Dialyzer for continuous countercurrent dialysis of large volume

Mandeles, S.

Woods, E. C.

English

NASA Technical Reports Server

CONTINUOUS COUNTERCURRENT DIALYSIS OF LARGE VOLUMES 
Stanley Mandeles and Ernest C. Woods, 
Space Sciences Laboratory 
University o f  Ca l i fo rn ia  
Berkeley, Cal i fornia  
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CONTINUOUS COUNTERCURRENT DIALYSIS 
I 1 
1 
-. ine aeterminat ion of  base Sequences i n  n u t i e i c  acids often invoives 
the chromatographic separation of large quan t i t i es  o f  o l igonucleot ides.  
The removal of s a l t  from these chromatographic f rac t i ons  by d i a l y s i s  poses 
a problem when the volumes t o  be dialyzed exceed several hundred m l .  We 
have constructed a d ia l yze r  from p l a s t i c  mater ia ls  tha t  removes so lutes 
more r a p i d l y  than the usual mul t i -s tep d i a l y s i s  method (1)  and compares 
favorably i n  e f f i c i ency  w i t h  the recent ly  described t h i n  f i l m  countercurrent 
d ia l yze r  o f  Craig and Stewart (2). 
M!!TER!ALS 4Nn FETurlrns 
I n  general, the d ia lyzer  consists o f  an inner d i a l y s i s  tube(through 
which the re ten ta te  flows) concentr ic w i t h  an outer  v i n y l  tube (through 
which the d i f f u s a t e  f lows). 
p l a s t i c  spacers a t  two foo t  in te rva ls  t o  minimize kinking. 
can be extended f o r  20 or  30 feet, and i s  terminated a t  each end w i t h  a 
p l a s t i c  connector w i t h  tubulature that  permits separate i n f l ow  and ou t f l ow  
o f  re ten ta te  and d i f fusate.  See Figure 1. 
The inner d i a l y s i s  tube i s  he ld i n  place by 
This assembly 
The fo l low ing  mater ia l  i s  required f o r  the const ruct ion o f  a s ing le  
d ia l yze r  u n i t  20 feet i n  length; 20 fee t  o f  tygon o r  v i n y l  p l a s t i c  tubing, 
1/2 J.D., 5/&'  0.0.;  2 fee t  o f  v iny l  tubing, 1/811 I . D . ,  1/4" O.D.; 25 fee t  
o f  6 nun d i a l y s i s  tubing; 12 inches o f  polyethylene tubing, 1/2It I .D . ,  5/8" 
O.D.; 4 inches of  polyethylene rod, 7/8" O.D., polyethylene sheet, 1/8" x 
41' x 4". 
Construct ion o f  the Dialyzer. The d c t a i l s  o f  const ruct ion are i l l u s t r a t e d  
i n  Figures 1 - 4. Tygon o r  v i n y l  tubing, 1/211 I.D. i s  cut  into t m - f o o t  
lengths. 
and shal low grooves are cu t  i n  the p l a s t i c  near the ends. 
The polyethylene tubing, 5/8i1 O.D., i s  cu t  i n t o  one-inch lengths 
Nine d iscs are 
c u t  from the 1/8'l t h i c k  p l a s t i c  sheet w i t h  0.51Il O.D. and a 
center, and s i x  or  e igh t  small per fo ra t ing  holes are d r i l l e d  
as shown i n  Figure 2. Each d isc i s  then pressed midway i n t o  
nector  so tha t  the plane o f  the d isc  i s  perpendicular t o  the 
p l a s t i c  connector tube. This i s  i l l u s t r a t e d  i n  Figure 2. 
/ & I  hole i n  the 
around the center 
a p l a s t i c  con- 
ax is  o f  the 
The end pieces are machined from the 7/8l p l a s t i c  stock according t o  
the dimensions shown i n  Figure 3. 
made of the 1/4@ O.D. v i n y l  p l a s t i c  tubing. 
tub ing  may a lso  be made of  1/41 v i n y l  tubing o r  machined as shown i n  Figure 
3.  
The re ten ta te  i n l e t  and o u t l e t  tubes are 
The d i f f u s a t e  i n l e t  and o u t l e t  ,, 
Assembly. 
connections are shown i n  Figure 4. 
ing the d i a i y s i s  tubing a!iernately ihroiigh the ?/2l v i n y l  tc;Sing end the 
polyethylene connectors. 
