Protein Processing in Food and Bioproduct Manufacturing and Techniques for Analysis - Food and Industrial Bioproducts and Bioprocessing - page 97

Gel filtration chromatography
Gel filtration chromatography has been widely used to purify biomolecules due to its
simplicity and efficiency (Pastorello and Trambaioli, 2001). The technique separates pro-
teins on the basis of their molecular size independent of the composition of the buffer used
as eluent. Different media are used to perform the molecular exclusion in packed columns.
The inertness and physicochemical stability of the chosen media are key requirements in
column selection. Details of the technique can be found elsewhere (Amersham, 2002). Gel
filtration is generally used in combination with the other chromatographic techniques
described (Neyestani et al ., 2003 ).
Ion exchange chromatography
Ion exchange chromatography separates biomolecules according to their net chemical
charge under determined experimental conditions. The degree of interaction between the
charged proteins and the opposite charges of the ionic groups of the ion exchange medium,
as well as differences in the charge properties of the proteins being separated, are key factors
influencing separation (Ishihara and Yamamoto, 2005). The elution method generally
involves a gradient of at least two buffers, such that the elution of the proteins is performed
after the disruption of the electrostatic bonds between the biomolecule and the ionic groups
of the chromatographic medium resulting from an increase in ionic strength or pH change
(Levison, 2003). Two ion exchangers are commonly used: anionic exchange columns are
generally used to separate acidic proteins, whereas cationic columns are more widely
utilized for the purification of basic proteins (Pastorello and Trambaioli, 2001).
Reversed phase chromatography
Reversed phase chromatography is a widely used chromatographic technique which offers
high resolution in separating proteins on the basis of their hydrophobicity (Pastorello and
Trambaioli, 2001). The stationary phase is generally composed of an n -alkyl hydrocarbon
that is able to interact with the proteins through hydrophobic interactions. The separation is
often performed using a gradient elution. Thus, after adsorption to the immobilized n -alkyl
hydrocarbon, the injected proteins are eluted due to the disruption of their hydrophobic
bonds with the stationary phase after decreasing the polarity of the mobile phase (Pastorello
and Trambaioli, 2001). Reversed phase chromatography has been extensively used in
analytical analysis as well as at the preparative scale for the purification of food and
recombinant proteins (Olson et al ., 1994 ).
Affinity chromatography
Affinity chromatography separates proteins according to their specific reversible interactions
with a chemical ligand attached to the chromatographic matrix; it might be a substrate
analogue, enzymatic inhibitor or activator, or a specific ligand of the targeted protein
(Chaiken, 1986). Hence, proteins could be separated based on their biological function and
specific structure.
Immunochromatography refers specifically to the immobilization of antibodies onto the
gel matrix for affinity chromatography and is a technique that has been widely used for the
isolation of high purity proteins (Riggin and Sportsman, 1993).
Proteins separated by chromatography using any of these techniques can be detected
using UV spectroscopy, refractive index, evaporative light scattering, fluorescence or mass
spectrometry detection.