High-pressure liquid chromatography (HPLC) #
HPLC can be interpreted as high-performance liquid chromatography or as high-pressure liquid chromatography. The former is often correct; the latter always is. For about 40 years HPLC instrumentation was designed to operate at pressures up to 40 MPa (400 bar or – in ancient units – about 6000 psi). During these years common particle sizes in packed HPLC columns decreased from 10 µm, to 5 µm, and then to 3 µm. Smaller particles allow faster analyses.
The most common column internal diameter (i.d.) remained 4.6 mm, although 2-mm i.d. columns and 1-mm i.d. column were introduced to save solvents and to enhance compatibility with mass spectrometry (MS). Reducing column diameters and reducing particle sizes necessitate smaller extra-column volumes, so as to minimize extra-column band broadening.
Ultra-high-pressure liquid chromatography (UHPLC) #
Some 20 years ago, LC took a big leap forward with the emergence of equipment for UHPLC, allowing pressures up to 100 (and later 150) MPa Concomitantly, the particle size was reduced to diameters below 2 µm (so called sub-2-micron particles). This allowed very fast analyses (well within 1 min) on very short columns (50 mm length or less).
A caveat proved to be that the high amount of dissipated frictional energy led to an increase of the temperature inside the column. A radial temperature gradient, which would arise if the outside of the column ere to be kept at a constant temperature, would have detrimental effects on the column efficiency. To remedy this, narrower columns (typically 2.1 mm i.d.) are typically used in UHPLC and forced cooling of the column is strongly discouraged.
As a result of the short and narrow columns, extra-column band broadening is a major threat in UHPLC. Extremely small extra-column volumes and connections without any dead volumes are as important as the high-pressure capability of the instrument.
