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Flow conductivity of solutions of hyaluronic acid : effects of concentration and molecular weight Lam, Luk Sang 1988

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FLOW CONDUCTIVITY OF SOLUTIONS  OF HYALURONIC ACID: E F F E C T S  OF CONCENTRATION A N D M O L E C U L A R WEIGHT by L U K SANG L A M B.S., The University of Oregon, 1984  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF T H E REQUIREMENTS FOR T H E DEGREE OF MASTER OF APPLIED SCIENCE  in  T H E FACULTY OF GRADUATE STUDIES DEPARTMENT OF CHEMICAL ENGINEERING  We  accept to  THE  this  the  thesis  required  UNIVERSITY  as  conforming  standard  O F BRITISH  September  © L U K SANG  COLUMBIA  1988  L A M , 1988  In  presenting  degree at the  this  thesis  in  partial  University of  fulfilment  of  of  department  this thesis for or  by  his  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  her  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be  or  for  It  is  granted  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department  of  Ghemlcal Engineering  The University of British Columbia Vancouver, Canada  Date  DE-6  (2/88)  September 30, 1988  ABSTRACT  Hyaluronic fluid  a n d solutes  hyaluronic the of  acid  acid  hydraulic the  i n the  flow of  flow  process  acid  of  tissues  available  not continuous.  regulating  of this  weight  factors  of  hyaluronic  of of  influence  A n experimental  weights  acid  acid  three  were  using  not reproducible, p a r t l y  Nevertheless,  transport  study on  the  work.  molecular  hyaluronic  These  tissues.  weight  the  a n d molecular  are different.  a n d molecular  were  in  of connective  different  T h e results  role  T h e concentration  is the subject  commercially  was  K',  concentration  conductivity  chromatography.  important  connective  conductivity,  Hyaluronic fractionating  an  interstitium.  in different  effect  hydraulic  plays  obtained  ion-exchange because  molecular  by  column  of the  elution  fractions (6.99  weight  5 to  11.1X10 ) were  obtained.  5 Hyaluronic obtained 1  by  hour  acid  of  lower  acid h y d r o l y s i n g  and  2  hours.  molecular  some A  weights  (0.454  of the chromatographed more  homogeneous  to  1.65X10  fractions  )  were  for 15 m i n . ,  hyaluronic  acid  fraction  5 ( M . W . = 1.96 X 1 0 ) hydrolysed  The calculated measured the  was  obtained  by  fractionating  hyaluronic  acid  materials  acid  for 15 m i n .  hydraulic from  the  flow  conductivity  sedimentation  b y ultracentrifugation.  molecular  weight  fractions  of  solutions  coefficient  of  the  of  hyaluronic solutions  Centrifugation experiments of  hyaluronic  n  acid  at  acid  at  determining  various  can  20 ° C , the S  concentrations  be  ^20' of were  therefore  undertaken.  concentration hyaluronic three  acid  of  M.W.  fractions. with  non-acid  fractions the  higher  results,  and  M.W.  and  However,  lower  the  is  non-acid  high  acid  with  the  hydrolysed  two  due  to  fractions  the  and  low S^Q For  higher M . W . S^Q  past  taken  than  literature  experimental are  a  and  data.  higher  with  of  that  fractions,  sedimentation  agree  probably  high  hydrolysed  consistently  not  function  concentration for  past  has  a  increased  indicating  in SgQ between  does  most  as  same  the  difference  i n results is  of  with  concentration,  the  for  finding  decreased  0  at  M . W . fraction  This  fractionated  is  agreement  the  lowest  fractions.  the  in  high  formed  molecules  fractions,  difference  when  is  S  curves  at  concentrations,  the  that  the  converged  network  M . W . , which  small,  Also,  between  hydrolysed  is  c,  molecular  At  showed  acid.  concentration,  entanglement  increased  results  hyaluronic  dimensional  extent  the  of  The  errors.  as  a  high  5 M.W.  group  ( M . W . = 6.99  to  11.1X10  )  and  the  acid  hydrolysed  the  curves  fractions  as  a  function  of  5 low  M . W . group  c of  the  low  agreement  fractions  all  HA M.W.  past  to  fall  1 . 9 6 X 1 0 ),  below  sedimentation  hydraulic conductivities  varied  compared for  M . W . group  with  The  ( M . W . = 0.454  well  the  inversely with  fractions  molecules  of  the  the  with results  converged high  those  of  the  calculated  concentration.  at  2  as  Q  a  M . W . group,  which  is  in  data.  (K'),  of  high  of S  Ethier high  M . W . group  f r o m S^Q  The  (1986).  log-log The  K'  data, plots  At  low  has  K'  than  group.  in  a  higher  of  versus  concentrations.  for  all K'  c  the  versus  c  relationships  concentrations, those  HA  of  the  the low  T A B L E OF CONTENTS ABSTRACT  ii  TABLE  iv  OF CONTENTS  LIST  OF TABLES  LIST  O F FIGURES  vi vii  ACKNOWLEDGEMENTS Chapter  1.  ix  INTRODUCTION 1.1.  1.2.  1.3.  1  Hyaluronic Acid 1 1.1.1. Conformation 3 1.1.2. Viscosity 6 1.1.3. Colloid Osmotic Pressure 9 1.1.4. M a s s T r a n s p o r t through H y a l u r o n i c A c i d Solutions . . . 11 T h e Components of the Interstitium 13 1.2.1. Proteoglycan 13 1.2.2. Collagen 17 1.2.3. T h e H y d r a u l i c Resistance of F l o w in the Interstitium 18 Objective of the Thesis 19  Chapter  2.  THEORETICAL BACKGROUND 2.1. Introduction 2.2. E n z y m e D e g r a d a t i o n Method 2.3. F i l t r a t i o n - C h a m b e r Method 2.4. Sedimentation M e t h o d 2.5. Sedimentation T h e o r y 2.5.1. T h e A n a l y t i c a l Ultracentrifuge 2.5.2. T r a n s p o r t in the U l t r a c e n t r i f u g a l Cell 2.5.3. M e a s u r i n g the Sedimentation Coefficient 2.6. Ion-Exchange C h r o m a t o g r a p h y T h e o r y  21 21 22 24 26 32 32 35 42 46  Chapter  3.  EXPERIMENTAL PROCEDURE 3.1. Introduction 3.2. H y a l u r o n i c A c i d Concentration  52 52 52  3.3.  3.4.  Determination  Fractionation of H y a l u r o n i c A c i d 3.3.1. D e s a l t i n g H y a l u r o n i c A c i d 3.3.2. P r e p a r a t i o n of D E A E - c e l l u l o s e Precycling Fines R e m o v a l Equilibration 3.3.3. P r e l i m i n a r y Fractionation 3.3.4. F r a c t i o n a t i o n 3.3.5. Purification and Isolation of H A A c i d H y d r o l y s i s of H A iv  54 54 55 55 56 57 58 65 66 67  3.5.  Fractionation  3.6.  3.5.1. 3.5.2. Dialysis 3.6.1. 3.6.2.  3.7. 3.8.  of A c i d  Hydrolysed  HA  Acid Hydrolysis Fractionation Preparation Dialysis  of D i a l y s i s  Tubings  Viscosity Sedimentation Velocity 3.8.1. Preparation of S a m p l e Cell 3.8.2. M a k i n g a Sedimentation Velocity R u n 3.8.3. T a k i n g Photographs 3.8.4. E n d i n g a Sedimentation Velocity R u n  69 69 69 70 71 72 74 77 78 83 87 88  Chapter  4.  RESULTS A N D DISCUSSION 4.1. Hyaluronic A c i d Concentration Determination 4.2. Fractionation of H y a l u r o n i c , acid 4.3. A c i d Hydrolysis of H y a l u r o n i c A c i d 4.4. Fractionation of A c i d H y d r o l y s e d H A 4.5. Sedimentation Coefficient 4.6. H y d r a u l i c Conductivity  90 90 92 98 98 102 117  Chapter  5.  CONCLUSIONS  123  AND RECOMMENDATIONS  REFERENCES  127  APPENDICES A. B.  Sample Calculations Experimental D a t a  v  134 134 146  List of Tables 2.1  Buffer  used  by  different  3.1  Results  of p r e l i m i n a r y fractionation  I  59  3.2  Results  of p r e l i m i n a r y fractionation  II  61  3.3  Results  of p r e l i m i n a r y fractionation  III  62  4.1  Results  of the  uronic  4.2  Results  of the  large  4.3  Intrinsic  viscosity  workers  25  acid carbazole scale  and  test  90  fractionation experiment  93  molecular weight of  15minAH,  lhrAH  and  2hrAH  Fractions  98  4.4  Results  of the  4.5  Sedimentation  4.6  Values  A.l  Viscosity  A.2  Results  A.3  c  of A  fractionation coefficient  and B  data for  a n d K'  for  for  a n d hydraulic conductivity  in E q n [4.4]  0.325M-9  sedimentation  data  of acid hydrolysed H A  for  Fraction  Run C l  0.325M-9  100 for  all H A Fractions  all H A Fractions  .. 103 117 136 139  Fraction  VI  142  List 1.1  1.2  C h e m i c a l structures of the glycosaminoglycans Schematic  demonstration  of  dominant  Figures disaccharide  units  of  various 2  of the  volume  occupied by  a  hyaluronic acid  molecule  4  1.3  D i a g r a m a t i c representation  1.4  Osmotic  1.5  Illustration  1.6  Structure of cartilage  1.7  Schematic  2.1  A  2.2  Sector-shaped  2.3  (a)  pressure  s y n o v i a l joint  of hyaluronic  of the  steric  acid versus  exclusion  structure  s y s t e m of macromolecules cell used  9  concentration  10  mechanism  14  manomer  15  proteoglycan  model of the  of cartilage  sedimenting  in  in all a n a l y t i c a l  proteoglycan  aggregates  an ultracentrifuge  ultracentrifuge  16 27 30  The concentration profile of a sedimentation r u n . (b) A Schlieren trace of the cell along the r a d i a l direction. T h e Schlieren peak represents  the  2.4  Schematic  2.5  The  2.6  G e o m e t r y of a  2.7  Solute  boundary position  d i a g r a m of a n  sector-shaped  concentration of a  profile  Demonstration of radial  2.9  Concentration profile Structures  of the  cell  35  up for the  3.2  Intrinsic  viscosity  3.3  T h e set  up for  3.4  T h e Ubbelohde  type  3.5  Position  torque  ultracentrifugation of  macromolecule,  a  typical  D= 0  38  dilution  DEAE  T h e set  of the  33  during  of the  3.1  32  cell  homogeneous  2.8  31  a n a l y t i c a l ultracentrifuge  analytical ultracentrifuge  solution  2.10  of a  39  sedimentation  and  C M cellulosic  equilibration of  plot of the  of a  the  0 . 3 M and  real  ion-exchangers  ion-exchanger 0 . 4 M Fractions  performing dialysis semi-micro wrench  43 48 57 64 73  dilution viscometer and  solution  vii the  cell  housing  75 80  3.6  Installing  the  rotor  82  4.1  Relationship between  the  absorbance  a n d the  concentration  of  glucuronolactone. 91  4.2  Intrinsic  viscosity  plot for the  H A Fractions  96  4.3  Intrinsic  viscosity  plot for  acid hydrolysed Fractions  99  4.4  Intrinsic viscosity plot of the unfractionated H A  4.5  4.6  the  1 5 m A H - 0 . 3 2 5 M Fraction  and 101  Sedimentation coefficient as a function of concentration 0 . 3 2 5 M and 0.325M-9 Fractions  for  Sedimentation  for  lhrAH,  coefficient  2hrAH  and  4.7  Sedimentation  coefficient  4.8  Sedimentation  data  4.9  Sedimentation  patterns  as  a  function of concentration  0.25M, 105 15minAH,  1 5 m A H - 0 . 3 2 5 M Fractions as  a  106  function of concentration  of L a u r e n t of the  et  al.  0.25M,  for all Fractions.  . 107  •.:  108  (1960). 0.325M  and 0 . 3 2 5 M - 9  4.10  Sedimentation  patterns  of the  acid hydrolysed H A Fractions  4.11  Sedimentation  patterns  of the  15mAH-0.25M  4.12  Sedimentation  pattern  and concentration  and  Fractions  110 Ill  1 5 m A H - 0 . 3 2 5 M Fractions. 112  profile of acid hydrolysed H A  Fractions  115  4.13  Hydraulic  conductivity  data  for  0.25M,  4.14  Hydraulic  conductivity  data  for  all acid hydrolysed Fractions  119  4.15  Hydraulic  conductivity  data  for  all H A Fractions  120  A.l  Sedimentation  A.2  The  photograph of R u n C l  sedimentation  plot  for R u n C l  0.325M  and 0.325M-9  Fractions.  118  138 140  viii  ACKNOWLEDGEMENTS  I  wish  understanding  and or  are  to  use  personnel the  other  of  Natural  also  my  the the  to  Model  E  Dr.  to  the  Bob  of  Joel  Bert,  in  the  Engineering  Cushley  of the  form of  Research  acknowledged.  IX  for  his  patience,  project.  of  S.F.U.  who  and to  Chemical Engineering  completion  assistance  and  the  Dr.  analytical ultracentrifuge,  Department  financial  Sciences  supervisor,  throughout  due  contributed  Finally, the  thank  a n d supervision  Thanks permission  to  who  has  the have  given  faculty, in  one  the staff way  program.  a  Research  Council  of  Assistanceship Canada  is  from  gratefully  C H A P T E R 1. INTRODUCTION  1.1.  HYALURONIC  ACID  Hyaluronic polysaccharide structure  ionized.  as  to  form  the  tissue  (Laurent, mg  contains  1-3  mg/mL  hyaluronic then  polymerized  membrane surgical High  is  into  aid  in  molecular  which  acid/g  (Sunblad,  the  the  fresh  the  membrane  ophthalmic  sterile  wt  chain  carboxylate  heparin acid  (amino  (see  vary  1980).  Recent  research  has  (Prehm,  1983,  chain  Interestingly,  (Miller  pyrogen-free  1  et  al.,  moves  a  while very  1977;  solutions  of  Pape  dermatan  sodium  1.1).  The  rooster  comb  synovial  fluid  content  revealed 1984). a  acid &  The  tissue  low  through  hyaluronic  known  from  F o r example,  has  the  fully  sugars).  Figure  plasma  membrane  /3(l->4)  disaccharide  widely  1970),  are  is  6-sulphate,  Human  cell  by  per  and  units  polysaccharides  chondroitin  (Laurant,  chemical  group  charge  hexosamines  1980).  weight  Its  disaccharide  similar  hyaluronic  and  space.  surgery  of  tissue.  polymer  negative  and  Tengblad,  Tengblad,  extracellular  weight,  sulphate  molecular  N-acetyl-D-glucosamine These  the  4-sulphate,  1965).  in  group contain  of  of  bond.  one  high  connective  units  is  a  all  length)  synthesized at  of  Laurent &  &  all  a  unbranched  distribution  heparan  hyaluronic  not  is  conditions,  member  (chain  (Laurent  acid  linear,  chondroitin  1970;  7.5  Mg/mL  are  if  disaccharide  long  (GAGs)  size  contains  = 0.3  a  sulphate,  and  most  physiological  is  polysaccharides  hyaluronate)  /3(l->3) glycosidic  charge  H y a l u r o n i c acid  keratan  in  a a  Under  Therefore,  concentration to  linked b y  bonds.  sulphate,  found  repeating  glycosaminoglycans  other  (synonym:  is  of  acid  together  glycosidic  unit.  which  consists  D-glucuronic linked  acid  is  hole a  of  that It  in  is the  valuable  Balazs, hyaluronic  1980). acid  Figure 1.1 C h e m i c a l structures of the dominant disaccharide units glycosaminoglycans. (Modified from Comper & L a u r e n t , 1978)  of  various  INTRODUCTION have a  been  used  therapeutic  and  to  a  effect  physicochemical referred  as  in  joint  reviews  for more  (Denlinger  diseases  of hyaluronic  recent  1978)  substitute  certain  properties  some  Laurent,  vitreous  Balazs,  (Asheim  acid are  (Laurent,  &  &  1980).  It  Lindblad,  summarized &  below.  1987;  Bert  dilute  hyaluronic  /  also  has  1976).  The  T h e reader  Pearce,  1984;  3  is  Comper  details.  1.1.1. Conformation  Detailed shown  that  the  1955;  Laurent  some  degree  groups atom  of of  1979).  al., in  of  the  in  of  Cleland  take  acid  counterion,  pH of  found  into and  the  of  the  is  (1984)  has of  review,  see  and of  a solid  Hadler  hydration.  In  threefold,  a  i.e.  stable three  &  that  has  the  to  hydroxyl  residue  hyaluronic  Gergely,  the  extra  have  However,  the  configuration  oxygen (Cleland,  degree  of  (Morris  et  acid  molecules  worm-like  chain.  studied  extensively  been  Napier,  influenced all  of  hexosamine an  &  1977).  H-bond  random-coil  state  is  is  One  hydrodynamic  fibres  acid  exist. a  solutions  (Laurent  provides  revealed  acid  Cleland,  in  H-bonding  stiffened  form  could  following  — the  coil  1970;  involved  next  the  films  degree  be  residue  random  Wang,  ordering  acid in  (for  hyaluronic to  &  inter-residue  of hyaluronic  hyaluronic  is  group  on  extended  Cleland  acid  conformation  crystallography  configuration  1960;  an  conformation  N-acetyl  can  studies  forms  glucuronic  Recently,  conformation  turn.  local  the  solution  helix  al.,  presence  1980).  X-ray  molecule et  the  The  stiffness  physicochemical  1977).  by  oriented  The  packing  the  nature  films  and  left-handed  helix.  Most  disaccharide  units  per  The  of  by of the  fibres,  the  commonly,  the  complete  helical  INTRODUCTION The  molecule  also occupies  a  very  large  h y d r o d y n a m i c volume  (Figure  /  4  1.2).  5 In  the  case  as  throughout  weight) space In  the  this  such  a  is  and  charges  on  On  the  work  by  the  unless  to  the  charged the  it  carbonyl  hand,  of  occupies  solvent  the  groups  is  the  of  weight  1,000  water  times  (Ogston  is  the  and by  water  repel by  one  ,  where M . W . ,  average  molecular  larger &  than  Stanier,  principal  polar bonds  polysaccharide  polysaccharide  retention  the  chain  H-bonds  on  (M.W. = 8 X 1 0  is  polysaccharide  domain,  the  fluid  stated,  hyaluronic acid by  backbone  other  volume  acid  synovial  otherwise  unhydrated  hyaluronic  held  groups  hyaluronic acid of  hydrodynamic  occupied  Water  coil.  of the  chains.  another  hyaluronic  to  acid  the  1951).  component. to  the  The  anionic  expand is  OH  found  the to  F i g u r e 1.2 Schematic demonstration of the volume occupied by a hyaluronic acid molecule. F o r comparison, some other macromolecules are shown. (Modified f r o m Comper & Laurent, 1978)  INTRODUCTION have  increased  (Fessler,  the  1957,  compression  1960a).  and  to  the  Hyaluronic conformation  and  solution  is  charges  on  charges  on  another.  The  reversible. changes  the  intrinsic  has  some  Wang,  biological  Changes  in  converted  to  the  have  a  Changes acid  in  solution  gyration 1977).  are This  alkaline  pH  decrease  in  pH  alter  is  treated  (less  energy  the  the  lowered, finding  but  a  from  the  form) result  a  It  of  the  Also,  one  of  alkali,  or  can  the  molecular  Comper  expect of  weight  than  in  neutral  ionization  acid pH of  negative  that  fully  hyaluronic  Laurent,  the  1978). can  polyion in  acid  When  1970)  (Mathews  has  a  be — a  pH  hyaluronic  &  radius of Decker,  higher  flexibility  extended  form).  hydroxyl  groups  will  solutions.  and  not  the  phenomenon  changes  residue.  is  one  observing  hyaluronic  (more  repel  molecules of  (Swann, is  the  the  &  its  negative  sodium  acid  contraction  viscosity  hyaluronic  the  that  glucuronate  that  of  believed  of  the  process  by  its  hyaluronic  to  The  of  properties the  tendency  solutions  1954;  to  that  the  screen  coil.  hyaluronic  expansion  of  monitored  is  in  Therefore,  greater  example,  1984).  to  other.  be  gel  both  concentration  strength  random  can  rheological  an  each  have  for  ionic  collagen  resistance  ionic  available  (Katchalsky,  on  demonstrates  is  are  a  5  hyaluronic acid solution.  the  the  in  polyelectrolyte  in  extended  of,  ionization  with  extended  viscosity  an  environment  on  As  chain  Cleland,  transduction.  effect  both  acid  provides  typical  conformation  implications  water  a  backbone  forms  1970;  mechanical  large  as  hyaluronic  through the  counterions  viscosity  ionic  chemical-mechanical  of water  counterion.  acid  in  of  of  changes  less  then  changes  (Cleland &  of  hyaluronic  polyion  acid  the  type  The  in  retention  polysaccharide  the  system  behaves  lowered,  the  a  bulk flow  with  the  of  The  acid  changes  environment acid  stability  /  in The  involved  in  INTRODUCTION H-bonding viscosity from  (Morris is  the  et  al.,  1980).  reduced, which  suppression  is  At  low  attributed  to  concentrations the  molecular  of intramolecular electrostatic  and  acidic  pH,  coil contraction  repulsion  (Morris  et  /  6 the  resulting  al.,  1980).  1.1.2. Viscosity  Hyaluronic can As  be  explained  mentioned  times  larger  order  of  1978).  1  At  form.  In  saline  hydrodynamic  the  mg/mL,  formed,  the  dependent An very  studies  on  hyaluronic  ~  the  at  a  of  and  1978).  viscosity  explanation large  acid  At  increases the  acid  of  volumes  solutions  concentrations,  about  different  the  same  is  have  at  at  with  on  (Comper  the with  shown  (above  those  at  is  rate  acid  each  (Laurent  to  isotonic The  in  solutions  acid  solutions a  gel  is  concentration & Stanier,  acid  molecules  other.  Sedimentation  low  concentrations,  at  sediment  mg/mL),  the  Laurent,  1984).  (Ogston  hyaluronic  in  in  which  strongly  1,000  expected  molecules  weights 2  is  hyaluronic  that  be  &  Pearce,  concentration  that  can  This  domain.  concentration  &  acid  acid  molecular  slow  acid  hyaluronic  below  entangle  of  sediment  studies  solution  network  (Bert  hyaluronic  observation  At  polymer  mg/mL  sharply  large  entangle  hyaluronic  molecules higher  to  concentrations  viscosity  for  of  a  concentrations.  hyaluronic  umbilical cord 2-5  low  has  of  continuous  of  at  molecule.  start  sedimentation  hydrodynamic  hyaluronic  will  entanglement  viscosity  acid  unhydrated  a  even  volume  undegraded  non-Newtonian the  of  concentration the  viscous  hyaluronic  molecules  solution  Laurent,  However,  molecules  volume  concentration,  a  the  &  rates.  the  supporting  (Comper  with  above,  gel  from  1953).  fact  than  that  very  the  fact,  evidence  are  by  higher  can  come  acid solutions  the et  at  different  hyaluronic  al.,  1960).  acid These  INTRODUCTION findings  indicate  continuous  The  cell,  acid is  In  al.,  frequency, to  fluid  acid  (Balazs,  molecular  may  of  properties  viscometric  study  heart  molecular tissue  stretching  of  high  weight  with  fact,  is  is  forces  Wang,  to  are  and  the  that  synovial  fluid  (Schurz  in  in  the  &  the  the  al.,  the  is  the  viscosities  the  polymer al.,  entanglement.  At  low  elasticity  directly  (Myers  is  hyaluronic rheological  are  1987). has  when  tissue  acid  and and  Moreover,  shown  in  properties  functioning  decreased,  fluid  1986).  mL/g  are  pericardial the  molecular  definition,  concentration  acid  on  0.0228M^'^''"^  mechanical  Ribitch,  hyaluronic  compliance  (Honda et  only  et  these  the  acid  the  By  c  of  diseases,  weight  a  [77] in  =  viscoelastic  concentration  the  770  molecular and  believed  [77]  1970).  in  in  form  through  their  viscosity  77 and  solutions  not  also on  equation:  role  molecular  and  &  but  respectively,  joint  plug  dependent  intrinsic  where  the  deteriorated  resiliency  the  behavior,  it  porous  important  acid  hyaluronic  or compressive  acid  attributed  mg/mL),  In  are  is  Since  an  • c,  0  acid  elastic  hyaluronic  rheological  high  which  have  1974).  weight  NaCl,  solvent,  hyaluronic  (1-3  a  molecules  7  molecules.  (Cleland  the  shows  high  as  Mark-Houwink  weight  concentration.  is  hyaluronic  the  and  1980) it  0.2M  0  addition,  sediment  hyaluronic  jl^Q(T}~v )/v  solution  W e l s h et  in  to  [77] =  polymer  synovial  acid  fitted  of  hyaluronic acid  hyaluronic acid molecules,  molecular  viscosity  proportional  joints  is  the  concentration.  of  individual  as  the  M  strain  than  of  intrinsic  1966;  and  environment  hyaluronic  the  network  solutions  For  the  rather  concentration,  of  hyaluronic  of  high  viscosity  weights.  where  at  polysaccharide  ultracentrifuge  solution  that  /  of the the a  that  the  could  provide  the  is  subjected  to  INTRODUCTION The  surface  hyaluronic  acid is  a  coil  random  1980).  The  different  &  important  in  the  from  synovial  postulated function of  13  in  Warburton's  in  cavity,  (Levick,  1983).  which  (1985)  surface  is  of  a  also  viscosity  by  high  acid  [77] form  [77]  form  existing  the  high  [77] form  in is  in  that  has  an  Cleland  with  very  alkaline  endothermic.  Warburton,  in  bulk  Therefore,  very bulk  of  is  thin  they  are  fluid  Levick  reports acid  the  (Kerr  acid  1.3)  to  exclude  viscosity in  of  found  temperature.  that  and  in  The  shift  when  be  an  neutral  the  from  to  concentration of K e r r  obvious  &  from fluid  proteins.  acid  solution,  However,  The  in  been  hyaluronic  non-alkaline  temperature.  to  the  is are  has  results (as  of  proteins  synovial  hyaluronic  very  viscosity  certain  as  film  hyaluronic  arthritis, the  phase  articular cartilage/synovial  considered  solution.  the  joints,  that  increasing  a  concentrations  the  shows  acidic  of  However,  the  on  the  than  a  Figure  increasing  in  &  proteins.  have  at  effect  be  (Kerr  in  enough  present  can  both  these  high  with  surface  rheumatoid  the joint.  (see  solution  normal  to  (1979)  solution  existing  in  exclude  interfaces  [77] increases  In  that  in  property  H y a l u r o n i c acid  present  [77] decreases  hyaluronic  but  could be  fluid  higher  revealed  exists  viscosity  rheological  necessary  show  acid  has  8  that  or  The  much  joints.  to  is  not  viscosity)  study  solution,  it  study  Temperature  the  barrier  manner,  synovium/synovial  that  surface of  interface  phase.  very  synovial  and  alkaline  hyaluronic  the  high  intrinsic  of  is  acid solutions  molecule  the  functioning  this  The  This  hyaluronic  polyanionic  surface  surface  a  of  at  the  the  solutions.  the  1985).  form  mg/raL,  property  the  fluid  to  active  concentrates  that  acid at  study  surface  rheological  Warburton,  excluded  a  and  from  hyaluronic  rheological  /  author  in  the very  interprets  equilibrium mixture solutions  the  low  temperature  and  a  [77] form is  low to  increased,  INTRODUCTION  /  9  solution  is  bone!  synovial  fluid j  . synoviunr articular  cartilage bone  Figure  the  1.3  low  D i a g r a m a t i c representation  [77] form is  shifted  to  the  of a  high  synovial joint.  [77] form,  and  the  [77] of  the  increased.  C o l l o i d Osmotic P r e s s u r e  1.1.3.  The the  osmotic  As  shown  considerably weight of  a  osmotic  behavior  pressure in  Figure  higher  50-100  non-ideal  rises 1.4,  than  times  it/RT  is  solutions  rapidly the  that  lower  polymer  of  of  than  c/M^  +  albumin  of  the  Ac  2  2  virial +  is  of  very the  non-ideal —  polysaccharide.  hyaluronic  acid  solutions  ( M . W . 67,500)  with  a  of hyaluronic  by  acid  concentration  pressure  serum that  hyaluronic  the  osmotic  described =  with  of  acid.  The  osmotic  is  molecular  pressure 7T  expansion  A c 3  3  +  [Ll]  INTRODUCTION  Figure 1.4 Osmotic pressure of hyaluronic acid versus comparison, the osmotic pressure curve for a l b u m i n is Comper & L a u r e n t , 1978)  where  R  is  the  concentration and  A , 2  A  physiological the done  of  universal the  conditions Ogston, non-ideal,  the  determine  is the  (e.g.  Cleland,  1963).  Since  some  workers  constant, in  T  g/mL,  virial  concentration,  dominating t e r m to  polymer  are  3  gas  its  coefficients.  the  term  c / M  A c .  Over  the  2  2  second 1984; the  virial Wik,  osmotic  believe  is  this  the  absolute  number  is  a  relatively  years,  a  large  1979;  behavior special  of  Cleland of  temperature,  average  Since  coefficient  10  concentration. F o r shown. (Modified from  has  n  /  generally  small  value.  acid  of  1970; acid  the  weight, at  Therefore  hyaluronic Wang,  is  large,  of work  hyaluronic  property  molecular  amount  &  c  has  in  been  various  Laurent is  hyaluronic  &  extremely acid,  along  INTRODUCTION with  other  GAGs,  Pappenheimer, a  fluid  then On in  loss  1963, from  developed the  to  GAG-containing  the  hand,  osmotic  contribute  the  Comper & Laurent, the  in  other  the  can  tissue,  a  due  to  1978).  tissue,  the  dilution in the  homeostasis In some a  large  non-ideal  tissue  in  vivo  conditions  osmotic  behavior  will cause  a  of  /  11  (Landis  &  when  there  is  restoring  force  is  the  polysaccharide.  relatively  small  decrease  pressure.  1.1.4. Mass Transport through Hyaluronic Acid Solutions  Since  hyaluronic  connective  tissues,  regulating  transport  transport  of  transport,  fascia  before  added.  Using  similar  finding  in  tissue  study at  resists  The  for  it.  upward  movement  same  the  al.,  Therefore,  concentration.  of  of water  the  which a  has of  is  a  are  slower  to  discussed  10  to  20  experiments it.  determine acid  below.  the an  In  show Also,  the  rate  of  hyaluronic  acid  that  of other  in  of  mouse degrades  enzyme  is  obtains  a  hyaluronic  hyaluronic  of  the  case  of  (1963)  from a  resist  on  the  the  that  and  role  which  after  network  than  its  matrix  enzyme  Hedbys  earlier,  matrix  permeability  times  method,  discussed  intercellular  hyaluronic  and immobilizes  sedimentation  solvent)  of  hyaluronidase,  These  polymer  the  performed  degradation  is  of  measured  increases  stroma.  