Reprinted from American Laboratory February 2010
Application Note
by William Foley, Beth Gildea, and Thomas E. Wheat
The Missing Link Between HPLC and UPLC Technology
O
ver the past 30 years, liquid chromatography has proven
to be the predominant analytical technology within
laboratory-dependent organizations around the world.
No other single technology supports a company’s analytical
needs from product discovery, research, all stages of development, scaling, and manufacturing to final testing for release
with the performance and efficiency of LC.
Yet even with its role as a central analytical tool, not too long
ago LC technology came dangerously close to becoming a scientific commodity where new systems and methods merely offered
incremental advancements with a relatively minor competitive
advantage. While the world of technology took great leaps
forward, analytical chemistry lagged behind. High-performance
liquid chromatography has not kept pace with the urgency of
today’s business demands to analyze more samples, faster, and
with better results.
Six years ago, a new category of chromatographic performance
emerged with the commercialization of sub-2-µm particle columns engineered in tandem with advanced instrument design
featuring modern fluidics modules that deliver very high performance. The result of smaller column material particle sizes
and reduced system dispersion has been significant improvements in analytical resolution and sensitivity while increasing
sample throughput.
Figure 1
The ability to form a quaternary gradient on the ACQUITY UPLC
Waters Corp. (Milford, MA) is widely recognized for fueling H-Class system improves the critical resolution for U.S. EPA Method 8315 Option 2
this LC revolution in 2004 with the introduction of Ultra- for the analysis of 15 aldehydes and ketones as 2,4-dinitrophenylhydrazine (DNPH)
Performance Liquid Chromatography ®, better known today
derivatives, with a UPLC separation in less than 10 min. (Figures courtesy of Mike
as UPLC ®. This disruptive technology, first released as the
Jones and Tanya Jenkins, Waters Corp.)
ACQUITY® UPLC system, has already replaced thousands
of HPLC systems, supported more than 500 peer-reviewed
papers, demonstrated reduced solvent consumption up to 95% for
quaternary solvent manager (QSM) and sample manager with flowgreener laboratories, and has served the needs of regulatory agenthrough needle (SM-FTN) design (see Figure 1).
cies around the globe.
The ACQUITY UPLC, the original system, features a binary
solvent manager (BSM) with fixed-loop sample manager. The
System description
BSM-based ACQUITY UPLC system is characterized by lower
Having proven its performance and reliability under extremely
system volume (dwell volume), minimized dispersion, and fast
rigorous conditions for the most demanding applications, UPLC
injection-to-injection cycle times. The system is well-suited
technology has brought routine benefits to increasing numbers of
for high throughput and particularly any MS-based applicascientists, so much so that today’s HPLC users, many of whom are
tion where its low system dispersion provides high resolution
not able to or prefer not to change their approach to LC, are lookand sensitivity. The system represents the lowest in solvent
ing for the benefits from UPLC technology in a way that is compatconsumption and highest-efficiency performance for researchible with their laboratory procedures. With that goal in mind, the
grade applications.
ACQUITY UPLC H-Class system was developed, which provides
UPLC performance with HPLC familiarity.
The ACQUITY UPLC H-Class system, with its quaternary solvent manager and sample manager, with flow-through ­n eedle
Achieving UPLC-quality separations without changing how HPLC
design, features a nearly identical work flow compared with a
operators work was identified by Waters customers as the key
traditional HPLC system. The system technology maintains
challenge to accelerating their adoption of UPLC. The answer to
the ACQUITY UPLC’s low dispersion characteristic, enabling
this hurdle came in the form of the ACQUITY UPLC H-Class’s
the chromatographer to achieve the same high-efficiency
Figure 3
Solvent composition is easily adjusted using the AutoBlend capability of the ACQUITY UPLC H-Class system. In this example, the optimum
concentration of TFA for a peptide map is identified simply by varying the percentage of flow taken from the D line. There is no need to make extra bottles of
solvent, and intermediate values can be tested with minimal effort.
back. In fact, the method transfer process can be accomplished in
three steps:
1. Select an ACQUITY UPLC column that is comparable to the
current HPLC column using the Waters reversed-phase column
selectivity chart.
2. Enter existing HPLC conditions into the ACQUITY UPLC
columns calculator. The tool guides users step-by-step to either
scale the gradient or further optimize for speed or resolution.
The best column dimensions are selected from the menu, and
the best method conditions are calculated.
3. Run the analysis.
The multisolvent blending capabilities of the ACQUITY UPLC
H-Class system also make it an effective platform for methods development. A series of method development kits consist of several
UPLC columns that encompass a range of selectivities to accommodate different method development approaches. The kits enable
methods to be developed efficiently and effectively on the system.
Figure 2
In this method transfer example using the related substances
test for galantamine, used in the treatment of Alzheimer’s disease, the USP
method (monograph: USP32-NF27 supplement: no. 2, page 4245) is demonstrated first using an HPLC system. The method is directly transferred to
the ACQUITY UPLC H-Class system using an HPLC column, maintaining
selectivity and resolution, then scaled to UPLC using the ACQUITY UPLC
columns calculator and optimized for the shortest analysis time at equal peak
capacity. The run time decreased by 46 min.
