Chromatography Forum

Hello everyone. I have a problem about peak in blank. HPLC cond. is gradient and MFA: %0,01 m H3PO4 and MF B: ACN. I tried a lot of cleaning procedures but I saw always the active peak in my blank injection. Did anybody see same problem before? I need suggestion. Thanx..

Many possible reasons for your carryover contamination.

(1) Your equilibration times may be too short.
(2) Your column wash procedures after EACH analysis may be doing a poor job of removing any remaining material (use solvents that are STRONGER than your mobile phase).
(3) You may be overloading the column with sample/standard.
(4) You may need to perform proper maintenance on your instrument. The injector valve's rotary seal may be worn (the stator may be scratched or thier may be damage to the inside of the valve). These parts wear out and require regular replacement.

This article may help you troubleshoot the issue.
"Carry-Over (Carryover) Contamination in HPLC and LC-MS Systems"; https://hplctips.blogspot.com/2015/02/c ... on-in.html

Thanks your replies. I changed instrument, used new column but that peak came again. I thought, maybe it came from mobile phase. I prepared new mobile phase, but it still came. Do you suggest me any strong washing procedures?

Can you please clarify which did you performed?

1. Method blank run? (Run hplc method without injection)
2. Matrix blank/diluent run?

I injected diluent which is given in method.

Can you please perform a method blank run and report back?

is your diluent different from mobile phase? Can you tell us what are they?

I performed blank run before. I didn't put vial on tray and injected. But that peak still came.
My diluent is ACN/0,01 M H3PO4 (1:1,5)(v/v). It is like mobile phase.

Sounds like mobile phase is contaminated. Can you please use very clean containers, use new bottle of the salt and acn, and prepare once more, a fresh mobile phase?

Can you also please wash your filtration set thoroughly with DI water, then rinse it with Methanol before using.

Also, how large is your peak?

Btw, try using a new bottle of ACN on your hplc, I had similar problem before and the ACN on my instrument was the culprit.

also i cleaned column and instrument with water then meoh. even i cleaned 3-4 hours with 40°C column temperature.
peak area is 4000-5000. but method criteria should be <LOQ and my LOQ injection area is 3000.

Try flushing your column with 90%/10% ACN/Water for 2 hours.

What column is it btw?

my column is RP-18-Lichrocart 5x4.0mmx3.0µm

What is your needle wash solvent btw? Did you also tried to clean your needle?

I cleaned needle with %70 ACN. I tried to clean %100 MeOH, diluent (ACN/0,01 M H3PO4 (1:1,5)(v/v)). Sometimes peak didn't come, i continued analysis. next time i did same washing procedure, peak came again.