form  effects  1952)  addition  1960),  been  particles  enzymatic  flow  constituent  have  The  permeability  concentration  through  major  studies  (1950,  corneal  the  a  globular  same  (Laurent et  high  and  after  the  of  is  tissue.  Day  and  acid.  lot in  water  water  hyaluronic  a  acid  acid  sedimentation acid  flow (or  molecules of  the  macromolecules  solvent rate of  of the  INTRODUCTION The  effect  estimated  by  of  hyaluronic  the  acid  co-workers  (Laurent  colloidal presence  of  particles.  In  in  the  decreases  hyaluronic addition,  data  are  1963; a  series  the  of  rate  of  (1955)  rate  of  and  solutions. the  to  follow  the  albumin.  They  of  particles  empirical  in the  and  Laurent  sizes  of  &  proteins  of  decreases  be  Laurent  found  rate  can  that  of  have  12  shown  1964;  studies  sedimentation  transport  particles  has  Persson,  of hyaluronic acid and increases  shown  globular  sedimentation  acid  decreases  of  Johnston  Laurent  hyaluronic acid  transport  sedimentation  done  through  the  method.  the  al.,  have  concentration  Their  et  1961)  particles  on  sedimentation  hyaluronic  Pietruskiewicz,  acid  /  and  that the  the  solute  with  increases  the  particles.  relationship:  i s/s where  s  and s  hyaluronic is  a  B  is  et  hyaluronic filtration The  al. acid  of  by  as  a  in  retarding  effect  et  al.  rate of  a  the  hyaluronic  The  authors  as  sieving  effect  increase acid  on  the  radius  the  is  in  of  of  The  sieving  formed  by  describe  the  hyaluronic  particles  can  respectively.  A  solute,  solute  particle.  is  from acid also  like  a  acid.  acid  been  of  has  behavior  a  slightly  partially the  as  decrease  concentration. be  in  hyaluronic  sedimentation  acting  and  movement  sedimentation  evident  in  process  hyaluronic  network  solute  the  particle  sodium  independent  three-dimensional  solution, on  meshwork in  macromolecular  depending  down  mechanism.  lysozyme  acid  The  with  slowing  sieving  of  2,  hydrodynamic  the  (1981).  through  sieve.  the  of the  or buffer  less t h a n  macromolecular  hyaluronic  protein  sedimentation  to  molecular the  [1.2]  Bc  coefficients  usually  describe  by  Ae'  c and in water  and  behavior  Wedlock  macromolecular  1  (1963)  particles  lysozyme  associated  sedimentation  proportional  sedimentation  studied of  exceeding  constant  Laurent  the  acid of concentration  constant a  are  0  =  0  explained  by  a of The the  INTRODUCTION steric the  exclusion  fact  time.  that  Figure  Figure  or  more  1.5  is  an  the  of  fibres  have  a  (Comper  two  1.5,  network may  mechanism  role  particles  space  greatly  in  of  for  Steric  the  can  particle  then  distribution  of  exclusion  same  phenomenon.  spherical  One  the  1978).  occupy  the  a  reduced.  regulating  Laurent,  cannot  illustration  available is  &  space  As to  plasma  13  refers  to  at  the  depicted  in  move  postulate  /  in  that  a  same C  of  random  hyaluronic acid  proteins  in  the  tissue  space.  1.2.  THE  COMPONENTS  In  order  connective  tissue  the  major  The  three  collagen. see  Laurent in  of  in  interstitium  the  and  previous  components of  Bert  water  vascular  and are  structure  &  Pearce  In  collagen  fibres  are  components  to  the  this  in the  interstitium,  and lymphatic  the  section.  and  major  flow  the  reviews  (1987)  the  INTERSTITIUM  outside  structural  recent  the  proteoglycans three  space  major  THE  understand  components  For  discussed  the  to  OF  their  properties  hyaluronic and  mentioned  h y d r a u l i c flow  of  structure  briefly.  Then  resistance  is  the  the  cells,  studied  first.  proteoglycan the  Hyaluronic  the  and  are  acid,  transport  (1984).  section,  systems  which  interstitium,  acid and  the  in the  and  has  been  properties  of  contribution  of  interstitium  is  discussed.  1.2.1.  Proteoglycan  Like body.  The  hyaluronic size  and  acid, proteoglycans  composition  of  the  are  found  proteoglycans  throughout vary  the  widely  tissues from  of  tissue  the to  INTRODUCTION  /  14  Figure 1.5 Illustration of the steric exclusion m e c h a n i s m . A: steric exclusion of sphere b from the domain of sphere a. Centre of b cannot come closer to centre of a than 2r and therefore is excluded from a volume (dotted) that is 8 times larger t h a n the volume of each sphere. B: steric exclusion of a sphere with radius r from the d o m a i n of a rod with the radius r a n d length /. E x c l u d e d volume is equal to a cylinder with the radius r + r a n d the length I. E n d effects have been disregarded. C: available space for a sphere in a random network of rods is equal to the volume (dotted) in w h i c h the centre of the sphere can move freely. (Modified from C o m p e r & L a u r e n t , 1978) r  r  s  r  INTRODUCTION tissue. their the  Usually, component  diversity  reviews 1986;  on  nasal  of  the  are  cartilage  a bottle  number  of  are  distinguished  glycosaminoglycans, the  group  chemical  Heinegard  properties  of  they  &  (Figure  chondroitin  Light  have  1.6).  and  1984).  below.  proteoglycan  and  the the  length  Only  chains  some  scattering  shown  that  has  type the  of  the  of  their  are  is  of  Due  to  to  macromolecules typical  takes  protein core attached.  the to  some (Poole,  structure  studies  on  and  bovine  conformation  which a  Many  15  length  core.  referred  viscosity  molecule a  and chain protein  reader  and  the  (GAGs)  of  the  structure  T h e molecule  sulfate  number,  proteoglycans,  properties  Paulsson,  discussed  brush  of  by  /  large  proteoglycans  keratin sulphate rich binding region  globular protein and protein core  Figure  1.6  Structure of cartilage  proteoglycan monomer.  INTRODUCTION form  large  Hascall  &  aggregate. the  aggregates Heinegard, There is  proteoglycan.  a  The  with  hyaluronic  1974a,  1974b).  binding site  for  aggregates  may  acid  (e.g.  Figure  Hardingham  1.7  is  hyaluronic acid contain  over  &  Muir,  an  illustration  on  the  100  /  of  globular  proteoglycan  16  1972;  such  an  protein  on  monomers,  5 each  with  hyaluronic the  M.W. acid  shortest  1979;  (1983)  has  Figure  1.7  .  Studies  oligosaccharides  hyaluronic  decasaccharide (Cleland,  2.5X10  HA, ,  acid  the  Nieduszynski et  shown  that  Schematic  the  model  with  al.,  the  which  subscript 1980).  minimum  of  the  1:1  proteoglycan  chain  where  0  of  A  chain  structure  complexes monomers  forms 10  a  refers  recent length  formed have  strong to  10  viscometric of  of cartilage  by  small  revealed  complex  is  that the  monosaccharides study  hyaluronic  proteoglycan  by  acid  Cleland for  the  aggregates.  INTRODUCTION , formation occupy of  of  a  large  bovine  nasal  Pearce,  1984).  hyaluronic the  large  cartilage  acid  of  a  by  &  1.2.2.  (1978)  is  structural  third unit  major  of  a  the  the  of  that  In  to  the  of  Proteoglycans  the  m L / g (Bert  less  The  core  proteoglycan  than  protein  the  native  molecules  exclusion  a  large  amount  structures  of  of  is  & of  that  limits  the  tissue,  the  passing  steric  structure  that  reason  by  branched  branched  .  0  140  1.2).  molecule.  or  of  much  the  localizing the  is  to  transport  2  example,  (Figure  G A G chains of  is  composed  three  giving  a  ropelike  stabilized  collagen  of  by  are The  by  is  molecule  peptidic  cross-linking  fibre.  stabilized  collagen  of  fibre  packed  The  of  the  is  electrostatic  a  interstitium  collagen protein  coiled  three  a-chains  interaction  and  of  through  properties. of  charges.  charge-packing  energy-storage  of  hydrophobic  collagen. which  molecular  together  molecules  is  molecule,  cylindrical structure  together,  side-to-side  the  the  a-chains  configuration.  molecules  collagen  component  collagen  A  be  occupies  ion-exchange  describes  For  H A  of  starch.  tropocollagen.  to  it  weight  capable  analogous  as  the  of  influence by  is  h y d r o d y n a m i c volume  weight  are  solution.  volume  number  dimer  17  Collagen  The  is  mean  either  (2:1)  in  molecular  given  structures  Laurent  and  a  matrix  proteoglycans  glycogen  a  aggregates  extracellular  Comper  large  acid  volume  hydrodynamic equivalent  occupied  branched  has  of  proteoglycan  Their  of  hydrodynamic  The  attachment  volume  the  proteoglycan-hyaluronic  /  to  (Light  the &  parallel to the  collagen  bonds  weight  form  of  is  a  The also  collagen  each  helix  other,  amino  is  It -  molecule  1980).  molecules  between  known  285,000.  triple  Bailey,  basic  to  The form  believed acids  on  INTRODUCTION the  outside  of the  cross-linked fibre  together  may  1968). the  be  It  molecules  is  from  found  passage  to  of  al.,  resist  changes  in  tissue  GAGs  (Aukland  &  Pearce,  higher  several  the  molecules  et  a  to  that  (Meyer  the  give  one  fibrils  (Bert  structure.  hundred  organization as  1977).  large The  1984).  of  the  plasma  main  function  & Nicolaysen,  and  The  collagen  diameter  microns  as  configuration  The  is  loose  proteins  volume,  of  (Schubert  fibres  of  fibres  18 then  collagen  Hamerman,  enough  between  to  permit  the  individual  fibres  are  that  proteins  and  immoblize  collagen exclude  are  the  &  /  they  1981).  1.2.3. The Hydraulic Resistance of Flow in the Interstitium  The  connective  networks  of  stiff  give  and  filled In  with  such  been  a  hydraulic  with  determine  It  is  high  often  to  in  the  reviews  a  offer  network,  by  immobilizing of collagen  the  the  Jain main  high  to  water,  hydraulic  The and  a  (e.g. and  of  to  to  water  flow  by  its  steric  is  mainly  and  resistance  amount  work  for  the  shown are  hyaluronic by  of  Nicolayen, have  has  exclusion from  its  flow high  1981). that  in  responsible  acid  forming  is  complexes.  hydrodynamic  interstitium  above,  relatively  interfibrillar space  &  (1987)  mentioned  are  responsible  Aukland  the  fibres  the  are  Levick  interpenetrating  proteoglycan  large  contribute  components As  acid  molecules,  of  collagen  G A G chains  (1987)  resistance  the  The  hyaluronic  that  consist  compartments.  component(s)  resistance.  to  fibres.  interstitium  by  three  hydraulic  interstitium  interpenetrating  suggested  all  the  collagen  inside  which  potential  contribution  and  trapped  tissue,  in  structure  to  recent  general the  solid  resistance  However,  for  a  structure  resistance.  a  polysaccharides  solvent  done  tissues  a  has  molecular  properties. steric  the  The  exclusion  INTRODUCTION properties.  Because  movement  of  acid  in  the  reduced. from  1.3.  hyaluronic  the  case  retention  Over  of  hyaluronic  acid  On  the  ions,  Levick,  contribute  to  h a n d , the  tissues  are  the  toward  disk  (Aukland acid  will  Silberberg, likely  to  molecular  (Szirmai, &  the  1974).  out Since  occur  in  weight  of  the  great  in  the  role  in  the  flow  available  to  the  for  the  of hyaluronic  conductivity flow  is  further  resistance  is  properties.  the  In  In  hyaluronic  membrane hyaluronic  layer  disk  is  of  is  few  of the  slices is  a  in  processes.  different  large  in  polysaccharide  within  not  in  different  acid  and  the  of  saline  other  of  center  fixed  tissues  its  content  in  of  tissue  hyaluronic (Meyer  compaction  the  has  concentration  the  amount  tissue, On  solvent  1978).  toward  is  of  that  Laurent,  Also,  incubated  mobile  &  Since  of h y a l u r o n i c  hyaluronic  increasing  and  believed  transport  vitreous,  acid  is  behavior  1965).  days,  it  (Comper  vitreous,  transport  interstitium.  space,  osmotic  hyaluronic  transport acid  the  the  regulation  (Balazs,  cord acid  in  animals  bovine  addition,  umbilical  the  in  distribution of the  center  a  etc.  interstitial  in  and  Over  interest  non-ideal  cortical tissue  1981). of  space  concentration  exclusion  proteins,  intervertebral  1970).  Nicolaysen,  diffuse  been  homeostasis  constant.  gradually  the  Also,  concentration not  in  the  contribution  steric  plasma  1987).  in the  glycosaminoglycans  has  important  highest concentration decreases  the  19  THESIS  there  an  Then  their  and their  fibres,  Therefore  increased.  major component  plays  (e.g.  other  connective  a  collagen  reduced. is  THE  water,  acid is  may  is  of  proteoglycans,  years,  hyaluronic  acid  acid  of  OF  the  distribution  solute  presence  of water  OBJECTIVE  and  the  interfibrillar space  In  the  of  /  is  hand,  (Section  &  very the 1.1).  INTRODUCTION Therefore  a  study  concentration better  Chapter  hydraulic  chapter  flow  of  of  relationship  between  hydraulic  acid,  different  molecular  with  of hyaluronic acid in the  the  present  of  hyaluronic  sedimenting  concluded  work,  with  in  the  a  brief  the  acid  real  weights,  methods  discussed.  ultracentrifuge review  of  conductivity will  and  give  a  tissue.  different are  flow  20  is  the  of  The  then  estimating  sedimentation  presented.  ion-exchange  theory  The in  chromatography.  Chapter  4.  role  conductivity  then  ion-exchange  techniques  2  particles  is  the  hyaluronic  picture on the  In  theory  of  of  /  used  The summary  3  discusses  in  in  obtaining  the  a n d conclusions  detail data. are  the The  experimental  results  presented  are  procedures  then  in Chapter  5.  discussed  and  the  in Chapter  C H A P T E R 2. T H E O R E T I C A L  2.1.  INTRODUCTION  As mass to the  discussed  exchange  estimate tissue  because  (e.g.  Jackson  has  been  of  extracellular  the  be extremely  the  hydrodynamic  the  weights  ion-exchange  resistance  velocity  will  mixed  or  The  then are  of  follow.  vivo  1986).  performed  not  enzyme  the  with tissue  degradation,  vitro  only  other  method as  as  studies.  component  amongst  the  In  study,  21  the  three,  acid  alone  hyaluronic  is  to  acid  problems,  are  which  the  samples  is  study.  sedimenting  h y a l u r o n i c acid  of this  discussed.  their  particles  T h e hyaluronic  chromatography. T h e theory  as  vivo  H o w e v e r , it  approach in the present  macromolecular In this  because  This  i n the  components.  can be made.  well  in  The hydrodynamic  are in  experiments  methods,  taken  investigated.  been  i n the  the- h y d r o d y n a m i c resistance  methods is  have  the contribution of hyaluronic acid  best  is  studies  methods:  entangled  in  three  method, theory  three  acid  an i m p o r t a n t role  Ethier,  of the whole  by  The  to  A l l three  difficult to estimate  chapter.  of  by  plays  many  1982;  hyaluronic  resistance  this  ultracentrifuge molecular  is  out.  in  discussion  that  singled  sedimentation  briefly  It  of the hydraulic  discussed  James,  estimated  fact  matrix.  be  &  and sedimentation.  will  cannot  1, hyaluronic acid  i n the interstitium. Therefore,  filtration-chamber  studies  in Chapter  the contribution of hyaluronic acid  resistance  is  BACKGROUND  in  A the  of different  fractionated  chromatographic technique  by  the  is  also  THEORETICAL 2.2.  ENZYME  DEGRADATION  This the  enzyme  before is  method  and  fixed  and is  end  rate  (0.85%(w/v) of  flow  addition a  an  across  hyaluronic acid  the  tissue.  If the  and  there  at  the  a  of  the  up  an  in  there  are  glass  of  Then  22  degradable  by  times  in  in  the  rate  of  tissues,  fascia.  in the  ten  several  potential  saline  rates  are  contribution  head  to  of  (1962,  1963)  have  stroma.  flow  quantified  drawbacks  in  the after  after  flow  that  times  They  the  of  of by  show liquid  this  is  of  that  through known,  hyaluronic this  also  addition  selected tissue of  saline  twenty  experiments the  of  observed  corneal  two  be  flow  the  He  permeability  resistance  tissue can  two  Mishima  regulating  the  flow  pressure  of hyaluronic acid in the  the  saline,  membrane  a  through  These  with  quantified.  under  the  membrane  The  tissue,  and  filled  measured  measured.  in  the  is  tissue  then  the  increased  water  tissues  When  Hedbys  enzyme.  is  is  be  is  selected  tube  mouse  of  role  The  then  flow  acid  selected  membrane  membrane  important  particular concentration  However,  16  enzyme.  can  the  flow  to  some  changes  flow  the  concentration changes  to  hyaluronic  solution.  hyaluronidase.  on  plays  the  membrane  through  of  The  other  acid-degrading  hyaluronic  no  no  that  measured.  measured  testicular  increase  is  are  NaCl)  study  the  tube.  again  resistance  of saline  of  through  is  there the  fact  hyaluronidase  1952)  of  glass  saline  (1950,  similar  observed  a  with  acid to  Day  of  of  if  the  administration  membrane  and  hyaluronic  the  permeability  the  compared  done  The  perfused  through  the  hyaluronidase.  flow  then  rate  of  one  at  the  use  the  /  METHOD  makes  after  BACKGROUND  acid  method.  approach.  As  THEORETICAL described  in  Chapter  superstructure addition also  of  in the  down Also,  concentration  tissue.  the  and  still  have  Chapter  1,  not  all  diffuses  out it  solution  of  is  the  during  conductivity between  K'  only  al.  the  that  has  on  where in  a  vitro  are  pressure,  fact,  many  6  and  tissues  (Levick, of  commonly  1978;  Urban  conductivity in  &  and  et  al.,  the  a  1984).  of  to  is  gradient  of  1980).  the  is  tissue  Due  to  protein  the  enzyme  hyaluronic &  A  the  power  in acid  Siberberg,  washed-out hand,  (Z).  low  into  the  hydraulic  law  equation  (1970): [2.1]  Jain  (1987)  may  erroneous  pointed  fibre  explains  vary  on the out  by  by  the  the  linear  just  a  weighted of  that result  during of  the  measurements.  applied pressure for Levick  (1987),  that  (Comper  relation (Levick  number  a  fact  concentration  Since  as  conductivity  dependent  complicated  the  will  mentioned  (Meyer  is  other  content  not  but  b  as  is  acid  and Fatt  this,  be  Since  saline  content  to  Moreover,  concentration across  aZ  lead  shown  in  as  a the  hyaluronidase  addition,  23  that  the  despite  /  form  acid,  therefore  that  tissue.  the  Bert  water  may  Maroudas, fibre  On  =  view  conductivity  have  conductivity  (Mow  been 1987).  tissue  tissues  tissue  In  therefore  conductivity has  interpretation  value  constants.  conductivity experiments,  applied In  and  in  hyaluronic  been developed b y K'  In  incubated  water  and  shown  and  pointed out  hyaluronic  performed,  fixed  test. the  have  the  activity.  is  tissue  conductivity dependent  steps  slices  some  (1982)  have  protease  cord  hyaluronic acid  superstructure,  (1984)  acid  to  hydrolyze  purification  umbilical  is  and Z  et  hyaluronic  the  (K')  not  detectable  possible  bind  Jackson and James  will  Knepper  used  proteoglycans  glycosaminoglycan  preparations  1974),  many  hyaluronidase  break  network.  1,  BACKGROUND  connective &  Laurent,  between 1987),  the  average  of  possible  the  hydraulic measured the  problems  whole in  the  THEORETICAL enzyme  degradation  estimates using  2.3.  of  this  the  method,  it  is  contribution  of  hyaluronic  In  this  are  used.  The  filters  in  solution  by  pressure  method  a In  a  &  Curry,  of  hyaluronic  partly evidence  from  constant  pressure,  variability  of  (Ogston  the  the by  work the  resistance  in  known  is  is  make the  calculation  in  accurate  whole  acid  of  place  through  applied  of  instead  held  forced  hyaluronic  the  solutions,  pressure  1982)  method.  the  and  the  tissue  the  the  two  hyaluronic  acid  in  resistance  whole  between  flow  concentration  of  buffer  flow  used  1,  the  p H of  the  solution  a  large  effect  on  the  is  the  Carr  have  studied  is  Curry  continuously  that  the  are  and the and  results  buffers  shown  their  in  with the  Hadler,  the  rate  are  tissue,  this  contributed  time.  have  by  2.1. of  used  As  hydrodynamic volume  in the  perfusion  conductivity considerably, This  is  reported  that  at  are  reason  hyaluronic of  experiments  the are  for  different.  mentioned  the  Adamson  1987).  Another  workers  Table  differ  (Levick, who  1980;  hydraulic  results  (1982),  ionic environment  the  &  time-dependent  falls  workers  shape  1961;  However,  of A d a m s o n &  results  Therefore,  the  & Sherman,  this  flow  the  and  & James,  buffers  molecules.  to  tissue.  with  the  solution  solvent  permit  Jackson  acid  because  with  acid  acid  The  head,  will  workers  1982;  hyaluronic  hyaluronic  conjunction  acid i n the  Some  pure  chamber.  presumably  hyaluronic  acid  difficult  24  METHOD  approach,  backing  measured.  very  /  method.  FILTRATION-CHAMBER  tissue,  therefore  BACKGROUND  in  The  Chapter  acid  hyaluronic affected  the  by  have acid the  THEORETICAL buffer  method  are  other  potential  over-estimates  hyaluronic  acid  membrane  the  matrix  (Levick,  1987).  polarization  the  chamber.  solution uniform  region  along  close  showed  to  that  concentration  conductivity. matrix,  and  sensitive  to  Table  2.1  (or  most  of  restraining  the  pressure was  compaction  therefore hydration  Buffer  the (Bert,  used  tubular  Using  drop  across  develop  a  reduces  the  amount  of  and  bj' different  rose  without  would  effective  tend  directly  and  from of  to  related  acid  at  solution  the  with  polarization; the  flow  hyaluronic  acid  conductivity a  in  E t h i e r (1986)  underestimate  to  filter  steeply  acid  Since  that  hyaluronic  method,  i n the  filter  shown the  concentration  porosity  hydration.  buffer  have  very  hyaluronic  This  restraining  (1984)  computational  that  is  the  upstream  but  method.  the  concentration  the  than  of  from  Winlove  the  experiments  1969)  because  chamber,  filter.  greater  filtration-chamber  can  experiment,  the  filtration-chamber  upstream  Parker &  compaction)  their  the  initially  together,  addition,  the  Also,  with  conductivity  In  In  polarization  therefore,  problems  moving  concentration  and  25  used.  There  was  BACKGROUND /  power  is  very  function  workers  Buffer  (M)  workers  pH NaCl  1.  Ogston  &  2.  Carr  3.  Adamson  4.  Jackson  Sherman  (1961)  & H a d l e r (1980) & Curry & James  (1982) (1982)  Na HPO, 2  KH PO« 2  0.2  0.0077  0.0023  7.3  0.1  not  shown  7.0  0.2  not  shown  7.25  0.  0.0077  0.0023  7.25  of  THEORETICAL water of  content  (Bert  compaction.  out  by  the  the  whole  pressure  matrix  the  its  disadvantage  of  is  that  acid  produced  for  the  whole of  cell.  this  The  cell  sedimentation conductivity  of  by  total  Chapter  1,  studies  of  molecules rather  Ogston a  porous  effective  then  a  et  presence is  of  the  flow  hard  to  is  forced  drop  across the  therefore  just  an  of buffer  filter  pointed  compaction,  and  is  26  presence  as  buffer  flow  drop the  the  pressure  non-uniform;  with are  the  total  pressure  acid  al. acid  acid at  pointed  From  buffer this  driving  solution  very  hyaluronic from (1960) that  3-dimensional  stationary  centrifugal  flow  the  matrix. F i n a l l y ,  out  point force  is  (Parker  the  average  is  affected  &  Winlove,  determine.  speed  acid  is  high  view,  on the  coefficient. their  network,  system to  the  of the  Ethier  particles  an  rpm) The  As  the  and  (1983)  hydraulic  whole  of has  the  buffer  reasoned  unit volume  is  acid  network,  cell.  sedimenting  in  velocity  hyaluronic  the  the  mentioned  ultracentrifuge  passage  and  sedimentation  particles  per  ultracentrifuge  50,000  concentration,  in  in  determined.  from  sedimented  analogous of  (>  sedimentation  concluded  a  contained  high  polysaccharide  that  was  the  at  individual molecules,  (1951)  plug.  of  calculated  Laurent  the  and  by  method,  experiments,  the  of interaction  centrifuged  hyaluronic  than  through  then  formed  the  hyaluronic  coefficient is  of  direction  affected  /  METHOD  method, is  Because  is  filtration-chamber  perfusion  hyaluronic  SEDIMENTATION  the  matrix,  solution  the  measurement  the  solution  along' the  data  in  T h e extent and method  In  a  1970),  measured.  interaction  1984).  &  is  gradient  measurement  2.4.  (1986),  hyaluronic  conductivity  by  Fatt,  Another  Ethier  through  &  BACKGROUND  Fessler together through  that  equivalent  the to  THEORETICAL the  pressure  2.1).  gradient  Consider  uniform  the  solvent.  created  by  the  sedimentation  of  The mass  m  flow a  of each  7  of  solvent  single  BACKGROUND  through  kind  of  the  particles  suspended  h y d r o d y n a m i c particle  /  27  (Figure  particles  in  a  is  n m  h  where  M  is  Avogadro's grams  of  factor  6\  the  molecular  number solvent  and  5^  associated  =  (1  weight  of  is  solvation  the  with  1  in  the  equation  macromolecules  like  hyaluronic acid.  to  determine.  centrifugal solvated  However,  force  particle  on  the  we  + 6, )M/JV  will  particle,  In  see 8y  unsolvated  gram  accounts  is  [2.2]  A  of  for  factor, the  the  general, later  macromolecules,  that  cancelled  which  unsolvated  fact 5  is  that is  :  in out.  the  is  solvent  is  or  of The  bound  very  derivation  T h e total  the  number  macromolecule.  unknown the  N  of  difficult the  volume  of  is  solvent  sedimenting macromolecules  Figure  2.1  A  system of macromolecules  sedimenting  in a n  to  ultracentrifuge.  net the  THEORETICAL v where  u  volume  is  2  of the  In a)  the  force  is  specific  ultracentrifuge,  a  angular  buoyant  Equation  specific  force  p VjG) r  per particle  effective  the  the  the  +  solvent  centrifugal  product  force  is  per unit  gradient,  from  v ^  is  the  specific  of  = F  X  (M/N  =  c cj r( 1  m^oi r,  where  M/N^  the  volume  1  -  D  -  2  =  -  the solvent  with  Opposing  density  p,  [2.4]  -  p ^ u , )  the  reciprocal  2  D  [2.5] of  the  solvent  2  P  1  )  [2.6]  is per volume  v p , ) XJV / v o l . p 2  number  [2.7] of  particles  the sedimentation  per volume coefficient,  v /a) r of as  the  c v {\  D a r c y ' s law, c a n be expressed  s,  is  just  is [2.8]  2  2  velocity written  respectively.  p , )  B y definition,  c a n be  is  volume  a n d the  sedimentation  p , u  1  2  2  2  [2.5] becomes  )cj r(  2  is  2  no. of particles  =  position,  by  p y,  2  A  per particle  )u r  h  -  A  s  2  and  [2.4],  (M/N )o r( per unit  2  v  y  density,  force  polymer concentration c .  where  [2.3]  A  then  p v  5,  u , . Therefore E q u a t i o n =  is  -  h  2  F  because  (m  A  solution,  volume,  exerted  2  x  =  force  a n d the r a d i a l  (M/N )a> r(l  F The  1  of the polymer,  [2.2] a n d [2.3] into  F = dilute  6,u )M/iV  centrifugal  velocity  F  For  +  2  volume  the  the particle. T h e net force  Substituting  (v  / 28  solvent.  a n d r are the  this on  the partial  =  h  BACKGROUND  2  2  polymer. -  Then,  v P\)/s 2  the  centrifugal  • T h e pressure  as:  dP/dy = -r\vjK  [2.9]  THEORETICAL where the  77 is  the  sedimenting  dilute  solution  solvent polymer,  viscosity,  v ,  is  and K  is  the  specific  =  v ,,  approximation v K'  where  K'  1971;  Ethier,  acid  is  solution  hydraulic  the  given  by  Ethier  sedimentation long  as  the  sedimentation hyaluronic by  the  an  conductivity  (1986).  This  is  as  at  acid  at  an  lower  the  In  is  to  Making  al.,  1965;  Mijnlieff  coefficient  known  the  [2.10]  of  physical  in  the  near  to  the  & Jaspers, a  hyaluronic  parameters,  in  is  an  the  centrifuge depends  wall  for  to  swell  the the  has  advantage  filtration-chamber  filtration-chamber  value  method  the  cell  only  the  studies.  As  bottom,  the  on  the  conductivity  the  (1986),  entire  there  frictional  may  layer  are  to  cell,  the  the  erroneous to the  local  obtained at  the  which  the  of solution.  