Saurav Datta

Department of Biotechnology, IITR

F-8, Old Biotechnology Building
Email: sdattfbt@iitr.ac.in
 Principles of Bioseparations Engineering – Raja Ghosh
 Bioseparations Science and Engineering– Harrison et al.
 Precipitation based bioseparation involves selective conversion
of a specific dissolved component to an insoluble form within
a complex mixture using appropriate physical or
physicochemical means
 Proteins precipitate because they become insoluble, hence
factors affecting the solubility of proteins play major role
 Often used for concentration of bulk proteins to reduce
volume of the process solution (all the proteins in a stream is
precipitated and redissolved in a smaller volume)
 Applied to fractionate proteins to some extent as well
(fractional precipitation is carried out to precipitate the protein
of interest and leave many of the contaminating proteins in
the liquor)
 Purification of blood plasma protein – consists of an array of
precipitation steps to recover various proteins at different
steps (Cohn Fractionation)
 Can be carried out with simple equipment
 Relatively inexpensive in terms of operating cost
 Leads to a form of the protein that is often stable for long
term storage

 Needs secondary separation step

 Slow process
 Larger footprint – higher capital cost
 Precipitation by external agents is governed by the following
equation at equilibrium
(1)

 The chemical potential of the precipitated solute is constant at

a given temperature
 The chemical potential of the dissolved solute is expressed as
(2)

 The standard reference state chemical potential is increased

by adding foreign substance (salt or solvent)
 Therefore, the solute concentration in the solution phase (x)
must decrease in order to satisfy the equation μppt = μliq
The most important factors affecting the solubility of proteins
are structure and size, charge, and the solvent/salt addition

1. Structure and size:

 In the native state, a protein in an aqueous environment
assumes a structure that minimizes the contact of the
hydrophobic amino acid residues with the water solvent
molecules and maximizes the contact of the polar and charged
residues with the water
 The major forces acting to stabilize a protein in its native state
are hydrogen bonding, van der Waals interactions, and
solvophobic interactions
 In aqueous solution, these forces tend to push the hydrophobic
residues into the interior of the protein and the polar and
charged residues on the protein’s surface
 The native structure is only marginally stable and can be
destabilized by relatively small environmental changes
  A study of the hydration of human serum
albumin found two layers of water around
the protein.
 These hydration layers are thought to
promote solubility of the protein by
maintaining a distance between the
surfaces of protein molecules a shown in
the Figure

 The size of a protein becomes important with respect to solubility when

the protein is excluded from part of the solvent
 E.g. addition of nonionic polymers results in steric exclusion of protein
molecules from the solution
 Juckes developed a model for this phenomenon that expresses the
solubility of the protein, S. This is based on the assumption that protein
molecules form solid sphere and the polymer molecules form rod

rs and rr are the radius of the protein solute and polymer rod, respectively, V = the partial
specific volume of the polymer, cp = the polymer concentration, and β’ = a constant

 Model reveals that larger proteins correspond to lower protein solubility

2. Charge:
 The solubility of a protein increases as its net charge increases –
a result of greater interaction with dipolar water molecules
 A repulsive reaction between protein molecules of like charge
further increases solubility
 A simple way to vary the charge on a protein is by changing the
pH of the solution
 The pH of the solution in which a protein has zero net charge is
called the isoelectric point
 The solubility of a protein is minimum at the isoelectric point

 The net charge of a protein is determined by the following

factors: total number of ionizable residues, dissociation
constants (or pKa values) of the ionizable groups, and pH of the
solution
 Besides, factors that can influence the pKa values are chemical
nature of the neighboring groups (e.g.. inductive effects),
temperature, chemical nature of the solvent as partially reflected
by its dielectric constant, and ionic strength of the solvent
3. Presence of solvent/salt:
 Presence of solvent/salt alters the polarity of the medium

