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Computer automation of a novel ion-exchange process for the simultaneous recovery of lysozyme and avidin… March, Alan Charles 1988

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COMPUTER AUTOMATION .OF A NOVEL ION-EXCHANGE PROCESS FOR THE SIMULTANEOUS RECOVERY OF LYSOZYME AND AVIDIN FROM CHICKEN EGG ALBUMEN  by ALAN CHARLES MARCH  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS IN APPLIED SCIENCE  IN THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIO-RESOURCE ENGINEERING  We accept t h i s  t h e s i s as conforming  to the r e q u i r e d standard  The U n i v e r s i t y of B r i t i s h  Columbia  August, 1988  (£)  Alan Charles March, 1988  In  presenting  degree  this  at the  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department publication  this or of  and study.  thesis for scholarly by  this  his  or  her  the  requirements that the  I further agree  purposes  representatives.  may be It  thesis for financial gain shall not  that  T^JC "/2^S<t>t4/2c£,  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  by the that  allowed without  permission.  Department of  advanced  Library shall make it  understood be  an  permission for extensive  granted  is  for  ^ur>//UgH^/V^-  head  of  my  copying  or  my written  i  ABSTRACT A three-column ion-exchange system was designed, f a b r i c a t e d and computer-automated  to accommodate a novel ' e l u t i o n  process developed by Dr. Tim Durance  looping'  (U.B.C. Department  of Food  Science) during h i s d o c t o r a l s t u d i e s on the r e c o v e r y of lysozyme and a v i d i n . This p r o c e s s i n g technique enhances recovery chicken to  of these two egg  handle  liters  pharmaceutically  albumen. The  throughput  per  day  of  small commercial ion-exchange  processing  rates  albumen  between to  important p r o t e i n s  system prototype was approximately f i v e  facilitate  both  s c a l e work. Very e f f i c i e n t  resin  due  to  a  the simultaneous  use  two-column  from sized  and  300  laboratory  and  i s made of the cascaded  feed  arrangement. The  processing  flexibility  columns  toward  allowing  to  handling  unattended  software  was  designed  to p r o v i d e  and ease of o p e r a t i o n i n s e t t i n g up new and  method f i l e s , of  control  use  productivity  f o r the s e l e c t i o n of any column  and  columns  operation.  existing  providing  a  fouled  with  This  approach  1  staged-shutdown' congealed attempts  of the system even when one  has become fouled with congealed albumen.  or group approach  albumen to  or two  during  maximize  of the  the  columns  ii Abstract Table of Contents L i s t of Tables L i s t of F i g u r e s Acknowledgements Introduction  TABLE OF CONTENTS  i i i i i i iv v 1  1. BACKGROUND Ion-exchange Chromatography Ion-Exchange Resins F a c t o r s A f f e c t i n g Exchange Dynamics Recovery of Lysozyme and A v i d i n  3 3 9 10 12  2. AUTOMATION OF LYSOZYME/AVIDIN RECOVERY Physical Plant Operating Sequence Nomenclature Flow Path Design Flow Handling Equipment Valves Pumps Columns Homogenizer Tubing Filters Pressure Transducers U l t r a v i o l e t Monitor E l e c t r o n i c Flow C o n t r o l Hardware DC Power Source System C o n t r o l I n t e r f a c e Analog to D i g i t a l (A/D) Board D i g i t a l Input/Output (I/O) Board Process C o n t r o l Computer  14 14 19 21 24 24 24 25 25 29 29 31 31 32 32 34 36 39 40  3. PROCESS CONTROL SOFTWARE Mandatory C o n t r o l Functions C o n t r o l Enhancements I n t e r a c t i v e input Visual display D e f a u l t d r i v e path F i l e handling r o u t i n e s Staged shutdown Column s e l e c t i o n Manual process c o n t r o l Alarms S t a r t time options Step time assignments Run completion s e t p o i n t Automatic column c l e a n i n g Program S t r u c t u r e and Operation  44 44 45 46 47 50 50 52 54 54 54 55 55 56 56 57  4. DISCUSSION AND FUTURE CONSIDERATIONS  66  References Appendix: System c o n t r o l s t a t e s  70 72  ill LJST OF TABLES 1.1  F u n c t i o n a l groups used f o r ion exchangers  9  iv  LIST OF FIGURES 1.1  Flowchart f o r a t y p i c a l  ion-exchange  procedure  8  2.1  Cascade sequence f o r columns A, B, and C as they operate i n the c a p a c i t y of primary and secondary feed, and r e g e n e r a t i o n  17  2.2  L i q u i d flow path, v a l v e and a n c i l l a r y equipment arrangement  22  2.3  A typical  l a b o r a t o r y s t y l e chromatography column  with temperature  c o n t r o l jacket  26  2.4  E l e c t r i c a l c o n t r o l system schematic  2.5  Darlington-connected N-P-N s i l i c o n power t r a n s i s t o r s . . 35  2.6  Card l a y o u t f o r DATA T r a n s l a t i o n DT2814 A/D card  2.7  DATA T r a n s l a t i o n DT2814 A/D card user connections  2.8  D i g i t a l I/O card backplane T r a n s l a t i o n DT2817  2.9  D i g i t a l I/O card user connections f o r DATA T r a n s l a t i o n DT2817  42  3.1  A t y p i c a l APC (Automatic Process C o n t r o l ) screen  48  3.2  General c o n t r o l s t r u c t u r e of process software  59  4.1  General system layout i n c l u d i n g p o t e n t i a l f u t u r e a d d i t i o n s f o r n e a r l y t o t a l automation of c o n t r o l  69  connections f o r DATA  33  37 .... 37 41  V  ACKNOWLEDGEMENTS  I  would  advisor,  like  to express  f o r h i s constant  my  appreciation  support  p r o j e c t . I would a l s o l i k e t o thank  and  to Dr. K.V. Lo, my  interest  throughout  this  Dr. S. Nakai and Dr. A. Lau f o r  s e r v i n g as members of my committee. T e c h n i c a l a s s i s t a n c e was g r e a t fully  gleaned  special computer  debt  from of  widow  Jurgen  Pehlke  gratitude through  and N e i l  for putting  the  many  Jackson. up  long  with  nights  To my w i f e : a the  life  of a  I  spent  that  e r a d i c a t i n g software bugs (or c r e a t i n g mutations). Funding  for this  project  Columbia  was  Agricultural  supplied Science  Marketing Agency.  by  grants  from  C o o r d i n a t i n g Committee  the  British  and the Canadian  Egg  1 INTRODUCTION  The novel  primary  goal  ion-exchange  i n producing  this  method, developed  work has  i n the  Science a t the U n i v e r s i t y of B r i t i s h Columbia the simultaneous albumen,  and  been to take  Department  of  Food  (Durance, 1987)  for  r e c o v e r y of lysozyme and a v i d i n from chicken to  develop  a  computer-automated  p r o d u c t i o n system to accommodate t h i s  a  egg  continuous-  process.  Sub-goals s u p p o r t i n g t h i s main goal make up the b a s i c d e s i g n s p e c i f i c a t i o n s f o r the system and 1. The  system  must  be  i n c l u d e the f o l l o w i n g :  capable  of  operating  continuously  i  a c c o r d i n g to the novel o p e r a t i n g sequence e s t a b l i s h e d by Durance 2. The  (1987).  system  should  provide  for  continuous  feeding  of  albumen to ion-exchange columns. 3.  The  column arrangement  should  efficiently  use  the  resin  beds i n order t o minimize the q u a n t i t y of r e s i n r e q u i r e d f o r a given p r o d u c t i o n 4.  The  physical  rate.  structure  of  the  recovery  system  must  promote h y g i e n i c o p e r a t i o n and ease of maintenance. 5. The  p h y s i c a l p l a n t should be compact and  p o r t a b l e f o r use  in research. 6. The it  o p e r a t i n g system must be "user f r i e n d l y " , e n a b l i n g to  be  invoked  knowledge of the 7. F i l e  by  an  operator  with  a  basic  for  the  process.  handling  reading/writing  easily  routines of  method  should files  allow  to/from  storage  media  2 (either  floppy  p r i n t e d output 8. There should  or  hard  disk)  as  well  as  providing  f o r record-keeping. be some degree  of f l e x i b i l i t y  which and how many columns are t o be used  in selecting i n order t o  accommodate both p r o d u c t i o n and r e s e a r c h needs. 9. Columns  should  be  easily  and  quickly  replaced  to  accommodate the use of columns of d i f f e r e n t  s i z e s and t o  allow  for rapid  the  should  congealed  in-process albumen  be  cleaning filtered  of  columns  out of the feed  flow stream by the r e s i n , r e s t r i c t i n g the flow r a t e . 10.  The  pump  rates may  capacities  t o accommodate be  times resin.  used  between  to  should cover a broad range of flow the v a r i o u s  allow  sizes  of columns  f o r the v a r i a t i o n  the process  liquids  of  that  contact  and the ion-exchange  3 1. BACKGROUND  Ion-exchange Chromatography  The  theory  efforts soils  of ion-exchange  t o understand  was  the mechanism  initially  studied  of n u t r i e n t  transport  . C l a y p a r t i c l e s were observed t o r e v e r s i b l y bind  nutrient cations  during in  inorganic  such as N a , K*, Ca *" and NH***" as i l l u s t r a t e d i n +  -  2  Equation 1.1 ( S a l i s b u r y and Ross, 1978):  2NH * + clay»Ca 4  I t was d i s c o v e r e d according  2  + clay»(NrU)  strengths  of t h e i r  charged bonding s i t e s .  t h i s pattern  1.1  a  that d i f f e r e n t c a t i o n s could  the r e l a t i v e  negatively  Ca*  attractions  The i n e q u a l i t y which  +3  right,  2  +2  describes  t o weakest a t t r a c t i o n s a r e shown from l e f t t o  charged  site  having  the  1.2  2  with K"*" and NrU*" e x h i b i t i n g  charged ion  2  strongest  negatively  for  i s c a l l e d the Hofmeister or l y o t r o p i c s e r i e s :  A l >H*>Ba* >Mn* >Ca >Mg* >(K*=NH^)>Na*>Li-*  The  be c l a s s i f i e d  will  sites. generally  a stronger  roughly equal a t t r a c t i o n s t o  A cation relinquish  residing  at a  i t s position  negatively t o another  e l e c t r o s t a t i c a t t r a c t i o n as i n d i c a t e d by  i t s p o s i t i o n i n the s e r i e s . This  i s an e q u i l i b r i u m r e a c t i o n which  can  the c o n c e n t r a t i o n  be reversed  strongly  by  attracted  increasing  ion.  I t i s due t o t h i s  fact  of the l e s s that  an  ion-  4  exchange  material  can  "regenerated" by  be  their  stripped  of  replacement with  so that the exchange process can begin Commercial reversal  of  softening  ion-exchange  equilibrium  exchange of  sodium  ions  systems  importance  to  is  the  involving proteins  which than  rely  upon  upon  stability. and  For  include  proteins  one  of  the  this  and  synthetic  as  i o n s . Due negative  Equation  wide  spread.  1.1.  of  called  soaps,  i n water  Of  are  The  greater exchanges  recovery.  protein  detergents  to prevent  ions  cyclic  magnesium ions  repulsion  reason they are  materials  of water  as  industries  the  Hydrophobic c o l l o i d s , on the other property  and  most  of  and  held  the  often" e x i s t as s o l s or c o l l o i d a l  electrostatic  such  involve  to e f f e c t t h e i r  affinity  ions  again.  such  for calcium  in order  the  the  equations  held  less strongly  processes  bio-processing  Ionic proteins  strongly  for  dispersions  water,  like  charges,  hydrophilic  soluble  et  f l o c c u l a t i o n and  soluble  al.,  hand, r e l y upon the settling  for  colloids  starch,  (Clark  rather  1977).  dielectric of  these  to the p o l a r nature of water molecules, c o l l o i d s with a surface  charge become surrounded with a  l a y e r of  water  molecules o r i e n t e d with the p o s i t i v e pole toward the s u r f a c e .  The  negative  the  particle  pole  of  water  molecule  points  r e s u l t i n g i n a n e g a t i v e l y charged  removed  from  repelled  by  the the  enough p r o x i m i t y them  each  together.  actual like  particle.  charges  before  Other the  f o r the a t t r a c t i v e Van Metal  oxides,  usually  hydrophobic s o l s (Clark et a l . , 1977).  away  "shell" similar  from  some d i s t a n c e particles  p a r t i c l e s are  in  are  close  der Waals f o r c e s to  bind  p o s i t i v e l y charged,  form  5 With a s i z e subject  range  to Brownian  of roughly 1-200  nm,  these p a r t i c l e s  motion caused by the uneven  distribution  collisions  with molecules of the continuous phase.  collisions  tend  sufficiently over  the  to  destabilize  close  contact  electrostatic  sols  that  by  Van  repulsion  pH  of the  aqueous  system.  These  Waals  forces  (charge  electrolyte  counter-ions) 'allowing  dominate  also  occurs  neutralizaton  by  are  the  reduced  attachment  by of  f l o c c u l a t i o n - (chemical  b r i d g i n g between p a r t i c l e s ) to occur f o l l o w e d by s e t t l i n g et a l . ,  into  i s n e u t r a l i z e d by a l t e r i n g  Repulsive forces  coagulation  of  random  particles  . Destabilization  when the s u r f a c e charge on a p a r t i c l e the  forcing  der  are  (Clark  1977). |  The pH a t which the e l e c t r o s t a t i c charge i s n e u t r a l i z e d i s called  the  isoelectric  pH  c o l l o i d , a t which p o i n t  or the  isoelectric  point,  p i , of the  i t s tendency t o remain d i s p e r s e d  i s at a  minimum. Lysozyme e x i b l t s a p i of 11.0 while a v i d i n has a p i of 10.0. by  While many p r o t e i n s  the  method  encountered the  protein  or  isoelectric  with t h i s  neutralizing use  of  was  chemical  method  i s that  the  becomes  often  (Durance,  ion-exchange groups  precipitation,  recovered  electrolytes,  processing  problems,  I n c l u d i n g lysozyme have been recovered  resins  c o v a l e n t l y bound t o an  stock  i t unfit  In  were  feed  major  order  with  overcome  with  insoluble  which the  f o r subsequent  to  devloped  problem  from  contaminated  making  1987).  the  such  charged  porous matrix.  Counter-ions could then be attached and r e v e r s i b l y exchanged with other  ions  (Pharmacia,  of  the  1980).  same The  charge  major  without  benefit  of  altering this  the  i s that  matrix specific  6 molecules, can  be  including  many  specifically  e f f i c i e n t l y secured the need to add lysozyme and the matrix, stock can  targetted  onto  be  to the  recovery,  displacing  the  processed  and  the charged  chemicals  avidin  pharmaceutical^  important  very  sites  effectively  of the matrix  feed s t o c k . In the  once these  initial  e x a c t l y as  proteins, and  without  i n s t a n c e of  have been secured  c o u n t e r - i o n s , the though the  onto  spent  protein  feed  extraction  had not taken p l a c e and with no l o s s of f u n c t i o n a l i t y i n terms of gel  strength, whipability  or n u t r i t i o n a l  value  (Li-Chan et a l . ,  1986). A typical  s e t of steps  i n the general o p e r a t i o n of an  ion-  exchange column i n c l u d e s :  1. I f the r e s i n be prepared involves,  f o r use  for  the  i s being used  f o r the f i r s t  f o l l o w i n g the manufacturer's D u o l i t e C-464, washing  with d e i o n i z e d water, with a l k a l i  and  bed be  until  d i r e c t i o n s . This resin  with  the predetermined  sites  are  breakthrough  diffusion solid  followed  1986).  