on  and  hyaluronic acid due ultracentrifuge  in  potential  effects  velocity, lead  method  been of  polysaccharide concentration  sedimentation  on  there  conductivity,  Ethier  The  boundary  fact,  that  not  the  average  the  sedimentation  b o u n d a r y . Therefore, the  related  by  out  the  from  frictional force  insignificant.  et  the  present  the  is  method.  hyaluronic  not  therefore  mentioned  may  the  is  relative  2  other  of  pointed  boundary  method  measured  acid  is  is  different  hyaluronic  wall  has  concentration  sedimentation  that  and  with  solvent  29  D p,)  sedimentation  discussion  which  velocity,  -  (Preston  along  the  hydraulic conductivity.  2  the  of  /  obtained.  He  solvent/solution  However,  shows  be  velocity  S/C (1  =  if  then,  in-depth  sedimentation  boundary.  the  measured,  method  acid  K/rj  Therefore,  conductivity c a n  Recently,  =  hydraulic conductivity  1983). is  2  the  BACKGROUND  the  problems  in  sedimenting  tendency results. presence  acceleration  of  the  Ethier of force  the is  THEORETICAL radial,  and  therefore  sector-shape  design  frictional  wall  visualized  by  swelling the  of  of  effect the  the  Schlieren  "sharp  sedimenting  to  This  criterion data  error  conductivity  shows  the  that  sedimentation conductivities  Base best the  among  which  conductivity  of the  the  the  2.2  at  swelling  show is  the  kept  problem  above  three.  sharp to  little  Therefore,  Sector-shaped  cell  a  will be  minimized the  peak In  following  — implying  by  are  used. the  2.3).  that  the  the is The  broadening  of  establishing  a  only  the  Therefore,  the  study  criterion,  the  boundary  conductivity,  fact,  this  a  30  reduce  (Figure  in  /  Therefore,  will  peak  result  calculating  scatter  2.2)  Schlieren  can  radii.  solvent/solution  minimum.  by  Ethier  from  the  past  data  are  true  matrix.  it  in  is  the  conductivity  used  the  Schlieren  a  discussion,  the  In  calculated,  show  as  along  (Figure  boundary  1986). a  move  cell  Also,  system  hyaluronic acid  approach in studying  Figure  acid  data  experiments  on  minimum.  (Ethier,  sedimentation in  a  hyaluronic  will  ultracentrifuge  Schlieren optical  peak.  peak"  the  molecules  BACKGROUND  clear  that  present  sedimentation  investigation,  of hyaluronic acid  in all analytical  it  method is  solutions.  ultracentrifuge.  is  adopted  the as  THEORETICAL  BACKGROUND  /  31  F i g u r e 2.3 (a) T h e concentration profile of a sedimentation r u n . (b) A Schlieren trace of the cell along the radial direction. T h e Schlieren peak represents the boundary position. (Modified f r o m M c C a l l & Potter, 1973)  THEORETICAL 2.5.  SEDIMENTATION  solution  instrument. in  analytical  at  Figure  an  electric  in  the  up  to  The 2.5.  motor.  solution  32  sample  of  Ultracentrifuge  ultracentrifuge 70,000  sample  The  /  THEORY  2.5.1. The Analytical  The  BACKGROUND  cell The  to  rpm.  solution is  then  is  device  Figure  is put  in  outward,  an force i.e.  capable  2.4  contained  ultracentrifugal  sediment  a  is in  a an  spinning  schematic  or  generated  titanium  causes  the  a  diagram  ultracentrifuge  aluminum  towards  of  cell rotor  the  bottom  of  as  shown  driven  of  the  cell.  Film holder Motor  lens  I Rotor lens Armored Cooline  Temperature control Vacuum pump L i g h t source  Figure 2.4 Schematic Cantor & S c h i m m e l ,  diagram 1980)  of a n  analytical  ultracentrifuge.  by  macromolecules  Camera  chamber  the  (Modified  from  At  THEORETICAL  BACKGROUND  Cell Screw ring (^^^)  Screw-ring gasket  K^Cj5^| Upper window holder (^^^)  Window gasket  e  Window liner  (^^^)  Centerpiece  Window Centerpiece gasket  Single-sector centerpiece  Aluminum centcrpeice Centerpiece gasket Double-sector centerpiece  ^ ^ ^ ^ Window Window line Window gasket Lower window holder Cell housing Housing-plug gasket Housing plug  Figure Manual,  2.5  The  Model  analytical  ultracentrifuge  cell.  E A n a l y t i c a l Ultracentrifuge,  (Modified from Beckman)  the  Instruction  /  33  THEORETICAL typical  rotor  enormous vacuum  amount before  precisely The  speeds,  by  an  the  optical  along  the  system,  for  {dn/dr)  with  radial  (Figure  The  design  The  rotation  (Figure  the  use  cell  of  The  Schimmel,  1980).  texts,  texts  force  below.  only  a  shown  in  is  Figure the  then  sends.  regulated  commonly  used  In  a  typical  concentration  2.3(a).  recorded  mixing. light  the  refractive  high  UV  Schlieren.  macrospecies,  for  the  analytical  when  viewed  r u n , the  generated  will not  (e.g.  and  is  an  a  convective  three  34  The  Schlieren  index as  a  gradient Schlieren  film.  cell  development  Therefore, given  cell  The  to  sample  which  /  generate  brought  single  variation  centrifugal  acceleration  standard  is  a  rotation.  is  will  avoid  system  v a r i a t i o n of  sector-shaped  During  mathematical in  the  the  the  air  the  to  interference  is  The  sample be  of  of  unit  optical  a  photographic  the to  of  and  chamber  control  Raleigh  r.  rotor  axis  detects  sector-shaped  presented  on  of  2.2).  the  the  rotor  temperature  an  direction  distance  has  because  to  run  example,  a  The  has  the  the  temperature  radial  2.3(b)) on  critical.  radii  started.  absorbance,  optical  the  is  parallel  are  between  Therefore,  sedimentation  distribution  peak  heat.  ultracentrifuge  sample  speed  friction  automatic  systems  high  of  spinning  analytical  through  the  BACKGROUND  of  result  the  Williams, simplified  ultracentrifuge parallel  molecules  is  in  in the  the  will radial  the  direction.  Fujita,  development  of  theory 1975; the  very  axis  sediment  s t i r r i n g of the  ultracentrifugal 1963;  to  is  of  along Only  sample.  has  been  Cantor  theory,  &  based  THEORETICAL  BACKGROUND  / 35  2.5.2. Transport in the Ultracentrifugal Cell  Figure its  position  r defines  2.6  relative  the radial  of  concentration  at  radii  surface  area,  is  an expanded to  the  of a  at  radius  r  solvent  denoted  as  can be written  is  from  the axis  determined, is  the  first  in the  the rate  of solute  Before  the rate bounded  of solute  transport  (1)  mass  a  mass simple  a n d solute transport  (2).  across  a  of  by  system  by  cell and  T h e coordinate  i n the volume  Consider  subscript  ultracentrifuge  ultracentrifuge.  of rotation.  species  studied.  by  rate  of a sector-shaped  of rotation  macromolecular  a n d r+dr  defined  view  axis  distance  r  components, which  is  change surfaces  across  the  with  two  T h e flux, surface  of  J , 2  unit  as J  2  = c (v ) 2  [2.11]  2  r b  Figure 2.6 Geometry of a position of the meniscus is r is at r = 0 .  m  sector-shaped ; the bottom  cell. T h e of the cell  radial distance is r^. T h e axis  is r, the of rotation  THEORETICAL where  c  is  2  the m a s s  average  velocity  flow  at  radial  that  the flow  and  on  concentration  of the molecules. distance  the  sedimentation  like the sedimenting of  coefficient,  s,  the  the concentration  of  =  velocity  molecules  all m o v i n g  at  v  =  the  flux  b y the angular  2  If  there  is  develop  a  where and  D  is  [2.13]  across  the  diffusion  gives  a surface  an  J  2  surface of  r  solute  is  the  is  J A, 2  mass  of the  the  flux  rate  r  The  [2.8]  is  c ,  a n d i f the  2  solute  molecules force,  then  is described by: [2.12]  a driving  the  force  caused  b y diffusion  will  by Fick's law: [2.13]  molecules.  rate  2  A  is  in the volume  which  of change  area, the  between  (f>rdrXa.  of m a s s  the  surface  of  Combining  mass  transport  Equations of  the  [2.12]  molecules  D(dc /dr)  2  total  rate  area.  -  Then  at radii  of  mass the  transport  total  rate  r a n d r+dr  at  of  divided by the volume  change  [2.15]  2  of the sector  of change  the  is given by  J A(r+dr)  is the area  T h e rate  [2.14]  2  surfaces  = J A{r)  the two surfaces is  -  2  per unit  where  between  sector,  rotation.  2  the  speed  as:  is given  = io rsc  2  2  volume  of  -D(dc /dr)  of  for  dm /dt The  on the rotor  a t radius r in the ultracentrifuge: J  Since  is dependent centre  obvious  2  at r,  constant  equation  force,  the  then  2  =  2  It  = u> rsc  2  due to diffusion J  causing  2  acceleration  concentration gradient  at r. T h e flux  the applied force is  36  is the  2  due to the applied centrifugal  2  J  and v  /  (dr/dt)/u r  at  o rs  surface,  of molecules.  from  =  2  2  solute  the  mass  defined  v /cj r  are  caused  molecules  is therefore s  If  per unit  2  distance  at  In the ultracentrifuge,  r is u r  velocity,  of molecules  BACKGROUND  of  between  times  concentration,  the  height  which  the two surfaces,  is is  THEORETICAL dc /bt 2  Substituting  =  [J A(r)  -  =  (-l/0ro)  (dJ A/dr)  =  -(l/r)[3(r«/ )/3r]  =  2  Equation  [2.17]  gives  general  system  in the  In 1975).  is called  solutions  the L a m m of  s  of the  and  D  observed  c a n be represented  is  derived  -  equation  the  are s  cj r sc ] 2  [2.17]  2  2  for the ultracentrifuge.  solute  mass  functions  of  transport  of  a  T h e equation  two  component  versus  the value  positive  of s  at  constants.  macromolecules,  the  c  solute  concentration,  relationships  2  for dilute  c  2  (Fujita,  macromolecular  empirically b y either  s  0  gives:  ultracentrifuge.  or s  [2.16]  2  s = S  where  •  2  [2.16]  / 37  ]/(<j>radr)  2  (]/r)-^[rD(dc /dr)  description  general  Most  J A(r+dr)  2  E q u a t i o n [2.14] into  dc /dt  a  2  BACKGROUND  = s  / ( 1 + K,c )  [2.18]  2  ( 1  0  infinite  On  dependence  0  -  2  )  2  dilution,  the  of D  Kc  other  on c  2  [2.19]  and  and K  hand, c a n be  for  are empirically  2  dilute  represented  solutions  by  of  a n empirical  relation: D where  D  is the value  0  constant.  Because  non-linear very  in  difficult.  numerically  c , 2  In  the  and fact,  dependence  the the  +  0  of D at infinite  of  i n certain  = D (\  limiting cases  D  of  s  and  and  D  treatment  equation only  [2.20]  2  dilution,  mathematical Lamm  & c )  has  (Cantor  is on of been  a n empirically derived  c , 2  Equation  Equation solved  & Schimmel,  [2.17]  [2.17]  becomes  analytically  1980).  is  or  THEORETICAL  from  For  the case  Fujita  (1975)  of s = c o n s t a n t = s  a n d D=0,  0  the  BACKGROUND  solutions  /  38  of Equation [2.17]  is c  2  (r,t)  = 0  c (r,t)  if r  = c e  2  2  (  J  S  Q  if  t  0  <r<r^ r,<r<r  2  where = and  c  is  0  Equation Figure  the  [2.21],  2.7  solute the  concentration  compaction  is a schematic  boundary is a n infinitely the  solvent-solution  plateau  region  particular in  the  time.  plateau  in  e  "  2  s  at  zero  solute  at  °  [2.22]  r  time the  sharp  which  T h e second  the  line r * , is solute  equation  decreases  because  of  A s shown  there  represented  throughout bottom  d i a g r a m of the results.  boundary,  region  of  r  the  the  cell  concentration of [2.21]  shows  exponentially  with  Equation is  time.  ignored.  T h e position of  [2.22].  independent that  is  In  in the d i a g r a m , the  is no diffusion. by  chamber.  the solute The  There  of  r  at  is  a  any  concentration  reason  that  F i g u r e 2.7 Solute concentration profile d u r i n g ultracentrifugation of a typical solution of a homogeneous macromolecule, D = 0. (Modified from Cantor & S c h i m m e l , 1980)  the  THEORETICAL solute it  concentration  decreases  the  cell a  molecules  v (r 2.8.  the  very  there  solution  at  thin are  r >r , 2  than  latter  those  time  molecules  stronger  centrifugal  between  the  number  of  two  solute  The t,  because  at  at  molecules  are  define  a  acceleration  at  all  times.  molecules  in the  time.  This  Equation  [2.22]  with  the  obtained  increases  volume  phenomenon second  is is  equation  (Tanford,  volume r  they  sr,.  called  V,  (Figure  have  as  a  an  result,  velocity in  the  shape.  t  Figure farther  the  to  a  volume  Since  the  concentration  dilution.  By  combining  equation  describing  the  1961): [2.23]  C2A0  time  The  subjected  hand,  of  layer of  sedimented  sector  radial  [2.21],  shown  been  other  the  2.8).  with  as  while  distance.  F o r another  have  2  the  of  fixed,  of  ty  39  area  radial  sedimenting  On  because  the  time  originally at because  also  at  /  r-independent  cross-sectional  with  2  r,,  layers  the  v (r, ) =co  a velocity  layers  both  r , >r^  molecules  two  maintained  increase  at  with  dilution is  solution  be  originally  decreases  radial  of  the  2  is  can  acceleration  the  1  region  time  sedimenting  2  a  with  layer  2  At  plateau  centrifugal  ) = c o s r > v ( r , ).  2  the  exponentially  and  Consider  in  BACKGROUND  time  t  2  F i g u r e 2.8 Demonstration of radial dilution. Solute in two equal volumes followed from an e a r l y to a late time during sedimentation.  are  THEORETICAL  Therefore, to  the  square  rotation; the  the solute  or  the  time  t,,  c  above,  1980).  is  are subject  Suppose  there  is  of the surface,  2  from  time  t  Equation  boundary  to the  during  the  axis  of  course  of  =  (0/2TT) [ 7Tr  =  U a / 2 ) [ r  surface  = dr /dt  2  [2.24],  region  at  by demonstrating that all  as  1  dilution (Cantor &  shown  in Figure  2.8.  2  r,  2  ]  [2.24]  of volume  = u> sr  V , is [2.25]  2  x  a  7Tr, ]a  -  2 2  plateau  are the same, a n d  -  2 2  the  t,)  r  located  at ^ ( f , ) ,  at time  t  2  [2.25] to give  = r,(t,)e° at  = r U,)e° 2  the ratio  } 2 s  °  { t 2  ~ ' t  [2.26]  )  r (t ), 2  }  2  s  }  Q  {  t  of volume  2  ~ ' t  V,  [2.27]  )  at time  t  2  to  the  volume  is  x  Vy(t )/V Ui) 2  =  y  = This  2  in the two volumes  originally located r (t )  Then,  40  inversely proportional  of radial  V = V  originally (at time  2  for a surface  degree  volume  at the  r,(* )  at  a  at  c a n be shown  to the same  obtained b y integrating Equation  Similarly,  is  constant  concentration  of r. T h i s  of the molecules  location  remains  2  solute  j>(r,) The  region  solvent-solution  %  the solute concentrations  velocity  plateau  the  and r  2  V,  The  the  from  the  is independent  in the cell  Schimmel,  of  at  /  experiment.  mentioned  particular volumes  the distance product  sedimentation  As  At  of  concentration  BACKGROUND  volume  ratio  is  ir (t ) -r,(t ) ]/[r (t,) -r,(t,) ] 2  2  2 e  "  2 s  2  2  2  2  2  2  oU -<i ) 2  independent  [  of  the  radial  distance,  r.  2 2  8  ]  Since  THEORETICAL V  2  (t  ) = Vf (t!  :  2CJ S  (t  2  e  -t  0  each the  , or  1  volume  V  a  2CJ  c e  0  deal  (in  &  which  realistic  maintained.  of  written  identical  D  [2.19],  Lamm  cases  of  V  (1  2  2  )/ V  n u m b e r of  of  a  In  volume  (t  2  )  y  fact,  if  we  concentration  solute  is  also  molecules  increase  particular  and r ,  time let  at  by  the  sector-shaped  they  cell.  in  a  in  is  the  same,  are  all  equal.  ti=0, later  and  c  time  Lamm  form  t  be  0  will  z  equation  s=s  0  and  s  ( 1 ~K c). 2  subjected  to  In their computations,  according  Equation  [2.18] or  briefly  mentioned  are  been  solved  and s  varies  very  below.  sector  in  J.  Archibald  with  constant  series,  with  Dishon  et  and  (in  al.  is  time  1938) s  complex  extended  Faxen's  concentration (in  1966-67)  in  constant  and  s  varies  used  an  to  exact  However, to  be  solution  to  accordance  for with  his  used  numerically  boundary conditions  =0,  2  D.  too  r  (2scj r<<1 )  found  and  Faxen  which  early  case  complex  H.  of  varies  [2.19].  infinite  have  constant  obtained  1956)  is  positive  solutions  (in  D  which  limiting  infinite  initial  Equation  obtained c a n be  W.  Fujita  a  an  ^=0,  b o u n d a r y position.  equation is  if  he  equation an  results.  constant,  i.e.  of  Also,  T h e solutions  the  2  the  are  considering  (2D/so) r<< 1 ). the  the  [2.21]. the  Lamm  and  equation  for  is  i n which D  the  results  Equation  the  Thus,  in  is  [2.22],  experiments. to  at  the  to  derivations  sedimentation  where  then  the  ratio  is  1980).  solution  evaluating  Equation  The  diffusion  analytical  the  volumes  situations  experimental  weak  solution,  is  limiting  solved  the  ratio  Because  the  region  Schimmel,  instead  predict  case  ,  more  1929)  r^ = <»,  and  2  concentration.  (Cantor  all  ).  2  and  concentration,  other  with  with  same,  identical to E q u a t i o n  The  volume  41  t  0  [2.26] is  in  solute s  2  the  ) = V , (f  2  the  plateau  original  (t  2  remains  concentrations  the  the  therefore,  /  )  2  Therefore,  be  ),  BACKGROUND  some  in the  with solved actual  concentration  THEORETICAL The show  that,  compared plateau 0,  the  the  in  more  As  driving  some  of  caused  the  solute  cases  by  diffusion,  are  The  of the  at  solutions  very  (high  centrifuge  speed)  is  boundary  and a  at the b o u n d a r y , D  is not  width  of  the  plateau  from  solution  is  is  decreases  Lamm are  actual s  boundary  region  for the  problem of measuring  in the next  a  T h e solutions  coefficient  42  above  the  complex.  /  discussed  obtained  does not require a general  is discussed  cases  there  gradient  concentration the  realistic  are strong  sharp.  sedimentation  treatment  forces  infinitely  mentioned,  the  more  a concentration  not  the  treatment  treatment  is  limiting  determining  This  force  is  Also,  studies  sedimentation  there  already  satisfactory  (1953). The  D.  the  the  boundary  treating  value  as  Since  on  time.  from  long  region.  and  with  as to  dependent  by  results  BACKGROUND  equation of  limited  experiments.  given  A  b y Goldberg  of the L a m m  equation.  section.  2.5.3. Measuring the Sedimentation Coefficient  The which  treatment  there  compared plateau  to  is the  region,  equation  a  of  plateau driving is  are zero.  Goldberg region.  force  of  independent  Then  is  valid When  for  any  the  sedimentation  diffusion,  of r.  E q u a t i o n [2.14]  there  Thus,  sedimentation  is  dc/dr  a n d [2.17]  a  experiment  forces  plateau  region.  and 3 c / 3 r 2  are  2  strong In  in the  become  2  P  Now plane unit  consider is time  a  fixed  a<j>r^. is  cross-sectional  Then  a<f>r J .  the  total  This  =  -2c  plane number  amount  of  s CJ  [2.29] [2.30]  2  P P at  the  Lamm  J = oi rs c P PP dc /dt  in  r  (Figure  of moles solute  2.9).  of solute  transport  The area crossing  across  the  of  the  the plane  plane  per  must  THEORETICAL  BACKGROUND  / 43  m Figure  2.9 Concentration profile of the sedimentation  equal  the decrease  in the  plane  at r . Therefore, P  total  amount  ad>r J p p  If  is  diffusion,  the  position  then  at  of  any  the  time  =  of solute  between  solution.  the  meniscus  a n d the  — 5 7 ( J* ^ad>rcdr) at  [2.31]  J  infinitely t,  of a real  sharp  the  total  along  the  boundary,  amount  of  as  solute  in  the  case  between  r*  of  no  and r  P with  no  diffusion  total  amount  (in  of solute  which  c  between r  and r  r J* ^a<j>rc dr  **  with  distance  =  the presence  c )  is  equal  to  the  of diffusion:  P  m  r  radial  P  = r  r j  m  ^cuprcdr  [2.32]  THEORETICAL The  left  hand  side of E q u a t i o n [2.32]  P  P  Substituting  this value  into  is  a  [2.34]  becomes  fixed  °t  coordinate,  rJ  = ~ i ( r  Equations  [2.29]  PP  Substituting we  an  experimental  plot  of  is  r„ )c  P  independent  r )(dc/dt)  ]  2  of  [2.34]  time.  Therefore,  Equation  + c rAdrJdt)  2  P  [2.35]  P  for J  and  p  dc^/dt  into  Equation  [2.35],  [2.33].  the optical  obtained,  However, system.  for example,  to be expressed integrating  r  a0f  P  r where  c  m  c  = m  is  available,  hand  the  c a n be obtained  K {dc/dr), o  of 3 e / 3 r  system  hand  side  of  from E q u a t i o n [2.32] a n d  c cannot  where  right  K o  is  be  measured  a n optical  measuring  in E q u a t i o n [2.32].  dn/dr.  directly  constant,  is  Therefore,  T h i s c a n be  achieved  side of E q u a t i o n [2.32] b y parts:  rcdr = £ a 0 ( c r  m  r  the Schlieren  i n terms  is the solute  so  versus  Then  Instead,  [2.36]  %  2  pp  ultracentrifuge, meniscus,  2  in the ultracentrifuge,  from  the right  = {dr^/dt) /u r  p  c  Equation  by  [2.33]  get  [2.32] is determined.  has  -  2  P  a n d [2.30]  Equation  by  it  -  2  P  s If  2  P  = --L[la4>(r  PP  r  - r* )c .  2  P  E q u a t i o n [2.31]  a<t>r J Since  / 44  is simply  r a<t>rc dr = ia<p(r r*  S  BACKGROUND  -  concentration  the  sedimentation  0-  Then,  c r  )  2  mm  iatff  at the meniscus.  boundary  combining  -  Equation  is  r P  r  2  r m  After  completely  [2.32]  {dc/dr) dr  [2.37]  t  a  finite  separated  a n d [2.37]  time  i n the  from  a n d solving  the for  THEORETICAL  BACKGROUND  / 45  r r*  =  2  (1/c ) /  p  r  r  r Also,  c a n be replaced b y /  P  r  P  r (dc/dr)dr  Equation the  [2.39]  = / r  2  (dc/dr)dr.  Therefore,  m  r  P  r (dc/dr) dr/S 2  (9c/3r)^r  P  t  [2.39]  r  c a n be used  sedimentation  t  m  r r*  [2.38]  2  to determine  experiment.  Since s  for each  Equation  =  [rf(ln  individual  photograph of  [2.36] c a n be rewritten as  r*)/dt]/a>  [2.40]  2  P therefore, time.  the sedimentation  T h e advantage  principle shape  o f this  method  This  of the b o u n d a r y .  However,  this  method  the  difference coefficient  c a n be  used  to  of  dc/dr  between  at  and  c a n be calculated  the Schlieren peaks  has  Equation  method  obtain  [2.41]  to be used.  will  r=r -dr is  s  therefore, is  if  very  the  it is based  on the  independent  o f the  time-consuming.  Schlieren  peak,  A  r ,  more  o f the  the boundary is s y m m e t r i c a l at its  a n d r=r +dr  small  is  versus  enough  so  are identical. that  the  Then  the  sedimentation  from s  of  method,  b o u n d a r y is s y m m e t r i c a l . W h e n  values  by plotting In ( )  of determining s is that  of mass.  sedimentation  If  c a n be obtained  of conservation  practical  peak,  coefficient  =  (dr  for sedimentation result  in a  /dt)/(J r  [2.41]  2  boundaries are m a r k e d l y skewed,  large  error.  Therefore,  Equation  the use  [2.40]  still  THEORETICAL 2.6.  ION-EXCHANGE  Several weight  are  methods  reported  cetylpyridinium in  aqueous  salt  with  aqueous  sodium  dissolve  of  the  highest  sucessive  A  et  fractions  of  NaCl  method.  salt.  more of al.,  DEAE-cellulose  with  molecular  In  fractionate  the of  forms of  aqueous  upon dilution of  a  Fractions  of  hyaluronate decreasing  basis  al.,  of  its  molecular  salts have  of  of  sodium  molecular  sulfate  as  weights  are  molecular  is  in  needed  to  hyaluronate  (of  sulfate,  a  fraction  precipitates  weights  with  cetylpyridinium  containing  obtained  salt  solubilities  the  sodium  1960),  solubility  different  different  weight  solution  is  et  molecular  cetylpyridinium  cetylpyridinium chloride and  weight  the  different  molecular  to  (Laurent  on a  formed  the  according  method  fractionated  salts  higher  concentration  weights),  one  cetylpyridinium  the  46  are  of  then  the  obtained  dilutions.  fractionation (Cleland  However, The  hyaluronate  hyaluronate  chloride,  Therefore,  molecular  In  Hyaluronate  When  sulfate.  salt.  cetylpyridinium in  sulfate.  the  of  hyaluronic acid is  formed.  higher the  different  literature.  cetylpyridinium  are  the  of  /  THEORY  fractionation  the  chloride.  weights  salt,  of  in  sodium  cetylpyridinium mixed  CHROMATOGRAPHY  BACKGROUND  rapid  hyaluronate 1968). are  A  mixed  increasing solutions the  and  of  efficient  according salt  free  together  molecular  present  hyaluronate.  more  to  molecular  solution and  weights  method  of  charged are  obtained  as  study,  above  with  use  chromatographic  chromatographic  on  hyaluronate a  concentration,  the  weight  to  increasing the  is  DEAE-cellulose  and  column. the of  a  slurry  Hyaluronate  column  is  stepwise  technique  of  is  eluted elution  used  to  THEORETICAL The  theory  is  referred to  reader 1976)  for more  of  another  different  strength  is  charges  association usually  the  commonly  materials.  the  consisting  positively  charged  interact  with  charged  functional  cations  on  DEAE-(or  the  on  Figure  a  low  molecules  ionic, to  molecules.  2.10.  The  The  of  under  molecular  relatively  factors  Morris  called  The  most  is  a  &  are  The  Morris,  cation  which cation  is  mild  allows buffer  used an  are  cellulosic  anion  bear  because  they  of  Also,  negatively  interact  with  ion-exchangers and  because  in  a  large they  to  which  bear  they  cellulosic  attached  structures  used  results  elution  conditions.  An  exchanger,  Their  widely  which the  because  The  bonding.  are  which  of  are  is  exchangers  exchangers  ionic  molecules  exchangers  anion  exchanger.  substitution,  density  molecules groups  of  different  considered.  biological  exchangers  commonly  be  hydrogen  called  molecules  distribution  and  ion  mixture  increasing  the  to  a  with  poly-electrolyte  functional  the  of  and  have  The  and  in  (adsorbent)  size  that  the  ion-exchangers  group  component  gradient  and the  celluloses.  molecules,  cellulosic  charge  47  below.  Poly-electrolyte  buffer  hydrophobic  groups  which  functional low  a  ion-exchangers,  are  one  charge.  separate  diethylaminoethyl-)cellulose,  level  charges.  when The  modified  groups  carboxylmethyl-)cellulose, in  i.e.  the  1970;  ion-exchanger  ion-exchanger  functional  anions  the  the  used  of  on  net  important  cellulosic  matrices  of  are  forces,  ion-exchanger  basis  eluant.  between  summarized  Peterson,  distinguishes  separated  molecules  secondary  the  the  (e.g.  is  /  technique.  adsorbed  then  as  forces  On  on  are  are  used  of  texts  chromatography  charges  They  chromatography  standard  of the  component  net  affinities.  some  details  Ion-exchange from  ion-exchange  BACKGROUND  low  are they  are  CM-(or shown have  density  of  poly-electrolyte have  an  open  THEORETICAL  _ +  O  / C2H5  rr + H  U  j  2.10  Structures of the  DEAE  C2Hl4 I  +  \  / C2H5  charged Figure  O  W  CzK<* +\  and C M cellulosic  BACKGROUND  C2HS  X C2H5  uncharged ion-exchangers.  /  48  THEORETICAL microstructure  which  provides  easy  accessibility  of  BACKGROUND  the  ionized  sites  /  to  49 the  molecules.  During on  the  are In  cellulosic  formed, some  long  is as  is  a  greater  becomes  large  enough  to  occur.  ionic It  has  to  solution.  noted  with  the  very  C h a n g i n g the  low.  equilibrium, Usually,  the  If  is  ions  to  and  an  is  equilibrium' dissociation than  The with  is  that  is  of the  the  raised.  unity,  the  buffer  that  bonds  to  part  of  of the  the  The  As  of  are long  as  molecules  carries the  to  the  migration rate  the  of  be  and  molecules.  the  all  the  of  is  in  ions  in and  simultaneously will the  disrupt  the  concentration. of the  increase  electrostatic will  then  in  'finite  molecules  is  ion-exchanger.  buffer  the  the  so  'tight  dissociating  probability the  and  termed  effectiveness  When  said  molecules.  ion-exchanger  to  the  bonds  exchanger  is  system  in  all  adsorbed  the  dissociate  the  increased.  probability  the  molecules  concentration, is  on  constantly  changes  more  dissociation  situation  the  charges  exchanger  molecules  are  As  and the  the  adsorption',  bonds  any  by  molecules  exchanger.  This  polyelectrolyte  binding  the  exchanger  adsorbed  same.  opposite  molecules.  simultaneous  'tight  of  between  between  remain  the  partners  adsorbed  less than  all  buffer  enough,  the  will  disrupted  bonding  high  of  the  in the  simultaneously  column.  slower  for  the  in  composition  change  increase  compete  dissociate  will  that  that  the  formed  electrostatic  probability  equilibrium is  concentration  the  the  bonds  so  formed  between  charged  Individual  but  of  are  poly-electrolyte  remains  be  reforming,  the  molecules  environment  equilibrium  buffer  The  bonds  affinity  number  the  there  and  the  adsorption'.  