y , Chicago 47, Illinois.
VOL. 2 0
(z956)
SNORT COMMUNICATIONS
4Z7
with a lower net negative charge yield diffuse zones on the ionograms, b u t dextran, which exhibits only a very low electrophoretic mobility in free solution, gave a s h a r p band.
Department of Biochemistry, The Graduate School and Stritch School o] Medicine, Loyola University, Chicago, IlL (U.S.A.)
EDWARD W. BERMES, Jr. HUGH J. McDONALD
1 H. J. McDoNALD, R. J. LAPPE, E. P. MARBACH, R. H. SPITZER AND M. C. URBIN, Ionography; Electrophoresis in Stabilized Media, Year Book Publishers, Chicago, Ill., 1955. Received F e b r u a r y 2r, st, i956
Elution apparatus for quantitative paper chromato9raphy I n the course of recent studies on p l a s m a corticosteroids it was necessary q u a n t i t a t i v e l y to elute large n u m b e r s of p a p e r strips 7 × 1.5 cm in a minimal volume of solvent. The a p p a r a t u s illustrated greatly facilitated the procedure. I n place of micropipettes, 1.5 m m bore glass capillary t u b i n g was used to collect t h e eluate, one end being b e n t up at a right angle and flared to receive the end of the p a p e r ; each t u b e was calibrated at o.i and 0.2 ml. The t a n k and inner t r o u g h are constructed in stainless steel'. The t r o u g h is m o u n t e d on a screw at each end, b y m e a n s of which its height is adjusted to a c c o m m o d a t e strips of different lengths. The slope of the tubes m a y be varied; t h e y are attached by clips to s h o r t sections of steel t u b i n g m o u n t e d on a 5 m m rod, and these m a y be rotated on the rod by slackening a fixing screw.
Fig. z. Papers are held between a single thin glass sheet below and small rectangles of 7 tuna glass plate above. The t a n k is covered by a glass lid which rests on a s p o n g e - r u b b e r rim. The b o t t o m of the t a n k is covered with the eluting solvent, and after inserting the papers the t r o u g h is half filled with solvent; it is unnecessary to await equilibration. Using this procedure 2 big cortisol was eluted into 0.05 ml absolute ethanol w i t h o u t detectable loss in ten recovery experiments.
Department o/Physiology, University o[ Cape Town (South Alrica)
BARRY LEWlS Received F e b r u a r y -,ist, t956
" The stainless steel a p p a r a t u s was constructed by the Marks Engineering Works Association, Capetown.