the  percentage  column  outlet.  essentially  of product At  occupied,  this  i s observed  p o i n t , the  except  in  p o r t i o n of the bed. The p r o p o r t i o n of unexchanged s i t e s at  acid,  feed stock i s then fed i n downflow through the packed  passing through  bonding  the  i t should  then water again  by e q u i l i b r a t i o n b u f f e r (Li-Chan et a l . ,  2. The  time,  depends l a r g e l y upon the  r a t e s f o r the  phases.  A  high  d e s i r e d product  feed  flow  rate  to  resin  the  lower  remaining  feed flow r a t e and  the  i n both  and  gives  a  the  low  liquid  contact  time  7 which reduces the the  feed  liquid  bed.  The  contact  balance  find  an  time should  Backwash the i n order  4.  exchange s i t e be  the  prior  chosen with care to optimize  the  e f f i c i e n c y of  deionized  an  used to monitor  p r o t e i n s . As  ultra-violet  the  concentration  Re-equilibrate  passing  equilibration  eluting  counter-ions of  product  a suitable  with  i o n s . The  product is  for  ion  the  the  frequently  next  feed to  appropriate  i s now  steps  2 to  5 continues  long  until  flow  chart  resin  i s no  longer  to d e t e r i o r a t i o n . F i g u r e form  p r e p a r a t i o n and  the  cyclic  for  ready to r e t u r n  i s exhausted, or a planned shut-down occurs,  f u n c t i o n due  cycle  by the the to  (Step 2).  c y c l e c o n s i s t i n g of  the  buffers  replace  feedstock run  elution  eluant.  column  most  process  column  nm wavelength between  the process  through the  resin.  of p r o t e i n i n e l u t i n g  column  buffer  the a p p l i c a t i o n of feed  This  the  the  spectrophotometer  the p r o t e i n - f r e e e l u t i n g b u f f e r and  capture  or  down through the  by comparing the absorbance of l i g h t at 280  5.  water  recovery.  to remove non-adsorbed p r o t e i n s from the  bonded  within the  column with  ionically  proceeds,  molecule  to e x i t i n g  Pass a s o l u t i o n of counter-ions  t'o e l u t e step  will  that a p a r t i c u l a r  between r a t e of production and  3. buffer  probability  sequence operation.  of  1.1  able  to  until  the  or i n the  carry  out  its  shows s c h e m a t i c a l l y i n  procedures  for  the  initial  8  Choose starting buffer  Choose ion exchanger  Swell gel if necessary and pack in suitable column  Set up equipment •  UV-monitor  •  Recorder  • Fraction Collector » Pump ® Gradient Mixer  Equilibrate (2—3 volumes buffer)  Apply sample  I  Equilibrate sample if necessary  Wash away unbound substances Optimize separation  1 Elute bound substances  Desalt  Regenerate gel  Analyze separation  FIGURE l . 1:  F l o w c h a r t f o r a t y p i c a l ion-.exchange procedure  9  Ion-Exchange Resins  The the  most  basic  counter-ions  negatively charged  distinction  between  ( i . e . exchangeable  charged.  ions)  N e g a t i v e l y charged  counter-ions  and  are  positively  charged  resins  are termed  anion exchangers.  v a r i o u s c o v a l e n t l y bound  with  resin  are  resins  called  types  W i t h i n each  positively  having  cation  negatively  i s whether  positively  exchangers  charged  while  counter-ions  type of exchanger,  ions can be c l a s s i f i e d  or  the  a c c o r d i n g t o the  s t r e n g t h with which they b i n d ' c o u n t e r - i o n s . Strong i o n exchangers maintain a s t a t e  of complete  while weak exchangers  ionization  are much more i n f l u e n c e d  pH on the degree of i o n i c d i s s o c i a t i o n Table 1.1,  over a wide range  of  pH  by the e f f e c t  of  (Pharmacia,  1980).  below shows a sampling of c o v a l e n t l y bonded  ionic  groups used i n modern r e s i n s to produce s t r o n g , moderate and weak exchangers.  Table 1.1  F u n c t i o n a l groups used f o r i o n exchangers 1980)  ANION EXCHANGERS  FUNCTIONAL GROUP  Aminoethyl  -OCH CH NH -  (AE-)  Diethylaminoethyl  2  (DEAE-)  Quaternary aminoethyl (QAE-)  2  3  -OCHaCHaN-H(CH CH ) 2  (CM-)  Phospho Sulphopropyl  2  2  2  -OCH COO2  -PO-oHa"  (SP-)  3  2  -OCH CH N*(C H ) CH CH(OH)CH  CATION EXCHANGERS Carboxymethyl  (Pharmacia,  -CH CH CH S0 2  2  2  3  B  2  2  3  10 Strong  exchangers are formed u s i n g s u l p h o n i c and  amino groups while and  quaternary  the phospho group i s of intermediate  strength  the others are considered weak exchangers (Pharmacia, 1980). Other  f a c t o r s t o be considered  when s e l e c t i n g  a resin  type  i n c l u d e the p o r o s i t y of the matrix as determined by the degree of polymer c r o s s - l i n k i n g , the change i n r e s i n volume d u r i n g i n pH, i o n i c s t r e n g t h and c o u n t e r - i o n , the chemical the  resin  i n the s o l v e n t  used,  changes  s t a b i l i t y of  the p h y s i c a l d u r a b i l i t y  of the  r e s i n , and the mesh s i z e and s i z e d i s t r i b u t i o n of the r e s i n .  F a c t o r s A f f e c t i n g Exchange Dynamics  The  degree of d i v i n y l b e n z e n e  polystyrene  resin  cross-linked while  the  molecules  determines  matrix small  provides  pore  cross-linkage i n a  the pore a  large  diameters  i n c l u d i n g most  size  proteins.  resins  structure.  These  were  developed  with  A  highly  of charged  the  In order  access t o i n t e r n a l exchange s i t e s , and thus capacity,  within.  number  prevent  sulphonated  entry  of  to allow  sites large  proteins  i n c r e a s e the exchange an  open  r e s i n s are g e n e r a l l y produced  macroporous  i n the form of  s m a l l s p h e r i c a l beads which are a v a i l a b l e i n a v a r i e t y of s i z e s u s u a l l y between about 16 and 400 mesh (U.S. standard The charge  size  sieve).  and shape of the molecule as w e l l as i t s s u r f a c e  distribution  are of great  importance  in biological ion-  exchange processes. The r e s i n pore diameter can be chosen so that only  particular  diameter  molecules  can enter  the r e s i n  which  are  matrix.  smaller  T h i s allows  than  the  pore  for separation  11 i n part by s i z e ionic  species  resin,  while  e x c l u s i o n as w e l l as by  can the  access myriad  ion-exchange s i n c e l a r g e  only s u p e r f i c i a l internal  sites  bonding  are  sites  reserved  on  the  for smaller  ions. Liquid-phase  diffusion  coefficients  for  feedstock  c o n s t i t u e n t s can be i n c r e a s e d by d e c r e a s i n g the l i q u i d which  is  in  turn  lowered  by  raising  more  the  temperature.  diffusion  rates  promote  therefore  allow  for  biological  l i q u i d s can reduce the v i s c o s i t y by exposing  higher  rapid flow  or coagulated p r o t e i n to high shear to  Gaulin  high  pressure  (6.9  MPa),  reason  congealed  f o r homogenizing  lumps of  packed r e s i n The  any  the  contact  time  between  particular  presence the  processes.  and  Homogenization  of  congealed  (1987) used  a  Manton-  homogenizer  to  to f e e d i n g t o the column.  The  egg  orifice  albumen  was  to prevent  the  ion  to  an  to  break  plugging  of  up the  exchange  The  resin  Since the  site  is  a  greatly  attachment stochastic  i s r e q u i r e d i n order to ensure  ions competing f o r bonding  of product  recovery  p a r t i c u l a r l y hindered  and  recovery.  of extraneous  efficiency  liquid  recovery e f f i c i e n c y .  a given l e v e l of product  hinders  kinetics  bed.  process, some minimum c o n t a c t time  The  Higher  f o r c e s which break them apart  p r o t e i n i n order  i n f l u e n c e s the product of  rates.  small  reduce the albumen v i s c o s i t y p r i o r major  exchange  f r e e - f l o w i n g i Durance  make them more  viscosity,  of  lysozyme  i n t h i s way  are markedly higher than those  recovery  sites  i n many ion-exchange and  since their  avidin  is  not  i s o e l e c t r i c points  of most other p r o t e i n s .  12  Recovery of Lysozyme and  Lysozymes, approximately and  basic  Avidin  proteins  15,000 d a l t o n s ,  insect  and  bacteriocidal  fungal  having  are  cells  produced  since  b a c t e r i a l c e l l w a l l s i n c l u d i n g those  this  assay,  with  a  a  slurry  causes  compared  with  suspected  of  turbidimetrically. an  they  can  of  I f mixing the  feed  containing  of  plants,  They  exhibit  lyse  certain  been developed.  containing  feed  i n the  size  of Micrococcus l y s o d e i k t i c u s  lysodelktlcus  increase  a  1987).  assay f o r lysozymes has  liquid  slurry  average i n animals,  (Durance,  characteristics  upon which a popular  an  lysozyme  and  liquid  transmission no  i s mixed  then  with  the  of  lysozyme,  observed dead  cell  visible  then  In  the  light percent  increase i n t r a n s m i s s i o n g i v e s a q u a n t i t a t i v e measure of lysozyme concentration The  ( J o l l & s et a l . , 1965).  dominant  commercial  source  of  albumen which c o n t a i n s approximately (Wilkinson 3.5%,  and  Dorrington,  1975).  88%  lysozyme water and  Lysozyme  while a v i d i n makes up only 0.05%  i s chicken 10.2%  accounts  egg  protein  for  about  of t h i s t o t a l p r o t e i n ( L i -  Chan et a l . , 1986). The 95%  using  (Li-Chan physical  recovery of lysozyme has been shown to be between 90 Duolite  make  exchange  et a l . , 1986). This high and  chemical  swelling/shrinking ionic  C-464 c a t i o n  strength, this  qualities  during  good  cyclic  chemical resin  resin  recovery  along  including changes  inertness a  in a  in and  a  with low  the  bed  desirable degree  liquid  physical good  packed  and  of  phase  stability choice.  13 E l u t i o n - L o o p i n g Technique  The  novelty  of  designed and b u i l t can  be  i s the way  the c o n c e n t r a t i o n  of a v i d i n .  column  process  for  Thus,  which  mentioned  lysozyme  of lysozyme  i f both  that  strength  solution  saline  lysozyme  that  than  were e l u t e d  after  i s almost t o t a l l y  required  each  masked  separate the  u s i n g a lower for avidin  (although the opposite case would be expected from t h e i r pi's  avidin  i s 70 times greater  can be e l u t e d  than  was  this  proteins  earlier  peak. In order to b e t t e r  peaks, the f a c t  system  in  f e e d i n g , the presence of a v i d i n  by the l a r g e  this  i n which both lysozyme and  recovered s i m u l t a n e o u s l y . As  section, that  the  two  ionic  elution relative  (Durance, 1987)) i s used to advantage. The  avidin  specified  number  is  allowed  to  remain  of  feed/regeneration  removed each time. As the q u a n t i t y increases which  with each  i s larger  and  cycle, more  on  cycles  the  while  for  lysozyme  of a v i d i n bound t o the  i t s delayed removal pure  column  than  that  from  produces a  a is  resin  a peak  single  cycle.  While the c l a r i t y and p u r i t y of t h i s delayed peak i n c r e a s e s with the number of c y c l e s between a v i d i n e l u t i o n s , the optimum has not yet been determined. This m o d i f i e d r e c o v e r y process which has of  termed two  "elution-looping"  commercialy  high i n i t i a l  promotes  important p r o t e i n s  p u r i t i e s and  recoveries.  Durance  the simultaneous r e c o v e r y on  a  single  column  with  14 2. AUTOMATION OF LYSOZYME/AVIDIN RECOVERY  Prior  to  automating  the  should  be  recovery  noted  total-system recovery  t h a t the  lysozyme  I t does  concepts  in  the  equipment  that  not  intent  and  experimentation  two of  to  The  used  operation  process  of  is particularly  and j commercial  modify  the  clogged  the  congealed  whatever column(s)  detail and  the  white  or  in  new the  to present, a  f o r both  laboratory  The  complete  capabilities allows  which  and  for  time  any  the  of the the  system.  unattended  software  will  column(s) which become  continue  processing  with  remain.  operating l o g i c  in  egg  the  many of the three columns are  o p e r a t i n g mode to exclude  with  The  during  radically  attempt  suited  for  a  elution-looping  production.  capability  system  software, the  to  proteins, i t  ion-exchange  I t does, however>  shutdown"  of  and  taken  i s to provide  introduce any  enhance the experimental  "staged  known  paper  using  f l e x i b i l i t y r e g a r d i n g which and how t o be  this  avidin  approach  well  hardware  attempt  general  that  particular  these  both  i s used.  package  the  of  prototype,  of  technique.  system  discussing  of the system w i l l  be e x p l a i n e d i n more  f o l l o w i n g s e c t i o n s d e s c r i b i n g the  the e l e c t r o n i c c o n t r o l  physical  plant  circuits.  Physical Plant  Lysozyme continuous  and  avidin  have been recovered  ion-exchange arrangements. While  i n both  batch  and  batch methods e x p l o i t  the be  least  expensive  relatively  optimum use accurate result  technology  labour  of  i n t e n s i v e and  resin.  contact  time between the  i s required  resin  to  they  tend  to  of  the  i n terms  i s used to ensure  r e s i n and  If less r e s i n  i n order  operation,  inefficient  If s u f f i c i e n t  i s a packed bed.  agitation  in their  the  feed l i q u i d ,  during  enclosed  as  the  agitation  insure a uniform  hand,  are  better  i n c l u d i n g contact  d i c t a t e s whether the process feed  input.  multiple  an  impeller,  contact  either  i n a pump. Packed column continuous  other  conditions  with  suited times.  to  The  systems  are  time  easily  open,  or  feed systems,  optimizing number  i s continuous  Multiple-column  the  i s used, then some form of  over a l l of the feed s t o c k . Ion-exchange r e s i n s tend to be damaged  an  of  on  operating  columns  used  or not on the b a s i s of generally  operated  as  i n d i v i d u a l columns which are fed i n sequence.  The  feeding  molecules  begin  of  to  a  given  break  column  through  into  continues the  until  column  product  effluent.  At  t h i s p o i n t , s i n c e the a d s o r p t i o n of ions i s a s t o c h a s t i c process, a  significant  portion  column  i s unsaturated.  