High Performance Liquid Chromatography (HPLC) is a workhorse technology that has stood the test of time. It has evolved to suit a myriad of applications with widespread usage throughout the research, clinical, processing, and a host of other fields. In essence, HPLC is ubiquitous throughout the analytical world, and for good reason: it is versatile, reliable, scalable, and the instrumentation involved has enjoyed a long run of compatibility, durability, and resilience. It’s clear that HPLC has no intention of fading from the limelight any time soon. There are a few limitations, though, that have begun to be addressed by an evolutionary step of technology.
HPLC, in it’s most basic form, is a technique used to separate, identify, and quantify each component in a mixture. The acronym HPLC originally highlighted the fact that high pressure was used to generate the flow required for liquid chromatography in packed columns. In the beginning, pumps only had a pressure capability of 500 psi [35 bar] -- so called high pressure liquid chromatography, or HPLC. The early 1970s newly developed HPLC instruments could develop up to 6,000 psi [400 bar] of pressure, and incorporated improved injectors, detectors, and columns. With continued advances in performance during this time, the acronym HPLC remained the same, but the name was changed to high performance liquid chromatography.
While instrumentation developments were important, the history of HPLC is primarily about the history and evolution of particle technology. After the introduction of porous layer particles, there has been a steady trend to reduced particle size to improve efficiency. However, by decreasing particle size, new problems appeared. The practical disadvantages stem from the excessive pressure drop needed to force mobile fluid through the column and the difficulty of preparing a uniform packing of extremely fine materials. Every time particle size is reduced significantly, another round of instrument development usually must occur to handle the pressure.
Let’s skip over the theories behind chromatographic separation for now and move to practical details.
Particle size - Most traditional HPLC is performed with the stationary phase attached to the outside of small spherical silica particles (beads). These particles come in a variety of sizes with 5 µm beads being the most common. Smaller particles generally provide more surface area and better separations, but the pressure required for optimum linear velocity increases by the inverse of the particle diameter squared. This means that changing to particles that are half as big, keeping the size of the column the same, will double the performance, but increase the required pressure by a factor of four. Keep this in mind…
Pore size - Many stationary phases are porous to provide greater surface area. Small pores provide greater surface area while larger pore size has better kinetics, especially for larger molecules. For example, a protein which is only slightly smaller than a pore might enter the pore but does not easily leave once inside.
Pump pressure – Pumps vary in pressure capacity, but their performance is measured on their ability to yield consistent and reproducible flow rates. Pressure may reach as high as 60 MPa (6000 lbf/in2), or about 600 atmospheres.
Modern HPLC systems have been improved to work at much higher pressures, and therefore are able to use much smaller particle sizes in the columns (<2 μm). These "Ultra High Performance Liquid Chromatography" systems or UHPLCs can work at up to 120 MPa (17,405 lbf/in2), or about 1200 atmospheres. The term "UPLC" is a trademark of the Waters Corporation, but is sometimes used to refer to the more general technique of UHPLC.
UHPLC has changed the game so to speak (again) by enabling chromatographers to combine the speed of shorter columns with the resolution of longer columns, thereby enjoying the best of both worlds.
The HPLC technology has become a household name by infiltrating every facet of analytical chemistry and many other fields as well. The ubiquitous adoption of the technology has been met with an equally impressive array of high end manufacturers and models. UHPLC is simply a measured evolutionary step towards addressing the conflict of speed versus resolution. It’s important one though.