Chromatography Forum

Morning,

Lately during my runs I have been seeing a massive spike in pressure when I flush the column with 100%ACN. Normally when my mobile phase is running (10%ACN/90%H2O) I have a backpressure of around 190-200bars but when I switch to 100%ACN the back pressure spikes to about 275-280bar and remains there until I revert back to the normal phase. Obviously this should not be happening but I do not know what could be causing it. Thoughts? Thanks

B

Do not connect the column outlet to the detector , and reverse flush the column.
Check if the pressure drops.

What is the column dimensions ? What is the column phase ( C-18 ? )

Column is an Agilent Poroshell120: 100mmx4.6mm, 2.7um particle size, C18

When I flip the column and run 100%ACN it is about half the pressure, 140bar. I flushed for an hour with a very minimum decrease in pressure, went from 140bar to about 138-139bar. When I put the column back in the correct direction the pressure is again at 280bar.

Possible causes are :
Blocked inlet frit of column ( which permits one way flow ).
The flow passage is partially blocked in the column head at the inlet side.

Also , you may cut an inch of the connection tubing ( if it is a PEEK tubing ) to the column , if there is a blockage in the tubing .

To remove any impurities blocking the column follow these steps :
1) separate the column from the outlet frit .
2) make the column drainage in a glass beaker and then wash the column with 60% methanol for about 60 min .
3) invert the column and repeat step (2) .

ph/Amr Tarek
Instrumental Unit Head at Otsuka Pharmaceutical Company

From the product manual:

Cleaning Your Column/Extending Column Life

For columns that can be backflushed (Poroshell 120, ZORBAX columns
with particles >1.8 µm and all Pursuit and Polaris columns), start with a
stronger (less polar) solvent.

1. Disconnect column from detector and run wash solvents into a beaker.
2. Start with your mobile phase without buffer salts (water/organic).
Run at 10 to 20 column volumes through.
3. Next, use 100% organic (methanol or acetonitrile).
4. Check pressure to see if it has returned to normal; if not, then,
5. Discard column or consider stronger conditions,
e.g., 75% acetontrile/25% isopropanol
6. Increase to 100% isopropanol, 100% methylene chloride or 100%
hexane (if you use methylene chloride or hexane, you will need to flush
the column with isopropanol prior to use and before returning to your
reversed-phase mobile phase).

I tried the 60% Methanol flush and that improves the pressure slightly. I've gone through the entire column flush procedure and that only provides minimal improvement as well. I just didn't know if there was something I was missing or not trying but I think with the content of the samples I'm running this will be a constant problem for me. Thanks for all your help!

As I mentioned earlier , it seems it is not a chemical contamination problem , so washing will not improve it.
It is a physical blockage somewhere and you have to locate it to get rid off.

If it is an inlet frit problem , you may try changing it but there is a risk of disturbing the packing.

You may try also remove and exchange the end-fittings ( inlet to outlet ) of the column to locate the problem.

I'm sorry I forgot to address that... I do not believe its a physical block in the system. When I place a new column on there is no issue in regards to the pressure. The problem only arises after I've run 50-60 samples.

I'm sorry I forgot to address that... I do not believe its a physical block in the system. When I place a new column on there is no issue in regards to the pressure. The problem only arises after I've run 50-60 samples.

That can be a problem with certain types of samples. It would be best to use a small guard column with prefilter. This would catch any contaminates that clog the main column and is much cheaper to replace than the main analytical column.

Guard Cartridge Holder

http://www.restek.com/catalog/view/3081

Guard Cartridge

http://www.restek.com/catalog/view/7005

Filter Frits

http://www.restek.com/catalog/view/920

These are what we use for analysis where dirty samples are a problem. The holder with filter frit has replaceable frits that are easy to change. Saves us a lot on column replacement when doing things like analyzing for Formaldehyde and PAH in tobacco samples.