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.

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