As a ligand, the following conditions shall be met:
① The complexes formed by them should be soluble in water, that is, they should exist in solution in the form of ions;
② The ligand does not interfere with the redox reaction of anode and cathode, and does not decompose and hydrolyze in solution;
③ The formed complex ions should have certain stability and overvoltage.
There are many kinds of ligands, which can be divided into inorganic and organic. The number of inorganic ligands is relatively small and the structure is relatively simple. There are many organic ligands with complex structures. Although its coordination ability can be inferred from the nature of the coordination atom, its structural factors often play an important role, such as the role of conjugated bonds, chelation, steric hindrance and the influence of substituents. [1]
1. Biological special effect ligand
Many biological macromolecules or small molecules can be used as biological specific ligands, such as:
① Nucleotides, nucleosides, nucleotides Oligonucleotide , RNA Deoxyribonucleic acid (DNA)； ② Amino acid, polypeptide, protein, enzyme;
③ Coenzyme, which is composed of vitamin PP (nicotinic acid and nicotinamide) or B vitamins, nucleotides and metal ions, such as coenzyme I [Co I, also known as Nicotinamide adenine dinucleotide (NAD)]； ④ Exogenous agglutinin (It can be extracted from lentils, wheat germ and castor bean); ⑤ Sugar [including monosaccharide, oligosaccharide, polysaccharide (such as heparin)];
⑥ Antigens and antibodies;
⑦ Avidin biotin;
⑧ Hormone;
⑨ Biological cells, biomolecular assemblies and microorganisms.
The above ligands have special biological affinity recognition function, and can couple with complementary biomolecules to form key complexes. [2]
2. Dye ligand
Use some Synthetic dyes The molecule has a chemical structure similar to that of nucleotide, and acts as a biomimetic ligand. Anthraquinone reactive triazine dyes, such as Ciba Blue F3G-A, naphthalene reactive triazine dyes, such as Blossom Red HE-3B and fluoro triazine dye Ciba Yellow F3R, have been widely used. Such ligands are cheap and easily available, and have been used in preparative affinity chromatography. [2] 3. Positioning metal ion ligands
Many organic chelating agents, such as Iminodiacetic acid (IDA), ethylenediaminetetraacetic acid, imidazole, thiourea 8-hydroxyquinoline , dithizone, etc., can be mixed with metal ions, such as Cu 2+ 、Zn 2+ 、Ni 2+ 、Mg 2+ 、Fe 3+ These chelates show a specific affinity for proteins and enzymes, and thus have more and more applications in affinity chromatography. [2] 4. Inclusion complex ligand
with β - cyclodextrin (β - CD) as the host, uses its special hole structure to form host guest inclusion complexes with guest biomolecules. In addition, crown ethers, cave ethers and calixarenes can also be used as mimic enzymes to form inclusion complexes with biomolecules, which have been used for the separation and analysis of affinity chromatography. 5. Charge transfer ligand
The charge transfer ligand can be an electron donor, such as 5,10,15,20-tetra (p-hydroxyphenyl) porphyrin, or an electron acceptor, such as copper sulfonated phthalocyanine, which can realize the separation of biomolecules by using their affinity for the positive and negative charge attraction between complementary biomolecules. [2]
6. Covalent ligand
Using pyridine with disulfide bridge (- S-S -) Disulfide And small peptides（ glutathione ）Coupling, as a ligand, can be used to separate sulfur-containing proteins, which is a special bioselective affinity. [2] 
Figure 1 Such as adenosine monophosphate AMP and Adenosine diphosphate The affinity stationary phase prepared by ADP and agarose coupled with aminohexyl or hexyl hydrazide can be connected in four ways, as shown in Figure 1. In the figure (a) N - (6-aminohexyl) - AMP agarose [N-AMP], (b) C - (6-aminohexyl) - AMP agarose [C-AMP], (c) P - (6-aminohexyl) - P - (5 '- adenosine) - pyrophosphate agarose [P-AMP], (d) ribosyl linked AMP [R-AMP]. [2] It can be seen from the above examples that when the selected ligand has several active action points that can be coupled with the spacer arm, and the binding mode of biological macromolecules and ligands is not clear, it is better to synthesize several affinity stationary phases to evaluate their affinity binding efficiency with biological macromolecules. For ligands with two or more connecting action points, the action points that are relatively sensitive to chemical derivation should be selected. For example, for the above AMP derivatives, because the preparation procedure of 8-position (C) substituted ligand is relatively simple and saves raw materials, while the synthesis route of 6-position N (N) or phosphoric acid (P) substituted derivatives is complex, and special organic synthesis training is required, the 8-position substitution scheme is mostly adopted from the perspective of preparation feasibility. It is generally believed that the larger the ligand molecule, the more active interaction points it has with complementary biomacromolecules, and the greater the freedom of synthesis routes available for preparing affinity stationary phases.
Adsorption/desorption in affinity chromatography
Affinity chromatography is based on biospecific adsorption, or the reaction of proteins with ligands with certain structures. Adsorption is not a common chromatographic action, it is carried out within the pH range of low salt or high salt width. The effect of coordination density on the retention characteristics and column capacity is different from other chromatographic operation mechanisms. The completion of desorption depends on the addition of ligands in the mobile phase, the change of pH range, and the change of ionic strength. The specific displacement agent is an ideal desorption agent in affinity chromatography. It can desorb the binding points between ligands and biological macromolecules. Generally, in the process of desorption, the protein recovery rate is reduced due to the destruction of its structure.
Immunoaffinity chromatography is widely used because it can keep bioactive substances unchanged during the separation and purification process. In particular, the use of monoclonal antibodies as ligands is more widely. Immunoaffinity chromatography has achieved satisfactory results in industrial purification of interferon. [3]
