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Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
A Simple Automatic Apparatus for the Concentration of Macromolecular Solutions William J. Hubbard
a
a
Department of Zoology , University of Georgia , Athens, Georgia , 30602 Published online: 05 Dec 2006.
To cite this article: William J. Hubbard (1977) A Simple Automatic Apparatus for the Concentration of Macromolecular Solutions, Preparative Biochemistry, 7:5, 313-319, DOI: 10.1080/00327487708061651 To link to this article: http://dx.doi.org/10.1080/00327487708061651
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [Michigan State University] at 10:06 25 February 2015
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-andconditions
PREPARATIVE BIOCHEMISTRY, 7(5), 313-319
(1977)
A SIMPLE AUTOMATIC APPARATUS FOR THE CONCENTRATION OF MACROMOLECULAR SOLUTIONS
Downloaded by [Michigan State University] at 10:06 25 February 2015
W i l l i a m J. Hubbard, Department o f Zoology, U n i v e r s i t y o f G e o r g i a , Athens, G e o r g i a 30602
ABSTRACT An a p p a r a t u s f o r t h e c o n c e n t r a t i o n of macromolecular s o l u t i o n s is d e s c r i b e d i n d e t a i l .
I t f e a t u r e s a u t o m a t i c volume c o n t r o l and
i s i n e x p e n s i v e and s i m p l e t o f a b r i c a t e .
The b a s i s f o r t h e d e v i c e
is n e g a t i v e p r e s s u r e d i a l y s i s . INTRODUCTION The problem o f r e s t o r i n g d i l u t e macromolecular s o l u t i o n s t o a workable c o n c entration occurs r e g u l a r l y i n biochemical r e s e a r c h . While v a r i o u s commercial c o n c e n t r a t o r s a r e a v a i l a b l e , t h e y a r e generally r a t h e r c o s t l y , e s p e c i a l l y those with automatic f e a t u r e s . Conversely, l a b o r a t o r y f a b r i c a t e d devices (e.g.,
pressure di-
a l y s i s ) , w h i l e s i m p l e and i n e x p e n s i v e , must b e watched c l o s e l y bec a u s e t h e y a r e n o n a u t o m a t i c a n d t e n d t o form c o n c e n t r a t i o n g r a d i e n t s due t o g r a v i t y . This report describes an inexpensive concentrating device t h a t c a n be f a b r i c a t e d from common l a b o r a t o r y materials.
In
addition the apparatus w i l l operate automatically; i t w i l l cease
313 Copyright @ 1977 hy Marcel Dekker. Inc All Rights Reserved. Neither this work nor any part may he reproduced or transmitted in any form or hy any meahs. electronic or mechanical, including photocopying, microfilming, and recording. or by any information storage and retrieval system. without permission in writing from the publisher.
314
HUBBARD
c o n c e n t r a t i n g t h e s o l u t i o n when i t i s r e d u c e d t o a p r e d e t e r m i n e d volume.
Operations a r e c a r r i e d o u t under g e n t l e n o n d e n a t u r a t i v e
c o n d i t i o n s e i t h e r i n t h e c o l d o r a t ambient t e m p e r a t u r e .
The d i -
mensions of t h e a p p a r a t u s c a n be v a r i e d t o s u i t t h e volume of
Downloaded by [Michigan State University] at 10:06 25 February 2015
f l u i d t o b e c . o n c e n t r a t e d o r t o c o n t r o l t h e rate of c o n c e n t r a t i o n . MATERIALS AND METHODS The b a s i c a p p a r a t u s c o n s i s t s of a g l a s s column e n c l o s i n g a l o o p of d i a l y s i s t u b i n g , as shown i n t h e s c h e m a t i c d r a w i n g ( F i g u r e 1 ) . The column is s e a l e d on e i t h e r end, w i t h t h e t o p conn e c t e d t o a vacuum r e s e r v o i r and t h e lower end h o l d i n g t h e d i a l y s i s tubing.
Tubular g l a s s e x t e n s i o n s are connected t o t h e
d i a l y s i s t u b i n g which f u n c t i o n as l i q u i d p i c k u p t u b e s .
These
?
I FIGURE 1.
