Abstract:
A method of fabricating a chromatographic column and columns produced by such a method are disclosed. The method includes: (a) obtaining a tube comprising two open ends and a lumen with a diameter of 75 microns or less extending between the two open ends; (b) depositing a liquid silicate composition into the tube lumen at a first open end of the tube; (c) forming a porous ceramic material from the composition in the tube lumen at a location near the first open end such that a space is formed between the ceramic material and the open end that is substantially free of the ceramic material; and (d) forming a tapered emitter having an orifice diameter of less than 3 microns at the first open end.

Description:
CLAIM OF PRIORITY 
     This application is the National Stage of International Application No. PCT/US2008/064845, filed May 27, 2008, which claims the benefit of U.S. Provisional Patent Application Ser. No. 60/932,734, filed on Jun. 1, 2007. The contents of these applications are hereby incorporated by reference in their entireties. 
    
    
     TECHNICAL FIELD 
     This invention relates to chromatography, and more particularly to chromatographic columns with porous frits and integrated single micron and sub-micron electrospray emitters, as well as methods of making them. 
     BACKGROUND 
     Liquid chromatography (LC) is a well-established analytical technique for separating components of a fluidic mixture for subsequent analysis and/or identification, in which a column is packed with a stationary phase material that typically is a finely divided solid or gel such as small particles with diameters of a few microns. A variety of different detectors can be used to detect the analytes separated on an LC column. Additionally, the separated components may be passed from the liquid chromatography column into other types of analytical instruments for further analysis, e.g., liquid chromatography-mass spectrometry (LC-MS) separates compounds chromatographically before they are introduced to an ion source of a mass spectrometer. 
     Mass spectrometry (“MS”) is an analytical technique used to measure the mass-to-charge ratio of gas phase ions. This is achieved by ionizing a sample and separating ions of differing masses and recording their relative abundance by measuring intensities of ion flux. Electrospray ionization (ESI) is a commonly applied ionization technique when dealing with biomolecules such as peptides and proteins. The electrospray process creates highly-charged droplets that, under evaporation, create ions representative of the species contained in a sample solution. 
     Mass spectrometry-based proteomics, particularly liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS), has recently become the technique of choice for rapid identification and characterization of proteins in biological systems. Moreover multiple approaches now exist to conduct these experiments in a semi-quantitative manner to monitor changes in protein expression and specific post-translational events as a function of biological perturbation. Despite its advantages discussed above, mass spectrometry suffers from limited dynamic range (e.g., the inability to detect peptides spanning an abundance ratio greater than 5000:1 in a single scan/spectrum) and finite acquisition rate (e.g., the inability to acquire MS/MS data at a rate sufficient to provide sequence information on all peptides in a complex mixture during a single LC-MS/MS run, regardless of their relative abundance). These limitations, combined with current trends towards analysis of increasingly complex mixtures (biomarkers, signaling pathways, etc) continue to push the limits of the ubiquitously used 75 μm and 100 μm I.D. (i.e., inner diameter) fused silica capillary columns packed with widely available 5 and 10 μm diameter reversed phase HPLC resins. 
     SUMMARY 
     The invention is based, at least in part, on the discovery that one can achieve exceptional LC-MS performance using chromatographic columns that include a porous yet robust silicate-based frit, a minimal dead space, and an integral electrospray emitter that has a diameter of, e.g., less than 3 microns. These columns can be used at very low flow rates, e.g., as low as 3-5 nanoliters/minute and even 1 nanoliter/minute. 
     In one aspect, the disclosure features methods of preparing robust fused silica-based capillary columns with integrated emitter tips, e.g., for high sensitivity LC-MS/MS analyses. The methods include: (a) obtaining a tube including two open ends and a lumen with a diameter of 75 microns or less extending between the two open ends; (b) depositing a liquid silicate composition into the tube lumen at a first open end of the tube; (c) forming a porous ceramic material from the composition in the tube lumen at a location near the first open end such that a space is formed between the ceramic material and the open end that is substantially free of the ceramic material; and (d) forming a tapered emitter having an orifice diameter of less than e.g., 3 microns at the first open end. 
