How to clean an HPLC column!

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

4 posts Page 1 of 1
If you have to clean an HPLC column see attached;

https://phenomenex.blog/2019/06/26/colu ... olumn-care
I would not recommend that anyone follow the Phenomenex promotional article, advertisement or suggested method to "clean" their typical HPLC column. Backflushing to wash an HPLC column (referred to as 'reverse flushing' in the advert) are best performed when other traditional methods have failed as the bed or packing material may be disturbed during a backflush. Diagnosing why the column needs cleaning or backflushing should lead you to realize that flushing the column with more appropriate solutions should be performed first.

One of the best ways to clean any HPLC column (which should of course be routinely performed after each analysis) is to put some thought into the analysis mobile phase properties used to retain and elute the samples first. A proper column wash or flush should incorporate solution(s) which are stronger than the analysis mobile phase and most importantly will fully dissolve ALL compounds which were run through the column. FULL solubility is important as you want to dissolve, then wash them all off. For example, if buffers are used, it is vital that you wash them off first using pure water (a bit of organic should be mixed in to, but keep the percentage low so you do not precipitate any salts). Methanol is usually better at dissolving salts than ACN is. Use of a stronger solvent (think polarity, less polar for RP, and better as dissolving material) should follow to dissolve and elute off any built up material Once this is done you can then re-equilibrate the column.

*Put some thought into your column wash methods and your columns will last much longer with less fouling (= better peak shape and %RSD). Leave column backflushing to when good chromatography techniques to remove fouling do not work well.
... it's a tool in the toolbox. Whether it's the right tool depends on the column and the nature of the contamination. With a modern C18 column, probably the bulk of what Phenomenex sell, the packing is so tight and reliable that reverse flushing won't cause any disturbance, and is perfectly safe. Phenomenex are column manufacturers - they're not going to recommend any procedure that causes their columns to perform worse. But on an old-fashioned irregular particle column, the column bed is less stable, and back-flushing might be a bad idea.
Similarly the nature of the contamination matters: if you are working on complex biological extracts then it is quite possible there will be components that are extremely strongly retained - to the extent that they take hours to move a few mm onto the column even in 100% strong solvent, and would take many days to get all the way through. In this situation, washing forwards with a strong solvent won't help (unless the wash keeps going for days...) - in fact if the dirt is stuck on the guard column and someone washes forwards without first changing the guard, the wash may merely move the contamination from the guard to the analytical column. Reverse flushing helps with very strongly-retained, but chromatographically well-behaved contaminants, because it reduces the distance the dirt has to travel, which means they come out quicker than in an isocratic wash.
Of course, ideally, samples containing that sort of dirt could be cleaned up by a quick SPE before analysis, but since the SPE will probably cost vastly more, per sample than a guard column, and will take extra sample-prep time, it's often more cost-effective to accept the contamination.
If there are trace amount of phosphoric acid in you mobile phase composition, you must wash column immediately after use the column.
4 posts Page 1 of 1

Who is online

In total there is 1 user online :: 0 registered, 0 hidden and 1 guest (based on users active over the past 5 minutes)
Most users ever online was 1117 on Mon Jan 31, 2022 2:50 pm

Users browsing this forum: No registered users and 1 guest

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food & Beverage, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry