Chromatography

What Is Chromatography?

Scientist with chromatography vials

Chromatography refers to a technique used to separate compounds from a mixture. During the chromatographic process, a mixture is dissolved in a gas or liquid solvent known as the mobile phase. The mobile phase moves the mixture through the chromatographic system (column, capillary tube, plate, etc.).

The chromatographic system contains the stationary phase. Different compounds in the mixture will have different affinities for the stationary phase and different retention times with the surface of this phase. Essentially, the compounds travel at different velocities in the mobile phase.

What Is a Stationary Phase?

In chromatography, a stationary phase can be solid or liquid applied to a surface, where compounds in the mixture are separated at different rates. During the process, stationary phases remain in one place, while the mobile phase moves.

Stationary phases are usually porous, allowing compounds to bind with different retention periods during the process. The type of stationary phase used depends on the kind of compounds in the sample.

Scientist in gloves with chromatography vials

What Is a Mobile Phase?

The mobile phase can be liquid or gas. In the chromatographic machinery, the mobile phase and sample mixture move through the stationary phase. The compounds of the sample are adsorbed at different rates through the stationary phase. Some of the most common types of mobile phases include acetic acid, acetone, alcohol, and water.

How Does Chromatography Work?

There are different chromatographic techniques used across various industries. Every chromatography technique needs a mixture (liquid or gas) that can pass over the surface of a stationary phase (liquid or solid). As the compounds go through the surface of the stationary phase, some of the molecules bind to the surface through adsorption.

Adsorption refers to the process where compounds temporarily are held inside the body of a surface. Adsorption rates vary by compound, allowing lab technicians to separate desired molecules through different chromatography methods.

Where Is Chromatography Used?

While chromatography techniques may not be common knowledge for many people, these techniques affect everyone’s everyday life. Chromatography is used to determine the chemical composition of substances by separating compounds into different components. Chromatography affects everything, from the production of food and beverages to the purification of pharmaceuticals.

Here are just a few ways chromatography is used:

  • Vaccine production - Chromatography can be used to find out which antibodies work against viruses and diseases.
  • Food testing - High-performance liquid chromatography with mass spectrometry is used to determine the composition of raw food samples.
  • Beverage testing - Drink manufacturers use HPLC-MS to ensure the composition of all beverages in bottles on the production line is the same.
  • Drug testing - Chromatography can help identify substances in a person’s bloodstream. It is commonly used to perform drug tests for performance-enhancing drugs on athletes.
  • Forensic testing - Chromatography is commonly used in forensic science to determine the guilt of criminals. Gas chromatography is used to analyze cloth and blood samples.

Essentially, chromatography is a common but not well-known technique that keeps people safe and healthy.

Affinity Chromatography

Affinity chromatography works by separating components in a sample based on their affinity to the stationary phase. The compounds that do not have a strong affinity to the stationary phase are removed with the mobile phase. The compounds that bind to the stationary phase are removed by altering the ionic strength, pH, and other factors.

Affinity chromatography is commonly used to separate proteins and enzymes. In addition, it is also used to remove contaminants from a mixture.

Adsorption Chromatography

In adsorption chromatography, different compounds are adsorbed to the stationary phase with different retention rates based on the adsorptive quality of the compounds.

Anion Exchange Chromatography

Anion exchange chromatography involves the binding of negatively charged molecules as they pass through the positively charged stationary phase (ion-exchange resin).

The anion exchange resin is moved through the column, and the negatively charged molecules attach to the anion exchange resin, taking the place of the positively charged resin. Then, different buffers are added to the column to separate the anion exchange resin and charged compounds.

Anion exchange chromatography is commonly used to separate amino acids from proteins, in water purification, and the separation of metals.

Cation Exchange Chromatography

In cation exchange chromatography, positively charged compounds interact with a negatively charged stationary phase (ion-exchange resin). During the process, the mixture with the charged compounds is moved through the column with negatively charged resin. The positively charged compounds adhere to the negatively charged resins.

Then, the cation exchange resin goes through the column, and the positively charged compounds bind to the cation exchange resin, taking the place of the negatively charged resin. Then, different buffers are added to the column to separate the cation exchange resins and charged compounds.

Cation exchange chromatography is used for various analytical and purification purposes, including water purification, rock and inorganic substance analysis, the separation of metals, and analysis of substances obtained from the hydrolysis of nucleic acids.

Column Chromatography

In column chromatography, compounds in a mixture are separated based on their adsorptive characteristics when passing through the stationary phase. Each compound in the mobile phase (liquid or gas) moves through the stationary phase (solid) at different rates.

In this technique, the chromatographic column is assembled by using a dried glass tube and coating it with a thin layer of the stationary phase (silica, cellulose). Then, the sample is added to the mobile phase and passed through the top of the column, allowing gravity to move it through.

The compounds that bind to the column are removed through the isocratic or gradient technique. Column chromatography is commonly used to remove contaminants from different biological samples.

Planar Chromatography

Planar chromatography refers to a chromatographic technique where the stationary phase, such as a paper or glass plate, is flat. Different molecules in the mixture pass through the stationary phase at different rates based on how they interact with it.

Supercritical Fluid Chromatography

Supercritical fluid chromatography is a user-friendly and powerful type of normal phase chromatography popular in the purification of molecules with low or moderate weights. This technique uses pressurized carbon dioxide as its mobile phase and moved through the machinery with a pump.

Flash Chromatography

Flash chromatography is a method that involves a stationary phase (gel particles) and pressurized gas to move the solvent through the column. Similar to column chromatography, the compounds are separated based on their adsorptive qualities.

In this method, the column is prepared by using a dried glass tube coated with a thin layer of stationary phase (silica, cellulose). The top and bottom of the column contain cotton wool to keep the gel from falling out.

Then, the sample is added to the mobile phase and added into the column from the top and pumped through using pressurized gas. As the mobile phase passes through, the compounds bind to the column and are removed through the isocratic or gradient technique. The elution solvent is added to move the solute down the column.