Chromatography is a widely used analytical technique used to separate components from a mixture. Since the 1990s, chromatography has been used in various industries for analytical testing and purification.

In the cannabis industry, it has been used to determine contaminant levels and remediate certain compounds from cannabis oil. In this guide, we will explore different types of chromatographic techniques and their applications across various industries.

What Is Chromatography?

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.

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.

Gel Filtration Chromatography/ Gel Permeation Chromatography/ Size Exclusion Chromatography

Gas filtration chromatography, also known as gel permeation chromatography or size exclusion chromatography, is a type of partition chromatography used to separate compounds of different sizes. If the mobile phase is an aqueous solution, it is known as gel filtration chromatography. If the mobile phase is an organic solvent, it is known as gel permeation chromatography.

During this process, semi-permeable, porous polymer gel beads with various pore sizes are placed in the column. The mobile phase and sample mixture are passed through the column from the top.

The compounds that bind to the stationary phase are removed with an elution solution via the isocratic or gradient technique. Lab technicians can determine the elution conditions needed to work with the desired compounds.

Gel filtration chromatography is commonly used since this technique can be performed in conditions where the compound’s stability and activity are not altered. This method has been used to purify peptides and proteins from various substances.

Gas Chromatography

Gas chromatography is one of the most common techniques used. Its mobile phase is inert gas like helium, nitrogen, or argon, while its stationary phase is a packed column. In this technique, the molecules separate based on their affinity to the stationary phase.

During the gas chromatography process, the sample is passed through the column and vaporized into a gas. The vaporized gas blends with the mobile phase and moves through the column. Then, the compounds bind to the stationary phase based on their individual affinity to the phase. Each compound reaches the machinery’s detector at different times.

Gas chromatography is commonly used to determine the concentration of various chemicals in a sample. It is also used to analyze oil spills, air pollutants, and biological samples in forensic science.

High-Performance Liquid Chromatography

Behind gas chromatography, high-performance liquid chromatography (HPLC) is one of the most popular techniques used today. This technique is a type of column chromatography. In this method, labs use a liquid mobile phase that moves the sample through the equipment using a high-pressure pump.

A high-performance liquid chromatography machine features a solvent reservoir that holds the mobile phase. The liquid sample and solvent mix together and move through the machine via a pump, bringing them to the stationary column phase, featuring various pore sizes.

As the mixture falls through the pores, some compounds will be left behind if they are too big. Lab technicians can monitor the sample and retention time of compounds. HPLC is commonly used for methods that require the separation of non-volatile mixtures. It is commonly used in chemical research, pharmaceuticals, environmental work, and biotechnology.

Thin-Layer Chromatography

Thin-layer chromatography (TLC) is a technique where the stationary phase (silica gel or alumina) is applied in a thin layer onto a plastic or glass plate. The mobile phase is liquid and can be one or several solvents mixed together.

In this technique, a technician uniformly applies the stationary phase on a solid support plate. Then, a small sample is applied to the plate using a micropipette. The plate is inserted into a sealed jar containing the solvent.

Each component in the sample will separate at different rates. Lab technicians analyze the sample by determining the distance traveled of the component.

This method is commonly used in labs to identify different liquid or solvent components in a mixture. Common industries that use this technique include food chemistry, pharmaceuticals, toxicology, and pesticide analysis in agriculture.

Paper Chromatography

Paper chromatography is one of the simplest methods of chromatography. In this technique, the mobile phase is a single or several solvents. The stationary phase is a specialized absorbent paper.

Simply put, paper chromatography starts with applying a sample to the specialized paper. The edge of the paper is then dipped into a solvent mixture of organic or inorganic solvents. The solvent is absorbed and moves up the paper until it reaches the sample, separating its components.

Generally, paper chromatography is widely used in forensic analysis, but also used in reaction monitoring, food coloring, plant pigments, and more.

Reverse-Phase Chromatography

Reverse-phase chromatography, also known as reversed-phase chromatography, is a type of liquid chromatography where compounds are separated through a hydrophobic interaction between the stationary phase and liquid mobile phase.

In reversed-phase chromatography, a column is fitted with a glass tube with a solid support such as silica gel, where hydrophobic groups can bind. The sample and mobile phase (organic and inorganic solvents) are passed through the column from the top.

The compounds with hydrophobic groups interact with the hydrophobic groups of the stationary phase. The compounds without hydrophobic groups pass through the column with the mobile phase. Then, an elution solution with decreasing salt gradient is moved through the column, removing the compounds that attach to the stationary phase.

Reverse-phase chromatography is commonly used with high-performance liquid chromatography to separate biomolecules and analyze drugs and metabolites. This method can also remove contaminants from environmental samples.

Hydrophobic Interaction Chromatography

Hydrophobic interaction chromatography separates compounds based on their hydrophobic qualities. In this process, the column features a glass tube with silica gel, where hydrophobic groups like octyl butyl and phenyl can bind to.

After the sample is mixed with the mobile phase, the sample is passed through the column from the top. The compounds with hydrophobic groups interact with the hydrophobic groups of the stationary phase.

The hydrophilic groups pass through the column with the mobile phase. Then, an elution solution with a decreasing salt gradient is moved through the column, removing the compounds from the stationary phase.

Ion Exchange Chromatography

Ion exchange chromatography is a method for charged compounds based on how they interact with an oppositely charged stationary phase (ion-exchange resin). In this process, a column is filled with positively or negatively charged resin as a stationary phase.

The mixture with the charged compounds is moved down the column to attach to the positively or negatively charged resins.

When a cation exchange resin is applied, the positively charged compounds adhere to the cation exchange resin, taking the place of negatively charged resin. When an anion exchange resin is applied, the negatively charged compounds attach to the anion exchange resin, taking the place of positively charged resin.

Then, different buffers are added to the column to separate the solution of charged exchanged resin, and charged compounds are separated. Ion exchange chromatography is commonly used to purify water and separate metals and inorganic compounds.

Partition Chromatography

Partition chromatography, also known as counter-current chromatography, has several different types of chromatography methods, including centrifugal partition chromatography (CPC), high-speed counter-current chromatography, and droplet counter-current chromatography. In partition chromatography, also known as liquid-liquid or gas-liquid chromatography, compounds are separated between two liquid phases, the original solvent and the solvent film in the column.

The stationary phase stops the liquid surface from moving, changing into a stationary phase. The compounds are separated right after the mobile phase moves from the stationary phase.

Partition chromatography can be used to detect color blends, isolate carbohydrates and amino acids, DNA/RNA sequencing, pharmaceutical analysis, and food and beverage purification.

Media Bros: Chromatography in Cannabis Processing

In the cannabis industry, chromatography methods have played a major role in the analytical testing and purification of cannabis oil derivatives. Media Bros uses the principles of chromatography and adsorbent media to remove stubborn colors from crude oil.

Whether you’re running a CO2, ethanol, or BHO extraction system, we have the right color remediation solution for your operations. Ready to see the Media Bros difference? Request a filter media sample, and one of our technicians will help find a solution for you.