Chromatography Forum

Hello everyone
I have collected phospholipid (PC, PE) peaks from the HPLC which is connected to a ELSD.
The expected peaks are eluted at 12 and 15 min.
After collecting the peaks, a known concentration of the peaks was re-injected into the HPLC to make sure the right peaks were collected. I was able to detect them on HPLC.

Then a known conc. of the collected PC and PE was dried and resuspended in chloroform and sent for mass spec. But we did not observe any spectra at their mol. wt.
The PC and PE peaks collected from HPLC were also analyzed on TLC, where we could detect both the phospholipids.

The problem is why cant we detect them on Mass which is the most sensitive method when compared to HPLC and TLC.

Can anyone suggest a solution.

Thanks
Sankella

Hello.
If we assume that the mass spectrometr not defective, putting the sample in the mass spectrometric and ionization are correct and the "hands are not the curves", then:
One t is a stupid question, and whether you have collected? ELCD nonselective detector.

Alternative solutions
1) Enter this or similar in structure to lipid mass spectrometer under the same conditions. If it is not detected a problem with mass spectrometry, for example you are using GC-MS, but phospholipid non-volatile.
2) If it is found - the problem with HPLC - to forcibly collect fractions every 30 seconds and check for the presence of phospholipids. At least an elementary reaction with molybdenum blue.

What kind of mass spectrometer did you try to analyze those and with what method? For example if you try to ionize those with ESI-MS, and your analytes are in chloroform, you won't see anything. Mass spectrometry polarity can play also a role. I would suggest to follow a published MS based method for the analysis of your compounds or provide more information so that we can help you...

Thanks for the input.

@Kostas Petritis
Yes, ESI-MS was used for detecting the peaks and the phospholipids were in chloroform. I dont understand why the peaks cant be detected.
The PC and PE standards were loaded on MS, which showed spectra at the right mol. wt. But the same phospholipids when collected from HPLC cant be detected on MS.


Thanks
Sankella

There might be several things happening here...

What is your mobile phase? If you are injecting compounds dissolved in chloroform and your mobile phase contains lower hydrophobicity solvents (on top of any non-miscibility problems) your injection solvent might be too strong.

If your mobile phase is something like chloroform as well, electrospray does not work well at those solvents... You mentioned that you infused? those in the mass spec and saw the compounds? What was the solvent and lipid concentration you used?