About the Structure and Working Principle of HPLC Columns

Structure of HPLC column

High-performance liquid chromatography (HPLC) column consists of a column tube, pressure cap, ferrule (sealing ring), sieve plate (filter), joint, screw (head), and column packing. The column tube is made of stainless steel and the thick-walled glass or quartz tube can also be used if the column pressure is no more than 70 kg/cm2. The inner wall of the tube requires a high degree of finish. It has a high degree of smoothness for the tube’s inner wall. It’s used for the packing of column packing. Made of stainless steel, the empty column is able to withstand the action of the solvent.

The column joints at both ends are equipped with sieve plates, which are sintered stainless steel or titanium alloy with a pore size of 0.2~20µm (5~10 µm) which depends on the particle size of the filler.

However, since chloride-containing solvents are corrosive to a certain degree, care should be taken when using them, and do not store them in the column for a long time to avoid corrosion. Sealing ring: The elastic ring located on the inner wall of the spiral ring of the connector, mostly made of PTFE, is used to seal the pressure cap at both ends and the outer wall of the column.

HPLC Column packing

The separation of liquid chromatography columns is carried out between the packing and the mobile phase, and the classification of the column is determined by the type of packing.

Normal phase HPLC column:
They mostly use silica gel as column packing. It can be divided into amorphous and spherical two kinds, with particle size in the range of 3-10μm, according to appearance. Another type of normal phase filler is the so-called bonded phase silica gel with functional groups such as -CN and -NH2 bonded on the surface of the silica gel.

Reversed phase HPLC column:
It is mainly a non-polar filler with silica gel as the matrix and octadecyl functional group (ODS) bonded on the surface. There are also amorphous and spherical points. Other commonly used reversed-phase fillers include bonding C8, C4, C2, phenyl, etc., and the particle size is between 3-10μm.

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Working principle of HPLC Column

HPLC column uses a high-pressure infusion pump to pump the mobile phase, such as a single solvent with different polarities, a mixed solvent of different proportions, or a buffer solution into a column packed with a stationary phase.

Then inject the sample to be tested through the injection valve, which is taken into the column by the mobile phase. The components in the column are separated into the detector, and the chromatographic signal is recorded by a recorder, an integrator, or a chromatographic workstation.

About the HPLC Column

High-performance liquid chromatography column(HPLC) is an important core component of high-performance liquid chromatography. The chromatographic column is connected and analyzed by the column material, the specifications of the structural column, etc. It is suitable for biological acids/alkalis, amines, antibiotics (polyether antibiotics are more common), and amino acids.

At present, it can give full play to its advantages in almost all analyses of macromolecular organics that need to be separated. The HPLC chromatographic column tube often uses a stainless steel tube whose inner wall is precisely processed and polished to obtain high column efficiency. The stainless steel tube is resistant to corrosion by solvents, water, and buffer solutions.

Before use, the chromatographic column tube is washed with chloroform, methanol, and water in sequence, and then the inner wall of the HPLC column is passivated with 50% hno3. During passivation, let hno3 stay in the chromatographic column tube for at least 10 minutes to form a passivated oxide coating on the inner wall of the column.