Sunday, March 13 – 1:20 pm; Presenter: Richard A. Henry
Co-Authors: Wayne K. Way, Carmen T. Santasania
Title: Studies on Stationary Phase Selectivity for Solid-Core Particles
Solid-core particles with a very narrow size distribution have produced columns with surprisingly high performance at low pressure drop. The van Deemter efficiency plots for 2.7 µm solid-core particle columns have been shown to virtually overlay those of 1.7-1.8 µm porous particle columns, while requiring only half the pressure. The lower flow resistance of these new UHPLC columns has permitted standard 400 bar HPLC instruments to be effectively used with only minor optimization. While high column efficiency at low pressure drop is very attractive, a range of different stationary phases is still needed for solid-core particles.
Selectivity for the new particles with C18, C8, pentafluorophenyl, RP-amide, and phenyl phases has been studied. Investigations for porous particles have shown that these HPLC phase chemistries cover a wide range of solute selectivity space. Selectivity will also be compared for C18 phases on solid-core particles having two different pore sizes. Probable phase-solute interactions will be discussed for each phase type and compared to previous results with classic porous particles. Test solutes will consist of acidic, neutral, and basic pharmaceutical compounds plus classic test probes that have become popular for mechanistic studies.
Results will demonstrate how knowledge of HPLC column phase structures and possible phase-solute interactions can aid in choosing the most selective columns for initial screening. High efficiency of solid-core particle columns can be combined with phase selection and low pressure drop to improve HPLC method development and assay speed. Selectivity results will be shown to correlate well between solid-core and porous particle columns.
Monday, March 14 – 3:55 pm; Presenter: Xiaoning Lu
Co-Authors: Michael Monko, Michael Ye
Title: High Recovery Method of HybridSPE-Phospholipid for Cleanup of Biological Samples Prior To LC/MS Analysis
Sample cleanup of high abundance proteins and phospholipids, present in biological matrices such as plasma and sera, is essential for reproducible and sensitive determination of low concentration analytes by LC-MS. It is well-known that phospholipids in biological samples not only cause ion-suppression in LC-MS analysis, but also reduce HPLC column life due to their accumulation in the column. HybridSPE-Phospholipid is a newly introduced technique for simultaneous cleanup of proteins and phospholipids present in biological matrices utilizing a procedure as simple as that for a standard protein precipitation method, either in 96-well plate or cartridge formats. This method has been proven effective for analysis of a wide range of pharmaceutical compounds in plasma and sera. However, there are occasions when lower recoveries are observed for compounds bearing certain functional groups, such as strong bases, strong acids and chelating groups. The low recoveries of such compounds are probably caused by their strong binding to the zirconia-coated silica employed in the HybridSPE-Phospholipid sorbent. Presently, thorough experimental studies are being performed to fully understand the non-specific binding. Additionally, new strategies and methods have been developed to circumvent many of the non-specific binding issues. The improved method gives high recoveries (>80%) for those difficult compounds.
Tuesday, March 15 – 9:00 am; Presenter: Richard A. Henry
Co-Authors: Hillel K. Brandes, Craig R. Aurand, Dave S. Bell, Wayne K. Way
Title: Affecting Reversed-Phase/MS Peptide Separations on High Performance Silica Particles
Reversed-phase chromatography of peptides on modern silica phases generally employs an acidic ion-pair reagent to affect pH and thus improve retention within the operational limits of the column. This had traditionally been done with TFA which coupled well with low-UV detection. However, the popularity of MS detection has dictated alternate choices regarding this formerly common practice for reasons of detector sensitivity. Formic acid or acetic acid has routinely been substituted for TFA in this case of coupling to MS detection. However, in the case of particularly basic peptides, it is not uncommon for peak shape to suffer dramatically in the presence of formic (or acetic) acid, in comparison to TFA.
This work will demonstrate examples in which chromatographic performance of peptides suffers commensurate with their basicity, and suggest simple modifications of chromatographic conditions to mitigate charge-charge interactions, that can dramatically improve peak shape. These modifications to the chromatographic conditions not only affect peak shape and selectivity, but also MS response. Attempts are made to understand the mechanistic reasons for the change in chromatographic performance and MS response.
Thursday, March 17 – 10:25 am; Presenter: Jauh-Tzuoh Lee
Co-Authors: William Campbell
Title: HPLC Enantiomeric Separations of Pharmaceuticals Using Polar Organic Mobile Phases
In pharmaceutical and biotech industries, chirality has long been regarded as one of the critical issues during drug discovery and development stage. An early understanding of pharmcological aspects of chiral drugs is imperative since each enantiomer possesses different biological properties in terms of absorption, distribution, metabolism and excretion (ADME).
In HPLC, macrocyclic glycopeptide phases are renowned for their usage of polar mobile phases while polysaccharide phases have earned their reputation in typical normal phase operation. However, polysaccharides were also found to have good selectivity towards racemic drugs in polar mobile phases. In this study, one vancomycin and one cellulosic phase were tested against a group of chemically diverse, basic pharmaceutical drugs. The mobile phases were also designed to aim for mass spectrometry detection using methanol-based eluents with some acid and base or volatile ammonium salts as additives. Optimization steps on resolution and sensitivity will be discussed. The complementary performance between these two types of CSPs will also be demonstrated.
Monday, March 14 – am
Authors: Hillel K. Brandes, Richard A. Henry, Dave S. Bell, Wayne K. Way, Frank Michel
Title: Optimizing Instruments for Modern HPLC Columns
Higher performance HPLC columns result in higher resolution chromatograms with very narrow peak widths that cannot be achieved by all HPLC instruments in the laboratory, independent of pressure limitations. For example, porous-layer particles can deliver peak widths comparable to sub-2 µm particles with flow resistance comparable to 3 µm particles so that columns can easily be operated within the pressure range of traditional HPLC instruments. However, UHPLC performance and narrow peak widths can only be observed with older, traditional instruments that have adequately low instrument bandwidth (i.e. dispersion). This paper will give an overview of the problem and describe some simple ways HPLC instruments can be qualified within their operating parameters, for use with modern, higher performance columns.
Typically, contributions to band spreading vary with different instruments and their factory configuration. Origins of band spreading will be examined in detail; including 1) dispersion within the column particle bed, 2) dispersion from column fittings, and 3) extra-column dispersion from HPLC instrument components. Techniques for estimating HPLC instrument performance will be shown with the objective of qualifying each HPLC instrument (establishing bandwidth and ranking performance) before it is used with modern HPLC column technology. Practical suggestions for lowering dispersion of older instruments will be offered. In many cases, an investment in new instrumentation is not required to substantially elevate HPLC speed, resolution and sensitivity. Porous-layer particle columns can yield the high analysis speed and sample resolution at the lowest pressures when matched with either UHPLC instruments or optimized, traditional HPLC instruments.
Monday, March 14 – am
Authors: Dave S. Bell, Carmen T. Santasania, Jennifer Claus, Wayne K. Way, Craig R. Aurand
Title: Impact of Reversed-Phase Chiral Chromatography on the LC-MS Analysis of Drugs in Biological Fluids
Macrocyclic glycopeptide and cyclodextrin-based chiral stationary phases (CSPs) often provide enantiomeric selectivity using polar organic and aqueous-organic mobile phases. As these mobile phases are highly amenable to mass spectrometry (MS) sources, separations intended for LC-MS detection may be improved over traditional chiral separations utilizing normal phase solvents.
In this study, chiral methods for several drugs and metabolites were developed utilizing both reversed-phase and normal phase conditions. The methods were then applied to LC-MS analysis in various biological fluids and the results were compared and contrasted. It is demonstrated that, when possible, chiral methods operating in reversed-phase mode, can greatly improve sensitivity in LC-MS.
Tuesday, March 15 – pm
Authors: Dave S. Bell, Carmen T. Santasania, Wayne K. Way, Craig R. Aurand
Title: Understanding Separations in HILIC Chromatography: We’re not in Water Anymore
Interest in chromatography using aqueous-organic mobile phases high in organic content (aqueous normal-phase, HILIC) has continued to build in recent years.(1,2) In this mode of chromatography, analyte retention increases monotonically with an increase in the organic component of the mobile phase. In previous studies, significant contribution of stationary phase chemistry toward the manipulation of retention and selectivity in ANP has been demonstrated.(3) The aim of this continuing study was to further enhance our knowledge of mechanisms of retention that dominate in this in this interesting mode of chromatography.