     In another aspect, the disclosure features chromatographic columns that include a silica tube including a tapered end forming an orifice having a diameter of, e.g., less than 3 microns; a lumen having a diameter of, e.g., 75 microns or less, and a porous frit comprising silicate. The porous frit is located adjacent the tapered end within about 1 to 5 mm from the orifice and has a length of from about 1 to 3 mm. 
     Embodiments may include one or more of the following features. The method can include packing the tube with a slurry of beads having a diameter of about 1.8 μm to 5 μm, e.g., after forming the ceramic material. The liquid silicate composition may include lithium silicate and tetramethylammonium silicate. The ceramic material can be formed by heating the composition. The ceramic material can be formed by heating the composition to 300° C. or higher. The tapered emitter can be formed by using a laser-based pipette puller. The tapered emitter can have an orifice diameter of 1.5 microns or less. The tapered emitter can be configured to be used as an electrospray emitter. 
     Embodiments can also include one or more of the following features. The silicate of the chromatographic column can include lithium silicate. The orifice can have a diameter of 1.5 microns or less. The orifice can have a diameter of 0.75 micron or less. The column can be capable of withstanding a pressure of 5000 psi or higher. The column can be capable of withstanding a pressure of 7500 psi or higher, e.g., 9000 psi or higher. The orifice can be dimensioned and configured for use as an electrospray emitter in mass spectrometry. The orifice can be dimensioned and configured to operate at a flow rate of, e.g., about 10 nL/min or less. The flow rate can be 5 nL/min or less, e.g., 1 nL/min or less. 
     As used herein, the term “single-micron” means a dimension of larger than 1 micron and smaller than 10 microns. The term “sub-micron” means a dimension of smaller than 1 micron. 
     Aspects and embodiments described herein may include one or more of the following advantages. This disclosure features fabrication of fused silica based capillary HPLC columns with integrated electrospray emitter tips, which can be used in proteomics-based liquid chromatography mass spectrometry experiments. The construction of fused silica based HPLC columns with integrated electrospray emitter tips decreases dead volume between the HPLC column bed and emitter tip and thus improves chromatographic performance. The in situ formation of column bed retention frits in fused silica capillaries of any size, especially of small I.D., facilitates construction of small-inner diameter HPLC capillaries with arbitrarily small electrospray emitter tips. In addition, the methods disclosed herein can be used in conjunction with so-called ultra-high pressure LC, which offers further improvements in chromatographic performance. For example, the new columns with integrated electrospray emitters made by the methods disclosed herein can withstand ultra high pressures, e.g., 5000 psi or higher, 7500 psi or higher, 9000 psi or higher, and even 10,000 psi or higher. 
     This disclosure also features columns made by the disclosed methods, which have arbitrarily small inner diameters and have porous retention frits that are mechanically stable, but still allow sufficient volumetric flow to facilitate packing of particulate LC beds and have mechanical stability sufficient to withstand ultra-high pressure LC and facilitate placement of integrated emitter tips close to the frit and with arbitrarily small orifice diameters. Particularly, devices with 25 micron-inner diameter capillary columns, packed with 3 micron diameter HPLC resin, terminating with an electrospray emitter tip of less than 1 micron diameter, at flow rates of less than 1 nanoliter per minute have been fabricated and operated successfully applying the methods disclosed herein. In comparison, for example, the smallest devices currently offered commercially, e.g., those offered by New Objective, Inc., have 50 micron inner diameters, are packed with 5 micron diameter resin beads, terminate with a 10 micron emitter tip, and are intended to operate at flow rates of 50-100 nL/min. 
