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WO2009086092A1 - An on-column frit-fabrication method for fused silica capilaries - Google Patents

An on-column frit-fabrication method for fused silica capilaries Download PDF

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Publication number
WO2009086092A1
WO2009086092A1 PCT/US2008/087663 US2008087663W WO2009086092A1 WO 2009086092 A1 WO2009086092 A1 WO 2009086092A1 US 2008087663 W US2008087663 W US 2008087663W WO 2009086092 A1 WO2009086092 A1 WO 2009086092A1
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Prior art keywords
column
capillary
silica
frit
packed
Prior art date
Application number
PCT/US2008/087663
Other languages
French (fr)
Inventor
Ebrahim Zandi
Ling-Chi Wang
Harold Kochounian
Original Assignee
University Of Southern California Usc Stevens
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Publication of WO2009086092A1 publication Critical patent/WO2009086092A1/en

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/22—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the construction of the column
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
    • B01D15/206—Packing or coating
    • 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
    • B01J2220/82—Shaped bodies, e.g. monoliths, plugs, tubes, continuous beds
    • 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
    • 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
    • G01N30/60—Construction of the column
    • G01N30/6004—Construction of the column end pieces
    • G01N2030/6013—Construction of the column end pieces interfaces to detectors
    • 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
    • G01N30/84—Preparation of the fraction to be distributed
    • G01N2030/8423—Preparation of the fraction to be distributed using permeable separator tubes
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G01N30/46—Flow patterns using more than one column
    • G01N30/461—Flow patterns using more than one column with serial coupling of separation columns
    • G01N30/463—Flow patterns using more than one column with serial coupling of separation columns for multidimensional chromatography
    • 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

