Proteomics/Protein Separations - Chromatography/Ultra High Performance Liquid Chromatography

Ultra High Performance Liquid Chromatography
High performance liquid chromatography has proven itself to very useful in many scientific fields, yet forces scientists to consistently choose between speed and resolution. Ultra performance liquid chromatography (UPLC*) eliminates the need to choose and creates a highly efficient method that is primarily based on small particle separations.

Ultra high pressure liquid chromatography, also known as ultra HPLC (UPLC), is a form of column chromatography used to separate, identify, and quantify compounds. It allows for separation and analysis of small particles both quickly and effectively. Liquid Chromatography is the process of passing a mixture of particles to be separated through a column. This allows the analyte, which was separated from the mixture, to be measured from other molecules. The columns are filled with a packing material, known as the stationary phase. In UPLC a pump pushes the mixture, known as the mobile phase, through the columns. As the mobile phase is passing through the stationary phase a detector shows the retention times of the different molecules. Retention time varies depending on the interactions between the stationary phase, the molecules being analyzed, and the solvent used.

The theory behind the development of UPLC is the van Deemter equation. The van Deemter equation is an empirical formula that shows the relationship between linear velocity, also known as flow rate, and plate height, also known as column efficiency. Since particle size is one of the variables of the van Deemter equation, the curve generated can be used to investigate chromatographic performance. As the particle size decreases to less than 2.5 μm, there is a significant gain in efficiency even when flow rates are increased or when linear velocities are increased.



Besides efficiency, the advantage of working with small particles is the fact that small particles can work at higher linear velocities. This allows for increases resolution and speed. Resolution is proportional to the square root of N, which is inversely related to both particle size and peak width. Peak width is inversely related to peak height. This means that the use of smaller particles will allow for narrower, taller peaks; greater resolution. It is also important to note that as particle size decreases the optimum flow increases. However, pressure is proportional to flow rate so smaller particles will require higher energy or pressure.

Current HPLC technology could not suffice for the new pressures associated with UPLC; a new system was designed by Waters called ACQUITY UPLC. Waters ACQUITY Ultra Performance LC systems have been developed to take into account all the advantages that small particle separations currently have over HPLC. This task was not easy. A new hybrid material was invented for the columns called Bridged Ethyl Hybrid (BEH). Also the columns needed a smoother interior surface. ACQUITY UPLC BEH columns also include microchip technology that captures the manufacturing information for each column, including the quality control tests and certificates of analysis called eCord™. The eCord database is also updated with real time method information, such as the number of injections, or pressure and temperature information, to maintain a complete continuous column history.

Many of these advantages are primarily based on the theories behind liquid chromatography. In general, increasing the efficiency of a separation will also increase its resolution. Since both efficiency and optimum flow rate are inversely proportional to particle size, a decrease in the particle size will increase efficiency and speed up the flow rate.

In the ACQUITY system, the particle size is decreased to 1.7 um compared to 3.5 um or 5 um. The particles were specifically designed to withstand wide ranges of pressure and pH, have a high load capacity, and improve efficiency. Other innovations to the chromatography method include a high pressure solvent delivery system, to take into account the smaller particle size, fast injection cycle sample management, and specialized detectors with fiber optic flow cell design.


 * UPLC is a term trademarked by the WATERS corporation. Elsewhere, the technique is generally referred to as Ultra High Performance Liquid Chromatography (uHPLC).

uHPLC vs HPLC
A comparison between Ultra High Performance Liquid Chromatography and High Performance Liquid Chromatography is shown in the table below:

The lower bead size is the true reason for uHPLC increased flow rate and resolution. This can be shown mathematically using deemter's equation: H = A + B/µ + Cµ. H being the plate height and µ being the particle size. The A constant remains constant independent of flow rate (it is referred to as the 'Eddy diffusion term'). The B constant is the diffusion coefficient, and C is the "analyte mass transfer" coefficient. As µ decreases, the A and C values of needed for a similar H value decrease, allowing for higher resolution. This also reduces the effect of the C value on the H value,  yielding faster separations for similar resolutions. Note uHPLC out classes HPLC in all aspects, and is expected to replace HPLC in the near future.

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Resources

 * 1) Waters - Ultra Performance Liquid Chromatography
 * 2) Waters - UPLC New Boundaries For The Chromatography Laboratory
 * 3) ISCO - UHPLC System Configuration