What Is the HPLC Column?

The high-performance liquid chromatography(HPLC) column is a chromatography consumable with the function of analysis and separation of mixture compounds. The working principle is to pump the analyte or say sample mixture into a solvent(mobile phase such as water) under high pressure, as the column with stationary phase inside with chromatography packing sorbents, the column can do separation and identify the compounds that exist in any sample that could be dissolved into a kind of liquid in trace concentrations with the value as low as parts of per trillion.

Due to its versatility, the HPLC column can be used in a variety of scientific and industrial applications, such as pharmaceutical analysis, environmental analysis, forensic analysis, and chemical analysis.

The sample retention(time) always depends on the interaction between the stationary phase(Column inner packing material), the molecules being analyzed, and the solvent, or solvents used(liquid phase).

As the analytical sample passes through the column, it will interact with the two phases(stationary phase and mobile phase) at a different rate, primarily due to the analytes’ different polarities. This means that the stationary phase with high polarity will interact with compounds with high polarity, so the molecules with lower polarity will have low retention, exit the column earlier, and appear to peak faster.

Both prepacked HPLC and empty HPLC Column, separately bulk sorbent available for your choice. (Empty Column picture as below for review)

Structure of HPLC Column

The empty column is assembled from column joints, column tubes, and filters.
The HPLC column joints are constructed with a low dead volume. The column joints are threaded assemblies at both ends, with 7/16-inch external threads on one end and 3/16-inch internal threads on the other end (standardized at home and abroad). The 7/16-inch external thread is connected to the 1/4-inch column tube (Φ6.35mm) and placed in the middle for sealing. The 3/16″ internal thread is connected to the 1/16″ (Φ 1.57mm) connecting tube, and a pressure ring is placed in the middle for the seal of the column joint. To minimize the dead volume outside the column, when installing the column, use a Φ1.57mm connecting tube to insert the hollow screw into the ring, and then tighten the hollow screw. The pressure ring is pressed and deformed by the hollow screw and then hung on the connecting pipe (the length of the pipe exposed after the connecting pipe passes through the pressure ring should be strictly controlled to 2.5 mm long or other fixed sizes).

In the column joints at both ends, a stainless steel filter (or strainer) is placed at each end of the HPLC column tube to block the column packing from being washed out of the column by the mobile phase. Each component of the empty column is made of 316# stainless steel, which can withstand the general solvent effect. However, since the chloride-containing solvent is corrosive to it, it should be noted that the solvent and the connecting tube cannot be stored for a long time to avoid corrosion.

Type of HPLC columns

Commonly used column packing agents are silica gel and chemically bonded silica gel, ion exchange resin, gel, or glass microspheres. Among the chemically bonded silica gels, cetyl silane-bonded silica gel is most commonly used, followed by octyl silane-bonded silica gel, and cyano or amino-bonded silica gel is also used.

In recent years, due to the rapid development of separation and purification technologies for biological macromolecules such as proteins, the application of ion exchange chromatography columns and gel chromatography columns has become more and more widespread.

Factors affecting column life

Mobile phase

When an aqueous solution is used as the mobile phase, microorganisms such as bacteria in the aqueous solution are easily grown. When the column is protected with an aqueous solution or an organic acid buffer, some mold may grow in the column and block the gap between the particles of the stationary phase.

Due to the advent of wet filling technology, the commonly used column packings are generally less than 10um in diameter, and the particulate impurities in the mobile phase are easily deposited on the column head and then slowly block the column.

The pH of the mobile phase also affects the column, especially for chemically bonded silica gel. The pH of the aqueous solution is between 2 and 7.5. When the pH is 8, the silica gel will release floccules and block the column. Recovery, the efficiency of the column is quickly reduced or even completely failed. When an organic solvent such as methanol or acetonitrile is added to the buffer, the solubility of the salt is lowered, salt precipitation is precipitated, and the column is blocked. At the same time, the organic solvent and salt in the mobile phase corrode the sieve plate of the column head, resulting in a stigma depression.

The polarity of the mobile phase also has an effect on the column. For silica gel columns, more polar substances such as methanol, water, and glacial acetic acid will destroy the filler. Conversely, for chemically bonded silica, the less polar substances such as n-butanol and dichloro Methane and the like also play the same role. The alkaline solution destroys the cation exchange resin column, and the acidic solution easily damages the anion exchange resin column.

Sample and stationary phase

If a small or small amount of the sample is not able to dissolve in the mobile phase, the column will be blocked by the stationary phase. Particulate impurities in the sample can also block the column. When sample components such as sugars, proteins, etc. pass through the column, irreversible adsorption occurs, so that the active sites of the stationary phase are covered.

The sample components also react with the stationary phase, especially for the amino column, because the amino group easily forms a Schiff condensation with the anhydride and the ketone to reduce the activity point and shorten the retention time. Column bonded phase group shedding, stationary phase decomposition, and reactive group denaturation as well as significant changes in column efficiency and retention time.

Most of the bonded phases are bonded to the silicon substrate via a silicon-silicon bond (Si-O-Si) to form a stationary phase with organic groups, Si-O-Si-R (R is CnH2n+1), silicon. The siloxane bond may be hydrolyzed in an aqueous solution to break, causing the bonding group to fall off.

Maybe, Hawach column C18 Alkaline HPLC Columns, Phenyl-Ether HPLC Columns, C8/C18 Standard HPLC Columns, and other HPLC-Columns you’d be interested in.