Punkscience just destroyed another £400 HPLC column.
I'm trying to quantify metallothioneins in my beasties according to a method adapted from these two papers. And its not working.
I can't seem to get a peak from the derivatised metallothionein, no matter what I do. The derivatising agent, 6-IAF, should bind happily to thiols such as those on the MT. I've tried pre-incubating the MT with cadmium to ensure the thiols are saturated, there's EDTA in the labelling mixture too which should pull those metals off and allow the label to bind. I've also tried adding a reducing agent, TCEP, so that the thiols aren't oxidised and unable to bind the metals/label. Still no peak.
I've varied the gradient, I've extended it to silly lengths just in case the MT peak was being masked by one of the other peaks that come out. Still nothing.
I'm using the gradient version of the HPLC protocol with acetonitrile/methanol and water and when I go to clean the columns after a busy day's cocking around and getting no peaks I ramp the organic eluent up to 100% to clean all the crap off the column. The first time I tried this with one of my bitch columns a load of cloudy white stuff came off as I ramped the organic phase back down again. Oh dear. That would be the stationary phase being stripped off the silica particles then. Exit first column.
I then tried using my uber-bad analytical column I bought a couple of years ago for my crab-urine work. This should have improved separation and resolution of any peaks. It didn't. No.
Instead, after taking great care not to ramp the solvents up and down too quickly and to rinse the column with milliQ I ran the wrong gradient program one day and got another nasty little white cloud in my waste bottle, just like before. I almost cried that time.
Then yesterday, using my last column- a random C8 one I 'acquired' nefariously- I switched from the stupid, shitty MT analysis to another piece of work I was planning, which also used an acetonitrile-water gradient and managed to fuck that column too, first time I tried to wash it.
Bastard fuckbollock goat cock. Now I am out of good RP columns and still have no data.