HPLC – Negative Peaks and Baseline Drift

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We had two cool questions come in recently, and I can solve both with one simple answer.

So, are you ready for this? We’re going to talk about negative peaks baseline drift in HPLC.

How To Correct Negative Peaks And Baseline Drift?

So both of those should be a fairly minor thing. Baselines in HPLC shouldn’t drift very much because when we do gradients, neither mobile phase absorbs anything. So, we should really have no major baseline drift in the HPLC world unless you’re using a refractive index (RI) detector. If you’re doing refractive index, well, that’s just part of your life. But in HPLC UV detection, we don’t get too much drift, and at the same time, we generally don’t get negative peaks. So let me deal first with the negative peak concept.

Negative Peaks

If you look in troubleshooting books, they will not help you at all. They will lead you astray because if you look at any book that talks about troubleshooting LC and if you look up negative peaks, it’ll say that your analyte is absorbing less than your mobile phase, which is totally impossible. First of all, your mobile phase should absorb zero, and anything you’re looking at can’t absorb less than zero unless it’s fluorescing or something like that. So, the bottom line is, we should not get a negative peak due to absorbance.

The reason we get a negative peak is a little complicated to answer. If you have a diode array detector, it’s common to use what’s called a reference wavelength. In fact, by default, the reference wavelength is turned on. It’s set to 360 nanometers plus or minus 100 or plus or minus 50. That is the standard setting for most diode array detectors for what’s called the reference wavelength. Now, reference wavelength is literally a subtraction wavelength.

Most compounds absorb at 254 if it has a benzene ring, 220 if it’s got an acid group, 280 if it’s a protein. Most things are down there below 300. So what we tell the software to do is to subtract everything up there at 360. Whatever you find up at 360, subtract it from my signal at 254. Now what are we going to find up there in the 360 range? Well, hopefully, nothing, but any kind of noise, whether it’s lamp noise or refractive index noise, is equivalent pretty much across all wavelengths.

So here’s a cool concept. We’re going to go to some high wavelength where my analytes do not absorb, and we’re going to subtract that, which is noise, from where my analytes do absorb. You’ll get a slightly better signal-to-noise ratio and slightly better sensitivity. That’s the name of the game. That’s why we do that.

Now let’s say you have a compound that actually absorbs at 360 nanometers. I get this call about once a year from somebody, and they say, “We’re getting negative peaks. It doesn’t make sense.” Now, first statement, if you get a negative peak at the beginning of the chromatogram, that’s t0, that’s totally normal. If you’re doing refractive index, you get a negative peak, well, that’s refractive index.

If you’re doing UV detection, like most of us are, you should never see a negative peak. So when you do, it’s usually because your compound is a colored compound. If your compound is bright red, orange, purple, or green, it means it’s absorbing up in the visible, which means it probably has a lot more absorption at 360 than it does at 254. So you’re actually creating a negative peak by subtracting more than your compound is absorbing.

In the software, there is a really simple way to fix that. It’s in the diode array. When you set up the diode array detector, it seems complicated; it’s really not that bad. We tell it what sample wavelength we want. 254 nanometers is by far the most popular wavelength. It’s good for benzene rings. Also, back in the 1970s, it was our only wavelength, which is why 254 is still popular. So, 254 bandwidth of 20, which means I’m looking at everything from 244 to 264, sort of adding all those diodes together, then averaging them to get a really good high signal, very low noise. Now, by default, I’m going to subtract everything up here at 360. So, why is it such a big bandwidth? Well, I don’t want to sort of subtract any one peak or band. I just want to subtract any noise.

So, the easy answer, if you’re getting negative peaks later on in your chromatogram, probably because you have brightly colored compounds, simply shut off the reference wavelength. By shutting it off, the problem goes away.

Now, a lot of people wonder, “Wait a minute, why did I have it on to begin with?” Well, most of us should leave it on. It gives us slightly better sensitivity. Now, I don’t know the exact numbers, but if your detection is one PPM, that’ll take it down to 0.7 PPM, which is fantastic if you’re doing trace analysis, but if you’re not doing trace analysis, it’s not nearly as important. More importantly, if you’re getting negative peaks, get rid of that stuff, so shut off the reference wavelength, negative peaks go away.

Baseline Drift

Okay, now the second question that I’m going to answer with the same answer is drifting baselines. We don’t usually see drifting baselines in HPLC, but the little drift we get is usually due to the refractive index change from the initial mobile phase to the final mobile phase. Since refractive index effects are equivalent across all wavelengths, all we got to do is turn on the reference wavelength. So, if you’re getting baseline drift, if your baseline is going up or down during a gradient run, then most likely, that means you have not turned on your reference wavelength. In that case, simply go in and turn it back on. You’ll find that in your detector screen. The default reference is 360, with a bandwidth of 100. You could just leave it at that.

Negative Peaks and Baseline Drift: A Quick Review

Now, if you have a situation where you’re getting negative peaks, just shut off the reference wavelength. It’ll solve all your problems. If you really want the correct answer, you need to move the reference wavelength to someplace where your compounds do not absorb. I would take the first approach. I would just shut it off because you don’t lose much sensitivity, but you’ll certainly correct those negative baseline peaks.

Hopefully, I’ve helped you out with negative peaks and baseline drift. It’s a little bit of a complicated answer, but hopefully, you stayed with it.

Here’s another great HPLC troubleshooting article and video. Peak Tailing. It’s one of the biggest problems in chromatography.

If you have other questions, contact Axion Labs here.

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