Protein Technology Topics: High Performance Liquid Chromatography High Performance Liquid Chromatography

High Rerformance Liquid Chromatography (HPLC) is also called high pressure, high speed, modern liquid chromatography, commonly called high performance liquid chromatography. It was a method for efficient and rapid separation of compounds that was established in the mid-1960s. It was widely used in the separation and purification of proteins in the late 1970s and has become one of the most effective methods for separating and purifying proteins.

Compared with classical atmospheric chromatography, it has the following characteristics:
1. The speed of HPLC separation and purification of proteins is fast. Usually it can be carried out once in about half an hour, which greatly shortens the time for purifying the protein;
2. The resolution is high. A 10cm long column can separate more than ten kinds of substances;
3, adapt to a wide range, flexibility, almost all proteins according to their differences in properties (isoelectric point, hydrophobicity, molecular weight, charge distribution, etc.) can be separated and purified by different HPLC methods;
4. High sensitivity. A wide range of highly sensitive detectors such as UV, fluorescence, and conductivity have been widely used in HPLC. The sensitivity of fluorescence can reach 10-9g;
5, the repeatability is good, in the analysis of protein analysis test, the error is generally not more than 5%.
Therefore, HPLC has an extremely important position in protein separation and purification.

Of course, HPLC also has its disadvantages. For example, the equipment is expensive. Although it has been used in preparative columns, the amount of preparation is limited. In some methods, the activity of the protein may be affected by the use of an organic solvent.

High-performance liquid chromatography is a special instrument for high-performance liquid chromatography. There are many types. From the type, it is divided into a comprehensive high-performance liquid chromatograph and a special-purpose instrument. It is functionally divided into analytical, preparative, analytical and preparative. type.

The general high performance liquid chromatograph has the following main components:

1. An infusion pump that delivers liquid to the column;
2. Sampler;
3. The column is separated from the sample;
4. The detector detects the separated sample;
5. Recorder (data processing and printing part), processing and recording the detected signal;
6, the program controller, program control of each part, such as input chromatographic conditions, parameters and so on. In addition, a liquid storage device, a degassing device, a branch collector, and the like are often provided.

High performance liquid chromatography is usually divided into the following modes depending on the separation mode:
1. High-efficiency exclusion liquid chromatography, separated according to the molecular weight of protein and peptide;
2. High-efficiency ion-exchange liquid chromatography, which is separated according to different charging of proteins and peptides;
3. High-performance reversed-phase liquid chromatography, which is separated according to the hydrophobicity of proteins and peptides;
4, high efficiency hydrophobic liquid chromatography, the principle is the same as reversed phase chromatography. The difference is that the surface hydrophobicity of the hydrophobic chromatography is not as strong as that of the reversed phase medium;
5. High-efficiency affinity liquid chromatography is a biological macromolecular separation method that utilizes the specific separation of biological macromolecules and stationary phase surfaces for selective separation.

High performance exclusion liquid chromatography

Also known as Size exclusion chromatography (SEC), molecular sieve chromatography (molecular sieve chromatography), gel filtration (gel filtration), etc., biochemical industry commonly used gel filtration. SEC is a chromatographic method that separates the volume of a solute molecule in a mobile phase solvent. The filler has a range of pore sizes. The macromolecule does not enter and exits the column, and the small molecule flows out. The traditional SEC filler used for the separation of biological macromolecules is mainly a polysaccharide polymer soft gel, which can only be used for slow separation at low pressure. Hydrogels (such as cross-linked agarose Superose 6 and 12), ethylene copolymers (such as TSK-Gel PW) and hydrophilic bonded silica (such as Zorbax GF 250 and Replaced by 450). Depending on the pore size of the filler used, the SEC can separate molecular weight fractions ranging from 10,000 to 2 million. For analytical separation or laboratory small-scale preparation, the average particle size is suitable for specifications of 3-13 microns, with good column efficiency and separation capacity. However, for large-scale preparation separation and purification, a coarser particle size can be used because cost and permeability are considered. HPSEC has a low loading, analytical HPSEC loading between 10-500 μg and a volumetric load of 1-2% of the total column volume.

In addition to protein separation, HPSEC is also used in molecular weight determination and purity identification of unknowns. It is faster than SDS-PAGE electrophoresis, and for proteins consisting of subunits, the measured value is the natural molecular weight. It can also be used for quantitative analysis with an error of less than 5%.

(1) Experimental purpose <br>Detecting nrhTNF

(2) Instruments and reagents

1, high performance liquid chromatography, Beckman Gold System

2, column: TSK-GEL G2000SW, Japan Soda (TOSOH) 7.5 × 300mm

3, 0.1mol/L PB-0.1mol/L NaCl pH6.8

(three) instrument parameters

Mobile phase 0.1mol/L PB-0.1mol/L NaCl pH6.8

Detection wavelength 280nm

Injection volume 20μl

Flow rate 0.5ml/min

(4) Sample preparation
nrhTNF purified by Q-Sepharose FF and SP-Sepharose FF

(5) Experimental operation (teaching)

1, buffer degassing: ultrasonic degassing; microwave degassing; suction filtration degassing

2, boot

3, input parameters

4, balanced column

5, injection

6, elution

7, stop the experiment, print and analyze the results

8, wash the column and save

(6) Analysis of results

1, retention time

2, peak type and purity (normalization method)

3, calculate the content

Attached: TSK-GELSW series gel filtration column data.

Size Exclusion Chromatography (SEC) is a branch of liquid chromatography. The separation process is based on the molecular volume (or molecular weight) of the analyte to be eluted in sequence to achieve a complex mixture. Separation purpose. Size exclusion chromatography is one of the important means of analyzing macromolecular compounds because the distribution of molecular weight and molecular weight is the most basic and important parameter of the compound. According to the nature of the sample to be separated, size exclusion chromatography is further divided into gel filtration chromatography (GFC, Gel Filtration Chromatography) suitable for separating water-soluble samples, and gel permeation chromatography (GPC, Gel Permeation) suitable for separating oil-soluble samples. Chromatography).

The packing of the TSK-FEL SW series column is based on a rigid spherical silica gel, and a hydrophilic compound is bonded to the surface by chemical covalent bonding. The packing of TSK-GEL SW series columns has the properties necessary for high performance size exclusion chromatography, ie low adsorption and good pore size distribution; its pH range is 2.5-7.5, and organic solvents that are completely miscible with water can be used. Such as acetonitrile, acetone, methanol or ethanol. The TSK-GEL SW series columns are suitable for the separation of protein and peptide samples, and many other applications have been reported in the literature. The most commonly used is a 10μm TSKgel 4000SW column; the TSK-Gel2000SWXL and 3000SWXL are 5μm, the TSK-Gel 4000SWXL is an 8μm high-efficiency packed column; the analytical column can be constructed of glass or stainless steel.