the  ion-exchanger  there  rare  dynamic  electrostatic  conditions,  molecules bonds  adsorption,  of  in  buffer buffer  bonds move  to  down  adsorption simultaneous  will a l w a y s  be  THEORETICAL Therefore, approach under  is  to  negative  state.  ions  positive  attractive weaker.  of  molecules  concentration equilibrium'  The the  effective  state  and  sample  overtaken  by  they  will  band  the is  move of  be  the  rear  the  at  the  rear of the  at  is  charges  of  ions  adsorption  and  will  be  will  the  column  at  the  of  general  example,  adsorbed  gradually  molecules  molecules  eluted the  column more  gradient.  The  same  rate  always move  as  by  either  eluant  on  the  a  'tight  increased,  the  exchanger,  and  molecules.  The  the  become  progressively  equilibrium' collected.  reach the  The  in  state.  As  the  'finite  'finite  the  higher faster.  the  The  buffer  adsorption  the  power  the  than That  lightly  will  gradually  becomes  bound  less  will  equilibrium'.  move  However,  Therefore,  of  m e d i u m surrounding  the  move  faster  Since  the  buffer. that  is  elution.  buffer.  molecules  eluant  stepwise  ion-exchanger  adsorption  than  or  concentration  molecules  eluting and  gradient  buffer  increases,  slowly  increasing  band will  'finite  molecules.  the  is  the  acid  50  a  For  originally  negative  and  column  more  c a n be  move  band  the  molecules.  hyaluronic  positive  to  /  eluted.  the  the  the  the  the  as  the  concentration  concentration  constantly  at  be  of  molecules  exchanger),  shield  method,  buffer  at  buffer  increased,  down  molecules  (anion  will  down  buffer  such  exchanger  molecules  the  molecules  until  the  elution  adsorption'  shield  chromatography,  poly-electrolyte  molecules  will  will  ion-exchange  of  'tight  buffer  immobilizing a  the  eluant  move  further  adsorbed  the  then  As  mixture  buffer  is  in as  the  between  gradient  increased.  the  If  molecules  will  the  ion-exchanger  of the  forces Some  the favor  the  ions  performing  charged  charged  adsorption'  slowly  that  negatively  positively  In  apply  conditions  consider  the  when  BACKGROUND  at  the  result  in  front, a  they  are  and  faster,  eluting  power  the  molecules  sharper b a n d ,  or  THEORETICAL higher  resolution  disadvantages. maker buffer  and is  in  a  the  elution  power  the  unadsorbed  molecules  in  a  simple  eluant  remains suitable  elution  higher  the  Some  time. in the  a  one,  series is  successively  other  molecules will  bound  eluting  move  are  that  because  needed.  is  of  the of  used.  lower.  to  a  smaller  volume  charges  rates.  column  Therefore, is  faster, of  in  the  the  until  is  are  used,  lower  eluting into  the  'finite of  the  subsequent  advantages  is  eluant  the  rest a  molecules  buffer  disadvantage  The  its  stronger  the  in  51  gradient of  bound molecules  net  the  a  each  increase  /  has  volume  solutions,  Each  different  method  primary  is  lightliest  at  include  the  buffer  different  method  since  eluate  the  The  elution which  Also,  applied.  (adsorbed)  power  gradient  equipment,  more  bring  tightly  are  more  method,  previous  state  concentration  and  the  concentration  elution  will  method  equipment  obtained.  collector,  stepwise than  However,  requires  molecule  equilibrium'  of  stepwise  method  the  state.  adsorption  obtained.  fraction  power  of  eluant  The  large,  In  is  BACKGROUND  of  the  collected and  only  resolution  is  C H A P T E R 3. E X P E R I M E N T A L  3.1.  INTRODUCTION  The  objective  concentration  and  of  Different  fractionation  with  was  obtained a  by  function  determining described.  the  Then,  of  unknown the  and  1947)  the  of  described  method.  Known  preparation  modified  ACID  method  procedure  used  The  Bitter  in  a  results  this  was &  concentrations of  velocity  of  chapter,  a  then  hyaluronate  followed  taking  of  viscosity  line.  method  the  were:  52  obtained  by  conductivity  of  hyaluronate method  solutions  in  the  first  molecular  description  in  presented  is  for  of  the  Finally, analytical  Chapter  4.  DETERMINATION  for  -  the of the  standard The  of  measurements.  are  of  solutions  according to  obtained  (1962) the  by  in  effect  flow  chemical  measurements  modification  Muir  calibration  uronic acid carbazole  a  the  velocity  study  were  solution  hyaluronate  CONCENTRATION  hyaluronate by  of  and  the  of  hyaluronate  cellulose,  this  for  sedimentation  discussed.  HYALURONIC  concentration  the  conductivity  of  fractionating is  experimentally  flow  DEAE-)  concentration  detail.  taking  the  fractions  In  This  dialysis,  The  on  investigate  sedimentation  for  of  3.2.  the  procedure  method  is  to  weight  concentration.  in  ultracentrifuge  weight  measuring  discussed  of  was  diethylaminoethyl-(or  is  method  work  molecular  weight  the  this  molecular  hyaluronate.  as  PROCEDURE  determination the  carbazole  modified  uronic  (glucuronolactone)  reagent  solutions  of  method acid were  made  up  unknown (Dische, carbazole used  for  for  the  EXPERIMENTAL 1.  0.025M gr.  di-sodium  1.84  0.125% carbazole  3.  glucuronolactone prepared  by  Aldrich)  standards  dilution  of  with  in  mL an  be  of  acid  bath  splashing  Then  bath  0.2  mL  swirled  again  then  heated  in  a  Varian  1  cm.  Model  For  triplicates.  of  standard  was  the  sp.  ag/mL  acid  (99%,  jug/mL,  of  bath  was  for  the  for  each  test  the  true  glass  the of  content for  in  10  follows.  test  tubes  standard a  or  pipette  stoppered  added  to 15  solutions at  into  as  and  the  min.  sample onto  the  swirled  tubes in  and  a  with  appeared  vigourously  temperature.  appeared  sample,  as  49.25  985  means  were  heated  reagent  or  taken  acid,  benzoic  described  of  by  the  room  spectrophotometer  absorbance  absorbances  water  absorbance 2390  each The  boiling  mL  tubes  then  is  pipetted  when  about  content  reaction  One  The  were  to  and  with  solution,  layered  stopped  carbazole  until the  the  The  of  4°C.  acid.  tubes  and cooled  were  temperature.  The  39.40  saturated  were  carefully  was  sulfuric  Aldrich).  reagent  the  in  53  (Stanchem).  29.55,  standard  about  was  Swirling  homogeneous.  acid  of  water  acid carbazole  to  solution)  cooling.  boiling water  uronic  sulfuric  ice  avoiding  continual  the  ethanol  19.70,  distilled  (99 + %,  for  9.85,  BDH)  /  BDH).  in absolute  D-glucurono-6,3-lactone  (hyaluronic  three  (KODAK)  stock  cooled  to  a n a l y t i c a l reagent,  a  Five  acid,  or  from  procedure  (AnalaR,  2  (AnalaR  2.  The  tetraborate* 1 O H 0  PROCEDURE  be  nm.  carbazole  was  and in  tubes.  then  color)  reaction and  The  tubes  tubes  were  to  then  of test  the  The  cooled  was  Pathlength  recorded,  absorbance.  the  homogenous.  min.  (purple 525  to  the  room  read cells  was  done  average  of  on was in the  EXPERIMENTAL 3.3.  FRACTIONATION  3.3.1.  Desalting  Fibrous numbers %  human  Since  commercially  a  Then,  60  mL  Since  HA  1000  To  added  to  occasionally then  the  HA  reprecipitated  with  for by  The  (Na  by  g  +  K  had to  be  +  +  )  the  flask  flask  600  m L of  gel-like  was  to  HA  1300 X g  for  to  free,  7 the  ethanol.  were  ethanol  added.  the  HA. in  a  (Stanchem)  The  precipitates  Lot  3  overnight  solution.  fibrous  of  dissolve  left  absolute  1,  salt  precipitation in  the  about  Grade  of hyaluronic acid  to  white  redissolved  540  mL  of  mL  of  salt,  was  dissolved  RB  The  flask  to  was  300  of  70  2.0779  the  at  content  desalted  H A , about  centrifuging  (Sigma,  for fractionation  water,  hours.  salt  added  containing  5  about  free  removed,  a  were  in  the  flask  essentially  Buchi)  water  slowly  collected  with  120,  had  acid  flask  which  5  min.  in  a  mL  of  water  was  formed  centrifuge  Centrifuge, U n i v e r s a l Model U V ) .  reprecipitated  flask.  hyaluronic  hyaluronic acid was  precipitate  collected  The  75F-0519)  distilled  swirled  (International  cord  m L E r l e n m e y e r flask,  of  54  ACID  hyaluronic acid used  dissolves  refrigerator.  were  the  /  Acid  umbilical  and  obtained  To  HYALURONIC  Hyaluronic  124F-0329  (w/w).  were  OF  PROCEDURE  was  remove  m L of  then  solvent  distilled  ethanol  in  ethanol. (90%  in  70  connected (ethanol  water  about  were  4-  to  60  The  v/v). mL a  The of  to  After  collected  resulting  distilled  rotary  water). added  HA  evaporator  dissolve  the  was  in  was  100  mL  (Rotavapor  RE  solvent residue  a  then  further  H A , which  water  all the  and  had  been  remaining  EXPERIMENTAL in  the  (Virtis) stored  flask.  The  for  days  3  desiccated  HA  solution  to  yield  in the  was  1.61  freeze-dried  g  of  white  in  a  fibrous  PROCEDURE  Unitrap  II  /  55  freeze-dryer  HA.  The  HA  HA  was  a  was  then  freezer.  3.3.2. Preparation of DEAE-cellulose  The reduced  D E A E - c e l l u l o s e used  cellulose  mEq/g.  The  hydrated  In  hydrogen become  cellulose  so  molecules.  its  the  dried  to  (treatment  DEAE-cellulose)  to  cellulose  the  large  expose  procedure  became of  the  form.  stated  Before  fully  caused  molecules. acid  ion-exchange  of  some  it  to  the  was  1.0 fully  charged  cellulose  the  then  fine  of  ion-exchange  Therefore, and  fibrous,  capacity  use  accessible  O H groups  which  HA  England),  dried  concentrated  these  used  DEAE-cellulose  flask.  many  with  a  sites  the  follows  W h a t m a n A d v a n c e d Ion  17.5  in  of  formed sites  cellulose  alkali  HA  to was  for  the  sites.  Precycling  The  then  state,  fractionation  Maidstone,  supplied  charged  within  inaccessible  the  Whatman,  was  that  bonds  precycled  for  (DE23,  for  g).  Each  portion  H C 1 solution added  to  the  Exchange  (DE23, of  (0.5N, flask  the  28.5  cellulose  method Cellulose  g)  was  was  described  to  diluted from concentrated (15  mL  of  liquor/g  Information  ( W h a t m a n , Clifton,  precycled  added  in the  dry  a  in 500  2  portions mL  volumetric weight  New  of  solution, ion  Jersey).  (11.0  plastic  Leaflet  g  and  Erlenmeyer BDH)  exchanger).  was The  EXPERIMENTAL content and  was  the  (judged  occasionally  wet by  was  to  until  flask. the  then  alkali  the  filtrate  Fines  filter  with  the  disc  The  to  slurry  at  of  wet 7.30 was  removed  by  times  remove  to  n e a r neutral.  the  filtrate  cellulose  was  (0.5N,  from  reagent  weight  min.  The  ("fines")  the  bottom of  wet  fine  The  liquor  was  at  grade  ion  pellets,  was  then  pH  4  500  mL  BDH)  was  The  filtered  washed  56  filtered  the  exchanger).  supernatent was  was about  transferred to  of  cellulose  suction  of  the  column It  NaH PO„  to  the  2  settle an  for  In  because was  (15 30  aspirator.  fines.  cellulose  column).  r e m o v a l of the  through  most  the  DEAE-cellulose  0.5M  allowed  of  packing.  precycled by  within  the  column  procedure for  The adjusted  was  supernatent  wet  30  repeated.  The  until  liquor/g d r y  was  performance  The  funnel  The  stirred for  fragments  non-uniformity fines.  hour.  /  content and  in  a  the  funnel  Removal  Small (the  mL  1  a  solution  (15  occasionally  for  in  Fisher).  NaOH  flask  treatment  washed  p H paper,  Erlenmeyer added  cellulose  stirred  PROCEDURE  could addition,  of  a  necessary,  mL min. The  dispersed  is  in  cloudy  above  they  therefore,  liquor/g d r y The  the  bed  could  tendency  ion exchanger  was  clog  to to  remove  the  below.  Na HP0 , 2  weight  was  pH  4  of  supernatent  procedure  interfere accentuate  described  2mM  support  cellulose). was  then  repeated  5  EXPERIMENTAL  exchanger  exchanger into  DEAE-cellulose the It  in  funnel  The  was  p H of  funnel  until  had  to  equilibrium  was  solution,  10.0. a  and  with  redispersed i n  measured  then  be  by  adjusted then  the  first  the  filtrate  a to  15  equilibrated the  volumes  was  of  bring  buffer.  57  7.30  by  was  at p H  (Orion  0.5M N a H P O 2  f l  .  2 m M phosphate  about  6.90.  the  weakly  ionizing  The  'fines  reduced'  buffer.  The p H  2 m M phosphate  Digital Ionalyzer/501  equilibrated with  filtrate  to  dilute  Phosphate  Research),  The  slurry  buffer buffer  (see was  7.30.  2mM  phosphate  buffer  i  -  precycled  filter  Figure  /  Equilibration  The  of  PROCEDURE  3.1  T h e set  up  for the  DEAE-cellulose  paper  equilibration of the  ion-exchanger.  was was  about filtered  Figure added  to  3.1). the  EXPERIMENTAL  PROCEDURE  /  58  3.3.3. P r e l i m i n a r y F r a c t i o n a t i o n  Preliminary described  by  Cleland  DEAE-cellulose was  used  free  solution  water)  in  75  column  was the  of  was  (about  fractionation  fines  hyaluronic with  eluted of  weights  obtained.  7.2  mL  mixture flow of  to  was  fine  pores  water.  in  the  increasing  charged  to  about  However, The  glass was  The  2  cm the  disc  fold  excess  of  was  then  charged  to  method  at  acid  cm  of i.d.  fractions  of  acid  the  slow  was  rate  of  by  22  15  probably  was  then  decanted  glass  filter  disc  as  of  eluate  NaCl. of  out  The  each  at  flow  salt  room rate  were  temperature  was  concentration  controlled was  The  2  washed  column  carried  H 0.  in  gravity  i n the  then  mg  by  T h e content  coarse  molecular  mL  of  a  increasing  72.5  bottom  with  column  HA/mL  column. T h e  the  column  the  a  — after  was  salt  occurred  then  slow  A  DEAE-cellulose  7.0  packing  very  flow  in  the  Water  mg  increasing  solution  DE23  column;  35  successively  T h e column was rate  (about  with  0.6. g  method,  equilibration step.  water  hyaluronic  long.  for  no  method  six  a  flow  was  and  about  slurry  a  reason  filter  concentration  stop-cock.  3.8  a  the  their  distilled  hyaluronic  with  In  to  in  mixture  I,  according  (1984).  Sections  there  elution  fractionation  acid solution.  elution  The  stepwise and  and  of  done  Cleland to  dissolved  slurry  mixed  bed  Kontex  Stepwise  a  a  noticed.  hyaluronic  by  was then  give  distilled  i.d.  0  was  flow  cm  2  step  water).  NaCl,  preliminary H  a  were  and  according  acid  by  concentration  In  (1968)  reduction  DE23/mL  was  were  al.  prepared  mixed  mg  et  experiments  min.,  because  clogged  the  to  collected  with  into bed  with  a  no the the 5.1  support.  successively  about in  mL  0.5  L/hr  1 fraction.  EXPERIMENTAL The  concentration  modified  uronic  fractionation  the  evaluation are  amount the  of  the  are  of  the  were  The  solution pH  of  could  be  there  was  no and  Therefore, However,  Table  the  they  3.1  Eluant  the  that  'tight  HA to  the  out  by  to  close the  7.  were  not.  A  in  Section  3.1.  sample  As  The  is  pH,  was  the  of  in  volume,  the  and  the  two  were  at  (pKa pH  tightly  p H when  of  leading  to  3.1,  a  mixed  9.5) 7.30)  large out  the  by HA  Therefore,  slurry and  slurry in  the  hyaluronate  adsorbent.  dissolved  adsorb the  cellulose  59  results  washed  cellulose  /  by  fractionation,  the  the  of  was  HA  cellulose  The  Table  just  charged  should change  pH  of p r e l i m i n a r y fractionation  Eluate  from  the  Before  3.2.  amount  adsorbent of  determinated  calculations  and  obvious  beginning  When  (negatively  molecules  described  water.  the  be  HA  then  adsorption equilibrium' with  which  HA  was  cellulose  the  together,  in  fraction  concentration  A.  solution,  change the  as  Table  at  washed  mixed  Results  [NaCl],  bound to  were  assumed  charged,  at  were  in  Appendix  suggests  H A molecules  HA  in  not  not  each  glucuronolactone  presented  This  in  method  tabulated  H A was  water.  molecules  hyaluronate  acid carbazole  the  collected  of  PROCEDURE  was  about  distilled  7.  water,  together,  would  the  and  if  be  positively  negatively  charged.  on  the  cellulose  the  H A solution  molecules. was  mixed  I  Glucuronolactone /ig/mL  HA  recovery,  M  mL  concentration,  0  50  39.63  25.5  0.1  420  5.51  5.3  0.2  381  4.78  4.2  0.3  446  6.45  6.6  0.4  449  3.61  2.7  0.5  440  1.80  1.8  mg  46.1  EXPERIMENTAL with  the about the  cellulose  7  to  pH  buffer  64%.  of  The  mixture  cm  long.  was  was  not  pH  lost  was  increasing by  at  presented tightly  column  each in  some  weight  of  to  the  moisture the  of  the  controlled  cellulose  large  61.4  pH to  mg  was  of  i.d.  and  H A charged  to  was  the  a  in  weak  with I  the was  HA-cellulose  A  pH Flow  collected of  to  HA  in  was  not  be  the  HA  column  dissolved about  15  mL  large  to  of  the  72%.  eluted. during be  was  of  bed  salt  cellulose molecules.  about  0.7 with  solutions  was  about  0.5  The  Probably, HA  handling. less  DE23  method  fraction.  Since  3  phosphate  HA  a  about g  2mM  elution  one  might  1.4  the  give  rate  in  excess  for  stepwise  The  could  the  in  column  buffer.  absorbed  If  Therefore,  fractionation of  from  became  adding  of  available  by  NaCl.  recovery  cellulose  slurry  was  eluted  phosphate  The  Section  HA)  out.  60  added.  equilibrated  transfer  solution.  adsorbent cm  &  by  H A were  3.3.2)  HA  5.1  of  A  Section  the  was  /  column.  7.30.  then  was  washed  the  concentration 3.2.  the  was  to  the  2mM  salt  Table  bound  and  concentration  adding  was  (cellulose  probably due  enough  was  species  the  pH  (pH  preliminary  with  into  two  and  II,  in  7.30)  buffer  in  according  poured  mentioned  HA  buffer,  that  as  phosphate  recovery was  fact,  s l u r r y changed  adsorbed  7.30),  The  In  unequilibrated cellulose  the  experiment,  mixed  ensure  7.30  eluate  true  was  The  hygroscopic, the  HA  phosphate  to  successively  are  of  s m a l l column to  7.30  used  The  one  processed  pH  at  and  amount  ImM  was  kept  the  p r e l i m i n a r y fractionation  (equilibrated, buffer,  of  weak  (2mM,  from the  pH  suspected.  a  The  hence  when  fractionation  In mL  the  (Section  mixture  10  changed  then  next  buffer  about  was  phosphate  just  was  (Equilibration), the  uncharged, the  slurry  PROCEDURE  than  L/hr. results  some was  HA very  Therefore, 61.4  mg.  EXPERIMENTAL From the in  Table  3.2,  H A was the  adsorbed  original  small.  In  molecular  this  weight  to the cellulose.  sample  because  fractionation  collected  in  one  was  collected  in  subfractions  elution  the  fraction.  volume.  There  In  in  The  was  was  H A recovery  experiment,  was  with  fractionation  the  the  order  next to  intrinsic  apparently  little for  eluate  the  low  for each  the  obtained.  low molecular  fractionation  study  PROCEDURE  [NaCl]  weight H A eluant  experiment,  viscosities  of  the  a  of  100.4  of  61  M o s t of  concentration  change  /  was  of  the  salt  eluate  H A concentration  fractions  were  also  determined.  In  preliminary  fractionation  4.0  m L 2 m M phosphate  20  mL  2 m M phosphate  column. NaCl. The  T h e column Flow  Eluant  then  was  about  of  HA  was  some  3.2  was  H A was  Results  [NaCl],  tightly  pH  eluted  0.5 at  solution  p H 7.30  buffer,  rate  recovery  because  Table  buffer,  III,  was  7.30. with  mixed The  with  successively  63%.  adsorbed  to  Again, the  H A dissolved  about  mixture  L / h r . T h e results least  mg  was  2.0  the  in  low  was  yield  in  to  the  concentration  presented  cellulose  DE23  charged  increasing  are  g  in  a n d couldn't  Table  of  3.3.  probably be  eluted,  of preliminary fractionation II Eluate  volume,  Glucuronolactone  ng/mL  HA  recovery,  M  mL  concentration,  0  15.5  0.015  0.2  0.1  378  0.014  0.6  0.2  373  0.033  1.6  0.3  407  0.204  11.3  0.4  373  0.372  19.3  0.5  413  0.203  11.4  mg  44.4  EXPERIMENTAL  Table  3.3  Eluant  Results  [NaCl], M  of p r e l i m i n a r y fractionation  Subfraction volume,  mL  23.6  PROCEDURE  /  III  Glucuronolactone concentration,  Mg/mL  H A recovery, mg  = 0  = 0  1.  100  = 0  = 0  2.  100  = 0  = 0  3.  100  4.  100  1.  100  11.09  2.5  2.  100  2.76  0.6  3.  100  4.  100  5.  33.6  1.  100  117.7  26.8  2.  100  33.28  8.0  3.  100  4.  100  5.  100  1.  100  74.10  16.9  2.  100  24.30  5.5  3.  100  4.  100  5.  69.6  1.  100  11.57  2.6  2.  100  2.22  0.5  3.  100  4.  100  5.  70.4 63.4  62  EXPERIMENTAL and  there  there 2.  was  The  3.3.5.  the  subfractions  and  0.3M  two  and  molecular Wang,  the  was  HA  0.4M  1970):  molecular sample  solutions  when  the  molecular  were  were  salt next  weight the  HA  large.  is  precipitates was  concentration  decreased  higher  eluant  fractionation  section.  In  fractionation  physical  measurements  were  still  salt  obtained with  of  experiment, More  the  reliable  viscosity  3.2.  0.2M  volume was  solutions  for  the  and  the  (Cleland  &  higher  molecular  weight  The were  not  were then  in  dialysed.  Nevertheless,  HA and  difference  recovered then  A  for  increased  large  performed in the  were  plot  the  added.  then  results  for  NaCl.  the  of  respectively.  5  lower  because  eluant  HA  4.63X10 ,  Figure  in  H A was  Section  viscosities  plot  were  viscosity  equation  H A solutions  concentration  experiment  made.  in  the  and  5  shown  to  m L / g , respectively,  viscosity  dissolved  fractionation  intrinsic  Mark-Houwink  of  eluate,  fraction  and the  63  subfraction  according  intrinsic  958  each to  NaCl  The  that  However,  0.4M  The  intrinsic  than  1  3.5.  4.49X10  the  behavior  not  3.2.  the  be  steeper  This  is  to  subfraction  and  Section  from  For  precipitated  m L / g and  material, is  from  sample.  in 0 . 2 M N a C l ,  Figure  934  8 1 6  was  to  in  HA  fraction  dissolved  according  presented  sample  original  fraction  determined  1961).  weight  eluant  is  the  NaCl  fraction w a s  polymeric  (Tanford,  each  each  [T?] = 0 . 0 2 2 8 M ° "  weight  molecular  0.3M  in  were  molecular weight  The  of  fractions  same  by  in H A concentration  measured  fractions  weights  the  absorbed  large decrease  solution  For  moisture  T h e H A of each  each  the  a  two  combined  for  was  PROCEDURE /  dialysed obtained.  scale next before  EXPERIMENTAL  PROCEDURE  /  EXPERIMENTAL  PROCEDURE  /  65  3.3.4. Fractionation  A  slurry  cellulose  in  containing  of  360  2mM  1.39  mL  phosphate  g  of  E r l e n m e y e r flask.  about  10  mixture long.  was  The  Stepwise with  most  then  elution  carried 7.30  out  by  at  of  was  was  increments  Results  0.075M  0.5M.  The  each  the  were  collected.  Elution  at  HA  concentration  concentration conducting was  was  the kept  fractionation  HA  for in  resumed  was  the  the  at  column  with  was  0.325M  at  bind  mL  of  Then  eluant  salt  least  dropped by  8  the  giving 2mM  40  mL  a  500  mL  a  that  after  cellulose.  The  bed  14.0  buffer.  0.10M  0.40M  at  final  step  of  solutions  was  adjusted  a  cm  phosphate  NaCl  subfractions  (0.5M  to  about 2  carbazole For  NaCl)  concentration  collecting  L/hour.  to  test  the  3  in  to  elution  was  to  pH  The  HA  of  eluate  and  then  3.2).  The  mL  by  0.325M  20  it  was  thought  column  was  left  However,  at  the  subfractions  ng/mh.  above  solution.  each  10-20  Although  the  5  mL  terminated  8.  and  500  which  subfraction /ig/mL,  about  was  (Section  elution  of  after  slow  in  of  showed  to  column  aqueous  step.  concentration  1.0  NaCl  with  the  acid  was  600  buffer  DE23  buffer.  had  stopped  eluate  out  would  processed  solution  phosphate  glass  about  uronic  about  i.d.  particular salt  determined  modified  acid  cm  each of  last  a  p r e l i m i n a r y experiments  with  pH  a  2mM  with  of  from  5.1  at  mixed  g  in  salt concentration, except  was  28.0  acid  carried  of  collected,  found  a  washed  were  rate  in  then  approximately  hyaluronic  adding 2 m M phosphate  For  eluate  the  packed  column  buffer  hyaluronic  plastic  min.,  containing  the  the  flow  solution,  concentration degradation  overnight.  beginning,  eluant  NaCl  HA that  when  the  Elution flow  rate  of was of  EXPERIMENTAL eluate  was  very  slow.  was  controlled  at  min.  Elution  was  concentration  lL/hr.  continued  when  the  eluant  above  the  higher  concentration  eluant  salt  concentration  on,  bed  less  was  10  for  9  was  (500  back  to  m L ) , the 14  the  regular level  collection  was  66  collected,  time in  and  was  85  which  the  terminated,  the  Mg/mL.  one  pipetted  was  rate  subfraction  than  elution  step  the  flow  until  salt  out  added.  concentration.  with  the  F o r subfraction  of H A was  Also,  the  Later  PROCEDURE /  before  This  Each  concentration fresh  uronic acid carbazole  salt  procedure  subfraction  was  solution  ensured  was  a  then  of  the  discrete  analyzed  next  rise  in  for  HA  test.  3.3.5. Purification and Isolation of H A  The  eluate  different  salt  rendering  the  concentrations, fractions  sedimentation subfractions  fractions  each  To salt  hyaluronate  evaporated  on  RE  A n exception  and  were  6  evaporated eluate IL  to  sub-fractions  round  120 was  evaporated another  bottomed  HA  the for  for  that to  a  flask.  large,  which salt  the  solution.  evaporation  Section  later  a  work, 85%  solution,  was  or  were  the done,  eluate, and  were low,  more  the  9  the  and or  subfraction  by  few  combined  1  portion  and  of  subfractions  of  thus  first  solution  volumes  in  (viscosity the  concentrated  NaCl  Since  very  measurements  concentration, to  3.3.4  were  contained  0.325M  concentrated  concentrated  were  for  evaporator  for  in  concentrations  materials  that  rotary  described  further physical  concentration,  eluted  dryness.  and  prepare  eventually  a  as  unsuitable  velocity). of  collected  to  to  4  was  combined  portion,  in  a  EXPERIMENTAL HA of  NaCl  was  present  in  with  salt,  few  salt.  a  concentrated  and  was  dryness  was  out  75%  in  little  as  water  bit  of  salt  containing  the  -28  to  0.25M,  named  the  a in  shown  phase  In  the  was  Hg  0.325M  30  min.  0.4M  In  could  other  not  HA  of  two salt  the  ethanol. to  three  went  into  decanted  and  The  was  be  content,  Therefore,  was  ethanol  subfraction  0.325M-9,  small.  flask.  connected  for  4  step,  ethanol, of  about  washed  bottomed  traces  after  volumes  supernatant  nearly of  steps.  four  lot  most  lower  latter  and  were  and  with  precipitate  water  in.  0.325M,  very  a  67  remove  that  was  with  round  residue  0.325M,  0.25M,  had  The  remove  -30  required to  fractions  Since  solutions  were  twice  (v/v).  in  the  solutions.  reprecipitation  precipitate.  flask  were  3  ethanol  fact  solution  the  and  To  at  for  precipitated  decanted  of  the  in  the  after  evaporator.  for  concentrated  precipitated  in  especially  first  then  Scientific)  and  HA  rotary  obtained  eluate  were  a  the  experiment of  HA  H A remained  the  (Central  fractions  of  redissolved  the  residue,  Pump  NaCl  the  H A and  on  amount  volumes  precipitate  which  HA  four  solutions,  precipitate  was  from  reprecipitation steps  cases,  solutions  with  solution, the  to  supernatant  times  the  some  fail  the  precipitation  amount  In  might  The  steps,  large  precipitated. HA  a  Preliminary  reprecipitation words,  precipitated out  was  saturated the  then  PROCEDURE /  solution,  evaporated  remained  to  a  or  more.  in  to the  Cenco-Hyvac The H A  9,  0.4M  and  0.5M  and  0.5M  Fractions,  respectively.  3.4.  ACID  The  HYDROLYSIS  HA  OF  fractions  HA  obtained  in  the  fractionation  experiment  (Section  3.3)  5 was  found  to  be  in  a  narrow  M . W . range  (6.99  to  11.1X10  ).  Smaller M . W .  EXPERIMENTAL fractions  were  hydrolysis Cleland  method  in  then  was the 1M  in  solution  the  HA  degradation  with  of  was  heated  HA  solution  HA  were named  In 0.325M-9 for  at  into  hydrochloric  H A followed  15  acid.  the  one  above.  