Please help me figuring out what is going on with my RP-HPLC protocol.
I am purifying a small peptide out of E.coli Culture, heat stable enterotoxin, its amino acid sequence is:
I am collecting culture supernatant and using amberlite XAD2 to concentrate and desalting the peptide in 80% and 99% methanol fractions. These fractions were subjected to flash evaporation. Then I am using on MCI gel CHP20P (glass column) for further concentration and removing unwanted proteins (polar and less hydrophobic ones) using stepwise elution with 20%, 40%, 60%, 80% and 100% methanol fractions. Each fraction is subjected to flash evaporation. A sample (1 ml) from each fraction (60, 80 and 100%) was then loaded to RP-HPLC using xbridge C8 preparative column (19x25 mm,10 micron, 130 angstrom) on a gradient system (solvent A: water/ solvent B: 80%methanol) initial-5 minutes 100 A, 5-10 minute 30% B, 10-65 minute 100% B, 65-80 minute 100% B at flow rate 2 ml/minute. The chromatogram showed a single large peak (3 AUF) at 25 minutes, B solvent around 45-50%.
I am wondering that the retention time is lower than that was reported before, 35 minute at 55-60% B (I followed almost the same protocol 5 years ago except using MCIgel methacrylate resin and Vaydc C8 column instead of mci gel CHP20P and xbridge C8 column). It is also very frustrating that I loaded 0.5 ml of the collected peak on Waters analytic C8 coulumn, 5 micron particle size and it elutes very early at 5-6 minutes (almost in the mobile phase A: water). My frustration comes from that I am using highly hydrophobic resin particle (MCI gel CHP20P) to capture hydrophobic molecules and loaded these fractions (60%, 80% and 100% methanol MCI gel) on C8 column and eluted so early.
I am sorry for this long message but I rely on your understanding. Any answer would be highly appreciated. Thank you in advance