Magnetic Stirrer
I
315
CONCENTRATION OF MACROMOLECULAR SOLUTIONS
s e t t h e d e p t h o f p e n e t r a t i o n below t h e l i q u i d s u r f a c e i n t h e v e s s e l c o n t a i n i n g t h e f l u i d t o b e c o n c e n t r a t e d , which i n t u r n d e t e r m i n e s t h e f i n a l volume a f t e r c o n c e n t r a t i o n . A l a r g e vacuum r e s e r v o i r is u t i l i z e d b e c a u s e i t r e q u i r e s l i t t l e
Downloaded by [Michigan State University] at 10:06 25 February 2015
a t t e n t i o n and w i l l c o l l e c t t h e f l u i d withdrawn from t h e d i a l y s i s t u b i n g , o r i n t h e e v e n t o f a n a c c i d e n t a l r u p t u r e of t h e t u b i n g , t h e r e s e r v o i r w i l l r e c o v e r t h e sample w i t h o u t l o s s . The d e v i c e used s u c c e s s f u l l y i n o u r l a b o r a t o r y w i l l b e d e s c r i b ed i n o r d e r t o s e r v e a s a b a s i s f o r d e s i g n .
A d e t a i l e d list of
materials is as follows: Item and d e s c r i p t i o n
-
Quantity required
Glass column l m x 28 nun I D , f i r e polished 2 . D i a l y s i s t u b i n g - k" ( p e n c i l size) 3 . g l a s s t u b i n g , 6mm OD 4. number 6% r u b b e r s t o p p e r s w i t h h o l e s f o r snug f i t w i t h 6mm tubing 5. s i d e arm f l a s k , 4 1 , w i t h s t o p p e r 6. s t o p c o c k , 2mm b o r e w i t h t e f l o n va 1ve 7. beaker, 1 l i t e r w i t h g r a d u a t i o n s 8. m a g n e t i c s t i r r e r and f l e a 9. heavy w a l l t u b i n g , 5/8" OD x 3/16" bore 1 0 . r u b b e r t u b i n g , 5 / 1 6 OD x 3/32 wall 11. s u p p o r t s t a n d ( w i t h clamps) 12. Nylon f i l a m e n t s t r a p p i n g t a p e (3M no. 880) 13. a s p i r a t o r , water powered 1.
1 ea a p p r o x i m a t e l y 2m a p p r o x i m a t e l y 40 c m 1 single hole, 1 do ub 1e ho 1e 1 ea 1 ea
1 ea 1 ea approximately 1.3 m 2-4 c m l e n g t h s
1 ea a p p r o x i m a t e l y 20 c m 1 ea
To a s s e m b l e t h e a p p a r a t u s , p l a c e t h e w e t t e d 2m l o o p o f d i a l y s i s t u b i n g i n t h e g l a s s column and i n s e r t t h e two c u t e n d s i n t o t h e 2hole stopper.
A d j u s t t h e l o o p so t h a t t h e u p p e r end w i l l l i e 3-4
cm s h o r t o f t h e upper s t o p p e r .
Take two 10 cm ( f i r e p o l i s h e d )
HUBBARD
316
l e n g t h s of t h e 6 mm g l a s s t u b i n g and i n s e r t them a p p r o x i m a t e l y 2 cm i n t o d i a l y s i s t u b i n g ' s c u t e n d s p r o t r u d i n g t h r o u g h t h e 2 h o l e
S l i p the g l a s s tube d i a l y s i s tubing u n i t s a t least half-
stopper.
way i n t o t h e two r u b b e r s t o p p e r h o l e s .
Downloaded by [Michigan State University] at 10:06 25 February 2015
I n t o t h e u p p e r ( s i n g l e h o l e ) s t o p p e r i n s e r t a l e n g t h of 6mm t u b i n g t h a t h a s been b e n t i n t o a "J" s h a p e , w i t h t h e l o n g a r m down. I t i s a d v i s a b l e t o s e c u r e t h e l o w e r s t o p p e r t o t h e g l a s s column
w i t h nylon filame n t t a p e .
P a s s t h e t a p e from t h e g l a s s o v e r t h e
s t o p p e r and t o t h e g l a s s on t h e o p p o s i t e s i d e .
Tape o v e r t h i s
l o o p , c o m p l e t e l y a r o u n d t h e c i r c u m f e r e n c e o f t h e g l a s s column. Du r i n g a l l o p e r a t i o n s t h e d i a l y s i s t u b i n g s h o u l d n o t b e a ll o wed t o d r y , s o i t i s a d v i s a b l e t o f i l l t h e d i a l y s i s t u b i n g promptly a f t e r s e c u r i n g t h e lower s t o p p e r .
Optionally
one can
p l a c e b u f f e r i n t h e column t o p r e v e n t d r y i n g i f i t is n o t t o b e use d i m m ed i at el y . no a i r b u b b l e s .
