US20090203146A1 - Narrow bore layer open tube capillary column and uses thereof - Google Patents

Narrow bore layer open tube capillary column and uses thereof Download PDF

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US20090203146A1
US20090203146A1 US12/306,232 US30623207A US2009203146A1 US 20090203146 A1 US20090203146 A1 US 20090203146A1 US 30623207 A US30623207 A US 30623207A US 2009203146 A1 US2009203146 A1 US 2009203146A1
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column
channel
stationary phase
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columns
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Barry L. Karger
Jian Zhang
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Northeastern University Boston
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
    • B01J20/28078—Pore diameter
    • B01J20/28085—Pore diameter being more than 50 nm, i.e. macropores
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/285—Porous sorbents based on polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B01J20/289—Phases chemically bonded to a substrate, e.g. to silica or to polymers bonded via a spacer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/327—Polymers obtained by reactions involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/328—Polymers on the carrier being further modified
    • B01J20/3282—Crosslinked polymers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G01N30/6052—Construction of the column body
    • G01N30/6073—Construction of the column body in open tubular form
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/80—Aspects related to sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J2220/84—Capillaries
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/80—Aspects related to sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J2220/86—Sorbents applied to inner surfaces of columns or capillaries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G01N2030/524—Physical parameters structural properties
    • G01N2030/528—Monolithic sorbent material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G01N30/724—Nebulising, aerosol formation or ionisation
    • G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/11—Automated chemical analysis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/11—Automated chemical analysis
    • Y10T436/117497—Automated chemical analysis with a continuously flowing sample or carrier stream

Definitions

  • Electrospray ionization-mass spectrometry has become a routine tool in proteomic studies, primarily due to its high sensitivity, broad dynamic range, and versatility for online coupling with capillary high performance liquid chromatography (HPLC) 1-3 .
  • High-resolution separation prior to MS detection allows complex mixtures to be characterized by extending both the dynamic range and detection level achievable in the analysis.
  • Capillary LC using 75-150 ⁇ m i.d. reversed-phase columns, offers the advantages of high resolving power, high mass sensitivity, and low sample and mobile phase consumption, and hence are widely used today.
  • LC-MS analysis of very low quantity samples e.g., cells from small tissue samples obtained using laser capture microdissection 4
  • More sensitive proteomic analysis methods are necessary to tackle many challenging biological problems.
  • volumetric flow rate is an important parameter influencing ESI sensitivity 5-10 .
  • Low flow rates resulting from the narrow bore columns lead to smaller electrospray droplet sizes, thus enhancing analyte ionization efficiency and reducing the effect of ion suppression, all leading to higher sensitivity.
  • the electrospray emitter attached to such a low flow rate column can be placed nearer to the MS inlet than in comparable configurations, which improves the sampling efficiency at low flow rates.
  • NanoESI at flow rates of ⁇ 30 nL/min, will, thus, significantly increase the MS response compared to conventional flow rates (>300 nL/min) 5,6,11 .
  • packing narrow-bore ( ⁇ 20 ⁇ m i.d.) columns with conventional microparticles can be technically difficult because the decreased ratio of column i.d. to particle size induces more frequent column clogging, and packing microparticles into narrow-bore ( ⁇ 20 ⁇ m i.d.) columns requires ultrahigh packing pressure (usually >10,000 psi) and special instrumentation.
  • the ratio of column i.d. to particle size should be greater than 10 to pack dense columns reproducibly. Recently, the preparation of 10 ⁇ m i.d.
  • silica-based monolithic columns 8,16 demonstrating sensitive and quantitative proteomic analyses at the very low flow rate of 10 nL/min 6.
  • preparation of the monolithic columns was difficult, in part due to the increased surface area to column i.d. ratio.
  • Porous layer open tube (PLOT) columns were introduced in 1960s to increase the sample loading capacity of the GC columns 19 .
  • PLOT capillary LC columns 20-23 success has been limited due in part to the following: 1) lack of a sensitive, universal, small dead volume detector 24 ; 2) lack of ability to generate effective gradient elution at very low flow rates; and 3) difficulties in the preparation of capillary columns with a uniform stationary layer reproducibly.
