1、 Ion exchange chromatography

Ion exchange chromatography separates compounds based on their net charge. Functional groups with negative or positive charges covalently bind to the solid carrier matrix, becoming cation exchangers and anion exchangers, respectively. When a charged molecule is added to an exchange agent with an opposite charge, it is adsorbed, while ions and neutral molecules with the same charge are eluted into the outer water volume of the chromatography column. The binding of charged molecules is reversible, and adsorbed molecules can usually be eluted using salts or pH gradients.

Ion exchange chromatography can have various applications, including separation and purification of biomolecules, separation of inorganic ions, deionization of reagents and water, salt conversion, and removal of metal ions, ethidium bromide, and SDS.

Separation strategy for amphoteric compounds. If the molecule is stable at pH above its isoelectric point, anion exchange resin can be used. If the molecule is stable at pH below its isoelectric point, cation exchange resin is used.

GE (formerly Amasya) chromatography packing (commonly used model) in the United States

2、 Affinity chromatography

During the affinity chromatography process, the ligand has a specific affinity with the molecule to be separated and covalently binds to the solid. When the sample mixture is added to the chromatography column carrier formed by these matrices, the target molecules (such as proteins, peptides, DNA fragments, etc.) bind to the ligands fixed on the matrix, while the non affinity components in the mixture precipitate. By changing the pH value, salinity concentration, using organic solvents, or adding molecules that compete with ligands, the target molecule can be eluted.

GE (formerly Amasya) chromatography packing (commonly used model) in the United States

Affinity chromatography packing:

Metal chelating filler:

17-0920-07 Packing name: IMACSepharoseHighPerformance

17-0921-09 Packing name: IMACSepharose6FF

3、 Molecular weight exclusion chromatography (also known as molecular sieve chromatography, gel filtration chromatography)

Molecular weight exclusion chromatography (SEC), also known as gel filtration chromatography, separates molecules according to molecular weight. The gel matrix is composed of spherical beads with specific size pores, and molecules with different molecular weights are blocked from passing through the pores to separate. Small molecules entering the pore will delay their passage through the chromatography column, while large molecules avoid the pore and wash out into the volume of water outside the chromatography column. Therefore, the samples are separated based on their molecular weight and eluted in descending order of molecular weight.

Select operating conditions and media based on the purpose of chromatography and the required resolution. A common method in molecular weight exclusion chromatography is group separation, which includes:

Desalination - target molecules are eluted, while small molecules remain in the gel pores. In order to achieve ideal separation, the molecular weight exclusion limit of the matrix should be smaller than that of the target molecule.

Fractionation - the separation of molecules in a certain molecular weight range within the gel matrix. When using this method, the target molecule should be within the range of gel fractionation.

The conventional applications of molecular weight exclusion chromatography include protein fractionation and molecular weight determination, nucleic acid separation, plasmid purification, and polysaccharide fractionation.

The chromatographic resolution depends on particle size, pore size, flow rate, column size, and sample volume. Generally, the highest resolution can be obtained with low flow rate (2-10cm/hr), slender column, small particle gel, small sample volume (1-5% of the total bed volume), twice the molecular weight difference and the same solution viscosity. If used for desalination, the product volume can reach 30-40% of the total bed volume, and short and thick columns can be used.

GE (formerly Amasya) chromatography packing (commonly used model) in the United States

Gel filter filler:

4、 Hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) separates compounds based on their hydrophobicity; The sample molecules with hydrophobic and hydrophilic groups were added to the HIC chromatography column in a high salt buffer, and the salt in the buffer reduced the solubility of the sample. Due to the decrease in total solubility, the exposed hydrophobic groups are adsorbed by the medium. The stronger the hydrophobicity of a compound, the less salt needs to be added to enhance its binding strength. Usually, the sample in the chromatography column is eluted using a decreasing salt gradient in ascending order of hydrophobicity, or mild organic regulators or detergents can be added during the elution process.

GE (formerly Amasya) chromatography packing (commonly used model) in the United States

Hydrophobic chromatography packing:

5、 Hydroxyapatite chromatography

Hydroxyapatite (Ca5 (PO4) 3OH) 2 is a type of calcium phosphate with unique selectivity and resolution for separating proteins that cannot be distinguished by other chromatography and electrophoresis techniques. Hydroxyapatite chromatography is suitable for any step from initial capture to the most complete one. Applied to the following separation and purification:

·Different types of monoclonal and polyclonal antibodies · Distinguishing between supercoiled and linear DNA

·Antibodies with different light chain compositions · Distinguishing between double stranded DNA and single stranded DNA

·Antibody fragments, viruses, and isoenzymes