Chromatography Forum

Actually I'm validating a HPLC method for analisys PAHs (polycyclic aromatic hydrocarbomns) in water by HPLC.
My problem is that the retention time for the last 6 PAH are diferent along the day, they decrease from the first inyections, but for the other PAHs that elute earlier the retention time isn't than different. The variations can be about 1.5 minutes for the first and the last.

The temperature of the room as the same all the time.
The mobile phase that I'm using is water:acetonitrile in gradient:

0 min 50:50 MeCN/Water
3 min 50:50 MeCN/Water
12 min 90:0 MeCN/Water
22 min 100:0 MeCN/Water
33 min 100:0 MeCN/water
40 min 50:50 MeCN/water (regenerate de initial mobile phase)

I also used other gradients and occur the same.

All the samples are clean standards without any matrix

I hope that anyone can help me.

Thank.

What is the size of your column? Are you controlling temperature? If so, what temp are you running? In what section of your gradient are the problem PAHs eluting? How long are you holding that 50/50 at the end of the run?

If your temperature isn't a problem, and you see this issue with other gradients, it looks like you're not re-equilibrating the column completely. But without more info it will be difficult to me completely sure.

Camino,

What degassing do you use?
Do you purge the water and MeCN before the analysis?
Can you display the "pump ripple pressure" on your system?
Perhaps your early peaks are varying too - you could calculate a % variation to check that.

Has this always been a problem because:
even if the temperature of the room is constant this doesn't mean the column temperature is always the same. You should invest in a column oven.

Best Regards

WK

Camino

I agree this could be a case of insufficient equilibration. Some suggestions.:
Don't rule out temperature variations and has been suggested use a column oven to be sure. Any possiblity of variability in gradient formation? May be slight variations in gradient formation by your pump system and or slight variations in flowrate due to air in the system. I would suggest running some standard gradient tests and as already suggested ensure solvent degassing is adequate. One point- if you are running from a premix of 50:50 ACN/H20 you may get some settling so would advise stirring to ensure proper equilibration.

after analysis i use a crecient gradient from minute 33 to 40 to obtain the intital mobile phase, and I use 15 minutes for the re-equilibration of the column after minute 40. The problem is in the last five PAH (minute 12 to 33 in the gradient program) and the % variation is different for the early peaks (in these the variantion is minimal). I always purge MeCN and water before analysis.

The colum is a C18, 25 cm and 3 cm ID. and I'm using a in-line degasser. The ripple is about 0.2%.

The resolution is more poor along the time in adition of the decrease in the retention times.

What is the stimated time needed for the re-equilibration for this column?
If more information is needed please tell me.

Thank you

Best regards

Hello Camino,

try also this:
take out the column and replace it with 2m 0,17id tubing.
take solvent A and solvent B. Make a special mixture of B ----> add 0.5Vol% Acetone. Program a gradient from 0% B at start to 100%B in the end, may be 10min run. Detector set to 250 or similar nm.
Make this run in the morning after equilibration and in the afternoon. Compare the runs by overlaying both "chromatograms". If the courves dosn't fit You have to control the pump mixing system.
If all is ok, than You it is a column problem.

Regards

Bernd

Camino,

please check also the void time (dead time) to in the morning and afternoon. Do You use piston clean flow?

Regards

Bernd

Sounds like a mixing problem to me. Do you have a mixer installed? The type of pumps you are using can make a difference too. Is this a high pressuer or low pressure gradient? You could try this on a different system if you have one available. Also, it looks like you are mixing with 100% ACN. I always like to do my gradient to 95% ACN: water. Having a little water in the "B" line helps it to mix better with the more aqueous "A" line.