  • ESI-MS has proven to be an ideal sensitive detector with zero dead volume, and current HPLC pumps can provide stable flow rate at low nL/min level after accurate splitting.
  • a new polymer-based PLOT column prepared by in situ copolymerization of a functional monomer, which usually contains the retentive chemistries, and a crosslinking monomer, which enhances the strength of the polymer matrix, is disclosed herein.
  • a functional monomer which usually contains the retentive chemistries
  • a crosslinking monomer which enhances the strength of the polymer matrix
  • styrenic based monomers such as styrene and divinylbenzene or meth/acrylic based monomers such as butyl or stearyl methacrylate and ethylene glycol dimethacrylate.
  • Columns of the invention can be prepared in a robust fashion with a very narrow i.d., e.g., 5-15 ⁇ m. Thus, they are suitable for commercial use in ultratrace LC/MS proteomic analysis.
  • columns according to the invention can be online coupled to other sensitive detectors such as fluorescence, electro/chemiluminence or nuclear magnetic resonance (NMR) for, e.g., detection of trace chemical or biological agents in chemical or biological defense applications.
  • sensitive detectors such as fluorescence, electro/chemiluminence or nuclear magnetic resonance (NMR) for, e.g., detection of trace chemical or biological agents in chemical or biological defense applications.
  • the invention is directed to a porous layer open tube capillary column, or channel in a microfabricated device, the column or channel including a capillary column or channel having an i.d. of 15 ⁇ m or less (preferably 10 ⁇ m or less); and a rigid porous layer separation medium comprising a highly crosslinked, macroporous, organic polymeric stationary phase layer attached covalently to the inner wall surface of the column or channel, wherein the organic polymeric stationary phase layer includes styrenic, methacrylic or acrylic monomeric units, or combinations thereof; wherein the organic polymeric stationary phase layer is from 0.5-3 ⁇ m in thickness; wherein the organic polymeric stationary phase layer is thermally stable to 250° C.; and wherein the reproducibility of retention time on comparable columns or channels during use varies less than 10%, and, preferably less than 5%.
  • a preferred capillary column according to the invention has a length of greater than or equal to one meter, and preferably greater than or equal to three meters.
  • the organic polymeric stationary phase layer is poly(styrene-divinylbenzene) or has (C4-C18) alkyl methacrylate monomer units, and the column or channel during use has a flow rate at 6000 psi or less of 5-50 nL/min.
  • the invention is directed to method of preparing a separation capillary column or channel in a microfabricated device, the column or channel comprising a porous layer open tube separation medium including a macroporous, organic polymeric stationary phase layer, said method including the steps of (1) providing an unfilled capillary column, or channel in a microfabricated device, the column or channel being open at both ends thereof and having an i.d.
  • the inner wall surface of the column or channel including a bifunctional anchoring or coupling agent suitable for covalent attachment of a macroporous, organic polymeric stationary phase layer as a porous layer open tube separation medium; (2) adding to the column or channel a mixture including a functional monomer selected from the group consisting of styrenic, methacrylic and acrylic monomers, and combinations thereof; a crosslinker compatible with the functional monomer, the crosslinker being capable of providing extensive crosslinking; a polar porogenic solvent; and an initiator for thermal or UV induced polymerization; and (3) polymerizing the mixture in the column to form the macroporous, organic polymeric stationary phase layer as the porous layer open tube separation medium attached to the inner surface of the column or channel.
  • a functional monomer selected from the group consisting of styrenic, methacrylic and acrylic monomers, and combinations thereof
  • a crosslinker compatible with the functional monomer the crosslinker being capable of providing extensive crosslinking
  • a polar porogenic solvent and an initi
  • the inner wall surface of the column or channel is silica and the bifunctional anchoring or coupling agent contains at one end a functional group reactive with silica and at the other end a functional group reactive with said functional monomer (an exemplary bifunctional anchoring or coupling agent being 3-(trimethoxysilyl)propyl methacrylate); the functional monomer in the polymerization mixture is styrene and the crosslinking agent is divinylbenzene.
