I have a RP-HPLC snag. While a sample containing 30% glycerol is being run through a Synergi MaxRP-C12 column with a linear acetonitrile gradient (water phase being Tris-0.05%TFA, pH 8.5) and the gradient reaches 45-50%, pressure goes down and a bubble forms blurring a substantial part of the picture. Glycerol-free samples run with no trouble. Glycerol is essential for solubility of proteolysis fragments in the sample, sample pH is 8.0 and is needed for proteinase K proteolysis. It would be great if someone could help sort this out. Thanks in advance.