Unknow peak detected in HPLC-UV

[wjthlapb]

I recently purchased the 2nd hand HPLC. but unknown peak is kept detected.

not sure if it is a hardware problem or simply due to my mistake.

Please help me figure out this unknown peak issue.


Condition:
I am running a gradient HPLC method on a conventional C-18 column(250x4.6mm, 5um)

*Detection wavelength: 227nm
*Eluent A: 1.2mM potassium dihydrogen phosphate, 0.05% phosphoric acid in water
*Eluent B: Acetonitrile






Whenever I run HPLC, this unknown high peak is detected at the exactly same RT and same height.

Other compounds are detected correctly but this unknown peak covers all adjacent RT compounds.

Even I run it with Pure water, this peak is still there.

So I assume that potassium dihydrogen phosphate or phosphoric acid in the eluent is detected.

has anyone else experienced something similar to my case?

I desperately need to know why and where it comes from.

[lmh]

could be anything, and it's not easy to be sure. There are some things you can do to check. If you can get your system to do a run without injecting anything at all (not moving the injection valve, not doing anything), this is worth trying. It rules out autosampler contamination issues. If the peak only happens when the autosampler is involved, then you know that it's come from some autosampler contamination.
If it happens without the autosampler, then it's a weird artefact either of something stuck on the column that comes off at a particular point in your gradient, or it's something in one of the solvents. For example, if you have a contaminant in the aqueous phase, it will build up on the column during equilibration and the early part of the gradient, and come off as a peak later. In this case, it will also be a bigger peak if you equilibrate for longer!
If it's autosampler contamination, then it can be tricky to sort out. Check the situation with your needle wash solutions. Some loop-injection systems use needle wash to fill whatever part of the loop isn't filled with sample, so if you have a contaminant in the needle wash, you'll get a lovely big peak. Otherwise, it's a matter of trying different needle-washes and multiple injections trying all sorts of solvent combinations to see if you can find conditions in which the peak gets smaller and smaller... and hoping that it's not something nasty lingering in a worn injection valve.
Others may have other advice. Good luck!

[Consumer Products Guy]

In your clean out part, I'd bump the ACN from 80 to 95%, and hold at 95% longer to see if that helped. Of course, you need sufficient equilibration time at the Time zero condition.

[TylerSmith123]

Hi wjthlap,

I don't think that you're detecting your buffer or acid, especially since you are using quite low concentrations of buffer (not sure how much of an effect it would have on your chromatography at this concentration), and a normal amount of acid which should not give you a peak this strong at that concentration. It would be much less uniform. Do you have any history on this HPLC you just purchased and what it may have been used for prior? Like Imh said, it really could be anything. To check and see if there's contamination, I would equilibrate your column for a 20 minutes using a high aqueous mobile phase (~80%) and then wash it and measure the unknown peak. Do this again but double the equilibration time and measure the peak again. If you get roughly double the peak height there is some sort of contamination in your system. By any chance, is the column also 2nd hand?
The mobile phase could also be an issue, even if you ran in pure water. Are your filtering your buffered mobile phase before using it? Is the buffered mobile-phase prepared everyday? What do you wash procedures look like? What type of samples are you injecting onto the column and does this weird peak share any similarities with what you're injecting?

Sorry I couldn't be more help, there are certainly a lot of places where contamination or ghost peaks can originate from-- especially if you're using a second hand machine.

[Andy Alpert]

Phosphate salts and phosphoric acid contain small amounts of impurities that can accumulate on columns in different modes of chromatography. They then elute during a gradient in a reproducible peak, whose amplitude is proportional to the amount of time that the column had been equilibrated with the phosphate buffer. Using HPLC-grade phosphate salts and phosphoric acid largely eliminates this problem. What purity grade reagents have you been using?

[wjthlapb]

Thank you to everyone who commented on my post.

In a nut shell, I just solved the ghost peak problem.

The problem was 3DW from water purification machine which was newly purchased.

There were some chemical left in the 3DW which was used for making mobile phrase... and it is detected as unknown peak.

Mechanic guy told me that I should have pulled enough amount of 3DW water out of the purification before I use it.....

Everyone, Thank you so much again for the help, and please remember to throw enough amount of water out whenever you change the filter in the purification..