The pressure increases suddenly with every injection from 120 to 300 bar and decreases to normal in 20 sec or by switching the valve to mainpass Why?
Agilent 1260 series HPLC
MP: MeOH:ACM:Water (48:48:4) isocratic
Column: Phenomenex Luna C18, 3 microns, 4.6 mm, 150 mm with a guard column
Column temp: 50C
Flow rate: 1 ml/min
Injection amount: 50 microliters with an autosampler.
Samples in ACN with a strong acid (TFAA)
Thank you.
Depends on your sample. 300 bar is not too high but should be your maximum. Typically, a radical change in your pressure is due to bypassing your column via an injector valve switch. What are your samples (remember other crap in your sample is being injected)? How are they prepared? Do you filter them beforehand (minimum 0.22 um)? Do you dilute them in your mobile phase before injection?
Two main causes:
(1) System was loaded with sample dissolved in an incompatible liquid (injection solution must match mobile phase !);
(2) Your injection port or rotary valve seal may be damaged (probably from clogging/fouling with sample, overloading, precipitation etc). * Inject pure mobile phase and see if the problem still occurs and/or if the high pressure problem appears or is now normal (a small change in pressure is normal during injection switching only).
If the system back-pressure is abnormal then: Perform routine service on the AutoInjector, inspecting and cleaning the stator, needle seat and capillaries and replacing the normal wear parts. Be sure to only use the injector with chemically compatible solvents too (we see a lot if new users running solvents that the injector is NOT compatible with, resulting in damage. For example, injectors that have PEEK rotors!). Once the valve has been repaired, then flush the flow path down in both the main and by-pass positions and verify correction system back-pressures while isocratic conditions exist (constant flow, constant composition and temperature).
Dear William, thank you for the invaluable information and your recommendations.
Kind regards
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