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Column Chromatography: The Definitive Guide

Column Chromatography

The Ultimate Guide to Column Chromatography

– The Definitive Guide

Column chromatography is a means of using pressure in a column (e.g. glass) to effectively separate the different components of a mixture.  When you are trying to isolate a certain desirable compound, this method of chromatography is essential.

Chromatography, in general, is a term that refers to a group of laboratory techniques that are used in numerous industries to separate components from one another in a mixture.

Some of the products we carry are for column chromatography, and others are for flash chromatography.

How does column chromatography work?


Chromatography Method
Since each component of a mixture has its own particular size, shape, polarity, and solubility, it is a matter of using those distinguishing characteristics to isolate and purify each individual component present within a mixture.

This can be accomplished in numerous ways, though the principle is always the same: Utilizing the affinity of certain molecules to others to slow their movement through a medium while other molecules in the mixture can pass easily through, allows for clear separation and purification of the components of a mixture, otherwise known as analytes.

In layman’s terms, certain molecules will pass through the column before others, separating some of the components contained within your glassware, for example.

Solvents are a critical part of this type of chromatography.

A mixture is initially combined with a suitable solvent or pair of solvents, which can either be a liquid or a gas – this is known as the mobile phase.

This mobile phase is then passed through or over the stationary phase, which may be either a liquid or a solid.

The equilibrium between the components of the mixture, the mobile phase, and the stationary phase determines the degree of separation.

Resolution is the measure of how well two components separate during chromatography.

Methods of performing chromatography:


Chromatography Techniques
Some of the more common laboratory methods in use today are:

  • gas chromatography,
  • high-pressure liquid chromatography,
  • column chromatography,
  • and thin-layer chromatography.

Gas chromatography.


Gas Chromatography
Gas chromatography is a method that utilizes a gaseous mobile phase and a solid stationary phase.

The sample is vaporized and passes through the coiled column that separates the components.

These molecules can be detected using various spectroscopic equipment, which is very useful in identifying and quantifying components in a sample.

High-pressure liquid chromatography.


Gas Liquid Chromatography
High-pressure (performance) liquid chromatography uses a liquid solvent that is pumped through a solid stationary phase with high pressure, causing separation.

Many different kinds of detectors may be used to quantify and identify the components of a sample.

Programmable parameters allow for precise variation in fractionation capabilities on a GC or HPLC system, and automation allows for high throughput.

Column chromatography in its basic form.


Basic column chromatography uses a liquid mobile phase that is passed over a liquid or solid stationary phase, using gravity to pass the mobile phase through the column.

This can be time-consuming, so a small amount of pressure can be applied to the top of the column, which forces the mobile phase to flow at a greater rate.

The technique of using positive pressure is also known as flash chromatography, like the machine we sell on this website.

Flash chromatography results in shorter run times and allows samples to be eluted quicker.

Thin-layer chromatography.


Gas Chromatography
Thin Layer Chromatography (or TLC) uses thin layers of silica on a backing plate, which is usually glass, instead of a column.

The sample is loaded, or “spotted,” on the plate, which is placed vertically in a TLC reservoir that contains the mobile phase.

The level of the mobile phase in the container is lower than the level of the spotted sample of the plate.

Capillary action causes the mobile phase to move up the TLC plate and separate (or not separate) the spotted sample.

If the sample and its separated components are not visible to the naked eye, a dying solution may be sprayed on the plate that causes the components to be visible with the naked eye and/or with the application of UV light.

A TLC test may be performed before running a sample on the HPLC to determine the correct solvent system to use.

TLC is used to determine whether the resolution (separation of the components) is going to be sufficient with the current system, or if the mobile and/or stationary phase need to be changed.

TLC is quick and allows for rapid experimentation and optimization of the system before using HPLC. Optimization includes using the least amount of solvent to achieve the desired resolution of components.

How do you choose a mobile phase?

The mobile phases used in any type of chromatography are generally chosen based on their polarities, inertness, ability to be recycled, and solubility of the sample.

Common mobile phases used in gas chromatography are:

  • hydrogen,
  • nitrogen,
  • or helium.

In liquid chromatography, the mobile phase is generally a mix of polar and non-polar, or less polar, solvents.

Common mobile phases are a mix of acetonitrile and water or water and methanol.

The water may be acidified or basified to optimize the method. Which solvents to use and when to use them are largely dependant upon the media inside the column, components in the mixture, and the relative solubilities of each in a given solvent.

Types of stationary phases:

There are many types and varieties of media that may be used as a stationary phase, and each type has its own chemical characteristics. The target molecule to be separated is matched with the appropriate media.

The most common types of media that are used as the stationary are silica-based, such as c8, c18, diatomaceous earth, celite 545 and functionalized silicates.

Note: These can be harmful to breathe in, so handling of any silica – especially powdered silica – should be done in the confines of a vent or fume hood. Many columns are obtained pre-packed, others have to be packed manually.

How do you pack a chromatography column?


Column Chromatography Packing
Proper packing of a chromatography column is done in a way that creates an even and level stationary phase, which ensures proper flow without streaking. Media may be wet or dry packed.

Dry packing.

Dry packing is when dry, powdered media is loaded into a column.

Solvent is then passed through the column until the stationary phase is evenly distributed through the length of the column.

Wet packing.

Wet packing is when the media is dissolved in a solvent and poured into the column as a slurry.

Solvent is passed through the column and the slurry settles, making a packed bed. Eliminating air bubbles within the media is more easily accomplished with wet loading.

Gentle tapping on the side of the column can also force the bubbles to rise to the top of the column and help settle the media. A small amount of vacuum can also be applied to the bottom of the column to further remove air bubbles that are trapped inside the silica matrix.

In some cases, axial compression is used to pack columns. Air or dry media in the column will cause an imperfect flow of the mobile phase, compromising separation.

After the stationary phase is packed, solvent flows through the column until the solvent is even with the media bed for loading of the sample.

Note: Care must be taken to never let the media run dry, even during loading, or the desired separation will not be achieved.

Loading for column chromatography.

Loading a chromatography column with the sample can be done two main ways:

  • Wet loading
  • Dry loading

Wet loading.

Wet loading involves dissolving the mixture into a minimum volume of solvent and adding it to the top of the column gently, as not to disturb the stationary phase.

Dry loading.

Dry loading involves dissolving the sample in a solvent, mixing with stationary phase media, then removing the solvent. This dry stationary phase media then contains the loaded sample, which is added to the top of the column.

The mobile phase then flows through the column and the components of the mixture spread through the column, then elute from the bottom.

Fractions are collected in different containers and then analyzed to see which contain the desired (or undesired) components. The fractions are kept separate based on their composition, then the desired fractions may be combined.

The solvent is then recovered, resulting in a purified sample. The undesired fractions can then be combined and their solvent recovered, as well. This recycling of solvents helps minimize the cost of operation.

Whether manual or automated, gas or liquid, chromatography is an easy way to separate desired, or undesired, components from a mixture and is a great addition to any lab.