The t u b i n g s h o u l d b e f i l l e d so t h a t t h e r e are I f b u b b l e s are p r e s e n t t h e r e s u l t w i l l b e d i s -
c o n t i n u o u s c o n c e n t r a t i o n and l o s s o f e f f i c i e n c y i n t h e apparatus.
After f i l l i n g , the g l a s s t i p s (connecting t h e tubing)
a r e immersed i n t o t h e 1 l i t e r b e a k e r f i l l e d w i t h s o l u t i o n . are two methods f o r t h i s m a n i p u l a t i o n .
There
The f i r s t i s t o s i m p l y
h o l d t h e column c l o s e t o t h e b e a k e r a n d q u i c k l y t i p i t i n t o t h e solution.
A l t e r n a t i v e l y and p r e f e r a b l y , s h o r t s e c t i o n s o f r u b b e r
t u b i n g c a n b e s l i p p e d over t h e t i p s o f t h e g l a s s t u b i n g p r i o r t o f i l l i n g , s o t h a t t h e r u b b e r c a n b e clamped o f f ( h e m o s t a t s work w e l l ) , and t h e n t h e column c a n b e l o w e r e d i n t o t h e s o l u t i o n an d unclamped.
317
CONCENTRATION OF MACROMOLECULAR SOLUTIONS With t h e column immersed and s e c u r e d i n a s t a n d , p l a c e t h e upper s t o p p e r - " J " vacuum h o s e .
t u b e assembly a t o p t h e column and c o n n e c t t h e
P r e p a r e t h e vacuum r e s e r v o i r by s t o p p i n g t h e t o p
and c o n n e c t i n g t h e s i d e arm t o a s h o r t l e n g t h of vacuum h o s e
Downloaded by [Michigan State University] at 10:06 25 February 2015
i n t o which i s i n s e r t e d t h e s t o p c o c k .
The s t o p c o c k w i l l p r o v i d e
f o r i s o l a t i o n o f t h e vacuum r e s e r v o i r f o r e v a c u a t i o n and connection t o t h e apparatus. E v a c u a t e t h e r e s e r v o i r w i t h a water-powered
aspirator.
The
s i m p l e water a s p i r a t o r i s recommended b e c a u s e t h e vacuum o b t a i n e d w i l l n o t be s o low a s t o damage t h e d i a l y s i s t u b i n g .
However, i f
o t h e r vacuum s o u r c e s a r e u s e d , make c e r t a i n t h a t t h e vacuum i s n o t l e s s t h a n 166 mm Hg ( 0 . 2 2 Atm). Connect t h e vacuum r e s e r v o i r t o t h e column and a p p l y t h e vacuum v e r y g r a d u a l l y .
Observe t h e s t a t u s o f t h e d i a l y s i s t u b i n g
c l o s e l y d u r i n g e v a c u a t i o n ; i f i t a p p e a r s t o be s t r e t c h i n g t o t h e b u r s t i n g p o i n t , r e l e a s e t h e vacuum and b l e e d some a i r i n t o t h e reservoir.
Reconnect t h e vacuum s o u r c e and r e p e a t t h e e v a c u a t i o n .
I t i s u s e f u l t o p l a c e t h e b e a k e r o f s o l u t i o n on a m a g n e t i c
s t - i r r e r t o obviate t h e formation of concentration g r a d i e n t s i n t h e bottom of t h e beaker.
The f o r m a t i o n o f s u c h g r a d i e n t s c a n be
used t o a d v a n t a g e , however, i f one w a n t s t o o b t a i n a n a l i q u o t o f c o n c e n t r a t e d m a t e r i a l p r i o r t o c o n c e n t r a t i o n of t h e e n t i r e b a t c h . I n t h a t r e g a r d , one c a n a l l o w a d e n s e , c o n c e n t r a t e d zone t o form i n t h e bottom of t h e beaker ( i n t h e absence of s t i r r i n g ) , and withdraw a n a l i q u o t from below t h e i n t e r f a c e w i t h a p i p e t t e .
HUBBARD
318
The d e t e r m i n a t i o n o f t h e a u t o m a t i c s h u t o f f volume is s i m p l y a m a t t e r o f l o w e r i n g t h e t i p s i n t o t h e b e a k e r t o a volume c a l i b r a t i o n mark.
When t h e s o l y t i o n r e a c h e s t h i s l e v e l , t h e l i q u i d
column i n t h e t u b i n g w i l l b e b r o k e n and t h e d e v i c e w i l l c e a s e t o
Downloaded by [Michigan State University] at 10:06 25 February 2015
c o n c e n t r a t e even though t h e vacuum i s s t i l l b e i n g a p p l i e d .
