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15 making the complex...simple iggy kass - waters
- 1. Making the Complex Simple: UPC2 UltraPerformance Convergence Chromatography Expanding Selectivity for the Chromatographic Laboratory ©2012 Waters Corporation 1
- 2. Evolution of Separation Technology Gas Chromatography Liquid Chromatography Convergence Chromatography GC HPLC SFC Capillary GC UPLC UPC2 ©2012 Waters Corporation 2
- 3. Separation Technology Overview Separation achieved by a temperature gradient •High efficiency [N] • Virtually no limitation on column length GC •Limited selectivity [α] • Limited stationary phase options Gas Chromatography Separation achieved by a solvent gradient •High efficiency [N] • Limited to pressure drop across column LC • Moderate selectivity [α] • Different modes: reversed-phase, normal-phase, SEC, IEX, affinity, ion pair, HILIC, GPC…etc. Liquid Chromatography Separation achieved by density/solvent gradient •High efficiency [N] CC • Very low viscosity enables longer columns and smaller particles •High selectivity [α] • Wide variety of stationary phase and mobile phase co-solvent and modifier options Convergence Chromatography ©2012 Waters Corporation 3
- 4. UltraPerformance Convergence Chromatography Convergence Chromatography is a category of separation science that provides orthogonal and increased separation power, compared to liquid or gas chromatography, to solve separation challenges. UltraPerformance Convergence Chromatography [UPC2] is a holistically designed chromatographic system that utilizes liquid CO2 as a mobile phase to leverage the chromatographic principles and selectivity of normal phase chromatography while providing the ease-of-use of reversed-phase LC. The ACQUITY UPC2 System is built utilizing proven UPLC Technology to enable scientists the ability to address routine and complex separation challenges while delivering reliability, robustness, sensitivity and throughput never before possible for this analytical technique. ©2012 Waters Corporation 4
- 5. The Advent of Convergence Chromatography UltraPerformance SFC Convergence Chromatography UltraPerformance Convergence Chromatography is the result of significant technological advancements in Supercritical Fluid Convergence Chromatography Chromatography instrumentation and chemistry design while providing exceptional increases in available selectivity Data courtesy of Davy Guillarme, Jean-Luc Veuthey LCAP, University of Geneva, Switzerland ©2012 Waters Corporation 5
- 6. Diverse Applicability of Convergence Chromatography Chemical Materials Pharmaceutical Impurity Profile Paracetamol and impurities Non-ionic surfactants A 4-hydroxyacetophenone H B p-nitrophenol C 2-hydroxyacetophenone D 4-chloroacetanilide E acetanilide F phenacetin G 3-aminophenol H paracetamol I 2-aminophenol A C D E G B I F Environmental Food and Beverage Chiral pesticides Vitamin E isomers Permethrin ©2012 Waters Corporation 6
- 7. Convergence Chromatography: Applied to Neutral and Polar Lipids Neutral and Polar Lipids Unlike RPLC and GC which can only analyze lipids within a single class, UPC2 provides a single methodology to separate complex lipids by class Exceptional separation of inter and intra class moieties PC Selective for a number of biological extracts TAG PE SM LPC ©2012 Waters Corporation 7
- 8. Convergence Chromatography: Applied to Neutral Lipids FFA ESI- TAG mix ESI+ CE mix All mix ©2012 Waters Corporation 8
- 9. Conclusion ACQUITY UPC2 Technology, using compressed carbon dioxide (CO2) as the primary mobile phase, is a separation tool that solves both routine and complex chromatographic problems, especially for samples possessing a wide range of polarities UPC2 offers scientists unique workflow, application, and environmental impact benefits compared to LC and GC platforms Because UPC2 is built utilizing UPLC Technology, customers are assured of its optimized performance through holistic design of instrumentation, detectors, software data systems and chemistries. UPC2 provides an exceptional increase in available selectivity, making this technology widely applicable to a diverse range of compound types – 80-85% overlap of compounds that can be analyzed by CC and RPLC – Any compound soluble in an organic solvent ©2012 Waters Corporation 9
- 10. ACQUITY UPC2 System ©2012 Waters Corporation 10
Editor's Notes
- Today we are going to discuss convergence chromatography, a new category of separation science that provides an exceptional increase in selectivity to the chromatographic laboratory
- There are many different forms of chromatography that are used in the analytical laboratory. However, there are 3 primary chromatographic techniques that are the most widely used, GC, LC and CC. The differences being: One is performed with a gas, one is performed with a liquid, and the other is performed with both a gas and a liquid. The important thing to note is that each of these chromatographic tools are at your disposal to address your separation needs. Each of these chromatographic techniques have gone through their own evolution to enhance the performance possible by each technique. Build..Significant performance improvements occurred in gas chromatography with advent of capillary GC. Build..Advancements in liquid chromatography were small and incremental until Waters changed the game with the introduction of UltraPerformance LC in 2004 providing an exceptional increase in performance and productivity to the chromatographic laboratory. To say the technology was disruptive to the industry, would be an accurate statement. Build..With convergence chromatography we are at a similar stage as we were with the early stages of adoption of UPLC Technology. In fact, UPC2 has a greater potential than UPLC to change our customers workflow and become an even greater disruptive innovation to the Separations Market
- Let’s take a step back for just a moment to take a look at the performance potential of each separation technique. In gas chromatography, the separation is achieved by varying the temperature of the column over a period of time. This technique provides a fairly high degree of efficiency (separation power), but is limited by the choices to actually manipulate the separation. Essentially GC is driven solely by the column chemistry with limited options in the carrier gas (mobile phase) which must be specifically paired up with the type of detection that is being used.In liquid chromatography, the separation is achieved by changing the concentration of the strong solvent. With the advent of UPLC technology, this technique also provides a high degree of efficiency, However, on a broader scale, the separation performance that can be achieved will be limited by the instrument performance. There are many modes of separation within LC, However, if we concentrate on the most common approach, which is reversed-phase LC, the solvent and stationary phase choices are limited.In convergence chromatography, the separation is achieved by manipulating the density of the mobile phase. Due to the very low viscosity of the technique, a high separation efficiency can be achieved. In addition, the number of stationary phase and mobile phase (solvent) options, there is a very large selectivity space that can be explored.