varies  directly  time.  If  capacity  as  a l l of of  the  of The  the the  the  resin  exact  rate,  resin  could  would  be  at  the  proportion  flow  column  bed  or  of  bottom  under-used  i n v e r s e l y as  be  fully  increased  the  loaded,  to  of  the resin  contact then  accommodate  the more  feed, or the volume of the column could be reduced while t r e a t i n g the same q u a n t i t y of This  end  was  feed. accomplished  cascade arrangement wherein the of the  first  column, and  by  operating  feed  i t s effluent  two  i s introduced feeds  columns i n t o the  i n t o the  top  of  in  a  top the  16 second  column. The f i r s t  saturation  while  the  product  captured  i n the  sampling  a  single  tall  not  having  to  while  column can then be allowed  second  ions  column.  which  This  column  a t i t s midpoint  worry  about  t h a t upon s a t u r a t i o n of the f i r s t  losing  ion). is  required.  with  for saturation  column, the feed  the product  regeneration,  that  ions  only three  the time  i s greater  due  to  inlet  can be  the f i r s t  one i s  the d e s i r e d form of  f o r the t h i s sequence t o continue, Provided  as  the columns separated i s  ( e l u t e d and r e - e q u i l i b r a t e d with  In order  are  effect  product  t o the top of the second column while  regenerated  through  i s the same  breakthrough. The b e n e f i t of the having  directed  break  t o approach  required  than  a third  t o load  the time  columns are necessary.  i s l e s s than t h a t r e q u i r e d f o r r e g e n e r a t i o n ,  column  a column  required for  I f the feed  time  then e i t h e r the feed  s t e p would have t o be i n t e r r u p t e d or an a d d i t i o n a l column would be needed. Two s e t s of names are used order  to r e f e r  function, order  each  to a particular i s given  to describe  function,  names  t o the columns. In  column r e g a r d l e s s  an absolute  a given  relative  in referring  column are  of i t s c u r r e n t  name: "A", "B" and "C". In with  also  respect used:  to i t s current "Primary"  (1°),  "Secondary" (2<>) and "Regenerating" (R). Figure operate operating  2.1  in this duties  illustrates cascade in a  r e c e n t l y regenerated  the  sequence. continuous  use The  of  three  three  cycle  such  columns  columns that  which  exchange the  most  column becomes the secondary one i n the two-  columns s e r i e s , the column that was the secondary becomes the  17 FEEJUUL  A  B  a) STAGE 1  R  BARREN FEED OUT FEEQ_JJL  1  L  B  b) STAGE 2  R  BARREN FEED OUT FF.Rn I N  ^  1  £T  A  B  t  R  c) STAGE 3  BARREN FEED OUT  FIGURE 2.1:  Cascade sequence f o r columns A, B and C as they operate i n the capacity of primary and secondary feed, and regeneration  18 primary, while the primary column begins the r e g e n e r a t i o n stage. During  the  cascade  feeding  columns, the remaining exhausted  of  the  primary  and  secondary  ( f u l l y loaded) column i s exposed  to the f o l l o w i n g r e g e n e r a t i o n s t e p s :  1. The  egg  with  white  starting  i n the buffer  column  i s removed  i n order  by  backwashing  to remove any  p r o t e i n p r i o r to s t r i p p i n g o f f the product  unadsorbed  ions.  2. A weak s a l i n e s o l u t i o n e l u a n t i s passed downward the  column  avidin  to 'remove' the  bound  to  the  technique mentioned 3. I f  the  specified  removing is  resin  number  through  as  per  while the  leaving  the  elution-looping  earlier.  o n l y the  passed  lysozyme  through  of  lysozyme, the bed  loops  have  been  performed,  then a s t r o n g s a l i n e e l u a n t  to remove the enhanced  avidin  peak. 4. I f  just  the  further strong  weak s a l i n e  equilibration saline  was  e l u a n t has  is used,  been  necessary, then  the  used,  then  however, resin  no  if  the  must  be  e q u i l i b r a t e d p r i o r to r e t u r n i n g the column to feed duty.  This  sequence of r e g e n e r a t i o n steps i s thus a p p l i e d  column l a b e l l e d 2.1.  R"  11  i n each  to the  of the three STAGES shown i n F i g u r e  19 Operating  The  Sequence Nomenclature  various  valve  unambiguously i n order The  operating to prevent  sequences  fundamental  division  they  are  used  to  labelled  sequences, beginning  and  comprehensive. These terms w i l l wherever  be  e r r o r s i n c o n t r o l programming.  f o l l o w i n g terms d e s c r i b e these  most  must  progressing  be  presented  indicate  to  with the  the most  in capital  letters  specific  logical  these  d i v i s i o n s as d e f i n e d below:  STEP:  The  term STEP i n d i c a t e s a p a r t i c u l a r  the  23  Each  flow  of  control valves,  these  units  energized  or  STEPs are  shown  assigned an  by  not.  The  3 pumps and  be  either  ON/OFF  i n Appendix  the  existing  can  operator  method  control setting  ON  or  patterns  A.  STEP  the  homogenizer.  OFF,  for  time  diskette  or  that i s ,  the  various  durations  e i t h e r through the  from  for  uploading  are of  through  the  direct  the  term  STAGE  i n t e r a c t i v e method. STAGE:  For  the  represents  column the  regeneration  being  total  process.  regenerated,  number The  of  STEPS  number  of  involved STEPS  in  in a  the  STAGE  v a r i e s dependent upon whether or not the a v i d i n as w e l l as the  lysozyme  i s to  column(s) being feed  control  homogenizer.  be  s t r i p p e d from the  column. For  the  f e d , the STAGE i s e q u i v a l e n t to the s i n g l e setting  The  actual  of  the  valves,  duration  of  the  feed  pump  STAGE  is  and the  g r e a t e r of the l e n g t h of time r e q u i r e d f o r the sequence of  20  r e g e n e r a t i o n STEPS or the s p e c i f i e d  feeding  CYCLE: I f three columns are being used, then the  columns  has  "secondary"  occupied  and  columns,  longer  regeneration.  the  cascade  CYCLE each of of  CYCLE  "primary",  and  STAGE  are  fewer than three columns s i n c e the  c o n t r o l s e t t i n g s are no and  i n one  position  "regeneration".  synonymous when running  feeding  the  time.  completely  In  feed  the  can  independent f o r  case  only  of  take  two  active  place  if  the  r e g e n e r a t i o n procedure i s completed f o r the second column. Since is  non-cascade feeding can  carried  out  regeneration the  forward  in  can  the  be  progress  second  carried  while  column,  out  the  in parallel  regeneration feeding but  d i r e c t i o n , as d e f i n e d by the normal  and  only  in  operation  of three columns. For a s i n g l e column, i t i s obvious  that  only  that  one  operation  f e e d i n g and than  in  can  set  considered  performed  r e g e n e r a t i o n must take  parallel.  combined  be  of  For  one  STEPs  for  or  at a  place  two  time,  in series rather  columns,  feeding  and  and  then,  the  regeneration  as a s i n g l e sequence, making redundant the  is use  of both of the terms, STAGE and CYCLE. Since CYCLE appears to for  better each  describe column,  the the  o p e r a t i o n of j u s t one The  completion term  or two  incorporation  of  that  the  a v i d i n can  be  STAGE  a l l required  was  dropped  in  STEPs the  columns. the  r e q u i r e s t h a t a count be kept so  of  elution-looping  technique  of the number of CYCLES run stripped  from  the  column(s)  21 a f t e r the a p p r o p r i a t e CYCLE number.  BLOCK: T h i s  counter  avidin  has  each  been  time  the  and  is  value  between time  simply i n d i c a t e s removed CYCLE not  during  the  from  the  counter  trivial  subsequent  the  avidin process  number column.  reaches  since  of  the  that  I t increments  the  predetermined  number  removals  can  via  routine,  the  times  be  of  CYCLES  varied  at  MPC  any  (manual  process c o n t r o l ) .  Flow Path Design  The  cascade  incorporated  approach  into  the  used  to  system  recover the  plumbing  target  arrangement,  ions  was  with  an  attempt made to minimize the i n t e r n a l volume of the system while accomodating columns.  a  wide  Figure  arrangement.  2.2  The  range  of  valves  flow  illustrates  arrangement  t h e i r ON/OFF s t a t e s was number  of  the  of  Of  liquid  these  of c r i t i c a l  required.  rates  the  for  different  flow path and  valves  with  size valve  respect  to  importance i n minimizing the 23  electric  solenoid  valves  used, s i x are simple two-way v a l v e s which e i t h e r permit l i q u i d to flow through them or block i t s path completely (valves 9-14).  The  other  17  two  input  streams  are  three-way and  allow  v a l v e s ) or can d i r e c t lines  (diverting  valves one  one  valves).  which  can  select  of them to e x i t  input These  line  into  three-way  either  the valve  either vlaves  of two  of  (mixing output  cannot prevent  l i q u i d s from f l o w i n g through them, but can only s e l e c t the  ro FIGURE 2.2:  L i q u i d flow path, v a l v e and a n c i l l a r y equipment arrangement  appropriate permit  flow  path.  flow through  I f one  of these  valves  i s operated  to  one of the two p o s s i b l e pathways, then the  a l t e r n a t e path i s blocked o f f . In t h i s way each valve can provide dual f u n c t i o n s a t any given time. Great care had t o be taken t o assure t h a t a given arrangement  of v a l v e s and p o s s i b l e flow paths  would  accommodate  every  allowing  cascade  feed  required  approach  and  flow  arrangement,  the  isolation  of  the  physical  be able t o f o r the  regenerating  column from the others through  i t s v a r i o u s s t e p s . T h i s flow path  i n t e g r i t y had t o be maintained  throughout  the r o t a t i o n of column  d u t i e s from STAGE t o STAGE. In certain  tracing  through  flow paths  sequence,  the flow  cannot  however,  the  system,  i t might  appear  that  be blocked o f f as r e q u i r e d f o r a given use  of  positive  displacement  pumps  e f f e c t i v e l y p r o v i d e s the system with three more two-way v a l v e s as they are turned on or o f f . Once flow path input  the v a l v e criteria,  ports  placements w i t h i n the system the matching of i n l e t  on the mixing  lines  v a l v e s , or o u t l e t  p o r t s on d i v e r t i n g v a l v e s was done t o minimize that  each  minimized transferred  valve  was  the heat  required  loading  t o the product  T h i s might be a s i g n i f i c a n t system were to be operated  to  from  be  with  lines  energized.  the s o l e n o i d s  source  particular with  This  output  which  in turn would  room.  be  environment.  t o be considered  in a refrigerated  the  the amount of time  or t o the surrounding heat  satisfied  i f the  24 Flow Handling Equipment  V a l v e s : The v a l v e s  used i n the prototype system are B u r k e r t  (West Germany) 24v D.C. e l e c t r i c for  low pressure  121-A),  two  diverting fittings  Pumps:  operation.  three-way  valves sized  to  diameter,  plastic  t u b i n g used.  Three  peristaltic,  are  to c i r c u l a t e  1/8  liquid squeeze  were  inch  positive  the l i q u i d s  adjustment  flow r a t e s ; the l i q u i d  used  valves  (121-E)  (Model and  i n the system  the (3.2mm)  1/4  inch  inside  15  with  (6.4mm) diameter  •  especially suited  infinite  valves  accommodate  outside  used  S i x two-way  mixing  (121-F)  s o l e n o i d v a l v e s designed  displacement pumps were through the system.  These  f o r low pressure systems and a l l o w  of r o t o r  speed  over a wide range of  Since the r o l l e r s through  c o n t a c t with the l i q u i d  the flow  on the pump line,  no  rotor direct  i s ever made, enhancing the l e v e l  of hygiene of the system. One pump moves feed l i q u i d (egg albumen) through the system; another p r o v i d e s backwashing b u f f e r , while the t h i r d  one does double duty by p r o v i d i n g  e i t h e r s t r o n g or weak eluent as r e q u i r e d with the a i d of a diverting  valve.  The pump speeds must be s e t manually  on the c o n t r o l l e r f o r each pump, while the ON/OFF s e t t i n g for  each was made c o n t r o l l a b l e by the software.  25 Columns:Quick d i s c o n n e c t  fitting  were used  columns to a l l o w f o r the r a p i d various  sizes  and aspect  diameter) d u r i n g process this 12"  on e i t h e r  replacement  ratios  of columns of  (ratio  of height  o p t i m i z a t i o n steps  s t u d y ) . The columns f a b r i c a t e d  end of the  (not p a r t of  f o r t h i s system were  (305mm) long, made from l"i.d.(25.4mm) c l e a r  tube,  selected  inertness liters  for  and s i z e d  i t s transparency roughly  selected  as a compromise  and  acrylic chemical  f o r the p r o d u c t i o n  per day of albumen.  This  between  o p e r a t i o n s which c o u l d provide  to  processing the minimum  f o r both  of 25  rate  was  s c a l e of  laboratory scale  r e s e a r c h runs and small i n d u s t r i a l s c a l e p r o c e s s i n g of up to approximately  250-300 l i t e r s  per day a t the l i m i t of  the pumps. F i g u r e 2.3 shows the c o n s t r u c t i o n of a t y p i c a l commercial for  the  column. resin  column  operation. F i l t e r s fines  from  prevent  Screens in  at either  escaping  the r e s i n  provide  into  both  the primary upflow  end serve  the flow  bed from being  and  support downflow  t o prevent  lines  blocked  resin  and t o h e l p with  congealed  albumen.  Homogenizer:  A l a b o r a t o r y s i z e Manton-Gaulin high  orifice in  homogenizer used by Durance  breaking  up congealed  pressure  (1987) was e f f e c t i v e  egg white and resuspending the  p r o t e i n , however, i t s s u b s t a n t i a l s i z e and weight made i t u n s u i t a b l e f o r use i n the p o r t a b l e s t y l e system t h a t was developed.  The major c r i t e r i a , besides p o r t a b i l i t y , f o r  26  Heavy wall _ glass column  Acrylic jacket  Interchangeable bed supports (Polyethylene, Nylon, Polypropylene, or TFE)  TFE o-ring shield provides a leak free seal \  Threaded acetal jacket cap  Threaded acetal end cap  TFE end fitting CTFE V4"-20 to  V4 "-28 thread adapter (TOP AND BOTTOM ENDS)  FIGURE 2.3: A typical laboratory style chromatography column with temperature control jacket (Kontes S c i e n t i f i c Glassware/Instruments)  the s e l e c t i o n of a homogenizer were the p o s s i b l e range of flow  rates,  exclusion  continuous  of  air  processing  from  the  capability,  product  while  and  the  processing.  