The Clog Blog
Clogging and over pressuring are definitely problematic for Liquid Chromatography columns. Back in 2005 John Dolan’s sage advice to chromatographers to make an LC column last forever was simple, “Never open the box!” As John pointed out, this is not practical and I have to agree. But as an LC column manufacturer I have seen remarks from customers regarding clogging as though it was it was a flaw in the column. Unfortunately, there is not really anything that we do when we manufacture a column that would cause one column clog faster than another. Typically, if a column clogs, it is due to particulate matter that is larger than the pores of the column frit getting stuck in the frit and not allowing the flow to pass through. As obvious and simple as this sounds, it can still be overlooked.
To understand LC column clogging, it is important to know how a modern LC column is built. It is actually pretty straight-forward. A metal tube has an endfitting attached to the bottom. This fitting has two purposes. It provides a means to attach your LC’s tubing to the column and it holds a porous filter or frit in place. Consider the frit as a semipermeable membrane. It has pores that allow the liquid mobile phase to pass through, but are not big enough to allow the column packing particles to pass through. I’ll talk more about the frit later, as it is very important to clogging.
Back to building the column. Once one of the endfittings, the bottom one, is attached to the tube, the packing material, or bonded particles (think of spherical silica of generally uniform diameter, coated with C18 ligands) are mixed into a solvent solution to make a slurry, and pumped into the column tube under pressure. Obviously, the solvent from the slurry passes through the frit that was fixed to the end of the tube with the endfitting and the particles are stopped by the frit. The slurry is pumped into the tube until the tube is filled to the top with particles. You can then remove the tube from the packing pump and affix another frit and endfitting to the open end of the tube. Now the particles that were packed into the tube cannot get out either end and you have an LC column. The particles are packed so tightly into the tube that there is no extra space between the packing material and the frit. Any extra space would be called a void and a void in a column will spoil your day. It can result in problems that will affect the efficiency and peak shape in your separations. For this reason, we recommend that the user never try to take off the endfittings from their column. You will never get a tight fit like a column manufacturer can. So if you take the fittings off the column it is now pretty much scrap. Even though I simplified the packing process here, it is actually a very precise process that takes a great deal of time to perfect. Different particles behave differently as do different phases, column lengths, and different internal diameters. Better to leave it to the professionals.
Back to frits. They are so important to clogging because just as they are the right size to let liquid through but not the particles in the column, they allow your HPLC mobile phase pass though when you connect the column to your HPLC. Now keep in mind the particles inside an LC column are very small. A typical HPLC column has 3 to 5 micron particles. If you look on the internet you will find that an average human red blood cell is about 5 µm in diameter. Now that is similar to a relatively large HPLC column particle. UHPLC columns typically have particles that are less than 2 µm in diameter. Remember the pores in the column frit need to be smaller than the particles inside the column. And when a column manufacturer says that a column has 3 or 5um particles know that that is an average. Silica is a natural product so there is a distribution of particle sizes inside that column. A 3um column could have, for instance, particles ranging from 2 to 4um in it. It is very typical for a column manufacturer to use a 2um average pore size frit for 5um particle columns and a 0.5um average frit for 3um or smaller particle columns. I say average for the frit pore sizes because of how they are made. Frits are typically sintered metal. Sintering is process of heating up small uniform metal bits to near melting point and then pressing them so that they fuse together. The spaces that are left between the fused metal bits are the pores. By being layered they create channels or pores in the material. Some may be larger or smaller than others but the process is controlled and repeatable to make a more or less uniform material. This porous metal material is cut into small discs that HPLC column manufacturers use as frits.
So when you are injecting your sample into the column consider what is in your syringe. Was your sample soil or water, blood or urine, or maybe a plant extract? Do you know what size particles are in your sample? Will they pass through the column frit or will they get stuck in the pores? There are some very easy ways to be sure that you won’t clog the frit on your column and give it a nice long operating lifetime. The easiest thing to do is prefilter your sample. If you know that your column has a 0.5um frit, you should prefilter it through a membrane that is smaller than 0.5um, perhaps a 0.45um or 0.2um syringe filter or filter vial. The better you can filter your sample (translation the smallest filter you can use) the better. Maybe you think that your sample is free of particles. Try centrifuging it. If you can see anything at the bottom of the tube, you have a dirty sample. But let’s face it, the human eye could never see a 1um or even a 10um particle in a solution. Of course there are other ways to protect your column’s precious frit. A precolumn disposable frit for example. Again choose one that is, ideally, smaller than the frit in your column. Or a guard column. A good quality guard column will have 2 frits on it just like your column. If the precolumn frit or guard column get clogged, just throw them away and get a new one that is what they are for. Your column is protected.
Your support team at Restek can help you with several different options to help you keep your column from clogging. Remember, if you want your column to last forever, keep it in the box. But if you want your column to maintain its operating pressure under use for a long time, you now know how it works so think before you inject.