LC-MS, HPLC: Analyte eluting throughout the gradient time

[sirus]

Hello,
I have problem of my analyte eluting throughout the gradient. I am running binary gradient with increasing percentage of organic (something like scouting gradient). I do not see any separation of my analytes (I have 4 analytes in a sample) instead my analytes are showing up throughout the gradient time.
Please let me know where I am missing? Is my column not retaining the analytes.
Suggestion are highly appreciated and will be a guide for me to troubleshoot the problem.

Thank you.

[sav]

Please let me know where I am missing?

We need info (analytes, column, detection, eluent, flow etc.) for suggestions...

[sirus]

Hi Sav,
The analyte is a mixture of two phospholipids, triacylglycerol, and diacylglycerol.

Mobile phases: Water: Acetonitrile (40:60) A
Acetonitrile: Isopropanol (10:90) B

Accucore C18 columm (100*4.6*2.6), qtrap 5500. Gradient increase of 10% per minute for 10 minutes.

[lmh]

do you get a signal all the while even when you don't inject anything whatsoever? (thinking: If you've injected a generous amount of something as sticky as a phospholipid, it could linger in the spray chamber for a while, and will ionise strongly given the high organic nature of your run.)

[sav]

Hi Sav,
The analyte is a mixture of two phospholipids, triacylglycerol, and diacylglycerol.

Are you using APCI or ESI? Are you see all target ions in mass spec of eluted peak (in case of APCI)? You must add ammonium ion (ammonium formate or ammonium acetate) to eluent for efficiently detecting these analytes via + ESI. Also, Water:Acetonitrile (40:60) is not good start point for gradient, especially with mid and high molecular weight lipids. I think you have detection (unappropriate ionization efficiency) and/or elution problems (some analytes not eluted).

[sirus]

Thank you all for your suggestions.
Still I am messed up. The blank shows noise through LC-MS but if I tap out the same solvent in the vial and inject directly to the mass spectrometer, there are no noise. So the same solvent shows noise through LC but not through direct injection. I do not know what is going on. It is difficult to figure out what is going wrong.
If my ionization is not good, how am I seeing all those unexpected peaks? I am running blank actually. I want to get rid of those ghost peaks.

I am using ESI.

Hi lmh,
I thought the same. However when you do direct injection I do not see any of those peaks. The interesting observation is when I take the same blank (that gives noisy peaks) from LC and inject directly to the mass spec, no noises are observed. But the noises are there through LC. So this should not be the contamination of Ionspray needle, I think.

[dr-statz]

We solve problems with ghost peaks in Gradient HPLC by using online cleaning columns. Installation of the Ghost-Guard-LC before the HPLC-Pump removes traces on impurities from the mobile phase which could cause such ghost peaks!
This is an easy way to get a good baseline in Gradient HPLC.

[sirus]

Hey dr-statz,
Can you please let me send the link to purchase ghost guard column?

Thank you.

[sirus]

We solve problems with ghost peaks in Gradient HPLC by using online cleaning columns. Installation of the Ghost-Guard-LC before the HPLC-Pump removes traces on impurities from the mobile phase which could cause such ghost peaks!
This is an easy way to get a good baseline in Gradient HPLC.

Can you please send me the link to purchase ghost guard column?

Thanks.

[lmh]

Can I clarify, do you have genuine chromatographic-looking peaks in your run, or do you mean you have masses in a spectrum, throughout your run?

If you have a clean "chromatogram" and clean spectra when you run solvent directly into the spray chamber without a column, but you have peaks and masses when you use the column, then one of two things has happened:

(1) You have a very dirty column and rubbish is coming off it.
(2) You have contaminants in the low-strength solvent. In this latter case, the typical symptoms are ghost-peaks of contaminants, whose size depends on how long the column was equilibrated. The argument is that the column collects and concentrates the contaminant, which then elutes during a gradient (yours is a gradient method; isocratic methods this shouldn't really happen).

Given the information so far, I would be looking suspiciously at your column.

[dr-statz]

Hey dr-statz,
Can you please let me send the link to purchase ghost guard column?

Thank you.

Here are the link the the Ghost-Guard-LC: "http:\\analyte.de"