7. Column
HPLC column is the reason we are able to separate compounds and measure them. Very shortly, it is a tube filled with material that retains chemicals at different rates, hence a mixture separates into individual components.
HPLC columns are typically classified by the application and chemistry of fill material.
There are 3 main classes of liquid chromatography columns based on application:
1. Preparative
2. Analytical
3. Guard
1. Preparative column has a larger volume and is used for separation of compounds from a mixture. A typical application is isolating compounds from a mixture at a larger scale – typically at a milligram scale. In comparison, the analytical column separates compounds on the order of a fraction of micrograms or nanograms. Separated compounds from the preparative column are usually collected individually and used either in research or for further testing. When used in an HPLC instrument, the technique is called “Prep-HPLC”. In comparison to the analytical HPLC technique, prep-HPLC usually runs at much higher flow rates – usually higher than 30mL/min.
2. Analytical HPLC column is a traditional application of HPLC – separation, identification, and quantification of chemical compounds. These columns come in various dimensions. We specifically use a reverse-phase C18 column that is 150mm x 4.6mm with 5um particles. This is a standard dimension for an HPLC column. Companies developing HPLC systems attempt to shorten the analysis time. One way of doing this is by using material that has smaller particle sizes (typically 1.8um, not 5um). These smaller particles are more tightly packed and have a significantly higher surface area allowing for more interactions with tested mixtures. This allows for using shorter columns, for instance, 100mm instead of 150mm, which results in a shorter analysis time because the mixture goes only through 100mm of the column instead of 150mm. This also comes at expense of higher pressures putting more strain on the pump and fittings with a higher potential for leaks. As a result, new generations of HPLCs are designed that operate under much higher pressures and are called ultra high-pressure HPLC (UHPLC).
3. Guard columns are of small dimensions, typically around 10mm in length and the same diameter and packing material as the main analytical column. As the name suggests, they serve to protect the main analytical column from damage. Guard columns are mounted before the main column and prevent any particles from entering the main column and plugging it. Guard columns are significantly cheaper than the main analytical column and are replaced regularly.
Guard columns usually have a cartridge holder and a replaceable cartridge.
There are 2 main classes of liquid chromatography columns based on chemistry:
1. Normal phase
2. Reverse phase
Normal phase columns are packed with polar material, such as silica (lots of polar oxygens on the surface), and non-polar solvents (hexane, heptane) are used as mobile phase. They are not as popular due to the limitations of surface modification and solvent variety.
Reverse-phase (RP) columns are the exact opposite, the material in the column is non-polar and solvents used are polar, such as water, methanol, and acetonitrile. These columns are more popular due to endless possibilities in modifying the packing material. As an example C8 material has eight carbons long hydrocarbon chain, that can be branched in many ways; C18 are the most popular columns. You will often find columns being called C18 – this being said, each company uses different technology to branch these hydrocarbon chains and they separate compounds differently.
On top of this, you can combine polar solvents in a way to increase or decrease the ability of the column to retain compounds by changing polarity + the ability to make modifications of pH or add different other supporting compounds into the mobile phase, there are many tools one can use to optimize the separation process.
Below is a representation of the two types of columns.
Below is an example of the interaction of Androsterone (a metabolite of testosterone) with the C8 reverse-phase column.
Beyond normal and reverse phase columns, there are many other types based on the interaction of the packing material chemistry and tested compounds.
How are HPLC columns made?
A typical HPLC column is rated for maximum operations pressure of 6,000 psi, hence is made from a stainless steel body. The column has a frit at each end and is packed with the material at 12,000 psi.
This packing causes small variations in the column behavior; typically showcases as a small variation in retention times. We test each of our columns before they go to the customer with all cannabinoids and supply the columns with retention times or updated methods to account for this variation. In the past, there were issues with the column packing process and the end cap frits, which did not allow to use the column in reversed flow, because this would loosen the packing material when the flow was reversed, damaging the column. It is no longer an issue on all modern columns. This being said, we recommend reversing the flow only to do the back-flush when cleaning the column if it is severely plugged.
Most cannabinoid tests are done on C18 columns. As mentioned before, companies manufacturing these columns have a big variation in terminal chemistries on the C18 hydrocarbon chain, which results in cannabinoids eluting in a different order. Below is an example.
