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Module-30: Fermentation Product Recovery and Purification-V:
Chromatography
Chromatography
 In many fermentation processes chromatographic techniques are used for isolation and
purification of relatively low concentrations of metabolic products.
 Chromatography is generally used for the separation of mixtures based on the differences in
their shape, size structure and/or composition.
 In chromatography, components of the mixture to be separated are distributed between
stationary phase & mobile phase.
 Components to be separated are carried by the fluid or gaseous phase is called mobile phase
which moves in a definite direction &carries them through a structure holding another
material which is termed as stationary phase.
 Some components interact tightly with the support & can move slowly through the support
than other components which interact weakly.
 Different components of the mixture move at different speeds, which finally get separated.
 Components are separated because of their differential partitioning between two phases i.e.
the mobile and stationary phases.
 For example, in liquid chromatography as liquid is passed through a column it allow passage
and separation of different solutes.
 Depending on the mechanism by which the solutes may be differentially held in a column ,the
techniques can be grouped as follows:
1. Adsorption chromatography
2. Ion –exchange chromatography
3. Gel-permeation chromatography
4. Affinity chromatography
5. Reverse phase chromatography
6. High performance liquid chromatography
 Chromatographic techniques are also used in the final stages of purification of a number of
products.
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1. Adsorption Chromatography
 Adsorption chromatography involves weak Vander Waals forces to bind the solute to the solid
phase.
 To pack columns different inorganic & organic materials can be used.
 Inorganic adsorbants utilized here includes active carbon, aluminium oxide, aluminium
hydroxide, magnesium oxide; silica gel etc.
 For example active carbon (0.25 to 0.5%) is used to remove pigments and other impurities
form penicillin containing solvents.
 Organic macro-porous resin may be used for same purpose.
 The first synthetic organic macro-porous adsorbants, the Amberlite XAD resins, were
produced by Rohm and Haas in 1965.
 These resins do not have any ionic functional groups but have surface polarities which differ
from non-polar to highly polar.
 Voser (1982) specified their best significant use in the isolation of hydrophilic fermentation
products.
2. Ion Exchange Chromatography:
 Ion-exchange chromatography is used for the separation of ions and polar molecules like large
proteins, small nucleotides and amino acids on the basis of their charge.
 Molecules of acidic, basic and neutral nature can be easily separated by this technique.
 In this separation, matrix-bound charged groups reversibly adsorb oppositely charged sample
molecules.
 Diffusion of the molecule through the matrix of the exchanger is dependent upon the degree
of cross-linkages of the exchanger and the ionic strength of the buffer.
 This diffusion process can control the rate of the whole ion-exchange process.
 However, desorption of the bound ion is achieved by change in pH or changing salt
concentration, or by adding competitive counter ions, since it tends to disrupt the electrostatic
interaction of the matrix with the bound molecule.
 Ion-exchange chromatography is perhaps the most frequently used chromatographic technique
for separation of proteins according to their net charges.
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 There are two types of ion exchange chromatography,
1. Cation exchange chromatography: the stationary phase contains a negatively charged
functional group which holds positively charged cations.
2. Anion exchange chromatography: the stationary phase contains a positively charged
functional group which holds positively charged anions.
 Cationic ion-exchange resins normally contain acidic active group like sulphonic acid,
carboxylic acid etc.
 Carboxy methyl cellulose is a common cation exchange resin.
 Positively charged components (e.g. certain proteins) will bind to the resin.
 Then by passage of buffers of increasing ionic strength or pH, deposited solutes are
successively washed away.
 Anionic ion-exchange resins normally contain a secondary amine, quaternary amine or
quaternary ammonium active group.
 DEAE (diethylaminoethyl) cellulose is a common anion exchange resin.
 Weak acid cation ion-exchange resins like Amberlite IRC 50 can be used in the isolation and
purification of antibiotics.
 For example to recover the streptomycin, sodium form of Amberlite IRC 50 is used.
 On to the column, by displacement of sodium ions the streptomycin is adsorbed.
RCOO-Na+ (resin) + streptomycin —> RCOO- streptomycin (resin) + NaOH
 The resin bed is now rinsed with water and eluted by hydrochloric acid to release the bound
streptomycin.
RCOO- streptomycin (resin) + HCl —>RCOOH (resin) + streptomycin + Cl A slow flow is used to ensure the highest recovery of streptomycin using the smallest volume
of eluent.
 In one step the antibiotic has been both purified and concentrated, maybe more than 100 fold.