While the range of flow r a t e s needed to be matched to the d e s i r e d range of p r o c e s s i n g r a t e s as d e f i n e d by the pump capacities,  the  the  stream  feed  denaturation at  the  style  resulting  critical  readily  encountered  were of  protein,  even  is  the  point  rotates  tube. narrow  The  albumen  gap  a can  between  (mortar).  The  amount  subjected  can  be  by  varying  the  to  vessel  shear  forces  tp  liquify  the  of  denaturing  the of  at  least  of homogenizer found, a mortar and  p r e c i s i o n bore be  congealed  of  the  pestle style unit  shear  to  Nemours &  Co.)  borosilicate  continuously rotor  within  fed  (pestle) which  the  glass  through and  r o t a t i o n and  product  diameter  p e s t l e with r e s p e c t to the f i x e d tube, and  the  stator  p r e c i s e l y c o n t r o l l e d over a wide speed  the  operation  sealed processing  (Reg.TM E . I . duPont de  within  due  molecules.  most s u i t a b l e type  the above c o n s t r a i n t s , was  rod  blendor-  of equipment might w e l l be much  larger individual protein  i n which a T e f l o n  f r e e energy  batch  high  in  protein  using  the  extremely  required  to  avoid  unsuitable  a i r and  continuous-flow  i n t h i s type  than  The  thus  a v a i l a b l e . The  greater  to  i n t e r f a c e . Equipment  entrainment  s i n c e no  entrainment  from the high s u r f a c e  r o t a t i n g blades  style,  of a i r bubble  was  gas/liquid  large-scale  was  prevention  is  range  of  the  the feed r a t e ,  28 which to  governs  pass  the  through  length  of time which  the shear  t h i s type of u n i t and  zone.  No  the  liquid  takes  a i r i s entrained  in  the shear f o r c e s can be maintained  a t a low enough l e v e l t h a t bubbles are not c r e a t e d due to cavitation.  Although  this  h i g h cost  (approx. U.S.  prototype  system.  The  unit  totally  chosen  enclosed  cooling  jacket.  was  the  unit  of  choice, i t s  $3000) precluded i t s use  was  an  ultrasonic  through-flow  Although  in this  horn  processing  with  cell  m i c r o s c o p i c bubbles  are  a  with formed  and c o l l a p s e d u r i n g e x c u r s i o n s of the v i b r a t i n g horn t i p , the  intensity  although  of  the  the  vibration  vigorous  tip  c o n s i d e r a b l e amount of heat jacket  can  be  used  can  be  controlled,  oscillations to the l i q u i d ,  to maintain temperatures  and  transmit the  a  cooling  below  that  which w i l l cause the p r o t e i n t o denature. Again, the flow r a t e of l i q u i d part, liquid  the  degree  period  of  heat  buildup i n a  i n large  g i v e n volume  of  f l o w i n g through the u n i t . Durance  feed,  through the shear zone d i c t a t e s ,  that  (1987) found, when u s i n g batch homogenized  the  albumen  tended  to  recoagulate  over  of time while w a i t i n g t o be processed. I t was  a to  a l l e v i a t e t h i s problem t h a t a continuous-flow homogenizer was  placed i n - l i n e  and the d u a l i n l e t  between the r e f r i g e r a t e d feed  filters.  feed storage  29 Tubing:  The (Reg.  flow t u b i n g used In the prototype  TM  Norton  Company)  flexible  plastic  diameter  and a 1/16 i n c h  tubing  tubing  i s excellent  R-3603  with  easy setups. for  (1.6mm) w a l l  f o r i t s smooth  clear,  (6.4mm) o u t s i d e thickness.  bore,  This  c l a r i t y and  c o l l a p s i n g , f o r quick and  I t i s a u t o c l a v a b l e a t 121 degrees  pressure,  has a u s e f u l o p e r a t i n g  ^45 C t o +74 C, and i t w i l l  maximum working pressure I  a  centigrade  30 minutes a t 15 pounds per square i n c h ( p s i ) (103.4  kPa) of  formulation,  a 1/4 inch  a b i l i t y t o bend s h a r p l y without  Is Tygon  kPa) a t an ambient  range  not age or o x i d i z e . The  f o r t h i s tubing i s 44 p s i (303.4  temperature  of 21  Instrument Company 1987-88 catalogue, The  temperature  C  (Cole-Parmer  p558).  l e n g t h of t u b i n g In the system was kept s h o r t by  expedient although determine  placement an  of v a l v e s  optimization  the v e r y  best  on the mounting  routine possible  could  be  arrangement.  frame,  used  to  For the  purposes of t h i s study,  the time r e q u i r e d t o s e t up such  a  aided  routine  f o r computer  optimization,  i n l i g h t of  the r e l a t i v e l y i n s i g n i f i c a n t r e d u c t i o n i n system  internal  volume, was deemed e x c e s s i v e .  Filters:  Since recongeal, two  chicken  egg albumen has a s l i g h t tendency t o  even a t room temperature a f t e r  filters  were  homogenizer  and  (Sartorlus  D-3400,  placed the  in  columns.  SM16508B),  homogenization,  parallel The  between  the  filter  holders  are c o n s t r u c t e d  of c l e a r  polycarbonate with a s m a l l i n t e r n a l volume. 50mm diameter Whatman  GF/D  filters  (Whatman  nm were used  r e t e n t i o n of 2.7  Ltd.)  with  a  particle  because of t h e i r high  flow  c a p a c i t y , adequate f i l t r a t i o n c a p a c i t y ( g e n e r a l l y used a prefliter  f o r membrane f i l t r a t i o n ) , and  Either f i l t e r changed the  either in  end  of the  the  resin  discs  The of  the  Secondary  of each r e s i n  one  lines.  while  filter.  p r e v e n t i n g the  low c o s t .  can be clamped o f f from the system and  independently  remaining  as  resin  process  continues  filters  are  from  becoming  prefilters  become ruptured  fines  from  escaping the  main  filter  supports  40x60 mesh s t a i n l e s s  r e s i n bed.  The  filter  synthetic  non-woven  l o c a t e d at  column. These p l a y a dual bed  cloth  of  and  i n preventing  w i t h i n the  m a t e r i a l used  role  f o u l e d should  column i n t o the  steel  with  screen  flow  columns  are  next  the  to  i n the columns was  fine  but  a  undetermined  p a r t i c l e s i z e r e t e n t i o n h e l d i n p l a c e from the other s i d e by  a disc  in  domestic  of coarse  s c o u r i n g pads  pads held the f i l t e r prevent the  leakage  s i d e s of the  filters columns  was  s y n t h e t i c sponge-like m a t e r i a l  c o n t r o l program.  washing. The  spongy  c l o t h t i g h t l y a g a i n s t the s c r e e n to  of r e s i n and/or congealed filter.  required  i n both  for dish  used  upflow  Support  since and  from  fluid  albumen around  both flows  downflow as  s i d e s of  the  through  the  r e q u i r e d by  the  31 Pressure Transducers: congeal  Due to the tendency of the egg white to  and f o u l  condition  of  the f i l t e r s  the  filters  and r e s i n ,  Is  monitored  the o p e r a t i n g to  alert  the  c o n t r o l program and the operator when a flow blockage i s imminent. each  The d i f f e r e n t i a l  of the three  filters.  The  pressure  columns  transducers  i s monitored  as w e l l  as a c r o s s  (Honeywell)  have  across  the i n l e t a  nominal  c a p a c i t y of measuring ± 5 p s i (34.5 kPa) a c c o r d i n g t o the u n i t s p e c i f i c a t i o n s , however, a l l four of the transducers were t e s t e d and found  to provide a u s e f u l s i g n a l  output  up t o about +7.3 p s i (50.3 kPa) and -5.0 p s i (34.5 kPa). The  requisite  DC  computer  interface  pressure  sensor  power while  was  signals,  program  with  terminate  voltage  by a separate  (A/D) converter board  f o l l o w i n g s e c t i o n ) v i a the screw  These  provided  the output  was monitored  the analog t o d i g i t a l a  source  once  processed,  information  the o p e r a t i n g  which  allows  routine  from  each  channel  of  (described i n  terminal  provide  from the  connector.  the  control  i t t o modify or  to s u i t  the p h y s i c a l  s i t u a t i o n w i t h i n the chromatography system.  U l t r a v i o l e t Monitor: U l t r a v i o l e t 280nm  i s absorbed  degree  of l i g h t  can  be used  which  contain  by  (UV) l i g h t a t a wavelength of certain  amino  acid  absorbance a s s o c i a t e d with  to indicate them.  the c o n c e n t r a t i o n  For the purpose  c o n c e n t r a t i o n of e l u t i n g  groups. these  The  groups  of p r o t e i n s  of observing  p r o t e i n s , an i n - l i n e UV  the  monitor  unit  (UV-1  Single  Single  Path  Monitor  Chemicals) was the two liquid  Path  placed  Monitor  Control  Optical  Unit  between the  Unit  by  and  UV-1  Pharmacia  Fine  processing  columns  and  s o l e n o i d valves which c o n t r o l the flow of e l u t i n g to waste or to the a p p r o p r i a t e  product r e s e r v o i r ,  depending upon whether or not the eluant c o n t a i n s p r o t e i n at  a  concentration  value.  The  analog  fed to the  A/D The  order  route  than  s i g n a l coming  board as  software. to  higher  an  from the  UV  threshold monitor  is  input s i g n a l f o r the c o n t r o l  c o n t r o l - program the  some s p e c i f i e d  eluting  monitors  liquid  to  this the  -signal i n appropriate  vessel. The  UV-1  output  s i g n a l i s i n the range 0-10  mv  DC.  E l e c t r o n i c Flow C o n t r o l Hardware F i g u r e 2.4  shows the general arrangement of components which  perform the e l e c t r i c and  e l e c t r o n i c c o n t r o l f u n c t i o n s r e q u i r e d to  t r a n s l a t e the computer output  DC Power Source: A 24 v o l t operate takes  an  the  flow  i n t o a working flow c o n t r o l system.  (v) DC power source control  valves.  i n r u s h c u r r e n t of approximately  and  a h o l d i n g c u r r e n t of 0.4  one  that was  One  (Power-One Inc.)  the  purpose. The  solenoid  i s r e q u i r e d to  A. The  output  valve  amperes  power source  a v a i l a b l e from a previous 7.2  1.1  Each  (A)  used i s  p r o j e c t , a Power-  A u n i t which proved adequate f o r  of t h i s u n i t was  used as an input to the  ELECTRICAL SOLENOID VALVES DT2817 32 c h a n n e l DIG I/O INTERFACE (4x8-bit . ports)  backplane connector IBM COMPATIBLE COMPUTER  115v  backplane connector DT2814 16 c h . ( S E ) A/D BOARD (12 b i t s )  VOLTAGE AMPLIF.  20 c o n d u c t o r ribbon connector  FIGURE  115v  2.4: E l e c t r i c a l schematic  UV  AC  UNIT  CONTROLLER  UV OPTICAL UNIT  control  system  AC  34 computer/system  interface  from the computer d i g i t a l switch  wherein  the c o n t r o l  signals  output l i n e s were used t o help  i n d i v i d u a l valves  on and  o f f according  to the  c o n t r o l program.  System C o n t r o l I n t e r f a c e : The i n t e r f a c e i s the power d i s p a t c h i n g center  of the system. I t s inputs  output  from  the power  source  c o n s i s t of the 24v DC  mentioned  above,  power used t o d r i v e the homogenizer and three pumps, and the LSTTL  115v AC  peristaltic  (large scale t r a n s i s t o r - t r a n s i s t o r  l o g i c ) s i g n a l s from the computer. The  2.4v (minimum) s i g n a l  input/output computer  (Dig I/O)  card  slots  supplied  board  arrives  by the d i g i t a l  mounted a t the  i n one interface  of the with  a  maximum 15.0 mA. This m i n i s c u l e c u r r e n t must be a m p l i f i e d in  order  t o switch  Darlington  on  the flow  t r a n s i s t o r arrangement,  2.5 f o r a s i n g l e channel, electrical board  control  the i n i t i a l  Darlington-connected  shown  i s used t o achieve  output. The d i g i t a l  supplies  as  solenoids.  i n Figure the d e s i r e d  output s i g n a l from the I/O  base  silicon  A  current  power  f o r the TIP120  transistors  (Texas  Instruments, Inc) which then passes 24v DC c u r r e n t a t the required  amperage  through  the  4N32  opto-isolator  (Motorola) to one of the 23 s o l e n o i d s . The  four  115v AC outputs are t r i g g e r e d i n a s i m i l a r  manner, but with for  the a d d i t i o n of a moving contact  each of the four channels.  relay  35  TYPES N-P-N  D A R L I N G T O N - C O N N E C T E D SILICON  TIP120, TIP121. TIP122 POWER  TRANSISTORS  D E S I G N E D FOR C O M P L E M E N T A R Y U S E WITH TIP125, TIP126, TIP127 •  65 W at 2 5 ° C Case Temperature  •  Min h F E of 1000 at 3 V , 3 A  •  5 A Rated Collector Current  •  50 mJ Reverse Energy Rating  device schematic  0>  -t  c <  op  COLLECTOR Q  r BASE O -  •» 150 n  Wv  to -* co ro  <  VA—  j  mechanical data THE COLLECTOR IS IN ELECTRICAL CONTACT WITH THE MOUNTING TAB  _ e J  J  MO  ALL  D I M E N S I O N S A R E IN I N C H E S  absolute maximum ratings at 2 5 ° C case temperature (unless otherwise noted) Collector-Base Voltage Collector-Emitter Voltage (See Note )) Emitter-Base Voltage Continuous Collector Current Peak Collector Current (See Note 2) Continuous Base Current Safe Operating Areas at (or below)-25°C Case Temperature . . . Continuous Device Dissipation at (or below) 25°C Case Temperature (See Note 3) Continuous Device Dissipation at (or below) 25°C Free-Air Temperature (See Note 4) Undamped Inductive Load Energy (See Note 5) Operating Collector Junction Temperature Range Storage Temperature Range Lead Temperature 1/8 Inch from Case for 10 Seconds "0TES:  1. 2. 3. 4. 5.  TIP121 TIP122 80 V 100 V 80 V 100 V 5V 5V 5 A •a 8A •• 0.1 A •»— See Figures 7 and 8 " 65 W •a 2W • — — — 5 0 mJ • — - 6 5 ° C to 150° C — * 6 5 ° C t o 150°C — • « 260° C • TIP120 60 V 60 V 5V •.  Thaia valuai appty whan the baaa-amittar dioda i l o p a n c i r c u i l a d . T h l i valua appllo* for l < 0,3 m i , duly cycla < 10%. Darata Mnaarlv to 1S0' C caaa tamperatura at tha rata of 0.62 w / * C or rafar to Diulpatlon Ooratl nfl Curva, Figure 0, Darata llnaarlv to 150*C fraa-alr tamparatura at tha rata of 16 rriW/°C or ratar to Diulpatlon Oa< aiing Curva, Figure 10. T h l i rating Ii tiaiod on tha capability of tha tramlitori to oparata lafaly In tha circuit of Figura 2. L - 100 m H , R n,2 - 100 f i . V - 0 V, Rg - 0.1 f l , V - 20 V . Energy » l c l - / 2 . w  ri  !  B  a  2  c  c  TEXAS I N S T R U M E N T S I M  IIHI'OH A I I  PO%1 O f F l C f BOH » 0 l l  FIGURE 2.5:  •  5375  II  DALLAI  rtXAt  »tll»  D a r l i n g t o n - c o n n e c t e d N-P-N s i l i c o n power t r a n s i s t o r s  36 Analog to D i g i t a l produce  (A/D) Board: Most system monitoring d e v i c e s an  analog  signal  based  on  either  v o l t a g e or  c u r r e n t l e v e l s . In order f o r d i g i t a l computers t o perform any  o p e r a t i o n s u s i n g t h i s data, i t must be converted to  binary  format.  