We are using a Zorbax HPLC PAHs column and we are observing a high variability in the retention time of PAHs, even in one same batch. Analysis is performed in a LC-MS/MS system, ionization source is APCI, mobile phase is composed by wateer and acetonitrile, flow of 0.5 mL/min and the temperature of column oven is 25°C. The column is relatively new (January 2020, circa 400 injections). Did someone can give us som tip or recommendation about the use of these columns?
What is the ratio water / acetonitril of your mobile phase?
And is the column temperature stabilised at 25 C?
What is the kind of sample you inject, what composition?
Define the RSD of retention time values.
How long you equilibrate the column before the start of analysis?
Can you post all details of you HPLC method? For example gradient program.
If possible also some examples of chromatograms.
Regards
GB
Dear Rodrigo, I performed PAH analysis in drinking water, and I never had this problem of variation in RT.
To try to better understand your problem I have some questions:
1) When the variation in RT occurs, are they at all peaks or just one or a few?
2) Are you sure there are no leaks? It is usually very noticeable when there is a leak, but it would be good to do a more detailed check.
3) Is the variation random or is it always shortening or increasing the RT? Normally if it is decreasing, the column may be reaching the end of its useful life, but this is also accompanied by an enlargement in the peaks
Best Regards
Thank you so much for your help, dear colleagues! We use a gradient starting with 40% of acetonitrile, increasing to 100% and returning to 40%. I attached a figure demonstrating the gradient curve. The equilibrtion time is 2 minutes (with 40% of acetonitrile). Our samples are mussels and oyster extracts, reconstituted with pure acetonitrile. The column is maintaned with a stable temperature of 25ºC. For instance, in a recent batch, for the internal standard Chrysene-d12, the retention time showed a RSD of almost 35%, with RT 4.00 at the beginning of the batch and decreasing to RT 3.55 at the end (see the 2nd attachment). Similar variation was observed for all analytes and internal standards. No detectable leak was observed and the pressure behaviour was the usual. Moreover, the peak shape remains the same, without noticable enlargement. Our column is relatively new (a Zorbax Eclipse PAH 2.1 X 100 mm, 1.8 micron).
Hello again Rodrigo Barcellos Hoff ,this behavior is very strange to me...
Can you check that the pump has a steady flow? It could be that during the runs the flow is increasing, which could create this effect.
Another thing, I would try would be to increase the stabilization time at the end of the gradient, around 2 minutes.
Best regards
Dear Moacir Brito thank you so much for your tips. I will try to increase the stabilization time and also to verify the pumps flow. Wish me luck!
Have you tried reconstitution of the samples with the mobile phase at the time of injection, hence 40 % acetonitrile.
I agree with Moacir Brito that it would be much better to elongate the stabilization time at the end of the run. In addition you could also consider making a less steep slope at the end, going back from 100 to 40% acetonitrile.
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