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After using an HPLC (C18) column several times it doesn't do what it has to do. When we use it the HPLC instrument shows a very high pressure and the process stops. We have washed with methanol, water, and acetonitrile, but none of them have been effective. What are we overlooking? What should we do to decrease the pressure of the column?

  • Available pump heads Max. flow rate in ml/min is 10
  • Max back pressure in bar 400
  • Recommended particle size is 5 μm
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    $\begingroup$ What's the pressure, mode (isocratic/gradient), column geometry, analyte/matrix and mobile phase? Was there a downtime before you noticed an issue with pressure? What does the column's manual say regarding washing and storing conditions? $\endgroup$
    – andselisk ♦
    Feb 16, 2022 at 8:36
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    $\begingroup$ You barely answered any of the questions. 1… what? ml/min, μl/s? What's the flow unit? What does "column C18 with nanometere column" mean? Are you doing nanoflow LC with a guard column, or what? What are the dimensions of the column (length, diameter) and particle size? Again, what mode you are working in? What are the specified maximum pressures for the column and for your LC system? What is the matrix and fructose concentration (could it crystallize)? What temperature is your column thermostat is set to? Feel free to edit these details into your question instead of posting comments. $\endgroup$
    – andselisk ♦
    Feb 16, 2022 at 9:00
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    $\begingroup$ It's OK, the problem is that HPLC troubleshooting is a multivariant task and we need as much info as possible. If the washing procedure described in column's manual didn't help, or there is none, as the last resort you could try washing the column in reverse using low flowrate, say, 0.05 to 0.2 ml/min. Modern columns (which model is yours, BTW?) should handle this all right. I'd strongly recommend though first to talk with your supervisor/TA or anyone on campus who has experience with LC and could come by and have a look at your machine. $\endgroup$
    – andselisk ♦
    Feb 16, 2022 at 9:38
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    $\begingroup$ By the way, are you sure the problem lies with the column? Have you tried to run the solvent with the column disconnected? A bit silly suggestion I should've probably started with, but still… $\endgroup$
    – andselisk ♦
    Feb 16, 2022 at 9:46
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    $\begingroup$ Well, if you disconnect the column and the pressure drops, then it is indeed the column giving you trouble. Alternatively, you could try to increase thermostat temperature to, say, 60 °C and wash with your MP again. Also, there are several regeneration guides for various columns such as one from Agilent (PDF). $\endgroup$
    – andselisk ♦
    Feb 16, 2022 at 10:13

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What your professor has to recognize is that an HPLC column is a consumable. It is not meant to last forever. The back-pressure shows that it has been mistreated in the past with careless handling. Also, the HPLC pump seal could be going bad and generating polymeric microparticles which are clogging the column. At both ends of a column you have very fine sieve called frits (micron sized, typically 2 micron pores).

If your solvent, sample had small particles they can easily clog the frits. Also, if someone were careless enough, a sample can precipitate at the head of the column due to solubility issues. It is always the inlet frit which causes problems.

Look at the schematic of a column:

LC column

Some standard ways to check the system:

  1. Prime the pump, which just means that you ensure pump is filled with solvents and there are no air bubbles.

  2. Remove the column and connect the injector to the detector with a long tubing. And start the flow, always start at low flow rates like 0.2 mL/min and then slowly change it to 1 mL/min. How much pressure is observed? There is negligible pressure like 5-6 bars, then the problem is in the column.

  3. HPLC columns have an arrow to show the direction of flow. Intentionally use the opposite direction and start the flow, again slowly. Do not connect the column to the detector. Starting at 0.1 mL/min and then go up to 0.5 mL/min. If it still stays at the same high pressure even after an hour, it is time to either toss it in the garbage.

  4. If your professor is more mechanically oriented, open the column (last resort). Sonicate the inlet frit in various solvents and fill in just bare silica if you accidently scraped a layer of stationary phase and put the cleaned frit back.

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