Definitions

  • the present invention is made, at least in part, with the support of grants from National Institute of Health (Grant GM65325). The government has certain rights in the invention.
  • the invention pertains to the field of chromatography technology. More particularly, it relates to a method for making on-column frits for fused silica capillary in microcapillary columns.
  • Fused silica based capillary liquid chromatography microcolumns are an essential component in high-resolution and high- sensitivity separation of peptides in Liquid Chromatography/Mass Sp ectro me try/Mass Spectrometry (LC-MSMS).
  • Microcapillary columns with pulled tips (10 cm in length and 50- 100 pm id) packed with one, two, or three independent chromatography phases are widely used [I].
  • Two limitations of these columns/spray tips are: 1) they have low capacity, and 2) the frequent clogging.
  • the end portion of an upstream fused silica buffer transfer tubing can be packed with one or more independent chromatography phases.
  • the on- column frit has to be sufficiently strong to retain the packing material and to resist the pressure applied for packing and flushing the column.
  • the frit also needs to be highly permeable for different solvents.
  • Figure 1 shows a microscopy of an exemplary on-column frit.
  • A Micrograph at IOOX magnification shows an overview of an on-column frit. The inner diameter of the fused silica capillary is 75 ⁇ m (measured by the manufacturer) and the length of the frit is approximately 0.3 mm. Scanning electron micrograph at 400X (8), 10,00OX (C), and 45,000 X (D) magnifications are shown.
  • the smooth slanted wall is the fused silica capillary tube, whereas the middle, rocky part shows the frit consisting of cross-linked silica.
  • C, D Silica resins were cross-linked by fibers (C) and these fibers were about 7 1.10 nm in width (D).
  • FIG. 2 shows an exemplary nanoflow electrospray ionization (ESI) chromatography.
  • a spray tip column was connected through a MicroTee to a frit-fabricated primary capillary column (PCC), which essentially replaces the liquid transfer line. High voltage was applied to the liquid through a high voltage pin lead which was also connected to the MicroTee. A divertfinject valve was used to apply sample or solution to the column at flow rate of 500 nl/mln.
  • B and C Total ion chromato grams of a 1-D (B) and a 2-D (C) chromatography using PCC are shown as time (x-axis) against relative abundance (y-axis).
  • Standard polyimide coated flexible fused silica capillary with different inner diameters 50, 75, 100, or 200 ⁇ m x 360 pm od, Polymicro Technology, Phoenix, AZ) were cut into 40 cm in length, washed with HPL C -grade methanol (J.T. Baker, Phillipsburg, NJ), and dried with ultra high pure grade compressed helium gas (Gilmore, South EI Monte, CA).
  • the fused silica capillary was dipped into dry lichrosorb 5 ⁇ Si 6OA resins (Varian, Palo Alto, CA) until approximately 0.5 mm resin was packed.
  • Frit fabricated capillaries were washed and tested with the following solutions introduced by a pressure injection platform (New Objective, Woburn, MA) at 400 psi of helium gas: 1 M ammonium nitrate (J.T. Baker, Phillipsburg, NJ), 1 N HCl (EM, Gibbstown, NJ), HPLC- grade water, and 100% HPLC-grade ACN. Frit fabricated capillaries were dried with helium gas and kept dry at room temperature for future use.
  • An advantage of using the lichrosorb particles is that the length of the frits can be controlled by the amount of the resin packed in the capillary.
  • This protocol was used to prepare frits of 0.2 - 0.5 mm in length for fused silica tubings with inner diameters of 50, 75, 100, and 200 ⁇ m.
  • the frit fabrication process did not result in any discemable deformity or shrinkage in the fused silica capillaries under light microscope.
  • the polyimide coat was intact and as a result fused silica columns maintain their flexibility, which is essential for making tight connections.
  • PCC is an inexpensive and simple way to increase the capacity, life, and versatility of the spray tip columns.
  • the schematic in Figure 2A shows the positioning of a PCC relative to a spay tip column.
  • a PCC replaces the buffer transfer capillary from divert/inject valve to the MicroTee.
  • a PCC can serve several purposes.
  • the PCC when there is no need to increase the capacity of a spray tip column, we have used the PCC as a pre-column, packed with a small amount of RP bed (e.g., 0.5 cm), which has increased the life of spray tip column significantly and prevented clogging. Second, when there is a need to increase the capacity and the resolution of peptide separation before MS, we have packed up to 30 cm of bed length of RP particles. Third, the PCC can be packed with different chromatography beds to allow two or multi-dimensional applications.
  • trypsin-digested peptides were separated either on a 10 cm RP-C18 PCC upstream to a 10 cm RP-C18 spray tip column (1-D set up) or a 5 cm SCX cation exchange PCC upstream to a 10 cm RP-C18 spray tip column (2-D set up) followed by mass spectrometry on an ion trap LTQ (see Figure 2 for relative positioning of the PCC to a spray tip column, and representative ion chromatograms of a 1-D and 2-D runs).
  • the SEQUEST program was used to match MS/MS spectra for peptide and protein identification. For the known protein mixture, the 1-D set up identified 56 peptides resulting in identification of 28 proteins.
  • the 20 set up identified 207 peptides resulting in identification of 44 proteins.
  • the 1-D set up identified 139 and 242 proteins in two independent runs. A total of 50 identified proteins were common between both runs.
  • Using the 2-D set up 906 and 1570 proteins were identified in each run. A total of 345 proteins were common between runs numberl and 2 in the 2-D set up.
  • the 2-D set up identified about 7.4-fold more proteins than the 1-D set up, which can be attributed to an increased resolution and capacity of the eptide separation.
  • This frit fabrication method allows the end section of a fused silica liquid transfer line anywhere along the path from an LC system to MS inlet to be used as a chromatography column.
  • the frit fabrication process does not damage the polyimide coating of the fused silica tubing and as a result tight connections are made without breaking the fused silica.
  • the frits can withstand pressures as high as 1500 psi on-line after packing with 30 cm 5 ⁇ resins.
  • Exemplary uses for PCC according to the present invention include on-line phospho- protein and other affinity chromatography enrichment applications.

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
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Abstract

A modified sol-gel method for a one step on-column frit preparation for fused silica capillaries and its utility for peptide separation in LC-MSMSA. The method of making a stable and permeable on-column frit-fabricated silica capillary comprises: partially packing a polymer coated flexible fused silica capillary with silica, dipping said packed capillary into a solution, and heating said capillary to generate a frit; wherein said packed silica crosslinks to the inner wall of said capillary and to each other. The on-column may replace the buffer transfer capillary from the divert/inject valve to the MicroTee. Also, the on-column may be used as a secondary column upstream of a spray tip column to expand resolution and capacity.