Solute descriptors important for chromatographic processes such as ionization constants (pKa/pKb) are often only available as measured in aqueous environments. When operating in HILIC mode, one deviates significantly from the aqueous environment and thus from these aqueous-based descriptors. It is therefore important to understand the state of the analytes and stationary phases in the HILIC environment. In this study, the influence of high organic content on basic and acidic pKa values as well as mobile phase pH is explored. The impact of the variation is related to real chromatographic separations and useful general trends are developed.
1. W. Naidong, Journal of Chromatography B 796 (2003) 209.
2. D.S. Bell, Jones, A. Daniel, Journal of Chromatography A 1073 (2005) 99.
3. D.S. Bell, Brandes, Hillel K., in 30th International Symposium and Exhibit on High Performance Liquid Phase Separations and Related Techniques, San Francisco, California, USA, 2006.
Wednesday, March 16 – pm
Authors: William R. Betz, Michael J. Keeler, Daniel L. Shollenberger, Leonard M. Sidisky
Title: Characterization of Polymer Carbon Sieves, Graphitized Polymer Carbons and Graphitized Carbon Blacks for Carbon Purification Processes
The use of carbons in processes requiring trace level purification of gas-phase systems and liquid-phase systems has become increasing effective due to the tailoring of high performance carbon adsorbents. The ability to synthesize spherical carbons with particle sizes ranging from 0.3 µm to 1000 µm, with pore structures ranging from ultramicroporous to macroporous is important for this optimization to meet specific process purification requirements. The use of packed carbon beds and carbons bonded to specific substrates, using two proprietary, patented adhesives, has provided for a wide range of purification processes to be realized.
For gas phase applications, the use of multiporous carbon adsorbents has become effective due to the kinetics of the pore structure. Multiporous particles have proven useful in both static and dynamic gas-phase purification processes. For example, high purity, spherical multiporous carbons have been determined to be effective for purifying gas streams containing hydrocarbon impurities ranging from C2-C20 hydrocarbons. Both microporous and multiporous 2.0 µm carbons have been utilized for bonding to metal and plastic surfaces for purifying gas streams as well.
For liquid phase applications, the use of large surface area microporous carbons have been used for the removal of organic pesticides and herbicides from aqueous matrices. Also, multiporous carbons have been used for the purification of pharmaceutical process streams. The surface chemistry of the carbons has also been modified to effectively remove specific impurities when required.
Nitrogen porosimetry, helium pycnometry and autotitration techniques were used to study the carbons. Adsorbent capacities and reversible adsorption characteristics have been determined using the respective sample preparation processes.
Wednesday, March 16 – pm
Authors: Alexander Rück, Christine Hellriegel, Rudolf Köhling, Jürg Wüthrich, Michael Weber, Matthias Nold, Vicki Yearick
Title: A New Generation of Certified Reference Materials by the Quantitative 1H-NMR Technique (qNMR)
Quantification of organic substances is usually performed using chromatographic techniques such as HPLC or GC. Over the past few years quantitative NMR (qNMR) has evolved not only in the pharmaceutical industry but also in many other fields. In the R&D labs of Sigma-Aldrich Buchs (Switzerland), a new Bruker Avance III 600 MHz Ultrashield NMR spectrometer was installed for high resolution qNMR measurements of organic substances, and the lab was fully accredited under both ISO/IEC 17025 and also ISO Guide 34 for the certification of organic reference materials using 1H-qNMR.
The most outstanding attribute of qNMR is that it is a relative primary method. The signal intensity is in direct proportion with the number of protons contributing to the resonance. Hereby the structures of the chemical substances are fully irrelevant. The signal ratio of two different protons can be measured with tremendous precision and only the integration of the signals is dominating the measurement uncertainty.
As a first series of new CRMs, proteinogenic amino acids have been chosen for an intended use as chromatography standards. These CRMs are comprehensively characterized by qNMR and other analytical techniques leading to highest accuracy and very low uncertainties of the certified values. They are traceable to internationally accepted references from NIST. A detailed certificate is attached to each CRM that is in accordance with ISO Guide 31, and contains comprehensive documentation, proper uncertainty calculation, lot-specific values, the expiration date, and storage information.
