HPLC and column maintenance

[Jerslatt]

I was hoping someone knowledgeable might be able to share their thoughts on the subject of washing/flushing.
We use Agilent 1200 gradient HPLC's using RP and various methods using mobile phases that contain buffer. A colleague put it to me that flushing instruments and columns before and after use actually has no effect on chromatography or operation of the system. The procedure for the instruments is to flush each channel with at least 30ml of a wash before using a mobile phase and regardless of whether all lines are used, and to wash the columns with specific wash and storage solutions depending on the samples used.
So is just leaving the mobile phase in the system and columns OK? This person commented that washing a columns does not really do anything, and that for the lines that are not used, what is the point in flushing.
Personally I don't do this as it is contrary to what I have been taught, however I cant really argue with this person, as they maintain they haven't encountered issues so far.
Obviously they seem like durable machines, and can take a lot, but at what point would you start questioning doing this? Or is just a case that flushing systems is not something that requires strict adherence, and if so *how much is too infrequent?
Appreciate any comments, thank you.

[Gerhard Kratz]

When you use buffer it is always recommended to flush your system, all lines, and for sure the column. Best is first wash with water. Reason is that buffer salts will destroy the packing of your RP column. After water you can wash with Acetonitril or Methanol. Life time of your piston seals and your RP column will increase.

[per_oxid]

I think the answer depends on the column and usually an instruction for column maintenance is provided.

I am also trying to figure out a good end of run procedure. I dont know the best procedure but this is what I do at the moment.

The column instruction is to store in 100% ACN. However I use 10mM ammonium acetate that is not compatible with 100% ACN so it is important to first clear all buffer. At the same time I also wish to eluta all my lipid analytes that would crash out in aqueous. For a C8 column i use this

1) 80%B= ACN and 20%A= MEOH:H20 (1:1) to elute lipids
2) then a gradient to to 100% A to elute buffer
3) then gradient back to 100% B to remove water from the column that can now be stored.
4) Then I use column bypass and 100% A for storing the LC system in MeOH as I have been told that ACN can polymerise in switch valves and a mix of MeOH H20 sould keep prevent precipitation and growth of microorganisms.


For our a ion exchange column they recommend storage in the mobile phase. So i just remove the column and flush the LC with 1:1 MeOH:H2O. I always have unvuffered ACN on B2 line and 1:1 MeOH:H2O as A2 for the end of run procedure and use A1 and B1 for the analysis gradients.

Any suggestions for improvement?

[James_Ball]

Another thing to keep in mind is that aqueous solutions sitting still in the lines will promote algae growth. If you get it growing in the solvent lines from the bottle that is easy to fix, when it is growing inside the degasser, not as easy.

The same is true for the column, if the buffer is not low or high enough pH to kill the algae it will grow. I like to flush the column with 100% water if I was using a buffer mobile phase to remove any salts that can precipitate out and clog the flow path, then flush with at least 50% Organic in water through the pump and lines, column and detector. Unless the column is specifically designed for 100% aqueous mobile phases you will have a long equilibration time when restarting the analysis after it sits for a long time in mostly aqueous mobile phase, so put organic in it for storage.

If the system will sit for more than a couple days, it is best to flush any aqueous solvent lines from the bottle to the pump, first with 100% water to remove buffers then at least 50% organic to store and prevent algae growth.

I have also made a method that pumps a constant flow of 0.2ml/min through the system at standby to keep things moving to help prevent growth between runs and prevent pump seals from sticking to the pistons, I don't know if it is necessary but it seems to work.