1  half  of  plate-stirrer to  one  described  100 The  of  68  The  acid  described  and Then  back  by  concentrated  HC1  Distilled  a  hot half  of  neutralized  in  the  250  another  as  described  1  hour The  and HA  1  the  HA  with  solid  7.0  with  mL  hour. above. the  and  plate-stirrer  solution  7.0  on  concentration  about  for  water  m L mark  pH  3.3.5.  beaker  Then  the  The  two  other  fractions  for  2  obtained  Fraction.  prepared  T h e H A fraction obtained  was  200  one  was  0.2M  solution  to  in  solution  on  mL  titrated  HA  Section  of  the  about  H A solution  HA  HA/mL  heated  for  m L of distilled HA  mg  1  per  solution.  about  hydrolysed  HA  mL  reached  pH  and 2 h r A H  solution  HA  the  50+2°C  about  acid  to  The  meniscus  the  at  beaker. 17  hour.  and  Fraction  About  stirred  pellets)  Fraction  min.  was  grade  as  in  of  The  solutions,  a  HA  beaker.  neutralized  lhrAH  added  0.325M  mL  mL.  until the  for  one  mg  250  a  hot  purified  a  were  of  50±2°C  the  Fraction  150  solution  remaining  addition,  about  grade)  HA  was  to  solution  reagent  on  to  (4.57  added  solution  decanted  (BDH,  neutralized  a  solution  were  the  The  PC351) was  described  to  giving  HC1. T h e  hours,  degrading  HA  reagent  added  HC1.  (Corning,  cone.  of  withj water  beaker,  above  ml  analytical  NaOH  for  concentration)  then  were  used  44  diluted  (BDH,  by  /  (1984).  About NaCl  obtained  PROCEDURE  water was was  by  dissolving  was then  about  acid hydrolysed neutralized  named  15minAH  and  120 as  mg  described  purified  Fraction.  of  as  EXPERIMENTAL 3.5.  FRACTIONATION  In  each  hydrolysing M.W.  of  the  H A , the  HA  then  OF  with  a  fractionating  ACID  HA  range  small the  HYDROLYSED  fractions of  range  of  resulting  HA  in  is  the  broad.  M . W . was  sample.  /  69  HA  obtained  M . W . of  PROCEDURE  done  T h i s is  previous An  by  attempt  acid  described  Section to  by  acid  obtain  hydrolysing  low  HA  and  in Section  3.4.  Fraction  and  below.  3.5.1. Acid Hydrolysis  The About 120  procedure  400  mg  beaker.  mg  0.325M-9 Then  HA  solution  and  stirred  then in  of  30  HA  on  solution  mg  Fraction) mL  a  acid  (50  of  hot  0.25M  were  NaOH  dissolved  precipitated  50 and  and  in  to  described  230  320  H C 1 were  at  pellets  of H A was  Fraction,  1 M concentration  plate-stirrer  with was  hydrolysis  concentrated  approximately  neutralized  the  for the  mg  mL  added  of to  in HC1. T h e 52.5°C  back  for  titrated  freeze-dried  as  0.325M  the  in  a  beaker  400  to  H A solution  15 to  water  mL  give  was  heated  m i n . T h e solution pH  described  about  7.0.  in Section  an  was  The  HA  3.3.1.  3.5.2. Fractionation  The An  HA  procedure  solution  of  for 324.4  phosphate  buffer  was  from  g  DE23  6.5  charged  to  of a  5.2  fractionation  cm  mg  acid  one  HA  a  slurry  with  as  described column.  the  hydrolysed  mixed  i.d.  followed  80 in  The  mL  Section mixture  described  dissolved of  3.3.2. was  in  in 10  Section mL  of  3.3.4. 2mM  D E A E - c e l l u l o s e prepared The  packed  mixture from  4.1  was cm  then to  a  EXPERIMENTAL constant  bed  column.  The  Stepwise  of  column  was  elution  and  0.5M  mL  each  10-20  height  NaCl were  3.2).  The  HA HA  (subfractions  1  0.25M  0.325M  and  to  15mAH-0.325M  3.6.  At  until  with  each the  the  were HA  about with  salt  air 50  pressure mL  0.1M,  concentration,  was  0.25M  in  were  as  top  of  phosphate  0.25M,  eluate  0.325M,  by  the  was  and  in  Section  0.4M  of  less  carbazole  1)  the  buffer.  subfractions  the  described  named  the  2mM  (subfraction  out  at  eluate  determined  eluate  precipitated fractions  of  0.175M,  H A concentration  concentration in  Fraction,  applying  70  250 than  method  0.325M  eluate  3.3.5.  The  Fraction  and  hyaluronic  acid  15mAH-0.25M  respectively.  DIALYSIS  Many (e.g.  physical  viscosity  order  to  and  compare  literature in  5)  by  carried out  solutions.  The  cm  washed  then  collected  Mg/mL.  (Section  was  2.6  PROCEDURE /  results,  0.2M NaCl.  3.4,  3.5)  mg/mL solutions were  was  sedimentation properties the  Each  in that  to  be  buffer  available velocity)  of the in  Since  in  the  have  as  30  m L of  there  dialysed  in  concentration.  was  of  prepared  H A fractions  salt  0.2M NaCl  NaCl in to  the  for  measured  the  and  purified  0.2M  literature  been  including estimates  H A solutions  dissolved  concentration. were  properties  in to  in  molecular used the  in  this  previous  give  a  0.2M  NaCl.  In  weight  with  the  study  Sections  H A solution  purified H A fractions,  make  sure  must  that  the  HA  be  (3.3.5, of  2-3  the  HA  solutions  EXPERIMENTAL 3.6.1.  made  Preparation of  The  dialysis  of  cellulose,  12,000-14,000 traces  of  before  being  and Some  Dialysis  tubing  Daltons.  washed  Tube  of  procedures  contaminants  from  were  as  treated  Procedure  the  tubings.  heavy  15  to  below  contained metal  20 20  were cut  molecular  and min.  to  then  and  glycerin  ions, 30  2.  Repeat  1.  3.  Soak  4.  Repeat  5.  W a s h in distilled  lOmM  2 L . of 50%  sodium carbonate,  3. water.  ethanol  and cm  71  remove  washed  to  lengths  (v/v).  for  a  therefore  most  1 hour.  (Spectrum)  cut  as  in length  employed  ImM EDTA  tubing  weight  I 1 hour in  step  tubing  material  The  and  membrane  follows:  S i m m e r for  in  for  listed  1.  step  width  and  this  in r u n n i n g water  vigorous  Spectrapor  dialysis  sulphurous compounds used.  was  flat  The  /  Tubings  used  25mm  PROCEDURE  at  plasticizer, was  treated  were  of the  remove of  off  cut  out  glycerin. the  dialysis  other tubing  EXPERIMENTAL Procedure 1.  Soak  2.  Stir  3.  Soak  for  /  72  II  1 hour i n  gently in  PROCEDURE  1% acetic  in distilled water 1%  sodium  acid for  (v/v).  15  carbonate,  min.  ImM  EDTA  for  15  min.  with  gentle  heavy  metal  stirring. 4.  Heat  to  5.  Repeat  6.  Soak  7.  Repeat  8.  Rinse  75 ° C step  2  in distilled water step  6  I  remaining  heavy  water  in  a  is  added  to  restrict  for II  metal  wide  2  the  to  75 ° C  min.  removal the  Unused  plastic  bacterial stored  15  for  ions.  -EDTA.  3  water.  for  is  mouth  then  Na CO  with fresh  Procedure  was  3  and heat  in distilled water  and  solution  Na C0 -EDTA.  4 with fresh  Procedure ions,  with  the  removal dialysis  bottle.  growth.  i n the  of  The  glycerin  of  few  bottle  some  sulphurous  tubing  A  and  was  drops  compounds  suspended of  and  and  the  stored  dichloromethane  containing  the  tubing  and  in  were water  freezer.  3.6.2. Dialysis  One one with and at  end  end  so  distilled HA the  that  the a  water  solution top  of  by  cleaned  dialysis to  was tieing  bag  check  for  pipetted two  dialysis was leaks.  into  knots,  the  tubing  was  formed. The  The  distilled  dialysis  leaving  sealed  a  bag. small  by  dialysis water The dead  tieing bag  was  tubing space  two  was then was  knots first  filled  poured then  above  the  at  out  sealed HA  EXPERIMENTAL solution of  surface  dialysis.  to  The  100  fold  volume  and  the  bag  Erlenmeyer  The least was hand  2  3.3  was  was  The  content  T h e set  of  Pasteur  up  for  of  dialysate the with  carried was  flow  solvent  suspended  in  covered  was  holding a  the  then  spinned  Dialysate the  for  excess).  dialysis  finished,  Figure  bag  was  flask  days.  while  allow  a  out  a  with  the  flask  of  stirred the  by  cold  a  bag  room  (see  during  dialysate  The Figure  removed  by  holding the  rubber bulb squeezed  performing dialysis.  NaCl,  of  the  3.3).  When  hours.  73  course  mouth  at  24  /  stirring bar,  controlled  about  the  (0.2M  magnetic  dialysate.  of parafilm  after was  a  of  piece  in  bag  pipet  was  vortex  changed the  in  into  PROCEDURE  4-7° C the  bag  in the  for  at  dialysis with  other  one  hand.  EXPERIMENTAL The  tip  of  plunged  the  into  centrifuge  3.7.  Pasteur  the  H A solution.  viscosities  viscometer  shown  in Figure  made  in the  stabbed  H A solution  determine  the  save  the  time  (Section  changed  after  addition  to  cleaned  by  Cleland  (1984).  the  was  viscometer  starting  of  The  prior  to  hours.  viscometer,  through  the  air  bubble  pipetted  out  and  stored  at  measurement Then and  concentration  the the of  of  use  were use  in  final  about  diagram  hyaluronic acid are of  to  the  be  and in  a  of  each  the  solution  was  of  in  solutions  and  10  min.,  a  a  the as  various  at  least  Dialysate  was  diluent.  Before were  recommended  was that  to  viscometer  dialysates  diluted  such  can  order  for  concentrated  solution  chosen  as  is  runs.  0.2M NaCl  used  diluted  dilution  solutions  successive  hyaluronic acid was  viscosity  that  measured  was  for  is  viscometer  determination.  dialysate HA  the  semi-micro  Cannon-Ubbelohde  against  started . with  of  of  viscometer  viscosity  2000Xg  was  HA  A  dialysed  dialysed  solution  Cannon-Ubbelohde  a n d rinsing between  The  the  a  this  concentrations  in cleaning  24  of  T h e viscosities  hyaluronic acid 3.6)  in  Co.).  feature  viscosities.  centrifuging  acid.  special  different  used  about  Viscosity hyaluronic  at  intrinsic  Solutions days  One  measured  Instrument  viscometer.  weights  the  The  were  (Cannon  3.4.  molecular  2  quickly  74  VISCOSITY  dilution  will  was  /  tube.  Solution  be  pipet  PROCEDURE  a  solution few  times  measured. the  by  of in The  viscosities  EXPERIMENTAL  Figure  3.4  T h e Ubbelohde  type  semi-micro  dilution  viscometer.  PROCEDURE  /  75  EXPERIMENTAL measured  for  recommended Preliminary but  the viscosity  HA  with  and  a  in  higher  (precipitates  of  HA  solution  was  diluted  concentrations,  the  became  F o r each  0  deviation than  of  5%,  viscosity  Then  the  by  term  0  was  sucking  filled  chromic  water,  the  with  and  was  then  air through  the  (T}-77 )/T} 0  of  as  0  »c  that  the  last  was  Also, the  of HA  at  different  the  intrinsic  to  solvent  viscosity  made.  If the  standard  closer  HA  The  choice  starting  determining  chosen.  of  lower M . W .  the  (for  diluted  of  concentrations),  viscosities  were  solutions  with  as  and  higher M . W .  the  T J became  thoroughly  acid,  acetone.  HA  with  solution  was  procedure  for  more taking  follows:  was  viscometer.  of  measurements  decanted.  with  solutions  the  same M . W .  for  salt  76  centistokes.  solution  with  chosen  higher  fact  the  /  in  concentration  solution was  4  the  for  same  the  measure  concentration  chromic  higher  fell  to  with  with  the  viscosity  «c  0.8  concentrations).  by  viscosity  described  the  was  the  for  salt  to  term  viscometer  acid  for  times  0  with  solutions  solutions  would be  H A eluted  lower  {T)-T] )/ri  starting  use,  4  which  concentration  of  solution  diluted  HA  higher  chosen  the  as  is  be  starting  few in  larger  measurements  the  distilled  a  error  another  viscometer  would  complicated  H A solution,  Before  solutions  with  was  for  HA  was  eluted  that  viscosity  lower  concentration  concentration  7j -  for  the  viscometer,  the  M . W . (precipitates  starting  viscosity)  a  the  and  shown  viscosity  general,  higher  have  and  M . W . , the  Therefore,  of  concentrations,  concentration;  different  solution  range  experiments  of different  higher  starting  PROCEDURE  cleaned  and it w a s  The  then  viscometer  The  viscometer  with left  was was  chromic for  rinsed dried  about  acid. an  thoroughly in  an  The hour. with  oven  or  EXPERIMENTAL HA into  the  into  a  solution lower  holder  Thermomix  (1  m L ) was  reservoir  and then  1420  J  of  the  inserted  heater  solution  to come  and  suction  was applied to tube  a n d finger  from  etch  mark  were  made  for  solution  The  to  reach  average  final  etch  constant  bath  was  at  finger  water time  4 measurements by  the  25.00  was  ±  time  1/10  into  20  over  to  tube  of bulb C . to  six  manufacturer)  move  dilutions  were  measurements  for each  time  B  reservoir J . T h e  minutes  before made  of efflux  the  placed  of the concentration  bath were  Five  G  allowed for  for the meniscus  measured.  77  .05 ° C b y a  were  placed  /  tube  then  reached the centre  diluted H A solution, the  through was  minutes  of dialysate  examined  of efflux  (determined  kept  A  was  amounts  with  measurements  F  pipette  T h e viscometer  the liquid  mark  F o r each  a  A p p r o x i m a t e l y 20  A until  solution  of the  from  removed. T h e efflux  equilibration  value  a water  temperature.  measured  examined.  Quadruple  viscosity  then  of the  temperature  taken.  were  D  into  the bath  b y pipetting  concentration initial  to  directly  viscometer.  (B. Braun).  the  Suction  charged  PROCEDURE  of the allowed were  concentration.  was multiplied with the to  give  the  kinematic  viscosity.  3.8.  SEDIMENTATION  The  VELOCITY  hydraulic  sedimentation  flow  coefficient  conductivity of a polymer solution c a n be related to its  by the equation: K'  The  sedimentation  analytical the  coefficient  ultracentrifuge  sedimentation  = K/ri of  (Spinco  velocity  = s/c {\ HA  was  Division,  using  -  2  this  U  2  p , )  measured  in  [2.10] a  Beckman  Beckman). T h e procedure device,  as  described  in  Model  E  for m e a s u r i n g the  Instruction  EXPERIMENTAL Manual cell,  supplied  making  a  sedimentation simplified  by  Beckman,  can  sedimentation  velocity  version  of  The  one  divided  velocity  run.  the  be  run,  4  parts:  taking  following  described  into  preparation of  photographs  description  in the  PROCEDURE  of  Instruction  and  the  /  78  sample  ending  procedure  is  a a  Manual.  3.8.1. Preparation of Sample Cell  A for  the  diagram  of  preparation of  1.  Check  that  2.  Place  a  holder. ends  Place of  arrow  is  in  on  the  cell  the  are  push  bottom  housing.  away  the  the  described  as  around  the  from is  arrow  with  the  Figure  2.5.  The  procedure  follows:  on  ridge  of  the  of  in  making  down  edge  of  the  window  window,  the  the  holder  sure  holder  one the  each  side  that  the  once  the  the  cell  Align  the  of  window. and  window  with  the  arrow  holder.  window aligned  and  keyway  the  into  holder,  keyway  the  keyway  window  window  Keep  in  liner  "key"  black  window  shown  window  liner  The  a  is  gasket,  face  is  cell  is  clean.  window  place.  There  assembly  sample  window  liner  pointing down, Slide  the  the  the  housing.  cell  all parts  white  window  3.  the  facing with  up,  the  into  key  the  (also  bottom in  steps  of  the  4  and  at  the  a  thin  5). 4.  Place top  two  a n d the  5.  A d d top  6.  After film  red  other  window  the of  centerpiece  cell  one  holder,  at  the  with  components  Spinkote  gaskets  (vacuum  are  in  the  aluminum  centerpiece,  one  bottom. the  window  properly  sealant)  to  facing  seated the  down.  in the  brown  housing,  screw  ring  apply gasket.  Place  EXPERIMENTAL the  gasket  thin  film  of  "Buttress ring  into  ring  is  in  the  use  assembly  screw  torque  Then ring.  press  on  the  upper of  the  window  the top  " B " side  inside  either at  lower  the  F i t the torque  down  on  wrench  raise  holder.  screw on  ring.  the  up, j u s t  the  which is  the  Then  The  screw  enough  shown  verticle  aligning  rod  79  apply  letter  ring. so  a  B  for  Screw  the  that  pins.  the  base  verticle  fits  wrench,  rod and  rotate  driver  the  wrench  with  sample.  into  The  align  the  cell  its  the  screw  socket  on  the  dial  until  the  a  hand  series  of  about  hole  top  of  the  slots  the  fluid  Make the  in  the  rod. Before  reads  turning  cell  at  slots  of the  needle  5  assembled  experimenter.  with  while  3.5.  the  housing  key  the  one  slide  filling  into  with in  the  in Figure  and  facing or' directly opposite  wrench driver  the  Turn  torque  wrench,  key  the  clockwise.  a  up,  the  the  threads  stamped with  with  should be  turning  is  the  on  /  place.  ring  that  bottom.  in  cell  screw  to  housing,  firmly  To  housing  Spinkote  the  the  sure  cell  Threads"  Tighten  cell,  the  PROCEDURE  0.  the turns  Then wrench  to  120  inch-pounds. Fill  centerpiece  Hold a  the  cell  1 m L syringe,  the  sample  prevent  corrosion.  screw  two  fingers, much,  Weigh  horizontal position, fill  the  overflows,  and  too  in a  the  it  centerpiece  immediately  Then  housing  place  a  plug  into  just  enough  will  distort the  filled cell  and  to  with wipe  the  about it  the a  0.75  away  hole  facing  plug  gasket  in  filling  hole.  Screw  the  good  seal.  If  up.  Using  m L of H A solution. the  housing.  counterbalance.  filling  from  red housing  provide cell  with  the  filling the  filling  plug  plug  hole  is  in,  If to  hole with  tightened  EXPERIMENTAL  PROCEDURE  F i g u r e 3.5 Position of the torque wrench and the cell housing. (Modified from the Instruction M a u n a l , Model E A n a l y t i c a l Ultracentrifuge, Beckman)  /  Note:  the  rating  of  Figure  analytical 67,700  3.6),  sample  cell  used  r p m . There  one  counterbalance.  rotor  for  The  the  Remove  counterbalance  b.  Balance  with  of  0.5  equal  g  of  two  sample  the  weight:  lighter.  In  are  /  A n - H type  a  speed  holes  on  cell  and  sample  tolerance  no case  2  the  sides  of  the  other  between  the  the  rotor  (see  for  the  one weights  of  the  g.  The  the  may  maximum  81  of rotor.  cell. -  with  two  limits +0.5  from Side  filled  PROCEDURE  tolerance  counterbalance  a.  be  are  acceptable  a n d the  is  EXPERIMENTAL  counterbalance  counterbalance counterbalance  and  cell  be  from  may be  heavier  must 0  than  to the  cell. c.  If  the  cell  replace  the  aluminum to d.  that  Clean  and  counterbalance  original  plug  of the the  that  will  in  the  make  the  the  acceptable  counterbalance weight  of  tolerance  with  the  a  limits,  brass  counterbalance  or  equal  cell.  rotor  holes  Spinkote  on  up,  into  Side  the  counterbalance  counterbalance  plug  exceed  the 2.  with  Kimwipes  counterbalance. Hold by  with  the  rotor  Slide  up  matching  and  and the  apply  the  corresponding  very  thin  counterbalance,  using scribe  a  the  cell  lines  lines  on  aligning  on  the  the  film  screw  side  tool,  align  bottom  bottom  of  of  of the  the rotor  hole. Note: HA  e.  For  all  solutions  the  which  Therefore,  steps a  Place  sample  The  the  sample  sedimentation have  a  to d are  in  very  done  cell in Side  chamber  velocity  the  once  experiments,  close  if  not  the  samples  identical  are  density.  only.  1 of rotor. centerpiece  is  sector-shaped  so  as  to  EXPERIMENTAL  F i g u r e 3.6 Installing the rotor. (Modified from A n a l y t i c a l Ultracentrifuge, Beckman)  the  Instruction  PROCEDURE  Manual,  Model  /  E  82  EXPERIMENTAL minimize in  the  wall rotor.  may  be  thin  film  end  up,  and  scribe the  It  is  of  may  the  the  on  sector facing  using  cell  the  bottom  on  bottom  of  the  cell  (Side  1)  aligning  rotor  of  the  of  the  the  cell  be  aligned  sector-shape in  Slide  with  tool,  cell  convection  housing.  in toward  the  the  produce  the  rotor hole  that  benefit  possibly  Spinkote  into  lines  important  Otherwise,  lost a n d  part of the up  effect.  PROCEDURE  the  the  the  centre  of  precisely  cell.  rotor. by  Apply  screw  plug  the  83  centerpiece  cell,  housing  /  or  the  cell  with  the  corresponding  ring  narrow  Hold  align  a  rotor  matching  the  lines  on  making  a  hole.  3.8.2. Making a Sedimentation Velocity Run  The  following  procedure  sedimentation  run.  along  description  1.  with  a  Turn  main  A  diagram  has of  of the  power  on  been  the  face  controls  is  and  the  chamber  by  applied panel  uniformly  of  the  contained  MAIN  analytical  in the  POWER  when  pilot  ultracentrifuge  Instructional light  (C-20)  Manual. will  come  on. 2.  O p e n the  vacuum  t u r n i n g the  vacuum  chamber  switch  (B-4)  to  OPEN. 3.  A t t a c h the Place  the  support the  rotor to rotor  fork  threads  clockwise coupling  (as stem  in  (see of  the the  drive  chamber,  Figure the  3.6).  rotor  viewed and  shaft.  lifts  from the  with Align  coupling above) rotor  the  rotor  the  threads  stem. until  off  the  support  Then it  of  the  the  the  fork.  resting  coupling  lower  engages  support  ring  nut  coupling  threads  Turn  on  the  in  the with nut the  coupling  EXPERIMENTAL nut  so  that  its  the  coupling  and  support  flat  wrench, fork.  counterclockwise the 4.  coupling  Close  the  vacuum 5.  Close  sides jaws  After  (as  switch  to  T h e n t u r n on  6.  Turn  on the  refrigeration  7.  Turn  on the  RTIC  Turn  the  pilot  light  switch will on  is  to  then at  the  8.  RTIC  Make zero.  automatic  function will  the  ZERO  (C-9).  time.  selector  come  is  sure  the the  then  the  VALVE  pump  over  in  it  is  Slide  the  place,  fork.  coupling  turn  tightened.  the  nut rotor  Then  remove  chamber  is  closed,  turn  the  by  turning  B-5  closewise  until  (B-ll).  temperature  on.  switch Allow  ADJUST  Activate  If  the  (C-10), unit  the  switch  for  drops  After  the  with  the  ZERO  to  the  up  for  black  turning  to  ADJUST,  warm  zero  by  the  unit.  to  unit  RTIC  circuit  on.  (C-ll) the  knob  temperature  turned  control  the  below  The  the  temperature  5  function  on  is  the  selector  RTIC  refrigeration  designated  RTIC  minutes.  needle  R E G U L A T E . The  heater.  the  unit  unit  is  temperature,  attained,  the  heater  off.  temperature  corresponds  first, is  support  the  (B-3)  relay  performed.  9.  vacuum  control  shutted  until  After  AIR  T h e n turn  all  end  of  84  SAFETY.  INDICATE.  heater  The  the  (C-12)  meter  side  wrench  above)  (B-4).  PUMP  tight.  RTIC  open  coupling  from  chamber  VACUUM  using  the  and  the  /  wrench.  chamber  Then  parallel to  down  viewed  vacuum  the  are  PROCEDURE  calibration  Sedimentation to  a  the  Otherwise,  experiments  B A L A N C E dial VOLTAGE the  diffusion  (C-14)  CONTROL pump  RTIC are  unit  performed  reading of knob  cannot  be  has  previously at  20 ° C ,  been which  5.619.  (A-12)  is  turned on.  counterclockwise  to  EXPERIMENTAL 10.  When the  the  pressure  diffusion  pump  counterclockwise  tight  switch  The  DIFFUSION  PUMP  pilot  pressure  Load  the  The  film  film  is  to drop to  photographic used  loaded holder  the  to  plate  read  is  into  O U T and position  and  pull  down  the  way  into  chamber. the  the  plate  holder  previous  to  8  of  the  The  slot  drive  slot.  16  operating  speed  plate  holder.  the  PLATE  water  on.  inlet  VALVE  the  Wait  15  to  (B-6)  valve  diffusion  with pump.  min.  cover  of the  hold the  plate  plate  After  the  for  dial  (A-14) The  is  holder  set  at  EXPOSURE on  the  of  then  the  10  the  sec,  loaded switch  should to  IN  holder the  all  rotor  clutch press  holder  and  until  the  is  close  in  in the  zero.  which  INTERVAL  concentration  and  plate  at  dial  facing  button  The  (C-23)  plate  engage  be  the  switch  in place  DRIVE  should  the  holder  8in.  DRIVE  DRIVE  holder  edge  position  placing  position  Push  plate  x  button  plate  HOLDER  HOLDER  dial  DRIVE  holder slot.  on  2in.  HOLDER  The  the  size  Before  HOLDER  PLATE  testings.  is  on  2556,  plate  button.  the  being  the  come  type  PLATE  teeth  m i n . , depending  H A solution  in  85  camera.  revolving.  the  the  the  T h e plate  exposure  m i n . or  stops  mechanism,  TIME  water  WATER  turn  will  film,  PLATE  for the  DRIVE  Then  in the  turn  Turn  that  the  (C-19)  ORTHO  the  with  release  EXPOSURE  by  5.  cover  the  slot,  holder  (C-5)  and  the  (B-10).  camera,  dial  HOLDER  past  The  0  slot  photographic  press  T o ensure  plateshift  PLATE  the  aligning  the  /  micron.  plate  the  micron, open PUMP  light  KODALITH  into  between  =1  = 50-100  DIFFUSION  and  PUMP  (C-22)  13.  until  is  turning  DIFFUSION  plate  12.  by  (C-l)  the  the 11.  gauge  PROCEDURE  is  switch  determined is  turned  a n d molecular  weight  tested. set  at  52,640  rpm  using  the  speed  selector  knob  EXPERIMENTAL (A-11). 14.  Set  Brake  timer  (A-15)  VOLTAGE greater 15.  When  than  0  turning  on  and  voltage  Maintain  16.  the knob  reduce  the  The is  timer  will  turned  be  activated  clockwise  (i.e.  when  when  the  voltage  is  the  micron,  the  needle  the  90%  in  the  ammeter  control  knob  12  (=47,000  to  5  amps.  speed  is  at  knob  dial  between  speed  drive  TIME  voltage  current  set  CONTROL  EXPOSURE  drive  current  = 1  VOLTAGE  turning  until  is  The  current  clockwise.  (A-14)  will  =5  The start  amps  timer  is  revolving  ( A - l ) stablizes,  increase  clockwise  to  12-13  amps.  with  the  voltage  and  13  amps  rpm  in  A-3)  automatic  to  speed  is  attained.  control  Then  system  will  over.  When light  the  rotor  source  by  counterclockwise (C-26) the  hours.  gauge  After  by  control  take  4  86  SLOW.  knob(A-12)  the  counterclockwise. the  at  /  volt).  vacuum  by  turned  for  CONTROL  the  (A-l)  (B-8) is set  PROCEDURE  is  at  LIGHT  switch  turning  (until  =20,000  the  tight).  camera.  SOURCE  switch  (B-13)  to  pilot  light  (C-18)  about  10  sec,  turn  light  to ensure  long  lamp  life).  Align  the  will  intensity  SOURCE  sure  (B-9).  H I and turn  SOURCE  LIGHT  Make  Schlieren  r p m , open  that  the  slot  Then  switch  to  After  inlet  to  WATER  VALVE  CAMERA  selector  the  SOURCE  on.  water  in the W A T E R  turn  LIGHT come  the  the  light switch  the  L O (start  light  the (B-7)  switch  VALVE  source on. T h e comes  with  intensity LIGHT on  for  o n H I , r u n on L O  EXPERIMENTAL  PROCEDURE  /  87  3.8.3. Taking Photographs  1.  As  the  rotor speed  PLATE  HOLDER  AUTOMATIC  back  TIME  dial  PHOTO While  off  steady  speed  is  the  at  2.  button plate  the  the  will  EXPOSURE  INTERVAL  m a x i m u m of  For  each  taken.  To  to  cover  for the plate  dial  PHOTO  butts slot  holder  on  ON.  the  (8  switch.  moving  hold off  plate  has  taken  HOLDER holder  PLATE out.  to  the  (when  IN.  PLATE  the  No.  the  1  rotor  PHOTO  switch  will  click  open,  taken.  The  next  specified  on the 10  to  by  photographic 15  the  PLATE  slot  the  turn  HOLDER  DRIVE  button  cover.  Pull  DRIVE  plate  plate.  photographs  holder,  HOLDER  Take  switch  min.)  plate the  AUTOMATIC  holder to time  clicks  EXPOSURE  push  been  experiment,  plate  the  shutter  16  turn  shutter  the  interval  m i n . or  Turn  the  DRIVE  The  the  PLATE the  until  AUTOMATIC  dial.  be  Then  steady,  the  the  after  a n d p u s h the stops  the  turn  dial  photographic  against  O U T position. T u r n  HOLDER  photograph  velocity  the  the  (C-5). A t zero  TIME  setting  the  rpm), t u r n  and  Then  move  photographs c a n  O U T . Push  holder  the  5  change  plate  to  taken  sedimentation  AUTOMATIC switch  be  to  TIME  (C-23)  first  the  shut  PLATE  rpm), t u r n  photograph  A  the  EXPOSURE  that  clicks  position.  position  to  (=35,000  counterclockwise  EXPOSURE  =35,000  release  indicating  turn  value  (C-22)  shutter  this  2/3  (C-21)  (A-14)  at  the  DRIVE on  switch  the  and  the  switch  dial  until  holding  position  and  TIME  switch  HOLDER  DRIVE  PHOTO  EXPOSURE open;  approaches  off  the  DRIVE until  the  down  the  button  holder  are  out  until and  EXPERIMENTAL place  another  Turn  on  the  HOLDER 3.  A  one  the  slot.  AUTOMATIC  DRIVE  procedure  photographic  in  switch  for  plate  Then PHOTO  step  switch  11  and  in  Section  turn  the  88  3.6.2. PLATE  to I N .  calculating is  repeat  PROCEDURE /  shown  sedimentation  coefficient  from  the  TIMER  dial  in A p p e n d i x A .  