Chromatography Forum

Dear Chromatographers,
I'm trying to reproduce a method for pyridoxal 5-phosphate (PLP) quantitation by LC after derivatisations with semicarbazide+glycine but I'm having some difficulties. After running a plasma sample (HPLC PLP.jpeg) we ran a mobile phase and we found peaks of contamination (Mobile Phase.jpeg). We believe it's carryover from the last run. Please, find attached two chromatograms. Any help will be much appreciated.
Conditions:
isocratic mobile phase for chromatography was 60 mmol/l disodium hydrogen phosphate containing 9.5% methanol (V/V) and 400 mg/l EDTA disodium salt, adjusted to pH 6.5. Column Luna C18 4.6×250 mm. Flow-rate of 1.5 ml/min. excitation (Ex) and emission (Em) wavelengths were 380 and 450 nm, respectively.
Thank you.
Guilherme

Alas, you can't post images directly on the Forum. The instructions for displaying images are here:
viewtopic.php?f=1&t=2617

Thanks Tom, here is fixed. Is it for all? I appeciate some reply. Thanks.

That's a lot of carryover!

What happens of you inject a series of blanks after the plasma sample? Do the peak sizes diminish exponentially or linearly and , more important, do the sizes of both peaks diminish at the same rate (hard to tell from just those first two runs)?

If both peaks behave the same way and especially if the decrease is exponential, it suggests "physical" carryover; a pocket of sample trapped somewhere in the injection system. If the two peaks behave differently, and especially if the decrease is more linear, that suggests "chemical" carryover with sample adsorbed to a surface somewhere in the system. That is far harder to diagnose and fix. Often passivating the system with nitric (people have also recommended phosphoric) acid will help.

By the way, comparisons are easier if you take the time to crop those images down so that only the relevant parts (the chromatograms) show!