Chromatography Forum

Can anyone help me out. I have Normal phase HPLC method for separation of Prednisolone and Methylprednisolone and chromatographic condition as below
column : silica column 25cmx4.6mm 10 micron
mobile phase : Butyl chloride : water saturated Butyl chloride : Tetrahydrofuran : Glacial acetic acd : Methanol (475:475: 60:30:35)
Now as per method I suppose to get RRT for prednisolone is 0.7 and for Methylprednisolone is 1.0 but when I injected my resolution soln, i found mehylprednisolne eluated first and prednisolon second. I guess method given in literature seems incorrect internal std i.e prednisolone it should be prednisone because looking at the molecule structure and chemistry, method is purely partition based chromatography and effinity towards stationary phase and t is not pH dependent as both molecule has similar molecules except methyl group. Please suggest my guessing is correct or any other reasons.

In NP more hydrophobic molecules elute before more hydrophilic molecules. Thus methylprednisolone should elute before prednisolone. I was wondering why acetic acid was needed for this application as both molecules are neutral and won't be affected by pH.

I believe a reversed-phase method using a C18 column can do a better job for this application.

That method must be at least 30 years old. With that "witches brew" of a mobile phase, it is probably partition chromatography, where the stationary phase is a sorbed layer of acetic acid and/or water. My first guess is that the methyl should elute first as you suggest. That said, I agree with XL that you you replace that method with something less complex and trouble-prone.