Hello Michel. There are several possible explanations for peak tailing. It would help answering some questions to narrow the possible reasons. a) Is this Kinetex a semi-preparative or an analytical column? b) Is the alkaolid the principal compound of your extract? c) Is it possible that you have exceeded the sample quantity that your column can separate? [try to dilute your sample and observe if tailing is reduced]. d) it is possible that the extract has a lot of analytical background (is too complex)? Several other consituents could also change the chromatographic behavior of your system [try cleaning your extract using a sample preparation cartridge]. e) Are the solvents (sample/extraction solven and mobile phase) appropriate to keep everyting fully diluted? f) Are the two solvents compatible (sample solvent and mobile phase) miscible? g) Are all conditions of your analysis within the range specified by your column (apart from mobile phase and sample solvent you should check the temp and pH). h) Other possible reasons are incomplete degassing of solvents, problems with pump pressure, problems with sample valve, etc. i) Did you test other samples / perhaps a pure standard of the compound you are separating?

You can also contact me directly at gliak(at)aua.gr.

Regards

GL

Hey Georgios, thanks for the detailed answer and your great input.

Let me answer your question and maybe you have some further ideas:

a) it is a semi-prep column

b)yes, its the principal compound, with 2 (and one isomer) more alkaloids (lupanine)

c) checked at 10mg and 5mg without any change. Going even lower would be possible, but running too much runs would be not economical in the long run

d)I run the complete extract in multiple runs through the column, collect the fractions, evaporated the solvent and run them again without any change. So I think there shouldnt be any disturbing background left.

e+f) My extract is (since the alkaloids are in their salt form) completly dissolvable in my eluents starting with 0.1%FA in water.

g) pH is in range and temp. should be aswell. We cant control it as much, as the column is outside the instrument at RT

h) any other purification runs dont have these problems, so I guess the instrument is running fine otherwise.

Hello Michel and thank you for the feedback. What is your organic modifier to the eluent? Are you using a gradient elution? What is your sample solvent? I suspect that starting with 100% aqueous is a but challenging for a RP column. Some stationary phases do not tolerate 100% aqueous eluent as they are very hydrophobic. I would try adding some organic to the mobile phase at the beginning, including the column equilibration phase before injecting the sample. Something like 2-5% should be enough. Let me know if that makes things better.

Regards

GL

Hey Georgios, thanks for the answer again.

Im starting at 2% ACN with 0.1% formic acid, which is quite standard in our lab. My sample is solved in 0.5% FA in water. you think this could already be a problem? Just missing of 2% ACN? for me that doesnt sound dramatically.

Since we may get another column (HSS T3) I will try this one aswell and writing back my results in this thread.

Thank you for your help so far :)

Hi Michel. Trying a second column if available is a good idea. Also, despite that your sample and mobile phase solvents are very similar, you could also try to follow the old strict rule that says: "the sample should be diluted in a solvent as the mobile phase composition during injection". Hope you get some improvement.

Regards

GL

A little updated, if anyone is interested:

changing to the HSS T3 C18 column shifted the retention time of the alkaloids by 5min, but one is still having the peak tailing, aswell as bad seperation of two of the alkaloids.