Help! I am new to the field of macromolecule analysis, and am trying to perform a gradient HPLC method for analyzing a protein of about 150 amino acids. I'm using a Vydac C4 column and an HP1050 HPLC system with a guard cartridge. The gradient is Solvent A = water with 0.1% TFA, Solvent B = 90% ACN with 0.1% TFA. The gradient is from a low %B to a high %B. When I make multiple injections, the retention time keeps shrinking until the protein is eluting with the injection front. I keep changing the gradient start and end concentrations, but the phenomenon persists. Is this a column conditioning issue, or something else to do with gradient HPLC of proteins?

One suggestion to troubleshoot your problem here. Did you allow the column enough time to re-equilibrate to the initial conditions after your run was completed? Normally that is about 20 column volumes of your initial mobile phase. If the column is not "regenerated", you could see something like you describe here. Adding 3% propanol to both solvents will help reduce the conditioning time. See Anal. Chem., 1990, 62, 16 by Cole and Dorsey.

You may also try running in isocratic mode to see if you get reproducibility there. That may help you decide if the problem is in your sample or your separation method. Good Luck!

AL's comment is worth checking out. A related possibility, discussed in LC.GC 8 (1990) 524 (see Fig. 7 and related discussion) is a sizable gradient delay time combined with too short a column equilibration. In this case, it is possible for the end of the preceding gradient to overtake the next sample injection. The results are a normal separation the first injection, followed by rapid elution of the sample for each subsequent injection. L. Snyder, LC Resources.

Column reequilibration time and gradient delay are usual culprits, but do not fail to verify that your pumps have a consistent flow, your injection volumes are consistent, and your sample is solubilized in solvent of the same pH, molarity, and hydrophobicity as your starting conditions. Also confirm that the pH and composition of your buffers are not drifting over time.