3.8.4. Ending a Sedimentation Velocity Run  1.  Set  brake  (A-15) 2.  3.  Turn  (B-8)  at  RAPID.  Terminate  to O F F . T h e diffusion off  light  source  turning  pump is  (B-9)  the  and  source  by  LIGHT  (until  tight).  Turn  off  the  REFRIGERATION  Turn  off  the  AUTOMATIC  plate  holder. W h e n  a  r u n by  turning  automatically shut  shut  the  SOURCE  switch  water  inlet  WATER  (B-3)  off. to  the  VALVE  and  light  clockwise  the  RTIC  and  take  system  (C-ll). 4.  5.  Set  6.  Turn  braking off  turning  7.  rate  the B-5  Turn  off  (C-21) stop,  go  to  out  step  the  PUMP  (B-ll)  Before  v a c u u m to be  DIFFUSION  PUMP  and  open  opening  released  (B-10)  the  the  air  Turn  VOLTAGE  9.  Open  the  5.  valve  vacuum  C O N T R O L knob (A-12)  rotor c h a m b e r  (B-4)  completely.  and close  the  counterclockwise  and remove  by  chamber,  water  valve  clockwise.  8.  the  SLOW.  counterclockwise.  2 m i n . for  turning B - 6  at  switch  rotor comes to complete  VACUUM  allow  the  the  PHOTO  the  rotor.  to  zero.  by  EXPERIMENTAL 10.  Close  11.  The  the  rotor chamber  sample  plastic  rod.  cell  then  is  water  cell The  is  a n d then  pushed  screw  taken  and t u r n out  ring  apart.  dried with  is  The  of  m a i n power Side  1 of  unscrewed different  acetone.  PROCEDURE  /  89  off.  the  rotor  by  means  by  the  torque  wrench.  parts  are  rinsed  with  of  a  The  distilled  CHAPTER  The  experimental  results  are  presented  a n d discussed  the  evaluation  of the results  data  4.1.  4. R E S U L T S A N D D I S C U S S I O N  obtained  i n this  for  the  chapter.  are presented  various  experiments  T h e sample  calculations  in Appendix A . Detailed  performed leading  to  experimental  are listed in Appendix B .  HYALURONIC  The determine  ACID  uronic the  data  error  are fitted  standards in  4.1  of are  absorbance  Results  Concentration  =  DETERMINATION  method  (Bitter  &  H A . Absorbance presented  represents  by the least-squares ABS  Table  carbazole  concentration  glucuronolactone indicated  acid  CONCENTRATION  -0.03554  in 2  method +  data  Table  to give  of the uronic acid carbazole  ±  1962)  for  4.1  standard  (0.01651  Muir,  5  and  was  used  to  concentrations  of  Figure  deviations.  The  0.00087)  X  c  test  fig/mL  average  0.  0.0186*  9.85  0.1265±0.0090  19.70  0.2984 + 0.0050  29.55  0.4423 + 0.0052  39.40  0.6116 + 0.0016  49.25  0.7831 + 0.0098  *read  of 3 measurements  against  cone.  at 525 n m  H SO(, 2  90  absorbance  [4.1]  Absorbance  *  The  the calibration line  *  of glucuronolactone,  4.1.  RESULTS  A N D DISCUSSION  /  O  .0  Concentration of Glucuronolactone, /xg/mL F i g u r e 4.1 Relationship between glucuronolactone.  the  absorbance  and the  concentration  of  91  RESULTS where  ABS  is  the  glucuronolactone  in  otherwise  stated,  Appendix  A).  The  glucosidic residues  the  which as  the  the  the  95%  of  then  the  react salt  of  acid  disaccharide  by  the  glucuronolactone  4.2.  as  consists  the  acid  of  with  boric  disaccharide  purple  When uronic  The  results  4.2.  The  in  the acid  (Eqn [4.1])  concentration  of the  unit  is  is  in  then  hyaluronic acid  OF  of  the  intrinsic  hyaluronic  carbazole  of  of  multiplied  units  of  cone.  is  same  D-glucuronic  acid  1965).  formula  the to  weight  The  weight  (176.1)  fraction  glucuronolactone acid  concentration  the  a  formula weight  glucuronolactone by  the  2  Glucuronolactone,  hyaluronic the  H SO ),  The  401.3.  factor  is  is  solution obtained  to  is from  concentration. 2.279  of  The  give  the  solution.  HYALURONIC  ACID  fractionation experiment viscosities  unless  Calculations,  Richardson,  acid  method,  terms  work  disaccharide  the  Therefore,  concentration  this  Sample  color.  unit the  polymer.  of  in  &  a  concentration  borate  (Hough  give  92  W h e n hyaluronic acid  acid  repeating  the  repeating  and  the  in  (see  broken  to  the  throughout  are  carbazole  residue  is  /  residue.  (sodium  residues  c  interval  with  FRACTIONATION  Table  error,  confidence  acid  boric  repeating  calibration line  concentration  and  anhydride of D-glucuronic acid, has  4 0 1 . 3 / 1 7 6 . 1 = 2.279.  the  nm  and N-acetylglucosamine  linking  glucuronic  determined  hyaluronic  complexes  sodium is  represents  bonds  complexes of  indicated  concentrated  form  525  the  acid residue  with  at  Mg/mL;  structure  D-glucuronic treated  absorbance  A N D DISCUSSION  [77] of  the  (Section hyaluronic  3.3.4) acid  are  presented  fractions,  in  which  RESULTS Table  4.2  Results  Eluant [NaCl],  M  of the large  scale fractionation  subfraction  H A recovery,  [77J  M.W.  mg  mL/g  X10  volume,  mL  612  0. 0.10  A N D DISCUSSION /  1.  505  2.  502  3.  500  4.  502  5.  500  experiment  5  = 0  = 0  6.. 501  0.175  0.25  7.  501  8.  500  1.  500  4.027  2.  500  3.896  3.  500  4.  500  5.  500  6.  500  7.  500  8.  500  1.  500  42.67  2.  500  33.60  3.  500  18.95  4.  500  15.20  5.  500  12.48  6.  500  7.  500  8.  500  9.  500  10.  500  1340  6.99  93  RESULTS 0.325  0.40  0.50  1.  500  300.35  2.  500  128.44  3.  500  74.73  4.  500  54.10  5.  500  46.61  6.  500  41.09  7.  500  31.97  8.  500  28.59  9.  500  315.53  10.  500  22.48  11.  500  7.947  12.  500  5.235  13.  500  14.  500  1.  500  60.27  2.  500  11.40  3.  500  11.59  4.  500  9.396  5.  500  6.  500  7.  500  8.  500  1.  500  46.63  2.  500  3.93  3.  500  4.  500  5.  500  A N D DISCUSSION  •1530  8.44  1830  11.1  1500  8.19  1880  11.6  2260  15.5  = 0  1331.1 unfractionated  HA  /  94  RESULTS were  obtained  by  extrapolating  For  comparison,  the  also  determined  and  for  [57]  values  and  was  by  shown  the  l^lgg  molecular  m  m  Mark-Houwink  Figure  4.2.  following  ^S  weights  of  raL/g,  Z  is  M  S  the  of  equation  =  hyaluronic  From NaCl,  Table  2mM  4.2,  phosphate  cellulose  ion-exchanger  fractions  was  the  0.175M  only the  on  NaCl  eluate  about  8.0  The  throughout was than major  the  negatively that  was  at  the  of  the  fractions  acid  in  elution charged.  was  The  very  carbazole  beginning at  cellulose  the  rate  was  0.2M  at  NaCl  zero  were  0  -  8  1  by  the  the  [4.3]  out  HA  small  test  (done  because  The  pH  was  out.  the  2  by  in  of  and  that  cellulose  was  each  of  the  material.  In  fact,  m L / g , which  of  triplicate)  eluate about  H A is  positively  of  was  to  the  low M . W .  was HA  below  for  weight  collected  the  average  substantially  0.175M  period.  Since  therefore,  and  collected  the  was  HA lower  [77] for  lower  in  decreased  7.30,  charged  from  performed  gradually 7.3  H A fractions the  (0.M  of H A collected  r e m a i n i n g elution  the  buffer  bound  amount  amount  the  weak  tightly  amount  the  of  The  the  in  1625  The  6  7.3  [77] of  rate.  evaluated  about  The  necessary  shear  to  the  (1970),  [4.2]  elution  9.5  was  authors:  the  is  4.2.  acid  Wang  of  period,  was  shear  &  95  Figure  hyaluronic  Cleland  viscosity  carried  from  small.  zero  washed  was  subfraction  to  in  1400)/6250  M  7.30),  obvious  unfractionated  collected  HA pH  shown  /  1970):  0.228  elution  stayed  DE23  the  as  eluate,  p H then  p K a of  before  eluate.  0.175M  no  buffer,  little,  0.1M  eluate. the  very  the  from  since  -  intrinsic  =  z s  to  ([7?]  are  unfractionated  According  (Cleland & W a n g , [T]]  c=0,  derived b y  1 +  corrected  to  the  correction  equation  / [ T J ]  •c  0  viscosity  in  1400  [ T J ]  where  0  intrinsic  above  done  (17—TJ ) /r\  A N D DISCUSSION  than  the the  RESULTS  AND  DISCUSSION  /  CO CO  1  1  0.0  0.3  '  1  '  0.6  1 0.9  1  1 1.2  Concentration of HA, mg/mL Figure  4.2  Intrinsic  viscosity  plot for  the  HA  Fractions  1  1 1.5  RESULTS [77]  value  of  degradation eluant  was  of  salt  decreased  the  degradation  of  may  been  which  then  height  of  At  Convective the  regions  bed  was  the  buffer  is  lower  attributed overall  the  of  to  were  then  to  9.5  the  packed  that the  recovery  by  a  of  was  acid  Also,  after of  the  elution,  eluate  might  be  buffer  were  the  fixed eluted. the  volume.  0.325M  at  of  since  might  not  least  in  why  the  the 9  and  the  95.7%  (i.e.  tightly  not  NaCl  column  g/1.39  the was  tightly  by  the  buffer.  and  the  HA  continued,  known  g).  of  eluant  unreached  0.4M NaCl  flow  The  not  elution  fully  fractions  was  previously  convective 1.33  very  packed.  beginning  reached  [77] for the is  weight  the  As  regions  was  left  adsorbed  column  be  was  H A originally  0.325M  eluted.  H A in  subfraction HA  not  9  NaCl  because  at  the  eluate  probably  not  cm  97  each  the  column  molecular  with  present  The  T h e reason  degradation  of H A was  bed  was  was  14.0  the  for  0.325M  subfraction  the  smaller  elution  column  by  of  column  the  in  reason  Some  about  the  and  in  with  /  Obviously,  general,  acid  Elution  collected  into  from  eluate.  acid.  In  hyaluronic  The  the  process.  collected  column.  bacteria  cm  beginning  unreached  than  the  of of  subfractions.  decreased  in  8  hyaluronic  unadsorbed.  regions  flow  of  hyaluronic  fractionation  volume  subfraction  degraded  about  unfractionated  the  the  occurred in  column  to  some  the  concentration  in  other  became  fractionation  packed,  to  HA  the  completed.  eluate  amount  compared  the  increase  after  The  have  for  H A occurred during  with  overnight.  mL/g  concentration,  stopped  large  2260  A N D DISCUSSION  of  but  in the by  fraction may  eluate.  be The  RESULTS 4.3.  ACID  HYDROLYSIS  Three acid  for  15  fractions weights As  HA  of  fractions  min.,  are  1  expected,  three  the  hydrolysis.  4.4.  FRACTIONATION  are  results  tabulated  Fraction Table  and  4.4,  tightly  and  acid  in  bound  the  and  4.4.  HA  were  The  intrinsic  Fractions  Fractions  of  acid solution  was  eluted  HA  mixed  by  98  the  are  with plot  for  and  the  the  tabulated with  hydrochloric  in  three  molecular Table  the  4.3.  duration  of  HA  viscosity  hydrolysed  viscosity  decreases  hyaluronic  intrinsic  HA  viscosities  HYDROLYSED  The  not  hydrolysing  intrinsic  HA  fractionation  unfractionated  when  ACID  by  The  weight of the  the  Table  4.3.  hydrolysed  OF  of  hours.  /  ACID  obtained  2  Figure  molecular  acid  The  in  HYALURONIC  were  hour  shown  the  OF  A N D DISCUSSION  is  with 2mM  acid plot  hydrolysed for  shown the  the in  15  min.  15mAH-0.325M  Figure  cellulose,  phosphate  for  4.4.  the  buffer.  HA Since  From were the  5 M.W.  Table and  of  4.3  the  unfractionated  Intrinsic  2hrAH  viscosity  acid  and  hydrolysed  molecular  weight  HA  of  (M.W. = 2.02X10 )  15minAH,  lhrAH  Fractions  Fraction  Intrinsic  viscosity,  mL/g  M.W. X10  15 min A H  413  1.65  lhrAH  233  0.819  2hrAh  144  0.454  was  RESULTS  AND DISCUSSION /  CO  O  to  d  HA Fraction = 15minAH x = lhrAH • = 2hrAH A  o O  d  -x—x-  o ,  o  o d  0.0  0.7  1.4  2.1  2.8  Concentration of HA, mg/mL Figure  4.3  Intrinsic  viscosity  plot for  the  acid  hydrolysed  Fractions.  3.5  99  RESULTS  Table  4.4  Results  Eluant [NaCl],  of the fractionation subfraction  M  volume,  mL  120 0.1  0.175  0.25  0.325  0.4  0.5  A N D DISCUSSION /  100  of acid hydrolysed H A  H A recovery,  [77]  M.W.  mg  mL/g  X10  5  = 0  1.  250  2.  250  3.  250  4.  235  = 0  1.  250  1.84  2.  250  1.46  3.  250  4.  238  = 0  1.  250  9.93  2.  250  4.59  3.  250  3.36  4.  250  2.52  1.  250  203.06  2.  250  27.58  3.  250  11.98  4.  227  7.46  5.  250  11.17  6.  250  1.71  7.  250  1.71  8.  244  = 0  1.  250  4.96  2.  250  1.49  3.  250  1.39  4.  250  = 0  1,  250  ==0  2-  250  = 0  3.  250  = 0  V  475  1.96  486  2.02  J  296.2 unfractionated  HA  RESULTS  AND  DISCUSSION  /  101  O  1  '  i  0.0  1.2  '  i  2.4  1  i  •  3.6  i  1  4.8  6.0  Concentration of HA, mg/mL Figure  4.4  Intrinsic  unfractionated H A .  viscosity  plot of  the  15mAH-0.325M  Fraction  r  and  RESULTS substantially  lower  than  that  of  the  A N D DISCUSSION  unfractionated  HA  in  the  /  102  large  scale  5 fractionation fraction of  (M.W. = 15.5X10  of  H A would be  H A eluted  by  amount  of  by  0.325M  the  adsorbed  HA  to  the  about  91.3%.  lower  than  HA  by  eluant.  that  the  elution  15mAH-0.25M experiment.  It  at  Fraction  As  a  and  above  were  result,  of  fraction  very  small  [NaCl].  collected  The  for  the  after  and  compared to  and  were  were  recovery  the  the  large amount  of  that  tightly  HA  was  slightly  degradation of  samples  of  sedimentation  15mAH-0.25M  the  desorbed  ( 1 5 m A H - 0 . 3 2 5 M ) is  Unfortunately,  discarded  the  H A molecules  experiment  a  small,  H A m a t e r i a l . T h e r e f o r e , the  carelessly [77]  the  that  However,  was  was  the  0.3M  extensive.  the  expected  eluants  appeared that  major  not  was  [NaCl] eluants.  NaCl  beginning  was  it  0 . 4 M eluants  unfractionated  was  low  0.175M  the  the  3.3.4),  the  therefore  [ T J ] of  of  by  and  environment  The  Section  0.25M  cellulose  ionic  during  eluted  0.1M  eluted  the  when  the  ,  Fraction  the  velocity was  not  days,  the  determined.  4.5.  SEDIMENTATION  After hyaluronic different of  each  each  dialysis acid  of the  4.5  as  function In  solutions  and  Table  0.2M  made  with  H A solutions  solution  Table  4.8.  with  concentrations  HA  a  COEFFICIENT  was  plotted of 4.5,  the  0  is  the  from  the  for HA  4.7.  Laurent  sedimentation  The et  least  The  2 were  The  results  plot  of  al.  of  are  is  the  to  velocity  coefficient tabulated  sedimentation  (1960)  coefficient  diluted  sedimentation  T h e sedimentation  E q n [2.41]. to  at  Fractions  dialysate.  measured.  from 4.5  all  NaCl  then  Figures  solutions  from  0.2M  was  concentration S „  up  calculated in  NaCl  for in  velocity  shown  in  Figure  solution  at  20°C.  RESULTS  Table of  4.5  Sedimentation  coefficient  and hydraulic  A N D DISCUSSION  /  conductivity  all H A Fractions  Run  Fraction  M.W.  Concentration,  10  ± J  XS  2 ( )  ,  lO XK', Lyj  XIO'^  mg/mL  s  cmVdyn-s  6.99  0.3542  4.18±0.16  36.311.4  A2  0.7084  3.15±0.07  13.7 + 0.3  A3  1.0626  2.67±0.06  7.7310.17  A4  1.4168  2.28±0.02  4.9510.05  A5  1.7710  2.10±0.05  3.6410.09  0.4601  3.65±0.11  24.410.7  B2  0.9202  2.47±0.04  8.2510.12  B3  1.3803  2.05±0.03  4.57 + 0. OS  B4  1.8404  1.9010.06  3.1710.10  B5  2.3005  1.55±0.02  2.0610.03  B6  2.7606  1.50±0.04  1.6710.05  0.4840  3.2210.13  20.510.8  C2  0.9680  2.4710.04  7.8510.12  C3  1.4520  1.8810.06  3.9810.13  C4  1.9360  1.7010.02  2.6910.04  C5  2.4200  1.6010.03  2.0410.04  0.5916  2.51 + 0.11  13.010.6  D2  1.1832  2.1210.06  5.5110.15  D3  1.7748  1.8610.03  3.2210.04  D4  2.3664  1.5210.08  1.9710.10  D5  2.9580  1.4210.04  1.47+0.04  Al  Bl  CI  Dl  0.25M  0.325M  0.325M-9  15minAH  8.44  11.1  1.65  103  RESULTS  El  lht-AH  0.819  A N D DISCUSSION  /  0.6167  1.6410.56  8.1912.78  E2  1.2333  1.9910.06  4.9510.16  E3  1.8500  1.7410.10  2.8910.17  E4  2.4666  1.4410.06  1.7910.07  E5  3.0833  1.4510.06  1.4510.06  0.6109  1.9910.79  10.013.95  F2  1.2218  1.65+0.12  4.1410.30  F3  1.8326  1.5210.06  F4  2.4435  1.3810.09  1.7310.11  F5  3.0544  1.2210.11  1.2310.11  1.0954  2.26±0.30  6.3510.85  G2  1.6431  1.7010.05  3.1910.09  G3  2.1908  1.5610.05  2.1910.07  G4  2.7385  1.44±0.03  1.6110.03  G5  3.2862  1.3010.07  1.2110.07  Fl  Gl  2hrAH  15mAH-0.325M  0.454  1.96  •  2.55+0.09  104  RESULTS  F i g u r e 4.5 Sedimentation coefficient 0 . 3 2 5 M and 0 . 3 2 5 M - 9 Fractions.  as  a  A N D DISCUSSION  function of concentration  for 0 . 2 5 M ,  /  105  RESULTS  O TT  AND DISCUSSION /  106  o 1  »0  o • x o  O  HA Fraction = 15minAH = lhrAH = 2hrAH = 15mAH-0.325M  <D O  o  CO  •fH  •a  CM  CO  0.0  0.7  1.4  2.1  2.8  3.5  Concentration of HA, mg/mL F i g u r e 4.6 Sedimentation coefficient as a function l h r A H , 2 h r A H and 1 5 m A H - 0 . 3 2 5 M Fractions.  of  concentration  for  lominAH,  RESULTS  DISCUSSION /  q  O TT  AND  1  lO  HA Fraction 0.25M • • = 0.325M A = 0.325M-9 O = 15minAH lhrAH 2hrAH 15mAH-0.325M —  Ifl ~  O  (L>  •PH  O  o O  CO  •s  o CO  0.0  0.7  1.4  2.1  3.5  2.8  Concentration of HA, mg/mL F i g u r e 4.7 Fractions.  Sedimentation  coefficient  as  a  function  of  concentration for  all  107  RESULTS  CO H  I  O  Figure  q  4.8  Sedimentation  data  of L a u r e n t  et  al.  (1960).  A N D DISCUSSION /  108  RESULTS Since  the  points  for  patterns  uncertainty the  of  three  the  in runs  direction  sedimentation  coefficient  et  al.  increases,  higher  high  the  are  resistance  to  sedimentation  In  of  HA  sediment  the  at  Fraction  the  same  would  concentrations,  sediment  the  rate,  H A molecules  molecules  sediment  hand,  at  concentrations,  entangle  with  molecules  will  concentrations Fractions  faster  (15minAH,  (>  2.1  at  coefficient  the  lhrAH  and  and the  and  at  than  form  4.11, in is  As  the  al.  (1960),  low  the  4.7,  the  agreement  HA  with  concentration  becomes  different  lower  greater. becomes  the  lower  the  HA  and  molecules  three  From  concentrations,  (low  two  lower  M . W . H A Fraction;  and  the  would  On  or  At the  4.7,  at  curves  for  non-acid  M . W . Fraction  low  higher  the  high  different  the  at  higher M . W .  network,  Figure  0.325M-9),  for  HA  therefore  coefficient  that  Fraction.  dimensional  rate.  of  the  M . W . molecules.  sedimentation  higher  expected  Fractions  M.W.  independently  — for  was  H A concentrations,  the  a  it  fractionated,  the  which  H A network  the  2hrAH),  data  sedimentation in  in  109  the  Figure  molecules  through the  the  slow  lower  and  high,  The  which  (1965).  et  sediment  found  to  shown  individual  than  the  figures.  4.9  As  was  /  lower.  same  At  were  0.325M  higher  al.  G l  the  concentration, et  since  mg/mL),  converging.  (0.25M,  than  other  sediment  are  sedimentation Fractions  each  faster  in  right.  mg/mL),  faster  and  Figures  Laurent 2  F l  of solvent  is  of  M.W.  high  flow  (above  slow  to  with  rate  results  concentrations  in  between  the  and the  view  left  Preston  entanglement  El,  included  shown  from  and  Runs  not  decreases  (1960)  the  Therefore,  is  for  were  Fractions  sedimentation  Laurent  S^Q  A N D DISCUSSION  other M.W.)  the  HA  high  HA  all  HA  trends  in  hydrolysed sediments  acid hydrolysed F r a c t i o n s  M . W . Fraction  sediments  faster  than  RESULTS  Fraction: c=  0.7084 t=  1.4168  r=  Figure  0.9202  0.25M mg/mL  1.3803  Sedimentation  patterns  1.3803  mg/mL  112 m i n  of the 0 . 2 5 M ,  0.325M  110  0.325M-9  mg/mL  0.325M  mg/mL  64 m i n  0.9202  112 m i n  0.325M  64 m i n  4.9  mg/mL  64 m i n  64 m i n  Fraction: c=  mg/mL  0.325M  0.325M  0.25M  A N D DISCUSSION /  0.4840  mg/mL  64 m i n  0.325M-9 1.4520  mg/mL  64 m i n  and 0 . 3 2 5 M - 9  Fractions.  RESULTS  Fraction: c=  1.1832 t=  Figure  15minAH mg/mL  Sedimentation  2hrAH  lhrAH 1.2333  64 m i n  4.10  A N D DISCUSSION /  1.2218  mg/mL  56  64 m i n  patterns  of the acid  hydrolysed  mg/mL min  H A Fractions.  111  RESULTS  A N D DISCUSSION  /  112  Fraction: 15mAH-0.25M  15mAH-0.325M  15mAH-0.325M  15mAH-0.325M  c=  1.0954  1.6431  2.7385  1 unit c  t— 64 m i n  mg/mL  mg/mL  mg/mL  64 m i n  64 m i n  64 m i n  15mAH-0.25M  15mAH-0.325M  15mAH-0.325M  15mAH-0.325M  c=  1.0954  1.6431  2.7385  Fraction:  t=  1 unit c 32 m i n  F i g u r e 4.11 Fractions.  Sedimentation  mg/mL  144  32 m i n  patterns  of the  mg/mL min  15mAH-0.25M  128  and  mg/mL min  15mAH-0.325M  RESULTS the to  .lower the  M . W . Fraction.  trend found  al.,  1965;  the  0.325M-9  Silpananta  difference at  1.4  the  between  molecules  non-acid may  Since  be  the  coefficient  curves  the  7  and  were the  4.8)  %  the  between  to  in  errors  of samples,  in  etc.  are  form  the  of L a u r e n t  curve  results  et  al.  the  (1960)  at,  dilution  the  the  of  (0.2M  NaCl), have  fractionated,  not  the  18%  and  known,  samples  when  HA  (about  the  is  et  However,  coils  for  opposite  and  large  random  113  curve  solutions  Nevertheless,  looked  is  /  (Preston  0.325M  mg/mL).  saline  is  workers  the  c=1.4  in isotonic  coefficient  other  0.325M-9  expected  and that  Fractions  and  at  difference  systematic  all  (Figure  T h e difference  curve  for  concentration of  al.  (about  Fractions  to  trend in sedimentation  Fractions were  fractions  attributed  measuring  small  reason  hydrolysed  et  1968).  0.25M  the  The  al.,  is  the  of  shape.  Laurent et  curve  mg/mL).  similar  by  T h e former  A N D DISCUSSION  and  but in  sedimentation  molecules  of  the  high  5 M.W.  Fractions  (6.99  to  11.1X10  )  sediment  faster  than  those  of  the  low M . W .  5 Fractions  (0.454  to  1 . 9 6 X 1 0 ),  which  is  in  can  be  agreement  with  Laurent  et  al.  sharpness  of  the  (1960).  From Schlieren varies  peak,  directly  non-acid and  the  coefficients  HA so  samples, the  low  use  However,  peaks which  it  the  Fractions  the  justified.  for  4.11,  concentration  Schlieren  concentration  to  therefore  with  symmetrical, was  4.9  and  hydrolysed  coefficient 4.10),  Figures  seen  sharpness and  Eqn for  the  broadened  out  explains  why  concentration  the  molecular  (Figure  of  of  4.9),  acid  samples  evaluate  quickly, uncertainty  (Runs  For  Schlieren  hydrolysed  very the  to  the  solvent/solution  weight.  the  [2.41]  that  Dl,  HA  the peaks the  E l  fractionated, were  the and  sharp  sedimentation  Fractions  especially in  boundary,  in  (Figure the  low  sedimentation F l  in  Table  RESULTS 4.5)  is  very  molecular acid  high.  weight  hydrolysed  peaks  are  directly the  of  acid  out  the  acid  form  the  quickly,  a  the  4.12  and  large.  Runs  In  acid  HA  tail  In  D4  fact,  the  hydrolysis  increases,  molecules  also  all  and  degree  of  the  sedimentation  in of  Table  average  runs  of  for  the  the  Schlieren to  vary  concentration.  M . W . decreases, the  increased  114  range  appears  with  Therefore,  skewness  the  4.5,  skewness  indirectly  increases.  that  sedimentation  D5  degree  the  imply  the  and  the  on  peak,  of  r  of  Figure  4.12  as  shown  in  [2.41]  sedimentation other  the  Since  molecules  of  infinitely where  the  4.12.  Runs  Also,  difference  distance  to  F5  which,  higher  trail  the  As  and  Schlieren  with  between  the r  axis  the  peaks  duration  r,  as  t  the  r  Table  degree and  of is  4.5  the  rotation in of  of  the  M.W.  HA  M . W . ones  and  position  r  Thus,  the the  position  (a)  The  and  (b)  with  time,  calculated  from  than from  hydrolysed  in  the  2.5.2.  in  #  of  the  Section  coefficients  acid  to  than  higher  profile  skewed  increases  calculated  skewness larger.  lower  higher  are  be  the  than  and  in  in  is  sides  sedimentation  can  peak  defined  two  concentration  Also,  of  the  M . W . molecules  when  the  profile.  between  theoretically,  the  faster  behind  the  on  and  Schlieren  sediment  from  areas  Therefore,  Dl  the  boundary,  shaded The  pattern  concentration  away  sharp  equal.  the  the  farther  coefficients  overemphasized. higher,  is  Figure  words,  HA  left  are  for  Fractions.  M . W . H A molecules  the r ,  is  #  HA  M.W.  lower  theoretical  position  depicts  higher the  Schlieren  In  right.  hydrolysis  hydrolysed  and  molecules,  Eqn  except  the  acid  is  peaks  /  hydrolysis.  right,  of  to  Schlieren  samples  duration of  of  Figure of  the  M . W . of  broadened  broad  Fractions,  the  duration  range  in  skewed  with  The  A N D DISCUSSION  the  Eqn  true [2.40].  Fractions  are  Schlieren  peak  is  difference  between  RESULTS  A N D DISCUSSION  F i g u r e 4.12 Sedimentation pattern and concentration profile Fractions, (a) time t and (b) time t , where t >t- . u  2  2  i  /  115  of acid hydrolysed H A  RESULTS the  measured  degree  of  with the  Fractions, than  the  trend  i.e.  the  the  the  of  skewed,  other  symmetrical  and the  Table  4.5)  the  for  has  to  or  of  a  F5,  peaks  the  for  higher to  higher  Schlieren  peaks  listed  with  the  molecular  acid  hydrolysed  sedimentation  be in  correct Table  116  with  decreases  coefficient  expected  Dl  Schlieren  skewness  hydrolysis,  is  Runs  2.5.3. error  acid  coefficient  although  4.5,  are  the  higher  the  For  15mAH-0.325M  peaks  very  close  to  in S^Q  Fraction  was  the  were  Fraction  with  the  In  slightly  true  was  not  for  general, has  5  at a  measured.  to  were  Gl  the  and right.  15mAH-0.325M  for reasons photographs  each  (Figure  (Runs  the  values,  were  Fraction  skewed  sedimentation  (M.W. = 1.96X 10 )  time  15mAH-0.325M  measured  the  high.  this  quickly  and  Fraction  concentration samples  coefficients  R u n G l , because  interval  of  lower  very  of  (15mAH-0.25M  15mAH-0.25M  out  peaks the  Fractions  the  coefficient  for  sedimentation to be  of  flattened  Schlieren  which  hydrolysed  peaks  peaks  except  in  expected  the  of  sedimentation  Fraction,  the  sharp,  Fraction  clear,  larger  M . W . Fraction  for  the  the  Section  the  sedimentation  hand,  and  were  acid  Schlieren  Nevertheless,  in  in  is  degree  fractionated  the  Therefore,  in  duration  S^Q  /  values.  for  markedly  G2  the  coefficients  true  the  Since  M.W.  15mAH-0.325M),  On  true  higher  lower  As  4.11).  the  found  the  sedimentation than  and  skewness.  increases weight,  S^Q  A N D DISCUSSION  discussed were  not  H A concentration,  lower  S  2  Q  than  the  5 15minAH  Fraction  finding  i n agreement  is  because 15minAH  the  (M.W. = 1.65X10  Schlieren  Fraction  with the peaks  are lower  are  ),  as  shown  in  discussion above skewed  than the  to  that  the  measured  Figures for the  right,  values.  the  4.6  and  15minAH true  S^Q  4.7.  This  Fraction, for  the  RESULTS 4.6.  HYDRAULIC  The Eqn  squares  and  is  method  conductivity,  listed  to  give  in  K',  Table  straight  for  4.5.  lines  El,  K'  F l  very  is  in  and  c m /dyn • s 4  Gl  were  high.  The  shown  in  Table  plotted  in  Figures  specific  values 4.6.  to  c  used A  and  The  =  4.15.  Laurent  Ac'  117  Ethier K,  of  et  al., al.,  is  fitted  by  from least  [4.4] The  conductivity  because  Eqn  [4.4]  as  a  (1986), HA  data  calculated  B  fitting  data  was  by  line in  solution  conductivity  mg/mL.  B  et  HA  in  conductivity  (Preston  1961;  is  for  conductivity,  data  Pietruszkiewicz,  of The  4.13  hydraulic  sedimentation  not  and  each  represented  K' where  /  CONDUCTIVITY  hydraulic  [2.10]  A N D DISCUSSION  for  (M.W. = 1  The  HA of  for  Runs  intervals  were  Fractions  are  concentration  are  Eqn  [2.10],  computed  to  13X10  )  Fessler,  1960).  error  the  function  using  1965;  the  data  results  1960b; were  from Laurent  fitted  by  the past & the  equation K  Table  4.6  Values  Fraction  of A  =  2.92  and B  M.W.  X  10"  1 6  in E q n [4.4]  X10  5  c" ' 1  for  [4.5]  4 7  all H A  10  1 0  XA  Fractions  B  0.25M  6.99  8.28  1.43±0.04  0.325M  8.44  7.50  1.50±0.07  0.325M-9  11.1  7.17  1.4610.10  15minAH  1.65  6.62  1.3610.14  lhrAH  0.819  6.59  1.3810.33  2hrAH  0.454  5.50  1.3210.22  15mAH-0.325M  1.96  6.44  1.3910.17  RESULTS  A N D DISCUSSION  F i g u r e 4.13 H y d r a u l i c conductivity data for 0 . 2 5 M , 0 . 3 2 5 M and 0 . 3 2 5 M - 9 Fractions. F o r comparison, the results of E t h i e r (1986) is included.  /  118  RESULTS  A N D DISCUSSION /  1  CO  x o v a •  = = = = =  HA Fraction 15minAH lhrAH 2hrAH 15mAH-0.325M Ethier (1986)  to  a  J  10 Figure  4.14  i  i  i  J  i i i  I  I  L  10 Concentration of HA, mg/mL Hydraulic  conductivity  data  for  all  acid  hydrolysed  Fractions.  10  1  RESULTS  A N D DISCUSSION /  120  oo o  HA Fraction o = 0:25M A = 0.325M + = 0.325M-9 x = 15minAH o = lhrAH v = 2hrAH a = 15mAH-0.325M • = Ethier (1986)  a  J  10"  i i i i iI  io°  1  Figure  4.15  j  i ' • '•  Concentration of HA, m g / m L Hydraulic  conductivity  data  for  all H A  Fractions.  