Mobile Phase vs. Stationary Phase in Chromatography

To understand chromatography, you have to understand the two basic functions of chromatography: the mobile phase and the stationary phase. There is often some confusion as to what the mobile phase and stationary phase do in the chromatography process, whether they are a certain type of chromatography themselves, and how they differ in various chromatography methods. The truth is, the stationary phase and the mobile phase are essential pieces to the puzzle of chromatography, but sometimes it can be a complicated topic if you are new to chromatography either inside or outside the extraction industry.

In this article, we will discuss the basics of the mobile phase and stationary phase in chromatography. We will break down each phase into its individual components, the various mediums and solvents used for each phase, the difference between the two, and how they are used in the chromatography process.

What is the Mobile Phase in Chromatography?

To put it simply, the mobile phase is the phase in chromatography where the solution being tested or separated is injected before it is passed through the stationary phase.

The mobile phase is often called an eluent or solvent that is pumped through the column filled with the stationary phase often at a specific flow rate. This can be used simply through gravity, or it can be assisted through a pump to vary the flow rate through the column.

In order to work properly, the solvent used as the mobile phase must be of a relatively low viscosity, low compressibility and the ability to solubilize the intended sample being tested or separated. The mobile phase must also be selected to have a favorable affinity to the material being used for the stationary phase in order to properly perform separation.

Eluents used for Mobile Phase

There are a number of common eluents used as a mobile phase in chromatography. Due to the various selectivity and affinity of the mobile phase to the stationary phase, different solvents may be more efficient than others depending on a number of factors. Some of the most common solvents are hydrocarbons like pentane, hexane or petroleum ether, and some of the co-solvent methods like ether/hexane, methanol/dichloromethane and others.

Choosing the solvent used as the mobile phase is particularly important depending on the specific molecules being separated in a solution. Just like in extraction, some solvents simply work more efficiently with particular materials as opposed to others. The polarity of a solvent and the molecules being separated are essential in order to perform separations properly. We’ll get into this topic in more detail later.

scientist using chromatography equipment

What is the stationary phase in chromatography?

The stationary phase or the media is the material being packed into a column in which the mobile phase will pass through in order to perform separation. The material used for the stationary phase are often:

  • Gel
  • Silica
  • Alumina
  • Sand

These materials allow for the mobile phase with the dissolved sample to pass through it. That material used can either be monodisperse, or polydisperse meaning that the particle size of the media is one size, or various sizes respectively. This can be useful to perform separation as the particular components in the sample will have specific affinity for certain particle sizes.

Pore Diameter, Polarity and Selectivity of Stationary Phase

The pore diameter of a particular media is also important to how well a separation can be performed during the chromatography process. The reason for this is the certain size of the particles of the intended sample need to be small enough to pass through the pores of the media used as the stationary phase. For example, cannabinoids like CBD and THC have a very small particle size and would require a small pore size in order to separate properly. Things like plant proteins, however, have a larger particle size and will not pass through the media allowing the desired cannabinoids to be separated from them.

Perhaps the most important factor, however, the selectivity of the media to the solvent being used as the mobile phase. This refers to the way the media used as the stationary phase interacts with the mobile phase on a molecular level. It’s this reason that some media will interact with the sample and mobile phase in a way that properly separates the compounds, while another sample or mobile phase eluent will not interact properly and therefore not separate.

There is also an issue of polarity in the solvent used as the mobile phase. Because some molecules are polar and others are nonpolar, the polarity of the solvent used will have an effect on the separation of the desired components. An example of this can be seen in cannabinoid extraction. Because cannabinoids are non-polar molecules, the solvent used to extract them will be more efficient if it is non-polar as well. While it is possible for polar solvents to dissolve a nonpolar substance, the efficiency of that separation is much less than that of a polar solvent. This rule translates to the chromatography process.