length o f  d i a l y s i s  tubing w i t h  water and then t i e  o f f  the ends. This semi- 
r i g i d  tube i s  moistened w i t h  water on the ou ts ide  and can be threaded through 
the v i n y l  tubing and polyethylene connectors more easi ly.  
v i n y l  tubing are then forced over the ends o f  the P.E. connectors. Care 
should be taken dur ing l h i s  proceaure t o  see thac rne a i a i y s i s  tubing i s  
not  twisted. 
the ten sections of  v i n y l  tubing and nine polyethylene connectors and the 
appropr iate connections made, the end pieces are prepared f o r  connection by 
forc ing a length o f  1/4 I )  O.D. v i n y l  tubing through the hole i n  the end p iece 
provided fo r  re ten ta te  flow. The 1/4" v i n y l  p l a s t i c  tubing should extend 
about an inch beyond each end o f  the end piece. The d i a l y s i s  tubing from 
the main assembly i s  then cu t  so tha t  i t  extends about one inch beyond the 
edge o f  the I/2l1 v i n y l  tube. The cu t  end o f  the d i a l y s i s  tube i s  then forced 
over the end o f  the 1/4' v i n y l  tube from the end piece. 
i s  secured i n  place w i t h  an 'lo" r i n g  previously mounted on the 1/4l' v i n y l  
tube f o r  t h i s  purpose or  by several turns o f  Tef lon covered 18 gauge copper 
wire. The 1 / 2 l  v i n y l  tubing i s  then forced over the end piece whi le  tak ing  
care t o  draw the 1/4l v i n y l  re ten ta te  tubing ou t  p a r t  way through the reten- 
t a t e  ho le  i n  order t o  prevent tw is t s  and kinks. The same procedure i s  f o l -  
lowed f o r  the opposi te end. I n  the instances where the d i f f u s a t e  o u t l e t  
ho le  i n  the end p iece has not been provided w i t h  a machined polyethylene 
tube, a simp!e snt!et tube may be made by fo rc ing  1/4l O.D. v i n y l  tubing 
t o  a depth of  between 1/4l and 3/8" i n to  the o u t l e t  ho le  made s l i g h t l y  
smal ler  than I/4'l* 
The assembled d ia l yze r  together w i t h  some o f  the d e t a i l s  o f  the 
The assembly procedure involves thread- 
I t  has been found convenient t o  f i l l  the 20 f o o t  
The ends o f  the 
When a l l  o f  the d i a l y s i s  tubing has been threaded through 
The d i a l y s i s  tube 
Because the d ia l yze r  i s  f l ex ib le ,  i t  may be arranged in  any conveni- 
en t  con f igu ra t i on  provided tha t  there are no k inks o r  tw is t s  tha t  w i l l  
occlude flow. One sa t i s fac to ry  arrangement i s  t ha t  o f  a h e l i x  12" i n  
diameter and a 3" pitch.  The h e l i x  permits the d i f f u s a t e  stream t o  be 
l ed  i n  through the bottom and out  through the top and t h i s  has been found 
h e l p f u l  i n  1) 
d i s t i l l e d  water l i n e  or the de-ionized tap water l ine,  and 2) mounting 
add i t i ona l  u n i t s  for increased capacity. 
g r a v i t y  fed o r  pumped i n  through the  top and ou t  through the bottom. 
a pump i s  used, the d i a l y s i s  can be made continuous by connecting the o u t l e t  
removing a i r  bubbles when the  source of  the d i f f u s a t e  i s  the 
$ 1  
The re ten ta te  stream can be 
When 
o f  the retentate t o  the i n l e t  o f  the pump. 
fo r  re tentate volumes tha t  are larger  than the capaci ty o f  the u n i t  being 
used (about 200 ml f o r  the 20 foo t  un i t ) .  
can be continuously monitored, usual ly a t  o r  near the reservoir ,  f o r  
changes such as decreases i n  conduct iv i ty,  or i n  v i s i b l e  o r  u l t r a v i o l e t  