The
device can s i t unattended f o r a long p e r i o d of t i m e a f t e r shuto f f w i t h no ill e f f e c t s .
RESULTS AND DISCUSSION For a n a p p a r a t u s w i t h t h e d i m e n s i o n s d e s c r i b e d h e r e i n , t h e c o n c e n t r a t i o n r a t e is a p p r o x i m a t e l y 0.35-0.5 mum a s p i r a t o r vacuum a t 4°C.
1 i n 10 h r s . , w i t h maxi-
The s o l u t i o n s under c o n c e n t r a t i o n
were d i l u t e f r a c t i o n s of serum p r o t e i n s i n b u f f e r .
It i s o b v i -
o u s t h a t t h e rate o f c o n c e n t r a t i o n is a n i n v e r s e f u n c t i o n o f t h e
a c t u a l c o n c e n t r a t i o n o f s o l u t e , s o from t h i s one c a n e x p e c t t h e r a t e of c o n c e n t r a t i o n t o d e c r e a s e p r o p o r t i o n a l l y a s t h e s o l u t i o n reaches h i g h e r l e v e l s of c o n c e n t r a t i o n . A n i m p o r t a n t f a c t o r t o c o n s i d e r when u s i n g t h i s d e v i c e i s t o
a v o i d o v e r - c o n c e n t r a t i o n of s o l u t e s , e s p e c i a l l y p r o t e i n s .
Simi-
l a r l y o n e s h o u l d n o t a t t e m p t t o c o n c e n t r a t e s o l u t i o n s which cont a i n denatured p r o t e i n such as f l o c c u l a r a g g r e g a t e s o r p r e c i p i tates.
A s w i t h any membrane s y s t e m , p a r t i c u l a t e s u s p e n s i o n s ,
e s p e c i a l l y "sticky" denatured p r o t e i n s , w i l l clog pores and g r e a t l y r e d u c e t h e c a p a c i t y o f t h e membrane t o p a s s s o l v e n t .
It h a s b e e n
o u r e x p e r i e n c e t h a t when t h e d i a l y s i s t u b i n g i s s u b j e c t e d t o s u c h c o n d i t i o n s i t i s r a t h e r d i f f i c u l t t o c l e a n and r e s t o r e s a t i s f a c t o r y
319
CONCENTRATION OF MACROMOLECULAR SOLUTIONS pe r f o r m an ce.
When t h i s c o n d i t i o n arises, i t is a d v i s a b l e t o d i s -
c a r d t h e d i a l y s i s t u b i n g and r e p l a c e i t .
The u s e f u l l i f e o f t h e
d i a l y s i s t u b i n g is q u i t e l o n g i f m a i n t a i n e d i n t h e c o l d w i t h s u i t a b l e a n t i m i c r o b i a l a g e n t s , and i f t h e a f o r e m e n t i o n e d c o n d i t i o n s o f
Downloaded by [Michigan State University] at 10:06 25 February 2015
p o r e c l o g g i n g are a v o i d e d . I t was o u r c o n c e r n t h a t t h e d i a l y s i s t u b i n g p o r e s may become
e n l a r g e d u n d er vacuum s o t h a t t h e y may become u n a c c e p t a b l y l a r g e . To t e s t t h e p o s s i b i l i t y o f p o r e e n l a r g e m e n t u n d e r vacuum, a n t i b o d y l i g h t c h a i n s ( a p p r o x i m a t e l y 25,000 d a l t o n s ) were c o n c e n t r a t e d u n d er
a vacuum o f 0.22 A t m .
No p r o t e i n w a s found i n t h e b u f f e r w h i ch
passed through t h e tubing.
T h e r e f o r e , a 25,000 d a l t o n lower mole-
c u l a r w e i g h t l i m i t f o r c o n c e n t r a t i o n would p r o b a b l y b e a s a t i s factory guideline f o r t h i s device. ACKNOWLEDGEMENTS I w i s h t o t h a n k D r . R. T. Damian f o r p r o v i d i n g r e s e a r c h f a -
c i l i t i e s an d c o n s u l t a t i o n .
S u p p o r t f o r t h i s work was o b t a i n e d from
a g r a n t f r o m t h e R o c k e f e l l e r F o u n d a ti o n and G r a n t 2 R22 AI-12065 from t h e U n i t e d S t a t e s - J a p a n C o o p e r a t i v e M e d i cal S c i e n c e Program a d m i n i s t e r e d by t h e N a t i o n a l I n s t i t u t e o f A l l e r g y an d I n f e c t i o n Diseases.
Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
A Simple Automatic Apparatus for the Concentration of Macromolecular Solutions William J. Hubbard
a
a
Department of Zoology , University of Georgia , Athens, Georgia , 30602 Published online: 05 Dec 2006.
To cite this article: William J. Hubbard (1977) A Simple Automatic Apparatus for the Concentration of Macromolecular Solutions, Preparative Biochemistry, 7:5, 313-319, DOI: 10.1080/00327487708061651 To link to this article: http://dx.doi.org/10.1080/00327487708061651
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [Michigan State University] at 10:06 25 February 2015
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-andconditions
PREPARATIVE BIOCHEMISTRY, 7(5), 313-319
(1977)
A SIMPLE AUTOMATIC APPARATUS FOR THE CONCENTRATION OF MACROMOLECULAR SOLUTIONS
Downloaded by [Michigan State University] at 10:06 25 February 2015
W i l l i a m J. Hubbard, Department o f Zoology, U n i v e r s i t y o f G e o r g i a , Athens, G e o r g i a 30602
ABSTRACT An a p p a r a t u s f o r t h e c o n c e n t r a t i o n of macromolecular s o l u t i o n s is d e s c r i b e d i n d e t a i l .
I t f e a t u r e s a u t o m a t i c volume c o n t r o l and
i s i n e x p e n s i v e and s i m p l e t o f a b r i c a t e .
The b a s i s f o r t h e d e v i c e
is n e g a t i v e p r e s s u r e d i a l y s i s . INTRODUCTION The problem o f r e s t o r i n g d i l u t e macromolecular s o l u t i o n s t o a workable c o n c entration occurs r e g u l a r l y i n biochemical r e s e a r c h . While v a r i o u s commercial c o n c e n t r a t o r s a r e a v a i l a b l e , t h e y a r e generally r a t h e r c o s t l y , e s p e c i a l l y those with automatic f e a t u r e s . Conversely, l a b o r a t o r y f a b r i c a t e d devices (e.g.,
pressure di-
a l y s i s ) , w h i l e s i m p l e and i n e x p e n s i v e , must b e watched c l o s e l y bec a u s e t h e y a r e n o n a u t o m a t i c a n d t e n d t o form c o n c e n t r a t i o n g r a d i e n t s due t o g r a v i t y . This report describes an inexpensive concentrating device t h a t c a n be f a b r i c a t e d from common l a b o r a t o r y materials.
In
addition the apparatus w i l l operate automatically; i t w i l l cease
313 Copyright @ 1977 hy Marcel Dekker. Inc All Rights Reserved. Neither this work nor any part may he reproduced or transmitted in any form or hy any meahs. electronic or mechanical, including photocopying, microfilming, and recording. or by any information storage and retrieval system. without permission in writing from the publisher.
314
HUBBARD
c o n c e n t r a t i n g t h e s o l u t i o n when i t i s r e d u c e d t o a p r e d e t e r m i n e d volume.
Operations a r e c a r r i e d o u t under g e n t l e n o n d e n a t u r a t i v e
c o n d i t i o n s e i t h e r i n t h e c o l d o r a t ambient t e m p e r a t u r e .
The d i -
mensions of t h e a p p a r a t u s c a n be v a r i e d t o s u i t t h e volume of
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f l u i d t o b e c . o n c e n t r a t e d o r t o c o n t r o l t h e rate of c o n c e n t r a t i o n . MATERIALS AND METHODS The b a s i c a p p a r a t u s c o n s i s t s of a g l a s s column e n c l o s i n g a l o o p of d i a l y s i s t u b i n g , as shown i n t h e s c h e m a t i c d r a w i n g ( F i g u r e 1 ) . The column is s e a l e d on e i t h e r end, w i t h t h e t o p conn e c t e d t o a vacuum r e s e r v o i r and t h e lower end h o l d i n g t h e d i a l y s i s tubing.
Tubular g l a s s e x t e n s i o n s are connected t o t h e
d i a l y s i s t u b i n g which f u n c t i o n as l i q u i d p i c k u p t u b e s .
These
?
I FIGURE 1.