measured  The A/D board  parameter  and  takes  then  a  'snapshot'  produces  a  of the digital  r e p r e s e n t a t i o n of i t s v a l u e . The A/D board s e l e c t e d f o r use i n t h i s system DT2814 by Data T r a n s l a t i o n size  board  channels,  including a  12-bit  hardware/software  (Marlboro, MA). I t i s a h a l f -  16 single-ended A/D  converter  programmable  pacer  (SE) analog and  input  a  combination  clock  t o s e t the  sampling frequency. The maximum sample throughput board  i s the  f o r the  i s 25 kHz. F i g u r e 2.6 g i v e s the g e n e r a l l a y o u t of the c a r d and  i t s c o n n e c t i o n t o the IBM PC s t y l e Bus (PC/XT/AT), while F i g u r e 2.7 shows the user p i n assignments. The g e n e r a l o p e r a t i o n of the board i s c a r r i e d out by writing  t o and r e a d i n g  computer  memory  Hexadecimal  at  from  the  220) and Base+1  the Base, or c o n t r o l / s t a t u s  two Base  registers address  (221H). register  located i n 220H ( i . e .  A WRITE command to i s used  t o s e t the  c o n t r o l b i t s . B i t s 0-3 are used t o inform the m u l t i p l e x e r which one of the 16 input channels  i s t o be sampled. B i t  4 (ENB) enables or d i s a b l e s the on-board continuous divisor  sampling,  while  bits  pacer c l o c k f o r  5-7 s p e c i f y  the decade  f o r the hardware-jumpered base frequency f o r  37  16  16SE Analog Inputs  Analog Inputs  10O ion  Sample & Hold  MUX  12-bit A/D  +12V -12V  Control & Status logic  Address Decode Clock Control  Address  B Data Line  IBM  FIGURE 2 . 6 :  PC BUS  Card l a y o u t f o r DATA T r a n s l a t i o n DT2814 A/D card  PIN FUNCTION Channel 0 Channel 1 Channel 2 Channel 3 Channel 4 Channel 5 Channel 6 Channel 7 Power Ground +12V Out FIGURE 2 . 7 :  Interrupt  PIN FUNCTION  PIN 1 3 5 7 9 11 13 15 17 19  2 4 6 8 10 12 14 16 18 20  Channel 8 Channel 9 Channel 10 Channel 11 Channel 12 Channel 13 Channel 14 Channel 15 Analog Ground -12V Out  DATA T r a n s l a t i o n DT2814 A / D card user connections  38 sampling The  rate. useable  sampling  frequency  range  i s 0.005 Hz t o  20 kHz, however, f o r the purpose of the c o n t r o l continuous  sampling  i s not r e q u i r e d  values are requested  the  with  a zero  value  Base address.  but r a t h e r  single  p e r i o d i c a l l y by the c o n t r o l program.  Within the program, a channel along  software,  i s s e l e c t e d and t h a t number  f o r B i t 4 (ENB) are w r i t t e n to  When t h i s  register  r e c e i v e s the value  sent, the on-board m u l t i p l e x e r (MUX) s e l e c t s the d e s i r e d channel and the sampling  sequence i s t r i g g e r e d .  To read the d i g i t a l v a l u e , a READ command should be sent  to  the  Base  address  which  now  returns  status  i n f o r m a t i o n r e g a r d i n g the data c o n v e r s i o n . B i t s 0-3 contain and  the s e l e c t e d channel  Bit 4  is still  the  number,  still  for verification,  enable/disable  flag.  B i t 5,  however, r e t u r n s a value of 1 i f the A/D c o n v e r s i o n i s i n progress if  an  result  or 0 i f i t i s complete. B i t 6 (ERR) r e t u r n s a 1  error  i s encountered  of too f a s t  the data r e g i s t e r a  clock  initiated  speed  during  a c l o c k speed  before  the  in a first  as a  to clear  c o n v e r s i o n . Too high  second one  conversion  or a f a i l u r e  p r i o r t o the next  results  a  is  conversion complete.  being Bit 7  (FINISH) i s s e t (1) i f the conversion i s complete and the data  register  conversion  value  is valid,  i s incomplete  or i s c l e a r e d  and the data  (0) i f the  register  value i s  invalid. Where samples are taken  i n the c o n t r o l program, the  selected bit  channel  i s written  i s monitored  register data,  two  separate the f i r s t  while the second 4 bits  second  s e t , at  (Base+1) double  required:  The  until  byte  READ  which  i s READ.  statements  yields  the most  time  the data  In r e a d i n g the to  BASE+1  significant  r e t r i e v e s the l e a s t s i g n i f i c a n t  occupy  byte  t o Base and then the FINISH  read  the most s i g n i f i c a n t a t Base+1.  8 bits 4 bits.  positions  In order  are  of the  to arrive  at a  s i n g l e meaningful value from the combination of these two values,  the f i r s t  one must  be« m u l t i p l i e d  by 16 ( i . e .  shifted  four b i n a r y columns t o the l e f t ) while the second  must be d i v i d e d by 16 i n order t o compensate f o r the f a c t that  i t occupies the l e f t  (most s i g n i f i c a n t ) h a l f  of the  second word read. S i g n a l s get from the sampling J)oard  via a  screw  terminal  instruments t o the A/D  (DT757)  and  20  conductor  ribbon cable.  Digital  Input/Output  (I/O) Board  This Data is  a half-size  programmable channels is  Translation card  factory  c o n t a i n i n g 32 channels.  for either  per port  (Marlboro, MA) board, DT2817,  input or output  f o r each  i n groups  of 4 p o r t s . The Base  s e t a t 228H, although and  These are  contains  of 8  address  i t can be changed v i a  on-board  jumpers,  the c o n t r o l  register.  Addresses  Base+1 t o Base+4 c o n t a i n the ON/OFF s t a t u s f o r  40 each of the 4 p o r t s . A WRITE statement ports  must  first  be  c o n t a i n i n g the I/O  sent  to the  Base address.  four l e a s t s i g n i f i c a n t b i t s are used BIT  0 value  of  0 sets  port  0  s t a t u s of the 4 Only  the  i n t h i s statement:  (channels  0-7)  as  a  input,  while a value of 1 s e t s them f o r output. The same p a t t e r n holds f o r BITS 1-3 For  input  ( l o g i c a l low) -0.2mA,  f o r p o r t s 1-3  signals  corresponds  while  corresponds  to  a  the  card,  a  BIT  value  of  to a minimum 2. Ov  at  1  20.0U.A. Output  signals  high s i g n a l must be a minimum of 2.4v  a t -15.0  ribbon  cable  the  system  with  pin  0,  while  a  mA.  to the IBM-PC/XT bus with p i n  assignments as shown i n F i g u r e 2.8. to  at  high)  logical  The DT2817 i s connected  0  (logical  24.0mA f o r a  connected  of  to a maximum v o l t a g e of 0.8v  value  must a maximum 0. 4v a t  (channels 8-32).  interface  assignments  The  32  I/O  lines  by  a  50  conductor  as  shown  in  are  Figure  2.9.  Process C o n t r o l Computer  The  computer  exchange  system  contains  a  extensively  used is  to  an  multi-purpose used  i n the  monitor  IBM  and  PC/XT  card  from  control  control  compatible which  the  the  ion-  unit.  It  clock  is  r o u t i n e s . The  on-board  memory c o n t a i n s 640 kbytes of random access memory (RAM). The  computer c l o c k operates a t the standard PC/XT  41  PIN Al  A2 A3 A4 A5 A6 A7 A8 A9 AIO All A12 A13  A14 A15 A16 A17 A18 A19 A20 A21 A22 A23  A24  A25 A26 A27 A28 A29 A30 A31 BI B2 B3 B4 B5 B6 B7 B8 B9 BIO Bll B12 B13 B14 B15 B16 B17 B18 B19 B20 B21 B22 B23 B24 B25 B26 B27 B28 B29 B30 B31  MNEMONIC  SIGNAL DESCRIPTION  -I/O CH CK +D7 +D6  No Connection Data Bit 7 (MSB) Data Bit 6 +D5 Data Bit 5 +D4 Data Bit 4 Data Bit 3 +D3 +D2 Data Bit 2 +D1 Data Bit 1 +DO Data Bit 0 (LSB) -I/O CH RDY No Connection +AEN Address Enable +A19 No Connection (MSB) +A18 No Connection +A17 No Connection +A16 No Connection +A15 No Connection +A14 No Connection +A13 No Connection +A12 No Connection +A11 No Connection No Connection +A10 - • Address Bit 9 +A9 Address Bit 8 +A8 Address Bit 7 +A7 +A6 Address Bit 6 +A5 Address Bit 5 +A4 Address Bit 4 +A3 Address Bit 3 +A2 Address Bit 2 +A1 Address Bit 1 +A0 Address Bit 0 (LSB) GND Ground +RESET DRV Reset Driver +5V +5 Volt Power +IRQ2 No Connection No Connection -5VDC No Connection +DRQ2 No Connection -12V No Connection RESERVED No Connection +12V Ground GND -MEMW No Connection -MEMR No Connection -IOW I/O Write Command I/O Read Command -IOR No Connection -DACK3 No Connection +DRQ3 No Connection -DACK1 No Connection +DRQ1 No Connection -DACKO 6MHz CLOCK +IRQ7 No Connection +IRQ6 No Connection +IRQ5 No Connection +IRQ4 No Connection No Connection +IRQ3 No Connection -DACK2 No Connection +T/C No Connection +ALE +5 Volt Pover +5V 14.318MHz +OSC Ground +GND  FIGURE 2.8:  D i g i t a l J / O c a r d backplane connections for DATA T r a n s l a t i o n DT2817  42  SIGNAL NAME  PIN NO.  1 Digital Ground 3 Port 0, bit 0 5 Port 0 bit 7 Port 0 bit Port bit "9 11 Port bit 13 Port bit 15 Port bit 17 Port bit +5V Out(lA max) 19 21 Dig tal Ground 23 Dig tal Ground 25 Dig tal Ground 27 Dig tal Ground 29 Digital Ground +5V 0ut(lA max) 31 33 Port 2, bit 0 35 bit 1 Port 37 bit 2 Port 39 bit Port 41 bit Port 43 bit Port 45 bit Port 47 bit Port rorr. u n / 49 Digital Ground  FIGURE 2 . 9 :  2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50  SIGNAL NAME Digital Ground Port 1, bit 0 Port 1, bit 1 Port 1, bit 2 Port 1, bit 3 Port 1, bit 4 Port 1, bit 5 Port 1, bit 6 Port 1, bit 7 +5V 0ut(lA max) Digital Ground Digital Ground Digital Ground Digital Ground Digital Ground +5V 0ut(lA max) Port 3, bit 0 Port 3, bit 1 Port 3, bit 2 Port 3, bit 3 Port 3, bit 4 Port 3, bit 5 Port 3, 'bit " 6 7 Port r o r i 3 J I bit uii / Digital Ground  D i g i t a l I/O card user connections for DATA T r a n s l a t i o n DT2817  frequency of 4.77  MHz.  The  only a d d i t i o n s  to the  system  were the two I/O cards used to i n t e r f a c e with the process monitoring and c o n t r o l equipment. The  control  possible  so  that  system was the  p u r p o s e l y kept as simple as  functionality  of the software  was  not overcome by requirements f o r expensive graphics cards and  massive  graphic  memory  display  desirable  than  developing  and  of  upgrades. the  text  While  physical only,  accomodating  unwarranted at t h i s time.  the  such*a  a  real-time  system costs  color  i s often  more  involved  in  d i s p l a y are high  and  44  3.  PROCESS CONTROL SOFTWARE  Mandatory C o n t r o l Functions The  most  include  monitoring  sensors, steps  fundamental  of the c o n t r o l  the c o n d i t i o n of the system  accounting  and  requirements  f o r the elapsed  providing  real  time  time  control  software  v i a electronic  of v a r i o u s  output  sequence  signals  t o the  p h y s i c a l flow c o n t r o l system. As i n any decision-making the-minute account the  state  pressure albumin  of  drop  of p e r t i n e n t f a c t s .  the  current  across  each  filters,  eluted  liquids. sensors  and Due  of  the  of the columns  column  f o r thermal  t o the c o s t s  flow  include  and a c r o s s  the  rates  involved  the  the i n l e t  of p r o t e i n s  undergoing and  i n d i c a t i v e of  process  or absence  the temperature  i n part  Those most  ion-exchange  the presence  fraction  procedure,  process, i t helps t o have an up-to-  i n the  regeneration  of the system  i n securing  extra  and v o l u m e t r i c or mass flow measurement and  i n p a r t t o the r e l a t i v e ease with which both the temperature and flow  rates  can be  u l t r a v i o l e t monitor Elapsed times  controlled,  some  the course degree  chemical  of  one. Event  sensors  and  f o r the v a r i o u s STEPs i n v o l v e d i n the process f o r t r i g g e r i n g STEP  of a run, c e r t a i n variation  due  to  parameters  changes  sequence control  i s i n theory allows  changes.Since  are s u b j e c t to  in physical  p r o p e r t i e s of the o r g a n i c feed l i q u i d  event-controlled timed  the pressure  were used.  were used as the b a s i c c r i t e r i a during  only  and the r e s i n , an  preferrable to a  f o r optimal  and/or  rigid,  use of the system  while timed c o n t r o l has the b e n e f i t of s i m p l i c i t y i n terms of the sensing  equipment  suitable  requirements  on-line  assay  and software  has been  lysozyme i n the albumin l e a v i n g  perfected  structure.  Since no  f o r the d e t e c t i o n  the primary feed  of  column, a timed  procedure becomes the d e f a u l t method of c h o i c e . Output  control  program d i s k e t t e during  the  each  fixed  STEP  procedure  are s t o r e d  the ON/OFF  I/O card  according  form  on the  i n t o computer memory  prior  as  four  status  8-bit  of  one  to  the  operator's  words output  values  with  each b i t  line  from the  t o the c o n t r o l i n t e r f a c e . These a r r a y values are t o the p h y s i c a l  structure  system and cannot be a l t e r e d from w i t h i n  Control  i n array  p a r t i c i p a t i o n i n beginning a r u n . The c o n t r o l  contributing digital  are s t o r e d  and a r e uploaded as a r r a y s  initialization  interactive for  signals  of the flow  control  the program.  Enhancements  The  b a s i c c o n t r o l of the ion-exchange system presented  herein  i s r e l a t i v e l y s t r a i g h t forward and simple as i s i n d i c a t e d  by the  modest length  of the p r e v i o u s s e c t i o n . At t h i s p o i n t , the c o n t r o l  program would clock  used  number  to t r i g g e r  with  information  operate  i n much the same manner a signal  that  i t s corresponding would  be  control  values  would  editing  the program  passed have  between  would  control  along  to  increment output  the STEP  values.  the o p e r a t o r ,  to be assigned runs.  as a mechanical  to f i x e d  However, s i n c e  and arrays  No all by  few operators  want to be bothered with having t o modify the software  f o r each  change  provides  of  conditions,  an  interactive  program  which  menus, prompts and v a l i d i t y far  checks  more u s e f u l v e h i c l e through  f o r r e q u i r e d input data  which t o accomodate r a p i d  isa set-up  procedures. The  following sections outline  which add t o the f l e x i b i l i t y  these  and other  enhancements  and u s e r - f r i e n d l i n e s s of the c o n t r o l  environment.  Interactive input: Interactive method  of  data  allowing  entry  provides  operating  a  parameters  fast to  and  be  safe  s e t and  modified by the operator without h i s / h e r having to modify the control  program.  potential  I t i s a quick  changes are presented  progression  and important  overlooked.  Information  safety  of data  screen  the input  untenable The  data  entry  e n t r y method  on the monitor  value  assignments  i s prompted i s augmented  data  against  pre-set  in a  logical  are not e a s i l y  f o r as since  since a l l  r e q u i r e d . The  the program can  limits  to  prevent  situations.  use of menu s t r u c t u r e s allows the operator t o s e l e c t  p a r t i c u l a r parameters f o r which t o a s s i g n values or simply to view  existing  entire of  parameter  options,  line  number  flexibility single  ones  without  list  t o progress  through the  each time. Once presented  with a menu  a selection  i s g e n e r a l l y made  to i t s l e f t . and speed  key s t r o k e  having  This  allows  by e n t e r i n g the  the operator  i n adressing p a r t i c u l a r  selections.  