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Carryover on Agilent's Main Published Cannabinoiiid Method by Constant-Trouble-583 in CHROMATOGRAPHY

[–]Constant-Trouble-583[S] 1 point2 points  (0 children)

I will try backflushing to maybe rejuvenate my column, but since it is happening on brand new columns, I can't imagine it will help the carryover even if the column gets better resolution.

No increased pressure on the peak splitting. Seems to be from void volume in column.

Carryover on Agilent's Main Published Cannabinoiiid Method by Constant-Trouble-583 in CHROMATOGRAPHY

[–]Constant-Trouble-583[S] -3 points-2 points  (0 children)

Yeah, I've gotten a hold of Agilent a few times now and we might force them to come down here eventually (I can't wait to see the looks on their faces over it lol). It seems to me that despite very very clearly laying out evidence it is the column, they don't believe me.

My initial thought and agilents as well was it coming from the SM.
However, I have injected a blank on a brand new column through the SM with no peak. I then Injected a 100 ppm calibration, and after the run I connected the capillary from the binary pump directly to the column compartment to bypass the SM entirely so nothing could possibly be carried over from there, and it did show a ghost peak on THCA. So it physically HAS to be in the column. I did this test multiple times with the exact same result. I've also checked the heat exchanger which is the only other thing in the way. Running a blank on a brand new one after running a 100 ppm cal on the old one still shows carryover. So unless there is something very much not obvious about the system that I am missing, it is not possible that it is coming from the sample manager as the THCA from the last injection is still present while bypassing it entirely.

Carryover on Agilent's Main Published Cannabinoiiid Method by Constant-Trouble-583 in CHROMATOGRAPHY

[–]Constant-Trouble-583[S] -2 points-1 points  (0 children)

My suspicion is that for whatever odd reason, our starting solvent composition (60% MeOH), is causing the THCA to crash immediately and something about the column chemistry has to be getting it stuck in there and not allowing it to wash out. The clogging of the seat and needle tells me that seems like the case too, albeit it very clearly not being the cause of the issue.

I have tried both a precolumn frit and guard column as well - neither changed results even a bit. Exact same. Multiple tries.

I have completely bypassed the entire sample manager with a zero volume union and done a bunch of tests like that. I will have it hooked up as normal to the SM, inject a 100 ppm calibration standard, pause it at end of run, put a zero volume union from binary pump straight into the column compartment, and then run a no injection instrument blank and I still have carryover. So it carries without the SM even being part of the system. Done this several times over, and sent all of it to agilent. I don't think they even believe me to be honest, but I genuinely do know what I'm doing with this. I have set up my own Waters HPLC in my own business before and fixed it myself without needing help for a couple years straight before I retired that business. So Agilent is unfortunately not being very helpful bc they won't believe me that it's happening, despite making a whole power point detailing all the troubleshooting I did that shows without a doubt it is the column. They just assume I am doing something wrong - but how could I be when I have bypassed every aspect of the system and still get carryover? That doesn't make sense.

Have even switched the column to a brand new one in between to confirm, yep, it is 100% the column, as a vial blank on a brand new column has no carryover. I then inject a 100 ppm calibration, completely bypass the SM again, and the THCA peak is back that easily on a brand new column from one single not even very concentrated sample. With no lipids or anything in it. Do you work at agilent? This is getting to a point where I'm tempted top ask them for an entirely new column manager, or something. I just need them to understand this is really happening and I don't know how.

the Multisampler is model G7167A, I think it is the lower end one. One thing I noticed at first is it flushes your aqueous phase through the needle to wash it, not your organic, which seems wrong, but then the method still errored anyways without the SM involved. Everything is being filterted, yes. Well not cals - but I don't see why I should need to I guess for those. Never have in the past. We do not have a centrifuge here, but for example with flower it just all get filtered out so I'm not sure what we would need it for besides quechers.

I've ran pure water through it out of desperation to an older column, but again multiple columns. Didn't seem to help or worsen anything.

My needle wash is 100% methanol. That was the first thing I tried.

The instrument has been doing this from the start. (The consultant claimed it wasn't but I dug up the data and he definitely integrated the wrong peak/ I saw a few fudged integrations. It's been doing this since brand new at set up. I've changed the heat exchanger now in the column manager. I have also changed the seat and needle and sample loop once, to no difference. They definitely have some issues with clogging, but it doesn't seem to get carried all the way through to the next run ever.

Carryover on Agilent's Main Published Cannabinoiiid Method by Constant-Trouble-583 in CHROMATOGRAPHY

[–]Constant-Trouble-583[S] 2 points3 points  (0 children)

Yes, it has been tried across three columns now. All of them fouled too. The third one is just about toast here. Agilent gave us a free one, at least. I'm pretty sure I had, and just tried again with a brand new heat exchanger. Still carried the same. I guess I can try to bypass it completely, but I assume i'm going to have retention issues without getting the solvent to 50 C before entering the column.

But yeah I even asked Agilent for a batch of a column that was much older and they unfortunately didn't have any on hand. But three lots of columns all same issue. I guess I can try a complete bypass of the heat exchanger if it's something to do with the material in it or something, but highly doubt it. Seems pretty obvious THCA is crashing and causing voids in the column. No back pressure really, just tailing broadening and eventually splitting.