HPLC – A Rapidly Evolving Technology
High-performance liquid chromatography is constantly evolving, driven by the needs of the many areas in life sciences and technology wherein it plays a key role.
High-performance liquid chromatography (HPLC) has undergone a pattern of breakthrough technological advances followed by periods of slower, incremental improvements. Adoption of mass spectrometers (MS) as detectors expanded the capabilities of HPLC even further by enabling the discrimination of small mass variants, particularly in biomolecules.
Unlike product sectors where companies grow but only at the expense of the overall market, HPLC is and remains a growth industry where rising tides raise all ships. A recent research study estimated the global HPLC market at USD 3763.9 million in 2017. It is projected to expand at a CAGR of 5.2 percent from 2018 to 2026. The key factors driving growth include high sensitivity and accuracy of HPLC techniques, growing popularity of LC-MS technique, growing importance of HPLC tests in drug approvals, and increasing life science R&D spending. In the coming years, the market is expected to witness the highest growth rate in the Asia-Pacific region. The high growth in the region can be attributed to the growth in biomedical and medical research in Japan, strategic expansions by key players in China, increasing government initiatives and growing pharmaceutical industry in India, and the favorable regulatory scenario in New Zealand and Australia.
Trends in HPLC systems
With ultra-high pressure liquid chromatography (UHPLC) firmly established as the modern HPLC platform for more than a decade, all major manufacturers have already introduced the first- and second-generation UHPLC systems. In the past few years, manufacturers continued to introduce line extensions to existing equipment, such as dual-path, intermediary-pressure, and bioinert systems. This year’s new introductions also appeared to be trending toward application-specific systems (e.g., cannabis analysis), front ends to mass spectrometry systems, micro- and nano-HPLC systems, biopurification systems, portable instruments, preparative LC systems, and integrated HPLC systems for quality control (QC) applications. New MS systems abounded, ranging from single-quadrupole and triple-quadrupole, to quadrupole-time-of-flight (Q-TOF) hybrid MS systems.
Hydrophilic interaction liquid chromatography (HILIC) broadens the range of target compounds to small polar molecules. Although HILIC has been discussed for a long time, the method itself has become more routine because of better tools to perform this technique with confidence. Comprehensive two‑dimensional liquid chromatography (LC×LC) will leave the research field and enter routine analysis if more tools are developed to make this powerful technology simpler. Higher peak capacities can now be obtained by hyphenating multidimensional liquid chromatography with ion mobility spectrometry, and high-resolution mass spectrometry is driving the omics scene.
A continued growth is expected in the use of liquid chromatography with tandem mass spectrometry (LC–MS/MS) technology where previously other types of analytical instrumentation would have been preferable. This is because the improvement in MS technology, in both hardware and software, allows highly selective and sensitive methods to be developed for applications including food safety, food authenticity, and security; environmental protection, including water quality testing; biopharmaceutical research and development; and clinical research, forensics, and omics-related research. The use of high-resolution accurate mass (HRAM) LC–MS in all these applications is a growing trend.
Automation has always been the trend in LC and LC–MS and has been driving the development of various analyzers for alleviating the need for optimization and streamlining the sample preparation workflow. This trend will continue but with increased sophistication. Now instrument vendors are beginning to provide fully automated, sample-to-result analytical platforms.
On the LC side the trend toward UHPLC using sub-2 µm particle size is ongoing, whereas for low-flow applications the trend splits into two directions: toward very low flow (sub-nanolitre) rates and toward capillary flow LC. Very low flow is required where applications demand extremely high sensitivity. The demand for increased sensitivity, throughput, and robustness has seen capillary flow LC becoming more important because of its ability to provide increased MS sensitivity compared to typical analytical flow LC–MS, with the additional advantage of lower solvent consumption and higher throughput while maintaining similar sensitivity as nanoflow LC. For routine and QC markets there is a demand for increased productivity, robustness, reliability, and accuracy with high selectivity and sensitivity; this can be addressed by LC–MS, with a continuing trend toward the use of HRAM MS within these environments.