  • the functional monomer in said polymerization mixture is methacrylate (e.g., (C4-C18) alkyl methacrylate, in particular, butyl or stearyl methacrylate), and the preferred crosslinking agent is ethylene glycol dimethacrylate.
  • Preferred porogenic solvents include C n H 2n+1 OH, wherein 1 ⁇ n ⁇ 4), wherein ethanol is particularly preferred, or acetonitrile.
  • the ratio of total monomer (functional monomer plus crosslinking monomer) to porogenic solvent in the polymerization mixture varies between 10-40% (V/V) while the ratio of functional monomer to crosslinking monomer varies between 1:1 to 1:3.
  • the invention is directed to a process of carrying out a chemical analysis method including the steps of providing the separation capillary column or channel of the invention; coupling the column or channel to a concentration sensitive detector; and carrying out the chemical analysis method.
  • the invention is directed to a system for carrying out a chemical analysis method, the system including the separation capillary column or channel of the invention and a concentration sensitive detector coupled with an interface to the exit end of the separation column or channel.
  • concentration sensitive detectors include a mass spectrometer, a fluorescence detector, an electro-chemiluminescence detector and a nuclear magnetic resonance detector.
  • a preferred interface is an electrospray ionization (ESI) interface or a matrix assisted laser desorption ionization (MALDI) interface.
  • the system of the invention further includes a preparatory precolumn coupled to the entrance end of the separation column or channel.
  • FIG. 1 is a schematic diagram of an exemplary embodiment of a 10 ⁇ m i.d. poly(styrene-divinylbenzene) PLOT column according to the invention in a microSPE/nanoLC/ESI-MS system according to the invention;
  • FIGS. 2A and 2B are scanning electron micrographs of the middle section (A) of the PLOT column of FIG. 1 and of an end section (B) of the PLOT column.
  • the end sections constitute roughly 5% of the approx. 5 m long capillary;
  • FIGS. 3A-3D are MS/MS spectra from four peptides of a BSA tryptic digest with 10 attomole injected directly onto the PLOT column;
  • FIGS. 4A-4E illustrate comprehensive analysis of a Lys-C digest of EGRF.
  • A Base peak chromatogram from nanoLC-ESI-MS analysis of 25 fmol of a Lys-C digest of EGFR injected on the 4.2 m ⁇ 10 ⁇ m i.d. PS-DVB PLOT column according to the invention; selected MS/MS spectra are shown for long (B), phosphorylated (C), and glycosylated (D, E) peptides of EGFR.
  • B Base peak chromatogram from nanoLC-ESI-MS analysis of 25 fmol of a Lys-C digest of EGFR injected on the 4.2 m ⁇ 10 ⁇ m i.d. PS-DVB PLOT column according to the invention; selected MS/MS spectra are shown for long (B), phosphorylated (C), and glycosylated (D, E) peptides of EGFR.
  • the peptide sequences and the extracted ion chromatograms are shown in
  • the triangle ( ⁇ ) and circle (•) represent mannose and N-acetyl glucosamine, respectively.
  • SA represents sialic acid and the square ( ⁇ ) represents galactose;
  • FIGS. 5 a and 5 B are chromatograms providing for the calculation of peak capacity for the column according to FIG. 1 .
  • FIG. 5A is the base peak chromatogram from the microSPE-nanoLC-ESI-MS analysis of a 4 ng tryptic in-gel digest of a single SDS-PAGE cut of M.
  • Acetivorans and FIG. 5B showns extracted ion chromatograms of six high intensity peaks used to calculate the peak capacity.
  • high-efficiency, narrow, e.g., 10 ⁇ m i.d., PLOT columns e.g., poly(styrene-divinylbenzene) can be repeatedly prepared in a single copolymerization step.
  • the polymer layer is covalently attached to the walls of the capillary, and there is thus no need for column frits.
  • Column-to-column retention time reproducibility is ⁇ 3. RSD, and, in terms of relative retention time, ⁇ 2% RSD.
  • the high permeability of the open structure allows long columns to be used at moderate pressure, which aids sample loading capacities.
  • the concentrated analyte that elutes from a PLOT column according to the invention combined with decreased ion suppression and enhanced ion collection efficiency at a flow rate of, e.g., 20 nL/min, significantly improves ESI-MS sensitivity.
