A key component of the evolution of HPLC technology has been the use of smaller stationary phase particles. Chromatographic resolution is improved by the use of small particles due to the reduction in the height equivalent of a theoretical plate (HETP) resulting from the decrease in the C term of the van Deemter equation (resistance to mass transfer term). Since the C term is proportional to the square of the particle size, the use of smaller particles leads to significant improvements in chromatographic efficiency. In addition to this increase in chromatographic resolution, the optimum linear velocity of the mobile phase increases as particle size decreases, reducing analysis time (3.3x faster analysis using 1.5 um particles rather than a 5 um particles). The first routine applications of HPLC used 25 um particles with 25 cm long columns, and the development of 10 um and then 5 um particles with 25 cm long columns gave significant increases in chromatographic resolution and a reduction in analysis time. The relatively recent development of 3 um and 1.5 um particles has lead to decreases in analysis time, but no increase in chromatographic resolution since the pressure limitations imposed by these small particles forces the use of shorter columns. The mobile phase pressure at the optimum linear velocity is inversely proportional to the cube of the particle diameter, thus going from a 5 um particle to a 1.5 um particles requires a 37-fold increase in mobile phase pressure. Professor Jorgenson has designed and fabricated HPLC pumps capable of generating mobile phase pressures of up to 80,000 psi. At 19,000 psi UHPLC has given separation efficiencies in excess of 300,000 plates for a 66 cm long column operated at the optimum linear velocity. The same column operated at 59,000 psi (3x optimum linear velocity) still gave 190,000 plates, but with a 3x reduction in total analysis time. Thus, UHPLC provides separation efficiencies equivalent to those obtained by capillary electrophoresis, with the sample loading capacity of LC columns, along with the "logical" control of the separation process offered by gradient elution. UHPLC offers both high speed analysis as well as high sensitivity analysis.
Electrospray is a concentration sensitive detector, which is why the use of smaller ID chromatography columns gives significant improvements in detection limits. The increase in concentration sensitivity is proportional to the square of the column ID, thus a 2 mm ID minibore LC column offers a 5-fold improvement in sensitivity over a 4.6 mm LC column. The 150 um ID capillary columns used in these experiments offer a 940-fold improvement in concentration sensitivity over a 4.6 mm LC column. In addition to the improvement in concentration sensitivity offered by capillary columns, this UHPLC system also offers improvement in concentration sensitivity from improved chromatographic efficiency obtained by the use of small stationary phase particles. The higher chromatographic efficiency resulting from the use of small stationary phase particles translates into narrower chromatographic peaks, thus a 10-fold improvement in chromatographic efficiency translates into a 10-fold improvement in concentration sensitivity.
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