HPLC, or high-performance liquid chromatography, has been around for more than 50 years. In that time, science has drastically improved the reliability and accessibility of this method.

But how does HPLC work? How do you know which column to use? Do you know the difference between what is C18 and C8?

Don’t worry, we’re here to help. Here’s our guide on the basics of HPLC columns.

How Does HPLC Work?

HPLC is used to separate, quantify, or identify components in a mixture. The components are separated using column chromatography and then analyzed by a computer.

All forms of column chromatography work similarly. You pass your mobile phase (the liquid containing your mixture) through a stationary phase (a solid). The components will travel through the solid phase at different rates, which allows them to be separated over time.

In HPLC, high pressure is applied so that this separation can occur much more quickly than traditional means. Additionally, using smaller particles in the column’s packing material permits more precise separation.

HPLC has three steps:

1. A small volume of your sample (in the liquid phase) is injected into your stationary phase. Your stationary phase is your HPLC column, which is a tube filled with particles <5µm in diameter.

2. A pump moves the liquid down the column using high pressure. As the sample moves through the column, there are interactions between the packing particles and molecules in your sample. This will cause these molecules to travel through the column at different rates.

3. As the individual components exit the column, a detector measures them. This output is sent to a computer to produce a liquid chromatogram.

In HPLC, the column you use is often considered the most important component. The physical and chemical characteristics of the column determine the degree of separation. But how do you know what column is right for your experiment?

Determining Your HPLC Column

First, you’ll need to establish if you’re performing normal-phase or reversed-phase HPLC. In normal-phase HPLC, the stationary phase is hydrophilic while the mobile phase is hydrophobic. But reversed-phase HPLC is far more common.

In reversed-phase HPLC, the mobile phase is hydrophilic while the stationary phase is hydrophobic. That means that molecules are eluted by decreasing polarity through the use of an organic solvent.

Now you’re ready to pick out your column. But what features are the most important?

Base Packing Material

Silica gel is by far the most common base packing material.

The surface has silanol groups, which are highly polar and can interact with non-polar molecules in your liquid phase. They can also serve as chemical bonding sites. And the large surface area offers a strong adsorptive capacity.

Silica particles are rigid, which helps them resist compaction. This is crucial when you’re using extremely high pressures for very small molecules. Low acidity silica is used to avoid interactions between bases and your liquid phase, improving peak shape.

If you need a column that can work at extreme pHs, polymer packings are a solution if you’re working on a smaller scale. They are stable and do not leach the liquid phase. Additionally, a variety of coating options offer more functions for your column.

Pore and Particle Size

The pore size you want is based on the molecular weight of your compound.

The smallest molecules should be less than 120Å. Polypeptides and compounds with several proteins should be about 200-400Å. Very high molecular weight proteins should be 1,000-4,000Å.

Particle size used to always be the standard 5 microns. In the mid-90s, this standard lowered to 3.5 microns.

Now, if you want faster or better resolution, you can choose even smaller particles (even as little as <2microns). But note that for these, your columns will need to be shorter for standard equipment. A lot of this has to do with the increase in backpressure, which increases with both column length and decreased particle size.

While longer columns are possible, they can only be used by machines that operate at a higher pressure.

Column Dimensions

Again, the column dimension used to be standardized along with particle size (4.6 mm x 100 mm). If a higher resolution was required, 150 mm or 250 mm columns were used.

Smaller column dimension sizes are preferred because they are cheaper and use up less of your sample. If you have small amounts of sample, consider nanocolumns (<1 picogram), capillary columns (picograms to nanograms) or microbore columns (nanograms to micrograms)

Reducing diameter can also result in much greater sensitivity. For example, halving the diameter can quadruple the sensitivity.

The last aspect you’ll need to consider is the bonded phase. These are the chains attached to the surface of the silica. Common bonded phases include C18, C8, and phenyl. But what is C18, and how does it compare to the other types of bonded phases?

What is C18?

C18, C8, and C4 are all linear alkylsilane phases. C18 is octyldecylsilane and contains 18 carbons bound to the silica. So they have more carbons and a longer carbon chain than C8 (8 carbons) or C4 (4 carbons).

Because of the extra carbons, C18 has a larger surface area that the mobile phase has to travel across. This offers more interaction time between the bonded phase and the elutes. Thus the sample elutes more slowly and has more separation.

In contrast, C8 (also called octadecyl) is better for shorter retention time and provides sharper peaks. These column types are better for small organic compounds, while C18s are better for long-chain fatty acids and complex molecules.

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