Ghost Peaks in HPLC – 5 common sources

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A common problem happening in HPLC is what we call ghost peaks.

Ghost peaks means there are peaks that showed up that were not expected. There is something there, so we have to figure out where that something is coming from.

You have a sample, there are some peaks that showed up that you were not expecting, so let’s go through this investigation. Where do those extra chemicals or where did that contamination come from? 

5 Common Sources Of Ghost Peaks

It could be in the sample. It could be in the solvent. It could be in the vial. It could be in the cap. It could be in the HPLC

So let’s eliminate one at a time.

First Step – Run A Gradient Blank

The first thing you’re going to run is what we call a gradient blank. A gradient blank means you’re going to run the full gradient but without any injection, without putting a vial in, without even rotating the valve. That’s important because this will tell us what’s inherently inside your instrument, most likely in your water bottle. Any contamination of the water bottle will show up in all of your samples, so if you run a gradient blank, anything in the gradient blank is something that’s inside your HPLC, most likely contamination in the water bottle.

Second Step – Test The Solvent And Vial

Once you’ve done that, let’s say we make sure that’s clean, the next step is to say maybe it’s in the solvent. So we take some solvent that you use for your sample, put it in the vial, and run that. Did the peaks go away? Then take a vial, rinse it with solvent three times, and then put solvent in it and run it again. So was it in the solvent or was it in the vial?

Continue Process Of Elimination

So you get the idea that we’re just eliminating one thing at a time to find out where those impurities came from. A very commonplace for contamination is the mobile phase bottle itself, the water bottle. Also, contamination usually comes from sample prep, so someone in the back is working with a pipette, and somehow we got some cross-contamination.

So that’s how you find out where those ghost peaks are coming from.

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