What is chromatography column?
Chromatographic column can be divided into packed column and open-tube column. Most
chromatographic columns are made of metal or glass. There are straight tube shapes, coiled tube shapes, U-shaped tubes, and other shapes. Liquid chromatography usually uses packed columns. The separation effect of chromatographic columns depends on the selected stationary phase, as well as the preparation and operating conditions of the columns.
Chromatography is a means of separation and analysis, and separation is the core, so the column, which is responsible for the separation, is the heart of the chromatographic system. The requirements for the
chromatographic column are high column efficiency, good selectivity, fast analysis speed, etc. Commercially available for HPLC is a variety of particulate packings, such as porous silica and silica-based bonded phase, alumina, organic polymer microspheres (including ion exchange resins), porous carbon, etc., while its particle size is generally 3,5,7,10μm, etc., the theoretical value of the column efficiency can reach 5 ~ 160,000 / m. The general analysis only needs 5000 column efficiency of the number of plates; homolog analysis can be as long as 500; more difficulty to separate the material pairs can be used up to 20,000 columns. Therefore, a column length of about 10~30cm is generally sufficient for the analysis of complex mixtures.
Column efficiency is affected by factors inside and outside the column, and to achieve the best efficiency of the column, in addition to a small dead volume outside the column, a reasonable column structure (minimizing the dead volume outside the packed bed) and loading technology are required. Even with the best loading technique, the filling in the center part of the column and along the wall part is always different. The part near the wall is looser, easier to produce trench flow, and faster flow rate, which affects the flow shape of the rinse agent and widens the spectral band, which is the wall effect. This tube wall area is about 30 times the thickness of the particle size counted from the tube wall inward. In a general liquid chromatography system, the effect of the off-column effect on column efficiency is much greater than the effect of the tube wall.
Even with the best loading technique, the filling in the center part of the column and along the wall part is always different. The part near the wall is looser, easier to produce trench flow, and faster flow rate, which affects the flow shape of the rinse agent and widens the spectral band, which is the wall effect. This tube wall area is about 30 times the thickness of the particle size counted from the tube wall inward. In a general liquid chromatography system, the effect of the off-column effect on column efficiency is much greater than the effect of the tube wall.
Structure of chromatography column
Chromatographic column consists of a column tube, pressure cap, ferrule (sealing ring), sieve plate (filter plate), joint, screws, etc. The column tube is mostly made of stainless steel, and when the pressure is not higher than 70 kg/cm2, thick-walled glass or quartz tubes can also be used, and the inner wall of the tube requires a high degree of finish. In order to improve the column effect, and reduce the wall effect, stainless steel column wall polishing. There are also people in the stainless-steel column wall coated with fluorine plastic to improve the inner wall finish, the effect is the same as polishing. There is also the use of fused silica or glass lining, for fine tube columns. The column joints at both ends of the chromatographic column are equipped with a sieve plate inside, which is sintered stainless steel or titanium alloy with an aperture of 0.2~20μm (5~10&μm), depending on the particle size of the filler, in order to prevent the filler from leaking out.
Packings for chromatographic column
Common column packing: carbon 18 column [1] (ODS/C18), carbon 8 column (MOS/C8), carbon 6 column (Hexyl/C6), carbon 4 column (Butyl/C4), carbon 1 column (Methyl/C1), anion exchange column (SAX), cation exchange column (SCX), phenyl column (Phenyl), amino column (Amino/NH2), cyanide-based columns (Cyano/CN/Nitrile).
Common adsorption column packing: silica gel column.
Type of chromatography column
Chromatographic column can be divided into analytical and preparative, according to their uses and different size specifications as follows.
a. Conventional analytical column (constant column), inner diameter 2~5mm (commonly used 4.6mm, domestic 4mm and 5mm), column length 10~30cm.
b. Narrow diameter column (narrow bore, also known as fine tube diameter column, semi-microcolumn), inner diameter 1 ~ 2mm, column length 10 ~ 20cm.
c. Capillary column (also called microcolumn), inner diameter 0.2~0.5 mm.
d. Semi-preparative columns, inner diameter >5mm.
e. Laboratory preparation columns with inner diameter of 20~40mm and column length of 10~30cm.
f. The internal diameter of production preparation columns can reach several tens of centimeters. The column inner diameter is generally determined based on the column length, packing particle size and folded flow rate, with the aim of avoiding the tube wall effect.