  • the PLOT column of the invention Due to its open porous layer structure, the PLOT column of the invention demonstrates high efficiency for the separation of large peptides, as well as peptides with phosphorylated and glycosylated modifications. The columns are well suited to extended range proteomic analysis.
  • a PLOT column having an inside wall layer thickness of ⁇ 1-3 ⁇ m will reduce the open tube i.d. to roughly 7-8 ⁇ m.
  • This column diameter has previously been shown to be sufficient to minimize radial band broadening, leading to high performance separations.
  • commercial HPLC pumps will be able to deliver sufficient flow by virtue of the open tube structure.
  • One point of significance for such a column when coupled to ESI/MS is that the flow rate will be in the range of 5-50 nL/ ⁇ m, more than an order of magnitude lower than results with 75 ⁇ m i.d. columns.
  • the reversed phase PLOT column according to the invention e.g., 10 ⁇ m i.d., yields robust high resolution separation with minimal ion suppression. Use of such columns would significantly impact peptide quantitation and, therefore, yield more comprehensive and accurate results for, e.g., biomarker studies.
  • the invention is directed to a procedure to reproducibly prepare ultra-narrow bore (i.d. ⁇ 15 ⁇ m) porous layer open tube (PLOT) capillary columns for liquid chromatography coupled with mass spectrometry or other sensitive detection techniques such as fluorescence, electro/chemiluminence or NMR detection.
  • PLOT porous layer open tube
  • the invention is also directed to the resulting columns and to their uses.
  • the retentive stationary phase is a porous polymer formed by, e.g., temperature induced or UV light induced solution polymerization.
  • Exemplary uses of PLOT columns according to the invention include high-sensitive, high-efficiency gradient and isocratic single or multi-dimensional nano-LC analysis of limited amounts of biological or medical samples by coupling the columns at low flow rates to mass flow-sensitive detectors (i.e., ESI-MS).
  • ESI-MS electrospray ionization mass spectrometry
  • MALDI-MS matrix assisted laser desorption ionization mass spectrometry
  • One embodiment of the method according to the invention is characterized in that the inner surface of the bare fused-silica capillary is pre-functionalized before polymerization with, e.g., an anchoring silane, which contains acryl or methacryl groups, enabling the reaction of the anchoring silane with monomers and crosslinkers thereafter.
  • an anchoring silane which contains acryl or methacryl groups
  • the polymerization solution is composed of a functional monomer, such as styrene or alkyl methacrylate; a crosslinker that provides a high degree of crosslinking, such as divinylbenzene or ethylene glycol dimethacrylate, at a typical quantity ratio of monomer/crosslinker of 1:1; and a polar porogenic solvent (or porogen), such as ethanol, methanol, propanol or acetonitrile.
  • a functional monomer such as styrene or alkyl methacrylate
  • a crosslinker that provides a high degree of crosslinking, such as divinylbenzene or ethylene glycol dimethacrylate, at a typical quantity ratio of monomer/crosslinker of 1:1
  • a polar porogenic solvent or porogen
  • the porogen chosen is one that has a negligible swelling effect on the resulting polymer formed and not one that would be a good solvent for the resulting polymer, such as the non-polar solvents toluene, chloroform, tetrahydrofuran or heptane.
  • the polymerization solution has a low viscosity; thus, it can be introduced into the pre-functionalized fused-silica capillary under low pressure, such as 100-psi or lower, for a capillary tubing length of several meters.
  • the retentive layer thus formed using the monomers and crosslinkers described above is ready for chromatographic separation without additional surface functionalization steps.
  • Another embodiment of this method is characterized in that the polymeric retentive layer, e.g., 0.5-3 ⁇ m thick, formed after polymerization is integrated to the fused silica capillary inner wall.
  • the layer's structure is rigid and characterized by a rugulose inner surface, which enhances surface area and, thus, loading capacity.
  • Another embodiment of the method according to the invention is characterized in that no evaporation of the porogenic solution is needed after polymerization, in contrast to other methods.
