HPLC Tips Peak Tailing

Unfortunately, peaks tail in HPLC. We don’t want them to tail. In theory we like to think of peaks as Gaussian shaped and perfectly symmetrical, but in reality just about everything in chromatography has a slight tail on it - about a 5% tail.

We can deal with that, but we don’t want the peak to tail too much. The more that the peak tails, the harder it is to quantify it - to measure the area. A tailing peak also starts to run into other peaks. That makes it harder to separate the peaks.

This is a major issue that we have to deal with. That’s why I think a lot about how to reduce peak tailing in HPLC.

What causes peak tailing in HPLC?

There are two major causes of peak tailing in HPLC. As you will see they are not related to one another. That will be important to know later when we talk about HPLC peak tailing troubleshooting.

One is what I call a physical problem - empty space in the system. One is what I call a chemical problem - acid base interaction.

Let’s talk about the physical problem - you have empty space in your problem. You have a void. Maybe the top of your column has empty space in it because the stationary phase has settled. Maybe you have too much tubing in your instrument. Maybe you have a fitting that is way too big and thus has empty space.

Why does that affect the peaks and cause peak tailing?

Imagine a peak as a billion molecules moving through the column, moving through the tubing as one tight little plug. If they hit an open space, let’s say that they travel through a big box, now the molecules in the peak start to mix with the mobile phase that is already occupying that volume. Some of those molecules will go on the column, some will stay in that box and mix with the incoming mobile phase. What is the result? Instead of a symmetrical peak we will get what is called a tail. We call that infinite dilution because you dilute the molecules more and more. That’s a bad thing. Physical problems like this are due to the instrument. Bad plumbing in your instrument is one of the causes of peak tailing in HPLC.

Now let me talk about the chemical problems. And then I’ll give you a real trick on how to tell the difference - because they look the same.

Again a chemical problem is what I call acid base interaction. Your stationary phase, a typical HPLC stationary phase, is based on silica.

The surface of silica is slightly acidic - that Si-OH is weakly polar and acidic. So the hydrogen nucleus can dissociate, leaving a negative site. That means that the surface of the stationary phase, or we can say more generally, your column has a natural negative charge to it.

Now if you have a basic compound - let’s say a protonated amine - what happens? Well there is a positive charge cruising through the column, I have a negative charge on the column, and when they interact we get adsorption. I say that adsorption is like velcro because it is easy to stick but hard to let go. And that is why we get the tail.

So what types of compounds will tail based on this mechanism? Bases are the worst; amines are by far the hardest compounds to run on HPLC. The second worst? Acids. Acid tail due to their active hydrogen. It’s a different mechanism but results in the same problem. Aldehydes can also be difficult and tail on HPLC.

How to reduce peak tailing in HPLC
So how do we solve peak tailing caused by a chemical problem? There is a real simple solution. You buy a column that has no negative sites on it. We call that a base-deactivated column. Of all the columns in HPLC, there is one stationary phase that dominates, it is a c-18.

So if you are going to buy a C-18 column, I always tell people, buy a good, base-deactivated C-18 column.

So we talked about two things that cause tailing. Chemical problems, acid-base interactions, and physical problems, or plumbing problems in your instrument.

Troubleshooting peak tailing in HPLC

How do you tell the difference between plumbing problems and chemical problems?

It’s hard to tell just by looking at the chromatograms because they both look about the same.

The way to tell the difference is by injecting something that is not an acid or a base. Neutral compounds should not tail; they cannot tail due to the acid-base interaction. So if you inject a neutral compound and it does tail that means that you have a physical problem.

Fix the tubing.

If the neutral compound does not tail but your peak of interest does, then you have an acid base problem. So in that case you need to buy a better column.

The other philosophy that I use in troubleshooting is, if all the peaks tail you probably have a physical problem.

If you have ten peaks and only one of them tails, even if nine of them tail and just one does not, that proves that the instrument does not have a physical problem.

The good news is that you are just one injection away from determining the root cause to your tailing problem and then solving it.