Use of chromatography column
Ion exchange chromatography
A. Installation
a. First, you should confirm that the connectors and piping of the column and instrument are matched. To reduce the dead volume, use a line with the smallest possible inner diameter for the connection between the injection valve, column, and detector, and control the length of the connection line between the injector, column, and detector. Before installing the column, confirm that the solvent in the flow path system is in order. For the analysis of more complex samples, it is recommended to install a protective column.
b. To make the best connection between the column and the instrument system, you should try to use the screw cap and tapered connector that matches the column interface. If the original connector has been matched with other types of columns for a long time, it is recommended that the matching should be checked before connecting the new column to avoid causing damage to the column or leakage caused by the column mismatch.
c. Use universal fittings, which only need to be tightened by hand without a specific wrench, using a pressure of 5000psi; the use of temperature must not exceed 100 ℃.
B. Mobile phase
a. Use a mobile phase of at least 20 times the column volume to fully equilibrate the column before performing sample detection. Always use a chromatographic grade solvent for the mobile phase. If you use the aqueous phase buffer should be prepared daily to keep fresh to avoid bacterial production.
b. The mobile phase should be filtered through a microporous membrane before use to eliminate damage to the chromatographic system and column from particles in the mobile phase. The buffer should be re-filtered after mixing with other mobile phases to avoid new precipitation caused by changes in solubility. Pure water should not be used as the mobile phase to rinse the C18 column to avoid damage to the column performance (adding 5% organic solvent to rinse the column can achieve the effect of cleaning the buffer salt at the same time. (It can also make the column easier to balance).
c. The mobile phase needs to be degassed and used to avoid the improper operation of the pump and detector due to air bubbles. If the mobile phase used in the test is significantly different from the mobile phase used in the preservation of the column, it should be equilibrated using the form of overdistribution. Avoid damage to the column and instrument system due to sudden changes in the mobile phase that cause excessive increases in column pressure or crystallization of mobile phase buffer salts. Normal phase columns require a longer equilibration time than reversed phase columns.
C. Sample preparation
a. The sample should be dissolved in a solvent that is mutually soluble with the mobile phase whenever possible. Unless otherwise specified, the resolution of the column will likely be reduced if a strong solvent is used to dissolve the sample.
b. The sample solution should be pre-filtered using a needle filter before feeding the sample. Frequent changes in mobile phase composition will accelerate the reduction of column efficiency.
c. The chromatographic column is loaded by a high-pressure homogenization method and can withstand high pressure. In order to obtain the best separation effect, please do not exceed 200kgf when using, and avoid sudden pressure rise or change, otherwise, it will cause the destruction of silica packing and reduce the service life of the column. The maximum operating temperature should not exceed 60°C unless otherwise required.
D. Save operation
a. If the mobile phase contains acids or inorganic salts, it should first be rinsed off with deionized water (20 times the column volume). Then the column is preserved with 100% acetonitrile or methanol. Finally, seal the column with its joints and store it in a stable environment.
b. The column should be avoided direct mechanical impact or dropping to avoid the degradation of column performance.
E. Regeneration of chromatographic columns
The columns were rinsed with a solvent equivalent to 20 times the volume of the column in the following order, and it is recommended to rinse in the direction of the arrow of the column, without backflushing if possible. The reference sequence of solvents used in the rinse is water, methanol, chloroform, and isopropanol.
How to buy chromatography column?
ANTITECK provide lab equipment, lab consumable, manufacturing equipment in life sciences sector. If you are interested in our chromatography column or have any questions, please write an e-mail to info@antiteck.com, we will reply to you as soon as possible.