Magnetic Stirrer
I
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CONCENTRATION OF MACROMOLECULAR SOLUTIONS
s e t t h e d e p t h o f p e n e t r a t i o n below t h e l i q u i d s u r f a c e i n t h e v e s s e l c o n t a i n i n g t h e f l u i d t o b e c o n c e n t r a t e d , which i n t u r n d e t e r m i n e s t h e f i n a l volume a f t e r c o n c e n t r a t i o n . A l a r g e vacuum r e s e r v o i r is u t i l i z e d b e c a u s e i t r e q u i r e s l i t t l e
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a t t e n t i o n and w i l l c o l l e c t t h e f l u i d withdrawn from t h e d i a l y s i s t u b i n g , o r i n t h e e v e n t o f a n a c c i d e n t a l r u p t u r e of t h e t u b i n g , t h e r e s e r v o i r w i l l r e c o v e r t h e sample w i t h o u t l o s s . The d e v i c e used s u c c e s s f u l l y i n o u r l a b o r a t o r y w i l l b e d e s c r i b ed i n o r d e r t o s e r v e a s a b a s i s f o r d e s i g n .
A d e t a i l e d list of
materials is as follows: Item and d e s c r i p t i o n
-
Quantity required
Glass column l m x 28 nun I D , f i r e polished 2 . D i a l y s i s t u b i n g - k" ( p e n c i l size) 3 . g l a s s t u b i n g , 6mm OD 4. number 6% r u b b e r s t o p p e r s w i t h h o l e s f o r snug f i t w i t h 6mm tubing 5. s i d e arm f l a s k , 4 1 , w i t h s t o p p e r 6. s t o p c o c k , 2mm b o r e w i t h t e f l o n va 1ve 7. beaker, 1 l i t e r w i t h g r a d u a t i o n s 8. m a g n e t i c s t i r r e r and f l e a 9. heavy w a l l t u b i n g , 5/8" OD x 3/16" bore 1 0 . r u b b e r t u b i n g , 5 / 1 6 OD x 3/32 wall 11. s u p p o r t s t a n d ( w i t h clamps) 12. Nylon f i l a m e n t s t r a p p i n g t a p e (3M no. 880) 13. a s p i r a t o r , water powered 1.
1 ea a p p r o x i m a t e l y 2m a p p r o x i m a t e l y 40 c m 1 single hole, 1 do ub 1e ho 1e 1 ea 1 ea
1 ea 1 ea approximately 1.3 m 2-4 c m l e n g t h s
1 ea a p p r o x i m a t e l y 20 c m 1 ea
To a s s e m b l e t h e a p p a r a t u s , p l a c e t h e w e t t e d 2m l o o p o f d i a l y s i s t u b i n g i n t h e g l a s s column and i n s e r t t h e two c u t e n d s i n t o t h e 2hole stopper.
A d j u s t t h e l o o p so t h a t t h e u p p e r end w i l l l i e 3-4
cm s h o r t o f t h e upper s t o p p e r .
Take two 10 cm ( f i r e p o l i s h e d )
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l e n g t h s of t h e 6 mm g l a s s t u b i n g and i n s e r t them a p p r o x i m a t e l y 2 cm i n t o d i a l y s i s t u b i n g ' s c u t e n d s p r o t r u d i n g t h r o u g h t h e 2 h o l e
S l i p the g l a s s tube d i a l y s i s tubing u n i t s a t least half-
stopper.
way i n t o t h e two r u b b e r s t o p p e r h o l e s .
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I n t o t h e u p p e r ( s i n g l e h o l e ) s t o p p e r i n s e r t a l e n g t h of 6mm t u b i n g t h a t h a s been b e n t i n t o a "J" s h a p e , w i t h t h e l o n g a r m down. I t i s a d v i s a b l e t o s e c u r e t h e l o w e r s t o p p e r t o t h e g l a s s column
w i t h nylon filame n t t a p e .
P a s s t h e t a p e from t h e g l a s s o v e r t h e
s t o p p e r and t o t h e g l a s s on t h e o p p o s i t e s i d e .
Tape o v e r t h i s
l o o p , c o m p l e t e l y a r o u n d t h e c i r c u m f e r e n c e o f t h e g l a s s column. Du r i n g a l l o p e r a t i o n s t h e d i a l y s i s t u b i n g s h o u l d n o t b e a ll o wed t o d r y , s o i t i s a d v i s a b l e t o f i l l t h e d i a l y s i s t u b i n g promptly a f t e r s e c u r i n g t h e lower s t o p p e r .
Optionally
one can
p l a c e b u f f e r i n t h e column t o p r e v e n t d r y i n g i f i t is n o t t o b e use d i m m ed i at el y . no a i r b u b b l e s .
