Optimization Strategies in HPLC for the Separation of Biomolecules

High performance liquid chromatography (HPLC) finds wide application for the separation of biomolecules, offering different operation modes, which separate by physiochemical properties like size, charge, or hydrophobicity. Optimization of a method often entails trade-offs, for example if high selectivity is the ultimate goal, the method will likely not be as fast as possible. Many chromatographic run parameters have profound impact on the quality of the separation and can be tuned to get the best possible outcome. Some chromatographic modes can greatly benefit from the optimization of the gradient. Some chromatographic modes benefit from the addition of mobile phase modifiers. High-speed methods require ultra HPLC systems capable of operating at extreme backpressure and column formats, which can withhold pressures of up to several hundred bars. A very effective way to improve the sensitivity of a HPLC method is to change the column dimensions. Multidimensional separation modes are commonly used to obtain increased resolving power in HPLC.