Common chromatographic separation method - Database & Sql Blog Articles

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Common chromatographic separation method

in

Biomacromolecule

Purification analysis especially

Protein purification

In the analysis, chromatography is a very important and commonly used technique.

one,

Gel filtration

Gel filtration, also known as molecular sieve chromatography, is due to the fact that the gel has a network structure in which small molecules can enter the interior while macromolecules are excluded. When a mixed solution is passed through a gel filtration column, the materials in the solution are separated by different molecular weight sieves.

two,

Ion exchange chromatography

Ion exchange chromatography is in

Ion exchanger

For the stationary phase, the liquid is carried out in a mobile phase system.

Ion exchanger

By

Matrix

Charge group and

Counter ion

Constituted.

Ion exchanger

versus

Aqueous solution

Medium ion or

Ionic compound

The reaction is mainly carried out by ion exchange, or by means of a charge group on the ion exchanger to the ion or ionic compound in the solution.

Adsorption

get on.

three,

Adsorption chromatography

1. Adsorption column chromatography

Adsorption

R

Solid

Adsorbent

For the stationary phase,

Organic solvents

Or a buffer is a chromatographic method for the mobile phase to form a column.

2,

TLC

Thin layer chromatography is a substrate coated on a carrier such as a thin plate or a polyester sheet, and the liquid is used as a stationary phase.

Mobile phase

a chromatographic method. This chromatographic method is

Adsorbent

Is coated with

Carrier

Form a thin layer on it and press

Paper chromatography

The operation is carried out.

3,

Polyamide film

Chromatography

The adsorption of a polar substance by a polyamide is due to its formation with the object to be separated.

Hydrogen bond

. The strength of this hydrogen bond determines the separation and

Polyamide film

The size of the adsorption capacity between. When chromatography, the layering agent and the object to be separated compete on the surface of the polyamide film to form

Hydrogen bond

. Therefore, selecting a suitable layering agent to separate the adsorption, desorption, resorption, and desorption of the surface of the polyamide membrane can lead to the separation of the separated materials.

four,

Affinity chromatography

Affinity chromatography is based on

Biomacromolecule

with

Ligand

between

Specificity

Affinity, connecting a ligand

Carrier

A chromatographic technique that separates as a stationary phase and separates biomacromolecules that specifically bind to a ligand. Affinity chromatography is the most efficient chromatographic technique for separating biomacromolecules with high resolution.

The principle of affinity chromatography and the well-known antigen-antibody, hormone-receptor and enzyme-one

Substrate

The mechanism of isospecific reactions is similar, each pair

Reactant

There is a certain affinity between them. As in the enzyme

Substrate

In the reaction, the specific waste (S'') can be combined with a certain enzyme (E) to produce a complex (E-S''). Specific in affinity chromatography

Ligand

Can have affinity with certain life macromolecules and produce complexes. and

Affinity chromatography

Enzyme

Substrate

The difference is that when the former reacts,

Ligand

(similar to the substrate) is the presence of a solid phase; when the latter is reacted, the substrate is present in the liquid phase. essentially

Affinity chromatography

Is a ligand L with recognition ability (the ligand for the enzyme can be a similar substrate,

Inhibitor

or

Auxiliary group

Etc.)

Covalent bond

Way to cure to contain

Activating group

On the matrix M (such as activated agarose, etc.), made into affinity

Adsorbent

M-L, or a solid phase carrier. The cured ligand still retains the ability to bind specific substances.

Therefore, when the surrounding phase carrier is loaded into a small column (a few milliliters to several tens of milliliters of bed volume), the sample liquid to be separated is passed through the column. At this time, the sample is

Ligand

Affinity substance S can be used

Electrostatic attraction

Van der Waals force, and structure

Complementary effect

The same effect is adsorbed onto the solid support, while the non-affinity or non-specifically adsorbed material is washed out by the initial buffer and forms the first

Chromatographic peak

. Then, by appropriately changing the pH of the starting buffer, or increasing the ionic strength, or adding a factor such as 3, the substance S can be dissociated from the solid support and form the Mth.

Chromatographic peak

(See Figure 6-2). Obviously, the active ingredient can be satisfactorily separated from the impurities by this procedure. If more than two substances in the sample solution have affinity with the solid phase carrier (the difference in size),

Selectivity

Buffer

Elution can also be carried out to separate them. The used solid phase carrier can be reused after being regenerated.

Introduced above

Affinity chromatography

Also

Specific ligand

Affinity chromatography. Moreover

Universal ligand

Affinity chromatography

. These two

Affinity chromatography

Compared to the former

Ligand

Generally complex macromolecular substances (such as antibodies, receptors and similar substrates of enzymes), which have strong

Adsorption selectivity

And a greater combination of strength. The latter ligands are generally simple small molecular substances (such as metals, dyes, and amino acids), which are inexpensive and have high

Adsorption capacity

Improve chromatographic resolution by improving adsorption and desorption conditions.