HPLC Versus UPLC

UPLC can be regarded as a new invention for liquid chromatography. Compared with conventional HPLC, UPLC can offer significant improvements in speed, sensitivity, and resolution, which also mean reduced solvent consumption and analysis cost. Figure 10.2 shows an HPLC versus UPLC separation comparison of a ginger root extract sample, where both speed and resolution are improved, and sensitivity is increased [57]. 

FIGURE 8.5 UPLC versus HPLC for a compound and its hydroxyl metabolite. A threefold improvement in speed is observed while improving resolution and sensitivity. UPLC (1.7 pm particle size), RT 0.44 min, peak width 2s FWHM. HPLC (4pm particle size), RT 1.3-1.5 min, peak width 6 s FWHM. (From Wainhaus, S. et al., Amer. Drug Discov., 2, 6, 2007. With permission.)... 

FIGURE 8.10 (a) UPLC versus (b) HPLC for a compound and two of its hydroxyl analogs. 

FIGURE 8.11 UPLC versus HPLC for analysis of glucuronide metabolites. UPLC clearly shows the existence of two glucuronide conjugates while HPLC only shows one. (From Plumb, R. et al., Rapid Commun. Mass Spectrom., 18, 2331, 2004. With permission.)... 

Nordstrom et al. [121] also compared the differences between UPLC and HPLC for metabolomic profiling. This time a Waters Acquity system was coupled to Micromass Q-Tof-Micro mass spectrometer and data were acquired in the m/z 100-1000 range with an acquisition of 2 spectra per second. About 20% more components were detected using UPLC versus HPLC and the length of the chromatographic separation was one of the most crucial parameters affecting the number of detected features. These examples demonstrate the importance of appropriate mass analyzers when utilizing UPLC. For more on UPLC see Chapter 8. 

This gain can be seen in plots of plate coimt versus analysis time [5, 6]. An example of such a plot for a high-performance separation is shown in Fig. 1. Note that the time axis is a logarithmic axis. The assumption in this plot is a retention factor of about 10, combined with a standard solvent composition with the same viscosity as water. The performances of three columns are shown a 25 cm as well as a 30 cm 5 gm column as examples of the performance achievable by classical HPLC, and a 10 cm UPLC column showing the gain in speed at the same performance level as the 30 cm 5 gm column. The maximum plate coimt that is achievable with the UPLC column is about the same as that on a 30 cm column packed with 5 gm particles. However, this high performance (about 25000 plates) can now be achieved in about one-tenth of the time needed to do the job with the classical HPLC column. Conversely, if we force this column to be used for faster analyses, the column performance drops drastically. The 30 cm classical column... 

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