  • the porogen is simply flushed out of the column after polymerization. Avoiding the use of a swelling porogen, such as toluene, chloroform, tetrahydrofuran, etc., which may remain in the network after polymerization and thus necessitate an evaporation step, diminishes the problem of clogging during the evaporation step, thus simplifying preparation and improving reproducibility.
  • a more hydrophobic column could be prepared by using stearyl methacrylate instead of styrene, or 2-acrylamido-2-methyl-1-propane sulfonic acid for ion exchange chromatography.
  • Other retentive groups if desired, could include alkyl chains, hydrophilic groups or affinity functions.
  • An exemplary column according to the invention is a long, high-efficiency polystyrene-divinylbenzene (PS-DVB), 10 ⁇ m i.d. porous layer open tube (PLOT) capillary column.
  • PS-DVB polystyrene-divinylbenzene
  • PLOT porous layer open tube
  • Fused silica capillary tubing with a polyimide outer coating was purchased from Polymicro Technologies (Phoenix, Ariz.). Styrene, divinylbenzene (DVB), ethanol, formic acid (HPLC grade), 3-(trimethoxysilyl)propyl methacrylate, 2,2′-diphenyl-1-picrylhydrazyl (DPPH), N,N-dimethylformamide (DMF) anhydrous, and 2,2′-azobisoisobutyronitrile (AIBN) were obtained from Sigma-Aldrich (St. Louis, Mo.). Acetonitrile (HPLC grade) and deionized water (HPLC grade) were purchased from Fisher Scientific (Fair Lawn, N.J.).
  • a standard tryptic digest of bovine serum albumin was from Michrom Bioresources, Inc. (Auburn, Calif.).
  • Angiotensin I insulin from bovine pancreas, HPLC standard protein mixture (ribonuclease A (13700 Da), cytochrome C (12327 Da), apomyoglobin (17600 Da), holo-transferrin (>70000)), ⁇ -casein from milk and human epidermal growth factor receptor (EGFR) from an A431 cancer cell line were purchased from Sigma-Aldrich (St. Louis, Mo.).
  • Achromobacter protease I (Lys-C) was obtained from Waco Chemical Co. (Osaka, Japan), and trypsin (sequencing grade) was from Promega (Madison, Wis.).
  • Fused-silica capillary tubing with a 10 ⁇ m i.d. ( ⁇ 5 meters) was first flushed overnight with 1.0 mol/L NaOH at ⁇ 1000 psi, washed with water and flushed with 1.0 mol/L hydrochloric acid, and then washed again with water and acetonitrile.
  • the capillary was dried with nitrogen at ⁇ 1000 psi to remove residue water and acetonitrile.
  • a polymerization solution was prepared containing of 5 mg of AIBN, 200 ⁇ L styrene, 200 ⁇ L DVB, and 600 ⁇ L ethanol. The solution was degassed by ultrasonication for 5 min and then filled into the silanized capillary. Both ends of the capillary were sealed with septa, and the capillary was heated at 74° C. for ⁇ 16 h in a water bath. The column was then washed with acetonitrile and was ready for use. In addition, 50 ⁇ m i.d. PS-DVB monolithic precolumns were prepared using protocols described previously 14 .
  • HPLC separations were performed using a Surveyor pump (ThermoElectron, San Jose, Calif.). Mobile phase A (0.1% (v/v) formic acid in water) and mobile phase B (0.1% (v/v) formic acid, 110% (v/v) water in acetonitrile) were used for the gradient separation. Samples were either bomb loaded onto the PLOT column or onto a 4 cm ⁇ 50 ⁇ m i.d. PS-DVB monolithic precolumn. A microSPE/nanoLC/ESI-MS system using a 10 ⁇ m i.d. PLOT column is shown in FIG. 1 . Referring now to FIG.
  • samples are first loaded manually off-line onto a precolumn 10 , which is then inverted and butt-to-butt connected to a 10 ⁇ m i.d PLOT column 12 using a PicoclearTM fluoropolymer core, clear elastomeric insert connector 14 (New Objective, Woburn, Mass.).
  • the sample is back-flushed from precolumn 10 onto PLOT column 12 .
