I am having an increase in pressure when I try to analyze samples on an Agilent 2.1 x 100 mm 1.8 micron column using a starting gradient of A1: 95% H2O w/ 10mmNH4Oac and B1: 5% Acetonitrile w/ 0.1% Acetic Acid. The gradient increases to 30% Acetonitrile. The pressure starts out, even on a new column fine, but increases to almost the max column pressure which is 600 bar. We have run this gradient successfully numerous times using this column. Even with analyses of blank runs, the pressure may start out low for the first 20 runs, then increases so that it may or may not be able to do all 50 blank runs. The problem appears to be connected to the column, but I'm not sure if it is the column itself. We have tried flushing the solvent lines with 100% IPA; without any buffers in water or acetonitrile; changing 0.12 tubing on the entire LC system, but nothing seems to be working. Any suggestions???