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- Posts: 3
- Joined: Tue Jun 26, 2018 8:25 am
I am having some problems with my Agilent Eclipse Plus C-18 RRHD (2.1x50mm) column, so I am hoping you can help me. I am a student at University of Rijeka, and I am doing my thesis on determination of metallothionein by liquid chromatography (UHPLC). I am currently working on my standard which is comprised of gluthatione dissolved in water and DTNB (Ellman's reagent) dissolved in methanol. For mobile phases I am using phosphate buffer (0,2 M, pH=2,5) and acetonitrile with 0,1% formic acid. A week ago, during my analysis, the pressure suddenly rose to 1000 bar and the analysis was immediately stopped. I checked all the components of UHPLC but the problem seems to be in the column. My assumption was that salts from buffer clogged the column so I tried backflushing it (1h water, then 1h acetonitril- water 50%-50%), but the pressure is still pretty high. I am hoping you can give me some advice as to what else I might do to better the performance of my column.