Chromatography Forum

There are three compounds A、 B and C ( A : Theino [3,2-c] -4,5,6,7-tetrahydropyrindine hydrocholoride ; B: Formine of 2-thiophene ethylamine ; C : 2-thiophene ethylamine ) to be separate useing HPLC method !
During my test , I find A and B having a good resolution ,but rentention time of B and C is same , then I try to change the kinds the mobile phase or the pH or the column (ALL COLUMN WE HAVE IN OUR COMPANY!) , I get the failure results at last!
Note : The column I had used mostly are ODS C18, but the column for the normal phase I had used too !
if any one has any good advice , plese tell me ! thank you!
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hi,
check out this Purospher RP-18, 250 x 4.0 mm, 5.0µm (Make: Merck)
perchlorate buffer with pH 2.0
having linear gradient programme.
good luck
vijay

Hi bily200677
You don’t state the pH values you’ve tried, but I have the feeling that compounds B and C are positively charged under the tested conditions and that would be a sensible explanation of the co-elution you’re experiencing.
Given the deficit of information provided, it is difficult to get a clear idea of the situation, but I would not be surprised if these 2 compounds eluted unreasonably early/fast.
If you haven’t tried mobile phase at high pH (e.g. 9 – 10) it might be worth trying, but check the compounds' pKa first. Also, please remember to choose suitable column chemistry (i.e. a column that’s stabile at high pH values). It could be polymer based or even better; silica/organic hybrid based.
Alternatively, but certainly not my first choice, you could explore the ion pairing technique - if everything else fails.

Good luck

If I read this correctly, compound 2 is the formate salt of compound 3. Is my interpretation correct?

If I read this correctly, compound 2 is the formate salt of compound 3. Is my interpretation correct?
REPLY:
Firstly, thank you for your help! the correct name of the compound2 should be Methylene -2-thiophene ethylamine !

If I read this correctly, compound 2 is the formate salt of compound 3. Is my interpretation correct?
REPLY:
Firstly, thank you for your help! the correct name of the compound2 should be Methylene -2-thiophene ethylamine !
Secondly, thank DANKO , Owe Nene and Upamaniah very much ! Your adivices are very useful for me , I hope get a successful result in furture!
Lastly , I have a little question for these three compounds , in which , B and C show some basic , so the pH of mobile phase down to 2~3 ,the condition is suitable for testing ? the sample solution stablilty in this pH ?

pka of B and C should be very similar if not identical. Have you tried gradient elution or are you trying only isocratically? A mobile phase containing TFA or higher perflourocarboxylic acids and gradient with ACN should to the job...

Hi bily200677,

If you choose to follow my suggestion, the pH should be relatively high (e.g. 9 – 10 ) in order to avoid the positive charge on the amine.
Actually the best approach would be to find the pKa for these two compounds and proceed from there. For instance, if the pKas are in the 7 – 8 region, then the above suggested mobile phase pH (i.e. 9 – 10 ) would be suitable. If you can’t find these pKas, you can just go for the “try and errorâ€

Thank you Danko again in here! As you said, the the pKa value of these two compounds I can't get , so I want to change the colum padding to try and try !
In addition, the PH up to 7~9.0,I had tried in the past , the gradinet elution method I also tried, but the results is same, the two copmnds B and C normally elute out very early, the rentention time is about 3~4min !
The mobile phase I used is made up of ACN and buffer solution ( include Potassium dihydrogen phosphate, acetate , heptane sodium sulfonate and triethylamine ) , the effect have not any change .
In any case, the advices provided for me give some inspiring to go on this test !
By the way, thanks a lot to Kostas Petritis and every one for your kindful help ! Best wishs send to you!
Bily

Maybe you use too much ACN? What is the buffer/ACN ratio?

Best Regards

Maybe you use too much ACN? What is the buffer/ACN ratio?

Best Regards

Reply:
Normally the buffer/ACN ratio is 87:13 under isocratic conditions , in the other hand , I start 5% ACN increast to 80% when I using the gradient method , or I use Methanol repalce ACN, the rentention time of two compounds have not happen obvious changes, that is to say, the polarity of solvent have no influence to the retention action for these two substances! My idea is right?
thank you Danko!

Have you thought about using HILIC as these are polar compounds.

If you do a search using HILIC you will find information on this site.

Yes, I agree.

ZIC®-HILIC is the suitable alternative for this problem.