Bad separation, intensity and smearing of peaks in H-class UPLC, Amino Acid Analysis

Hi, have you ever seen this kind of chromatogram appearance in amino acid analysis?
We are currently working on amino acid analysis; it worked good before, until we closed the system. Once reopening the system, we had problems of resolution and intensity and received this kind of chromatogram. We tried changing the reagents used for mobile phase and for the samples making without success. Also we tried injecting ready samples made elsewhere and got the same results. In addition we switched several columns, and their positions in the system but it did not help. When using the same system for a different method with a different detector (PDA-TS instead of PDA), we received good results. Do you have any suggestions of what can cause this problem?
I am adding pictures showing previous results of the same concentration compared to current results.


  • Hi Gama,

    Amino Acid analysis can be sensitive to very small changes in temperature and MP composition.  I'd suggest making sure that the column heater is set appropriately, working correctly and that the column is seated in the plastic holders that keep the column from touching the sides of the heater.

    We have also seen some success in reducing the gradient delay time (if your instrument allows that.)  The delay time between the gradient start and the injection step can be too short. Consequently, the most hydrophilic amino acids are not well trapped on the column and are eluted too quickly, resulting in poor chromatographic resolution.  

    Decrease the timing in the inlet method (QSM) in section "Gradient start before injection" can be helpful. For instance, set 0.5min instead of 0.8min.

    Here are some KB articles that may also be helpful - Link

    Good luck!

  • Hi Marc, thank you so much for your answer and your time. We have not changed the running program, used newly arrived solvents and the heater works good. The analysis worked well for us before with the current program so we are trying to figure out what can be the cause for such change. I will have a further look at the articles you sent. thank you!