Ammonium sulfate precipitation

1
Ammonium sulfate precipitation

• Two approaches
• Addition of solid ammonium sulphate to sample
• preferred in small scale applications
• localized high salt concentrations
• uneven precipitate formation.
• Addition of ammonium sulphate solution to sample
• large-scale applications
• samples mixed with saturated ammonium sulphate
  solutions
• Ammonium sulphate-cut
• 50 ammonium sulphate cut 50 saturation
• Differential ammonium sulphate cuts

2
Mechanisms of precipitate formation

• Precipitate formation is time dependent process
• Involves formation of tiny particles due to
  association of macromolecules followed by their
  increase in size
• Precipitation can be tracked by measuring
  turbidity

Turbidity
Time
3
Mechanisms of precipitate formation

• Four stages of precipitation
• Mixing
• Nucleation
• Diffusion limited growth
• Convection limited growth

4
Mixing

• Formation of a homogeneous mixture
• Mixing is not instantaneous
• Takes a finite amount of time which depends on
  the properties of the components as well as on
  the processing conditions, i.e. mixing intensity
• Mixing time
• ? (le2 / 4 D)
• The average eddy length scale depends on the
  volume, density and viscosity of the medium as
  well as on the power input for mixing

5
Nucleation

• Involves formation of very minute particles
• Multimers
• Nucleation starts at regions having localised
  supersaturation
• Extent of supersaturation determines the nature
  of the precipitates
• High supersaturation gives gelatinous precipitate
• Controlled supersaturation gives amorphous
  precipitates which are easy to filter and
  centrifuge

6
Diffusion limited growth

• Increase in size is diffusion dependent
• Bigger particles typically of submicron to
  several micron range are formed
• Rate of particle formation is dependent on the
  physicochemical properties of the protein and the
  liquid medium alone
• The rate of formation of particles of a
  particular size is governed by second order
  kinetics
• dn/dt - ? n2
• n is the number of particles of a given size (-)
• ? is a rate constant which depends on the
  diffusivity and diameter of the protein (/s)

7
Convection dependent growth

• Particles grow in size by collision
• Particles are typically a few to several hundred
  microns in size
• Convection dependent
• More mixing higher frequency of collision
• Rate
• dn/dt - ? n2
• ? is a rate constant which depends on the size
  and sticking tendency of the particles, the
  volume, density and viscosity of the medium in
  addition to the power input for mixing