Description

AN ON-COLUMN FRIT-FABRICATION METHOD FOR FUSED SILICA
CAPILARIES
The present application claims the benefit of the filing date of U.S.
Provisional Application No. 61/015,516 filed December 20, 2007, the disclosure of which is incorporated herein by reference in its entirety.
STATEMENT OF FEDERALLYSPONSORED RESEARCHAND DEVELOPMENT
The present invention is made, at least in part, with the support of grants from National Institute of Health (Grant GM65325). The government has certain rights in the invention.
FIELD OF THE INVENTION
The invention pertains to the field of chromatography technology. More particularly, it relates to a method for making on-column frits for fused silica capillary in microcapillary columns.
BACKGROUND OF THE INVENTION
Fused silica based capillary liquid chromatography microcolumns are an essential component in high-resolution and high- sensitivity separation of peptides in Liquid Chromatography/Mass Sp ectro me try/Mass Spectrometry (LC-MSMS). Microcapillary columns with pulled tips (10 cm in length and 50- 100 pm id) packed with one, two, or three independent chromatography phases are widely used [I]. Two limitations of these columns/spray tips are: 1) they have low capacity, and 2) the frequent clogging. To increase capacity and versatility and to reduce the frequent clogging of a pulled microcapillary column, the end portion of an upstream fused silica buffer transfer tubing can be packed with one or more independent chromatography phases. To pack such columns, reproducible on-column frit fabrication is required. The on- column frit has to be sufficiently strong to retain the packing material and to resist the pressure applied for packing and flushing the column. The frit also needs to be highly permeable for different solvents. Although various combinations of sol-gel technique for on-column frit preparations for fused silica capillaries are known in the art [2-4], they suffer from the defect that they are either overly complicated or they destroy the polyimide coating in the process of on-column frit preparations or both [2j.
Therefore, there is still a need for a simple and reproducible method in which mechanically stable and permeable frits can be fabricated without damaging the polyimide coating in the process.
The above-mentioned and other features of this invention and the manner of obtaining and using them will become more apparent, and will be best understood, by reference to the following description, taken in conjunction with the accompanying drawings. The drawings depict only typical embodiments of the invention and do not therefore limit its scope.
BRIEF DESCRTPTION OF THE DRAWINGS
Figure 1 shows a microscopy of an exemplary on-column frit. (A) Micrograph at IOOX magnification shows an overview of an on-column frit. The inner diameter of the fused silica capillary is 75 μm (measured by the manufacturer) and the length of the frit is approximately 0.3 mm. Scanning electron micrograph at 400X (8), 10,00OX (C), and 45,000 X (D) magnifications are shown. (B) The smooth slanted wall is the fused silica capillary tube, whereas the middle, rocky part shows the frit consisting of cross-linked silica. (C, D) Silica resins were cross-linked by fibers (C) and these fibers were about 7 1.10 nm in width (D).
Figure 2 shows an exemplary nanoflow electrospray ionization (ESI) chromatography. (A) A spray tip column was connected through a MicroTee to a frit-fabricated primary capillary column (PCC), which essentially replaces the liquid transfer line. High voltage was applied to the liquid through a high voltage pin lead which was also connected to the MicroTee. A divertfinject valve was used to apply sample or solution to the column at flow rate of 500 nl/mln. (B and C) Total ion chromato grams of a 1-D (B) and a 2-D (C) chromatography using PCC are shown as time (x-axis) against relative abundance (y-axis). (B) For 1-D chromatography, peaks with higher abundance occurred within the gradient of 5 - 50 % ACN over the first 100 minutes. (C) For 2-D chromatography, higher relative abundance peaks are at 70% ACN after each salt elution, which were at 110, 220, 330, 440. and 550 minutes.
DETAILED DESCRIPTION OF THE INVENTION
Conventional sol-gel method of polycondensation of potassium silicate and formamide with different ratios is unable to produce stable and permeable frits. The inventors of the present invention have overcome the problems of the conventional sol-gel method of polycondensation of potassium silicate and formamide by including Iichrosorb silica particles to the process. Although the inventors do not wish to be bound by any particular theory, it is believed that a stable and permeable frit would be generated if potassium silicate would crosslink the silica particles to the inner wall of the glass and to each other. Accordingly, the following exemplary protocol was developed for frit fabrication.
The following examples are intended to illustrate, but not to limit, the scope of the invention. While such examples are typical of those that might be used, other procedures known to those skilled in the art may alternatively be utilized. Indeed, those of ordinary skill in the art can readily envision and produce further embodiments, based on the teachings herein, without undue exp erime ntation. Example
Standard polyimide coated flexible fused silica capillary with different inner diameters (50, 75, 100, or 200 μm x 360 pm od, Polymicro Technology, Phoenix, AZ) were cut into 40 cm in length, washed with HPL C -grade methanol (J.T. Baker, Phillipsburg, NJ), and dried with ultra high pure grade compressed helium gas (Gilmore, South EI Monte, CA). The fused silica capillary was dipped into dry lichrosorb 5 μ Si 6OA resins (Varian, Palo Alto, CA) until approximately 0.5 mm resin was packed. A mixture of 170 μl Kasil 1 potassium silicate (PO, Valley Forge, PA) and 20 μl formamide (EM, Gibbstown, NJ) was vortexed for 1 minute and centrifuged on a table top centrifuge at maximum speed for 1 minute. The supernatant (1 μl) was spotted on a small piece of parafilm, and the lichrosorb packed fused silica capillary was dipped into the solution for 5 seconds with the resin side toward the solution. To generate stable frits, several heating temperatures at different times were tested. For capillaries with 75 and 100 μm inner diameter, relatively low temperature of 1200C for 24 hrs generated mechanically stable and porous fits without damaging the polyimide coat. For tubing with 200 μm inner diameter, 1500C for 48 hrs was required. It is important to note that this process reproducibly generated frits with similar permeability and stability. The frit fabricated capillaries were washed and tested with the following solutions introduced by a pressure injection platform (New Objective, Woburn, MA) at 400 psi of helium gas: 1 M ammonium nitrate (J.T. Baker, Phillipsburg, NJ), 1 N HCl (EM, Gibbstown, NJ), HPLC- grade water, and 100% HPLC-grade ACN. Frit fabricated capillaries were dried with helium gas and kept dry at room temperature for future use.
