Chromatography Forum

One of the earlier methods is water acetonitrile gradient, the other is water THF isocratic. The first one works well, the second one sometimes gives the same problem; not so quickly as the new method; sometimes it is possible to perform a complete analysis (about one day) without splitting the peaks, sometimes the column does not resist.

Multiple blank injections between sample injections did not help.

I still have to try injectind more diluted sample, rising the temperature (although the column manufacturer told that with higher temperatures the permitted THF quantity decreases) and to filter the mobile phase one hour after the preparation.

I will get back as soon as I have the answers.

I have not tried either using buffer solution instead of water. Since the compound is not ionic, I developed the method without buffers. However, it could be another thing to try.

The mixing of THF/Water creates a warm mixture, and I often see a slightly turbid solution. Hence my preference to premix and filter aqueous mixtures of solvents like methanol and THF. I wondered whether sample and mobile phase temperature changes were contributing to the insoluble material and split peaks.

Other questions I have relate to needle wash solvent ( do you use one, if so, what? ), and do you use a pump seal wash, such as IPA/Water?

The reason I suggested using a buffer ( besides desperation ), is because it's not clear to me that it's your known non-ionic sample, or the THF, that are causing the split peak and pressure problems, but perhaps unknown mobile phase interaction.

Good luck,

Bruce Hamilton

Bruce,

The needle wash and seal wash we use is CH3CN / H2O = 90 / 10. The product is soluble in CH3CN.

Kati

I am sorry if the question sounds silly but are you filtering your samples before injecting? From what I have read and understood, I dont think your THF is the problem, I think its sample prep and some rubbish going into the column thats causing your problems.

Also, do you use a guard column?

If you are filtering the samples, then what filters are you using?



Salma

Kati,
I'm begining to think that the problem is dirty samples. What is your sample matrix and what is your sample prep?

like the majority of here, I am sure the columns were fed by junk after some injections. So the question is what the junk is, where it comes or how it forms.
I have the following q which may or may not be related to answere the above q:
1) what will happen if you added 0.1% H2O2 to your mobile phase?
2) What will happen if you treat THF with an THF stablizing agency?
3) How many column you were using gave the splitting? how many old or new? Can you recover these bad column? If you can, do these recovered column work fine when they were used in other projects?
4) how many LC gave this splitting? Does these LC work fine with other projects which has THF in the mobile phase?

5) did you have your LC changed their tubing from your validation time to the bad time? Make sure you have good tubing which is compatible with THF.
6) what is your API structure, impurity, excipients? Make sure nothing contaminate your sample during the sample preparation, such as filter membrane was partially dissolved into the sample solution. Find all possible degradation. Get organic chemist to help.
7) can you try other brand c18 such LUna c18;
sorry I got to go. let me know if you can share with your detail sample information and wnat me to help abnout the possible degradation. good luck
7)

If Kati doesn´t approach this problem systematically, eliminating possibilities which have been disproven, etc. instead of following every whim and irrationality she will still be on this problem at pension time.

Hans, thank you to put down what I had intended to.

Hi Kati,
It seems you have already done lot to find the cause.

Just one request. Could you please post HPLC chromatogram of split and normal peak ?

JM

Hi JM,

Sorry, I do not know how to do it. If you explain me, of course I can post examples.

Thanks,

Kati

It is explained in this topic by Tom Jupile

Sticky: Embedding Chromatograms and reports

It is fourth from the top of of the page.

I posted my first chromatogram last week and the instructions made it very easy.

Regards, Pat.