Chromatography Forum

Dear everyone

I made wrong flow direction of HPLC column. What will happen? and What should I do.


Thank you

Pirapon

Do not worry nana. It wont destroy the column Stop panicking and from next run, make sure that you check it before you run. After all, dont tell your seniors, some seniors are crap and will make big issue

I made wrong flow direction of HPLC column. What will happen? and What should I do.

If this was for a cGMP or GLP assay, just document that the column was installed backwards for that assay. On the majority of modern columns, it will not make a technical difference.

Do not worry nana. It wont destroy the column Stop panicking and from next run, make sure that you check it before you run. After all, dont tell your seniors, some seniors are crap and will make big issue

Not strictly true, some columns contain warnings about use when installed incorrectly as the head and tail of the columns are not identical.

However, if you document the fact and correct the problem, you should be able to tell from system suitability, calibration and QC data in the sequence if there is a problem.

Check with you manufacturer and see if you can reverse the column.

Some column cannot be reversed not because of the packing uniformity issue , but because of the different frits used in the column ends. If the same frits are used for both ends of the column you should be fine.

Remember to document it though, and if there's a problem you can see from system suitability:)

I agree with rwang,
mainly the flow direction indicated on the column label shows the direction of packing the column.

A major problem by reversing a column , you may crack the packing , if the column has a slight void at the inlet side.
The slight void may not cause a significant problem on the chromatograms , but if the column reversed , you may move the packing and channeling may occur.
This will absolutely affect your chromatograms.

The vast majority of HPLC columns can be backflushed without any problems. A few use different frits on the inlet and outlet, so it is a good idea to check with the manufacturer.

If a column has a headspace, then it is *already* damaged. In that case, backflushing should not make things worse and may actually help by "fluffing" the bed of packing (which is, at best, a temporary fix!).

Yes changing of directions doesnt really matters until you have chosen old column.
The reason is that sample matrix sits on the inlet side and there is flow direction effect on packing mediums due to flow rates the packing materials breaks into more pieces 5 microns to 3 microns with the passage of time.
So once you reverse the column direction first the column will make some extra backpressures, the packing material takes time to settle down and frits on the other end takes time to flush out .

faree...., what is your evidence for 5µ particles being degraded to 3µ?

Wow! So I may convert my 5µm column to 3µm just by reversing it and flushing for some time? I guess with higher flow rates, I may be able to convert the 5µm to sub-2µm! No need to spend the extra money for all these extraordinary STM-UPLC-UHPLC-RRHD columns any more!!!

SCNR

Hey !!
The packing breaks from the inlet side side due to flow rates not the whole column packing bed. Flow rate is a continuous process but it wont be a Micron CRUSHER for you. just joking.

Wow! So I may convert my 5µm column to 3µm just by reversing it and flushing for some time? I guess with higher flow rates, I may be able to convert the 5µm to sub-2µm! No need to spend the extra money for all these extraordinary STM-UPLC-UHPLC-RRHD columns any more!!!

SCNR

I still doubt that with a moderately modern column you will be able to crush the silica beads just by reversing the column and using high flow rates. Silica is AFAIK mechanically very stable and with a good packed column there shouldn't be any free space for the particles to move and create friction.
There are discussions that over the lifetime of the column you might mobilize silica fines in the bed that move to the frits and clog them resulting in increasing backpressure. But these fines stem from the production process of the silica and the packing of the column, they're not created by degrading the silica particles.
Anyhow, it doesn't make sense that the silica should be degraded by high flow rates only after reversing the column. When experiencing bad peak shapes (shoulders, splitting peaks) I usually reverse the column and flush (if it's not forbidden by the column care manual). Sometimes it helps to reestablish good peaks, sometimes it doesn't, but I had no issues with a general increase of backpressure after reversing the column.

Let me repeat what I said earlier :

The vast majority of HPLC columns can be backflushed without any problems. A few use different frits on the inlet and outlet, so it is a good idea to check with the manufacturer.

Unless the manufacturer tells you otherwise, the flow and pressure limitations will be the same in either direction. If the manufacturer does tell you otherwise, listen to them!

Trust me on this (I have a bit of experience in LC).