Hello,
I've been a experiencing an issue with decreasing peak response (both peak area and peak height) when changing the initial conditions of my solvent composition. I have a single analyte (which I unfortunately cannot provide any details of) in my method, which in some ways makes troubleshooting easier, though makes it trickier in other ways.
Please see the below table for general method and results comparison, and chromatogram. Method 1 is isocratic and starts at a high percentage of organic as this was a method I was using with a field service tech to troubleshoot some hardware issues. Once those issues were resolved, I adjusted the method by reducing the starting composition to 60% organic, but in doing so, get less 53% peak area and 40% peak height. Standard-to-internal standard ratios are similar between the two methods.
| Method 1 | Method 2 | |
| Injection Vol. (uL) | 20 | 20 |
| Flow Rate (mL/min) | 0.6 | 0.6 |
| Pump (Isocratic) | 80% ACN | 60% ACN |
| Separation Temp (degC) | 40 | 40 |
| Scan Type | dMRM | dMRM |
| RT of Analyte (min) | 0.7 | 1.4 |
| Avg Peak Height of Analyte (n = 3) | 108393 | 43170 |
| Peak Height Repeatability (n = 3) | 2.3% | 2.2% |
| Avg Peak Area of Analyte (n = 3) | 304464 | 162487 |
| Peak Area Repeatability (n = 3) | 2.4% | 1.1% |
| Avg STD:ISTD (n = 3) | 12.3 | 12.2 |
| STD:ISTD Repeatability (n = 3) | 0.8% | 0.7% |
Am I missing something obvious? Does this point to a method/application problem or a hardware problem? Any insight would be greatly welcomed!
