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James P. Grinias - One of the best experts on this subject based on the ideXlab platform.
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Two-Dimensional Separation Techniques Using Supercritical Fluid Chromatography.
Journal of separation science, 2020Co-Authors: Alexander S. Kaplitz, Mahmoud Elhusseiny Mostafa, Samantha A. Calvez, James L. Edwards, James P. GriniasAbstract:High Resolution Separation systems are essential for the analysis of complex mixtures in a wide variety of application areas. To increase Resolution, multidimensional chromatographic techniques have been one key solution. Supercritical fluid chromatography provides a unique opportunity in these multidimensional Separations based on its potential for high solvent compatibility, rapid duty cycles, and orthogonality to other Separation modes. This review focuses on two-dimensional chromatography methods from the past decade that use supercritical fluid chromatography because of these advantages. Valving schemes and modulation strategies used to interface supercritical fluid chromatography with other LC and GC techniques are described. Particular applications of multidimensional Separations using supercritical fluid chromatography for the analysis of oils and chiral Separations of pharmaceutical compounds are highlighted. Limitations of and a potential trajectory for supercritical fluid chromatography in this field are also discussed. This article is protected by copyright. All rights reserved.
Ying Ge - One of the best experts on this subject based on the ideXlab platform.
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online hydrophobic interaction chromatography mass spectrometry for top down proteomics
Analytical Chemistry, 2016Co-Authors: Bifan Chen, Andrew J Alpert, Santosh G Valeja, Ying Peng, Ying GeAbstract:Recent progress in top-down proteomics has led to a demand for mass spectrometry (MS)-compatible chromatography techniques to separate intact proteins using volatile mobile phases. Conventional hydrophobic interaction chromatography (HIC) provides high-Resolution Separation of proteins under nondenaturing conditions but requires high concentrations of nonvolatile salts. Herein, we introduce a series of more-hydrophobic HIC materials that can retain proteins using MS-compatible concentrations of ammonium acetate. The new HIC materials appear to function as a hybrid form of conventional HIC and reverse phase chromatography. The function of the salt seems to be preserving protein structure rather than promoting retention. Online HIC-MS is feasible for both qualitative and quantitative analysis. This is demonstrated with standard proteins and a complex cell lysate. The mass spectra of proteins from the online HIC-MS exhibit low charge-state distributions, consistent with those commonly observed in native MS. ...
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three dimensional liquid chromatography coupling ion exchange chromatography hydrophobic interaction chromatography reverse phase chromatography for effective protein Separation in top down proteomics
Analytical Chemistry, 2015Co-Authors: Santosh G Valeja, Zachery R Gregorich, Huseyin Guner, Ying GeAbstract:To address the complexity of the proteome in mass spectrometry (MS)-based top-down proteomics, multidimensional liquid chromatography (MDLC) strategies that can effectively separate proteins with high Resolution and automation are highly desirable. Although various MDLC methods that can effectively separate peptides from protein digests exist, very few MDLC strategies, primarily consisting of 2DLC, are available for intact protein Separation, which is insufficient to address the complexity of the proteome. We recently demonstrated that hydrophobic interaction chromatography (HIC) utilizing a MS-compatible salt can provide high Resolution Separation of intact proteins for top-down proteomics. Herein, we have developed a novel 3DLC strategy by coupling HIC with ion exchange chromatography (IEC) and reverse phase chromatography (RPC) for intact protein Separation. We demonstrated that a 3D (IEC-HIC-RPC) approach greatly outperformed the conventional 2D IEC-RPC approach. For the same IEC fraction (out of 35 f...
