Peak in blank

[Marian.Deacon]

I have a peak in my blank at aprox 2.5 mins that I do not know the cause of. I have removed all the reagents from my mobile phase and it remains.

separation conditions: ACE C18 3um column, 45:55 MeOH:H20, flow rate 1ml/min, temp 55C, 215nm, 75ul injection

I have tried

System: tried both Waters and Agilent system- Agilent system was new to the lab and had not been previously used.
Column: changed to a new column
Mobile Phase: changed supplier of MeOH and Water
Vials: agilent and waters vials, washed out agilent vials with blank

Blank: have taken directly from column exit into vial and injected
injected 100% water
injected 100% MeOH
The peak increases with increased injection volume
THe peak may vary in size from run to run but within a run it is constant.

[dblux_]

carry-over problems ? (dirty needle port, contaminated switching valve)

Wojtek

[Uwe Neue]

at 215 nm you will see methanol. The peak could be nothing else but the difference in the methanol content of the sample and the mobile phase.

[ym3142]

Uwe is saying "it is solvent front.", coming around your void time

[tom jupille]

Uwe is saying "it is solvent front.", coming around your void time

Or, more likely, it is a slightly retained "system peak" for methanol.

What are the dimensions of your column?

[Bruce Hamilton]

If you saw the peak increasing in size with pure water injection volume increase ( as you imply ), I believe it's less likely to be methanol absorbance, especially if the peak size is inconsistent.

Is your mobile phase premixed and degassed?, if not, try that first.

As the column is at 55C, and the peak is inconsistent, it's possible you are seeing the effect of some dissolved gases becoming undissolved on the column, or some sort of thermal/desorption effect from the column.

You could drop the column temperature and inject mobile phase blanks to see if the peak goes away, and reappears if you return to 55C.

You could also warm a mobile phase blank vial to around 60C immediately before injection of 100 ul, and look for a consistently larger peak. If seen, it's likely that it's some consequence of the difference in temperatures.

Bruce Hamilton

[danko]

Hi Marian,

You’ve injected 100 % water and 100 % MeOH. So the natural troubleshooting extension would be to inject 45:55 MeOH:H20 – preferably taken directly from your mobile phase reservoir. If your mobile phase isn’t premixed, then just collect some mobile phase at the outlet of the detector under equilibration conditions.

Best Regards

[HW Mueller]

My bet would also be that this is a system peak, one seldom bothers with these. You should probably just do your chromatography after that.
Incidentally, what happens if you just activate your injection mechanism ithout injecting anything?

[rhaefe]

Hi Marian,

You’ve injected 100 % water and 100 % MeOH. So the natural troubleshooting extension would be to inject 45:55 MeOH:H20 – preferably taken directly from your mobile phase reservoir. If your mobile phase isn’t premixed, then just collect some mobile phase at the outlet of the detector under equilibration conditions.

Best Regards

It was my understanding that she did inject mobile phase (effluent from the column collected in vial and injected)
cheers

[danko]

Hi rhaefe

I think you’re right. It’s not perfectly clear what the MP composition is at the collecting time, but 45:55 is most bribable. Actually my preference for collecting point would be, as mentioned, at the detector outlet, so I made sure I didn’t collect some peak/s without realizing that. But anyway thank you for bringing it to my attention.

Best Regards

[Marian.Deacon]

Thank you all for your suggestions. My mobile phase is premixed and degassed. I tried matching the sample and column temperature but no joy. If I activate the injection mechamism without injecting I do not get the peak. The peak appears in both MEOH and Water injection, however the peak from water is 1/10 the area of the peak for MeOH for an equivalent size of injection. This tells me that the MeOH perhaps mainly responsible. I have tried 2 suppilers of MeOH. Any suggestions?. [/quote]

[ym3142]

Dear Marian.Deacon:
Could you let us know what is preventing you from telling us your column length?

[Marian.Deacon]

Column length is cm so the solvent front is at 1 min and the blank peak in question has an RRT 2.5

[ym3142]

Dear Marian.Deacon:
Thank you for sending us the calculation test. My answer is that the column length is 50cm, is this right? DO you have other tests for us?
Thank you again,

[Marian.Deacon]

It was not meant to be a test the num lock was on the computer. Column lenght is 10cm