The advent of ultrahigh-pressure liquid chromatography (UHPLC) and its successful commercialization in the last few years
has brought forth a modern high performance liquid chromatography (HPLC) platform capable of higher speed, resolution, precision,
and sensitivity. Currently, all major HPLC manufacturers offer some type of low-dispersion UHPLC products with upper pressure
limits ranging from 15,000 to 19,000 psi (1000 to 1300 bar). This installment describes a number of popular myths or half-truths
in UHPLC and provides data that contradict or even repudiate some of these commonly held beliefs.
For five decades since the 1960s, the pressure limits of high performance liquid chromatography (HPLC) systems remained stagnant
at 6000 psi (400 bar). This pressure limit was appropriate for the available column packings, which continuously trended towards
smaller particle sizes during that period (that is, from 30, 10, 5, to 3 Ám). There appeared to be no concerted efforts to
increase the system pressure ratings during that period with the exception of an exploratory study published in 1969 (1).
The "breakthrough" in ultrahigh-pressure liquid chromatography (UHPLC) came in 1997 with proof-of-concept research by James
Jorgenson (2) and follow-on studies by Milton Lee (3). These early studies demonstrated spectacular performance (column efficiency,
N= 200,000 plates) at very high pressures (>60,000 psi) in research systems using capillary columns. However, the impacts of
their discoveries for typical practitioners and for routine applications were only possible after the debut of commercial
UHPLC equipment with reliable autosamplers and gradient capabilities.
In 2004, Waters Corporation introduced the first UHPLC system — the Acquity UPLC (Ultra-Performance LC) system with an upper
pressure limit of 15,000 psi (1000 bar) together with Acquity UPLC columns (1.0 and 2.1 mm i.d.) packed with sub-2-Ám hybrid
particles (4–8). Although this pressure rating was modest in comparison to that achieved using the early research systems,
the new UHPLC system generated considerable excitement and established higher performance benchmarks and expectations. These
early systems enjoyed immediate acceptance in research applications despite some initial concerns over injection precision
and other issues in quality control (QC) applications (7,9). Other manufacturers quickly followed with their own UHPLC systems.
By 2010, the transformation from HPLC to UHPLC was essentially complete with UHPLC product offerings available from most major
vendors. Today, all UHPLC systems have reduced system dispersion and dwell volumes as well as improved precision and sensitivity
The fundamentals, benefits, potential issues, and best practices of UHPLC are well documented (5–7,9–15). Some of the key
benefits are as follows:
- Faster analysis with good (or acceptable) resolution — the primary incentive for new users in high-throughput screening (HTS),
liquid chromatography–mass spectrometry (LC–MS), routine testing, and method development.
- Superiority in high-resolution separations of complex samples — peak capacities of 600–1000 are now possible in a reasonable
time (<60 min under gradient conditions). This capability is transformative in life science research and the analysis of complex
pharmaceuticals, filling an unmet need for QC applications (13–15).
- Other benefits of UHPLC versus conventional HPLC include substantial solvent savings (5–15-fold), increased mass sensitivity
in UV detection (3–10-fold), and improved precision for both retention times (2–3-fold) and peak areas (<0.1% RSD).
In the last few years, UHPLC has evolved from a scientific curiosity for early adopters in research and high-throughput screening
into a modern standard HPLC platform. As the saga of UHPLC unfolded, a number of myths or half-truths have emerged. The goal
of this column installment is to describe some of the more interesting myths and provide evidence to delineate or repudiate
these widely held misconceptions. The myths:
- You don't need an expensive UHPLC system — high-temperature LC or core–shell columns will get you there.
- Viscous heating is a "huge" issue for sub-2-Ám particle columns.
- A 2.1-mm i.d., sub-2-Ám column is the best choice for UHPLC.
- Gold-plated fittings with double ferrules are needed in UHPLC.
- A binary high-pressure mixing pump is a "must."
- UHPLC provides substantially higher UV sensitivity than conventional HPLC.
- Method transfer between UHPLC and HPLC is very easy ("a piece of cake") and method revalidation is not needed.
- Lower-dispersion UHPLC systems are better.