The t u b i n g s h o u l d b e f i l l e d so t h a t t h e r e are I f b u b b l e s are p r e s e n t t h e r e s u l t w i l l b e d i s -
c o n t i n u o u s c o n c e n t r a t i o n and l o s s o f e f f i c i e n c y i n t h e apparatus.
After f i l l i n g , the g l a s s t i p s (connecting t h e tubing)
a r e immersed i n t o t h e 1 l i t e r b e a k e r f i l l e d w i t h s o l u t i o n . are two methods f o r t h i s m a n i p u l a t i o n .
There
The f i r s t i s t o s i m p l y
h o l d t h e column c l o s e t o t h e b e a k e r a n d q u i c k l y t i p i t i n t o t h e solution.
A l t e r n a t i v e l y and p r e f e r a b l y , s h o r t s e c t i o n s o f r u b b e r
t u b i n g c a n b e s l i p p e d over t h e t i p s o f t h e g l a s s t u b i n g p r i o r t o f i l l i n g , s o t h a t t h e r u b b e r c a n b e clamped o f f ( h e m o s t a t s work w e l l ) , and t h e n t h e column c a n b e l o w e r e d i n t o t h e s o l u t i o n an d unclamped.
317
CONCENTRATION OF MACROMOLECULAR SOLUTIONS With t h e column immersed and s e c u r e d i n a s t a n d , p l a c e t h e upper s t o p p e r - " J " vacuum h o s e .
t u b e assembly a t o p t h e column and c o n n e c t t h e
P r e p a r e t h e vacuum r e s e r v o i r by s t o p p i n g t h e t o p
and c o n n e c t i n g t h e s i d e arm t o a s h o r t l e n g t h of vacuum h o s e
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i n t o which i s i n s e r t e d t h e s t o p c o c k .
The s t o p c o c k w i l l p r o v i d e
f o r i s o l a t i o n o f t h e vacuum r e s e r v o i r f o r e v a c u a t i o n and connection t o t h e apparatus. E v a c u a t e t h e r e s e r v o i r w i t h a water-powered
aspirator.
The
s i m p l e water a s p i r a t o r i s recommended b e c a u s e t h e vacuum o b t a i n e d w i l l n o t be s o low a s t o damage t h e d i a l y s i s t u b i n g .
However, i f
o t h e r vacuum s o u r c e s a r e u s e d , make c e r t a i n t h a t t h e vacuum i s n o t l e s s t h a n 166 mm Hg ( 0 . 2 2 Atm). Connect t h e vacuum r e s e r v o i r t o t h e column and a p p l y t h e vacuum v e r y g r a d u a l l y .
Observe t h e s t a t u s o f t h e d i a l y s i s t u b i n g
c l o s e l y d u r i n g e v a c u a t i o n ; i f i t a p p e a r s t o be s t r e t c h i n g t o t h e b u r s t i n g p o i n t , r e l e a s e t h e vacuum and b l e e d some a i r i n t o t h e reservoir.
Reconnect t h e vacuum s o u r c e and r e p e a t t h e e v a c u a t i o n .
I t i s u s e f u l t o p l a c e t h e b e a k e r o f s o l u t i o n on a m a g n e t i c
s t - i r r e r t o obviate t h e formation of concentration g r a d i e n t s i n t h e bottom of t h e beaker.
The f o r m a t i o n o f s u c h g r a d i e n t s c a n be
used t o a d v a n t a g e , however, i f one w a n t s t o o b t a i n a n a l i q u o t o f c o n c e n t r a t e d m a t e r i a l p r i o r t o c o n c e n t r a t i o n of t h e e n t i r e b a t c h . I n t h a t r e g a r d , one c a n a l l o w a d e n s e , c o n c e n t r a t e d zone t o form i n t h e bottom of t h e beaker ( i n t h e absence of s t i r r i n g ) , and withdraw a n a l i q u o t from below t h e i n t e r f a c e w i t h a p i p e t t e .
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The d e t e r m i n a t i o n o f t h e a u t o m a t i c s h u t o f f volume is s i m p l y a m a t t e r o f l o w e r i n g t h e t i p s i n t o t h e b e a k e r t o a volume c a l i b r a t i o n mark.
When t h e s o l y t i o n r e a c h e s t h i s l e v e l , t h e l i q u i d
column i n t h e t u b i n g w i l l b e b r o k e n and t h e d e v i c e w i l l c e a s e t o
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c o n c e n t r a t e even though t h e vacuum i s s t i l l b e i n g a p p l i e d .
