The most desirable features of High Performance Liquid Chromatography a user looks forward to are:
- Highest resolution
- Greatest achievable sensitivity
- Fastest analysis speeds
These became reality with the introduction of Ultra High Performance HPLC (UHPLC).Column efficiency was known to increase with reduction in particle size of stationary phase. Particle sizes in the range 3-5μm were used in conventional analytical separations. The sub-2μm barrier was broken in 2003 and at Pittcon in 2005, columns packed with sub- 2μm particle size were demonstrated. This development heralded the era of UHPLC.
Let us first appreciate the essential differences between you UHPLC and normal HPLC.
UHPLC columns are generally shorter and narrower (2.1 mm ID X 50 mm length) compared to analytical columns (4.6 mm id X 250 mm length)
UHPLC systems are capable of handling high pressures (15,000 – 18,000 psi) in comparison to normal analytical range High Performance Liquid Chromatography (5000 – 6000 psi). Pumps are able to withstand such conditions with minimum wear and tear of components.
UHPLC separations require lower flow rates (250 µL / minute – 750 µL / minute) in comparison to normal High Performance Liquid Chromatography (1 – 2 ml / minute). Such low flow rates require pumps with accurate metering and gradient reproducibility.
Separations on UHPLC columns often produce narrow peaks (few seconds or even less). Conventional detectors have slow response and generate compressed peaks due to lesser number of data points. UHPLC detection requires high-speed response (> 100Hz)
UHPLC provides a big advantage in terms of time-saving and increased laboratory throughput. Analytical separations using conventional size particles take on an average 10 – 30 min or more. In comparison UHPLC separations are mostly completed in less than 10 min and some even in less than 2 min
Improvement in Resolution
The peaks obtained by UHPLC are much better resolved due to benefits detailed above and that too in a fraction of time as compared to normal analytical separations.
Reduced Operational Costs
High Performance Liquid Chromatography is an expensive technique involving use of high purity grade solvents. UHPLC offers saving on operational costs because the chromatographic runs are completed in a fraction of time and at lower flow rates in comparison with the analytical High Performance Liquid Chromatography.
UHPLC has gained popularity in many laboratories working on new drug development, pharmaceutical quality control, life sciences and environmental analysis. The advancements in column packings, such as, core shell technology combined with LC-MS – MS are destined to make UHPLC even more popular and further extend the scope of applications.
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