washing hplc column

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

3 posts Page 1 of 1
Hi,

I analyse sugars in a wide range of food samples using hplc- rid, eqquiped with kromasil 100-5-NH2 (l 250 mm* i.d.4.6 mm). Mobile phase(75%ACN/25%H20), flow rate 1.4ml/min
I wash the column with the same mobile phase, but with reduced flow rate 0.4 ml/min.
Should I add an extra washing step?

Thank you for your interest
Do you incorporate the wash as part of your instrument method? Or do you just allow the system to flush for a while (couple of hours?) with the 0.4 mL/min?
"Have you tried explaining it to the rubber duck?"
A separate wash step should always be used after one or more sample analysis are complete, followed by a separate column re-equilibration step. This is critical when running isocratically, as you are, because column fouling is often the result of NOT washing the column. If possible, break this process down into three separate steps, as the "pros" do. (1) Column Wash method; (2) Column Equilibration Method and (3) Analysis. Use a gradient mode when "washing" the column to maximize the effectiveness.

Note: Column Washing should be performed at the same flow rate, not a reduced flow rate.

For your method example, the wash solution should consist of one of more liquids which are known to dissolve the sample types used fully, so they are eluted off the column quickly and cleanly. Usually, water plus some organic solvent. A few suggestions: (1) Consider using a gradient wash to start off with ~ 50% water/50% methanol, ramped up to 90% methanol/10% water for a few column volumes. Methanol/Water is a good solvent combo with better properties for dissolving many sugars when compared to ACN/Water. (2) If ACN is working well in your application, then you could use a gradient of water and ACN to perform a similar column wash. Be sure to use a ramp where the amount of ACN used in the WASH mode is higher than what is used in analysis mode, but just a bit (e.g. 80 to 85% ACN max). (3) Incorporating some IPA in the wash solution (10%) is often a good idea when amino columns are used. Amino columns often foul quickly so regular washes are needed to increase their lifetime. (4) Lastly, one trick that our lab uses with these columns is to wash them occasionally with some freshly prepared 0.05% formic acid in the water as an initial wash, then run a gradient with water and Methanol to clean them off (10% water/90% MeOH at ramp max). Seems to work really well for regenerating NH2's.

Try out a few solutions to see what works best.
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