I f data  input  great  parameters v i a i s called for  once the s e l e c t i o n has been made, values are prompted f o r on  47 the  screen,  program  and  once  integrity.  in the event  entered,  they  Appropriate  are  tested  to  ensure  e r r o r messages are d i s p l a y e d  of i n a p p r o p r i a t e e n t r i e s .  Parameters  which  can  be  assigned  values  prior  to  or  information  in  during a run are d e s c r i b e d i n the f o l l o w i n g s e c t i o n s .  Visual display: Just order  as  to  system, state  the  software  provide  the  so  appropriate  operator  that  requires  also  he/she  can  certain  control  must  sequences  remain  informed  make knowledgeable  for as  the  to i t s  adjustments  to  the c o n t r o l parameters as they are needed. System simplest system  information  case,  would  i s displayed  although be  more  in text  format  as  a g r a p h i c a l d i s p l a y of the p h y s i c a l  aesthetically  p l e a s i n g . The  monitored  and c a l c u l a t e d parameter values are updated s e v e r a l times second.  Figure  3.1  process c o n t r o l At  the  system  is  alternative (MPC)  top  which can  shows a  of  the  The columns  screen,  actively  automatic be  screen  the  title  controlled control  accessed  time d u r i n g the course of the  typical  during  per  automatic  (APC).  being to  the  by  indicates that  by  the  i s manual  process  p r e s s i n g the  of the run as noted  program;  "M"  key  the the  control at  any  i n the bottom l i n e  screen. second  line  operating  (described  provides  and  whether  information or  not  a  on  the  staged  number  of  shut-down  i n a f o l l o w i n g s e c t i o n ) i s i n progress, as w e l l as  48  AUTOMATIC PROCESS CONTROL  VER 1.0 AUG/88 TIME:00:35:42  OPERATING MODE NORMAL BATCH: 4 OPERATOR: ACM BLOCK LENGTH: 2 c y c l e s STEP : 2 STAGE: 3 BLOCK CYCLE: 2 1 DIFFERENTIAL PRESSURE (ps'id) VALVE STATE PUMP STATE VALVE STATE Threshold: 7.0 OFF 1 OFF OFF 13 1 2 OFF Column 1 0.2 OFF 14 ON 2 Column 2 -0.5 OFF OFF 15 3 ON . 3 Column 3 0.4 ON ! OFF 16 4 Filters 0.2 OFF 17 ON 5 OFF Flag 0 OFF 18 6 OFF UV PEAK DETECTION SONICATOR 7 ON 19 OFF OFF Threshold (Au): 2.0 8 20 OFF OFF 21 Reading (Au): 0.0 9 ON OFF 22 OFF 10 OFF OFF 23 BATCH VOLUME: 25.000 (L) 11 12 OFF FEED FLOWRATE: 5.000 (L/HR) BLOCK RUN STEP STAGE CYCLE 0: 0.5 0: 4.4 SET DURATION: 0:13.2 0: 0.1 0: 3.3 0: 1.9 REMAINING TIME: 0: 0.4 0: 1.1 0:11.3 0:21.1 ELAPSED TIME: 0 21.1 Press M to enter MANUAL PROCESS CONTROL FIGURE 3.1: T y p i c a l APC (Automatic Process Control) screen  49 the c u r r e n t time. Line  3  shows the  number  of  avidin  (as  section  2 of t h i s paper),  and  elution  values  per  cycles  the  of  BLOCK  per  length,  column  elution-looping the  is  the  removals  of  discussed  in  between concept  Operator  Identification  label,  the Batch Number. Line 4 shows the c u r r e n t values  BLOCK, CYCLE, STAGE and The line  left-most  17  are  two  the  equipment. The  of the sequence  t h i r d s of  state  the  screen  indicators  of  them read  the  from  for  by  the  operator,  although  software.  Appendix The  to  displayed values,  ON/OFF, i s  I/O  feature  card  output  intended  values  is invisible  i t i s a useful trouble-shooting  comparing the a c t u a l outputs  5  controlled  from the d i g i t a l  This  line  all  l a t c h e s r a t h e r than simply a s s i g n i n g them the harboured  indicies,  STEP.  accuracy  ensured by having  of  which  to the  intended  to  the  t o o l when  ones as shown i n  A. r i g h t t h i r d of the screen d i s p l a y s the c u r r e n t  a l l monitored  v a r i a b l e s as  well  as  the  values  threshold  values  which when exceeded are used to t r i g g e r some form of c o n t r o l event to o v e r r i d e the normal timed sequences. Below these listed  the  Batch  information controlled well  be  only by  the  expedient  Volume since  and they  next  four  are  Flowrate neither  program. In a commercial to  monitor  for both system l o g g i n g and The  Feed  lines  of  the  various  for  general  monitored operation  liquid  are  flow  nor  it  may  rates  c o n t r o l purposes. the  d i s p l a y show the  allotted,  50  remaining  and elapsed  time  f o r each of the c o n t r o l  sequence  times. While  this  optimized  screen  arrangement  f o r the u s e f u l n e s s  speed with  which  is  not  of a l l data  necessarily  displayed  or the  i t can be d i g e s t e d by the operator,  it  is  f u n c t i o n a l and adequate f o r t h i s v e r s i o n of the program.  D e f a u l t d r i v e path: 1 asks  Menu  the operator  to s p e c i f y  what  type  of d i s k  d r i v e the computer uses, and from t h i s the d e f a u l t d r i v e path is  assigned  Drive  A for single  floppy drives,  dual  f l o p p y d r i v e s , and D r i v e C f o r hard  path  can be changed  procedure  or d u r i n g  default  path  storage  and  a t any time the program  is specified, retrieval  s p e c i f y i n g the method f i l e is  no path  specified,  path  the pre-run 2.  v i a Menu  operations  B for  d i s k systems. T h i s  during  i t can  Drive  be  Even  when the  overridden  on  method  i n response  s e t up  during  files  by  t o the prompt. I f  then the d e f a u l t path  i s used.  F i l e handling r o u t i n e s : In  most  chromatographic  optimize  process  products  from  parameters  a given  parameter values  work,  feed  i s called  some  attempt  f o r the r e c o v e r y stock.  A  listing  i s made to of  specific  of these  run  the "method". When a method i s to  be used more than once, i t i s very d e s i r a b l e to s t o r e i t i n a method f i l e  on a computer d i s k e t t e f o r subsequent  This not only speeds the set-up  procedure  retrieval.  g r e a t l y , but a l s o  51 prevents  the i n c o r p o r a t i o n  of t y p o g r a p h i c a l  errors  i n t o the  f o r the  storage,  method. Three  routines  have  retrieval  and  output  routines  are  accessed  been  to  written  printer from  of  method  the  third  files. menu  These  in  the  i n i t i a l i z a t i o n s e c t i o n of the program.  the  Upon invoking  e i t h e r the storage  option  to view  exists  or r e t r i e v a l  the method  names  prior to  e n t e r i n g the name of the s e l e c t e d f i l e . The operator  may a l s o  d e c l i n e t h i s option and enter the  the d r i v e path and f i l e name a t  prompt. I f the d e f a u l t d r i v e path  outset  (as e s t a b l i s h e d a t the  of the program) i s to be used, then only the f i l e name  needs t o be entered. then  file  routines,  a  search  I f the v i e w - f i l e s option  i s made  of  the method  i s selected,  file  disk  i n the  s p e c i f i e d d r i v e path. Only f i l e s having the extension, (indicating search. about  "set-up"  The operator  does  the d i r e c t o r y  ".SET",  they  alphabetizing from  which  are  search piped  through the method storage operator  since  produces to  a  file  simply  with  routine  for  i n t o a temporary  file  SORT  i n blocks  has been i d e n t i f i e d ,  wants to avoid  storage  to be s t o r e d .  names  for display  routine,  him/herself  ending  DOS  If this  directory  the method  i t to any f i l e s  are r e c a l l e d  the operator.  in a  t o concern  and are then r e d i r e c t e d  they  i n by  selected  not have  attaches  names. Once the d e s i r e d f i l e keyed  are  the use of the extension  routine automatically When  data)  ".SET"  routine  then  of 25  i t s name i s  was  accessed  i t may be that the  overwriting  an e x i s t i n g  file  52 or  to ensure  o v e r w r i t i n g the c o r r e c t one. I f the method  files  are viewed v i a the r e t r i e v a l r o u t i n e , then once the f i l e name has been entered, the contents of the f i l e assigned to the a p p r o p r i a t e parameter In  are up-loaded and  labels.  order t o prevent a c c i d e n t a l l y o v e r w r i t i n g method  and/or  " c r a s h i n g " the program  error  handling  interrupts  program's c o n t r o l  structure.  in an e x i s t i n g  file  program  alert  will  alternatives  of DOS f i l e  , a wide v a r i e t y  have  been  accomodated  Thus, even  files  i n the  i f an operator  keys  name when down-loading a new method, the him/her  to  the  of c o n f i r m i n g the a c t i o n ,  fact  and  offer  the  a s s i g n i n g a new  file  name, or a b o r t i n g the procedure. If  an attempt  existent  file,  options  of  procedure The  i s made to up-load  the operator  re-entering  a r e presented  data  i s alerted  the  file  method  from  to the f a c t ,  name  for alternate  s t o r e d i n these  a method  or  a nonand the  aborting  the  action.  files  i n c l u d e s the STEP  time v a l u e s f o r a l l system o p e r a t i n g modes, and the values of the process constants presented  Staged  i n Menu 4.  shutdown: The  concept  of a system which can continue to operate i n  the event t h a t one or two of the three columns becomes f o u l e d with  congealed  promote  albumin  the h i g h e s t  was i n c o r p o r a t e d i n t o  possible production  system. Such a system could be l e f t day with o n l y a s i n g l e work s h i f t  the program to  from  t o operate  an  unattended  24 hours per  to maintain i t .  Rather total  than p r o v i d i n g a system with a simple alarm and  shutdown  when a  blockage  the impending blockage the  pressure  drop  filters  exceeds  partial  blockage  threshold  regenerating and  i s not  column  i s ready  regeneration  one  lower  i s reached,  to  of  the  columns  overpressure  column  removed  has  and  completed albumen  complete,  to  be  modified  control  remaining of  control  the  passed  process  high  with  i s then passed  sequence  required  to  for  the  the  two  columns  i s badly  from  operation  t r a c k of the  service,  becomes f o u l e d or a t o t a l  by a programmed endpoint  passed  buffer  the to  to a r o u t i n e which uses a  to  two  the  sequence  of  the  elapsed  The  the o f f e n d i n g or  one  of  the  shutdown i s e f f e c t e d  or a manual i n t e r v e n t i o n . f o u l e d , then  two  number  e l u t i o n - l o o p i n g procedure.  returned  the  the  phosphate  column  remaining  until  which causes  columns u n t i l  and  If  bed.  permit  keeping  inlet  i t . Once  is  process continues with these two i s cleaned  the  overpressure  through  control  from the r e s i n  columns while  CYCLES  or  i t s regeneration  backwashed  remove the feed l i q u i d Process  brings  threshold.  normal 3-column o p e r a t i o n to the r o u t i n e APC fouled  software  then the feed pump i s stopped  have  is  the  to the a t t e n t i o n of the operator when  across a  occurs,  a  a sequence  I f one of  of  events  occurs which i s s i m i l a r to that f o r the t r a n s i t i o n from three columns to two, The  l e a v i n g one column s t i l l  staged shutdown method of o p e r a t i o n thus p r o v i d e s f o r  a w e l l c o n t r o l l e d system response filter  active.  failure  due  to  fouling.  i n the event of a column or This  control  strategy  is  optimized  f o r continuous  production  work with  a  minimum  of  operator i n t e r v e n t i o n .  Column s e l e c t i o n : At the beginning for  the  selection  of the program, Menus 2 and of  three columns, any  the  initially  combination  active  of two,  2.1  provide  column(s).  or any  single  can be s e l e c t e d . T h i s f l e x i b i l i t y f a c i l i t a t e s both  All  column  commercial  and r e s e a r c h work.  Manual process  control: j  This  mode i s entered  routines  by  available remains main MPC be  while  fixed  monitor  as  the  and/or  for  any  the  i t was  valve, be  of  of  the  key. the  when the  control  was  pressed.  key  process  or  s e t , and  ON/OFF  the a DOS  From  the  constants  can be  homogenizer,  the  UV  shell  e x i t i n g the c o n t r o l program. The  permits  r e t u r n of c o n t r o l c a l l was  hardware  can  run without  which the MPC  are  status  using the SYSTEM command to a l l o w other DOS  the  control  options  flow  equipment  pump  automatic  Several  d u r a t i o n s and  changed,  b a s e l i n e can  "M"  status  menu, STEP time  viewed  toggled  depressing  from any  can  be  set  up  to  be  activities final  to the automatic  selection  routine  from  made.  Alarms: Both a u d i b l e and operator  to  v i s u a l alarms are provided to a l e r t  overpressure  conditions  within  the  system  the in  55  order  to a l l o w time  staged  shut  down  for correction  takes  place.  A  of the problem before a warning  message  i s also  presented a t the bottom of the s c r e e n . The a u d i b l e alarm can be shut the message remains u n t i l "beeps" are a l s o  used  o f f by a keystroke,  the s i t u a t i o n  to i n d i c a t e  data e n t r i e s p r i o r to reprompting  while  i s remedied. Audible  incorrect  or out of range  f o r correct data.  S t a r t time o p t i o n s : This  f e a t u r e allows  immediately start  the process  or a t some s p e c i f i e d  option i s l i k e l y  who o f t e n f i n d  to be i n i t i a t e d  date  and time.  to be most b e n e f i c i a l  i t difficult  t o conduct  either  The delayed  to researchers  chromatographic  runs  w i t h i n the bounds of normal working hours. With t h i s o p t i o n , a run can begin unattended any  date  analysis  so t h a t . the products as soon as the s t a f f  c a p a b i l i t y could speed  Step time As  a t any time of day or n i g h t and on of the run can be ready f o r arrives  i n the morning.  This  up r e s e a r c h i n v o l v i n g long run times.  assignments: selected  durations function  to  be  i n each  from  Menu  2, o p t i o n  assigned  to  of the three  3 allows  a l l STEPs  process  having  o p e r a t i n g modes  the  STEP same  (one, two or  three columns a c t i v e ) s i m u l t a n e o u s l y . When t h i s s e l e c t i o n i s made,  Menu  reference these  2.3,  STEP  descriptions,  to any p a r t i c u l a r  durations  are then  operating  written  i s presented mode.  