1. Precipitation

2. important traditional method for purifying proteins and nucleic acids.
Involves selective conversion of a specific dissolved component of a complex mixture to an insoluble form using appropriate physical or physicochemical means.

3. Factors utilized for precipitation
Cooling/heating
pH adjustment
Addition of solvents such as acetone and ethanol
Addition of anti-chaotropic salts such as ammonium sulphate and sodium sulfate (intermolecule)
Addition of chaotropic salts such as urea and guanidine hydrochloride (intramolecule)
Addition of biospecific reagents as in immunoprecipitation

4. Anti-chaotropic expose hydrophobic patches on proteins by removing the highly structured water layer which usually covers these patches in solution
Chaotropic solutes increase the entropy of the system by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects.

5. Cooling
solubility of A is more sensitive to the lowering of temperature than B.
Hence, the separation of A and B could be carried out by lowering the temperature of the solution to the point where A is largely precipitated while B is still largely in solution

6. Temperature induced precipitation
Based on the assumption that that denaturation follows first order chemical kinetics with an Arrhenius dependence
[p] is the dissolved protein concentrations
Rate constant k is given by
E can be manipulated by changes in pH or solvent

7. Example
Two of these dehydrogenases have the precipitation rate constants You have a solution containing equal activities of each enzyme. What will be the activity be after 10 min at 20oC? What will it be after 10 min at 50oC?

8. Solution By integrating we see that 20oC 50oC kA 8.3 x 10-11 1.6x10-4kB
4.0x10-10
3.0x10-3
By integrating we see that
20oC
50oC
[A]/[Ao]
>0.999
0.91
[B]/[B0]
0.17

9. Additives
Precipitation by using additives is governed by the thermodynamic equation:

10. Additives
When a solute is being precipitated from its solution, the precipitate is mainly composed of the solute.
So its chemical potential at a given temperature is constant.
On the other hand the chemical potential of the dissolved solute depends on its concentration and is given by:

11. Additives
The standard reference state chemical potential can be increased by adding substances such as salts and solvents.
In the presence of such additives, the solute concentration in the solution phase must decrease in order that the chemical potential of the solute in solution be the same as that in the precipitate.

12. Additives-Organic solvents
precipitate proteins and other macromolecules by reducing the dielectric constant of the medium

13. Additives-Organic solvents
Higher concentrations of organic solvents can denature proteins.
Organic solvents usually bind to specific locations on the protein molecules and thus disrupt the hydrophobic interactions which hold the protein structure in place.
very small amounts of organic solvent are used in precipitation processes and these are carried out at low temperatures to minimize denaturation

14. Example
The solubility of ovalbumin in water is 390 kg/m3. When 30 ml of ethanol was added to 100 ml of a 50 mg/ml ovalbumin solution in water, 33% of the protein was found to precipitate. How much protein would precipitate if 100 ml of ethanol were added to 100 ml of a similar protein solution at the same temperature? Assume that the dielectric constant of the medium varies linearly with volumetric composition of the two solvents.

16. Additive-pH
the minimum solubility being observed at its isoelectric point
Separation of the two proteins could be carried out by maintaining the solution at isoelectric point of B or at isoelectric point of A

17. Addition of anti-chaotropic salts
Anti-chaotropic salts such as ammonium sulphate and sodium sulphate expose hydrophobic patches on proteins by removing the highly structured water layer
hydrophobic residues on a protein molecule can interact with those on another and leads to aggregation/precipitate

19. The constant B is the natural log of the theoretical solubility of the protein in salt free water.
The Cohn equation is valid only in the salting-out region of the precipitation process.
The constant 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.

21. Addition of biospecific reagents
relies on antigen-antibody recognition and binding.
multivalent antigens react with antibodies in solution they form large molecular networks by cross-linking which eventually precipitate (precipitins)

22. Protein Solubility –Rules of thumb
Larger proteins are less soluble than smaller ones
Protein whose surface is rich in hydrophobic amino acids are less soluble
Denatured proteins are less soluble
pH close to the isoelectric point decrease solubility
The higher the dielectric constant the lower the solubility
The higher the ionic strength the lower the solubility

23. Mechanism of precipitate formation
precipitation is taking place as a function of time
A precipitation process has the following stages:
Mixing
Nucleation
Diffusion limited growth
Convection limited growth

24. Mixing takes place over a finite amount of time.
the amount of time depending on the properties of the components as well as on the processing conditions (mixing intensity and temperature)

25. Initial Mixing
Initial mixing is the mixing required to achieve homogenity after the addition of a component to cause precipitation
By assuming that mixing between randomly dispersed eddies is instantaneous and the mixing within eddies is diffusion limited
Where ρ is liquid density, v is the kinematic viscosity and P/V is the agitator power input/unit volume of liquid

26. For spherical eddies of diameter le, this becomes
It is necessary to mix until all molecules have diffused across all eddies. This time can be estimated from From Einstein diffusion relationship
Where δ is the diffusion distance and D is the diffusion coefficients
For spherical eddies of diameter le, this becomes
The average eddy length (Kolmogoroff length) scale depends on
volume of the system,
the density and viscosity of the medium,
the power input for mixing

27. nucleation step formation of very minute particles
initiates at regions having localised supersaturation
high degrees of supersaturation result in formation of gelatinous precipitates
Controlled supersaturation produces amorphous precipitates which are both easy to filter and centrifuge

28. Diffusion limited growth
leads to the formation of bigger particles
the rate of particle formation is dependent on:
the physicochemical properties of the protein
the liquid medium
The rate constant K depends on the diffusivity (D) and diameter (d) of the protein.

29. The constant k could be determined as
Integrating gives
Stokes-Einstein equation can be used to estimate the diameter of globular protein:

30. Example
We wish to precipitate the protein α2- macroglobulin contained in a 100 liters of aqueous solution at 20oC in a tank at a concentration of 0.2 g/liter. α2-macroglobulin is a globular protein with a molecular weight of 820,000 and diffusion coefficient of 2.41 x cm2/s at 20oC. The precipitate particles have a density of 1.3 g/cm3. The solution is stirred with 75 W motor. Calculate the concentration of nuclei at the end of the ‘initial mixing’ period

33. Convection limited growth
The greater the extent of mixing, the greater is the frequency of collision.
The rate of precipitate formation of a particular size in convection limited growth is given by:
The rate constant φ depends on:
the size and sticking tendency of the particles,
the volume of the system,
the density and viscosity of the medium
the power input for mixing.