l i g h t  absorption. 
D ia l ys i s  o f  Solut ions. 
d i a l y s i s  o f  so lu t ions commonly encountered i n  nuc le ic  ac id  sequence deter- 
minations: K C I  I M ;  urea, 7M; adenosine, 0.1 mgimi; o i igo-adenyi ic  acid ~f 
chain lengths A7 t o  A1 1, 0.1 mg/ml of  0.01 M NaHC03. In  each case, 220 
m l  o f  so lu t i on  was d ia lyzed i n  a s ing le  u n i t  w i t h  a capaci ty o f  200 m l .  
re ten ta te  o u t l e t  was connected t o  a reservo i r  which was i n  ser ies w i t h  a 
va r iab le  speed p e r i s t a l t i c  pump. The pump o u t l e t  was connected t o  the 
re ten ta te  i n l e t  so tha t  the retentate could be cycled continuously through 
the d ia lyzer .  
in terva ls ,  1 - 3 m l  o f  re tentate was removed a t  the reservo i r  f o r  analysis. 
The concentrat ions o f  each so lute was determined as fo l lows:  K C I  by conduc- 
t i v i t y ,  urea by weighing, adenosine and o l igo-adenyl ic  ac id  by spectrophoto- 
metry a t  260 mpr 
wh i le  tha t  o f  the d i f f u s a t e  was 30 - 40 m l / m i n .  
A reservo i r  may be provided 
The piogtess of t h e  dia!ysis 
The d ia lyzer  was tested by observing the ra te  o f  
The 
The d i a l y s i s  was car r ied  ou t  a t  23OC and a t  designated 
The f low r a t e  o f  the re ten ta te  was approximately IO ml/mln, 
RESULTS 
The resu l t s  o f  a l l  four  experiments are shown i n  Figure 5. I n  t h i s  
graph, the log o f  the percent concentrat ion o f  so lu te i n  the retentate 
i s  p l o t t e d  versus t ime i n  hours. The concentrat ion o f  so lu te a t  0 time i s  
set  equal t o  100% It can be seen that  the r a t e  o f  d i a l y s i s  i s  essen t ia l l y  
a s t r a i g h t  l i n e  i n  t h i s  p lo t ,  i n  accordance w i t h  theory. Oligo-adenyl ic 
ac id  i s  v i r t u a l l y  not d ia lysab le  i n  e igh t  hours, wh i l e  99% o f  the adenosine 
escaped i n  7 hours. The rates f o r  l M  KCI  and 7M urea were greater:  99% 
o f  these solutes escaped i n  2 1/3 hours (140 minutes). 
D I S C U S S I O N  
The continuous countercurrent d ia lyzer  described above i s  a simply 
fabricated, e a s i l y  assembled device. 
o f  a machine shop i s  necessary i s  i n  the fashioning o f  the end pieces. 
have found, however, t ha t  su i tab le  end pieces can be fabr icated o f  p l a s t i c  
T-tubes, l/2' O.D., and var ious sized v i n y l  tubes telescoped t o  provide the 
The on ly  instance where the f a c i l i t i e s  
We $ ,  
same func t ion  as the machined end pieces. 
used i n  a number o f  d i f f e r e n t  configurations, 
voiumes of retentate. We h a w  found, hewever, that increasing the volume 
o f  the re ten ta te  wi thout augmenting the d i a l y s i s  capaci ty by add i t iona l  u n i t s  
r e s u l t s  i n  a p ropor t iona l  increase i n  the time of  d ia l ys i s .  The d ia lyzer  
i s  s tab le  and may be used many times over. On the o the r  hand, the d i a l y s i s  
tub ing used w i t h  the apparatus can be chemically t reated i n  order t o  a l t e r  
the s e l e c t i v i t y  o f  the membrane ( 3 ) .  
The device i s  portabie, can be 
and i s  expandible f o r  various 
In  regard t o  the e f f i c i e n c y  u; i ; ~ e  apps;a;uz, J = c ! ~ p = r ! s ~ c  msy he made 
w i t h  the e f f i c i e n c y  o f  the d ia lyzer ,  described by Cra ig  and Stewart (2) ,  
t h a t  can c lea r  over 99% o f  a l M  NaCI so lu t i on  i n  a s ing le  pass through the 