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effective protein Separation by coupling hydrophobic interaction and reverse phase chromatography for top down proteomics
Analytical Chemistry, 2014Co-Authors: Santosh G Valeja, Andrew J Alpert, Ying GeAbstract:One of the challenges in proteomics is the proteome’s complexity, which necessitates the fractionation of proteins prior to the mass spectrometry (MS) analysis. Despite recent advances in top-down proteomics, Separation of intact proteins remains challenging. Hydrophobic interaction chromatography (HIC) appears to be a promising method that provides high-Resolution Separation of intact proteins, but unfortunately the salts conventionally used for HIC are incompatible with MS. In this study, we have identified ammonium tartrate as a MS-compatible salt for HIC with comparable Separation performance as the conventionally used ammonium sulfate. Furthermore, we found that the selectivity obtained with ammonium tartrate in the HIC mobile phases is orthogonal to that of reverse phase chromatography (RPC). By coupling HIC and RPC as a novel two-dimensional chromatographic method, we have achieved effective high-Resolution intact protein Separation as demonstrated with standard protein mixtures and a complex cell ...
James P. Landers - One of the best experts on this subject based on the ideXlab platform.
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An integrated sample-in-answer-out microfluidic chip for rapid human identification by STR analysis
Lab on a Chip, 2014Co-Authors: Delphine Le Roux, James P. Landers, Brian E. Root, Jeffrey A. Hickey, Orion N. Scott, Anchi Tsuei, Jingyi Li, David J. Saul, Luc Chassagne, Philippe De MazancourtAbstract:A fully integrated microfluidic chip for human identification by short tandem repeat (STR) analysis that includes a unique enzymatic liquid preparation of the DNA, microliter non-contact PCR, and a polymer that allows a high-Resolution Separation within a compact microchip footprint has been developed. A heat-activated enzyme that digests biological materials is employed to generate the target yield of DNA from a buccal swab or FTA paper. The microfluidic architecture meters an aliquot of the liberated DNA and mixes it with the PCR reagents prior to non-contact IR-mediated PCR amplification. The products of PCR amplification are mixed with a sizing standard (ladder) and the 18-plex STR amplicons are separated in an effective length (L eff) of just 7 cm. The development, optimization and integration of each of these processes within the microfluidic chip are described. The device is able to generate genetic profiles in approximately 2 hours that match the profiles from the conventional processes performed using separate conventional instruments. Analysis is performed on a single plastic microchip with a size similar to that of a 96-well plate and only a few mm thick with no pretreatment of any of the functional domains. This is significant advancement in terms of ease of fabrication over glass microdevices or polymeric systems assembled from multiple components. Consequently, this fully integrated sample-in-answer-out microchip is an important step toward generation of a rapid micro-total analysis system for point-of-collection human identification based on genetic analysis.
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direct capillary electrophoretic detection of carbohydrate deficient transferrin in neat serum
Electrophoresis, 2000Co-Authors: Amy Trout, Rajani Prasad, David Coffin, Andrea Dimartini, Tonya Lane, Cynthia R Blessum, Neeta Khatter, James P. LandersAbstract:Transferrin, an iron transport protein found in serum and cerebrospinal fluid, is known to be microheterogeneous with respect to its carbohydrate and sialic acid content. The forms of transferrin deficient in sialic acid and/or carbohydrate, termed carbohydrate-deficient transferrin (CDT), have been of clinical interest for almost two decades as a result of the initial finding that elevated CDT concentrations are associated with chronic, excessive alcohol abuse. We demonstrate the utility of capillary electrophoresis for examining the CDT sialoform profile via the direct electrophoresis of serum. The need for negligible preelectrophoresis sample preparation and absence of postelectrophoresis processing dramatically decreases analysis time compared to slab gel-based Separations. Using a fluorocarbon-coated capillary containing a hydroxyethyl cellulose/borate buffer, the high Resolution Separation of serum components is effected in less than 30 min. Under these conditions, the beta region proteins (including transferrin) are well resolved from the alpha-2 and gamma zone proteins in a window where the individual transferrin sialoforms can be detected. The usefulness of this method is demonstrated with the electrophoresis of serum from subjects known to be either non-alcoholic and alcoholic.