The future of LC–MS is the continued growth and adoption of the technology to solve challenging analytical problems. The technology involved in mass spectrometry development over the past 20 years or so has meant smaller, faster, more selective, and more sensitive instrumentation being designed and implemented for analytical assays that can be considered as extremely complicated. The analysis of multiple analytes with minimal sample preparation or the identification of protein structure is no longer a complex challenge. One of the biggest challenges going forwards is processing the data and understanding what all this data means to scientists.
Although much effort has been put into simplifying data processing and review, these factors still remain far from full automation, consuming a lot of time and analytical expertise. It is easy to envisage the implementation of artificial intelligence as an integral part of an analytical platform to assist data processing and review, as well as enabling self–diagnosis, self–tuning, and self-maintenance to further reduce human intervention in a routine operation.
In the future, LC will continue to be a separation technique of choice, and expected to see increasing connectivity with MS, in particular for workflows in the routine applied markets and QC. MS, and in particular HRAM MS, provides an additional level of confidence that most optical detectors simply cannot provide. In addition, with recent technological advances, both triple quadrupole (QqQ) and HRAM MS can now provide a level of sensitivity that opens doors for replacing other technologies, for example, costly immunoassays in clinical laboratories. This does, however, require very easy‑to‑use, robust instruments
and workflows providing reliable, high-quality data, regardless of the user’s experience.
Now is a great time for life science organizations to invest in HPLC technology. As the demand for HPLC systems increases in the life science industry, manufacturers are looking to develop systems that are tailored to the needs of life science research and manufacturing organizations. When bringing an HPLC system into a lab, it can be helpful to pair it with modern automation software.
Considering the costs of investing in HPLC. There is no doubt that HPLC can provide significant advantages for disease researchers, drug developers, and pharmaceutical manufacturers, but many life science organizations are concerned about the high cost of investment – including both up-front costs and the long-term requirements for maintaining and managing the system. One way to deal with the latter challenge is to adopt a software system that can automate routine calibration and maintenance workflows. That way, companies can avoid the expense of hiring a tech to maintain the HPLC, and they would not have to contend with the loss of valuable research time when regular lab members have to add manual HPLC maintenance tasks to their list of things to do. Over the next decade HPLC will continue to evolve, with productivity being at the center of all developments and enhancements, whether that is in hardware, software or columns, and chemistries.
A Brief Insight To HPLC
Dr S Tasleem Raza
Professor and In Charge – Central Research Lab, Department of Biochemistry,
ERA’S Lucknow Medical College
HPLC (high-performance liquid chromatography, formerly referred to as high-pressure liquid chromatography) is a chromatographic technique used to separate components of a mixture, for their identification, and their quantification. It is basically a highly improved form of column liquid chromatography. In general, the method involves a liquid sample being passed over a solid adsorbent material packed into a column using a flow of liquid solvent. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres making it much faster. Each analyte in the sample interacts slightly differently with the adsorbent material, thus retarding the flow of the analytes. If the interaction is weak, the analytes flow off the column in a short amount of time, and if the interaction is strong, then the elution time is long. All chromatographic separations, including HPLC operate under the same basic principle; separation of a sample into its constituent parts because of the difference in the relative affinities of different molecules for the mobile phase and the stationary phase.
There are different variants of HPLC like normal phase HPLC, reverse phase HPLC, size-exclusion HPLC, ion-exchange HPLC; which differ depending upon the stationary phase used in the process. A basic HPLC instrumentation includes a pump, injector, column, detector/integrator, and display system. The heart of the system is the column where separation occurs. The information obtained by HPLC includes resolution, identification, and quantification of a compound and this technique thus holds immense scope of applications in both academic and industrial laboratories requiring identification and quantification of mixtures of organic compounds. The other applications of HPLC include pharmaceutical quality control, detection of phenolic compounds in drinking water, bio-monitoring of pollutants, quantification of drugs in biological samples, identification of steroids in blood, urine, measurement of quality of soft drinks and water, sugar analysis in fruit juices, preservative analysis, antibiotics analysis in blood.