Problem with a ghost peak

[Pippo&Pic]

Hello everyone, I'm new to this community and generally to HPLC. In my lab, we use a C18 Rezex ROA-organic acid H+ column with a mobile phase consisting of an aqueous solution of H2SO4. We typically monitor organic acids from biological fermentations with UV detector. Lately, a peak always appears at a certain retention time, even when running a blank with just the eluent (prepared fresh). I've tried washing the detector cells with isopropanol and cleaning and regenerating the column according to the manufacturer's settings, but this peak continues to appear at the same retention time.
Do you have any suggestions for resolving this issue? In your opinion, is this likely a column problem or a detector problem? Thank you very much.

[vmu]

If this peak does not interfere with the peaks of the analytes, you have no problem here.

[Pippo&Pic]

Unfortunately it have the same RT than butirric Acid

[TylerSmith123]

Posting a picture could help us identify this issue. You'll need to isolate this issue, and, as you see this peak in your blank injection, it is likely originating from inside your system (assuming fresh, proper mobile phase preparation). However, if it is originating from your solvent, run a wash program that begins with a high aqueous portion for different lengths of time (5, 10, 20 mins) and then immediately followed by a strong organic portion to elute the troublesome peak and note the intensity of this peak. If it is growing with the length of time then the problem is likely your aq phase.
Another experiment will likely have you remove the column for a clean union and repeating the blank injection to see if the peak still appears.
I'm not so sure about your set-up, but if you have two pumps you can isolate one and strictly run your pre-mixed mobile phase through one pump at a time in order to isolate them.
Another source of these peaks could be from your frits in the solvent and you may want to exchange them and see if that fixes your ghost peaks.
There's a lot of potential sources for your ghost peak. It may even be from your sample-prep conditions. Perhaps you're not cleaning up your sample enough prior to injection and that is getting stuck on your column. Without more information regarding your analysis it can be hard to guess. Troubleshooting by isolation will be your best friend here, however once you identify the source it may be worthwhile trying to understand why this problem manifested in the first place. Please do let us know regarding the results of your multiple experiments: ie, did any of your washing steps actually affect this peak? What's the retention factor of this peak, uv spectrum? When you say you typically monitor organic acids from biological fermentation, does this mean that this system has been used for something else lately?
Hope this helps in some way and I hope you can get down to the bottom of this issue!

[Multidimensional]

A few comments about some of the posted comments for our new user.

"Another experiment will likely have you remove the column for a clean union and repeating the blank injection to see if the peak still appears." - Please do not do this. Doing so will lead to many problems and misleading data. To correctly troubleshoot if the column is part of the problem you must replace the column with a restrictive capillary line to provide the required system back-pressure. An HPLC pump requires a certain amount of back-pressure to operate properly (often ~ 40 bars, but this depends on the mobile phase and specific make/model of pump). Installing a properly sized restriction capillary (a "tool" that all experienced chromatographers should have and use) will provide the needed back-pressure and allow the rest of the flow path to be checked.

"If this peak does not interfere with the peaks of the analytes, you have no problem here." - This approach does not follow good chromatography principles. Any, all, unexplained "peaks" need to be investigated to find out their source. As a new user, this is the best time to learn the basics. Often, unexpected peaks are an indication of a real problem requiring a solution. That problem may be a hardware problem, software problem or training problem.

Finally, as others noted, it is important to include an actual chromatogram (with all scales) and the full method to offer any constructive advice. The 'peak" may be the injection peak or solvent peak (many new users do not know how to identify these). What you describe is vague, and could be many things including sample CARRY OVER (which would most likely be caused by a fouled column OR a worn injector rotary seal). Carry over contamination, as its name implies, will show a peak again-and-again, even when a true blank (mobile phase) is injected. You can read more about it at this link: "Carry-Over (Carryover) Contamination in HPLC and LC-MS Systems"; https://hplctips.blogspot.com/2015/02/c ... on-in.html

[vmu]

Any, all, unexplained "peaks" need to be investigated to find out their source. As a new user, this is the best time to learn the basics. Often, unexpected peaks are an indication of a real problem requiring a solution. That problem may be a hardware problem, software problem or training problem.

My experience tells me that new users often like to investigate the peaks near the void time that do not influence their results at all : )

[Multidimensional]

'VMU' wrote: My experience tells me that new users often like to investigate the peaks near the void time that do not influence their results at all : )
[/quote]

New and experienced users miss-identify many peaks at or near the column void time as "sample" or "ghost" peaks. Many do not understand the HPLC flow path or basic chromatography fundamentals (e.g K prime, valve operation, column fouling, back-pressure etc) so need to consult an experienced chromatographer who does understand these things to put their observations in correct context. We should never suggest that anyone ignore such findings.

[Pippo&Pic]

Update: Tests conducted have prepared a new mobile phase with distilled H2O and bi-distilled H2O, a new syringe wash solution (30% methanol), and a column change. The peak is still there, both with the UV detector at 209 nm and with the RID. I will try to do new washes of the system with isopropanol, do you have any other solvents to recommend?

Thank you all for the advice.

[Pippo&Pic]

I'm tring to add a picture, but i don't know how to do it

[LuccasLN]

Hi Pippo,

i propose a different approach. Considering that this is a ghost peak and that this has been appearing lately but not always (definition of ghost peak). try to remember what changed comparing past and present. did you change a reagent/solvent/other consumables (gloves, syringe filters,...) brand? do you by any chance reuse vials? how do you clean glassware in general? what is the chance of cross contamination in your lab considering samples and instrument?

PS: it is never a wise idea to reuse vials, but it is worth checking since we are trying to troubleshoot

thanks

best regards,