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Proteomics/Protein Separations - Chromatography/Ultra Performance Liquid Chromatography

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Ultra High Performance Liquid Chromatography

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   High performance liquid chromatography has proven itself to very useful in many scientific fields, yet is limited in either speed or resolution. Ultra high performance liquid chromatography (UHPLC) increases throughput and resolution, primarily based on the use of smaller particles for separations.

   The advantages are primarily based on the theories that underly 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 both efficiency and flow rate.

   For example, the particle size can be as small as 1.7um compared to 3.5um or 5um that is typically used in HPLC. The particles must be able to withstand high pressure, extreme pH, and have a high load capacity in order to 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.

UHPLC vs HPLC

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

Attribute HPLC uHPLC
Pressure 6000 Psi 100,000 Psi
Particle Size 5μm 1.7μm
Flow Rate Milliliters per minute Liters per minute
Max Resolution Relatively low Relatively high

Resources

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  1. Waters - Ultra Performance Liquid Chromatography
  2. Waters - UPLC New Boundaries For The Chromatography Laboratory

References

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  1. Leandro CC, Hancock P, Fussell RJ, Keely BJ. Comparison of ultra-performance liquid chromatography and high-performance liquid chromatography for the determination of priority pesticides in baby foods by tandem quadrupole mass spectrometry. J Chromatogr A. 2006 Jan 20;1103(1):94-101. *
  2. Michael E. Swartz, Ultra Performance Liquid Chromatography (UPLC): An Introduction www.chromatographyonline.com*
  3. Nicholas Ellor, Frances Goryki, Chung-Ping Yu Increasing sensitivity and throughput for LC/MS/MS-based bioanalytical assays using UPLC*
  4. Kate Yu, David Little, Rob Plumb HT Quantitative analysis for a drug mixture by LC/MS/MS:UPLC/MS/MS and HPLC/MS/MS compared*

.* Denotes Free Article

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