Pressure problems ONLY in H2O for reverse phase c18 column

[maisymouser]

I am baffled by my pressure issues as of late!

I run samples derived from clarified lysate on a C18 HPLC column. I do a hard spin of my samples, filter them, and also dilute them. However, I know some protein gets on the column. I ran some samples on the column without a guard column (tsk tsk). Everything went just fine- no clogging during the runs whatsoever, which involve both a 5% and 95% acetonitrile portion for at least 3-4 minutes.

However, I recently have had a lot of trouble with pressure only when I use water (isolated to the column, it is not a pressure problem in the rest of the system). With acetonitrile, the pressure is perfect, but there is a clear issue when I increase the water component. Figuring it might be protein, I performed a 6M urea flush, which has cleared out proteins really well for me in the past. However, I'm still getting higher-than-normal pressure when I increase % water.

I find this really strange! Has anyone had problems with their solvent A delivering higher pressure than normal? I'm going to prep fresh solvent but I am not confident that it will solve the problem.

[KM-USA]

Most C18 columns don't tolerate 100% water.

[tom jupille]

Water has a higher viscosity than ACN. And I think mixtures can have higher viscosity than either.

[Gerhard Kratz]

In addition lets assume that with 100% water the C18 alkyl chains will collapse!!!!!!!!!!! At the first 4 cm of the column the space between the particles will be reduced and packing density will be increased what will lead to increased back pressure. To avoid such problems C18 columns are available on the market who will tolerate 100% water. Even if you will go back to minimum 2-5% ACN in your mobile phase you will see now shoulders on your peaks, split peaks - with other words you have now a void volume on the column top. Time to order a new column with a different surface chemistry!

[KM-USA]

...see why I waited and let you two yell at Maisy....I think I learned on Day 1 not to run 100% water through C18 columns...

[TwistedHelix]

You could try regenerating the column packing, but it's doubtful to work. Running 100% water isn't problematic short term, i.e. trying to rid the column of buffer solutions to stop precipitation, however after a few column lengths one needs to start using less polar mobile phases and gradually work downwards to the DCM's etc.

This may restore your column, depends what one it is. Some C18's die completely with 100% water, some just get temporary damage that can be regenerated. Try regeneration, again, polar-non polar and back to polar. Always store RP C18's in Acetonitrile or Methanol.

It depends entirely on your compounds but we find 50:50 Methanol:Water works wonders.

P.S. We have some C18's that have run for years with a good 50,000 injections under their belts. It can be done with proper care, and not using harsh solvents and pressures.

[M Farooq]

In addition lets assume that with 100% water the C18 alkyl chains will collapse!!!!!!!!!!! At the first 4 cm of the column the space between the particles will be reduced and packing density will be increased what will lead to increased back pressure

This is an interesting observation. Is there any experimental evidence that C18 chain collapse leads to the reduction of the inter-particle distance in a packed column? I was under the impression that the spherical particles are so tightly packed that there is no movement possible until and unless there is a physical breakage or chemical dissolution of silica in highly aqueous mobile phases.

Thanks.
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The original poster can try reversing the column direction (consult manual first) to remove precipitated material at the inlet.

[HPLCaddict]

I'm with Farooq here. Even if you mistreated your hydrophobic C18 with pure water and experienced phase dewetting (formerly supposed to be phase collapse) it's usually no problem to get it back to life with a nice ACN flush. In hard cases, you might add some backpressure regulator after the column to increase the pressure and thus force the mobile phase back into the pores.