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- Posts: 2
- Joined: Tue Feb 18, 2020 7:49 am
I am currently optimising a UPLC purity/content method for a PEGylated peptide drug product. To elute small N-terminal fragments, the gradient at the front was adapted, starting from a lower percentage of acetonitrile (0.5% instead of 5%). The slope remained the same.
The following set-up is used:
-Waters Acquity UPLC H-Class with 425 µL mixer
-Flow rate: 0.30 mL/min
-Column: Waters Acquity CSH C18, 130A, 1.7µm, 2.1 x 100mm
-Detector: UV or PDA at 215 nm
-Mobile phase A: 0.05% TFA in water (high grade TFA is used in 1mL ampoules)
-Mobile phase B: 0.04% TFA in ACN
Using this gradient on a degraded drug product sample containing the N-terminal fragments, we get a desirable separation of the degradants, however the baseline is not flat.
Although we are already using a larger mixer, we hypothesised the mixing of the mobile phases had something to do with it. Therefore, we prepared a premixed mobile phase with 99.5% H2O and 0.5% ACN (with 0.05% TFA).
Using the premixed mobile phase, we already get a better result, but still the baseline is not really under control.
We additionally tested:
- other supplier of TFA: no influence
- slower gradient (50% slower): no improvement
- longer equilibration at 0.5% ACN at start (2.5min instead of 0.5min): no improvement
- mobile phase B (ACN) without TFA: same 'jump' visible
The same method was run in a different lab on Agilent 1290 UPLC system and there the baseline was much better.
Is this something system related? What could possible solutions be? As we are working in a GMP context we do not have much room to change the set-up of our equipment unfortunately.
Thanks a lot in advance!
all relevant images:
EDIT: embedding image did not work, this is the link to the images:
https://imgur.com/a/YlUTfn3
The following set-up is used:
-Waters Acquity UPLC H-Class with 425 µL mixer
-Flow rate: 0.30 mL/min
-Column: Waters Acquity CSH C18, 130A, 1.7µm, 2.1 x 100mm
-Detector: UV or PDA at 215 nm
-Mobile phase A: 0.05% TFA in water (high grade TFA is used in 1mL ampoules)
-Mobile phase B: 0.04% TFA in ACN
Using this gradient on a degraded drug product sample containing the N-terminal fragments, we get a desirable separation of the degradants, however the baseline is not flat.
Although we are already using a larger mixer, we hypothesised the mixing of the mobile phases had something to do with it. Therefore, we prepared a premixed mobile phase with 99.5% H2O and 0.5% ACN (with 0.05% TFA).
Using the premixed mobile phase, we already get a better result, but still the baseline is not really under control.
We additionally tested:
- other supplier of TFA: no influence
- slower gradient (50% slower): no improvement
- longer equilibration at 0.5% ACN at start (2.5min instead of 0.5min): no improvement
- mobile phase B (ACN) without TFA: same 'jump' visible
The same method was run in a different lab on Agilent 1290 UPLC system and there the baseline was much better.
Is this something system related? What could possible solutions be? As we are working in a GMP context we do not have much room to change the set-up of our equipment unfortunately.
Thanks a lot in advance!
all relevant images:
EDIT: embedding image did not work, this is the link to the images:
https://imgur.com/a/YlUTfn3
