Column testing and cleaning

With your new column do not rely on the column manufacturer's test. They usually test their columns only to check an efficiency, and to demonstrate that it is OK.

You have to test the column to check its selectivity, inertness (absence of the accessible silanoles), correct installation, and for the last it's efficiency.

It is a good idea to run your test mixture once in a while and keep test-chromatograms in the special file. If you are using your column intensively you may run your test once a week. Comparison of several test-chromatograms will give you a complete picture of your column current conditions.

You can use practically any test-mixture, just use the same all the time, than you could even compare performance of different columns. After some time you even will be able to predict how particular column will behave at the certain conditions just on the basis of your test-chromatogram appearance.

I would recommend you to include pyridine in your test-mixture. Pyridine is a very convenient and very sensitive probe for accessible silanoles. It has aromatic structure, so it is easily detectable with any UV detector; it contains nitrogen with nonpaired electron, so it is polar and small enough to access residual silanoles if the stationary-phase bonding density is not very high.

In MeCN/Water 70/30 as eluent pyridine will elute shortly after the void volume. It is important that it is actually slightly retained, this show it's ability to interact with the stationary phase and eventually could penetrate between chains and see silanoles. More polar components like uracil usually get excluded from the surface and won't be able to see silanoles.

Typical test chromatogram is shown on the Slide 19. This test has been done on the Prodigy-ODS2 column (on my opinion it is the best real reversed-phase column, despite that Phenomenex does not like it because it is very hard to pack). As one can see pyridine elute very close to the dead volume (V0=1.7 ml), and shows slight tailing. Fig. __ represent two chromatograms of the same mixture on the new good columns, but pyridine simply disappears there. These columns definitely are not suitable for the analyses of polar compounds, but they may work well for the hydrophobic ones.

Column cleaning

It is a very good idea to clean you column eventually. In most of the textbooks and monographs on HPLC you will find different cleaning procedures. Here I am going to suggest you another one and explain why it is better than all others.

When you are cleaning the column your main goal is to get rid of most of the impurities accumulated in your stationary phase and anywhere inside your column.

You probably have heard about column equilibration before the analysis, it is recommended that after each change of the eluent type or composition you better pump a new one for the certain amount of time to achieve an equilibrium. When your task is to clean the column you better disturb the equilibrium as much as possible, this will shake your stationary phase and ease the release of the trapped molecules. How to do it, how to disturb the equilibrium? Do something completely opposite to the equilibration process - sharply change the eluent composition. It is actually better just jump from one solvent to another, let solvent front pass the column (2 min) and jump back. On the Slide 20  we had shown the solvent profile for fast rigorous cleaning (total 30 min) and continuing detector absorbance profile at 210 nm.   Most  impurities trapped on the stationary phase are coming out at the front and in the couple of  cycles.

It is actually a good idea to clean even a brand new column. I have not heard that column manufacturers had ever thought about column cleaning after the packing. They mainly just wash it with their shipping solvent, run their test, and store or ship it. But their slurry is a very complex mixture, which they specifically accommodate for any particular adsorbent, and, believe me they are not using HPLC grade solvents for the packing. So stationary phase adsorbing all these slurry components and impurities and store it for you.

Slide 18
Slide 19
Slide 20