We have a developed a method for relating substance to compounds which have almost 30 impurities, out of which one impurity (0.25% level) is eluting at non-polar region.
As a part of validation all the impurities spiked at 1 % level, but due to a change in concentration, the said impurity RT has changed. Keeping in view of difficulty and nature of impurity, we are now developing a new gradient method with below conditions:
Column: Ascentis RP-Amide (250 x 4.6, 3.5 um),
Mobile phase: 0.1 % TFA in Water: MeOH (90:10)
0.1 % TFA in ACN:MeOH (90:10).
With this method we were able to obtain good reproducibility.
Validation was initiated for this method, when we used different columns with same lots numbers as part of an intermediate precision, we are getting RT shift (tried 3 same make of columns), RTs are different from one another.
I am running out of ideas, so I was wondering if anybody has experienced the same or similar problem in the past.
Thanks in advance
Retention times should not be very different. However it is very unlikely that the RT, absent changes, since it can not guarantee that the packaging is exactly the same, and would think that you should not compare retention times, and instead, compare the resolution between peaks.
0.1% TFA in water or in acetonitrile....It is difficult to get exactly the same retention time even with the same column. If the % becomes 0.12 or 0.08; pH will change and the retention times will change. You have 30 impurities. The longer the retention time more will be the difference in the retention time. How much is your total analysis time? Later eluting peaks will show more change in retention. Don't see only retention times; see relative retention times.
Dear Mr.Rao,
There are many reason in lc that operator only knows , and please cross check the below points
1)if available,inject the specific CRM to find out the RT.
2)Do performance check of the instrument,
3)Double check the method settings
4)Do check the Characteristic of the compound.
regards
Sekar.M
I am sure you resolved this issue a few years ago, but for anyone reading this thread after-the-fact, I hope they provide more information in their post so others can help. Lots of missing info. For example:
What is the exact HPLC method you are running (specific time/% composition values)? That is critical info for an HPLC question. What pH value are you trying to use? Maybe you do not need TFA. Unless you are working near pH 2, don't use 0.1 % TFA as it is a very strong ion pairing agent and can change the surface chemistry of the column (when the column surface chemistry is changed, so are your separation results). Try a less aggressive acid (e.g. Formic, Acetic) and make sure you are starting your method with some organic (not pure water) content (e.g. 5%) and ending your method with a high percentage of organic (but not 100%!) to make sure everything has eluted off the column.
Also, even columns from the same exact batch/lot can vary, but you have not provided any data regarding what the measured retention time shift variation was (% or Rt). It may be related to your method, your training or your column wash methods (run after every analysis method) too.
Here is an article which addresses reasons for retention time variations and how to troubleshoot them. "HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it."
http://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
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