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This work seeks to explore the capabilities of stationary phase gradients (SPGs) for the large-molecule chromatographic separations of proteins and peptides through a combination of experimental studies and simulations. Continuous SPGs are fabricated on commercial Phenomenex Jupiter C4 columns using the time-based infusion of trifluoroacetic acid through the column. The resultant gradients are characterized using smallmolecule chromatography and physical gradient profiling via thermogravimetric analysis. The gradient columns are then tested using two different protein analyte mixtures and a peptide standard analyte mixture to examine the efficacy of the separations. Collected experimental data from these tests are used to establish fitted parameters for the Linear Solvent Strength (LSS) model to computationally predict the retention behavior of the analytes on the gradient columns. Successful chromatographic separation of all three analyte mixtures was achieved. The re-parameterized LSS model successfully simulates the analyte retention on the gradient columns with only minor deviations from observed experimental retention data. Collectively, this work demonstrates the ability to create a C4 continuous stationary phase gradient starting with a uniform commercial column that can be effectively used to separate mixtures of biomolecules, and the ability to accurately simulate and predict retention times. The computational model described herein could be used to effectively predict retention behavior in silico, significantly reducing the time needed for experimental design.