id  RESULTS where  K  is  in  cm  and  2  c  is  E q n [ 4 . 5 ] can be  viscosity,  also  K'  is  for  [4.6].  the  Fractions  The  hydraulic  concentration larger,  and  converging the  the at  high  with  c  each other  the  is  in  4 . 1 5 . From  the  2  other  at  the  For  Figures with  comparison,  4 . 1 3 to the  with  solvent  the  have  Also,  is  as  more  or  i.e.  less  as  becomes  the  the  Eqn  because  lines  expected  three-dimensional molecular  M . W . group  by  molecules  higher.  is  conductivity  represented  HA  which  [4.6]  Eqn  4 . 1 5 , the  line  concentrations,  higher  the  concentration,  is  mg/mL),  molecules  a  is  [4.6]  solvent  high  rj  1A1  between  of  (>  HA  concentrations,  c'  0  slopes  flow  where  form  decreases  M.W.  form  1  K'=K/i\,  mg/mL.  entanglement  to  to  Since  10"  similar  the  lower  each  lower  X  concentrations  with  to  conductivity  resistance  and  entanglement  At  and  have  increases,  higher  entangled  7.51  in F i g u r e s 4 . 1 3 to  plotted  lines  =  cmVdyn'S  in  g/mL.  converted  K' where  in  / 121  A N D DISCUSSION  because  the  HA  are  same  molecules  network.  5 11.1X10 )  ( 6 . 9 9 to  has  a  5 higher  hydraulic  finding from this  is  which result  M.W.  that  than  i.e. at  high  higher the  consistent K' is  can that  molecules,  solvent, is  conductivity t h a n  for  same  resistance  the  trend  high  behave  low  in  as  they in  a  of  S^Q  obtained  shown  may more  concentration, the  solvent  With is  are  more  less  discrete  of  by  there  therefore  more  low  ).  al.  This (1960)  explanation than  to  Another  the  the flow  for low of  interpretation  M . W . molecules  between  entanglement higher.  et  compact  manner. are  1.96X10  One  resistance  entanglement  more  to  Laurent  Eqn [2.10],  in  have  since  degree  M . W . molecules. flow  M . W . group ( 0 . 4 5 4  low  M . W . molecules  therefore  molecules,  low to  the  determined  the  may  M.W. the  be  and  they the  with  the  molecules  between  is  molecules,  RESULTS In  addition,  hydrolysed Schlieren acid  the  sedimentation  Fractions  are  higher  peaks,  the  hydraulic  hydrolysed  HA  Fractions  values. for  HA  since  the  It  is  acid  between the  therefore hydrolysed lines  of the  expected  than  the  be  the  M . W . group  higher  true  lower  true  in (and  measured  values  evaluated  therefore  that  Fractions lower  coefficients  conductivities, are  A N D DISCUSSION  because  from than  positions Figure  of  for  Eqn  /  the of  [2.10],  for  true  the  conductivity  Ethier's line)  and is  the  even  acid  skewed  the  4.15,  122  the  conductivity lines  difference larger.  C H A P T E R 5. CONCLUSIONS AND RECOMMENDATIONS  In  this  study,  chromatography with  NaCl  with  that  and  column  each  NaCl  eluate  acid  DEAE-cellulose.  solutions  revealed the  hyaluronic  by  molecular was  stepwise  The  concentration,  the  by  been  fractionated  hyaluronic  elution  fractionation  controlled  has  was  ion-exchange  adsorbed  method.  Preliminary  attained  when  2 m M phosphate  amount  acid  by  the  buffer  of  had  the  within  of hyaluronic acid eluted  eluted  experiments  pH  to  was  7  eluant  to  decreased  8.  At  with  the  volume.  In  a  partially  formal  successful.  experiment  could  continuous. hyaluronic average  Because acid  hyaluronic  be of  the  in  reproduced,  partly  because  long  which  time  was  at  each the  taken  evident  fractionation  M . W . of  molecular  obtained  the  eluted  experiment,  results  the  occurred,  acid  and  The  not  [ T J ] after  volume,  fractionation  the  for  from  H A fraction  elution  the the  increases  decreased with  the  was  not  degradation  drop  general,  was  fractionation  process  elution,  large  In  concentration  fractionation  preliminary  the  experiment.  NaCl  weight  in  the  the  of  weight  amount  with NaCl  the  of  eluate  concentration 5  of  the  eluant.  determined  from [77] data,  Three acid  T h r e e distinct  molecular  were  obtained  hyaluronic acid fractions  hydrolysing  the  previous  weight  fractions,  from the  of lower  fractions  for  M . W . = 6.99  11.1X10  ,  fractionation.  molecular  15  to  min.,  1  weight hour  were and  2  obtained  by  hours.  The  with  the  5 molecular duration  weight of  acid  of  the  hydrolysis.  fractions Also,  (0.454 from  to  1.65X10 )  sedimentation  123  decreased  experiments,  the  range  of  CONCLUSIONS M.W.  in  the  acid  hydrolysed  fractions  AND RECOMMENDATIONS  increased  with  the  duration  /  124  of  acid  hyaluronic  acid  hydrolysis.  Another hydrolysed fraction.  fractionation  for  15  This  min. The  was  until the  not  was  performed  hyaluronic acid was  finding showed  chromatography cellulose  experiment  that  the  consistent.  results  found to  of  the  H y a l u r o n i c acid  ionic environment  in  the  on  column  be  eluted  in one  fractionation  by  was  adsorbed  was  tightly  above  major  ion-exchange to  0 . 3 M [NaCl].  the  From  5 sedimentation  experiments,  the  narrow  of molecular  weight.  range  Sedimentation that  sedimentation  observation  is  molecules,  in  and  experiments  accordance  therefore  the S^Q  concentration,  because  at  high  and  the  were  low  found  decreased M.W.  the  —  with  for  the  T h e former  curves  of  all  concentration, between  However,  for  the  trend in S  because  at  acid  a  to  the  ( M . W . = 1.96X 10 )  H A fractions  indirectly  resistance  fractionated,  M . W . and  all the  expectation  entanglement  M . W . fractions.  obtained  on  varies  hydraulic  the  and  performed  with  Also,  formed  fraction  coefficient,  concentration.  was  major  with the  entanglement flow,  fractions  was  the  lower  concentrations,  non-acid  hydrolysed  hydrolysed  does not  agree  increases  same two  past  for  high  network the  trends  high  in S^Q  fractions,  S^Q  increased  with  fractions,  with  with  at  molecular  HA  This  between  converged  three-dimensional  a  revealed  concentration.  solvent  molecules  have  has  sedimentation  data.  The  Au reason  is  fractionated ( M . W . = 6.99  unknown non-acid to  and  may  hydrolysed  11.1X10 ) 5  and  be  attributed  to  experimental  fractions  are  the  hydrolysed  acid  treated  as  a  fractions  errors.  high as  M.W. a  low  If  the group  M.W.  CONCLUSIONS  A N D RECOMMENDATIONS  /  125  M . W . group  fall  5 group  ( M . W . = 0.454  below  those  of  sedimentation  to  the  data.  high  In  fractions  were  skewed  fractions  were  higher  between  the  will be  even  curves  The been  as  the  varied  the  lines  the  The  for  all  the  reasons  for  and  the  M . W . molecules. the  relationships should  fall  to  there  high  the  to  network  is  this  less  may  of  The  the  is  low  in  peaks  agreement of  calculated  the  for  Therefore,  the the  and  those  the  hyaluronic  all  results  revealed  concentration. molecular  the  fractions  M . W . group  compact  determined  S^Q values.  K',  with  resistance  high  of  Due  the  of  which  M . W . group  increases,  molecules  for  curves  2 Q  Schlieren  true  data.  indirectly  molecular  lower  high  S^Q  three-dimensional  low  right,  conductivities,  from  and  conductivity  group.  the  the  the  concentration  increases  the  S  group,  since  than  of  hydraulic  fractions  that  M.W.  fact, to  the  of  with  acid  hydrolysed  acid  hydrolysed  actual  the  past  low  difference  M . W . group  higher.  calculated  HA  1 . 9 6 X 1 0 ),  is  flow  be  of  formed. a  entanglement Consequently,  At  higher  that  the  between the  the  K'  reason  for  this  solvent at  high  than  high the  for  of  to  the  the  flow  of  a  HA  low M . W . are  more  M . W . molecules the  is  The  because  M . W . molecules  resistance  the  increases.  concentrations,  high  all  molecules  concentration  those  have  finding  between  therefore  lower  K'  fractions  that  entanglement  converged  has  The  acid  than  solvent  is  M . W . molecules.  skewness  from  the  optical  for  the  acid  below  the  of  the  Schlieren  measurements, hydrolysed  relationships  shown  peaks, are  fractions in  the  sedimentation  overestimates. which  Figures  4.14  As  exhibited and  4.15.  coefficients, such  this  the  K'  skewness  Nevertheless,  CONCLUSIONS the  K'  calculated  From hyaluronic  are  the  acid  believed  results  to  be  close to  obtained  appears  to  be  results  of  the  in  a  this  A N D RECOMMENDATIONS  the  actual  study,  function  of  126  values.  the  both  /  hydraulic  conductivity  concentration  and  of  molecular  weight.  Since  the  reproducible, therefore  to  Also, interstitium.  and  at  different  a  the  of  of  of  is  major  Therefore,  it  proteins  (e.g.  study  p H on  temperature  solvent  molecular  degradation  performance  effect  lower  experiments  further  the  the  Another  proteins.  experiments  that  improve  investigating  fractionation  like  considerable  recommended  undertaken include  and  fractionation  to  not  is  weights  be  the  only  in  carried  that  a  the  and  it  method studies  or is be  might  performing  the  bacteria.  transported macromolecular  future  hyaluronic acid out.  by  component  in  occurred,  Future  pattern,  degradation  is  consistent,  fractionation  method.  elution  not  molecules  the  transported  suggested  albumin)  the  of  reduce  the  component  of  HA  were  study,  of different  in  the  species  sedimentation concentration  REFERENCES  Adamson,  R.  hyaluronic  acid.  H.  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Calibration Line for the Glucuronolactone Concentration  The was  results  tabulated  in  glucuronolactone fitted  straight  obtained Table  were  line  from  the  4.1.  fitted  uronic  The  by  absorbance  the  is represented  least  x is  y  is  the  absorbance,  the concentration  A  is  and  =  from  SS  A = =  SS and  n is  x  the n u m b e r of d a t a  SS SS and  .2 1 =  substituting  these B  -  I'I  =  .2  i  =  y  x. 1  points.  Then,  y  =  0.45238  x  =  29.55  =  970.225  = into  B  is  the  slope  of  least  squares  the  line a n d  M e n d e n h a l l (1987), [A. 2]  x Bx  [A.3]  2 z . ) ( .2 y . ) / n 1= 1 l i  -  2  1  5  xy  the  of  [A.4]  =  =  x  If  concentrations  /SS  (  n  values =  x.y.  1  method.  5  3.2)  [A.l]  the y-intercept,  of glucuronolactone,  xy  for  (Section  Bx  xy  SS  data  method  by  B  where  carbazole  squares  y = A + where  acid  ( .2 x. ) / n 1=1 l 2  for the calibration line,  16.02004 Eqns  [A.2] a n d [A.3],  16.02004/970.225  134  =  0.01651(17)  [A.5]  / A Therefore,  the  least  =  0.45238  squares  -  X  0.01651(17)  calibration  29.55  line  for the  +  0.01651  =  135  -0.03554  glucuronolactone  concentration  is ABS where  ABS  is  the  =  -0.03554  absorbance  at  525  nm  X c  and  c  [4.1] is  the  concentration  of  glucuronolactone.  A.2. Amount of Hyaluronic Acid in an Eluate Fraction  To  determine  concentration [4.1].  in  the  the  amount  fraction  T h e concentration  glucuronolactone  with  Consider  the  of H A in a n eluate  was  of H A was  the factor  0.325M  NaCl  (Table  4.2).  absorbance  (of  measurements)  0.5598.  concentration  =  (0.5598  amount  +  the  HA  =  3  6  from  -  0  6  X  2  -  2  of H A i n the subfraction is =  82.18  =  41.09 m g  7  9  subfraction subfraction  6  in  was  500  after  =  the  36.06  was  8  2  -  1  8  M  g  therefore  Mg/mL  absorbance  in the subfraction  =  X  glucuronolactone by E q n  concentration  of  4.1).  subfraction  sufbraction  the  multiplying the  0.03554 ) / 0 . 0 1 6 5 1  of H A in the  C  The  eluate, of the  of  by  (Section  T h e concentration of glucuronolactone c  The  T h e volume  evaluated  obtained  2.279  experiment  3  first  fraction, the  500 m L  /  m  L  the  fractionation  ml. The  carbazole was  Mg/mL  average  test  was  / A.3.  I n t r i n s i c V i s c o s i t y , [77]  The c=0,  intrinsic 7j  where  respectively. time HA  of  from  multiplying  example,  the  time  time  is  time  by  for  the  by  the the  of  solvent  0.325M-9 viscometer Data,  2  with  2  The  746.96 s. X  each  concentration  is  shown  solvent  =  77  is  Table  of  the  A . l . The  determined was  efflux  by  the  calculated  by  A t concentration = 0 . 8 0 6 7  77, is  3.1193  (0.2M NaCl)  as  H A . The  in  to  c  solution,  at  constant.  T h e viscosity,  0.004176  HA  of  viscosity  the viscometer  and  Fraction  Cannon-Ubbelohde viscometer cm /s .  (77-770)/VQ *  extrapolating  viscosities  B-Experimental  746.96 efflux  obtained  the  0.004176  efflux  the efflux  are  measured  of  was  the  for  was  Appendix  constant  manufacturer  17  and  0  Consider,  Fraction,  mg/mL,  viscosity  H A solution  viscometer  The  136  cm /s 2  220.26.  The  solvent  viscosity,  J?o> is 220.26  Table  A . 1 Viscosity data  Concentration,  for  X  0.004176  =  0.9198  cm /s 2  0.325M-9 Fraction  Efflux  time,  V,  (•n-"Qo)/vo * > c  mg/mL  s  cm /s  mL/mg  0.8067  746.96  3.1193  2.9643  0.4034  424.81  1.7740  2.3021  0.2689  347.56  1.4514  2.1493  0.2017  311.81  1.3021  2.0607  0.1613  291.97  1.2193  2.0187  0.08067  254.31  1.0620  1.9164  2  Therefore,  (77-770 ) / T ?  *C =  0  (3.1193  = The  data  intrinsic mL/mg  for  (77-770) / T 7 - C  or  versus  0  viscosity  obtained  2.9643  by  -  /  137  4.2.  The  0 . 9 1 9 8 ) / 0 . 9 1 9 8 • 0.8067  mL/mg  c were  plotted  extrapolating  as  shown  (77-770 ) / r j  -c  0  in  Figure  to  c= 0  was  1.830  1830 m L / g .  A.4. Molecular Weight  The again  the  viscosity  molecular  weight  0.325M-9  at  zero  Fraction.  shear,  this  value  HA  Since  [*?]__, was  [ T } ]  Substituting  of  Z  S  into  =  [ 77  was [77]  first  evaluated was  using  above  Eqn  1400  mL/g,  ] <1 +  -  ([77]  1830( 1 +  =  1956  intrinsic  1400)/6250)  (1830  -  1400)/6250)  mL/g  E q n [4.3],  where  1956  =  0.0228  M  =  [ log ( 1 9 5 6 / 0 . 0 2 2 8 ) ] / 0 . 8 1 6  M  =  1.11  then  the  Consider  calculated u s i n g E q n [4.2].  =  log  [4.3].  M  is  M ° '  X  10  the 8  1  molecular weight  of H A ,  6  6  A.5. Sedimentation Coefficient  The  sedimentation  sedimentation magnification not  the  photograph in the  actual  coefficient, Run  C l  optical s y s t e m ,  the  distance.  of  S  The  actual  , is  was  calculated  shown  distance  distance  in  Figure  measured between  using  on  the  Eqn  A . 1.  [2.41]. Because  A of  the  photograph  was  inner  reference  edge  H E Figure  CiRE  in  A . l Sedimentation  Figure  manufacturer, measured cm.  A.l)  was  by  a  RE  the  cm.  The  magnification M  The  distance  outer  from  manufacturer).  1^  The  to  the  distance,  between  (Oj^g)»  the  two  (Micro-Master M-150,  as  r , between  was  measured  the  3.248/1.60  centre  r  by  ,  the  Schlieren peak r  Eqn  [2.41] can be  rewritten S  of  =  the  reference  2  can  then  be  evaluated  edges  was  V.W.&R.)  S  to  be  3.248  between  2.03  rotor  the  was  5.70  Schlieren  cm  peak  travelling  a n d the =  (given  and  by  the  on  the  Kill  5.70  microscope.  centre +  r  of  mS  the  Then rotor  the  actual  distance,  was  /2.03  [A. 7]  as  2 Q  =  [d(\n  r )/dt]/a>  2  s  2.303[d{log SQ  the  given  factor =  F  edge  mS photograph  by  :  reference  distance  travelling microscope  Therefore, the  138  photograph of R u n C l .  and  1.60  /  from the  slope  of  r the  )/dt]/a>  [A. 8]  2  plot  of log  versus  time.  In  / all  the sedimentation r u n s ,  or  u  = =  Eqn  [A. 8]  then S  The  2  Q  52640 r p m X  5512.4)  =  7.579  r^/dt]  r ,  X  10"  [ci(log  8  determined  Table  A . 2 . T h e plot of log r  calculated  from  for  s  [A.9]  2  R u n C l . T h e results  E q n [A. 7],  versus  2  time  is  a n d the shown  from  Appendix  B  are  tabulated  in  A . 2 . T h e slope  of  log r ,  in F i g u r e  straight line was calculated using E q n [A.2] to SS SS slope  Table  s  rad/s  s  with  and  1 min/60  2 . 3 0 3 [ d ( l o g r )/dt]/(  were  least squares  X  to  along  the  2irrad/s  =  distances the  co= 52640 r p m ,  5512.4  reduces  =  x  xy B  5.28  X  10  =  1.3465  =  1.3465/5.28  [A.5]:  3  X  10  3  =  2.55  X  10"  min"  4  A . 2 Results of sedimentation R u n C l .  Time,  min  139  mc:>  r  c  m  r  c>  c  m  Log < r  0  0.427  5.9103  0.7716  8  0.485  5.9389  0.7737  16  0.538  5.9650  0.7756  24  0.588  5.9897  0.7774  32  0.646  6.0182  0.7795  40  0.729  6.0591  0.7824  48  0.754  6.0714  0.7833  56  0.822  6.1049  0.7857  64  0.885  6.1360  0.7879  72  0.949  6.1675  0.7901  1  /  Si  °  f  0.0  1  1  1  15.0  1  1  30.0  1  45.0  Time, m i n Figure  A . 2 T h e sedimentation  plot for R u n C l .  1  1  60.0  >1  75.0  140  / Then,  the sedimentation S  2  coefficient  Q  at  =  (7.579  =  3.22  20°C,  X  using E q n [A.9] is: X  10" )(2.55 8  10"  X  141  10" )/60  s  4  s  1 3  A.6. Hydraulic Conductivity  From  Section  2.4, K'  At  20°C,  [2.10]  then  / C  2  1.0068 g / m L  v  =  0.67  reduces  sedimentation  0  =  ( 1  -  D  2  p,)  mL/g  [2.10]  (from Preston  et  a l . , 1965)  to  K' For  2  p,  2  Eqn  = S  = S  2 Q  /0.3254c  [A. 10]  2  Run C I , S c  Therefore,  K'  2  =  3.22  =  0.484  10"  X  1 3  s  mg/mL  =  3.22  X  10"  1 3  =  2.05  X  10"  1 2  =  2.05  X  10"  9  / ( 0 . 3 2 5 4 ) (0.484) s« mL/mg cm'/dyn.s  A.7. Equation for K' as a function of c  Consider the  hydraulic  Table method.  A . 3. The  R u n C I to  conductivity, The  log  slope  K'  and  C 5 for the along  with  versus  log  the  0.325M-9  the c  y-intercept  values  data for  Fraction. of their  were  the  fitted  least  T h e concentration a n d logarithm, are by  squares  the  listed  least  straight  in  squares  line  were  / obtained  142  from E q n [A.2] to [A.5]: y  =  -9.2908  x - 0.100676 SS SS Then  =  0.3047 -0.443751  xy B  =  -0.443751/0.3047  A  =  -12.2906  -  =  -1.456  (-1.456) (0.100676)  -9.1442 Therefore  log K' K'  -9.1442  ,.-9.1442 ll) 7.17  A.8.  Errors  There temperature  Table c,  in S  on  A . 3 c a n d K' mg/mL  X  X  1.456 v  x  10-  -1.456  c 1 0  log c  c  -  L  4  5  6  a n d K'  are m a n y control  -  potential  of the  data  errors in m e a s u r i n g  ultracentrifuge.  for 0 . 3 2 5 M - 9  Log  c  However,  S g Q , such the  primary  as  errors due to  source  of error  Fraction  10 XK', 10  Log  K'  cm"/dyn«s 0.484  -0.3152  20.5  -8.6882  0.968  -0.01412  7.85  -9.1051  1.452  0.1620  3.98  -9.4001  1.936  0.2869  2.69  -9.5702  2.420  0.3838  2.04  -9.6904  is  thought  centre  of  to the  be  from  rotor,  measuring  the  The  in  r .  error  distance r  S of  the  Thus,  plot the  of log  the  log  task  versus is  error  manifested  Schlieren in  the  peak  error  of  from  the  the  slope  to  time,  which  determine  the  is  used  error  for  the  interval of  evaluation  the  slope  of  of the  plot  that  the  time.  determining  in r  the  143  S  r  here  versus  In errors  of  is  of  /  the  a n d therefore interval  error  interval  in log  r  calculated  of  are  is  the  the  slope,  it  is  assumed  approximately n o r m a l l y distributed. 95%  confidence  interval  based  Also,  on  the  Student's t-distribution.  For points,  the  the  least  squares  95% confidence  for is  a  (t 2>0.025) 95%  the  is  n  xy  SS  interval  equations  and  for the  x  are the  true  =  2  B  y  slope (SS  A  cr^ /  +  =.S  y . 1=1/1  as  defined B  B -  2  for example,  derived  be  X  SS  data  [ A . 11]  with  n-2 the  degrees  of  freedom  interval shown,  a„ a  expression:  )/(n-2)  [A.12]  x.) /n  [A. 13]  xy  (.2  n  1987)  x  within  by the  from  2  1=1 l in  can  t-distribution test  Bx  /SS  JJ  will  given -  y  slope  slope  Eqns  Run C l :  [A.4]  therefore  statistic  1987).  Consider,  =  (from Mendenhall,  X  O  the  SS and  is  ,0.025) n-A  s t a n d a r d deviation of the  SS  error  (t  B  y  t-distribution test statistic  probability that  in which and  ±  the  a  line  interval for  B where  straight  be  obtained  and  [A.5],  calculated from  a  respectively. from  t-table  the  The above  (Mendenhall,  / n SS ss ss  =  10  =  3.443  =  5.28  =  1.3465  y X  xy  X  a  From and  t-table  B  =  [(3.443  X  =  3.43  10"  (Mendenhall,  therefore,  from  = To  calculate  X  the  10"  B  X  error  2.306  9  95%  X  10"  X 10'  X  for  ,0.025)  X  4  =  confidence  3.43  0.109  interval  4  2.55  (t the  ±  io-  -  4  3  1.3465)/8 ]  0 , 5  4  E q n [A. 11],  2.55  10  X  1987),  ±  10"  X  B = 2.55  144  interval  10" /(5.28 4  for  X  the  10 )°" 3  slope  is  5  4  S ,  first  1.09  X  NN  2.306,  the  % error  in  the  slope,  % E  0  , is  obtained: %E„  = =  From  E q n [A.9],  Therefore,  the  S  error  2  interval S =  Similarly,  for K',  X  in S^Q  is  ±  2 Q  3.22  ±  it  is  assumed  that  c  2  is K'  = The  error  2.55  X  10"  4  X  100%  10"  X  8  B/60  4.27% 0.14  10"  X  s"  1 3  1  since K'  and  /  5  4.27%  7.579  =  Q  10"  intervals  in  2.05 the  =  S  2 Q  /0.3254  accurate, + ±  slopes  the  c  error  [A. 10]  2  interval  in K'  is  4.27% 0.09 for  X the  10"  9  plot  cmVdyn-s " 1  of  absorbance  versus  concentration  / (carbazole a  reaction test)  a n d for  the  plot of log K'  versus  log c were  145  obtained in  similar manner.  'The  values  different error  of  from  the the  intervals  error values  were  interval listed  evaluated  for  in  S^Q  Table  from  and 4.5.  values  It of  K' is  calculated because  SS ,  SS ,  x calculated were  by  rounded  computer off.  a n d were  not  rounded  off,  whereas  here in  are  Table  etc,  slightly 4.5,  which  the were  y i n here,  those  values  B.  EXPERIMENTAL  B.l.  Viscosity  DATA  / 146  Data  Fraction: preliminary fractionation III, 0.3M Concentration, mg/mL 0.2M NaCl 0.7347 0.4898 0.3674 0.2939 0.1837 0.07347  Efflux  time,  V. fs  cm 220.26 403.69 339.20 304.39 286.16 259.95 235.69  2  (Tlo  )/lo  mL/mg  0.9198 1.6858 1.4165 1.2711 1.1950 1.0856 0.9842  1.1335 1.1025 1.0396 1.0180 0.9813 0.9530  Efflux time  V  ( ' T ' J o )/Vo ' C  646.52 473.28 397.40 355.82 330.17 299.35 282.32 249.54  2.6999 1.9764 1.6595 1.4859 1.3788 1.2501 1.1790 1.0421  1.4551 1.2955 1.2093 1.1569 1.1257 1.0800 1.0594 0.9997  Efflux time  V  (')-i)o)/'7o-c  1007.47 503.07 389.01 340.21 313.56 264.09  4.2072 2.1008 1.6245 1.4207 1.3094 1.1028  2.5226 1.8125 1.6222 1.5375 1.4946 1.4041  Fraction: preliminary fractionation III, 0.4M  1.330 0.8867 0.665 0.532 0.4433 0.3325 0.266 0.133  Fraction: 0.25M  1.4168 0.7084 0.4723 0.3542 0.2834 0.1417  Fraction: 0.325M Efflux 0.9202 0.4601 0.3067 0.2301 0.1840 0.09202  time  739.50 420.59 342.95 308.42 288.70 252.89  n 3.0882 1.7564 1.4321 1.2880 1.2056 1.0561  2.5619 1.9768 1.8160 1.7397 1.6887 1.6103  Fraction: 0.325M-9 Efflux 0.8067 0.4034 0.2689 0.2017 0.1613 0.08607  time  746.96 424.81 347.56 311.81 291.97 254.31  V 3.1193 1.7740 1.4514 1.3021 1.2193 1.0620  2.9643 2.3021 2.1493 2.0607 2.0187 1.9164  Fraction: 0.4M Efflux 0.6315 0.3158 0.2105 0.1579 0.1263 0.06315  time  504.68 338.21 294.83 274.97 263.79 241.53  V 2.1075 1.4124 1.2312 1.1483 1.1016 1.0086  ( VVo  )/i)  2.0447 1.6959 1.6083 1.5733 1.5649 1.5288  0  'C  Fraction: 0.5M Efflux 0.8432 0.4216 0.2811 0.2108 0.1686 0.08432  time  833.90 444.29 355.63 317.26 295.82 256.36  1 3.4824 1.8554 1.4851 1.3249 1.2353 1.0706  (trio'/lo 3.3041 2.4127 2.1864 2.0893 2.0345 1.9444  Fraction: unfractionated HA Efflux 0.5594 0.2797 0.1865 0.1399 0.1119 0.05594  Fraction:  Efflux time  V  597.25 455.75 392.11 324.49 295.80 279.33 248.52  2.4941 1.9032 1.6375 1.3551 1.2353 1.1665 1.0378  (J?-7j  0  )/T)  0  3.4573 2.8107 2.5193 2.4154 2.3545 2.2992  0.5786 0.5422 0.5276 0.4800 0.4638 0.4534 0.4337  lhrAH Efflux  3.0833 2.0555 1.5417 1.0278 0.7708 0.6167 0.30833  Fraction:  V 2.6987 1.6429 1.3519 1.2305 1.1621 1.0381  15minAH  2.958 1.972 1.479 0.986 0.7395 0.5916 0.2958  Fraction:  time  646.23 393.42 323.74 294.67 278.29 248.58  time  n  413.66 343.71 310.37 277.35 262.09 253.37 236.37  1.7275 1.4353 1.2961 1.1582 1.0945 1.0581 0.9871  Efflux time  n  330.58 293.18 274.33 254.80 245.65 240.37 230.13  1.3805 1.2243 1.1456 1.0640 1.0258 1.0038 0.9610  (v-no  )/lo  0.2848 0.2727 0.2654 0.2522 0.2464 0.2438 0.2373  2hrAH  3.0544 2.0363 1.5272 1.0181 0.7636 0.6109 0.3054  Fraction:  0.1640 0.1626 0.1607 0.1540 0.1509 0.1495 0.1467  15mAH-0.325M Efflux  5.4770 2.7385 1.8257 1.3693 1.0954 0.5477  time  1480.82 651.83 472.38 395.56 355.31 282.25  n 6.1839 2.7220 1.9727 1.6519 1.4838 1.1787  1.0449 0.7155 0.6270 0.5813 0.5598 0.5139  Fraction: unfractionated acid hydrolysed H A c 4.770 2.385 1.590 1.1925 0.9540 0.4770  Efflux  time  1238.22 582.33 432.52 370.48 336.83 274.58  n 5.1708 2.4318 1.8062 1.5471 1.4066 1.1466  0.9689 0.6892 0.6061 0.5719 0.5548 0.5169  B.2. Sedimentation Data  Samp Is: 0.2SM Fraction HA Concentrations 0.3542 mg/mL From sedimentation velocity experiment: Tm i e (min) Distance from peak to Actual dist. (Y) of peak LOG V left ref. edge (cm) from centre of rotor (cm) 0.0 0.469 5.9310 0.7731 8.0 0.542 5.9670 0.7758 16.0 0.614 6.0025 0.7783 24.0 0.684 6.0369 0.7808 32.0 0.768 6.0783 0.7838 40.0 0.831 6.1094 0.7860 48.0 0.923 6.1547 0.7892 From the graph of LOG Y vs Time : SL0P£= 0.33107E-03/min Y-INTERCEPT;; 0.77306 CORRELATO I N COEFFICIENTS 0.99944 SSXs 0.17920E*04 SSYs 0.19663E-03 SSXYs 0.59328£«00 SSE= 0.21908E-06 SG I MAs 0.20932E-03 95% confidence interval for SL0PE= 0.12713E-04 Sedimentation coefficient at 20.0 deg.= 0.41819E-12 s. K/AETA= 0.3628E-11 sm ' L/mg Permeability. K=0.36278E-10 cm"2 Sample: 0.25M Fraction HA Concentrations 0.7084 mg/mL From sedimentation velocity experiment: istance from peak to Actual dist. (Y) of peak LOG Y Time (min) D from centre of rotor (cm) left ref. edge (cm) 0.0 5.8872 0.380 0.7699 B.O 5.9172 0.441 0.7721 16.0 5.9463 0.500 0.7742 24.0 5.9744 0.557 0.7763 32.0 6.0010 0.611 0.7782 40.0 6.0246 0.659 0.7799 48.0 6.0552 0.721 0.7821 56.0 6.0828 0.777 0.7841 64.0 6.1089 0.830 0.7860 From the graph of LOG Y vs Time : SL0PE= 0.24906E-03/min Y-INTERCEPT= 0.77013 CORRELATO I N COEFFICIENTS 0.99967 SSX= 0.384006-04 SSYs 0.23836E-03 SSXY= 0.9S640E*00 SSEs 0.15664E-06 SIGMAs 0.14959E-03 95%  confidence interval for SL0PE= 0.57091E-05 Sedimentation coefficient at 20.0 deg.= 0.31460E-12 s. K/AETA= 0.1365E-11 smL/mg Permeability. K=0. 13646E-10 cm"2 -  Sample: 0.25M Fraction HA Concentrations 1.0626 mg/mL From sedimentation velocity experiment: Time (min) Distance from peak to Actual dist. (Y) of peak LOG Y left ref.edge (era) from centre of rotor (cm) 0.0 0.487 5.9399 0.7738 8.0 0.544 5.9680 0.7758 16.0 0.590 5.9906 0.7775 24.0 0.632 6.0113 0.7790 32.0 0..688 6.0389 0.7810 40.0 0. 727 6.0581 0.7823 48.0 0.770 6.0793 0.7839 56.0 0.820 6.1039 0.7856 64.0 0.865 6.1261 0.7872 72.0 0.918 6.1522 0.7890 80.0 0.976 6.1808 0.7910 88.0 1.028 6.2064 0.7926 From the graph of LOG Y vs Time : SL0PE= 0.21157E-03/min Y-INTERCEPT= 0.77393 CORRELATO I N COEFFICIENTS 0.99944 SSX = 0.91520E*04 SSYs 0.41011E-03 SSXYs 0.19363E«01 SSE= 0.46188E-06 SG I MA= 0.21491E-03 95% confidence interval for SLOPEs 0.50052E-05 Sedimentation coefficient at 20.0 deg.s 0.26724E-12 s. K/AETAs 0.7728E-12 sm ' L/mg Permeability. KsO. 77278E-11 cm"2  /  148  Sample: 0.25M Fraction HA Concentrations 1.4168 mg/mL From sedimentation velocity experiment Distance from peak to Actual dist. (Y) of peak LOG Y left ref. edge (cm) from centre of rotor (cm) 0.0 0.414 5.9039 16.0 0.495 5.9436 32.0 0.578 5.9847 48 .0 0.661 6.0256 64 .0 0. 734 6.0616 60.0 0.819 6.1034 96 .0 0.909 6. 1478 112.0 0.987 6.1862 126.0 1 .067 6.2256 144 .0 1. 157 6.2700 a6 p6 hE-0 o3 f/m LOiG SLOPEtshe0.lg 8r0 n vsYTime : Y-INTERCEPTS 0.77119 CORRELATO I N COEFFICIENTS 0.99992 SSXs 0.21120E»05 SSY= 0.68944E-03 SSXYs 0.38156E«O1 SSEs 0.11489E-06 SG I MAs 0.11984E-03 95% confidence interval for SLOPE- 0.19016E-05 Sedimentation coefficient at 20.0 deg.= 0.22820E-12 s. K/AETAs 0.4949E-12 s-mL/mg Permeability. K=0.49492E-11 cm2  0.7711 0.7741 0.7770 0.7600 0.7826 0.7656 0.7887 0.7914 0.7942 0.7973  -,  Sample: 0.25M Fraction HA Concentration^ 1.7710 mg/mL From sedimentation velocity experiment: Distance from peak to Actual dist. (Y) of peak LOG Y left ref. edge (cm) from centre of rotor (cm) 0.0 4 38 5.9158 .7720 16.0 519 5.9557 .7749 32.0 595 5.9931 .7777 48.0 675 6.0325 . 7805 64.0 753 6.0709 .7833 60.0 831 6.1094 .7860 96.0 887 6.1369 . 