How to Clean an HPLC Column in Daily & Deeper Cleaning

In this video, we are going to teach you how to clean your column. 🧼
This is especially important to do if you add buffers or additives to your mobile phase. For instance, most people use ammonium acetate, formic acid, etc. Over time, if the column isn’t flushed out regularly, precipitates can form and can plug your column up.
We are going to show you 2 techniques to extend your column life as much as possible.
The first method is your daily maintenance method. If you have a solenoid valve in your pump, you can select up to 4 solvents. A and B can be used as your mobile phase for regular operations. C and D can then be selected to be used as your solvents for cleaning. C and D would normally consist of water and some kind of organic solvent like methanol. You can simply do a gradient after your last run of 5% to 95% methanol for over an hour.
If you feel like your column needs a deeper cleaning, it would be best to do a reverse flush. The method for reverse flush would be to
95% water and 5% methanol for 10 column columns
Do a gradient of 5 to 95% methanol for 5 column volumes
Hold 5% water and 95% for 10-column volumes
You want to run this at half your normal flow rates for your method.
The way that you know when to do deeper cleaning is to monitor the pressure build-up on your column. Always note the back pressure on your method when you first start to use your column and if pressure kicks up around 15-20% it’s probably a good idea to use the reverse flush.
When you have questions or have a topic you want to be covered let me know in the comments! 😎
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#biotech #biology #science #hplc #clanning

[@fakhrulnawawi9681]

Really helpful thankss

[@TheJoshtheboss]

Excellent video

[@Filtrous]

Thank you very much!

[@thehealer8330]

I have purchase new hplc c18 colums. I want to know how to use a new colum??. Or just start using it for routine analysis. I want your say on this

[@user-nu6dx7sf8v]

how about the flow rate of 10% Methanol???

[@kristabanks4523]

This is a good general usage recommendation, but what about for specialty columns? For instance, I sometimes use phenyl columns or HILIC columns. About 80% of what I analyze uses either C8 or C18 Reverse-phase columns, and I would definitely employ this cleaning method, but is there a better cleaning format for the lesser used types of columns?

[@arielhu4569]

I have the exact same question too

[@MuhammadUsmanAkram-usman101pak]

I’m using RP18 250 cm column with 90% 1%acetic acid and 10%MeOH on gradient where B maximum is 65%. After about 80 hours of constant use, I found peaks are moving forward for about half minute, even for standards. Can you tell me the reasons and solution. Column can work between 1-9 pH, n A phase has pH around 3.5

[@Filtrous]

you're probably not equilibrating your column long enough. you might want to hold it at B longer at the end of the run or even kick it up higher and then when you bring it back to the starting conditions - let that go for a bit before ending the run.

[@ifeoma4517]

Is 100 present ACN not good enough for flushing columns?

[@Filtrous]

not for all situations no.

[@hoa4300]

Can you write a detailed program to clean column C18 with this method (95% water and 5% methanol for 10 column columns. Do a gradient of 5 to 95% methanol for 5 column volumes. Hold 5% water and 95% for 10 column volumes?) Tks
Note: I use pharmaceutical analysis with HPLC Dionex Ultimate 3000 autosampler and Water E2695 autosampler.
Example: I used ACN: KH2PO4 0,01M (40:60) pharmaceutical analysis, so How I can clean column C18 with this method?

[@DavidYazdi1]

You need to remove your current mobile phase and replace with methanol and water. It's important to remove the salt in your column. Hopefully you have a binary or quaternary pump where you can do a gradient. You first start with a hold of 5:95 methanol to water. Then you do gradient. Finally you hold 95:5 methanol to water.

[@hoa4300]

@@DavidYazdi1 Tks so much

[@dhananjaygangadhare8333]

100 %Water is not good for column

[@danielwatson7386]

Do not follow this video. Always follow the column manufacturers manual and call their technical support if you have specific application questions. Anyone who would tell you to reverse flow through a HPLC column is basically telling you to piss way $1500.
Source: I'm a field service engineer for Thermo Fisher and I work in IC/LC chromatography.

[@Filtrous]

Hey Daniel, this is for a reverse phase column and this is in the column care guide that comes with the column. Yes, you are right that this does not apply to all columns but it does apply to this column as it is reverse phase.

[@abbesdimiri8839]

I invite everyone to read coran the last book of god on earth and judge it