Game-changing Liquid Chromatography
A combination of microfluidics and electronic integration results in simpler sample analysis.
At Pittcon 2014 in Chicago, Waters Corp. had a dual celebration of two game-changing technologies—the 10th anniversary of its introduction of the ACQUITY UltraPerformance LC (UPLC) System and the introduction this year of its ionKey/MS System. “In 2004, we changed the game,” says Art Caputo, President of the Waters Division. “High performance liquid chromatography (HPLC) wasn’t keeping pace with business demands to analyze more samples, faster and with better results. The introduction of the ACQUITY UPLC in 2004 transformed laboratory science and the analytical science industry.”
UPLC has had a profound effect on labs worldwide, both scientifically and from a business standpoint, according to Caputo. The novel ability of Waters’ UPLC system to perform separations on 1.7-µm particle technology coupled with the ability to operate at 15,000 psi opened new possibilities for analytical testing. The narrow concentrated peaks of UPLC improved ionization efficiencies. Enhanced resolution and robustness led to high-quality, reproducible analyses in significantly less time, reducing LC run times by up to a factor of 10 compared to traditional HPLC technologies. Through this increased productivity, it has been shown that one UPLC system typically displaces multiple HPLC instruments.
Other benefits of the UPLC include reduction of energy costs, less solvent usage and a reduction in waste products. According to an infographic from Waters, laboratories using UPLC systems save about 222 L of solvent per year, or 5.5 million L over the 10-yr life span of the UPLC, which equates to more than $500 million for customers.
Today, Waters’ ACQUITY UPLC system is joined by similar UHPLC systems offered by PerkinElmer (Flexar FX-10 and Flexar FX-15 systems), Dionex/Thermo Scientific (Ultimate 3000 systems), Agilent Technologies (1290 Infinity Binary LC), Shimadzu Scientific Instruments (Nexera) and Jasco (X-LC system).
Numerous other companies also supply UHPLC columns and accessories, including Phenomenex and Restek.
Redefined MS interface
At Pittcon, Waters Corp. introduced the ionKey/MS System, a new approach for obtaining increased sensitivity, robustness and ease-of-use out of mass spectrometry for a broad range of applications, including bioanalyses, pharmacokinetic studies, food safety and environmental analyses. ionKey/MS was designed for use with Waters’ ACQUITY UPLC M-Class System and Xevo TQ-S (tandem quadrupole) Mass Spectrometer by integrating a UPLC separation into the mass spectrometer (MS), enabling researchers to obtain a high-performance separation and detection of compounds.
“With the ionKey/MS, we’re enabling any scientist to operate the most sensitive LCMS analysis on the market, eliminating many variables and simplifying the user experience,” says Ian King, VP of Separations Technology, Waters Division. “This has a big impact by reducing training requirements and standardizing operations. With the ionKey/MS we made it possible for scientists to reduce solvent and sample sizes. This results in a more environmentally, ethically and economically sound operation by reducing costs and solvent waste and reducing the amount of sample needed for each analysis.”
The system has a simple plug-and-play operation. Scientists do not have to manipulate and align delicate fittings and columns or concern themselves with extra column dispersions and the variability and band broadening that comes with that. Typical applications that can make use of the ionKey/MS include drug discovery and development laboratories, contract laboratories, food and environmental testing laboratories, academic research laboratories and core/central laboratories.
The ionKey/MS’s iKey Microfluidic Separation Device is the heart of the system. About the size of a smart phone, the iKey contains the fluidic connections, electronics, ESI (electro-spray ionization) interface, column heater, eCord Intelligent Chip Technology and the 1.7-µm UPLC-grade particles packed inside a 150-µm inside diameter channel to perform hundreds of UPLC separations reproducibly and reliably without degradation in performance. The iKey separation device is designed so that it only takes a few seconds for the user to insert it into the source of the MS and with the turn of a key, make the fluidic and electric connections and lock it firmly and accurately into place. Besides storing the iKey’s device dimensions, chemistry type, serial number, manufacturing information and QC history, the unique eCord chip captures and records information on the operator’s name, instrument name, sample set name, date of first and last injection, total number of injections, observed back pressures and operating temperatures. This data is tied to each iKey device and is accessed with Waters Empower CDS (Chromatography Data System) software and MassLynx MS software.