7880 112.0 967 6.1764 0.7907 128.0 1.045 6.2148 0.7934 144.0 1. 128 6.2557 0.7963 From the graph of LOG Y vs Time : SLOPEs 0.16604E-03/min Y-INTERCEPT= 0.77232 CORRELATO I N COEFFICIENTS 0.99953 SSX = 0.21120E»05 SSYs 0.58278E-03 SSXYs 0.35067E-»01 SSEs 0.54321E-06 SIGMAs 0.26058E-03 95%  confidence interval for SLOPEs  0.41348E-0S  Sedimentation coefficient at 20.0 deg.= 0.20973E-12 s. K/AETA= 0.3639E-12 s"mL/mg Permeability. K=0.36388E-11 cm2 -,  Sample: 0.325M Fraction HA Concentrations 0.4601 mg/mL From sedimentation velocity experiment: (Y) of peak Time (min) Distance from peak to Actual.dist. from centre of rotor (cm) left ref. edge (cm) 0.7746 5.9512 510 0.0 0.7772 5.9867 582 8.0 0.7796 6.0197 649 16.0 0.7619 6 . 0 5 1 7 . 7 1 4 24.0 0.7841 6.0833 .776 32.0 0.7863 6 . 1 1 3 6 . 8 4 0 40.0 0.7886 6.14 58 .905 48.0 From the graph of LOG Y vs Time : SL0PE= 0.28895E-03/min Y-INTERCEPTS 0.77482 CORRELATO I N COEFFICIENTS 0.99966 SSXs o . 1 7 9 2 0 E » 0 4 SSYs 0.14972E-03 SSXYs 0 . 5 1 7 8 0 E « 0 0 SSEs 0.10291E-06 SIGMAs 0.14346E-03 95%  confidence interval for SLOPEs 0.87131E-0S Sedimentation coefficient at 20.0 deg.s 0.36498E-12 s. K/AETAs 0.2438E-11 9 -mL/mg Permeability. KsO . 24375E- 10 cm"2  /  149  Sample: 0.325M Fraction HA Concentrations 0.9202 mg/mL From sedimentation velocity experiment: Distance from peak to Actual dist. (Y) ofpeak left ref.edge (cm) from centre of roto(c rm) 0.0 0 378 5.8862 6.0 0.432 5.9126 16 .0 0.474 5.9335 24.0 0 . 5 1 8 5.9552 32.0 0.563 5.9773 40.0 0.608 5.9995 46.0 0.644 6.0172 56.0 0.666 6.0379 64 .0 0. 735 6.0621 72.0 0. 777 6.0828 60.0 0.823 6.1054 ea.o 0 . 8 6 3 6.1251 96.0 0.907 6.1468 104 .0 0.966 6.1759 112.0 1.009 6.1970 From the graph of LOG Y vs Tm ie : SL0PE= 0.19565£-03/min Y- N I TERCEPTS 0.77010 CORRELATO I N COEFFICIENTS 0.99971 SSXs 0.17920E>05 SSYs 0.68635E-03 SSXYs 0.35060E»01 SSEs 0.39360E-06 SG I MAs 0.17400E-03 95X confidence interval for SLOPEs 0.28076E-OS Sedimentation coefficient at 20.0 deg.s 0.24713E-12 s. K/AETAs 0.8252E-12 sm ' L/mg Permeability. KsO.82523E-11 cm2  LOG Y 0.7698 0.7718 0.7733 0.7749 0.7765 0.7781 0.7794 0.7809 0.7 826 0.784 1 0.7857 0.7871 0. 7886 0. 7907 0.7922  ,-  Sample: 0.325M Fraction HA Concentrations 1.3803 mg/mL From sedimentation velocity experiment: Tm i e (min) Distance from peak to Actual dist. (Y) of peak LOG Y left ref. edge (cm) from centre of rotor (cm) 0.0 0.533 5.9626 0.7754 16.0 0.602 5.9966 0.7779 32.0 0.681 6.0355 0.7807 480 0.745 6.0670 0.7830 64.0 0.818 6. 1030 0.7855 80.0 0.897 6. 1419 0.7883 96.0 0 960 6. 1729 0.7905 112.0 1.055 6.2197 0.7938 128.0 1. 124 6.2537 0.7961 144.0 1. 163 6..2828 0.7982 160.0 1.268 6..3246 0.8010 176.0 1.346 6.3631 0.8037 192.0 1.434 6.4064 0.8066 208.0 1.526 6.4517 0.809 7 From the graph of LOG Y vs Tm ie : SLOPEs 0.16262E-03/min Y-N I TERCEPTS 0.77526 CORRELATO I N COEFFICIENTS 0.99964 SSX= 0.58240E*05 SSY= 0.15412E-02 SSXYs 0.94709E'0I SSE= 0.1I055E-05 SIGMAs 0.30352E-03 95X  confidence Interval for SLOPEs 0.27405E-05 Sedimentation coefficient at 20.0 deg.s 0.20541E-12 s. K/AETAs 0.4573E-12 S -mL/mg Permeability. KsO.45727E-11 cm"2 Sample: 0.325M Fraction HA Concentrations 1.8404 mg/mL From sedimentation velocity experiment: Tm i e (min) Distance from peak to Actual dist. (Y) of peak LOG Y left ref. edge (cm) from centre of rotor (cm) 0.390 5.8921 0.7703 0.0 0.447 5.9202 0.7723 16.0 0.498 5.9453 0.7742 32.0 0.582 5.9867 0.7772 48 .0 0.640 6.0153 0.7793 64.0 0. 737 6.0631 0.7827 80.0 0.804 6.0961 0.7850 96.0 0.874 6.1306 0.7875 112. 0.925 6.1557 0.7893 1.005 6.1951 0.7920 144 1 .066 6.2251 0.7941 160 1 . 129 6.2562 0.7963 176. 1 . 188 6.2852 0.7983 192 From the graph of LOG Y vs Tm ie : SLOPEs 0.IS023E-03/min Y- N I TERCEPT^ 0.77008 CORRELATO I N COEFFICIENTS 0.99891 12e  SSX = 0.46592E-05 SSYs o.10538E-02 SSXYs 0.69995E-01 SSE= 0.22950E-05 SIGMAs o.45677E-03 95t  confiaence interval for SLOPEs 0.46575E-05 Sedimentation coefficient at 20.0 deg.s 0.18976E-12 s K/AETA= 0.3168E-12 s *mL/mg Permeability. KsO. 3168 3E- 11 cm"2  /  150  Sample: 0.325U Fraction HA Concentrations 2.3005 mg/mL From sedimentation velocity experiment: distance from peak to Actual dist. (Y) of peak loft ref.edge (cm) from centre of rotor 0.0 0.543 5.9675 16.0 0.604 5.9975 32.0 0.650 6.0202 ta. .0 0. 708 6.0488 64.0 0.752 6.0704 80.0 0.820 61 .039 96.0 0.666 6.1266 112.0 0.932 6. 159 1 128.0 0.983 6.1842 144.0 1.040 6.2123 160.0 1. 105 6.2443 176.0 1. 153 6.2660 192.0 1.210 6.2961 208.0 1.270 6.3256 224.0 1.341 6.3606 From the graph of LOG Y vs Tm ie : SL0PE= o.l2232E-03/min Y-INTERCEPTS 0.77573 CORRELATO I N COEFFICIENTS 0.99976 SSXs 0.71680E«05 SSY= 0.1073OE-02 SSXYs 0.87681E«01 SSEs 0.51736E-06 SG I MAs o.19949E-03 95% confidence interval for SLOPEs 0. Sedimentation coefficient at 20.0 deg.s 0.15451EK/AETAs 0.2064E-12 s«sL/mg Permeability, IO0.20638E-11 cm"2  /  0.7758 0.7 7 80 0.7796 0.7817 0.7632 0.7856 0.7872 0.7895 0.7913 0.7933 0.7955 0.7971 0.7991 0 .8011 0.8035  -  Sample: 0.325M Fraction HA Concentrations 2.7606 mg/mL From sedimentation velocity experiment: Tm i e (min) Distance from peak to Actual dist. (Y) of peak LOG Y left ref.edge (cm) from centre of rotor (cm) 0.0 0.395 5.8946 0.7705 16.0 0.461 5.9271 0.7728 32.0 0.510 5.9512 0.7746 48.0 0.565 5.9783 0.7766 64.0 0.601 5.9961 0.7779 60.0 0.685 6.0374 0.7809 96.0 0.719 6.0542 0.7821 112.0 0.767 6.0778 0.7837 128.0 0.821 6.1044 0.7856 144.0 0.869 6.1281 0.7873 160.0 0.939 6.1626 0.7898 176.0 0.998 6.1916 0.7918 192.0 1.053 6.2167 0.7937 From the graph of LOG Y vs Time : SL0PE= 0.11868E-03/«in Y-INTERCEPTS 0.77070 CORRELATO I N COEFFICIENTS 0.99907 SSXs 0.46S92E.05 SSYs 0.6S752E-03 SSXY= 0.55298E»01 SSEs 0.12193E-05 SG I MAs 0.33294E-03 95% confidence interval for SLOPEs 0.33949E-05 Sedimentation coefficient at 20.0 deg.s 0.14992E-12 s. K/AETAs 0.1669E-12 sm ' L/mg Permeability. K=0.16687E-11 cm2 -,  Sample: 0.325M-9 Fraction HA Concentrations 0.4840 mg/mL From sedimentation velocity experiment: Tm i e (min) Distance from peak to Actual dist. (Y) of peak LOG Y left ref. edge (cm) from centre of rotor (cm) 0.427 0.0 5.9103 0.7716 0.485 6.0 5.9389 0.7737 0.538 16 .0 5 . 9 6 5 0 0 .7756 0.588 24.0 5.9897 0.7774 0.646 32.0 6 . 0 1 6 2 0 .7795 0. 729 40.0 6.0591 0.7824 0.754 48.0 6 . 0 7 1 4 0 .7833 0.822 56.0 6.1049 0.7857 0.885 64.0 6 . 1 3 6 0 0 . 7879 0.949 72.0 6.1675 0.7901 From the graph of LOG Y vs Tm ie : SLOPEs 0.25503E-03/min Y-INTERCEPTS 0.77154 CORRELATO I N COEFFICIENTS 0.99870 SSXs 0.52800E'04 SSYs 0.34430E-03 SSXYs 0.13465E-01 SSEs 0.89197E-06 SIGMAs 0.33391E-03  95% confidence interval for SLOPEs 0.10597E-O4 Sedimentation coefficient at 20.0 deg.s 0.32214E-12 s. K/AETAs 0.2045E-1 I s m ' L/mg Permeability. KsO.2045 IE - 10 cm*"2  151  Sample: 0.325M-9 Fraction HA Concentrat1on= 0.9680 mg/mL From sedimentation velocity experiment: Tm i e (min) Olstance from peak to Actual dist. (Y) of peak LOG Y left ref.edge (cm) from centre of rotor (era) 00 0.408 5.9010 0.7709 e.0 0.454 5.9236 0.7726 16.0 0.495 5.9438 0.774 1 24.0 0.551 5.9714 0.7 761 32.0 0.586 5.9867 0.7773 40.0 0.633 6.0118 0.7790 46.0 0.67 1 6.0305 0.7804 56.0 0.717 6.0532 0.7820 64.0 0. 760 6.0744 0.7835 .72.0 0.799 6.0936 0.7849 80.0 0.860 6.1236 0.7870 88.0 0.897 6. 1419 0.7883 96.0 0.938 6.1621 0.7897 104.0 0.987 6.1662 0.7914 From the graph of LOG Y vs Tm ie : SLOPEs 0. 19S73E-03/«l1n Y- N I TERCEPTS 0.77105 CORRELATO I N COEFFICIENTS 0.99969 SSXs 0.14560E.05 SSYs 0.55816E-03 SSXYs 0.28499E«01 SSEs 0.34661E-06 SG I MAs 0.16995E-03 95% confidence Interval for SLOPEs 0.30691E-05 Sedimentation coefficient at 20.0 deg.= 0.24724E-12 s. K/AETAs 0.7848E-12 s"mL/mg Permeability. KsO. 78481E-11 cra"2 Sample: 0.325M-9 Fraction HA Concentrations 1.4520 mg/mL From sedimentation velocity experiment: me (min) Oistance from peak to Actual dist. (Y) of peak LOG Y left ref. edge (cm) from centre of rotor (cm) 0.0 0.352 5.8734 0.7689 8.0 0.394 5.894 1 0.7704 5.9113 16.0 0.429 0 .7717 5.9276 24.0 0.462 0.7729 5.9365 32.0 0.480 0 .7735 5.9631 40.0 0.534 0.7755 5.9734 0 . 7762 48.0 0.555 5.9697 0.7774 56.0 0.588 6.0089 0 . 7 788 64.0 0.627 6.0202 0.7796 72.0 0.650 6.0399 0 . 7 810 80.0 0.690 6.0601 0.7825 88.0 0.731 6.0709 0.7833 96.0 0.753 From the graph of LOG Y vs Tm ie : SLOPEs 0.14873E-03/min Y-INTERCEPTS 0.76914 CORRELATO I N COEFFICIENTS 0.99888 SSX= 0.11648E«05 SSYs 0.25825E-03 SSXY= 0.17324E.01 SSE= 0.58062E-06 SG I MAs 0.22975E-03 95% confidence interval for SL0PE= 0.46854E-05 Sedimentation coefficient at 20.0 deg.s 0.18787E-12 s K/AETA= 0.3976E-12 s"mL/mg Permeability. K=0.39757E-11 cm2 -,  Sample: 0.325M-9 Fraction HA Concentrations 1.9360 mg/mL From sedimentation velocity experiment: ctual dist. (Y) of peak LOG Y Oistance from peak to A from centre of rotor (cm) Tm i e (min) left ref. edge (cm) 5.8872 0.7699 380 0.0 5.9177 0.7722 442 16.0 5.9473 0.7743 502 32.0 5 . 9 7 5 4 0.7764 5 5 9 48.0 6.0084 0.7768 626 64.0 6 . 0 3 7 9 0.7809 6 8 6 80.0 6.0665 0.7829 744 96.0 6.0931 0.7848 798 112.0 6.1256 0.7871 864 128.0 6.1571 0.7894 928 144.0 From the graph of LOG Y vs Tm ie : SLOPEs 0.13434E-03/min Y-INTERCEPls 0.77000 CORRELATO I N COEFFICIENTS 0.99986 SSXs 0.21120E0 '5 SSY= 0.38125E-03 SSXY= 0.26372E»01 SSEs 0.10908E-06 SG I MAs 0.11677E-03 95% confidence Interval for SLOPEs 0.18529E-05 Sedimentation coefficient at 20.0 deg.s 0.16969E-12 s. K/AETAs 0.2693E-12 s"mL/mg Per«eat> i 1 i ty. KsO . 26932E-11 cm"?  /  152  Sample: 0.325M-9 Fraction HA Concentration= 2.4200 mg/mL From sedimentation velocity experiment: Time (min) Distance from peak to Actual dist. (Y) of peak LOG Y left ref. edge (cm) from centre of rotor (cm) 0.0 0.398 5.8961 0.7706 16.0 0.457 5.9251 0.7727 32.0 0.508 5.9502 0.7745 48.0 0.571 5.9813 0.7768 64.0 0.624 6.0074 0.7787 80.0 0.686 6.0379 0.7809 96.0 0.738 6.0635 0.7827 112.0 0.796 6.0921 0.7848 From the graph of LOG Y vs Time : SL0PE= 0.12689E-03/min Y-INTERCEPTS 0.77060 CORRELATO I N COEFFICIENTS 0.99983 SSXs 0.10752E»05 SSYs 0.17318E-03 SSXYs 0.13643E*01 SSEs O.S7283E-07 SG I MAs 0.97709E-04 95% confidence interval for SLOPEs 0.23058E-05 Sedimentation coefficient at 20.0 deg.s 0.16028E-12 s. K/AETAs 0.2035E-12 s-mL/mg Permeability. KsO.20352E-11 cm"2 Sample: 15mn iAH Fraction HA Concentrations 0.5916 mg/nL From sedimentation velocity experiment: Time (min) Oistance from peak to Actual dist. (Y) of peak from centre of rotor (cm) left ref. edge (cm) 0.7720 0.437 9153 0.0 0.7737 0.484 9384 8.0 0 .7752 0.526 9591 16.0 0.7767 0.568 9798 24.0 0 .7784 0.616 32.0 From the graph of LOG Y vs Time : SLOPEs 0.19835E-03/min Y-INTERCEPTs 0.77201 CORRELATION COEFFICIENTS 0.99969  SSXs 0.64000E+03 SSY= 0.25195E-04 SSXYs 0.12694E.O0 SSEs 0.15660E-07 SIGMAs 0.72249E-04 95%  confidence interval for SLOPEs 0.90874E-05 Sedimentation coefficient at 20.0 deg.s 0.25054E-12 s. K/AETA= 0.1301E-11 S-mL/mg Permeability. KsO.13013E-10 cm2 -,  Sample: ISminAH Fraction HA Concentrations 1.1832 mg/mL From sedimentation velocity experiment: Oistance from peak to Actual dist. (YI of peak LOG Y Time (min) left ref. edge (cm) from centre of rotor (cm) 5.9182 0.7722 0.443 0.0 5.9419 0.7739 0.491 8.0 5.9542 0.7748 0.516 16.0 5.9749 0.7763 0.558 24.0 0.590 5.9906 0.7775 32.0 0.633 40.0 6.0118 0.7790 0.668 48.0 6.0291 0.7802 0.706 56.0 6 . 0 4 7 8 0.7816 0. 749 64.0 6 . 0 6 9 0 0 .7831 0.786 72.0 6.0872 0.7844 From the graph of LOG Y vs Time : SLOPEs 0.!6783E-03/min Y*N I TERCEPTS 0.77227 CORRELATO I N COEFFICIENTS 0.99941 SSXs 0.52800E.04 SSYs 0.14889E-03 SSXYs 0.86613E*00 SSEs 0.17548E-06 SIGMAs 0.14810E-03  95% confidence interval for SLOPEs 0.47001E-05 Sedimentation coefficient at 20.0 deg.= 0.21199E-12 s. K/AETA= 0.5505E-12 S-mL/mg Permeability. K=0. 55053E-11 ctiT'2  Sample: 15mn iAH Fraction HA Concentrations 1.7748 mg/mL From sedimentation velocity experiment: ctual dist. (¥) of peak LOG Y Oistance from peak to A from centre of rotor (cm) Time (min) left ref. edge (cm) 5.9148 0.7719 0.436 0.0 5.9473 0.7743 0.502 16.0 5.9823 0.7769 0.573 32.0 - 6.0099 0.7789 0.629 46.0 6.0448 0.7814 0.700 64.0 6.0798 0.7839 0.771 80.0 6.1084 0.7859 0.629 96.0 6.1429 0.7884 0.699 112.0 0.971 6.1783 0.7909 128.0 1.040 6.2123 0.7933 144.0 From the graph of LOG Y vs Time : SL0PE= 0.14731E-03/min Y-INTERCEPTS 0.77196 CORRELATO I N COEFFICIENTS 0.99985 SSX= 0.2U20E*05 SSY= 0.45647E-03 SSXY= 0.311!3E*01 SSEs 0.13448E-06 SG I MAs 0.12965E-03 95% confidence Interval for SLOPEs 0.20573E-05 Sedimentation coefficient at 20.0 deg.s 0.18608E-12 s. K/AETA= 0.3222E-12 S-mL/mg Permeability. K=0.32216E-11 c<n"2 Sample: 15mn iAH Fraction HA Concentrations 2.3664 mg/mL From sedimentation velocity experiment: Time (min) Oistance from peak to Actual dist. (Y) of peak LOG Y left ref. edge (cm) from centre of rotor (cm) 0.506 5.9493 0.7745 0.0 0.528 5.9601 0.7753 8.0 0.573 5.9823 0.7769 16.0 0.589 5.9901 0.7774 24.0 0.614 6.0025 0.7783 32.0 0.647 6.0187 0.7795 40.0 0.667 6.0286 0.7802 48.0 0.696 6.0429 0.7812 56.0 0.719 6.0542 0.7821 64.0 0.755 6.0719 0.7833 72.0 From the graph of LOG Y vs Time : SLOPEs 0.12028E-03/min Y-INTERCEPTS 0.77454 CORRELATO I N COEFFICIENTS 0.99794 SSX= 0.52800E*04 SSYs 0.76706E-04 SSXYs 0.63509E*00 S S E s 0.31578E-06 SIGMAs 0.19868E-03 95%  confidence interval for SLOPEs O.63050E-05 Sedimentation coefficient at 20.0 deg.s 0.16193E-12 s. K/AETAs 0.1973E-12 s-mL/mg Permeability. K=0. 19728E-1 1 cm"2 Sample: 15mn iAH Fraction HA Concentrations 2.9580 mg/mL From sedimentation velocity experiment: Distance from peak to Actual dist. (Y) of peak left ref. edge (cm) from centre of rotor (cm) 0.506 0.0 5.9493 0.7745 0.560 16.0 5.9759 0.7764 0.608 32.0 5.9995 .7781 0.666 48.0 6.0291 .7802 0.715 64.0 6.0522 .7819 0.803 96.0 6.0956 .7850 0.872 112.0 6.1296 .7874 0.921 128 .0 6.1537 . 7891 0.965 144.0 6.1754 . 7907 1.016 160.0 6.2005 .7924 From the graph of LOG Y vs Time : SLOPEs 0.11215E-03/min Y-INTERCEPT= 0.77461 CORRELATO I N COEFFICIENTS 0.99949 SSX= 0.28160E*05 SSY= 0.35455E-03 SSXYs 0.31581E»01 SSEs 0.36333E-06 SIGMAs 0.21311E-03 95%  confidence interval for SLOPEs 0.29285E-05 Sedimentation coefficient at 20.0 deg.s 0.14166E-12 s. K/AETAs 0.1472E-12 s m ' L/mg Permeability. KsO . 1 4 7 16E - 1 1 cm"2  /  154  Sample: lhrAH Fraction HA Concentrations 0.6167 mg/mL From sedimentation velocity experiment: Distance from peak to Actual dist. (V) of peak LOG Y Time (min) left ref. edge (cm) from centre of rotor (cm) 0.7701 0.386 5.8901 0.0 0.7704 0.393 5.8936 8.0 0.7720 0.439 5.9163 16.0 0.7728 0.461 5.9271 24.0 0.7741 0.496 5.9443 32.0 From the graph of LOG Y vs Time : SLOPEs 0.13019E-03/min Y-INTERCEPTS 0.76982 CORRELATO I N COEFFICIENTS 0.98336 SSX= 0 . 6 4 0 0 0 E » 0 3 SSY= 0.11219E-04 SSXY= 0.83325E-01 S S E s 0.37022E-06 SIGMAs 0.35129E-03 95%  confidence interval for SLOPEs 0.44165E-04 Sedimentation coefficient at 20.0 deg.s 0.16446E-13 s. K/AETAs 0.8194E-12 s mL/mg Permeability. Ks0.81940E-11 cm 2 -  ,-  Sample: lhrAH Fraction HA Concentrations 1.2333 mg/mL From sedimentation velocity experiment: istance from peak to Actual dist. (Y) of peak LOG Y Time (mini D from centre of rotor (cm) left ref. edge (cm) 0.7714 5.9074 8.0 421 0.7726 5.9241 16.0 455 0.7739 5.9414 24.0 490 ' 0.7753 5.9601 32.0 528 0.7767 5.9798 40.0 568 0.7779 5 . 9 9 6 6 48.0 602 0.7790 6.0123 56.0 634 0.7802 6.0281 64.0 666 0.7813 6.0443 72.0 699 From the graph of LOG Y vs Time : SLOPEs 0.15715E-03/min Y-INTERCEPTS 0.77019 CORRELATO I N COEFFICIENTS 0.99937 SSXs 0.38400E-04 SSYs 0.94949E-04 SSXYs 0 . 6 0 3 4 4 E » 0 0 S S E s o.12013E-06 SIGMAs 0.13100E-03 95%  confidence interval for SL0PE= 0.49996E-05 Sedimentation coefficient at 20.0 deg.s 0.19850E-12 s. K/AETAs 0.4946E-12 sm ' L/mg Permeability. K=0.49455E-11 cm2 -,  Sample: lhrAH Fraction HA Concentrations 1.8500 mg/mL From sedimentation velocity experiment: istance from peak to Actual dist. (Y) of peak LOG Y Time (min) D left ref. edge (cm) from centre of rotor (cm) 0.467 8.0 5.9 300 0.7731 0.506 5.9493 16.0 0.7745 5.9566 0.525 24.0 0.7751 5.9773 0.563 32.0 0.7765 5.9877 0.584 40.0 0.7773 6 . 0 0 4 9 0 . 6 1 9 46.0 0.7785 6.0172 0.644 56 .0 0.7794 6.0384 0.687 64.0 0.7809 6.0547 0.720 72.0 0.7821 From the graph of LOG Y vs Time : SLOPEs 0.13753E-03/min Y-INTERCEPTs 0.77198 CORRELATO I N COEFFICIENTS 0.99785 SSXs 0.38400E-04 SSY= 0.72950E-04 SSXYs 0.52813E'00 SSE= 0.31400E-06 SIGMAs 0.21180E-03 95%  confidence interval for SLOPEs 0.80832E-05 Sedimentation coefficient at 20.0 deg.s 0.17373E-12 s. K/AETA= 0.2885E-12 6mL/mg Permeability. K=0. 28855E-11 cm"2 -  /  155  Sample: lhrAH Fraction HA Concentrations 2.4666 mg/mL From sedimentation velocity experiment: Time (min) Oistance from peak to Actual dist. (Y) of peak left ref. edge (cm) from centre of rotor (cm) 5.9532 0.514 5.9695 0.547 5.9793 .567 5.9946 .538 6.0064 .622 6.0158 .641 6.0300 .670 6.0424 .695 6.0567 .724 From the graph of LOG Y vs Time : SL0PE= 0.11378E-03/min Y-INTERCEPTS 0.77398 CORRELATO I N COEFFICIENTS 0.99902 SSXs 0 .38400E*04 SSYs 0 .49811E-04 SSXYs 0 .43692E*00 SSE= 0.97357E-07 SIGMAs 0 .11793E-O3 95% confidence Interval for SLpPE= 0.45009E-05 Sedimentation coefficient at 20.0 deg.s 0.14372E-12 s. K/AETAs 0 .1790E-12 s-mL/mg  LOG Y 7748 7759 7767 7778 7786 7793 7803 7812 7822  Permeability. KsO.17904E-11 cm2 ,-  Sample: lhrAH Fraction HA Concentrations 3.0833 mg/mL From sedimentation velocity experiment: Time (min) Distance from peak to Actual dist. (Y) of peak LOG Y left ref. edge (cm) from centre of rotor (cm) 0.775! 5.9576 8.0 523 0.7759 5.9685 16.0 545 0.7770 5.9847 24.0 578 0.7778 5.9951 32.0 599 0.7784 6.0039 40.0 617 0.7795 6.0187 48.0 647 0.7804 6.0315 56.0 673 0.7812 6.0419 64.0 694 0.7819 6.0522 72.0 715 0.7834 6.0729 80.0 757 0.7844 6.0872 88.0 786 0.7852 6.0985 96.0 809 From the graph of LOG Y vs Time : SLOPEs O.11486E-03/min  Y-INTERCEPTS 0.77405 CORRELATION C O E F F I C I E N T S , 0 . 9 9 8 1 3 SSX= O.91520E*O4 SSYs 0.12123E-03 SSXYs 0.10513E*01 S S E s 0.45388E-06 SIGMAs 0.21305E-03  confidence interval for SLOPEs 0.49617E-05 Sedimentation coefficient at 20.0 deg.s 0.14510E-12 s. K/AETAs 0.1446E-12 S -mL/mg Permeability. K=0. 1446IE-11 cm"2 95X  Sample: 2hrAH Fraction HA Concentrations 0.6109 mg/mL From sedimentation velocity experiment: Time (min) Oistance from peak to left ref. edge (cm) 8.0 0.452 16.0 0.500 24.0 0.529 32.0 0.559 From the graph of LOG Y vs Time :  Actual dist. (Y) of peak LOG Y from centre of rotor (cm) 5.9227 0.7725 5.9463 0.7742 5.9606 0.7753 5.9754 0.7764  SLOPEs 0.15732E-03/min  Y-INTERCEPTs 0.77146 CORRELATION C O E F F I C I E N T S 0.99169  SSXs 0.32000E*03 SSY= 0.80536E-05 SSXYs 0.S0344E-01 SSEs 0.13328E-06 SIGMAs 0.25815E-03 951 confidence interval for SL0PE= 0.62096E-04 Sedimentation coefficient at 20.0 deg.s 0.19872E-12 s. K/AETAs 0.9995E-12 sm ' L/mg Permeability. KsO.99955E-11 c«r-2  j  Sample: 2hrAH Fraction HA Concentrations 1.2218 mg/mL From sedimentation velocity experiment: ctual dist. (V) of peak LOG Y istance from peak to A from centre of rotor (cm) Time (min) D left ref. edge (cm) 7706 5.8961 398 8.0 7717 5.9113 429 16.0 7727 5.9246 456 24.0 7 737 5 . 9 3 8 9 485 32.0 7756 5.9645 537 48.0 7 770 5 . 9 8 4 2 577 56.0 From the graph of LOG Y vs Time : SL0PE= 0.13041E-03/min Y-INTERCEPTS 0.76953 CORRELATO I N COEFFICIENTS 0.99861 SSXs 0.17173E*04 SSY= 0.29289E-O4 SSXYs 0.22396E*00 SSE= 0.81156E-07 SIGMAs 0.14244E-03 95%  confidence interval for SLOPEs 0.95416E-05 Sedimentation coefficient at 20.0 deg.s 0.16473E-12 s. K/AETAs 0.4143E-12 s raL/mg Permeability. K=0.41428E-11 cm"2 -  Sample: 2hrAH Fraction HA Concentrations 1.8326 mg/mL Actual dist. (Y) of peak LOG Y isvelocity tance from peakentto FromTim sedim e1nnt)ationO (m left ref. edegx eper(cim m) : from centre of rotor (cm) 5.9059 0.7713 0.418 8.0 16.0 0.443 5.9182 0.7722 24.0 0.468 5.9305 0.7731 32.0 0.495 5.9438 0.7741 40.0 0.528 5.9601 0.7753 48.0 0 . 5 4 6 5 . 9 6 9 0 0.7759 56.0 0.573 5.9823 0.7769 64.0 0.607 5.9990 0.77B1 72.0 0.632 6.0113 0.7790 From the graph of LOG Y vs Time : SLOPEs 0.12036E-03/min  Y-INTERCEPTs 0.77026 CORRELATION C O E F F I C I E N T S 0.99916 SSXs 0.38400E*04 SSYs 0.55725E-04 SSXYs 0.46219E.00 S S E s 0.93754E-07 SIGMAs 0.11573E-03  95% confidence interval for SLOPEs 0.44168E-05 Sedimentation coefficient at20.0 deg.= 0.15204E-12 s. K/AETA= 0.2549E-12 sm ' L/mg Permeability. Ks0.25492E-11 i  Sample: 2hrAH Fraction HA Concentrations 2.4435 mg/mL From sedimentation velocity experiment: istance from peak to Actual dist. (Y) of peak Time (min) Dleft ref. edge (cm) from centre of rotor (cm) 16.0 424 9089 0.7715 24.0 444 9187 0.7722 32.0 475 9340 0.7733 40.0 488 9 4 0 4 0 .7738 48.0 520 9562 0.7750 56.0 547 9 6 9 5 0 .7759 64.0 570 9808 0.7768 72.0 590 9906 0.7775 From the graph of LOG Y vs Time : SLOPEs 0.10915E-03/min  Y-INTERCEPTS 0.76970 CORRELATION C O E F F I C I E N T S 0.99803 SSXs 0.26860E*04 SSY= 0.321S3E-04 SSXY= 0.29341E*00 SSE= 0.12673E-06 SIGMAs 0 . I 4 5 3 3 E - 0 3 95%  confidence interval for SLOPEs 0.68593E-05 Sedimentation coefficient at 20.0 deg. 0. I3788E-12 K/AETAs 0 .17346-12 sm ' L/mg Permeability. KsO.17338E-11 cm-"2  s.  /  157  Sample: 2hrAH Fraction HA C o n c e n t r a t i o n s 3.0544 mg/mL From sedimentation velocity experiment: Time (min) Distance from peak to Actual dist. (Y) of peak left ref. edge (cm) from centre of rotor (cm) 0.7714 .421 9074 16.0 0.7722 .443 9182 24.0 0.7728 .461 927 1 32.0 0.7739 .489 9409 40.0 0.7744 .503 9478 48.0 0.7754 .533 9626 56.0 0.7763 0.557 9744 64.0 0.7765 0.564 72.0 From the graph of LOG Y vs Time : SL0PE= 0.96461E-04/min Y-INTERCEPTs 0.76987 CORRELATO I N COEFFICIENTS 0.99580 SSXs 0.26880E»04 SSYs 0.25223E-04 SSXYs 0.25929E'00 SSEs 0.21163E-06 SIGMAs 0.18781E-03 95X  confidence interval for SL0PE= 0.88640E-05 Sedimentation coefficient at 200 deg.s 0.12184E-12 s. K/AETA= 0.1226E-12 S-mL/mg Permeability. K=0 . 12258E-11 cm"2 Sample: 15mAH-0.325M Fraction HA Concentrations 1.0954 mg/mL From sedimentation velocity experiment: Time (min) Distance from peak to Actual dist. (Y) of peak LOG Y left ref. edge (cm) from centre of rotor (cm) 16.0 0.545 5.9685 0.7759 24.0 0.603 5.9970 0.7779 32.0 0.621 6.0059 0.7786 48.0 0.739 6.0640 0.7828 56.0 0.762 6.0754 0.7836 64.0 0.819 6.1034 0.7856 72.0 0.832 6.1099 0.7860 80.0 0.861 6.1241 0.7870 From the graph of LOG Y vs Time : SL0PE= 0.17920E-03/min Y- N I TERCEPTS 0.77339 CORRELATO I N COEFFICIENTS 0.99113 SSXs 0.37680E*04 SSY= 0.12317E-03 SSXY= 0.67522E+00 SSEs 0.21744E-05 SIGMAs 0.60200E-03 95X confidence interval for SLOPEs 0.23998E-04 Sedimentation coefficient K/AETAs 0.6349E-12 s m ' L/mg 20.0 deg.s 0.22635E-12 s. Permeability. Ks0.63495E-11 cm" Sample: 15mAH-0.325M Fraction HA Concentrations 1.6431 mg/mL From sedimentation velocity experiment: Time (min) Distance from peak to Actual dist. (Y) of peak LOG Y left ref.edge (cm) from centre of rotor (cm) 8.0 0.507 5.9498 0.7745 16.0 0.538 5.9650 0.7756 24.0 0. 56 3 5.9773 0.7765 32.0 0.595 5.9931 0.7777 40.0 0.620 6.0054 0.7785 48.0 0.653 6.0217 0.7797 56.0 0.679 6.0345 0.7806 64,.0 0.714 6.0517 0.7819 72.0 0.742 6.0655 0.7829 60.0 0.789 6.0887 0.7845 86.0 0.808 6.0960 0.7852 96.0 0.835 6.1113 0.7861 1 0 4 . 0 0 . 8 7 2 6 . 1 2 9 6 0.7874 From the grapn of LOG Y vs Time : SLOPEs 0.13485E-03/min Y-INTERCEPT= 0.77331 CORRELATO I N COEFFICIENTS 0.99919 SSX= 0.11648E*05 SSYs 0.21217E-03 SSXYs 0.15708E»01 SSEs 0.34474E-06 SIGMAs 0.17703E-03 95X confidence interval for SLOPEs 0.36103E-05 Sedimentation coefficient at 20.0 deg.s O.17034E-12 s. K/AETA= 0.3185E-12 sm ' L/mg Permeability. KsO . 3 1855E - 11 cm"2  /  158  Sample: 15mAH-0.325M Fraction HA Concentration= 2.1908 mg/mL From sedimentation velocity experiment:  Actual dist. (Y) of peak from centre of rotor (cm) Time (mini O istanref. ce fe ro ea left dm ge p te nk) to 7746 5.9517 16..0 0.511 7751 5.9581 7761 5.9724 240 0.524 777 1 5.9857 32.0 0.553 7785 6.0054 40..0 0.580 7794 6.0177 48.0 0.620 7600 6.0251 56.0 0.,645 7808 6 . 0 3 6 9 64.0 0.660 0.7825 6.0606 72.0 0.684 0.7831 6.0690 80.0 0.732 0.7840 6.0808 88.0 0.749 0.7845 6.0887 96.0 0.773 0.7860 6.1099 104.0 0.789 0.7877 6.1335 112.0 0.832 0.7881 6.1394 120.0 0.880 0.7894 6.1576 128.0 0.892 0.7901 6.1675 136.0 0.929 144.0 0.949 From the graph of LOG Y vs Time : SL0PE= 0.12383E-03/min Y-INTERCEPTS 0.77229 CORRELATO I N COEFFICIENTS 0.99811 SSXs 0 .26112E«05 SSY= 0.40191E-03 SSXYs 0 .32334E*01 SSEs O .15202E-05 SIGMAs 0 .31835E-03 95% confidence Interval for SL0PE= 0.41983E-05 Sedimentation coefficient at 20.0 deg.s 0.15641E-12 s. K/AETAs 0.2194E-12 s *«L/mg Permeability, K=di21938E-11 cm"2  Sample: 15mAH-0.325M Fraction.HA Concentrations 2.7385 mg/mL From sedimentation velocity experiment: Time (min) Distance from peak to Actual dist. (Y) of peak LOG Y left ref. edge (cm) from centre of rotor (cm) 16.0 0.530 5.9611 0.7753 32.0 0.576 5.9837 0.7770 48.0 0.621 6 . 0 0 5 9 0 .7786 64 .0 0.673 6.0315 7804 80.0 0.728 6 . 0 5 8 6 7824 96.0 0.783 6 . 0 8 5 7 7843 112.0 0.831 6 . 1 0 9 4 7860 128.0 0.889 6.1379 7880 144.0 0.943 6 . 1 6 4 5 7899 160.0 0.990 6 . 1 8 7 7 7 915 176.0 1 .038 6.2113 7932 From the graph of LOG Y vs Time : SLOPEs 0.11360E-03/min Y*INTERCEPTS 0.77331 CORRELATO I N COEFFICIENTS 0.99973 SSXs 0.28160E-05 SSY= 0.36490E-03 SSXY= 0.32047E»01 SSEs O.20O45E-06 SG I MAs 0.14924E-03 95% confidence Interval for SLOPEs 0.20117E-05 Sedimentation coefficient at 20.0 deg.s 0.14375E-12 s K/AETA= 0.1613E-12 s-mL/mg Permeability. KsO.16129E-11 cm2 ,-  Sample: 15mAH-0.325M Fraction HA Concentrations 3.2862 mg/mL From sedimentation velocity experiment: Time (min) Oistance from peak to Actual dist. (Y) of peak LOG Y left ref.0.ed ge from ce5 n.t9 re f rotor (cm)0.7753 16.0 53 0 (cm) 61o1 32.0 0.592 5.9916 0.7775 48.0 0.618 6.0044 0.7785 64.0 0.649 6.0197 0.7796 80.0 0.710 6.0498 0.7817 96.0 0.757 6.0729 0.7834 112.0 0.804 6.0961 0.7850 128.0 0.852 6.1197 0.7867 144.0 0.895 6. 1409 0.7882 1 6 0 . 0 0 . 9 6 0 6 . 1 7 2 9 0.7905 From the graph of LOG Y vs Time : SLOPEs 0.10252E-O3/min Y-INTERCEPTS 0.77363 CORRELATO I N COEFFICIENTS 0.99766 SSX= 0.21120E»05 SSY= 0.22303E-03 SSXYs 0.21653E>01 SSEs 0.10442E-05 SIGMAs 0.36129E-03 95% c o n f i d e n c e i n t e r v a l  f o r SLOPEs  0.57328E-O5  S e d i m e n t a t i o n c o e f f i c i e n t at 2 0 . 0 d e g . s 0 . 1 2 9 5 0 E - 1 2 K/AETAs 0.1211E-12 s'mL/mg P e r m e a b i l i t y . KsO.12109E-11 c n T ' 2  s.  /  159  B.3. Log K' Vs Log c Plots  F r a c t i o n : 0.2511 F r o e th« p l o t o f LOG K v« LOG C : Slopes -1.432614 Y - I n t e r c e p t at LOG C=0. U -9.0810 C o r r e l a t i o n c o e f f i c i e n t * -.99969 SSXs 0.304700 SSYs 0.625503 SSXYs -0.436518 SSEs «.00014156 SIGMAs 0.00686933 95X c o n f i d e n c e I n t e r v a l  for S l o p e s 0.039S9842  F r a c t i o n : 0.325M From the p l o t of LOG K vs LOG C : Slopes -1.498351 Y - I n t e r c e p t at LOG CsO. It -9.1247 C o r r e l a t i o n c o e f f i c i e n t s ..99935 SSXs 0.414094 SSYs 0.930866 SSXY«s -0.620458 SSEs 0.00120232 SIGUAs 0.01733721 95% c o n f i d e n c e I n t e r v a l  for Slopes  0.07479099  F r a c t i o n : 0.32SU-S F r o a the p l o t o f LOG K vs LOG C : Slopes -1.456352 Y - l n t e r c e p t et LOG CsO. Is -S.1442 C o r r e l a t i o n c o e f f i c i e n t s -.99924 SSXs 0.304700 SSYs 0.647242 SSXYs -0.4437S1 SSEs 0.00098411 SIGMAs 0.01811182 95% c o n f i d e n c e I n t e r v a l  for Slopes  0.10440607  F r a c t i o n : 15«1nAH From the p l o t o f LOG K ve LOG C : Slopes -1.361907 Y - 1 n i e r c e p t at LOG CsO. Is -9.1792 C o r r e l a t i o n c o e f f i c i e n t s -.99839 SSXs 0.304700 SSYs 0.566974 SSXYs -0.414973 SSEs 0.00181898 SIGMAs 0.02462372 95% c o n f i d e n c e I n t e r v a l  f o r S l o p e s 0.14194410  F r a c t i o n : lhrAH From the p l o t o f LOG K vs LOG C : Slopes -1.376716 Y - l n t e r c e p t at LOG CsO. Is -9.1810 C o r r e l a t i o n c o e f f i c i e n t s ..99694 SSXs 0.088552 SSYs 0.168869 SSXYs -0.121911 SSEs 0.00103258 SIGMAs 0.02272203 95% c o n f i d e n c e I n t e r v a l  for Slopes  0.32856356  F r a c t i o n : 2hrAH Fro» the p l o t o f LOG K vs LOG C : Slopes -1.317213 Y - l n t e r c e p t at LOG CsO. Is -9.2594 C o r r e l a t i o n c o e f f i c i e n t s -.99853 SSXs 0.088544 SSYs 0.154081 SSXYs -0.116632 SSEs 0.00045181 SIGMAs 0.01503010 95% c o n f i d e n c e I n t e r v a l  for S l o p e s 0.21734643  F r a c t i o n : 15»AH-0.325U F r o - the p l o t of LOG K vs LOG C : Slopes -1.391124 Y - l n t e r c e p t at LOG CsO. i s -9.1912 C o r r e l a t i o n c o e f f i c i e n t s -.99923 SSXs 0.050529 SSYs 0.097936 SSXYs -0.070292 SSE= 0.00015142 SIGMAs 0.00870122 95% c o n f i d e n c e I n t e r v a l  for S l o p e s  0.16656450  /  160  


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