I have run 300 uM of tryptophan in isocratic 50% methanol with 40 mM sodium citrate buffer pH 2.25, 400 uM SDS in a 40 min run at 35 degree and absorbance at 280 nm. During two reps the RT changed (chromatograms attached).
Column Zorbax Eclipse XDB-C18 4.6X250 mm
Flow rate: 1 mL/min
System Agilent 1260 infinity series.
You have not provided enough information to provide constructive suggestions. You are running in isocratic mode so there is a strong chance you have not properly flushed and re-equilibrated your column in between analysis runs. Temperature change, pH change, evaporation are other common reasons that may apply to your specific situation, but usually it is training based. Reproducible retention times are a product of following good practices in chromatography. The good news is that the reasons for such unwanted changes are often very simple to correct for with some basic training.
I suggest you start your troubleshooting by reviewing this free article at the link below; "HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it" *It was written to specifically help answer questions just like yours.
http://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
A change in the target molecule's microenvironment. This could be temperature (sun coming around), mobile phase (evaporation of the Methanol)....
You have not provided enough information to provide constructive suggestions. You are running in isocratic mode so there is a strong chance you have not properly flushed and re-equilibrated your column in between analysis runs. Temperature change, pH change, evaporation are other common reasons that may apply to your specific situation, but usually it is training based. Reproducible retention times are a product of following good practices in chromatography. The good news is that the reasons for such unwanted changes are often very simple to correct for with some basic training.
I suggest you start your troubleshooting by reviewing this free article at the link below; "HPLC Retention Time Drift, Change, Variability or Poor Reproducibility. Common Reasons for it" *It was written to specifically help answer questions just like yours.
http://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html
I agree with Bill. Try running a blank between two sample runs if you are in your initial phase of standardization. This will help you determine the run time, and stabilization time for the column prior to injection.
Thanks all for your responses, I tried running the same sample again considering all these points and got the same RT. I am in initial phase of standardization.
Why is there SDS in you mobile phase?
In my experience, running SDS in mobile phase during RP separations is strongly no recommended as it leads to poor and variable peak shape and may be at the root of your problem. Get rid of the SDS if at all possible.
is the system well equilibrated and does it pass system suitability prior to sample analysis?
Best of Luck!
This brings up one of the rules of troubleshooting - a problem that only happens once is not a problem.
I agree that the sodium dodecyl sulfate (SDS) is the likely culprit. The SDS is working as an ion-pairing agent in the method (ion pairs with amines to give longer retention times). Typically, you want the level of the ion pairing agent considerably higher than the concentration of your analyte(s). Otherwise reproducibility is very problematic. Equilibration with ion-pairing reagents also takes more time than a typical HPLC gradient without ion-pairing. If SDS is critical to your runs, you might try increasing the concentration in the mobile phase and allowing a longer equilibration between runs (this suggestion assumes you are washing the column with higher amounts of organic between runs). Alternatively you could add a slightly higher amount of SDS directly into the sample prior to injection. I have found that this will frequently provide reproducible retention times.
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