I'm attempting to use a Waters Acquity UPLC prior to mass spec and I'm seeing really weird chromatograms. When I inject water, the PDA detector shows a steadily increasing signal (looks like a ramp) that levels off and never comes back down to baseline. I see the same phenomenon when I inject BSA (0.1 ug total). What am I missing here?
MPA: Water + 0.05% FA
MPB: ACN + 0.05% FA
Method: 0-100% in 15 minutes, flow rate: 0.3 ml/min
Using a reverse phase column.
For any questions related to "HPLC Analysis" you really should consult your teacher at your school for help. Troubleshooting HPLC questions is hard to do via the web, esp for students who are new to the technique. It takes many years to learn just the basics, so for us to help you, we need some basic info. Please provide us with the basic HPLC details and setting used for your analysis.
Let's start with the column dimensions used (L x ID x particle size). Next, what is the exact gradient mobile phase program used (Time 0, A : B..... To end of run)? You state flow rate is 0.300 mL/min so we have that. What is the INJECTION Solvent (what did you dissolve your sample in, exactly!)? What is the injection volume? Now, here are some critical questions. Please state the exact wavelength and bandwidth used for this analysis. We can not tell what you are monitoring from the three examples provided so please tell us. Also, when do you have the autobalance set to run (at what time)? What is the scale shown for absorbance in your example (in units, such as mAU)?
Some early solution guesses would be your system is not equilbrated before analysis, you did not set the absorbance scale correctly and/or your injection solution is very different than the mobile phase causing the signal to go off scale after Tzero time, when it reaches the detector. However, you have not provided enough basic info to really know so guessing is a waste of time. Please provide the basic analysis details and I am sure what you have observed can be easily explained.
check your extra column column . Try to short tubing between column and detector . Best of luck
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Column