If you have the suspicion that your C18 column isn’t working as well as it did, you may be wondering if cleaning it will help.
The good news is that you can clean a C18 column. I’ll give you a couple easy things to do that cleans the column well.
But first, there are a couple of things that can actually destroy an HPLC column.
For example, if you physically damage the column, if you bent it in half, if you ran it at pH 14 overnight (you dissolved all the silica), or if you ran it at pH 0 and you chopped off all the C18 chains, then those are permanent damages. But, good news is those kinds of things rarely happen.
The most likely cause of a legitimately dirty column is a dirty or clogged frit, or dirty column packing.
Dealing with a Dirty or Clogged Frit
Why Frits Get Clogged
Usually, what happens to a column is two things. First, the top frit gets dirty. Why does that happen? Because people are not filtering their samples. So, if you have particulates in the sample, they’ll clog the frit at the top of the column. Your pressures will increase, and you’ll start seeing doublets and splits on the peaks. That’s an indication of a dirty frit. You could avoid that just by filtering your samples. Watch this webinar to see how we properly prepare a sample for HPLC.
The Controversial Cleaning Method
If you have a dirty frit, this is a controversial topic. At great risk to myself, I’m going to give you my best solution: Switch the column backwards and run it upside down. It will clean the frit immediately. You will go from a clogged frit to a perfect one in seconds. But, it’s controversial because column companies say “don’t ever change the flow of the column”. I agree with them. I used to pack columns and you should always use them in the same direction. That’s the best answer. But if you’ve clogged the frit and you have split peaks, you’re not getting any good use out of that column. If you flip it around, you will see an immediate improvement.
Steps to clean a C18 column?
So, let’s clean a dirty column. This column might be dirty because you’ve been injecting a bunch of chicken fat samples. The chicken fat is accumulating and building up on the C18 column because it’s very non-polar. Now, you want to clean the column.
Step 1: Flushing with Water
The easy answer is to wash it with a very high percentage of organic, either 100% methanol or 100% acetonitrile. But before we do that, especially if we’ve been using buffers or unknown polar compounds, I’d like to wash the column with a very high percentage of water. I know it sounds backwards. Water is weak, but in this case, we’re going to use a 90:10 water-acetonitrile or water-methanol ratio. This will flush out all the buffers and get rid of really polar compounds.
Step 2: Use 100% Organic Solvent
Then, we’re going to step it up to 100% methanol or 100% acetonitrile. I like to do my column cleaning at 60 or 70 degrees Celsius, so crank up the temperature a bit and let it run for an hour. There are very few compounds that’ll stick to a C18 column for an hour under those conditions – a very strong mobile phase and elevated temperature.
Summary
So, that is how you clean any reverse-phase C18 column in the world. You can’t do any damage to it. Simply get rid of the buffers first with a high percentage of water, then go to a very high percentage of acetonitrile or methanol. The third solvent, THF, is stronger than the other two, but I personally don’t go to THF. If you have something that has polymerized on the column, THF helps, but I find 100% acetonitrile or 100% methanol will clean up just about everything.
