Improving separations, making discoveries, and hyphenation of liquid separations with mass spectrometry are the milestones achieved in the journey of 50 years of high-performance liquid chromatography (HPLC). HPLC, born half a century ago, has become one of the most widely used analytical techniques in the world. The demand for higher separation efficiency, faster analyses, and greater throughput has been the driving force for extraordinary advances in instrumentation.

Remarkable improvements in the speed of HPLC over the last decade, due to the development of columns containing sub-2 m particles and the concept of ultra-high performance liquid chromatography have revolutionized the HPLC markets with unprecedented performance. The versatility of stationary-phase chemistry, or surface modification, allows for the design of efficient and selective packing materials for the separation of nonpolar, polar, ionizable, or chiral analytes, small molecules, or large biomolecules. Particle sizes have decreased from 50 m to less than 2 m, while core-shell particles have become the favorite packing materials for many users. Silica-based monolithic columns have become a popular alternative for efficient separations at moderate pressure drops. Many exciting new developments in the field of monoliths can be foreseen, as the short columns developed for fast HPLC can be operated at higher pressures than the conventional silica-based monoliths. The ongoing researches focusing on 3D printing of chromatographic columns were the highlight of 2016, promising further breakthrough improvements in the columns.

Dedicated HPLC systems for micro- and nano-LC applications with extremely low extra-column volumes have gained traction in the HPLC markets. Furthermore, it is now possible to reproducibly inject tiny volumes of only a few nano-liters. In addition, a few manufacturers are now offering miniaturized HPLC columns with an inner diameter of < 1 mm. The combination of MS and nano-LC has moved from being cutting edge to a routinely used technology. Ongoing advances in nano-LC technology are now enabling research that promises to be transformative in terms of moving proteomics from basic research, to large-scale translational research, and finally to the clinics, where it can greatly advance the quality and precision of medical care.

Nano-LC and Proteomics

The technology of nano-LC has evolved to become more robust and reliable since the commercial launch of "splitless" nano-LC systems. Researchers are pushing the boundaries of scientific research, multiplexed quantitation of low-abundance peptides in complex matrices, characterization of positional isoforms of intact proteins, protein structure characterization, and deep mining of post-translational modifications. High-sequence coverage in combination with a good dynamic range will allow collection of information about isoforms and PTMs; information, which will be important to understand the function of proteins.

The ultra-high-performance LC (UHPLC) nanoflow systems have a considerably higher tolerance for pressure and therefore allow for the use of longer columns that deliver far superior separation for larger numbers of peptides. The combination of nano-LC and MS has proven a major boon for efforts to thoroughly dissect proteomes in both basic and clinical research settings. As the technology continues to evolve, user-friendly chip-based systems are emerging that can minimize the amount of tinkering required to get an experiment underway.

There has been a great deal of technological developments in the last twenty years or so in both nano-LC and MS, which has enabled today's capability to characterize the human proteome at a practical depth in less than a day. The low flow rate and the narrow column dimensions (75 m internal diameter) of nano-LC offer benefits both in terms of LC separation and MS data quality. Innovations in column technology have also raised the bar for nano-LC performance. Longer columns with greater peak capacity and resolution have resulted in an increase in the number of peptides that can be separated and detected, leading to higher protein identification rates and improved identification of low-abundance peptides. These advancements in nano-LC have arrived parallel to
major strides in high-performance instruments, enabling proteomics researchers to identify hundreds of thousands of peptides with much greater ease and speed, producing powerful data from 30 minutes to an hour.

Future Advances

Advancements in separation science are driven by the demands of biological, pharmaceutical, environmental, and forensic scientists, and are realized by developments in technology. Materials science, electronics, and engineering are each contributing to the technology truly becoming smaller, faster, and smarter. With the ongoing advancements, 3D printing of micro devices for liquid handling, interfacing various devices, and separations is gaining momentum. It will be interesting to see where this concept can take separations in the future.

The perfect marriage of ultra-high-pressure liquid chromatography (UHPLC) and MS instruments continues to be the megatrend in chromatography in recent years. As separation methods in combination with advanced MS technology bring us closer to the capability to characterize the complete human proteome, advancing these methods to achieve high sequence coverage will be essential.

Liquid chromatography-mass spectrometry (LC-MS) is firmly established as the technique of choice for small molecule-based diagnostics, and is emerging as a tool for protein-based diagnostics. Future precision medication will require comprehensive sensitivity challenging metabolome and proteome analyses. With improvements in HPLC and UHPLC every year, HPLC and MS are expected to come closer together in the future.


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