UHPLC Troubleshooting: Loss of retention with DMSO in samples
Hi everyone, I’m having an HPLC issue and could really use some help.
I’m analyzing PET degradation products (TPA, MHET, BHET) from enzymatic reactions (PETases).
I recently had to switch my sample preparation protocol because the products are precipitating in samples from high-yield reactions.
The new protocol is supposed to solve this, and it also works for my colleagues using the same system and method, but for me, all retention is completely gone. everything elutes in the void/injection peak.
Here are the method details:
System: Dionex Ultimate 3000
Column: YMC Triart C18 at 40 °C
Mobile Phase: Isocratic, 20% Acetonitrile / 80% Water (acidified with 0.05% TFA)
Flow Rate: 0.4 mL/min
Detection: UV (254 nm)
Injection Vol: 15 µL (I also tried 1 µL, didn't help)
Now for the Sample prep:
The "Old" Protocol (Works, but products precipitate in water at high conc.):
12.5 µL reaction supernatant added to 50 µL Acetonitrile (with 1% TFA). Let sit briefly at RT, then centrifuge to pellet proteins. Transfer supernatant into 150 µL MilliQ H2O.
Result: Good separation of TPA, MHET, and BHET.
The "New" Protocol:
Dilute the enzymatic reaction directly 1:10 in DMSO (10 µL sample into 90 µL DMSO) to keep products in solution.
I froze this DMSO stock until further preparation. (Note: We also freeze 1M TPA standards in 100% DMSO without issues).
Take 12.5 µL of the thawed DMSO-mix and add to 50 µL AcN (with 1% TFA). Centrifuge, take supernatant, add to 150 µL MilliQ H2O.
Result:
No separation. Everything co-elutes as one massive peak at the void volume. I suspected strong solvent effect, but the same method has worked before on the same HPLC system, same column, mobile phase and vials...
Following things I have already tried:
Diluting the final sample with 20% AcN and also injecting less volume (1 µL instead of 15 µL), but it didn't solve the issue.
I also tried making fresh preparations from the DMSO sample stocks, but still got the same result.
Do you have any suggestions what could be the issue here or what else to try?
I really hope to be able to somehow salvage my DMSO-diluted samples so i can avoid to repeat the whole experiment...