Principle and Adsorbent of HPLC Chromatography Column

HPLC column chromatography, also known as chromatography. It is a distribution method with a distribution balance as the mechanism. The chromatography system contains two phases, one is the stationary phase and the other is the mobile phase. When the two phases are moving relative to each other, the difference in the distribution and balance properties of the components contained in the mixture is used repeatedly to achieve the purpose of separation from each other.

Chromatography has a history of more than 80 years since its invention. It is a common method for the purification and separation of organic or inorganic substances. Among them, the chromatogram with fixed phase polarity greater than the mobile phase is normal phase chromatography, and the opposite is reverse phase chromatography. According to the principle of similar miscibility: the substances with high solubility in the stationary phase will come out of the column and have a long retention time, which is difficult to elute.

Principle of HPLC Chromatography Column

An HPLC chromatography column separates compounds based on their interaction with a stationary phase and a mobile phase. Analytes partition between the two phases, leading to differential retention times. Factors like column chemistry, particle size, and dimensions influence the separation process. Column efficiency and selectivity are key considerations. Maintenance, pressure, and temperature control are crucial for optimal performance. Specific column types cater to different separation needs, such as reversed-phase or ion exchange chromatography. Understanding these principles is essential for effective chromatographic separations.

  1. Separation Mechanism:
    • HPLC separation is based on the differential interaction of sample components with a stationary phase and a mobile phase. The stationary phase is typically packed inside the chromatography column, and the mobile phase (a liquid or a combination of liquids) moves through the column, carrying the sample components with it.
  2. Adsorption and Partitioning:
    • The separation occurs through a combination of adsorption and partitioning. Adsorption involves the interaction of sample molecules with the surface of the stationary phase, while partitioning involves the distribution of molecules between the stationary phase and the mobile phase.
  3. Retention Time:
    • The time it takes for a particular component to travel through the column and reach the detector is called the retention time. Components with stronger interactions with the stationary phase have longer retention times.


HPLC Chromatography can be divided into column chromatography according to the fixed state. Plate chromatography and rod chromatography are the most commonly used in the laboratory are column chromatography and thin-layer chromatography, and the cooperation between them.

Adsorption column chromatography

The principle of adsorption chromatography: Under certain conditions, the role of silica gel and the substance to be separated, this role is mainly two kinds of physical and chemical effects. The physical effect comes from the van der Waals force between the surface of the silica gel surface and the solute molecules.
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Mainly due to the hydrogen bonding between the silicon hydroxyl group on the surface of the silica gel and the substance to be separated. The chromatographic tube is a hard glass tube with a uniform inner diameter and a narrower lower end. The lower end is plugged with cotton or glass fiber, and the tube is filled with an adsorbent. The particles of the adsorbent should be kept as uniform as possible to ensure a good separation effect.

Unless otherwise specified, particles with a diameter of 0.07 to 0.15 mm are usually used. The size of the chromatographic column, the type and amount of adsorbent, and the flow rate at the time of elution are all in accordance with the regulations under each item.

⑴ Filling of adsorbent
① Dry the adsorbent into the chromatography column at one time, vibrate the tube wall to sink it evenly, then slowly add eluent along the tube wall, or connect a piston to the outlet of the lower end of the column, add an appropriate amount of eluent, and unscrew The piston makes the eluent slowly drop out, then slowly add the adsorbent from the top of the tube to make it evenly wet and sink, forming a moderately tight adsorption layer in the tube. During the operation, sufficient eluent should be kept on the adsorption layer.
②Wet method Mix the adsorbent with the eluent, stir to remove air bubbles, slowly pour into the HPLC chromatography column, and then add the eluent to wash off the adsorbent attached to the wall of the tube to make the column surface flat. When the eluent used to fill the adsorbent naturally flows down from the chromatography column and the liquid level is equal to the column surface, the test solution is added.

⑵ Join the test product
Unless otherwise specified, dissolve the test product in the eluent used at the beginning of the elution, and slowly add it along the wall of the chromatography tube, taking care not to turn up the adsorbent. Or dissolve the test product in a suitable solvent, mix it with a small amount of adsorbent, and then volatilize the solvent to make it lose, and add it to the prepared chromatography column. If the test product is insoluble in common solvents, the test product and the appropriate amount of adsorbent can be ground and mixed in a mortar and added.

⑶ Elution
Unless otherwise specified, the type and ratio of the eluent are usually changed in increments of the eluent’s elution capacity, and the effluent is collected separately, until the content of the effluent is significantly reduced or no longer contained, then change the washing Varieties and proportions of agents. During the operation, sufficient eluent should be kept on the adsorption layer.

Partition column chromatography

The method is basically the same as that of adsorption column chromatography. Before packing the column, first, mix the carrier and the fixing solution, then move it into the chromatographic column and compact it with a flat glass rod; the test product can be dissolved in the fixing solution, mixed with a small amount of carrier, and added to the pre-made chromatographic column Upper end. The eluent should be mixed with a fixative solution to saturate it to avoid the change of the two-phase distribution during the elution.