Once  t o the STEP  without entered,  time  array  variables  f o r each  modes can  be  altered  STEPs  in  similar durations  can  of  the  without  any  also  modes. STEP affecting  other  simply  times  mode.  be  the  The  viewed  for  time  individual d u r a t i o n of  current  STEP  f o r a l l or  any  time single  o p e r a t i n g mode without being queued f o r changes.  Run  completion s e t p o i n t : The  endpoint  f o r a run can be p r i o r to beginning the  run  or a t any time w i t h i n the run by s p e c i f y i n g the BLOCK, CYCLE, STAGE and these  STEP number a t which t e r m i n a t i o n should occur. When  values  d i s p l a y the show  match process  whether  malfunction.  the By  the  corresponding  i s h a l t e d and  shutdown setting  was  the  ones  from  a message  planned  endpoint  or  the  screen  i s d i s p l a y e d to occurred  prior  to  due  beginning  to a  delayed s t a r t run, the process can be made, f o r i n s t a n c e , to s t a r t a run d u r i n g the n i g h t and BLOCK,  whether  operator,  so  or  not  the  t h a t analyses  stop a f t e r  apparatus  can  be  is  one  CYCLE or  attended  undertaken  one  by  an  immediately  as  personnel are a v a i l a b l e .  Automatic  to  column c l e a n i n g :  When any  column(s) i s (are) r e t i r e d  fouling,  it  (they)  buffer  to remove any  stored  at  periods  of  room time.  is  albumen and  temperature The  (are)  from the process  backwashed thus  i n the  same treatment  prevent  resin  bed  with  i s accorded  columns at the normal t e r m i n a t i o n of a run.  phosphate  i t from for  due  being  prolonged a l l active  57  Program S t r u c t u r e The  physical  and Operation operation  of the system  involves  five  basic  steps: 1. Turn on power t o a l l system components  including:  - computer - printer - DC power source - control  interface  - pumps (ensure c o r r e c t pumping - homogenizer (ensure c o r r e c t  speed)  intensity)  - d i f f e r e n t i a l pressure transducer - UV monitor d e t e c t o r  and c o n t r o l l e r  2. Place the program d i s k e t t e floppy  3.  module  i n d r i v e A f o r s i n g l e and dual  drives.  (a) For f l o p p y d r i v e s , simply type i n "IX" and then press "ENTER" (or "RETURN" depending upon the keyboard), and the  program, which r e s i d e s  i n the d i s k e t t e  file  "IX.EXE",  w i l l begin as soon as i t i s read i n t o the computer memory. (b) For hard disk d r i v e s having the program i n s t o r a g e , p r e f i x the program name with the a p p r o p r i a t e  drive  path  (see DOS manual f o r d e t a i l s ) .  4. Enter the run data r e q u i r e d  v i a the i n t e r a c t i v e menus  58 5. S t a r t  the  automatic  c o n t r o l p o r t i o n of the program v i a  the " S t a r t time o p t i o n s " s e l e c t i o n from the Main Menu.  The  general  shown i n F i g u r e of  control  control 3.2 with  flow.  Lines  s t r u c t u r e of the computer  program i s  arrow heads r e p r e s e n t i n g the d i r e c t i o n having  arrows  on  both  subroutine CALL and r e t u r n sequences. Included  ends  indicate  i n the f i g u r e are  the most prominent menu- and non-menu s u b r o u t i n e s . The menu-, or i n t e r a c t i v e p o r t i o n s of the program are shown with the a v a i l a b l e selections comprised of  as found  i n the program.  of the i n i t i a l  The non-menu r o u t i n e s are  d e c l a r a t i o n statements  and the a s s i g n i n g  d e f a u l t values t o c o n t r o l , STEP d u r a t i o n and process  constants  arrays. At the s t a r t the  operator.  of a new run, the Main Menu i s  The seven  options  are presented  presented t o  i n the order i n  which they w i l l g e n e r a l l y be used i n the course of a r u n . The  first  o p t i o n , "System C o n f i g u r a t i o n " , allows the user t o  s p e c i f y what type and  of d i s k d r i v e arrangement i s being used, which  how many columns are t o be used, whether or not the columns  are to be e q u i l i b r a t e d a f t e r saline  the s t r i p p i n g  s o l u t i o n , and how many CYCLES are t o be run p r i o r  removal of a v i d i n from the column(s). are: all  of lysozyme with weak  the previous  The d e f a u l t values provided  e n t r y f o r d r i v e type  three columns, no e q u i l i b r a t i o n ,  to the  (stored a f t e r  and the previous  each r u n ) , entry for  the e l u t i o n - l o o p i n g CYCLE number, r e s p e c t i v e l y . Option  2, " F i l e s  ( R e c a l l ; S a v e ; L i s t ) " , provides access t o the  f i l e handling r o u t i n e s through  which method f i l e s  can be up-  MAIN DECLARATION STATEMENTS INITIALIZE CONTROL AND STEP TIME ARRAYS  BEGIN CALL BLOCK. CALL MENU 1 CALL MENU 2 CALL APC CALL QUIT ERROR HANDLING ROUTINES END  MENU 1: ESTABLISH DISK DRIVE CONFIG.  MENU 2: MAIN MENU 1. PROCESS CONFIG.  MENU 2.1  2. METHOD FILES  MENU 2.2  3. STEP DURATION  MENU 2.3  4. PROCESS CONSTANTS  MENU 2.4  5: START TIME OPTIONS 6. SHELL (to DOS)  MENU 2.5  Interactive portion of program  7. QUIT L  FIGURE 3.2: General control structure of process software  60 loaded  t o the program a r r a y s  f o r use i n the c u r r e n t  loaded  from the run a r r a y s t o d i s k f i l e ,  or l i s t e d  run,  down-  to the p r i n t e r  (LPT1). Option current modes, can  3, "STEP d u r a t i o n s " , allows  length from  normal  be changed  values  of STEP  will  times  operation  by d i r e c t  be used  and staged  keyboard  t o save  t o view the  i n any or a l l of the o p e r a t i n g  input  i n the automatic  program, but i n order  the operator  shutdown  modes. Values  at this  time.  control  them f o r f u t u r e  The new  portion use they  of the must be •i  s t o r e d on d i s k using o p t i o n 2. Option and  their  and  4, "Process current  values.  'Run number',  allow  varied. from  others  The A/D and Dig I/O card  users  will  never  values  change  of parameters  such as 'Operator ID'  strictly  f o r the sake  such as the pressure  f o r the s e n s i t i v i t y  the f a c t o r y - s e t  shows a l i s t  Some of these,  are provided  documentation while values  Constants",  and UV t h r e s h o l d  of the c o n t r o l  system  t o be  base addresses can be changed  should  i t become  necessary.  the d e f a u l t s e t t i n g s s u p p l i e d  program, however, when the method  of run  file  Most  with the  i s stored  v i a o p t i o n 2,  provides  entry  the c u r r e n t address values are i n c l u d e d . Option  5,  "Start  Time  Options",  t o the  automatic c o n t r o l r o u t i n e s . The run can be s t a r t e d immediately or at  any time s p e c i f i e d Option  allows  using the d e l a y e d - s t a r t o p t i o n .  6, "SHELL", i s not r e q u i r e d t o be used, however, i t  the operator  COMMAND.COM with  to temporarily  which t o run other  install  a  second  DOS software  while  copy of keeping  the c o n t r o l program r e s i d e n t and ready to go. As o u t l i n e d i n the  61 DOS  documentation,  the  resident  confirmaton and  allows  t h i s data  7,  "Quit",  of  this  f o r the  can  be  reentered  b r i n g s up  another  a c t i o n before last  menu which prompts  allowing  minute s a v i n g  the  of the  program  end,  before  c o n t r o l passes to the s u b r o u t i n e ,  control),  which  selects  the  proper  the run  description,  the  c o n s i d e r e d . The  other  follow a similar the  to  provide the  (automatic  operating  mode based  the  three-column  configuration  o p e r a t i n g modes, f o r two  or one  process will  be  column(s),  procedure.  operation  of three  subroutine,  "NM"  columns, then, (normal  times are  initialized  STEP v a r i a b l e s as a "DO  c o n t r o l would  mode), which would  f o l l o w i n g general sequence of events  - Elapsed and  normal  has  APC  upon which column(s) are to be used. For the sake of t h i s  passed  for  is lost.  been i n i t i a t e d ,  For  to  method f i l e  When a l l of the run parameters have been s e t and  process  by  prompt f o r the second l e v e l C0MMAND.COM.  t y p i n g "EXIT" at the DOS Option  program  f o r RUN,  BLOCK, CYCLE, STAGE  LOOP" i s entered  f o r each of  four parameters ( r e c a l l t h a t STAGE i s  eliminated  i f fewer than three columns are used). The inner-most,  while  then  for c o n t r o l :  the l a t t e r  loop i s the  be  i t c o n t a i n s a STEP time  STEP loop  in which the b a s i c c o n t r o l of the system i s l o c a t e d . - Within the STEP number loop, but p r i o r to the s t a r t of the time loop, the STEP number i s monitored to ensure that the a v i d i n removal STEP i s not executed  unless the c u r r e n t  CYCLE number matches the number of CYCLES s p e c i f i e d for e l u t i o n - l o o p i n g . As w e l l , the t o t a l time f o r the  62 r e g e n e r a t i o n STEPs i s compared with the cascade duration  (cascade or non-cascade  feed  time, depending  which column i s being f e d , f o r two columns;  upon  non-cascade  time f o r one column) to determine the t o t a l STAGE d u r a t i o n (or CYCLE f o r fewer than three columns). - The flow c o n t r o l output a r r a y values are then sent to the Dig I/O board to e s t a b l i s h the c o r r e c t l i q u i d and pump/homogenizer assignments first  flow paths  f o r the f i r s t  STEP of the  STAGE (column A i s primary, B i s secondary, C i s  regenerating). - The s t a t i c p o r t i o n of the run time v i s u a l d i s p l a y i s e s t a b l i s h e d to which the v a l u e s to be updated with each pass of the c o n t r o l loop w i l l be superimposed.  As w e l l ,  the c o l u m n / f e e d - f i l t e r s overpressure alarms are initialized. - The STEP time loop i s entered w i t h i n which a l l of the automatic c o n t r o l f u n c t i o n s are executed f o r the c u r r e n t STEP. The loop i s e x i t e d when the STEP time i s complete or when the r e g e n e r a t i n g column i s ready to become the secondary column f o r the next STEP, which ever r e q u i r e s the most time. - W i t h i n t h i s STEP time loop the v i s u a l d i s p l a y of the p r o c e s s i n g system parameter  values i s updated with each  pass through the loop. - The ON/OFF s t a t e of the feed pump i s monitored as the routine controls  i t so that the elapsed feed time, r a t h e r  than the elapsed c l o c k time, s i g n a l s the end of the loop  63  and  the beginning of the next STEP and  system c o n t r o l  i t s corresponding  output.  - The pressure t r a n s d u c e r s are next t e s t e d a g a i n s t the s e t l i m i t s to check  to overpressure c o n d i t i o n s . If an e r r o r  c o n d i t i o n e x i s t s , then an a u d i b l e as well as a v i s u a l alarm i s turned on. While the a u d i b l e alarm can be turned o f f with a keystroke as per the prompt, the v i s u a l message remains  on the screen u n t i l the c o n d i t i o n  i s c o r r e c t e d or  the end of the STAGE at which time program c o n t r o l would be t r a n s f e r r e d back to APC  from which an a l t e r n a t e  o p e r a t i n g mode would be s e l e c t e d . . - I f the c u r r e n t STEP r e q u i r e s a column to be e l u t e d , the UV monitor  then  output i s t e s t e d a g a i n s t the a p p r o p r i a t e  t h r e s h o l d value to determine  the presence or absence  p r o t e i n peak. I f a peak i s present then the  of a  liquid  c o n t a i n i n g the p r o t e i n f r a c t i o n i s routed to the a p p r o p r i a t e storage c o n t a i n e r f o r e i t h e r lysozyme  or  avidin. - The presence of an e l u t i n g peak w i l l o v e r r i d e the STEP time as the c o n t r o l l i n g  factor  i n the event that the STEP  time has elapsed p r i o r to the end of a peak to prevent the l o s s of product. I f an extended  STEP time i s r e q u i r e d ,  both a u d i b l e and v i s i b l e alarms are invoked. - Keyboard  input i s t e s t e d to see i f "M"  has been pressed,  t r a n s f e r r i n g c o n t r o l to MPC  (Manual  in the event of an extended  STEP d u r a t i o n due to peak  elution,  Process C o n t r o l ) , or  i f any key has been depressed to stop the a u d i b l e  64  alarm. - A test  i s made to see, when the c u r r e n t STAGE time i s  complete, i f the elapsed  feed time equals  time or i f feed time was  l o s t due  the cascade feed  to f i l t e r maintenance  down time. - The  STEP time loop i s t e s t e d f o r completion.  completion,  the time loop i s e x i t e d and  loop increments  by one  and  Upon  the STEP number  r e s t a r t s the STEP time loop.  When the STEP number loop i s e x i t e d , the STAGE number loop i s incremented by one. increments  The  completion  of the STAGE  the CYCLE loop, which when completed  increments  the BLOCK loop which i s e s s e n t i a l l y an i n f i n i t e unless an endpoint  has been s p e c i f i e d v i a  loop  '  MPC.  - At any p o i n t w i t h i n the process, the run i s terminated when a s p e c i f i e d end overpressure  p o i n t has been reached  or when  c o n d i t i o n s f o r c e a shutdown, e i t h e r staged  immediate depending upon whether the source was  The  the columns or the i n l e t  operation  of  modes SI  feed  and  S2  down, r e s p e c t i v e l y , i s very s i m i l a r  for  the  operation  NM  routine.  during  a  As  each  staged  fouled  e q u i l i b r a t i n g b u f f e r to remove the egg If  three  shutdown three  columns are is  would  forced be  being  due  cleaned  to  used the  out  in  (NM  feed  and  two  columns  to t h a t d e s c r i b e d above column  shutdown,  of the problem  filters.  for one  shut  or  it  is  is  removed  from  backwashed  with  white from the r e s i n routine)  filters  sequence  and  an  immediate  plugging,  prior  to  bed.  then a l l  the  system  65  s h u t t i n g down. This gives added microorganisms  even  though  p r o t e c t i o n a g a i n s t the growth of  the lysozyme  i s an  anti-bacterial  agent.  Ending a r u n : When a run i s completed the  APC r o u t i n e  after  clearing  indicating  or has been terminated prematurely,  passes c o n t r o l  back  to the MAIN program  the s c r e e n and d i s p l a y i n g  b r i e f l y the cause  an end-of-run  of the t e r m i n a t i o n . At t h i s  segment message point,  the  next keystroke produces a small end-of-run menu which a l l o w s  the  operator to s t a r t a new run, t o save the c u r r e n t method t o a  disk f i l e ,  and/or to e x i t the program.  66  4.  