Precipitation

1
Precipitation

• Protein and nucleic acid separation
• Example Cohns fractionation of plasma proteins
  
• Selective conversion of dissolved components of
  complex mixtures to insoluble forms using
  appropriate physical or physicochemical means
• Precipitates are generally amorphous
• Crystallization Precipitate is obtained in
  crystalline form

Supernatant
Sample
Precipitate supernatant
Gravitational field
Precipitating agent
Precipitate
Centrifugation
2
Factors utilized for precipitation

• Biological macromolecules can be precipitated by
• Cooling
• pH adjustment
• Addition of solvents such as acetone and ethanol
• Addition of anti-chaotropic salts such as
  ammonium sulphate
• Addition of chaotropic salts such as urea
• Addition of biospecific reagents as in
  immunoprecipitation

3
Cooling
S
B
A
Difference in solubility
Temperature
4
Precipitation using additive

• Basic thermodynamic equation
• ?ppt ?liq
• In theory precipitate has one component, i.e.
  solute its chemical potential at a given
  temperature is constant
• Chemical potential of the dissolved solute is
  given by
• ?liq ?liq(0) RT ln (x)
• Precipitants act by increasing ?liq(0)

5
pH
Solubility difference
A
B
S
pIA
pIB
pH
6
Immunoprecipitation

• Antigens can be precipitated
  using antibodies
• Antibodies can be precipitated
  using antigens
• Antigens can be precipitated using insoluble
  forms of antibodies

Based on precipitin formation
7
Chaotropic salts

• Chaotropic salts denature proteins
• Examples urea, guanidine hydrochloride
• Disrupting intra-molecular hydrogen bonds and
  hydrophobic interactions in proteins
• Not used for protein bioseparation
• Mainly used for protein refolding, particularly
  in the processing of inclusion bodies
• Chaotropic salts are used in DNA and RNA
  purification since they precipitate proteins but
  leave nucleic acid molecules unaffected

8
Organic solvents

• Organic solvents precipitate by reducing the
  dielectric constant
• Governing equation
• ln (S/Sw) (A/RT) ((1/ew) (1/e))
• Dielectric constants
• water 78.3, ethanol 24.3
• Organic solvents can denature proteins
• Hydrophobic interactions are disrupted
• Small amounts are used
• Low temperature minimizes denaturation
• Examples Acetone, methanol, ethanol, propanol
  and butanol
• Denaturation by aliphatic alcohols depends on the
  o chain length, i.e. butanol causes more
  denaturation than ethanol.
• DNA and RNA are precipitated using ethanol at low
  temperatures
• Major application Cohn fractionation method for
  human plasma protein purification

9
Anti-chaotropic salts

• Anti-chaotropic or kosmotropic salts
• Structure forming or stabilizing
• Example ammonium sulphate, sodium sulphate
• Solubility of proteins is affected by salts

A
S
Salting in Salting out
B
Ammonium sulphate concentration
10
Anti-chaotropic salts

• Salting-in effect salts providing the
  electrostatic double-layer surrounding the
  proteins which are needed to keep them in
  solution
• Salting-out effect
• Electrostatic shielding
• Association of salt with water
• Cohn equation
• ln S B Ks Cs
• Valid only in the salting-out region
• B depends on the protein, the temperature and the
  solution pH
• Ks is independent of the temperature and pH but
  depends on the salt and the protein
• Precipitation is carried out at low temperatures
  (e.g. 4 degrees centigrade)
• Lower protein solubility
• Enhanced protein stability