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structurally related peptide agonist partial agonist and antagonist occupy a similar binding pocket within the cholecystokinin receptor rapid analysis using fluorescent photoaffinity labeling probes and capillary electrophoresis
Journal of Biological Chemistry, 1999Co-Authors: Maoqing Dong, James P. Landers, Xiqin Ding, Delia I Pinon, Elizabeth M Hadac, Robert P Oda, Laurence J MillerAbstract:The molecular basis of ligand binding to receptors provides important insights for drug development. Here, we explore domains of the cholecystokinin (CCK) receptor that are critical for ligand binding, using a novel series of fluorescent photolabile probes, receptor proteolysis, and rapid high Resolution Separation of peptide fragments by capillary electrophoresis. Each probe incorporated the same fluorophore and a photolabile p-benzoylphenylalanine at the amino terminus of the pharmacophoric domain (residue 24 of CCK-33) of CCK analogues representing full agonist, partial agonist, and antagonist of this receptor. Each was used to label the CCK receptor expressed on Chinese hamster ovary-CCKR cells, with the labeled domain then released by cyanogen bromide cleavage. Capillary electrophoresis with laser-induced fluorescence detection achieved an on-capillary mass sensitivity of 1.6 attomoles (10(-18) mol), with an excellent signal-to-noise ratio. Each of the biologically divergent, but structurally similar probes saturably and specifically labeled the same receptor domain, consistent with conservation of "docking" determinants. This had an apparent mass of 2.9 kDa, most consistent with the first extracellular loop domain. An additional probe having its site of covalent attachment in a different region of the probe (residue 29 of CCK-33) labeled a distinct receptor fragment with differential migration on capillary electrophoresis (third extracellular loop). Identification of the specific receptor residue(s) covalently linked to the amino-terminal probes must await further fragmentation and sequence analysis.
Alexander S. Kaplitz - One of the best experts on this subject based on the ideXlab platform.
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Two-Dimensional Separation Techniques Using Supercritical Fluid Chromatography.
Journal of separation science, 2020Co-Authors: Alexander S. Kaplitz, Mahmoud Elhusseiny Mostafa, Samantha A. Calvez, James L. Edwards, James P. GriniasAbstract:High Resolution Separation systems are essential for the analysis of complex mixtures in a wide variety of application areas. To increase Resolution, multidimensional chromatographic techniques have been one key solution. Supercritical fluid chromatography provides a unique opportunity in these multidimensional Separations based on its potential for high solvent compatibility, rapid duty cycles, and orthogonality to other Separation modes. This review focuses on two-dimensional chromatography methods from the past decade that use supercritical fluid chromatography because of these advantages. Valving schemes and modulation strategies used to interface supercritical fluid chromatography with other LC and GC techniques are described. Particular applications of multidimensional Separations using supercritical fluid chromatography for the analysis of oils and chiral Separations of pharmaceutical compounds are highlighted. Limitations of and a potential trajectory for supercritical fluid chromatography in this field are also discussed. This article is protected by copyright. All rights reserved.
Pat Sandra - One of the best experts on this subject based on the ideXlab platform.
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development of a supercritical fluid chromatography high Resolution Separation method suitable for pharmaceuticals using cyanopropyl silica
Journal of Chromatography A, 2008Co-Authors: Claudio Brunelli, Yining Zhao, Melissahanna Brown, Pat SandraAbstract:Abstract A method has been developed for the analysis of a broad spectrum of pharmaceuticals using packed column supercritical fluid chromatography (pSFC) on a cyanopropyl silicagel stationary phase. Five 25 cm × 4.6 mm I.D., 5.0 μm columns were coupled to generate ca. 100 000 plates. The selectivity was tuned by varying the nature and concentration of various modifiers and additives in the carbon dioxide mobile phase. It was noted that pressure influences both efficiency and selectivity of the chromatographic process. Final method conditions are: outlet pressure 100 bar, flow 2.0 mL/min, temperature 40 °C, organic modifier program from 5% (1 min) to 40% at 2.0%/min, organic modifier composition methanol:acetonitrile in a ratio of 3:1 (variable according to sample composition) with peak symmetry additives trifluoroacetic acid and diisopropylamine both at levels of 0.5%.