Backed by a range of ACQUITY UPLC columns that includes
three particle substrates in 11 chemistries, all of which are scaleable between HPLC and UPLC particle sizes, the ACQUITY
UPLC H-Class system supports a broad range of application
needs right from its introduction (see Figure 2). It makes the
productivity and chromatographic performance of UPLC accessible to HPLC users who need the flexibility of a quaternarybased system.
Peptide mapping separation
separations as the original system. The QSM module offers
expanded solvent blending capabilities, and the SM-FTN features an intuitive injector. The result is seamless upgrade of
chromatographic capabilities.
In addition to simplifying the use of UPLC technology in HPLC
applications, consistent separation chemistry is required throughout the transfer of methods. Method transfer kits from the manufacturer facilitate choosing an appropriate column to maintain the
integrity of a separation when scaling from HPLC to UPLC and
Early ACQUITY UPLC H-Class evaluations indicate applicability
across a broad range of routine analysis applications, including the
food, environmental, petrochemical, and life science industries. As
an example, an important bioseparation application will be examined. The most common chromatographic technique utilized for
peptide mapping is reversed-phase HPLC (RP-HPLC), in which
resolution is a primary criterion for success.
The gradients for RP-HPLC tend to be very shallow (<1% change
in organic/min) and run times are typically hours. A binary system
is usually selected based on the perception that its pump is better
able to run shallow gradients with minimal delay volume and accurate mixing. Some typical solvent compositions are 0.1% TFA in
water/acetonitrile or 0.1% formic acid in water/acetonitrile. Typically, columns are long (150 and 250 mm), are 2.1 mm in diameter,
and contain 1.7 and 3 µm (130 Å or 300 Å) silica-based C8 or
C18 particles. Usually these separations are run with flow rates of
0.1–0.4 mL/min.
Selectivity can be adjusted with the choice of mobile phase modifier, with the concentration of that modifier, and with the selection of the organic solvent. Evaluating the combinations of these
parameters with a binary gradient system requires manual preparation of many combinations of solvents.
With the ACQUITY UPLC H-Class system, multisolvent blending eliminates manual solvent preparation. The solvent management system delivers either pure solvents or concentrated
modifier solutions. The gradient proportioning valve then blends
these components into the proportions that are required. This
process is managed with AutoBlend technology, which automatically blends eluents in accurate proportions in any sequence of
combinations. Programming different proportions from each line
can generate a full range of solvent selectivities that are useful in
peptide mapping, allowing separation development experiments
to be executed in fully automated, unattended runs. This efficient
method development approach reduces the risk of undetected
modification to the protein.
This convenience in separation development is only useful if the system is robust and reliable. Chromatographic reproducibility is required
for quantitative peptide maps; UPLC has been shown to provide good
reproducibility while preserving peak shape and resolution of trace
components. The ability of UPLC to improve peptide mapping separations has been demonstrated in the literature; it delivers greater resolution in combination with improved sensitivity compared to HPLC.
The ACQUITY UPLC H-Class has been evaluated for reproducibility with gradients typical of peptide mapping. The system has
been used in both a binary gradient mode with premixed solvents
and in a quaternary mode with on-line multisolvent blending.
The results have been evaluated for consistency of retention time,
preservation of peak shape and resolution, and reproducibility of
quantitation. The binary and multisolvent preparations gave identical results. The reproducibility of retention time and resolution
ensure unequivocal identification of each peak in the mixture with
reliable relative quantitation.
Temperature can be used for additional selectivity alteration
for method development. Once a temperature is chosen for the
execution of a peptide map, it is critical that the temperature
is consistent. The system’s column heater, with an active inlet
preheater, provides exacting control of the temperature in the
column. This makes any peptide mapping run on the system more
accurate, predictable, and transferable.
Finally, the flow-through needle design in the ACQUITY UPLC
H-Class sample manager, in which the entire gradient flow specified for the analytical method flushes directly through the needle,
reduces the risk of changing the composition of the sample or of
losing sample as compared to systems that use the needle as a transfer device (see Figure 3).
ACQUITY UPLC family
The flagship ACQUITY UPLC platform no longer describes a
single system. In addition to the ACQUITY UPLC system and
the ACQUITY UPLC H-Class, the growing family of UPLC
systems also includes the nanoACQUITY UPLC system and
PATROL UPLC process analyzer.
The nanoACQUITY UPLC is designed for nanoscale, capillary,
and narrow-bore separations to attain the highest chromatographic resolution, sensitivity, and reproducibility, especially for
sample-limited analysis. The PATROL UPLC process analyzer
moves UPLC analysis from off-line quality control laboratories
directly to the manufacturing process, resulting in significant
improvements in production efficiency.
The technology strategy is clearly focused on accelerating industry
adoption of UPLC technology through a diverse range of systems
based on a common, proven platform. Benefits of this approach
reach well beyond targeted or individual laboratories. They are
at the heart of today’s business, academic, and regulatory agency
needs, ensuring that scientific, operational, sustainability, and profitability goals are met.
Mr. Foley is Director, LC Product Marketing; Ms. Gildea is Marketing Manager,
Biopharmaceutical Business Operations; and Mr. Wheat is Principal Scientist, Systems Laboratory, Waters Corp., 34 Maple St., Milford, MA 01757,
U.S.A.; tel.: 508-482-2000; e-mail: [email protected].