Waters Introduces UPLC System for nano- to Microscale Separations

Waters Corporation introduced the new Waters® ACQUITY UPLC® M-Class System, an industry-first nano- to microscale UltraPerformance LC® (UPLC®) system rated for 15,000 psi operation. Coupled to Waters mass spectrometers, the system delivers the sensitivity to quantify and to identify vanishingly small concentrations of key molecules. The ACQUITY UPLC M-Class System is ideally suited for a broad range of applications including proteomics, metabolic profiling, metabolite identification and pharmacokinetic studies. Its new 15k psi-capable ACQUITY UPLC M-Class Columns tap the potential of sub-2-µm particle technology, yielding faster separations, greater peak capacities and increased levels of sensitivity.

The ACQUITY UPLC M-Class System was introduced at WCBP 2014 Symposium, a conference with a focus on analytical methods for biotechnology pharmaceuticals. Waters expects to commence shipments of the new system in the current fiscal quarter. 

“When scientists doing proteomics or investigating new biopharmaceuticals talk to us about the work they do, they often speak of protein coverage or being able to quantify and identify incredibly small amounts of key molecules usually in the presence of much higher amounts of other molecules. This is difficult work without the appropriate tools. With the ACQUITY UPLC M-Class System, they will have the capability to peer into the unseen and achieve their research goals,” said Art Caputo, President of the Waters Division. “As an LC inlet to mass spectrometers, the ACQUITY UPLC M-Class System empowers more scientists to reliably employ microscale chromatography in order to solve their most complex analytical challenges.” 

Nano- to microscale LC is defined by flow rates of 200 nL/min. to 100 μL/min. with columns having an internal diameter of up to 1.0 mm. The advantages include the conservation of sample and solvent, increased sensitivity and a flow rate compatible with high sensitivity electrospray ionization (ESI) mass spectrometry (MS).

The innovations incorporated into the ACQUITY UPLC M-Class System are led by its internal low-volume design and newly redesigned fluidics that minimize dispersive and adsorptive losses during a chromatographic separation. The non-reactive materials for its internal fluid pathways ensure the integrity of samples and maximize the recovery of biologically important molecules. In order to preserve peak capacity, the ACQUITY UPLC M-Class System has a new micro ESI probe specifically optimized for microscale separations, along with low-dispersion flow cells that retain chromatographic resolution into the detector.  The increased pressure envelope enables the ACQUITY UPLC M-Class System to exploit fully sub-2-µm particles in longer columns  - up to 25 cm in length – resulting in faster separations and better resolution of individual chromatographic peaks.

With the introduction of the ACQUITY UPLC M-Class System, Waters is introducing five new chromatographic columns with internal diameters ranging from 75 µm to 1.0 mm. They are: HSS T3 (1.8 µm, 100Å pore size); BEH C18 (1.7 µm, 130Å and 300Å); BEH C4 (1.7 µm, 300Å); and CSH C18 (1.7 µm, 130Å); and Symmetry® C18 (5 µm, 100Å). Waters is complementing these new columns with a new line of 300 µm internal diameter steel columns packed with sub-2-μm particles.

Monitoring Protein Conformation with HDX Technology

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is used to study the structural dynamics and conformational changes of proteins and to better determine their higher order structure. Deuterium is a heavy isotope of hydrogen; as such, deuterium uptake can be measured or “weighed” by mass spectrometry.

Scientists use information about protein conformation from an HDX-MS study to compare a protein in one state, for example before its interaction with a drug candidate, to a new state after it is bound to a drug. These changes in protein conformation can be measured by the amount of deuterium that is taken up or released when compared to a control. HDX-MS is capable of monitoring domain interaction, localized protein dynamics, ligand binding and folding and unfolding in the solution phase.

High efficiency separations are essential to deliver the data quality required to detect small changes in protein conformation. Owing to its increased operating pressure range, the ACQUITY UPLC M-Class System can rapidly separate peptic peptides for MS analysis. Furthermore, the automated HDX-MS system integrates all the steps of HDX-MS from sample preparation to actual sequence determination, deuterium uptake curves, and comparison plots with Waters’ DynamX™ HDX Data Analysis Software. 

To take HDX-MS to another level, Waters has developed a UPLC-capable protein digestion column, the Waters Enzymate™ BEH Pepsin Column. The ACQUITY UPLC M-Class System together with Waters SYNAPT® G2-Si or Xevo® G2-S QTof mass spectrometers, delivers the quality spectral data required to resolve the small mass changes typical of HDX-MS. Together, these innovations combine to provide the only complete HDX-MS analysis system on the market. 

Expanding the Dynamic Range of Analyses with 2D Separations

Two-dimensional LC is a strategy for tackling the challenges of dealing with sample complexity and the dynamic range of the sample’s proteins or peptides. With this technique, a first-dimension separation of peptides or proteins is fractionated, and the fractions are trapped onto a second-dimension column that has different selectivity in order to multiply the peak capacity of each separation dimension.  

The ACQUITY UPLC M-Class System streamlines 2D-LC separations with an intuitive menu-driven method setup, standardized separation chemistries, and highly reproducible chromatography. Innovative valving strategies increase sample throughput while maximizing data quality.