 The resin column is regenerated to the sodium form by passing an adequate volume of NaOH
slowly through the column and rinsing with distilled water to remove excess sodium ions.
RCOOH (resin) + NaOH —> RCOO-Na (resin) + H2O
 Commercially, it is not economic to regenerate the resin completely; therefore the capacity
will be reduced.
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 In practice, the filtered broth is taken through two columns in series while a third is being
eluted and regenerated.
 When the first column is saturated, it is isolated for elution and regeneration while the third
column is brought into operation.
 Ion-exchange chromatography may be combined with HPLC in certain cases.
3. Gel Permeation Chromatography
 This technique also recognized as size exclusion and gel filtration Chromatography.
 The separation of the components in the sample mixture is based on their molecular weight,
shape and size.
 Generally it is used to determine the molecular weight of the molecule.
 Gel filtration is also an important preparative technique in the purification of proteins,
polysaccharides and nucleic acids.
 The stationary phase consists of porous glass granules or gel particle which is in equilibrium
with mobile phase.
 The smaller molecules having lower molecular weight can move into the gel faster than the
larger ones, and penetrate the pores of the gel to a greater degree.
 As a result of this, the larger molecules which are completely excluded from the pores of the
gel will be eluted first when elution is started.
 Intermediate sized molecules elute in the middle of the large and small molecules as they can
fit inside few but not all of the pores in the beads.
 However, molecules of small size are completely included.
 They get distributed between the mobile phase inside and outside the molecular sieve and
elute last in a gel filtration separation
 Various gels are available like cross-linked dextrans (Sephadex and Sephacryl) and cross
linked agarose (Sepharose) with various pore sizes depending on the fractionation range
required.
Application
 One early industrial application, although on a relatively small scale, was the purification of
vaccines.
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 This technique harvests a pure portion of vaccine which is then concentrated ten times by
removing the eluent buffer (Na2HPO4) by pressure dialysis.
4. Affinity Chromatography
 Affinity chromatography is used for separation and purification of most biological molecules
on the basis of their function or chemical structure.
 This technique depends on the highly specific interactions between pairs of biological
materials such as enzyme-substrate, enzyme-inhibitor, antigen-antibody, etc.
 Separation is based on the binding affinity of biomolecules with their respective biologically
active ligands (this ligand is bonded covalently or by physical absorption to the solid matrix)
which interacts with its analyte as a reversible complex.
 Eluent is then passed through the column to release desired molecule from the reversible
complex in highly purified and concentrated form.
 The major components required for an affinity chromatography procedure are bead matrix,
ligand, and a solution containing the substrate to be isolated, a wash to elute the non-bound
impurities in the solution, and a final wash to elute the bound substrate from its ligand.
 For efficient separation of product, the pore size and location of the ligand must be
corresponding to the size of the product.
 Coupling procedures have been developed using cyanogen bromide, bisoxiranes, disaziridines
and periodates, for matrixes of gels and beads.
 Four polymers which are often used for matrix materials are agarose, cellulose, dextrose and
polyacrylamide.
 Most common support used for the coupling of amino ligands is agarose gel activated with
cyanogen bromide.
 An effective alternative to gel supports found in affinity chromatography is silica based solid
phases.
 Purification may be several thousand folds with good recovery of active material.
 The method can however be quite costly and time consuming, and alternative affinity methods
such as affinity cross flow filtration, affinity precipitation and affinity partitioning may offer
some advantages.
 Affinity chromatography was used initially in protein isolation and purification, particularly
enzymes.
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 Since then many other large scale applications have been developed for enzyme inhibitors,
antibodies, interferon and recombinant proteins, and on a smaller scale for nucleic acids, cell
organelles and whole cells.
 In the scale-up of affinity chromatographic processes bed height limits the superficial velocity
of the liquid, thus scale-up requires an increase in bed diameter or adsorption capacity.
5. Reverse Phase Chromatography (RPC)
 Most liquid chromatography ("normal phase chromatography") uses a silica or alumina resins
as solid support stationary phase ("column") which is hydrophilic in nature.
 Thus, the hydrophilic molecules in the mobile phase which have strong affinity for the
stationary phase tend to adsorb to the column.
 At the same time the hydrophobic molecules are eluted by passing through the column.
 Opposite to that "reversed-phase chromatography" uses a hydrophobic stationary phase,
where solid support is covalently bonded to alkyl chains.
 Generally eight or eighteen carbons containing alkyl groupings are used (C8 and C18).
 A hydrophobic stationary phase has a stronger affinity for hydrophobic compounds.