At the scale of the iKey microfluidic-based chromatography, the savings in solvent approaches 90% and the ease-of-use that it provides make it a game-changer in how LCMS is performed.
Waters currently offers several 1.7-µm UPLC-grade particles for the ionKey/MS System available in 5-cm and/or 10-cm pathlengths. These include: C18 BEH (bridged ethyl hybrid) 300 Angstrom; CSH (charged surface hybrid) and; C18 BEH 130 Angstrom.
Scientists are driven to obtain lower limits of quantitation (LOQ) as many are limited by the smaller sample volumes available for analysis or are challenged to detect more potent analytes in biological matrices. This has led scientists to investigate microflow liquid chromatography as an alternative to standard flow LC (2.1-mm column format), as this technique has shown to increase sensitivity and ionization efficiency as well as reduce ion suppression. At the reduced flow rates, sensitivity gains of 10 to 20x have been shown to be achievable.
At flow rates greater than 100 µL/min, a significant portion of sensitivity is lost due to poor ionization efficiency and limited sampling efficiency. An electrospray plume generated from conventional LC flow rates can be quite broad and divergent. The inlet to a MS only has the ability to sample a portion of the electrospray plume. Most commonly, this is done by positioning the electrospray probe orthogonally to the inlet and sampling on the edges of the plume where fine droplets are present. As the solvent flow rate is reduced, however, the electrospray plume decreases in size and becomes more convergent. This allows the inlet of the MS to become more efficient and capture a greater percentage of the plume. This results in an ion signal. The increase in sensitivity or ion signal is roughly proportional to the decrease in column diameter and flow rate.
This behavior is similar to that obtained in a UV (ultraviolet) detector, which responds to the concentration of the analyte in the mobile phase rather than the absolute amount or mass of the analyte. This has led electrospray to be characterized as a concentration-sensitive technique, even though it has been generally understood that electrospray is a mass-sensitive phenomena.
For the ionkey/MS System, sensitivity gains were observed by Waters developers for both small molecules and peptides by comparing equal injection volumes with reduced mobile phase flow and column diameter, from the traditional 2.1-mm to 150-µm inside diameter. The signal enhancement realized at µL/min flow rates in the development program ranged from 2 to 83x and is molecule-dependent. The 150-µm iKey separation channel dimension was found to offer a unique balance between enhanced sensitivity and optimal throughput.
Numerous applications of the ionKey/MS system were performed by Waters developers and can be found on the website, www.waters.com. A brief summary of the dramatic results of three of these application notes are provided below.
Multiplexed analysis of steroid hormones: A highly sensitive multiplexed assay was developed for quantitation of five steroids in human serum. The use of the ionKey source and the 150-µm iKey separation device yielded a 100 to 400x increase in on-column sensitivity while at the same time decreasing solvent usage by 150x as compared to standard flow methods. The increased on-column sensitivity allowed researchers to simplify the steroid extraction procedure, which, in turn, streamlined the sample preparation requirements and reduced the per sample assay cost.
Improved SPE-LCMS/MS for quantification of Bradykinin in human plasma: Robust and sensitive analysis of peptides challenges chromatographic separations and MS. A 2x reduction in sample volume and a 10x increase in sensitivity for the ionKey/MS System allowed multiple injections to be performed for improved accuracy and to meet the incurred sample reanalysis guidelines. A 50x reduction in the required solvent reduced the overall cost of the analyses. Solid phase extraction (SPE) using mixed mode SPE reduced the matrix interferences and enhanced the selectivity of the extraction.
Pesticide screening of food samples: A 10x reduction in solvent consumption over the traditional 2.1-mm ID chromatography methods was obtained in analyses for pesticides in baby formula. Microliter-scale sample and internal standard consumption (of pesticide standards) resulted in additional lab cost savings. The ionKey/MS also resulted in improvement of signal-to-noise ratio for pesticides, with an average of 8x in sensitivity over conventional 2.1-mm ID chromatography methods.