I'm separating four components on RP-HPLC peak. A retention time is 2 minutes, peak B's RT is 4 minutes, peak C is 5.8 minutes and peak D at 12.5 minutes, but the peak D's RT changes in consecutive injections and the rest all elute at the same RT i.e peak A,B,C's RT is not changing but only peak D's RT is shifting from 12 minutes to 9 minutes. Why does this happen?
Flow is 1ml/min
column_C18_250x4.6mm
mobile phase is phosphate buffer:MeOH(50:50)
(buffer :: KH2PO4 8.5gm+TEA 5ml +OPA-2.5ml)
Any ideas?
Dear robin thanks for rply
but what will happen if i will delay injection time?????
This should not happen that RT of one compounds is changing.
When one injection will complete then give 3-5min delay to next injection. It will equlilibriate your column with mobile phase for next injection because some time during gradient some compounds did not get complete stablised with the mobile phase. So to complete stablisation of mobile phase with column and compounds give some delay time. Try it otherwise I will give u some other solution
Gudluck
:)
Dear robin thanks but dear my system is ISOCRATIC...not gradient...
Then there is some problm of standard because it may react with your buffer.
Try another mobile phase, use column temperature. But I think it is problem of your standard with mobile phase.
Same problem I m facing Mr Jagdish with my 3 component standard, I think the peak that is moving comparatively faster than other two is having pKa closer to the pH of the mobile phase. I am also working right now on same issue. Kindly co-operate
You may want to try at a lower flow rate and see if the RT changes as sometimes longer stay in the column may make to column -sample interaction becomes better, but be careful, sometimes the peak shape may be broader too.
Just out of curiosity, are you limited to HPLC for this project? Or do you have a GC option? Good luck with your work.
The mobile phase contain TEA, is component D a basic compound with amine group? Which is the pKa of the compound and the final pH of the mobile phase?
I have had compounds hopping around as you do. I selected a pH (buffered) at a point wher only one form (degree of ionization) predominates. I would also try increasing your MeOH % and run the compounds out faster. At a RT of 2 min, you can speed this up if there are no other interferences. It also may help stabilize Cmpd D's RT. At 12.5 min RT, that is a costly (time perspective) analysis. Third, check the col specs to see if column has been end capped. That could be very important. And try some different vendors. Not all RP columns are created equal. There are subtle (and some times not so subtle!) column chemistry differences. Bona Fortuna!
Dear friends thanks for your precious reply
@ Jonathan,peak shape is not disturbs but only problem is of peak shift.
@ silvia pH of mobile phase is 4.6
@ Okorie, we do have GC option but HPLC is convenient for us thats Wy we r trying to develop on hplc.if hplc wont work then will shift to GC development
@ mustaf sir, I tried at lower flow rate but facing the same problem.
@ william , I will try end capped column
thxxx
Greetings Mr. Jagdish,
Just wonder have you ever tried to run these standards in gradient setting instead of isocratic? There is a possibilities that some standard 'leftover' in the column due to its interaction with the packing material. As suggested by Mr. Robin Joshi, you can try to equlibrate the column with higher percentage of solvent at the end of the run. Afterall your column is a 250mm X 4.6 dimension. You will need a longer time :-) Good luck yeah
Mr. Chandra
thanks for your valuable comment.
i will try wtever u said here n get back to u with result
if u have any more suggetion then alwayz welcome.
thxxxxx
Dear Jagdish
You are using TEA in the mobile phase, I hope you are washing the column after the day's analysis with about 15 column volumes of washing procedure.
If you are washing the column with different column volumes, this TEA will be an issue.
For example one day the washing volume is 45 ml and another day about 30ml will differ the amount of TEA available in the stationary phse and it results RT differences.
Try with constant columes washing for the used column
Satyanarayana
I am also getting the same problem. My compound is basic, pKa 9.5, and I am using mobile phase pH 7. I also add 0.1% triethylamine in the phosphate buffer and final ratio is Acetonitrile:Buffer (20:80). The problem is there is the shift of the last column in repititive experiment (not in consecutive injection). I used 150*4.6mm column with guard column. I am doing pharmacokinetic study in plasma samples. Pls anyone can help solving this problem????
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