  • a PEEK tee Upchurch Scientific Inc., Oak Harbor, WA
  • the precolumn 10 /PLOT column 12 assembly is attached to arm 18 of the splitter.
  • NanoESI-MS was performed on an LCQ Deca XP or an LTQ ion trap mass spectrometer (ThermoElectron).
  • PLOT column 12 was carefully butt-to-butt connected to a coated ESI spray tip 26 (360 ⁇ m o.d., 20 ⁇ m i.d. fused silica with 5 ⁇ m i.d. tip, 2-3 cm in length, New Objective) using a PicoclearTM connector 14 .
  • Electrospray voltage 28 was applied directly on the spray tip 26 to direct droplets of generated sample ions to the MS inlet orifice 30 .
  • SEQUEST standard database searching algorithms
  • Lys-C digests of ⁇ -casein and EGFR and an in-gel tryptic digest of Methanosarcina acetivorans were used as test mixtures to evaluate the performance of the nanoLC-ESI-MS.
  • Lys-C digestion of ⁇ -casein was performed as follows: Lys-C was spiked into the ⁇ -casein (at 10 pmole) in a 1:40 (w/w) ratio and incubated for 4 h at 37° C. (pH 8.5).
  • Lys-C digestion of EGFR 10 ⁇ g lyophilized powder of EGFR was dissolved in 100 ⁇ L of 6 M guanidine hydrochloride and 0.1 M ammonium bicarbonate in water. Reduction was conducted with 40 mM dithiothreitol for 30 min at 37° C., followed by alkylation with 80 mM of iodoacetamide for 1.5 h in the dark at room temperature. The buffer was subsequently exchanged to 0.1 M ammonium bicarbonate buffer, pH 8.5, to remove additional salts and reagents. Lys-C (1:20 w/w) was added to digest the protein for 4 h at 37° C. (pH 8.5). The mixture was acidified with 1% formic acid to quench the digestion, followed by storage at ⁇ 20° C.
  • M. acetivorans cells grown in methanol, were cultured as previously described 37 . Protein extraction, SDS-polyacrylamide gel electrophoresis (PAGE) fractionation and in-gel digestion were performed using protocols reported previously 37 . The concentration of the whole-cell protein extracts, determined by the Bradford assay (Bio-Rad, Hercules, Calif.), was 3.0 mg/mL. Roughly 45 ⁇ g of total protein was loaded on the gel, and after electrophoresis, the gel lanes were cut into 5 fractions. The in-gel tryptic digest of a fraction of M. acetivorans proteins (M>70 kDa) was used to evaluate the performance of the PLOT column. In addition, all 5 in-gel digested fractions were combined together to represent a global proteomic analysis for characterization of the PLOT column.
  • PAGE SDS-polyacrylamide gel electrophoresis
  • organic polymeric stationary phases provide several advantages, e.g., improved chemical stability over an extended pH range and the absence of silanol groups that can cause irreversible adsorption of peptides and proteins.
  • the exemplary PS-DVB porous layer was prepared and attached to the silanized capillary wall in a single in situ copolymerization step. Selection of a suitable solvent for the copolymerization step is key to successful preparation of repeatable, high efficiency PLOT columns.
  • the polymer should precipitate from solution at an early stage of the polymerization process, forming a thin porous layer at the capillary wall, while leaving open the main section of the capillary tube.
  • a porogenic solvent in which the resulting polymer, e.g., PS-DVB, is not very soluble is, therefore, desirable for the preparation of the PLOT column.
  • the surface layer of a PLOT column prepared from ⁇ 5 meter of 10 ⁇ m i.d. fused silica capillary tubing using 60% ethanol, was then examined at different sections of the column using scanning electron microscopy (SEM). Referring to FIG. 2A , it can be seen that the porous layer was observed to be uniform throughout most of the capillary. Portions at the ends of the capillary ( ⁇ 5% from each end) contained relatively large globules, as shown in FIG. 2B . These ends are cut to produce a column of length ⁇ 4.2 meters. From the SEM picture, the thickness of the surface inner layer of the PLOT column was estimated to be between 0.5 and 1 ⁇ m.
  • the permeability is 15-fold higher than a recently introduced 10 ⁇ m i.d. silica monolithic column 16 .
