Chemical Forums
General Forums => Generic Discussion => Topic started by: enahs on January 28, 2008, 06:18:27 PM
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Cleaning a HPLC column is so boring.
Just thought I would mention that...
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Any job, well done, is its own reward. Well, maybe not. But, personally, I like tinkering with equipment. What dirtied it, and how are you cleaning it?
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Yea tell me about it. Replacing any HPLC parts can be tedious but also educational.
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What dirtied it, and how are you cleaning it?
Well, it is a C18 column. It is rated for 300-500 injections, but we have gotten over 5,000 and still getting good separation. And sure it is educational, but I have done it many times before.
To clean a C18 column and the typical injections we do.
95% H2O : 5%Methanol, for at least 25 equivalent volumes of the column. Ramping the flow rate up and down through-out the process. Today to maintain a safe pressure this was at ~ 0.7mL/min. And so a 1.2mL column took ~35 min. You can automate it, but you can get better results by manually ramping up and down the flow rate (to watch pressure) and see how much, if any comes out and making adjustments, or going more then 25 column equivalents.
Then 100% Methanol for 25 column equivalents. Again, ramping and lowering, making adjustments, going longer if needed, etc.
Then 100% 2-Propanol (Isopropyl alcohol) for 25 column equivalents. Ramping and increasing number of equivalents as needed.
Then 100% mixture of Hexanes. 25, ramping. However, so much was coming out with the Hexanes today
I did over 200 column equivalents.
Back to 100% 2-Propanol. 25, or as needed, ramping, etc. Never ever mix the Hexanes and Methanol directly, unless you want to destroy the column!
Then 100% Methanol, 25, or as needed, ramping.
Then 100% assay system (normal mobile phase being used in analysis), 25 equivalents, ramping, etc.
The HPLC I was doing it on only has two pumps, so there was a lot of switching. And between each switching of mobile phase you have to purge the pumps; with this system means tedious physical work. And you can not just go from 100% A to 100% B, it has to be a smooth gradient. So that makes switching out take longer too.
Before, the assay I was trying to run the pressure would get around 4000 (103) PSI. Afterwards it was back down to 2500 (103) PSI! With excellent separation again.
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Ah, very nice. Lots of people don't realize the best way to clean a column is with a gradient. Thorough priming, while time consuming, is important -- it doesn't save time if the pump ends up failing.