device. The f low rates reported f o r  t h i s  d i a l y s i s  r a t e  were between 0.5 and 
1.0 m l / m i n .  This means that, i n  o r d e r  t o  c lea r  99% o f  so lu te  from 200 ml 
o f  1M NaCI, between 200 and 400 
d i a l y z e r  described here c lears  t h i s  amount i n  140 minutes, prov id ing an 
advantage f o r  la rger  volume solutions. The disadvantages are tha t  f i v e  t o  
t e n  times as much d i f f u s a t e  i s  required t o  accomplish the same e f f i c i e n c y  
as the d ia l yze r  of Craig and Stewart. In  addit ion, the membrane area (1230 
cm2) i s  about three- fo ld  greater  than the t h i n  f i l m  d ia l yze r  membrane area, 
so tha t  the oppor tun i ty  to denature very f r a g i l e  so lutes i s  less i n  the 
t h i n  f i l m  dialyzer, I t i s  possible tha t  smaller volumes may be handled and 
a h igher  r a t e  of d i a l y s i s  achieved w i th  d i a l y s i s  tub ing o f  diameter smaller 
than 6 mm. 
minutes o f  d i a l y s i s  time i s  required. The 
SUMMARY 
A simply fabricated, e a s i l y  constructed continuous countercurrent d i a l y -  
zer  i s  described. 
solutes. 
l a rge  volumes 
The performance o f  t h i s  d ia l yze r  was tested w i t h  various 
It was found t o  be very e f f i c i e n t  f o r  the  d i a l y s i s  o f  comparatively 
ACKNOWLEDGEMENTS 
We are pleased to  acknowledge the exce l len t  technica l  assistance of 
Mr. Frank Fearney. 
Supported by grants NsG 479, and NsG 600, by the National Aeronautics , ,  
and Space Administrat ion and grant GM 12158-02, by the National I n s t i t u t e s  
o f  Health, to  the Space Sciences Laboratory, Un ive rs i t y  o f  Cal i forn ia ,  
Berkeley, Ca l i fo rn ia .  
REFERENCES 
1. Stauffer,  R.E., i n  Physical Hethods of Orqanic Chemistry (Technique o f  
Organic Chemistry, Vol. 1 1 1 1 ,  A. Weissberger, Ed., 2nd Ed., Interscience, 
New York - London, 1956, Part  1 .  
2. Craig, L.C., and Stewart, K., Biochemistry, 4, 2712 (1965). 
3. Craig, L.C., and King, T.P., Methods o f  Biochemical Analysis, 10, i75, 
( 1 962) 
. l  
Figure 1. 
shown. Outer tube made o f  v i n y l  tubing, inner tube i s  6 m d i a l y s i s  tube. 
Figure 2. Dimensions of perforated disc and p l a s t i c  connector. Cross-sec- 
t i o n  o f  connector shown w i t h  d isc  i n  place. 
Assembled d ia lyzer .  Cut-away o f  one csiiiieetar and one end piece 
sur= 3. 
de view, and end view. 
gure 4. 
D?mPncians o f  polyethylene end piece shown i n  cross-sect ion o f  
Cut-away and cross-section o f  connections i n  f i n a l  assembly: (A) 
1/4# O.D. polyethylene tubing; (B) end piece; (C) 1/4' O.D. v i n y l  tubing; 
(0) O-ring or Teflon-covered 18 gauge copper wire;  (E) connector ; (F) 6 mm 
d i a l y s i s  tubing; (G) 1/211 I .D. v i n y l  tubing. 
ous solutes. Ordinate: percent o f  o r i g i n a l  
Abscissa: t ime o f  d i a l y s i s  i n  hours. 
Figure 5. Rate o f  d i a l y s i s  o f  var 
so lu te  concentrat ion i n  retentate.  
D i a l y s i s  c a r r i e d  o u t  a t  23OC. 
I 
RET 
RET ENTAT E 
'EN TAT E 
01 FFUSATE Y 
C@NT!N!J@!JS COUNTER - CURRENT DIALYZER 
L- PERFORATED DISC 
1/16's HOLES 
* 
CROSS SECTION OF CONNECTOR 
WITH DISC IN PLACE 
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I O 0  
w 
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Q. I 
A OLIGOADENYLATE 
x ADENOSINE 
0 KCI 
0 UREA 
2 3 4 5 6 7 
DIALY.SIS TIME (HOURS) 
8 


Continuous countercurrent dialysis of large volumes

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