How to Test an LC Column 

Using Probes to Test LC Column Properties 

Yes, you can test a column. In the hands-on class that we do here at Axion, we have a nice lab where we talk about how to test a column. We have different, what I call “probes” that can tell us different things about your column. For example, I can tell the acidity of your column by injecting dimethyl aniline. I can tell the metal content of the column by looking for metal impurities by injecting something like quinizarin. So, we do have cool probes that we use to look for different properties of your column.

The Better Approach: Using Your Daily Standards 

But let me take a big step back and say there’s a much better way to test a column. Whatever sample you run, you probably have a standard that you run every day. That is your best test pick because that’s the sample you care about and those are the components you care about. So, let’s run a known; let’s run a standard. I want you to measure the efficiency of the peaks.

How to Measure Column Efficiency 

Now, efficiency—if you don’t know how to measure it—you can look it up on the internet or check out my other online courses. It’s retention time divided by peak width (quantity squared) times 5.545. That gives you efficiency, which is the best measure of the “goodness” of the column. When we have high efficiency, we know the column is well-packed, with good fittings and tubing, and is mechanically sound.

Evaluating Peak Shapes 

Then, we can also look at the peak shapes. Especially if you’re interested in acidic or basic compounds, inject a base like aniline. If aniline tails, it’s an indication that the column is not good for basic compounds. We could do the same with an acid like phenol. In my experience, acids and bases tail the same way, and that’s the tailing of bases, which is much worse than anything else. So, if a base looks good on a column, everything else will look good on the column; neutrals don’t care.

Testing a LC Column with a Neutral Compound 

So, how do we test a column? Just inject something you already have around. Column companies typically inject toluene; they say that efficiency is best measured on a well-retained, well-behaved peak. What does that mean? A peak with a capacity factor of at least five, and something that’s not an acid or a base. So, toluene is well-retained, or you could use any one of the millions of other neutral compounds out there. Measure its efficiency, see what the number is, and compare it with what the column should be or what the column was when it was new.

Understanding the Column’s Certificate of Performance 

By the way, when you get a brand new column, inside the box is a little certificate that shows you the exact efficiency of the column. When you get this, it’s not a bunch of marketing material; maybe other stuff is, but this is not marketing material. This is not junk. This is a unique, one-of-a-kind chromatogram that was run on your column—not on your batch, not on the day your column was made, not on a column that looked like yours—on your exact serial number column. It has the serial number, and it also has the number of plates.

What to Expect Over Time 

So, I have a new column here and the theoretical plates on this column are over 11,000 plates. I’m saying, “Woohoo, that is a good column.” This is a 5-centimeter, 1.8-micron column, so we’re in the 10,000-12,000 plate range. You look at that number; they got 11,000 plates the day they made the column. When you install it, you should get 11,000 plates, plus or minus 5%. Don’t read too much into it, but you should get about the same number. As the column ages, that number will drop. So, a month from now, if that column is down to 10,500, am I going to throw it away? Absolutely not. That’s the same column; it’s a great column. When it gets down to 6,000 plates, I’ll be like, “Oh my gosh, I’ve lost 40-45% of the efficiency of my column.” Yeah, time to get rid of the column.

Conclusion: The Ultimate Measure of a Good Column 

We’re going to use this efficiency measure as a measure of the “goodness” of the column. The ultimate measurement of a good column is: does it resolve your peaks of interest? So, you should have a standard; it should be your separation mix, your demonstration of what you can separate to the world. It should contain your major compound, your compound of interest, and all the impurities and everything you’re ever going to look for. You should have them all in this mix. Let’s say there are seven of them, and as long as you prove to the world that you can separate all seven, that you have a resolution greater than 1.5 between all seven of them, then that, to me, proves that the column is still good.

Final Thoughts 

So, yes, we can test the column. We can come up with an exotic probe mix, but an easier answer is just to inject something you have sitting around the lab—your standard that has more than one compound in it. This way, we can measure things like efficiency, resolution, capacity factor, and selectivity.

Hope that clears it up or helps out. Let us know what questions you want answered. Check out our YouTube channel; we try to keep it well-filled with answers to your most popular questions. So, come back and visit!