Please help: Baseline problem in separation of PGE2 and PGF2

[ru yan]

PGE2 and PGF2 standards were separated under the following conditions:

Column: waters Symmetry C18, 5um, 4.6x150mm
Mobile phase: ACN:H2O:Acetic acid (30:70:0.1)
Keep isocratic at a flow rate of 1 ml/min
Temperature: ambient
Monitoring: dual wavelenths at 195 and 210 nm
Injection volume: 50 ul

Result:
PGE2 and PGF2 were sharp, symmetric and well separated. But the baseline before each peak was much higher than the baseline after the peak.
The problem still existed when 0.2% acetic acid was added into mobile phase.

[Uwe Neue]

Don't know what your analytes are. At 195 and 210, you see a lot of things, nearly everything.

Could be displacement of stuff from the mobile phase that is displaced by your analytes.

Could be partial elution of the analytes, if they are dissoved in too strong a solvent.

[danko]

In addition to what Uwe wrote, you should consider (seriously) changing the detection wavelengths towards the red region, provided your analytes absorb energy elsewhere. Alternatively, you might consider a substitution of acetic acid with another acid – e.g. phosphoric.

Best Regards

[ru yan]

Thank you very much for suggestions.

The analytes are prostaglandin D2, E2 and F2. They have several double bonds but not conjugated double bond. They also have carboxyl group.
Their UV absorption was poor, thus I chose 210 and 195 nm to monitor.

Based on my experience, I don't think it's a problem of mobile phase. Actually, the company who provided us the standards have run the sample under the same conditions: 210 nm, ACN-H2O-Acetic acid (35:65:0.1), 1 mL/min, C18, 5um. The only difference is the column they used is longer than mine (250 mm vs 150 mm). Their chromatogram showed no problem in the baseline.

Do you think the length of the column might affect the baseline in this way?

Thanks again!

[ru yan]

One more detail:

The standards the company provided was disolved in methyl acetate. We made injection of 50 ul without any dilution to obtain suitable UV absorption.

[ym3142]

did your front high base line showed like a shouder to your peaks?
can you also see your vendor C-gram base line well? or if you expand them, will be theirs like yours?
is there a possibility that the materials you tested was not pure enough or the mobile phase not pure enough?
good luck

[chromatographer1]

First check and see how much water can mix with methyl acetate. If possible then try diluting your standard solution with 30%-70% water and inject a larger amount to compensate.

I am betting that 50µL of methyl acetate is too non-polar to mix immediately with the mobile phase you are using and is causing your problem.

best wishes,

Rod

[Uwe Neue]

The apparent consensus is that the cuase of the problem is the injection solvent. This would play less of a role for the longer column. You could inject less, if the sensitivity is still OK. Or you could dilute the sample with as much water as you can, maybe together with ACN, and inject a larger volume.

[Jackus]

PGE2 and PGF2 standards were separated under the following conditions:

Column: waters Symmetry C18, 5um, 4.6x150mm
Mobile phase: ACN:H2O:Acetic acid (30:70:0.1)
Keep isocratic at a flow rate of 1 ml/min
Temperature: ambient
Monitoring: dual wavelenths at 195 and 210 nm
Injection volume: 50 ul

Result:
PGE2 and PGF2 were sharp, symmetric and well separated. But the baseline before each peak was much higher than the baseline after the peak.
The problem still existed when 0.2% acetic acid was added into mobile phase.

We analyze these prostaglandins too (with slightly different conditions). The injection volume seems to be quite high. What is your sample concentration? (We use: 1mg/ml @ 210 nm, inject volume 10ul - Agilent 1100 VWD, Formic acid instead of Acetic).

[ru yan]

It was the problem of methyl acetate--The more I diluted the sample with mobile phase, the more better peaks and baseline I got. In the last injection I made, I had 15% methyl acetate in 100 ul of injection.

Thanks again for help!