Thursday, March 17 – am
Authors: Ingrid Hayenga, Michael Jeitziner, Nicola Stäheli, Caspar Demuth, Don Hobbs, Ingo Haag, Shyam Verma
Title: Selectophore products – New ion-selective sensor materials for food, environmental, biomedical and industrial applications
Ion-selective sensor analysis has been a well-established routine technique for many years. Ion sensors provide accurate, rapid, sensitive, on-line, and non-destructive analyses. Their field of application is widespread and covers food and environmental monitoring as well as biomedical and industrial applications.
Chemical sensors are often used in an electrode format which is then immersed in the sample to be analyzed. They consist of an ionophore and auxiliary compounds (polymers, plasticizers, additives). Ionophores are lipophylic, electron-rich complexing agents that are capable of reversibly binding ions and transporting them across organic membranes by carrier translocation.
Ion-selective polymeric membranes usually contain about 30-33% (w/w) polymer and 60-66% plasticizer. Films with such a high plasticizer amount demonstrate optimum physical properties and ensure relatively high mobilities of their constituents. Some of the common plasticizers are dioctyl phthalate (DOP), dioctyl sebacate (DOS), and 2-nitrophenyl octyl ether (o-NPOE). Ionic additives produce significant improvements in selectivity and decrease membrane resistance and electrode response time.
Selectophore from Sigma-Aldrich is the most comprehensive product line available to fulfill requirements for the preparation of sensor membranes for ion-selective potentiometric and optical devices. It is essential that the ionophores are free of disrupting impurities such as metal ions or surfactants. However, the specific purity requirements are a function of the analytes, with some analyte-ionophore combinations that are more sensitive to impurities than others. For this reason, all Selectophore ionophores are application tested.
We present the newest ionophores for the following application fields:
· Food and Beverage
· Pharmaceutical and Medical
Thursday, March 17 – am
Authors: James Desorcie, Kristen Schultz, Frank Michel, Katherine K. Stenerson
Title: Benefits of Radial Passive Samplers
Passive samplers allow users to conduct high-resolution measurements of numerous gaseous compounds for industrial, indoor, outdoor, and personal exposure applications, economically in many different locations. The radial symmetry of the radiello® passive samplers overcomes limits set by common axial or planar shaped samplers. Radiello samplers require no electricity, have no moving parts, and are simple to use. The system provides increased reproducibility (due to minimal reverse diffusion) and significant increases in uptake rates relative to traditional passive/diffusive samplers. The current radiello product line allows analysts to sample aldehydes, VOCs and BTEX, NO2 and SO2, HF, HCl, O3, phenols, H2S, NH3, and anesthetic gases and vapors.
Thursday, March 17 – am
Authors: Leonard M. Sidisky, Greg A. Baney, Yizeng Ni, James L. Desorcie, Katherine K. Stenerson
Title: A Comparison of Ionic Liquid and Polymer Based Capillary Columns for the Analysis of FAME Isomers
Analyses of fatty acid methyl esters (FAMEs) are continuing to gain importance as more research is focusing on their biomedical impact. This includes the analyses of saturated and polyunsaturated FAMEs along with the positional geometric (cis & trans) FAME isomers. Traditionally, FAME analyses have been performed using silicone polymer or polyethylene based stationary phases that yield typical elution patterns. Analysts performing the task of analyzing the fatty acid composition of food have a wide choice of capillary column selectivities available for resolving the fatty acids as fatty acid methyl esters, depending upon the information they require from their analyses. Nonpolar methyl silicone columns provide a boiling point separation of the FAME isomers with limited resolution of polyunsaturated isomers. Polar polyethylene glycol columns resolve the isomers by degree of unsaturation with minimal overlap of the carbon chain lengths. The highly polar cyanosilicone columns will resolve cis and trans isomers along with possibly providing positional geometric isomer separations depending upon the column type.
A new class of stationary phases based on Ionic Liquid technology has now been developed and these phases have demonstrated unique elution patterns for FAME isomers compared to the traditional silicone or polyethylene glycol based polymer phases. We will compare and contrast the selectivity of the ionic liquid phases with the polymeric based phases for a variety of FAME samples. Both traditional and high-speed applications will be investigated.