An advantage of using the lichrosorb particles is that the length of the frits can be controlled by the amount of the resin packed in the capillary. This protocol was used to prepare frits of 0.2 - 0.5 mm in length for fused silica tubings with inner diameters of 50, 75, 100, and 200 μm. As shown in Figure IA, the frit fabrication process did not result in any discemable deformity or shrinkage in the fused silica capillaries under light microscope. In addition, the polyimide coat was intact and as a result fused silica columns maintain their flexibility, which is essential for making tight connections. Scanning electron microscopy of the frits showed that silica particles were cross-linked to each other and to the inner wall of the capillary through a three dimensional network of fibers, which were about 71 nm in width (Figures IB- D). The frits did not generate excessive backpressure during packing or chromatography. Routinely, the columns [hereafter called primary capillary column (PCC)] were packed with 0.5-30 cm bed length with 5-10 u reverse phase (RP), ion exchange, or titansphere TiO resins using a high-pressure injection "bomb" at 400 psi. The PCC can be packed with one or more independent phases for multidimensional chromatography applications. Packing 10 cm of bed often requires 30-60 min. Home made and commercially available spray tip columns such as Picofrit (New Objectives) are widely used in LCMSMS applications. The PCC is an inexpensive and simple way to increase the capacity, life, and versatility of the spray tip columns. The schematic in Figure 2A shows the positioning of a PCC relative to a spay tip column. Essentially, a PCC replaces the buffer transfer capillary from divert/inject valve to the MicroTee. Depending on the requirements of a particular experiment, with this arrangement, a PCC can serve several purposes. First, when there is no need to increase the capacity of a spray tip column, we have used the PCC as a pre-column, packed with a small amount of RP bed (e.g., 0.5 cm), which has increased the life of spray tip column significantly and prevented clogging. Second, when there is a need to increase the capacity and the resolution of peptide separation before MS, we have packed up to 30 cm of bed length of RP particles. Third, the PCC can be packed with different chromatography beds to allow two or multi-dimensional applications.
We tested the utility of combining the PCC and a spray tip column by analyzing a complex mixture of 48 known proteins (Sigma Universal Proteomics Standard set; Sigma Aldrich, St. Louis, MO), and a more complex mixture of unknown proteins derived from 10 ml conditioned medium of MEF feeder layers, which support the growth and self-renewal of human embryonic stem cells in vitro. For both experiments, trypsin-digested peptides were separated either on a 10 cm RP-C18 PCC upstream to a 10 cm RP-C18 spray tip column (1-D set up) or a 5 cm SCX cation exchange PCC upstream to a 10 cm RP-C18 spray tip column (2-D set up) followed by mass spectrometry on an ion trap LTQ (see Figure 2 for relative positioning of the PCC to a spray tip column, and representative ion chromatograms of a 1-D and 2-D runs). The SEQUEST program was used to match MS/MS spectra for peptide and protein identification. For the known protein mixture, the 1-D set up identified 56 peptides resulting in identification of 28 proteins. The 20 set up identified 207 peptides resulting in identification of 44 proteins. For the MEF conditioned media, the 1-D set up identified 139 and 242 proteins in two independent runs. A total of 50 identified proteins were common between both runs. Using the 2-D set up, 906 and 1570 proteins were identified in each run. A total of 345 proteins were common between runs numberl and 2 in the 2-D set up. The 2-D set up identified about 7.4-fold more proteins than the 1-D set up, which can be attributed to an increased resolution and capacity of the eptide separation. We have described an improved sol-gel method for the fabrication of an inexpensive, simple, highly reproducible, and durable on-column fit. This frit fabrication method allows the end section of a fused silica liquid transfer line anywhere along the path from an LC system to MS inlet to be used as a chromatography column. The frit fabrication process does not damage the polyimide coating of the fused silica tubing and as a result tight connections are made without breaking the fused silica. The frits can withstand pressures as high as 1500 psi on-line after packing with 30 cm 5 μ resins. Exemplary uses for PCC according to the present invention include on-line phospho- protein and other affinity chromatography enrichment applications.
Many modifications and variation of the invention as hereinbefore set forth can be made without departing from the spirit and scope thereof and therefore only such limitations should be imposed as are indicated by the appended claims. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
References
The following references are incorporated by reference in their entirety.
1. Link, A. J., Eng, J., Schieltz, D. M., Carmack, E., et al., Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 1999, 17,676- 682.
2. Piraino, S. M. and Dorsey, J. G., Comparison of frits used in the preparation of packed capillaries for capillary electrochromatography. Anal. Chem. 2003,75,4292-4296.
3. Cortes, H. J., Pfeiffer, C. D., Richter, B. E. and Stevens, T. S., Porous Ceramic Bed Supports for Fused Silica Packed Capillary Columns Used in
Liquid Chromatography. Journal of High Resolution Chromatography & Chromatography Communications 1987, 10,446-448.
4. Schmid, M., Bauml, F., Kohne, A. P. and Welsch, T., Preparation of On- Column Frits in Packed Fused Silica Capillaries by Sol-Gel Technology. Journal of High Resolution Chromatography 1999,22,438 - 442.

