clogged column

Basic questions from students; resources for projects and reports.

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Hey everyone!
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.
Phosphate will precipitate when the ACN concentration is above ~50%. Reversing and washing the column with water will help.

Then, try washing the column (at least 10X column volumes) with 100% Methanol, 100% IPA, 100% Hexane and back again.
Hi elizabeta.rus,

You said,

For mobile phases I am using phosphate buffer (0,2 M, pH=2,5) and acetonitrile with 0,1% formic acid.

Is the phosphate concentration Really 0.2 M? If so, perhaps it may be worth some time to see if a lower concentration of phosphate could afford both good peak shape and separation.

I agree with HPLC Chemist's suggestions--warming the water for the backflushing may help to remove salt from the column, as well as using a low flow rate (<100 uL/min) to allow time for the water to dissolve the phosphate salt.

EDIT: This link describes some buffer solubilities: ... -97119.pdf
Thank you HPLC chemist!
Thank you very much mattmullaney!
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