The
device can s i t unattended f o r a long p e r i o d of t i m e a f t e r shuto f f w i t h no ill e f f e c t s .
RESULTS AND DISCUSSION For a n a p p a r a t u s w i t h t h e d i m e n s i o n s d e s c r i b e d h e r e i n , t h e c o n c e n t r a t i o n r a t e is a p p r o x i m a t e l y 0.35-0.5 mum a s p i r a t o r vacuum a t 4°C.
1 i n 10 h r s . , w i t h maxi-
The s o l u t i o n s under c o n c e n t r a t i o n
were d i l u t e f r a c t i o n s of serum p r o t e i n s i n b u f f e r .
It i s o b v i -
o u s t h a t t h e rate o f c o n c e n t r a t i o n is a n i n v e r s e f u n c t i o n o f t h e
a c t u a l c o n c e n t r a t i o n o f s o l u t e , s o from t h i s one c a n e x p e c t t h e r a t e of c o n c e n t r a t i o n t o d e c r e a s e p r o p o r t i o n a l l y a s t h e s o l u t i o n reaches h i g h e r l e v e l s of c o n c e n t r a t i o n . A n i m p o r t a n t f a c t o r t o c o n s i d e r when u s i n g t h i s d e v i c e i s t o
a v o i d o v e r - c o n c e n t r a t i o n of s o l u t e s , e s p e c i a l l y p r o t e i n s .
Simi-
l a r l y o n e s h o u l d n o t a t t e m p t t o c o n c e n t r a t e s o l u t i o n s which cont a i n denatured p r o t e i n such as f l o c c u l a r a g g r e g a t e s o r p r e c i p i tates.
A s w i t h any membrane s y s t e m , p a r t i c u l a t e s u s p e n s i o n s ,
e s p e c i a l l y "sticky" denatured p r o t e i n s , w i l l clog pores and g r e a t l y r e d u c e t h e c a p a c i t y o f t h e membrane t o p a s s s o l v e n t .
It h a s b e e n
o u r e x p e r i e n c e t h a t when t h e d i a l y s i s t u b i n g i s s u b j e c t e d t o s u c h c o n d i t i o n s i t i s r a t h e r d i f f i c u l t t o c l e a n and r e s t o r e s a t i s f a c t o r y
319
CONCENTRATION OF MACROMOLECULAR SOLUTIONS pe r f o r m an ce.
When t h i s c o n d i t i o n arises, i t is a d v i s a b l e t o d i s -
c a r d t h e d i a l y s i s t u b i n g and r e p l a c e i t .
The u s e f u l l i f e o f t h e
d i a l y s i s t u b i n g is q u i t e l o n g i f m a i n t a i n e d i n t h e c o l d w i t h s u i t a b l e a n t i m i c r o b i a l a g e n t s , and i f t h e a f o r e m e n t i o n e d c o n d i t i o n s o f
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p o r e c l o g g i n g are a v o i d e d . I t was o u r c o n c e r n t h a t t h e d i a l y s i s t u b i n g p o r e s may become
e n l a r g e d u n d er vacuum s o t h a t t h e y may become u n a c c e p t a b l y l a r g e . To t e s t t h e p o s s i b i l i t y o f p o r e e n l a r g e m e n t u n d e r vacuum, a n t i b o d y l i g h t c h a i n s ( a p p r o x i m a t e l y 25,000 d a l t o n s ) were c o n c e n t r a t e d u n d er
a vacuum o f 0.22 A t m .
No p r o t e i n w a s found i n t h e b u f f e r w h i ch
passed through t h e tubing.
T h e r e f o r e , a 25,000 d a l t o n lower mole-
c u l a r w e i g h t l i m i t f o r c o n c e n t r a t i o n would p r o b a b l y b e a s a t i s factory guideline f o r t h i s device. ACKNOWLEDGEMENTS I w i s h t o t h a n k D r . R. T. Damian f o r p r o v i d i n g r e s e a r c h f a -
c i l i t i e s an d c o n s u l t a t i o n .
S u p p o r t f o r t h i s work was o b t a i n e d from
a g r a n t f r o m t h e R o c k e f e l l e r F o u n d a ti o n and G r a n t 2 R22 AI-12065 from t h e U n i t e d S t a t e s - J a p a n C o o p e r a t i v e M e d i cal S c i e n c e Program a d m i n i s t e r e d by t h e N a t i o n a l I n s t i t u t e o f A l l e r g y an d I n f e c t i o n Diseases.
