1. Affinity Chromatography
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2. PRINCIPLE1
Affinity chromatography is the most specific chromatographic method. The interaction is biochemical in nature, e.g.
The highly specific nature of these interactions is due to the fact that the two participating compounds are ideally suited to each other both spatially and electrostatically.
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4. Fig. 17. 1 Principle of affinity chromatography. L, ligand; S, sample
Fig Principle of affinity chromatography. L, ligand; S, sample. δ+ and δ - represent partial charges (less than one elementary charge).
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5. If other sample components are present in the solution, e.g.
then they do not match the ligand and are not adsorbed.
The sample
is specifically retained by the stationary phase,
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6. The sample is now freed from all other impurities. It cannot
whereas the other molecules (proteins, enzymes. etc.) are removed by the mobile phase.
The sample is now freed from all other impurities. It cannot
be isolated until it has been separated from the stationary phase, this being done by elution with a solution containing a product with a great affinity to the ligand or even by a change in pit or ionic strength.
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7. As any biochemical activity can take place in just one specifically defined medium, it is clear that a p14 or concentration gradient may break the specific intact ion.
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10. 17.2 AFFINITY CHROMATOGRAPHY AS A SPECIAL CASE OF HPLC
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11. Affinity chromatography differs from other chromatographic modes in that a suitable stationary phase can specifically ‘catch’ either a single or several components out of a random mix of products owing to a naturally occurring biospecific bond. A suitable elution process then provides the pure compound (s).
No column is required for isolation purposes. hence affinity chromatography is frequently carried out in open Systems. e.g. suction filtering. Classical columns with a hydrostatic eluent feed offer a further possibility. This chapter. however, is confined to a description of separations with high- performance stationary phases (10 mm and below ) with high rapid chromatography can be achieved. Very small columns may be used.
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12. No column is required for isolation purposes
No column is required for isolation purposes. Hence affinity chromatography is frequently carried out in open Systems. e.g. suction filtering.
Classical columns with a hydrostatic eluent feed offer a further possibility.
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13. Very small columns may be used.
This chapter however, is confined to a description of separations with high- performance stationary phases (10 mm and below ) with high, rapid chromatography can be achieved.
Very small columns may be used.
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14. Stationary phases for affinity chromatography can he prepared by the user himself, often starting from diol- or amino-silica.
However, it is much simpler to buy an ‘activated’ gel which allows the desired ligand to be bound according to well known methods. For separation problems which require common ligands, ready-to-use stationary phases are commercially available.
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15. Most phases are built up with a long-chain group between the silica and the ligand, the so-called spacer to guarantee free accessibility of the sample molecules to the bonding site at the ligand.
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16. The bonded sample can be eluted by a gradient (pH, ionic strength or competing sample) or by a ‘pulse’ &. the latter being a typical affinity chromatography characteristic.
The bond-breaking compound is injected whilst the mobile phase passes through the column unchanged.
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17. The sample size is restricted only by the bonding capacity of the column. The yields of biologically active protein frequently reach 100%, which means that denaturation and irreversible adsorption are negligibly low in many cases.
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18. Anti-IgG3 ligands bond all antibodies of the lgG class specifically.
17.3 APPLICATIONS
Anti-IgG3 ligands bond all antibodies of the lgG class specifically.
The stationary phase synthesis required for the separation of lgG shown in Fig. 17.3, was described by the authors in the reference cited in the caption. The mobile phase starts off with a pH of 7.4: a switch to pH 2.2 breaks the antigen-immunoadsorbent bond and the IgG is eluted.
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19. As the characteristic lgG fluorescence is quenched at pH 2. 2
As the characteristic lgG fluorescence is quenched at pH 2.2. the pH of the column effluent had to be raised by adding a buffer of pH 8. The yield of active lgG was in excess of 97% . The stationary phase remains active for a long time provided that the column is stored at 4 0C when not in use.
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20. Lactins are plant proteins which can specifically bond hexoses and hexosamines.
Concanavalin A is a lectin widely used in the affinity chromatography and can isolate glvcopmoteins, glvcopeptides and glycolipids. Elution is triggered by a pulse of sugar solution.
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21. Separation of the enzyme peroxidase (a lycoprotein) is shown in Fig. 17.4.
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25. Detection is effected at two wavelengths simultaneously: 280 nm (non-specific for all proteins) and 405 nm (peroxidase-specific owing to the protohaemin group).
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26. Cibacron Blue is a popular ligand as it can hind a large number of enzymes and also some blood proteins. It is often referred to as a pseudo-affinity ligand’ because it is a synthetic triazine dye and not a naturally occurring biomolecule.
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27. This greatly reduces the activity of the eluted enzyme in most cases
This greatly reduces the activity of the eluted enzyme in most cases. One advantage of Cibacron Blue is its low price. The separation of isoenzymes H4 and M4 from lactate dehydrogenase (LDH). eluted in sequence by a gradient of reduced nicotinamide adenine dinucleotide (NADH). is shown in Fig A post-column reactor for measuring enzyme activity was used as a detector (see Section 20.5).
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28. Affinity Chromatography
Retention due to biospecific interaction using a ligand molecule chemically coupled to a dextran or cellulose matrix - (see 3.4 above for diagram).
Hence may be able to isolate analyte from complex mixture. Examples
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29. Analyte species Bound ligand
enzyme 
substrate ??, coenzyme, reversible competitive inhibitor 
antigen 
antibody 
antigen, cell fragment 
hormone 
receptor, binding protein 
receptor 
effector molecule, eg hormone, neurotoxin 
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31. Elution
Elution can be by displacement with ligand molecules in free solution. But analyte then eluted as complex with ligand. Better to elute by change of pH to weaken binding.
Chemistry of ligand coupling to matrix using cyanogen bromide.
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