DISCUSSION AND FUTURE CONSIDERATIONS  While no albumen the  various  has been run through the system thus f a r ,  operating  modes,  alarms  and  control  transfer  c o n d i t i o n s have been t e s t e d u s i n g dyed water and found to perform adequately. solution  As  to  in  the  any  project  stated  of  this  objectives  kind,  often  raises  Pandora's Box of program r e v i s i o n s and "new to add to the e x i s t i n g subroutines  that  were  architecture  was  code, i Such  programs  requirements, program  usefullness. times  while  laid  but  which  framework.  may  Parts  been of  others  the and  carefully program  not  been  routines  of more and more  wnen  the  immediate  simplicity  reduced have  to  been  so c o l s e l y  a  l i d on  program  leads to a patchwork q u i l t  grace  this  have  The a d d i t i o n  fulfill  the  the  and u s e f u l "  considered  out u s u a l l y  lack  has  not  approaching  of  processing of  the  a  mature  kernel  reworked  of  several  scrutinized.  When  working with a moderately long program such as t h i s , the constant threat of  to ever completing  ideas  the i n i t i a l  f o r improvements  version  i s the w e l l s p r i n g  to what has a l r e a d y been committed to  code.  meet  The  physical  and  exceed  plant  and  the b a s i c  control  design  software  designs  specifications  together  listed  i n the  i n t r o d u c t i o n of t h i s paper. Not only does the software promote an orderly allows to  shutdown  i n the  case  of  column  f o r the removal of the o f f e n d i n g  run what  is left  of the  fouling,  but  column while  system. This  i t also  continuing  approach attempts to  maximize production with a minimum of s u p e r v i s i o n .  67 The  delayed  operating  flexibility,  reasearch. the  run/start  The  MPC  operator  to  feature  which  routine modify  the  m a t e r i a l l y a f f e c t the course While included  a  in  refinements useful  host the to  in  of  of  i s e s p e c i a l l y d e s i r a b l e f o r use  in  values  smaller  the  great  flexibility  of  operating  there  considered.  tracking  great  a  most  are  One  behavior  such  new  change of o p e r a t i n g  system  parameter during  the  have  that  been  run  and  would  over  unattended p e r i o d i s a l o g g i n g r o u t i n e that notes and every  which  functions  addition  the  allowing  process.  enhancements  always  of  in  parameters  of events w i t h i n the  software, be  deal  gives  provides  a  be long  time stamps and  makes i s  easy to dump to the p r i n t e r as a run r e p o r t . While form, the  the  program provides  marketing  necessitate  a  display  essential relegated single  such  complete  enhancements which time  of  adequate c o n t r o l i n i t s c u r r e n t  a product  rewrite,  be  information,  redesigned with  keystroke.  The  of the i n t e r a c t i v e and  i n t o account  a l l of  to to  the  to a secondary screen  use  taking  have been add-ons  should  for commercial  this  a e s t h e t i c s and  only  important  which could  be  of  the  data  called  uniformity  data d i s p l a y screens  the  v e r s i o n . The  provide  less  would  up  runmost being  with  a  presentation  would a l s o need to be  improved. Other the  future  process  purification  considerations  itself step  might for  module to concentrate the  accompanying  i n terms of  include  the  proteins  the and  the  addition an  enhancement of  The  salt  solutions  secondary  ultrafiltration  the p r o t e i n f r a c t i o n s without  salts.  a  of  (UF)  concentrating  recovered  using  UF  68 could be r e c y c l e d as shown i n Figure 4 . 1 . Two required and  pH  to r e c i r c u l a t e  the  liquids,  meter shown would a l l o w the  proper  levels  of  more s o p h i s t i c a t e d concentration dispensing  salt  the c o n d u c t i v i t y  operator to ensure  c o n c e n t r a t i o n and  pH  were  pH for  by  incorporating  concentrated  meter  that  the  maintained.  sytem again would have automatic  and  pumps  and  more pumps would be  control  A  over  computer-controlled  eluant  and  acid  and  base  have  been  developed  solutions. Since  the  physical  plant  and  software  e x p r e s s l y to accomodate the recovery of a v i d i n and  lysozyme  using  the e l u t i o n - l o o p i n g technique, the general market appeal f o r such a  system  itself,  likely  to  be  however, can  be  used  procedure For  is  quite  limited.  with v i r t u a l l y  hardware and a moderate r e s t r u c t u r i n g be  performing  Immobilized  (IMAC) for the r e c o v e r y of The  use  processing, greatly  physical  plant  any  chromatographic  i f accompanied by the a p p r o p r i a t e l y m o d i f i e d software.  example, with a very minor m o d i f i c a t i o n  could  The  and  benefit  of  to the  of the software,the  Metal  Affinity  control  in  the  b i o - p r o c e s s i n g i n g e n e r a l , has the  producer  and  the  control.  Chromatography  field the  consumer  enhancement of p r o d u c t i o n process e f f i c i e n c y , and q u a l i t y  system  immunoglobulins.  automatic  both  flow c o n t r o l  cost  of  food  potential through  to the  effectiveness  "  S T R O N G  turn*  ©  KEY T O WASTE  © F R A C T I O N COLLECTOR COND. METER  0  P E R I S T A L T I C PUMP  ^  HOMOCENIZER  0  FILTER  <JP pH METER UV  A  NOTE:  2  8  0  PRESSURE  HONITOR  TRANSDUCER  ULTRAFILTRATION  UNIT  " * " i n d i c a t e s components n o t i n p r e s e n t Rvit-pm h u r h p i n t > r o n s i d e r a d f o r f u t u r e s t u d y  KATEB.  UF  A V I D I N  CONC.  FREEZE DRIER  DCIONIZED  WATER.  BARREN FEED  W A S T E  1m/EE ..r~|  • 1 _  MANUAL E  FIGURE  4.1:  General system layout including potential future additions for nearly total automation of control  l  I  TO DRAIN  70 REFERENCES  C l a r k , J.W.,  W.Viessman, J r . , and M.J. Hammer. 1977. Water Supply and P o l l u t i o n C o n t r o l P u b l i s h e r s . New  . 3 Ed. Harper &  Row,  York.  Cole-Parmer Catalogue. 1987-1988. Cole-Parmer Instrument Company. 7425 North Oak Park Avenue. Chicago, Illinois  Durance, T.D..  1987.  60648-3884 U.S.A..  I s o l a t i o n of A v i d i n and Lysozyme from Egg  Albumen. D o c t o r a l t h e s i s . U n i v e r s i t y of B r i t i s h Columbia.  Ion  Exchange  Chromatography:  priciples  and methods. 1980.  Pharmacia Fine Chemicals AB. Uppsala, Sweden.  Jolles  ,P., D. Charlemange, J. J o l l e s . Bull.  J.F. P e t i t ,  A.C. Maire and  1965. Biochimie comparee des lysozymes.  Soc. Chim. B i o l . 47:2241.  Li-Chan, E., S. Nakai, J . Sim, D.B.  Bragg, and K.V.  Lo..  1986.  Lysozyme S e p a r a t i o n from Egg White by C a t i o n Exchange Vol  Column Chromatography.  51 No.4,  S a l i s b u r y , F.B. and CW.  J.of Food  Science.  1032-1036.  Ross. 1978 . Plant P h y s i o l o g y , 2 ed.  Wadsworth P u b l i s h i n g Co., Inc.. Belmont,  CA.  71  Wilkinson, B.R. and R.E. D o r r l n g t o n . 1975. Lysozyme from Waste Egg White. Process 1975, 24-25.  (Muraraldase)  Biochemistry. March,  72  APPENDIX  OPBRATIOSAL STATES FOB BORMAL AHD DISABLED SYSTEM MODES SSQUKBCB Bo.  PUMPS  V1L¥B 1UMBEB  colaati  BOMOG-  DESCRIPTION  BHllEB  A B C  1  BOBHAL MODE  2  3  4  5 6 7  8 9 10 11 12 13 14 15 16 17 1 » 19 20 21 22 23  1 2 3  (3-coluan c y c l e : 2-coluan cascade)  a  I  b a c k - i i n s e C to barren  0 0 0 1  0 0 0 0 0 0 1  1  0  0 0  0 0  1  0  1  1 0  0  t>  I  1 2 R  back-rinse C to waste  0 0 0 1  0 0 0 1  0 0 0  1  0  0 0  0 0  1  0  1  1  0  0  0  1  c  I  apply weak s a l i n e to C  0 1  0 1  0  0 0 0  0 0 0 1  0 0 0  0 0  1  0  1  0 1  0  1  1 0  d  I  apply strong s a l i n e to C  1 1  0  0  0 0 0 0 0 0  0 0 0  0 0  1  1  «  I  equilibrate  0 0 0 1  0 0 0 1  0 0 0  1  0 0 0 0 0 0 0  M  C  C i s idle  1  1  1  1 0  1  0  0 0 0  0  0  0  0  0  0  0  0  1  1  0 0 0 0 0 0  1  0  1  0 0  0  1  1  0  0  c  I  apply veak s a l i n e to A  0  1  0 0 0 0 0 0 0  1  0 0  0 0  0  1 0  1  1 0  d  I  apply strong s a l i n e t o A l O O O l O O O O O O O l O O O O O l O l l l  e  I  equilibrate  1  I  * is idle  |  2  R 1  0 0 0 0 0 0 0  1  0 0 0  0  1  0  0 0 0 0 0  0 0 0 0 0 0 0  0 1  0 0  0 0 0 0 0  c  I  apply weak s a l i n e to B  0  0 1  0 0 0 0  d  I  apply strong s a l i n e to B 1  0 1  0 0 0 0 0 0 0 0 0  I  eqoilibrate B is idle  SBUTOOW MODE: S l ( a )  B  0 1  0  1  0 0 0 0 0  1  0  0  0  O i l  0 1 0  0  1  0  0  1  1  0  0  0  1  0  0  0 1  1  1  0  0  0  1  0  1  0  0  0  1  0 0  0  0 0  0 1 1  1  1 0 1 1 0  1  1  0 1  1  1  8 1  0  1  0  1 0  O i l  0  0  1  1  1  0  1  1  0  0  1  1  1  1  1  1 0  0  0  0 0 0 0 0 1  0 0 0 0 0  0  1  0 1  1  0  0  0  1  1  0  0 1  0  0 0 0 0 0 0 0 0 0 0 0  0  1  0 0  1  0  0  0  1  0  0  0  1 0  1  1  fl  0  0  0 0  1  0  0  0 0  0  1  0  0 0  1  0  0  1  0 0  0  0  0 1  1  0 0 0  1  0  0  1  0 0  0  0  0  1  1  0 0  (Cohan A i s down; 2 - c o l . intermittent cascade)  a  i  «  1  2  cascade B-C  0 0 0 0  b  I  *  B  1  backwash B to barren  0  0 0 0 0 0 0 0 0 1  c  I  *  B 1  backwash B to waste  0  0 0 0 0 0 1  0  d  I  *  8 1  apply weak s a l i n e to B  e  I  *  B 1  apply strong s a l i n e to B 1  f  I  *  B 1  equilibrate  g  I  *  1  backwash C to barren  8  0  0 1  0 0 0 0 0 0 1  I  0  1  back-rinse B to barren  1  1  0 0 0 0 0 0  back-rinse B to waste  e  0  0 1  1 1  0 0 0 0 1  0  0  0  1  0  1 0  0  0  1  O i l  0  0 0 0 0  0 0 0 1  1  1  back-rinse A to barren  0 0 0  0  0  0  back-rinse A to waste  0  0  1  0 1  I  A  0  0  0 0 0  I  I  2  1  0  a  *>  1  1  0 0 0 1  i>  a  K  1  O i l  B  1  0 0 0 0 0 0 0 0 0  0 0 0 0 0  0 1  0 0 0 0 0 0 0 0 0  0  0  0 0 0 0 0 0 0 0 0  0 0 0 0  0  0  0 0  0  0 0 0  1  0 0 1  0 0 0 0 0 0 0 0  0 0 0 0 1  0 0 0 0 0  1  0  0 0  h  I  *  1  R  backwash C to waste  0 0 0 0 1  0 0 1  i  I  *  1  8  apply weak s a l i n e to C  0  1  0 0  0 0 0 0 0 0  j  I  *  1  R  apply strong s a l i n e to C 1  1  0 0 1  0 0 0 0 0 0 0  0  k  I  *  1 R  equilibrate  1  0 0  0 0 0 0 0 0  0 0 0  C  0  1 1  0 0 0 0 0 0 0 0 0 0  0  0  1  1  1  1 0  1  1 0  0  0  1  1  1  1 1  1  1 0  0 0  0  1  1  1  1  0  0  1  1  1  0  0  1  0  0  0  1  1  0  0 1  1  0  0 0  1  0  0  1  0  0 0  0  0  1  0  0  0  1 0  0 0  0  0  1  0  0  0 1  0 0  1  0  0  0  1  1  1 0  1  1  1  0  0  0  1  1  OPBRATIOBAL STATES FOR BOBHAL ABD DISABLED SISTEH MODES ( c o n t ' d ) SEQUEHCE Bo. c o h a n A B C  VALVB NUMBER 1  SHUTDOWB MODE: S l ( b ) 1 R S R R R  PUMPS  DESCRIPTIOB 3  4  5  6  7 8  ( C o h a n B i s down; 2 - c o l . i n t e r m i t t e n t  cascade C-A 0 backwash C t o b a r r e n 0 backwash C t o waste 0 a p p l y weak s a l i n e t o C 0 apply strong s a l i n e to C 1 equilibrate C 0 backwash A t o b a r r e n 0 backwash A t o waste 0 a p p l y weak s a l i n e t o A a p p l y s t r o n g s a l i n e to A 1 equilibrate A 0  SHUTDOBB MODE: S l ( c )  2  0 0 0 0 0 0 0 0 1 1 1  0 0 0 0 0 0 0 0  0 1 1 1 1 1 0 0  1 0 0 0 0 0 1 1 0 0 1 0 0 1 0 0 1  0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0  ( C o h a n C i s down; 2 - c o l . i n t e r a i t t e n t  cascade A-B backwash A t o b a r r e n backwash A t o waste a p p l y weak s a l i n e to A  0 0 0 0 a p p l y s t r o n g s a l i n e to A 1 eqoilibrate A 0 backwash B t o b a r r e n backwash B t o waste 0 a p p l y weak s a l i n e t o B 0 a p p l y s t r o n g s a l i n e to B 1 equilibrate B 0  0 0 0 0 0 0 0 0 0 0 0  0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 1 1 0 0 1 1 0 0 1 1 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0  9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 cascade) 0 0 0 0 0 0 1 0 0 0 0  0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 1 1 0 0 0  0 0 0 0 0 0 0 0 0 0  0 1 1 0 0 0 0 0 0 0 0  0 0 0 0 0 0 1 1 0 0 0  1 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0  1 1 1 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 1 0 1 0 0 0 1 0 0 0 1 1 0 0 1 1 1 0 0 1 0  0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 1 1 1  cascade) 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0  0 0 0 0 0 0  1 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0  0 0 0 0  0 0 0 0 0 0 11 1 1 1 1 0 0 0 0 1 i 1 1 1 1  1  2  HOMOGENIZES 3  0PB8ATI0JAL STATES FOR HOBHAL ADD DISABLED SISTER MODES (cont'd) SEQUENCE Bo. cohin A B C  S8UTD0W MODE: S2(a-b)  1  I I | | | |  1  i 1 R 8 a  TOTAL SBOTDOW NODE: S3  I I I I  1 2 3 4 5 6 7 8 9 10 11 12 13 11 15 16 17 18 19 20 21 22 23  1 2 3  0 0 0 1 0  0 0 0 1 1 I  0 0 0 0 0 0  0 0 0 8 0 0  1 0 0 0 0 0  0 0 0 0 0 0  0 0 0 0 0 0  0 0 1 0 0 0  0 0 0 0 0 0  0 0 0 0 0 0  0 1 0 0 0 0  0 0 0 0 0 0  0 0 0 0 0 0  0 8 0 0 0 0  0 0 0 0 1 0 oooo 0 oooo 0 oooo 0 0 0 0 0 0 0 0 0 0  8 0 0 0 0 0  0 1 1 0 0 0  0 0 0 0 0 0  0 0 0 0 0 0  0 1 0 0 0 0  0 0 0 0 0 0  0 0 0 0 0 0  0 0 0 0 I! 0  0 0 0 0 0 0  0 0 0 0 0  0 1 1 0 0 0  0 0 10 10 0 1 0 1 0 1  0 0 0 0 1 0 0 1 0 0 0 0 1 0 0 0 oooo 0 oooo 0 oooo  0 0 0 0 0 0  0 0 0 1 1 0  1 0 0 0 0 0  0 1 1 0 0 1  0 0 0 0 0 0 1 0 1 1 1 0  0 0 0 1 1 0  1 0 0 0 0 0  G 1 1 0 0 1  0 0 0 1 1 0  1 0 0 0 0 0  0 1 1 0 0 1  0 0 1 0 0 1 0' 0 1 0  0 0 0 0 0 0 0 0 0 0  1 1 0 1 1 0 1 1 0 0  0 0 1 1 1  0 0 0. 0 1 0  0 0 0 0 0 0  0 0 0 0 0 0  1 0 0 0 0 0  0 0 0 8 0 0  0 0 1 0 0 0  0 1 0 0 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  (C ( A down; B operating alone)  feed B backwash B to barren backwash B to waste apply weak saline to B apply strong saline to B equilibrate B  59 9h 9i 3j  HOROGBB11B8  (B i C down; A operating alone)  feed A backwash A to barren backwash A to waste apply weak saline to A apply strong saline to A equilibrate A  SHUTD0S1 NODE: S2(c-a)  9a I 9b | 9c I 9d I 9e I 9f I  PUMPS  (A i B down; C operating alone)  feed C backwash C to barren backwash C to waste apply weak saline to C apply strong saline to C equilibrate C  SHUTDOfl MODE: S2(b-c) 8a 8b 8c Id 8e 8f  VALVB BUMBS8 DKSCEIPTIOH  0 0 0 0 1 0  0 0 0 0 0 0  0 0 0 1 1 1  0 0 0 0 0 0  0 0 0 0 0 0  0 0 0 0 0 0  0 0 1 0 0 0  0 0 0 0 0 0  0 0 0 0 0 0  0 1 0 0 0 0  0 0 0 0 0 0  0 0 0 0 0 0  0 0 0 0 0 0  0 0 0 0 0 1 0 0 0 0  0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0  0 1 0 0 0 0 0 0 0 0  0 0 0 0 1 0 0 0 0 0  0 0 0 0 0 0 0 1 0 0  1 0 0 0 0 0 0 0 0 0  0 0 0 1 0 0 0 0 0 0  0 0 0 0 0 0 1 0 0 0  0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0  oooo oooo 0 1 1 1  0 1 1 1  0 0 0 0 0 1 0 0 0 0  0 0 1 0 0 1 1 1  (all coluins down)  backwash A to barien backwash A to waste equilibrate A backwash B to barren backwash B to waste equilibrate B backwash C to barren backwash C to waste equilibrate C a l l power off  0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 1 0  0 0 0 0 0 0 0 0 0 0  1 1 0 0 0 0 0 0 0 0  0 0 0 1 1 0 0 0 0 0  0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0  0 0 1 0 0 1 0 0 1 0 0  0 0 0 0 0 0 0  tn  

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