Thursday, March 17 – am
Authors: Shyam Verma, Katherine K. Stenerson
Title: The Utility of Headspace Grade Solvents in the Analysis of Organic Volatile Impurities
The analysis of organic volatile impurities (OVIs) sometimes requires the use of organic solvents for dissolution and/or extraction of samples. Consequently, these solvents must be free from contaminants that could possibly interfere with GC analysis by coeluting with peaks of interest in the sample. This study presents data on two commonly used solvents for OVI analysis: dimethylsulfoxide (DMSO) and dimethylformamide (DMF). The impurities in conventional grades of these solvents will be compared with headspace grade, The compatibility of the later for use in the analysis of many of the OVIs listed in United States Pharmacopeia (USP) Method <467>, European Pharmacopoeia (EP) Method 2.4.24, and the International Conference on Harmonization (ICH) guidelines will be demonstrated.
Thursday, March 17 – am
Authors: Robert E. Shirey, Craig Aurand, Yong Chen, Katherine Stenerson, Len Sidisky, Dajana Vuckovic, Janusz Pawliszyn, Daniel Vitkuske
Title: SPME-LC Fibers for a Variety of Applications
A newly designed SPME fiber has been developed that will allow extraction of small analytes directly from biological fluids and other complex matrices. A metal-based fiber core is coated with bonded silica embedded in a proprietary binder. The binder is biocompatible, in that it tends to repel macromolecules such as proteins and phospholipids while enabling smaller analytes to be retained by the polymeric phase bonded to the silica. Because of biocompatible properties, minimal sample pretreatment is required prior to extraction. The bonded phases include C18 and ion exchange resins. These coated fibers are solvent compatible and do not swell when placed in organic solvents.
Two devices have been designed to house the fiber. The first design is a probe that contains a coated fiber housed within a hypodermic needle that enables the use of in-vivo sampling of lab animals such as mice and rats. The probe can be inserted into a shunt device for direct sampling of blood, or it can be inserted into tissues to monitor metabolites of animals. The second design is a fiber in a pipette tip for in-vitro sampling of fluids. This unique pipette tip design enables compatibility with common laboratory robotics. Since multiple samples can be extracted and desorbed simultaneously, sample prep time is reduced.
This presentation will show the broad range of applications includes biological fluids, tissue, and water samples. Some of the applications will be compared to other commonly used extraction methods.
Thursday, March 17 – am
Authors: Leonard M. Sidisky, Greg A. Baney, Yizeng Ni, James L. Desorcie, Katherine K. Stenerson, Steve Cecil
Title: New Developments with Ionic Liquid Capillary Columns
Ionic liquids have received considerable interest as new green solvent systems in the areas of organic reactions (1) and separation technologies (2,3) along with a number of other areas. These materials are a class of non-molecular solvents that consist of essentially organic cations and anions. They have been found to possess negligible volatility, non-flammability, high thermal stability, and low melting points. There are numerous combinations of cations and anions possible, so tailoring the material to a specific application or function is a potential benefit of these materials.
Recently, a series of geminal dicationic and polyionic ionic liquids have been prepared for use as stationary phases in capillary gas chromatography. These materials are known to provide higher thermal stability for gas chromatography, broader liquid working ranges and broader selectivity ranges than monocationic ionic liquids (4,5,6).
The purpose of this study is to determine the selectivity of the ionic liquid stationary phases and then to utilize them for the analysis of various petrochemical, food & beverage and environmental samples. In addition we will also demonstrate the stability of the ionic liquid phases using air as a carrier gas compared to a typical polar stationary phase based on polyethylene glycol.
1. M. Freemantle, C&E News, November 8, 2004.
2. C. Poole, J. Chromatography A, 2004, 1037, 49-82.
3. J.L. Anderson, R. Ding, A. Ellern, D.W. Armstrong, J. Am. Chem. Soc., 2005, 127, 593-604.
4. D. W. Armstrong, Patent Pending, US Patent Application 2006/0014955 A1.
5. J. Ding, T. Welton, D.W. Armstrong, 2004, Anal. Chem. 76, 6819-6822.
6. T. Payagala, Y. Zhang, E. Wanigasekara, K. Huang, Z.S. Breitbach, P.S. Sharma, L.M. Sidisky, D.W. Armstrong, 2009, Anal. Chem. 81, 160-173