- So let’s start out with some definitionsConvergence Chromatography is a category of separation science that provides orthogonal and increased separation power, compared to liquid or gas chromatography, to solve separation challenges.UltraPerformance Convergence Chromatography [UPC2] is a holistically designed chromatographic system that utilizes liquid CO2 as a mobile phase to leverage the chromatographic principles and selectivity of normal phase chromatography while providing the ease-of-use of reversed-phase LC.The ACQUITY UPC2 System is built utilizing proven UPLC Technology to enable scientists the ability to address routine and complex separation challenges while delivering reliability, robustness, sensitivity and throughput never before possible for this analytical technique.In case someone asks:So what is the difference between SFC and Convergence chromatography? Quite simply, the underlying technology of convergence chromatography is SFC. The exception being the added reliability, robustness and reproducibility of UPC2 brings together the promise of SFC with reliability, robustness and reproducibility of liquid chromatography, and the higher performance lower dispersion instrumentation and smaller particles, making UPC2 a suitable analytical tool for routine use.
- Which brings us to what where we are today: enabling the evolution of SFC to UltraPerformance convergence chromatography. We have taken everything we have learned about improving performance of HPLC to yield UPLC performance and applied that knowledge to fulfill the promise of SFC technology and make it a high performance, reliable and robust separations tool. Here is a van Deemter curve provided to us by one of our collaborators at the university of Geneva in Switzerland. If we compare analytical HPLC and SFC, we can see that equivalent efficiency can be achieved, but in SFC, that same efficiency can be achieved in a much faster analysis time.With significant developments in both instrumentation and column chemistry, we can now harness the improved separation performance of lower dispersion instrumentation and smaller particles, in combination with the improved diffusivity, and therefore speed of analysis of supercritical fluids, to yield UltraPerformance Convergence Chromatography. And in addition, exploit the factors that control selectivity to develop successful separations.
- In addition to those areas in which we were certain this technology fits, we conducted further testing and discovered how widely applicable convergence chromatography really was. From pharmaceuticals to environmental as well as chemical materials and food and beverage. Essentially, this technology is applicable to nearly all market spaces and customers that we serve
- The analysis of complex lipids historically is a challenging task that may require a variety of analytical techniques. Lipids are generally recognized as hydrophobic compounds, but the properties of complex lipids containing phosphorus, sulfur, sugar and nitrogen have a diverse range of polarity, often making analysis by a single chromatographic technique not feasible..Utilizing the ACQUITY UPC2 system in combination with a Synapt G2 QTof MS easily provides the necessary chromatographic separation of complex lipid mixtures while acquiring highly specific MS information to properly characterize the lipidomic profile.In this example, the lipid profile of this mouse heart extract was explored by UPC2 coupled to Synapt G2. This is just one sample of a study performed to examine the efficacy of dosing diabetic mice with new drug entity. Observations of the lipidomic profile changes between control and dosed mice facilitates understanding of effects of the drug. In this example, a UPC2 BEH column separated the lipids by class, providing a comprehensive profile. From this comprehensive lipid profile, focused protocols are used to interrogate the intra-class separations using a UPC2 HSS C18 SB columns, as illustrated on the next slide.
- Within this example, the focused protocol is used for the determination of the free fatty acids and neutral lipids. A UPC2 HSS C18 SB column provided the intra-class separation of triglycerides, cholesterol esters and free fatty acids. Although, some co-elution occurred with the neutral lipids, the use of UPC2 and a hydrophobic stationary phase provides inter- and intra- class separation of these entities. The MS provided specificity by using Electrospray negative ionization detection of the free fatty acids and electrospray positive ionization of the neutral lipids. The benefit of utilizing UPC2 is realized by performing these experiments on the same instrument, the same solvent system, and the same sample preparation while only performing a simple column change by means of the UPC2 column manager. Based on the results of this data, our collaborators at the University of Northern Texas plan on using this protocol to screen their library which consists of thousands of lipid samples searching for the presence of free fatty acids.Method details: 2-10% methanol gradient over 5 minutes. 1500 psi backpressure. Make-up solvent: 0.1% formic acid in MeOH. If only looking at FFA, 0.1% ammonia in MeOH will provide greater sensitivity.
- In closing, we have described how ultraperformance convergence chromatography is an analytical platform that utilizes supercritical carbon dioxide as the primary mobile phase to solve routine and complex separation challenges and is widely applicable to a number of sample types.In comparison to other chromatographic techniques UPC2 dramatically streamlines the entire workflow of sample analysis due to the ability to directly inject high organic extracts that result from a majority of sample preparation techniques.Built upon prove UPLC Technology, customers can be assured of the optimized performance of the ACQUITY UPC2 system through holistic instrument, detector, informatics and chemistry design.With that, I thank you for your attention.