High-performance liquid chromatography (HPLC) has undergone a pattern of breakthrough technologic advances followed by periods of slower, incremental improvements.
Waters’ 2004 debut of UPLC® leveraged sub-two-micron (s2m) stationary-phase particles, low instrument dispersion, and pressures up to 15,000 psi. Eventually other major vendors developed similar products, generically known as UHPLC (ultra high-performance liquid chromatography).
In 2006 Advanced Materials Technology (AMT) introduced Fused-Core® technology, with particle sizes of between 2 and 3 microns. Fused-Core, and its follow-on products (known as core-shell, superficially porous, porous-shell, etc.), promised performance approaching that of UHPLC but with much lower backpressures.
Adoption of mass spectrometers (MSs) as detectors expanded the capabilities of HPLC even further by enabling the discrimination of small mass variants, particularly in biomolecules.
The best technology depends on the needs and workflows of a particular organization.
“We’ve recognized, over the last several years, that needs exist for all levels of chromatographic performance in the pharmaceutical, biopharmaceutical, food, and materials industries,” notes Eric Grumbach, director, product marketing, chromatography systems at Waters. “Some customers require the highest resolution that is only achieved with higher-end UPLC systems and sub 2 µm particles. Other customers are quite happy with mid-tier instruments in the 9,000 to 10,000 psi range and superficially porous particles. And even with these higher-performing options many organizations continue to run legacy methods that utilize traditional HPLC systems with 5 micron particles. The best technology depends on the needs and workflows of a particular organization.”
As high-pressure systems were gaining followers, Waters and its UHPLC competitors underestimated the pull of routine analytical methods for common products, particularly among highly regulated industries.
“When we launched UPLC in 2004, we aspired to replace HPLC technology over time. Yet traditional HPLC Systems still remain a very large part of the analytical laboratory, particularly in routine analysis and QA/QC,” Grumbach tells Biocompare.
Very little crosstalk occurs among HPLC platforms. Methods developed for a particular analyte on a specific instrument type tend to stay put, although analysis sometimes develop methods on higher-throughput UPLC and transfer them to HPLC for routine use. “Organizations are unlikely to invest in altering methods that they have been running for 15 years or more. They usually adopt more modern technologies for new products.”
Nevertheless, since the advent of U(H)PLC, customers have been concerned with the analytical transfer of methods from older platforms to newer ones. In 2015, Waters introduced the ACQUITY Arc UHPLC system, which employs a selectable dwell feature to enable users to easily replicate established HPLC methods, independent of the system of origin, while preserving the profile of their method.
Existing on a technologic plateau is not the same as standing still. HPLC reliability and robustness have improved across the board, at every level, while performance has held steady or improved. To reduce peak dispersion on older instrument platforms manufacturers have eliminated unnecessary volumes through greater precision of fittings and connectors, and by reducing column diameters.
Choices, choices, everywhere
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 
.
“Every major HPLC system manufacturer offers both HPLC, UHPLC, and mid-pressure systems, both in fully porous and core-shell platforms,” says Jeff Layne, senior technical marketing manager at Phenomenex, a provider of HPLC columns. “All these technologies have a role to play. Rather than telling customers ‘buy this!’ we present them with alternatives. So many choices exist and before they buy anything they should see what works best for their applications.”
Layne adheres to the “plateau” theory of HPLC advancement. “Waters’ UHPLC and subsequent core-shell technologies bumped us up to new levels of sophistication. But it’s hard to say where the next big HPLC development will come from, or when. We are past due.”
Current customers are satisfied, Layne says, adopting and adapting current technologies to fit their needs. “In the meantime, incremental improvements make products a little better until we hit the next quantum leap.”
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Layne suggests that the next big improvement might result in response to the growing need for robust analytical methods that serve therapeutic and industrial biotechnology. If one believes the hype surrounding new protein-based therapeutic entities (e.g. biospecifics and antibody-drug conjugates), then the frontiers of biopharmaceutical HPLC will continue to expand. Biopharma's current quality binge will also play into this, particularly in the analysis of post-translational modifications of monoclonals (e.g. of protein-bound carbohydrates).
