Liquid chromatography, an instrumental technique of analytical chemistry, has found applications in a multitude of industries including clinical applications, forensics, research, and manufacturing. Over time, high performance liquid chromatography (HPLC) systems have delivered dramatic increases in analysis speed, sensitivity, and throughput.Â HPLC is a well-established separation technology that continues to evolve to meet the identification and purification challenges posed by pharma, biotech, and other life science applications.
The current trends in HPLC systems are integrated, cross-compatible platforms that offer higher separation efficiency, shorter analysis time, and improved IT tools.
Second-generation UHPLC systems. Second-generation ultra-high performance liquid chromatography (UHPLC) systems are being steadily introduced into research laboratories. Improved second-generation UHPLC systems that offer higher operating pressure ratings, lower dispersion, innovative ovens or auto-samplers, and faster cycle times. A large number of manufacturers are offering a single instrument platform with multi-tiered pressure ratings or configurations (modular versus integrated). Modular systems typically have higher system performance, while integrated systems generally have lower pricing for quality control applications. New systems can be brand new platforms or upgraded versions of existing systems. They can also be specialized application systems. While hardware specifications remain important, purchasing decisions continue to be dominated by chromatography data system.
Advances in HPLC detectors. Current HPLC detectors frequently use deuterium lamps as a light source because of the high light output in UV wavelengths as well as excellent stability of light and relatively long life. HPLC manufacturers select deuterium lamps because of the high stability of light output through the duration of a measurement as well as the relatively high light output of the lamps at their HPLC-relevant UV wavelengths.
Advancements in light emitting diode (LED) technology are also providing alternative solutions that match or exceed the performance of traditional ultraviolet C-range (UVC) lamps, while reducing system costs for instrument manufacturers. UVC LED solutions have enabled HPLC manufacturers to replace deuterium lamps and offer lower cost detectors for fixed wavelength applications. Systems using UVC LEDs can match and sometimes exceed the performance of systems using UV lamps, while at the same time delivering higher efficiency and reduced costs for fixed wavelength applications. The comparable performance allows manufacturers to offer longer instrument life, higher reliability, and increased productivity. These new devices are driving innovations in instrument design for life sciences to address key market trends around productivity, cost reduction, and miniaturization.
Superficially porous columns. The use of superficially porous particles (SPPs) in the manufacture of HPLC columns has become prominent in recent years. Over the course of the past decade most major manufacturers have built column lines around the technology. A major goal in HPLC over the past decade has been to develop particle technologies that provide enhanced separation efficiency, with the introduction of sub-2-Î¼m particles, along with advances in HPLC instrumentation. SPP technology provides nearly similar efficiencies to the sub-2-Î¼m particles, but at a lower cost of back pressure. The lower back pressure has allowed the technology to be used across a wider array of HPLC systems. Since its introduction, SPP technology has been embraced by most major column manufacturers and HPLC practitioners.Â A few major trends that have been observed in case of SPP include expansion of particle sizes and pore geometries; additional chemical surface modifications; and application beyond small molecule reversed-phase analyses.
Multi-dimensional workflows. The current trend of employing orthogonal chromatography techniques has given rise to the possibility of multidimensional workflows wherein these new methodologies can be put together and implemented much more quickly. Complete characterization workflows can now be undertaken on a single instrument at high throughput level. The software capabilities available have made it relatively easy to perform the analysis. Many multidimensional systems have complicated plumbing requiring experienced analysts. The use of a fractionating autosampler and the ability to select different columns is a simple solution. Any peak from any column can be collected for further analysis without changing the plumbing configuration.Â The benefits of increased resolution and selectivity using multidimensional workflows are promising. However, the huge drawback has been the complexity of the system setup and time taken to perform. These high-resolution systems now have the column chemistries to give the throughput required for complete characterization in a short time.
Advances in column chemistry. Advances in instrument technology have been aligned with similar advances in column chemistries to bring large molecule analysis into the realms of UHPLC. It was a necessity to improve several chemistries alongside the instruments as the analysis of bio-therapeutics requires several FDA-regulated characterization protocols using an array of techniques which are quite different from those used in small molecule analysis.
Utilization of high-resolution chemistries and more efficient sample preparation accomplishes the goal of higher throughput without increasing the effort required. Ion exchange, reverse phase peptide mapping, and intact protein analysis have been improved here to show highly selective resolution of different types of molecular variants to provide global methodologies.Â Many of these techniques however have eluent systems that are not compatible with mass spectrometry (MS) detection. The initial positive identification of each variant peak from all of these chromatography methods must be achieved through mass spectrometry.
Combinations of ion exchange, hydrophobic interaction chromatography, and reverse phase HPLC/MS can facilitate positive identification with accurate fractionation of the UHPLC peaks followed by reverse phase desalting. Method development can be greatly facilitated by understanding the interactions that different stationary phases provide and applying that knowledge to the separation task at hand. As the main differences of the set of analytes to be separated, a column or a set of columns can be judiciously chosen to screen for effective retention and selectivity.