Chromatography Forum

Hi,

For my separation, my mobile phases are DI + 0.1% TFA and ACN + 0.1% and I am using a 0 - 100% gradient, with the absorbance at 216 nm. For recent chromatograms the absorbance has been shifting down as more acetonitrile is added. I have run the exact same method, column, and sample using the above solvents and have seen the typical chromatogram for the previous runs. I was wondering if anyone knew the causes of this downward shift?

Thanks!

I suspect you may be using HPLC for the first time. It is very helpful to be aware of the properties of the various solvents and additives used in chromatography so you can tell if a "change" is from a problem or is in fact normal and expected. You are probably observing normal changes in absorbance (at low UV) due to changes in solvent composition. Small changes in absorbance are normal, but I am unsure how much drift you observed overall, relative to time so provided the system is fully equilibrated and the temperature stable, it might be normal. Water absorbs more light energy than pure ACN (because ACN is more transparent at low UV ranges). *This is one of the reasons why ACN is such a commonly used solvent in liquid chromatography. As the TFA ages, it also absorbs more and more light resulting in more dramatic changes or shifts, relative to pure ACN alone. The TFA used may be degraded causing changes in absorbance.

Three suggestions: (1) If possible, never use 0.1% TFA in a method. TFA at even 0.1 % concentration is a VERY strong acid. Try 0.05% or less, or another acid, if applicable to the method. Less TFA equals less interference and less absorbance resulting in better baseline quality and stability. (2) Use fresh acid(s) and RO water for mobile phase solutions with HPLC methods. (3) Do not run a gradient from 0-100% ACN on a column. Use only the composition range needed or, if scouting for the method, start at 5 or 10% ACN and run to no more than 95% ACN. Be sure to wash the column with a stronger solvent after the analysis too, to prevent (reduce) column fouling and buildup. Allow enough time for re-equilibration between methods.