Biomolecules are large, polymeric chemical compounds produced by living cells that serve as fundamental building blocks of living organisms and carry out many biological processes essential to life. The major biomolecule types include proteins, peptides, polynucleotides, carbohydrates, lipids, vitamins, and coenzymes. The nature of large molecule and biomolecule structure, their chemical diversity, the necessity to consider biological activity, and complex matrices demands an efficient characterization technique. Although there are many separation and analysis techniques available, high performance liquid chromatography (HPLC) is most commonly used. Due to their many functional groups and multiple conformations, HPLC biomolecule analysis is based on different modes such as reversed phase, size-exclusion, or ion exchange. Irrespective of the separation modes applied, efficient column packing and consistent stationary phase particle chemistry are critical for accurate and reliable biomolecule separation.