 Thus, the hydrophobic molecules in the mobile phase tend to bind to the column, while the
hydrophilic molecules pass through the column.
 To allow elution of hydrophobic molecules from the column, an organic (non-polar) solvent
of decreasing polarity is used which reduces hydrophobic interactions.
 This allows the separation of molecules according to their hydrophobicity.
 Wide variety of molecules can be separated by RPC.
 But it is not generally used for separation of proteins, because sometimes proteins get
denatured by the organic solvents used in RPC.
 Sometime RPC can be combined with other chromatographic techniques for the separation of
specific molecules.
6. High Performance Liquid Chromatography (HPLC)
 HPLC is an abbreviation for High Performance Liquid Chromatography.
 It has also termed as High Pressure Liquid Chromatography.
 HPLC uses smaller, more rigid and more uniform beads.
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 Such type of beads can also be produced by improving the nature of column packing materials
for a range of chromatographic techniques (e.g. gel permeation and ion-exchange).
 This allows packing of columns with minimum spaces between the beads which results in
minimizing peak enlargement of eluted species.
 Because of that resolving power of HPLC will be always superior at the time of separation of
mixtures.
 So it is a high resolution column chromatographic technique.
 HPLC uses better-quality media (in terms of their selectivity and physical properties) for the
solid (stationary) phase through which the mobile (fluid) phase passes.
 The stationary phase used in HPLC contains high surface area/unit volume, even size and
shape of that is resistant to mechanical and chemical damage.
 However, it leads to high pressure necessities and cost.
 In terms of principle, LC and HPLC work the same way.
 But HPLC is much superior in terms of the speed, efficiency, sensitivity and ease of operation.
 This may be acceptable for analytical work, but not for preparative separations.
 Thus, to reduce operating and capital costs in preparative separations by HPLC, some criteria
are often compromise like instead of small, larger stationary-phase particles are used.
 But at the same time for the separation of very high value products, significant HPLC
columns containing analytical media have been used.
 Affinity techniques can be combined with HPLC to get benefit of both i.e. the selectivity of
the affinity chromatography & the speed and resolving power of the HPLC.
References
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Principles of Fermentation Technology: (2nd edition, by Peter F. Stanbury, Allan Whitaker and Stephen J.
Hall, Butterworth-Heinemann, An imprint of Elsevier Science.)
Industrial Microbiology: (By Casida L. E.New Age international (P) ltd publications)
A Text Book of Industrial Microbiology: (2nd edition By Wulf Crueger & Anneliese Crueger)
Biotechnology: Food Fermentation Microbiology, Biochemistry & Technology Vol. 1 & 2:(By V.K. Joshi &
Ashok Pandey)
Manual of Industrial Microbiology and Biotechnology: (2nd Edition by Arnold L. Demain and Julian E.
Davies, Ronald M. Atlas, Gerald Cohen, Charles L. Hershberger, Wei-Shou Hu, David H. Sherman, Richard
C. Willson and J. H. David Wu)
Project control No: RE-02091011297, Christ College, Rajkot, Gujarat, India
NMEICT-MHRD (Govt. of India) Project on - Creation of e-Contents on Fermentation Technology
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Industrial Microbiology-An introduction: By Michael J. Waites, Neil L. Morgan, John S. Rockey and Gary
Higton)
Comprehensive Biotechnology-The Principles, Applications and Rugulations of Biotechnology in
Industry, Agriculture and Medicine: (By Mrray Moo Young)
Fermentation Technology : Up Stream Fermentation Technology- Vol-I: (By H. A. Modi-Pointer
Publications)
Fermentation Technology : Down Stream Fermentation Technology- Vol-II: (By H. A. Modi-Pointer
Publications)
Industrial Microbiology by Prescott and Dunn's: (4th edition, edited by Gerald Reed, CBR publications)
Fermentation Technology: (By M.L. Srivastava, NAROSA publications)
Industrial Microbiology: (By A.H. Patel)
International student edition: Microbiology- A laboratory Manual: (4th edition. By James G.
Chappuccino & Natalie Sherman)
Bacteriological Techniques: (By F.J. Baker)
Introduction to Microbial Techniques: (By Gunasekaran)
Mannual of Industrial Microbiology and Biotechnology: (2nd Edition by Arnold L. Demain and Julian E.
Davies, Ronald M. Atlas, Gerald Cohen, Charles L. Hershberger, Wei-Shou Hu, David H. Sherman, Richard
C. Willson and J. H. David Wu)
Web references
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lum%20Preparation%20and%20Development%29.pdf
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