  • the column according to the invention is characterized by relatively high permeability in comparison to a similarly sized packed column.
  • very long columns according to the invention e.g., of 4 m or greater, can be operated successfully with commercially available HPLC pumping systems that have a pressure limit of 6000 psi.
  • the loading capacities of the PLOT column were determined by measuring peak width at half-height (w 1/2 ) as a function of injected amounts of angiotensin I (1296.5 Da) and insulin (5733.5 Da).
  • the maximum loading capacity is defined as the amount of sample injected when the corresponding w 1/2 is increased by 10% over the peak width at low sample amounts.
  • NanoLC-ESI-MS was conducted with a 20 min gradient, and the w 1/2 for each analysis was determined from the corresponding extracted ion chromatogram.
  • the loading capacities of the PLOT column, prepared using 60% of solvent, were ⁇ 100 fmol for angiotensin I and ⁇ 50 fmol for insulin. Given that 10 ⁇ m i.d. columns were used, these values represent relatively high loading capacity.
  • the loading capacities of the PLOT column prepared using 70% ethanol decreased to ⁇ 50 fmol for angiotensin I and ⁇ 20 fmol for insulin.
  • the higher percentage of ethanol resulted in the PS-DVB polymer phase separation occurring at an earlier stage of polymerization, likely leading to less polymer coated on the tubing wall and thus a lower loading capacity.
  • the ultimate goal of narrow bore LC-ESI-MS is to achieve low detection limits without sacrificing separation performance.
  • the detection level achievable with the PLOT column of the invention was evaluated using a tryptic digest of bovine serum albumin (BSA). Ten attomoles of a BSA tryptic digest was bomb loaded directly onto the 10 ⁇ m i.d. PLOT column and detected by the linear ion trap MS. Four peptides that provided good MS/MS fragmentation and high SEQUEST scores were confidently identified, as shown in FIGS. 3A-3D .
  • the extracted ion chromatogram of the peptide (YICDNQDTISSK) with the highest MS response (signal-to-noise ratio ⁇ 210) is shown in the insert to FIG. 3B , indicating that the detection limit for this peptide can, in principle, be in the hundreds of zmole range.
  • FIG. 4A shows the base peak separation of ⁇ 25 fmol Lys-C digest of EGFR on the 4.2 m ⁇ 10 ⁇ m i.d. PLOT column.
  • FIG. 5A shows a 3.5-h gradient separation of only 4 ng of an in-gel tryptic digest sample of a gel fraction (>70 kDa) of M. acetivorans on the 4.2 m ⁇ 10 ⁇ m i.d. PLOT column.
  • FIG. 5A illustrates both the complexity of the sample and the high resolving power of the system, with symmetrical peaks being observed throughout the entire separation.
  • the peak capacity of the gradient separation using the PLOT column was estimated by examining the extracted ion chromatograms of individual components throughout the separation window ( FIG. 5B ) 47 .
  • the 2 ⁇ values of six high intensity peaks over the wide gradient range are between 0.21 and 0.30 s, leading to an estimation of peak capacity of ⁇ 400. Even higher peak capacities are anticipated with full system optimization.
  • a total of 689 unique peptides and 238 proteins (single-hit peptides excluded) were identified from this very small sample.
  • the peptides and proteins were identified by automated searching of MS/MS spectra of the M. acetivorans database.
  • the number of identified peptides and proteins increased significantly, from 689 and 238 to 1793 and 512, respectively, as the injection amount was increased from 4 ng to 50 ng. Given that the sample was prepared by in-gel digest 47 , 50 ng is still a relatively limited amount of material.

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US8506803B2 (en) 2011-03-01 2013-08-13 Wisconsin Alumni Research Foundation Integrated electrospray ionization emitter and detection cell for parallel measurements by fluorescence and mass spectrometry
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WO2017117004A1 (fr) * 2015-12-28 2017-07-06 Restek Corporation Colonnes plot en croissant et procédés de préparation de colonnes plot en croissant
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US10948465B2 (en) * 2015-12-28 2021-03-16 Restek Corporation Crescent plot columns and methods for preparing crescent plot columns

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