Choices
If you’re still confused about which LC platform to adopt, welcome to the club.
“Laboratories should invest in whatever drives their lab’s efficiency and productivity,” says Jason Link, marketing manager, SM, GPC, and preparative HPLC columns, at Agilent Technologies. Competition across market segments has been good news for consumers, who now have more choices than ever before.
But choices, while desirable, involve decisions, which are sometimes difficult.
“Labs need to strike a balance between current needs and potential future needs. With system lifetimes still being between five and ten years, buyers should examine the flexibility of a vendor’s portfolio in terms of ease of reconfiguration and upgrades,” Link adds. “For example will a system lock the lab into an application space? As there is no black and white answer, labs need to consider these variables when making purchasing decisions.”
Link believes that core-shell delivered on its promise of UHPLC-level results without the high backpressures. “Shortly after the introduction of modern superficially porous particles from AMT in 2006, and from other vendors including Agilent, labs rapidly adopted these columns.” Chromatographers recognized the benefits of migrating from legacy 5-micron methods to modern 2.7-micron superficially porous columns.
“That being said, while the initial draw for the 2.6–2.7-micron column was high performance with lower backpressures, many superficially porous column options, such as Agilent’s InfinityLab Poroshell 120, now include sub-2 micron versions, enabling even higher performance than their previous-generation counterparts. And, while running modern superficially porous columns on a UHPLC system isn’t necessary per se, more advanced systems may offer other benefits such as lower dispersion, which can benefit separations using higher efficiency superficially porous columns. With columns and instruments working more in sync with one another, chromatographers now have the ability to realize higher pressures when using columns, supplies, and systems designed for one another.
So where on the HPLC technology continuum has the dust settled? According to Link it hasn’t.
“We currently see a preference for the 600 bar [8500 psi] systems, but the main growth is coming mostly from the >1000 bar systems, as well as other systems such as two-dimensional LC, bio-inert systems, supercritical fluid chromatography (SFC), and high-throughput systems that increase productivity. Another variable to consider is that as regulations are changing around the world, the need for higher sensitivity, faster analyses, and more automation is becoming more common. Because of this, more analytical methods are being developed on LC/MS instruments, especially in the strong growing area of biopharmaceutical development.”
Improving Access to Ultrapure Water for HPLC
High-purity eluants are essential for HPLC analysis. The eluant used must have high physical and chemical purity, and may not contain suspended mechanical particles or any dissolved substances that can be released by the column and generate a signal. Water to be used as an eluant must also be free of microorganisms.
Unfortunately deionized or distilled water often contains organic substances. So scientists performing HPLC often have to purchase the ultrapure water required for HPLC. A water purification system is a new more-affordable option that allows labs to produce ultrapure water when and where it is needed.
One such system is the arium® pro VF from Sartorius. The arium pro VF system has been designed to produce ultrapure water from pretreated drinking water by removing contaminants. Production of ultrapure water requires continuous recirculation and a constant water flow rate, which is achieved using a built-in pump system with controlled pressure. The conductivity of the water is measured at the fed water inlet and also at the downstream port.
The system features two different cartridges. These are filled with an active carbon adsorber and mixed-bed ion exchange resins in order to produce ultrapure water with a low TOC content. In addition the system includes a UV lamp that has an oxidizing and bactericidal effect.
The arium pro VF system also has built-in ultrafilter modules that are used as a crossflow filter. The ultrafilter membrane incorporated within this filter retains colloids, microorganisms, endotoxins, RNA, and DNA.
Sartorius says that the arium pro VF system can produce ultrapure water on demand for HPLC analysis in medical, chemical and biochemical research as well as during in-process quality control testing in pharma and biotech facilities.