 The solvent/salt affect the solubility of proteins primarily

through two parameters, hydrophobicity and ionic strength.
 Hydrophobicity: Single-phase solutions of water and
monohydric alcohols cause protein denaturation at room
temperature. Could be avoided at lower temperature.
 Alcohols with longer alkyl chains bind more effectively to non-
polar groups on the protein, weaken the intra-protein
hydrophobic interactions, and thus lead to denaturation.
methanol < ethanol < propanol <butanol
 Ionic Strength: The ionic strength of the solvent/salt can have
both solubilizing and precipitating effects – well known as
salting-in and salting-out, respectively
1. Temperature (Cooling):
 Solubility is a strong
function of temperature
 Temperature is lowered to
precipitate out one
component over the others
utilizing solubility curve
 Is a preferred parameter to
control, because biological
macromolecules are more
stable at lower temperature
 Often used in conjunction
with other parameters,
such as solvent or salt
addition
2. pH adjustment:
 Solubility is a strong
function of pH as well
 Proteins achieve minimum
solubility at the isoelectric
point
 Precipitation is often
carried out at the
isoelectric point of one
protein to preferentially
separate it out over the
other proteins, which
largely remain in solution
3. Solvent addition:
 Used widely for precipitation of proteins. Also used for
DNA and RNA precipitation
 Ethanol and acetone are most common solvent used for
precipitation
 Solvents precipitate proteins by promoting hydrophobic
interaction and reducing the dielectric constant of the
solution
 Dielectric constant of a solvent is the measure of it’s
ability to insulate charge. Higher dielectric constant
indicates higher polarity
 Organic solvents denature protein at room temperature,
hence it is usually carried out at low temperature
4. Anti-chaotropic salt addition:
 Anti-chaotropic agents (such as
ammonium and sodium sulfates)
expose hydrophobic domains on
proteins by disrupting the
structured water layer
surrounding the protein
molecules
 Hydrophobic domains of the
protein molecules interact with
each other, form aggregate and
precipitate
 Solubility of different proteins is
reduced to different extent by
salt addition
 Anti-chaotropic salts do not
cause denaturation of the
proteins
 Salting-in vs. salting-out

 “Ammonium sulfate cut” is often

used
5. Chaotropic salt addition:
 Chaotropic agents (such as urea and guanidine hydrochloride)
disrupt the intra-molecular hydrogen bonding and the
hydrophobic interactions, thereby reducing the stability of the
native state of proteins
 Chaotropic salts cause denaturation of the proteins

6. Immuno-precipitation:
 Relies on antigen-antibody recognition and binding
Hofmeister Series or Lyotropic Series

Hofmeister Series or Lyotropic Series arranges various ions based on

their ability to precipitate (salt-out) proteins from aqueous solution

Ctr3-,
Salting-In Effect : Kirkwood Model:
Solute: size, shape, dipole moment; (3)
Solution: dielectric constant, ionic strength
Physical parameter: temperature

where Sp = the equilibrium solubility of the dipolar ion at ionic strength I,

So = the equilibrium solubility of the dipolar ion in the absence of salt,
Ki = the salting-in constant, and Ks = the salting-out constant

Ionic strength is defined by,

where ci is the molar concentration of any ion and zi is its charge.

The salting-in and salting-out constants can be related to other

variables as follows:

where ε = the dielectric constant of the solvent, T = temperature,

Ve = the excluded volume of the dipolar ion, u = dipole moment

Higher polarity leads to better salting-in characteristics

Salting-in effect tends to predominate in relatively non-polar solvents,
while the salting-out effect is more dominant in aqueous solvents.
 Salting-Out Effect – Cohn Equation: (4)

S is the equilibrium solubility of the protein at ionic strength I, Ks is a

salting-out constant characteristic of the specific protein and salt that
is independent of temperature and pH above the isoelectric point
The constant β, the natural logarithm of the theoretical solubility of
the protein at zero ionic strength. It depends only on temperature and
pH for a given protein and is a minimum at the isoelectric point

The Kirkwood equation for the solubility of dipolar ions can be

arranged to give

which is also identical in form to the Cohn equation, with

1. Data were obtained for precipitation of a protein by ammonium
sulfate. The initial concentration of the protein was 15 g/L. At
ammonium sulfate concentrations of 0.5 and 1.0 M, the
concentrations of the protein remaining in the mother liquor at
equilibrium were 13.5 and 5.0 g/L, respectively. From these
information, estimate the ammonium sulfate concentration needed to
achieve 95 % precipitation of the protein.

