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- Posts: 4
- Joined: Fri Jun 06, 2025 9:54 am
Hi,
We have a new-ish (2023) Buchi Pure C850 system in our organic chemistry lab that is working _fine_ (ahem, it's already had its pump, collecting arm, touchscreen, and software-related issues, some of which happened halfway through runs causing product loss, and they were quite expensive to fix) but it is generally working OK with normal phase -it does need like 2x the solvent, test tubes and time our ancient Biotage Isolera (2008) takes for the same level of separation of similar samples, but hey ho.
However, I keep finding a problem when doing reverse phase: For RP I start with a few volumes of weak solvent (water) only, then I proceed to increase the ACN gradient. And here's where my problems start:
When purifying similar amounts of the same mixture of 6-7 products and using the same C18 cartridge, my target molecule comes out 95+% pure at around 40% ACN (halfway through the run) in the biotage (the rest of molecules in the mixture elute separately between 25 and 70% ACN).
When separating the same mixture in the Buchi, every product tends to come out mixed with each other during the first 2-3 volumes of 100% water, or maybe a few products come out mixed first, and other few products are eluted as a mixture too in the next tube with like 99-98% water as an eluent.
I am using a 6g C18 Biotage Sfar cartridge for 100-150 mg of crude which should be enough. This is always the same one (I move it from the Buchi to the Biotage and the other way around), so it doesn't seem to be a cartridge-related issue. Increasing the size of the cartridge to 12g or/and using Buchi C18 cartridges hasn't improved things either.
I've had this issue since day one of trying to do RP, and from time to time when my product doesn't come out in the first water only fractions, then it might not come out at all until the end of the run with 100% ACN in which case I will get 2-3 different fractions, with mixtures of a couple products each. Sometimes nothing will come out at all during the run, and then everything will elute together _after_ having reached 100% ACN, ran a few volumes of it, and when equilibrating the cartridge back to 75% ACN for storage.
Something else I've noticed is, I never recover as much sample as I inject (only around, say, 70%).
Only one or two every 10 attempts have resulted in the products being eluted more or less accordingly to the gradient or at least while the gradient is happening (not with water only, not with ACN only, not post-run), but the separation is still quite poor. There's been no changes in the conditions of gradient, cartridges or pressure, and the odd "still bad but at least during the gradient" separation seems to happen at random and very seldom.
Over the last 2 years I've tried different cartridges, pressures (within the limit of course), flowrates, slower and faster gradients... nothing seems to improve the issue.
Having discussed this with the Buchi people who came to install the instrument, engineers, vendors etc, they basically "blamed" it on us for wanting to use the same conditions we use with the Biotage (again, there's no "conditions" when the separation is running with water only, if the products come out _before_ the "conditions" even start!). They even brought their "test mix" (which is for NP separation only, oh well) to show us how everything comes out exactly when it should according to the datasheet. During one of these visits I did an RP run of one of my crude products in front of them and showed them everything eluting at once by the 3rd volume of water (and compared it to how different things eluted differently over a gradient in the Isolera): they said it might be that the liquid injection valve was a bit loose and they tightened it. But the problem persists.
Our interactions with Buchi about this are becoming problematic as our contacts are getting increasingly defensive whenever we mention it, they always hint at us doing something wrong, and actually there's not much use discussing it anymore as none of them are chemists anyway and they don't see anything strange in the retention times we are getting, or in the fact that very fatty-like molecules coelute with hydrophylic ones. They keep insisting that their NP "test mix" is being separated according to the datasheet and that's it.
I never really liked this instrument for many reasons, and it is obvious that even an ancient Biotage system performs much better, but at least I would expect some RP separation from the Buchi, even on the poor side...What might be happening there? Could it be that the Buchi Pure is, for whatever the reason, just not great for reverse phase? Has anyone noticed similar issues with a Buchi Pure instrument on RP? What are your thoughts?
We have a new-ish (2023) Buchi Pure C850 system in our organic chemistry lab that is working _fine_ (ahem, it's already had its pump, collecting arm, touchscreen, and software-related issues, some of which happened halfway through runs causing product loss, and they were quite expensive to fix) but it is generally working OK with normal phase -it does need like 2x the solvent, test tubes and time our ancient Biotage Isolera (2008) takes for the same level of separation of similar samples, but hey ho.
However, I keep finding a problem when doing reverse phase: For RP I start with a few volumes of weak solvent (water) only, then I proceed to increase the ACN gradient. And here's where my problems start:
When purifying similar amounts of the same mixture of 6-7 products and using the same C18 cartridge, my target molecule comes out 95+% pure at around 40% ACN (halfway through the run) in the biotage (the rest of molecules in the mixture elute separately between 25 and 70% ACN).
When separating the same mixture in the Buchi, every product tends to come out mixed with each other during the first 2-3 volumes of 100% water, or maybe a few products come out mixed first, and other few products are eluted as a mixture too in the next tube with like 99-98% water as an eluent.
I am using a 6g C18 Biotage Sfar cartridge for 100-150 mg of crude which should be enough. This is always the same one (I move it from the Buchi to the Biotage and the other way around), so it doesn't seem to be a cartridge-related issue. Increasing the size of the cartridge to 12g or/and using Buchi C18 cartridges hasn't improved things either.
I've had this issue since day one of trying to do RP, and from time to time when my product doesn't come out in the first water only fractions, then it might not come out at all until the end of the run with 100% ACN in which case I will get 2-3 different fractions, with mixtures of a couple products each. Sometimes nothing will come out at all during the run, and then everything will elute together _after_ having reached 100% ACN, ran a few volumes of it, and when equilibrating the cartridge back to 75% ACN for storage.
Something else I've noticed is, I never recover as much sample as I inject (only around, say, 70%).
Only one or two every 10 attempts have resulted in the products being eluted more or less accordingly to the gradient or at least while the gradient is happening (not with water only, not with ACN only, not post-run), but the separation is still quite poor. There's been no changes in the conditions of gradient, cartridges or pressure, and the odd "still bad but at least during the gradient" separation seems to happen at random and very seldom.
Over the last 2 years I've tried different cartridges, pressures (within the limit of course), flowrates, slower and faster gradients... nothing seems to improve the issue.
Having discussed this with the Buchi people who came to install the instrument, engineers, vendors etc, they basically "blamed" it on us for wanting to use the same conditions we use with the Biotage (again, there's no "conditions" when the separation is running with water only, if the products come out _before_ the "conditions" even start!). They even brought their "test mix" (which is for NP separation only, oh well) to show us how everything comes out exactly when it should according to the datasheet. During one of these visits I did an RP run of one of my crude products in front of them and showed them everything eluting at once by the 3rd volume of water (and compared it to how different things eluted differently over a gradient in the Isolera): they said it might be that the liquid injection valve was a bit loose and they tightened it. But the problem persists.
Our interactions with Buchi about this are becoming problematic as our contacts are getting increasingly defensive whenever we mention it, they always hint at us doing something wrong, and actually there's not much use discussing it anymore as none of them are chemists anyway and they don't see anything strange in the retention times we are getting, or in the fact that very fatty-like molecules coelute with hydrophylic ones. They keep insisting that their NP "test mix" is being separated according to the datasheet and that's it.
I never really liked this instrument for many reasons, and it is obvious that even an ancient Biotage system performs much better, but at least I would expect some RP separation from the Buchi, even on the poor side...What might be happening there? Could it be that the Buchi Pure is, for whatever the reason, just not great for reverse phase? Has anyone noticed similar issues with a Buchi Pure instrument on RP? What are your thoughts?
