Hello,
I just changed to a new HPLC column and running the same procedure as on the previous column my peak showed splitting and multiple peaks appeared over the run where previously there was just one.
The new column was found in its box in the store room and dated 2017 and we think it was unused. I checked a forum that said age of column (if new and stored in acetonitrile shouldnt be a problem).
Experiment method
Solvent: Isocratic, 50% acetonitrile 50% water
Sample: Pure paclitaxel at known concentrations in acetronitrile
Injection volume: 50uL
Retention time (old): ~13mins
Rentention time (new column: ~8mins with additional peaks over 30 min run
The columns are both the same:
Specs: C18 5um, 250x4.5mm
Manufacturer: restek
My colleague ran a dexamethasone acetate sample (65%methanol-45%water) prior to me and her peaks did not show splitting but the retention time was also shorter compared to the old column. She built a concentration curve and the R2 value was v low, only 0.8. On the previous column she did not do this (as the column itself sprung a leak and had to be discarded) but I did for my sample before it died and it came out with .9994.
I tried running with 20uL and 10uL injection volume but still split and multiple peaks.
I have attached images of the sample run on the old column and the peak splitting on the new column.
Does anyone have any ideas on what could be happening?
Thank you in advance.
Hello, if you are not using another injection/autosampler unit, the only possiblity that cause such a problem is probably a clogged column. Since the only variable in this scenario is the column, the problem is probably occuring because of it. Try to rinse it with a proper solvent and connect the column in reverse direction. It helps to eliminate clogging. Let rinse it in reverse for 30 m. then give it another shot.
Please note: if the problem is really stemming from clogging your collegaue also would have the same problem with his/her peaks. If rinsing doesnt work, probably the integrity of the column is lost.
When installing a column, verify its operation with a standard compound. Periodically test the column for changes in peak shape and retention time.
I see a change in retention time- it appears the peak is eluting earlier on the new column. Verify that the solvents, including any modifiers such as trifluoroacetic acid are made correctly. Make sure you are using the correct injection solvent.
Thank you! Okay sounds like its just blocked then. I'm trying the back flow now and saw a couple of big peaks so hopefully it will work after that.
You must clean the column with methanol and then inject sta. Sample and see the peak of it before injection your sample
Still no change unfortunately, I've done back flow (0.1mL/min) and forward flow cleaning (between 0.1 and 1ml/min). I've cleaned with 90% methanol, 90% acentonitrile, 95% water, 90% isopropyl alcohol...Its been cleaning for a good 5 days and overnight too with these various mobile phases.
I just tried running my paclitaxel at my colleagues wavelength at 239nm and it still picked up the PAX split peak, there were slightly less multiple peaks... I thought perhaps whatever is coming out is not picking up at 239nm but just at 228nm. Perhaps its just not picking up in methanol phase. Hers are all still coming out at earlier times but she could still get a concentration profile to .99 R2 value whereas mine is 0.8 (old column with same samples was 0.994)...
So, at a loss as to what to do next, keep cleaning, or purchase a new column...?!
Not sure if anyone has any further suggestions?
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