Principle
Affinity chromatography is based on the inherent specific interactions of biomolecules for highly selective sample separation. Specific interactions include the binding of alcohol energy to substrates, inhibitors, coenzymes, etc. Binding of antibodies to antigens: binding of lectins to cell surface antigens and some sugars.
The separation principle is shown in the figure:
The molecules that could have specific interaction with the target separated molecules were fixed on the stationary phase matrix. Because the molecules in the complex sample matrix did not have such specific interaction, they had relatively weak interaction with the stationary phase and were eluted first, while the target molecules were eluted last.
Affinity chromatographic stationary phase can be divided into special type and general type depending on the interaction between ligands and samples. Affinity ligands can be either directly coupled to the matrix or indirectly connected via spacer arms.
An appropriate spacer arm can effectively overcome the steric effect of the matrix surface and make the ligand more easily bind to the separated substance. The AFC stationary phase with spacer arm often has better chromatographic performance. The spacer arm plays a more important role in the separation of large molecular affinity stationary phase with small molecules as ligands. According to the structure type, the spacer arm of AFC stationary phase mainly includes hydrocarbons, chained polyamines, peptides, chained polyethers, etc. The ammoniacyl compound NH2(CH2)nR, R is the commonly used spacer arm, where R can be carboxyl group, hydroxyl group, amino group or the ligand itself. The spacer arm with hydrophobicity may have non-specific interactions with ligands or samples and interfere with affinity separation. In order to eliminate non-specific adsorption interference, functional groups such as imino and hydroxyl groups can be coupled to some atoms along the long axis of the spacer arm molecule to increase hydrophilicity.
The ligands fixed on the stationary phase of affinity chromatography include dye ligand, metal ion ligand, inclusion complex ligand, specific civilian group, charge transfer ligand and covalent ligand. The structure of triazine reactive dyes is close to the natural substrate of enzymes and can be used in affinity chromatography by binding to the active sites of enzyme active proteins. Cu2+, Zn2, Ni2+, Fe2+, Fe3+ and other metal ions are fixed on the matrix or spacer arm by organic functional groups with integration effect. The specific affinity between metal ions and biomolecules in chelates can be used to separate or purify biomolecules. Commonly used chelating agents include iminodiacetic acid, iminodiacetaldehyde, oxime, thiourea, pyridinimidazole, 8-hydroxyquinoline, etc.
The inclusion complex ligand is formed by the special affinity force between the subject and the guest molecule. The main compound has a ring or hole, and the guest molecule can be included in the ring or the hole to form the inclusion compound. Common main molecules include cyclodextrin, calixarene and so on.
Phase of mobility
In order to maintain the activity of biomolecules, mild elution conditions are generally required. In affinity chromatography, a buffer solution with different pH values is usually used as the mobile phase. The buffer system consists of inorganic or organic weak acids, weak bases and their salts. In order to maintain the activity of the molecule: P value is maintained between 6 and 8. There are few punching systems satisfying this condition, including acid salts, Tris-HCI, borate, etc. Phosphate buffer solution has a small buffer capacity, and is easy to precipitate with high-priced cations, and plays an inhibitor role in many metabolic systems. When pH<7.5, Tris-HCl system has a small buffer capacity and strong reactivity, and also has an inhibitor effect on many metabolic systems. Borate system can affect the separation of many biological organic compounds into complexes. The buffer capacity of glycylglycine was very strong at pH>8, but there was almost no retarding effect below pH=7.5.
The Goods Series of flushers, developed by Good et al., includes 12 buffering solutions with strong buffering capacity and low sub-strength, and can be used in biological and physiological research at pH of 6.1 to 10.7.
The interaction between the general affinity ligand and the solute is usually not strong. In most cases, the non-selective mobile phase can complete the separation of different components. When the biolecule and ligand form a complex with a small stability constant, under the condition of equal degree, the buffer solution with weak elution ability and different pH values can be used to dissociate the complex to achieve elution. When there are non-specific interactions caused by electrostatic attraction, hydrogen bonding force or hydrophobic interaction in addition to coordination, the interference can be eliminated by changing the P-out ionic strength, mobile phase polarity and adding dissociative sequence reagents. The dissociative sequence reagents can change the structure of biomolecules and denature proteins, thus destroying affinity. Rapid elution can be achieved by using low concentration out-of-liquid sequence reagent while maintaining the molecular structure of egg self-plasmic as much as possible.
The special affinity ligand has strong affinity with the target solute, so it is necessary to use the mobile phase containing specific components with super washing ability for elution. In this case, another free ligand is usually added to the mobile phase to replace the ligand on the stationary phase to bind to the target compound for elution. In addition, elution can be achieved by selectively breaking the chemical bond between the stationary phase matrix and the ligand. Since the target molecule is still attached to the ligand after elution, appropriate methods (such as changing pH value or adding denaturing agent) should be used to free it.
Development
In affinity chromatography, the objects of separation and purification are amino acids, peptides, proteins, nucleotides, nucleotides, oligonucleotides, ribonucleic acids, deoxyribonucleic acids, enzymes, coenzymes, oligosaccharides, polysaccharides and other biomolecules, most of which are polar compounds, many of which have biological activity. Therefore, when elution from stationary phase, dilute buffer solution with H value close to neutral should be used to maintain their biological activity under relatively mild elution conditions. Before affinity chromatography separation, the stationary phase must be balanced. The pH, ionic strength, temperature and chemical composition of the equilibrium buffer solution should produce strong interaction between the ligand and solute to facilitate retention. Temperature is an important condition for affinity chromatography separation, and the affinity intensity usually decreases with the increase of temperature. Different temperatures can be used for adsorption and desorption. The interaction between ligands and biomolecules is slow, so the sample should be loaded at a slow speed, and the flow rate of mobile phase should not be too fast. For the stationary phase with strong affinity, the sample volume has little influence. When the affinity is weak, the sample volume should not be too large. For gradient elution, temperature gradient, pH gradient or ionic strength gradient can be used.
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