Claims

What is Claimed is:
1. A method of making a stable and permeable on-column Mt- fabricated silica capillary comprising: (a) partially packing a polymer coated flexible fused silica capillary with silica,
(b) dipping said packed capillary into a solution and;
(c) heating said capillary to generate a frit; wherein said packed silica crosslinks to the inner wall of said capillary and to each other.
2. The method according to claim 1, wherein said polymer is polyimide.
3. The method according to claim 1, wherein said silica is lichrosorb.
4. The method according to claim 1, wherein said solution comprises potassium silicate and formamide.
5. The method according to claim 1, wherein said frits are stable and permeable under conditions of 400 psi of helium gas and 1 M ammonium nitrate.
6. The method according to claim I1 wherein said capillary has an inner diameter of 50, 75, 100, or 200 μm.
7. The method according to claim 1, wherein said frit is from about 0.2 to 0.5 mm long.
8. The method according to claim 1, wherein said frit is stable under pressure of 1500 psi.
9. An on-column frit-fabricated silica capillary produced according to the method of claim 1, wherein said on-column maintains flexibility.
10. The on-column according to claim 9, wherein said on-column is used as a primary column in Liquid Chromatography/Mass Spectrometry (LC/MS).
11. The on-column according to claim 9, wherein said on-column is packed with reverse phase resin, ion exchange resin, titanium dioxide silica, titansphere TiO resin, or other resins for affinity purification in on-line LC/MS.
12. The on-column according to claim 9, wherein said on-column is packed with one or more independent phases.
13. The on-column according to claim 9, wherein said on-column is packed with different stationary phases for two- or multi-dimensional LC applications.
14. The on-column according to claim 9, wherein said on-column replaces the buffer transfer capillary from the divert/inject valve to the MicroTee.
15. The on-column according to claim 9, wherein said on-column may be used as a pre-column to increase the life span of spray tip columns.
16. The on-column according to claim 9, wherein said on-column may be used as a secondary column upstream of a spray tip column to expand resolution and capacity.
PCT/US2008/087663 2007-12-20 2008-12-19 An on-column frit-fabrication method for fused silica capilaries WO2009086092A1 (en)

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US61/015,516 2007-12-20

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