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How Mobile Phase and Stationary Phase Works in Chromatography

Now that we’ve reviewed what the mobile and stationary phases are in chromatography, let’s discuss how these two essential components of the chromatography process work together to perform separation. We will look at the basic methods used and how these phases work in all forms of chromatography.

Although we briefly covered some of the basics of chromatography, we’ll get further into the mobile and stationary phases now. Both the mobile and stationary phases in chromatography are absolutely essential and chromatography separation cannot be accomplished without both of them. So, here is how it works.

First, let’s break down the chromatography process in simple steps:

    • A column is prepared with a filter material, followed by a packing material, then the stationary phase is added.
    • The mixture that is intended to be separated is suspended in a small amount of solvent and added above the stationary phase.
    • More packing material is added above the mixture solvent solution followed by the rest of the solvent used as the mobile phase
    • Using simple gravity, or some kind of vacuum pump, the mobile phase is passed through the mixture, pulling it through the stationary phase.
    • As each component is separated by passing through the stationary phase, they are collected in separate vessels. The chromatography process is then essentially complete.

Now that we’ve established the basic steps of chromatography, let’s get into it in more detail. We’ve established that the mobile phase is a solvent or eluent that is fluid in that it is able to move freely through a chromatography system using a pump. As a solvent, it is also important for the mobile phase to dissolve the solution being tested or separated so that the individual components can be identified and remediated. This is why some eluents or solvents work more efficiently than others depending on the substance being separated as mentioned previously.

The material used as the stationary phase is always some sort of porous solid that remains fixed in a column that allows for the eluent used as the mobile phase to pass through it. This is where the actual chromatography process takes place. As the mobile phase passes through the stationary phase, the various components of the solution will move through the material at various rates depending on the affinity they have to that material.

So, if a particular material passes through the stationary phase more quickly, then it will be seen first on a chromatograph, or be collected first in a containment vessel. Then, the next fastest material will be seen or collected and the process continues until each individual component is identified and separated once the slowest material passes through. As they pass through the stationary phase, they can be identified in a chromatogram for testing purposes, or collected into separate vessels for separations purposes.

There are a number of factors that can cause variation in the efficiency of a chromatography separation process, but the basic principles remain the same. The mobile phase and stationary phase can be as simple as water and a coffee filter, or as complicated as a co-solvent solution and a silica gel, but the process of separation itself remains the same. By adding a solution to a mobile phase, and passing it through a stationary phase, you can effectively separate the individual components in a solution so that they can be identified, remediated and isolated altogether.

Mobile vs. Stationary Phase Mobile Phase Stationary Phase
Composition Liquid or gas Solid material
Material Solvent (pentane, hexane or petroleum ether, etc.) Porous Solid (silica, alumina, gel)
Purpose Solvate and carry mixture through stationary phase Separate mixture into various components as they pass through it
Post processing Solvent removal needed Remains in column
lab testing vials for chromatography

Final Thoughts

So, hopefully the issue of mobile phase and the stationary phase of chromatography is clearer after reading this article. To reiterate, the thought that either mobile phase or stationary phase are their own form of chromatography is just a simple misunderstanding. The mobile phase and the stationary phase are separate but equally essential parts of chromatography that allow the separation process to happen. So, no mobile and stationary phases means no chromatography.

If you have yet to consider an efficient chromatography system in your process, now is the time. Testing and separations have become essential pieces of the hemp and cannabis industry as purity and potency demands have increased. While there are other methods that can be useful for separations and testing, none of them come close to the efficiency of chromatography.

extraktLAB prides itself in being a resource not only for equipment, but also valuable information that can help you succeed in this industry. Our pure99 HPLC system is one of the most efficient chromatography systems on the market with a highly efficient throughput, closed loop solvent recycling system, and a myriad of process control systems that make it the most streamlined HPLC system in the industry. If you are looking to add the pure99 system to your workflow, contact us to discuss the best options for you today and take your separation process to a new level.

 

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