Chromatography Forum

I run a method on Waters Alliance W2489 with a UV detector and I would like to know what's the best way to wash down the system following a run. For Mobile Phase A I have pH of 2.9 for a 6g/litre solution of monobasic ammonium phosphate, Mobile Phase B is 100% Acetonitrile. My seal wash is 90:10 Water: Acetonitrile and needle wash is 80:20 Acetonitrile:Water. The column is an Interstil ODS 4.6 x 250mm particle size 5 micron with L1 packing.

The gradient goes from 70:30 A:B from Initial to 60 mins, 22:78 A:B at 61 mins and from 61 to 70 mins back to 70:30 A:B, all curves linear at 6. Detection wavelength is 228nm. Column temp is 35 Degrees, sample temp 21 degrees and flow rate 1ml per minute.

I set up a method to washdown the system by pumping 100% seal wash at 0.5ml per min through the system for 2 hours followed by 0.3 mls per minute of 100% Acetonitrile for 30 mins, then switch off. But the last 2 runs for the blank injections have been quite noisy and had a lot of peaks in it. Does this washdown seem appropriate for the conditions above? I'm thinking to use high aqueous to wash out the buffer from the system followed by storage in acetonitrile for 30 minutes. Everyone seems to do it differently, is there a general guide for washing down HPLC runs? Thanks in advance.

Best way to wash down a HPLC post-run?

I assume that you mean after a sequence, or series of runs/injections.

So I'll chime in with what we did with reverse phase. On gradient runs, after the last peak of interest eluted, we'd ramp up the organic to clean up the column for as long as that took, then move the settings back to the initial conditions to equilibrate for the next injection. Remember that equilibration is really a factor of column volumes, not time.

After a sequence, we'd run mobile phase made up without the salts, and ramp up the organic to clean out, and store that way.

Hi EmpowersBane,

I'm agreeing with CPG here on the washing intermittently, like an integrated wash within all of your methods. This could ramp up to 70-80% ACN and remove most of the stuff still on your column (however, depending on your buffer you may want to check the highest % organic that is possible without precipitation), followed by an equilibration for your next/same method for however long you need to eq. Something tricky that I've seen when it comes to pumping full organic is the sticking of check valves or accidental precipitation. Both of these issues will be realized pretty quickly and I don't believe that is what is happening in your instance. The last run, in your case, may be the only chance most of your contaminants have of being removed from the column/HPLC due to the high percentage of ACN. H2O is wonderful for removing buffers, but you really do need to ramp up the organic in order to clean off contaminants. This, along with a potentially contaminated injector, could be the reason you see so many peaks in your ACN wash.

If the samples are not dirty I usually flush with 100% water to remove buffers then at about 20% water 80% organic before shutting down.

If it will be shut down for weeks or months then I remove the water and pump that line full of Methanol diverting it to waste at the prime valve instead of into the column, to prevent any growth of algae in the aqueous lines.

Thanks to all for the replies. I think a sensible approach is to flush out the buffer from the column after the run with water and then 80 20 ACN Water as suggested. I cant add in equilibrate lines mid run as we are tied to validated methods so i have to keep it at after the run. Thanks again.