2. Ammonium sulfate is used to precipitate human monoclonal antibody

from 10 L of cell culture media. The initial concentration of the
antibody is 0.5 mg/mL. Solid ammonium sulfate is added to the
liquid such that the concentration of the salt is 1.5 kmole/m3. This
results in the precipitation of 90 % of the antibody. When the
ammonium sulfate concentration is raised to 1.75 kmole/m3, a
further 76.5 % of the remaining antibody is precipitated. Predict the
ammonium sulfate concentration needed for total antibody
precipitation. What is the solubility of the antibody in ammonium
sulfate free aqueous medium?
 Characteristics of the protein precipitate:
 Size: Small size fraction is undesirable for downstream
separation, so is wide size distribution
 Density: Low density is undesirable due to high bulk volume
 Mechanical strength: Low mechanical strength materials
(gels/colloids) are difficult to handle
 Precipitation time
The sequential steps of precipitation are as follows:
 Initial mixing

 Nucleation

 Growth by diffusion

 Growth by convection (bulk fluid motion)

Besides these two additional steps – particle breakage and

ageing (storage of precipitate without mixing), are also
observed during precipitation
Required to form homogeneous mixture of the components (solvent,
macromolecules and precipitating agent) in precipitation
Diffusion limited approach of the molecules and eventual collision

Molecules must travel across an eddy to meet and form particles

The time needed for initial mixing, t, is calculated using isotropic

turbulence model and Einstein’s diffusion relationship as follows,

(5)

le is the length of the eddies

Mean length of eddies, also known as “Kolmogoroff length”, le, is expressed as

(6)
Generation of particles of ultramicroscopic size
The solution must be supersaturated with respect to the solute,
i.e. the concentration of the solute in solution should be greater
than the equilibrium solubility
Rate of nucleation increases exponentially with supersaturation
(up to the maximum level of supersaturation)
High supersaturation typically has negative consequences in
precipitations – the precipitate tends to be in the form of a colloid
or gel, which is difficult to separate
Controlled saturation produces precipitate with the required
mechanical strength
Immediately after nucleation, the growth of the precipitate particles is
governed by a diffusion controlled mechanism that enables formation of larger
particles
Less than a micron to few micron size precipitate particles are typically
observed for this precipitate mechanism
Assumption: All particles behave identically and form bigger particles of
uniform size
(7)
The particle number concentration, N can be expressed as

(8)

N0=initial number conc. of dissolved solute molecules

Lmol, the diameter of the globular molecules, which are modeled as spheres

K = Boltzman constant, T = absolute temp.,

(9)
μ = liquid viscosity

M = molecular weight of particles at time t,

(10)
M0 = molecular weight of the solute

(12) (11)
3.

4. For the previous problem, calculate the time for the particles to reach a size of 1
micron, assuming that the growth is governed by diffusion only up to this particle size.
Also, calculate the number concentration of the 1 micron particles.
Growth of the precipitate particles is governed by fluid motion after they
reach a critical size, typically few micron in size
Particles tend to grow by colliding and then sticking together. Typically,
tens to hundred micron size precipitate particles are observed for this
precipitate mechanism

(13) (14)

Equation 14 developed by Camp and Stein.

5.

(Assume α = 0.05)
Precipitate particles grow bigger and become susceptible to breakage
during collisions
Rate of breakage also depends on the shear rate and particle
concentration
At the end, rate of particle formation becomes equal to the rate of
breakage
Aggregate size could be mathematically predicted by the Displacement
Model as follows:

(15)

Where, Le is the equilibrium aggregate

diameter; rate constant, k, is a function of
volume fraction of particles (ϕ) and shear
rate (γ)

Aggregate diameter as
a function of time for
soy protein particles
When a precipitate is stored after precipitation without agitation, a
process called aging takes place
Some change in aggregate size and distribution take place

It facilitates separation of precipitates from supernatant

Typical aging duration is 6-12 hrs.