     Without being bound by theory, it appears that no one has successfully demonstrated a method to fabricate columns that allow experimental exploration of ultra-small scale LC-MS regimes to date, because of the difficulties discussed below. Generally, if columns interfaced to electrospray ionization benefit significantly by having an integrated ESI emitter on the column, preferably immediately adjacent to the column frit. Electrospray ionization efficiency increases as effluent flow rate decreases. However, stable electrospray at very low flow rates (e.g., 10 nL/min or less) requires exceedingly small diameter orifices (e.g., less than 10 μm). Further, the Van Deemter equation predicts that at flow rates below Van Deemter plot minimums, longitudinal diffusion is expected to lead to band broadening. Moreover, the introduction of dead-volume between the frit and emitter tip further contributes to band broadening. As a result, the chromatographic band broadening may be expected to cancel out any gains realized by improved ionization efficiency. Surprisingly, however, use of the new columns with integrated single- or sub-micron electrospray emitters described in this disclosure has led to significantly increased LC-MS performance. 
     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. 
     Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is an enlarged cross-sectional view of a portion of a chromatographic column. 
         FIGS. 2   a - 2   e  are schematic representations in cross-section of intermediate columns during various steps of an exemplary method of making chromatographic columns with integrated electrospray emitters. 
         FIGS. 3   a - 3   e  are representations of intermediate columns during discrete steps for in situ formation of a frit in a fused silica tube.  FIG. 3   d  is an electron micrograph of an integrated electrospray emitter formed on the fused silica tube, and  FIG. 3   e  is an electron micrograph of a cross-section of the column of  FIGS. 3   a - 3   d  showing the porous frit formed in the tube. 
         FIG. 4  is a graph of mass spectral peak intensity plotted as a function of column size and column flow rate for 400 common peptides detected using the columns described herein. 
         FIG. 5  is a graph of chromatographic peak width plotted as a function of column flow rate generated using the columns described herein. 
         FIG. 6  is a histogram of number of peptides identified by MS/MS plotted as a function of column size and column flow rate. 
         FIG. 7  is a series of Venn diagrams that show the reproducibility of peptide identifications observed for LC-MS/MS analyses. 
     
    
    
     DETAILED DESCRIPTION 
     This disclosure features a simple strategy to construct robust fused silica-based capillary columns with integrated, porous silicate-based frits and single- or sub-micron emitter tips for high sensitivity LC-MS/MS analyses. This approach can use commercially available, economical starting materials, along with a simple and rapid procedure, which enables rapid column construction, e.g., in less than 30 minutes. These columns can be readily used with conventional- and high-pressure (e.g., 5000 psi or higher) binary HPLC systems. LC peak widths (e.g., full width at half maximum, “FWHM”) of less than 30 seconds at separation flow rates of about 3-5 nanoliters per minute (“nL/min”) can be routinely observed from these HPLC column assemblies. When appropriately stored, columns of 25 μm I.D. with integrated ESI emitters of 1.5 μm or smaller diameter can be used for several weeks without failure. Further, analysis of tryptic peptides derived from whole cell lysate clearly demonstrates the improved MS performance (e.g., 10-fold or higher increase of signal-to-noise ratio) realized through the combination of miniaturized column assemblies and ultra-low LC flow rates. 
     Chromatographic Columns with Porous Frits and Integrated Electrospray Emitters 
     The new columns are the result of carefully balancing a variety of parameters to achieve significant performance enhancements. The columns have a small inner diameter ranging from about 5 to about 75 microns, e.g., about 10 to 25 microns. The columns are filled with small resin particles or beads of 1.8, 2, 3, 4, or 5 microns. The columns include an integral emitter having an orifice of under 3 microns, e.g., 2, 1.5, 1.0, or even smaller. These combinations of dimensions and the use of a robust, silicate-based porous frit enable the new columns to be used at extremely low flow rates, e.g., under 10 nanoliters/minute, e.g., under 7.5, 5.0, 4.0, 3.0, 2.0, 1.0, and even less than 1.0 nanoliter/minute. In addition, the new columns are surprisingly robust, operating well at high pressures, such as pressures at or above 5000, 7000, 9000, 9500, and even 10,000 psi. 
     As shown in  FIG. 1 , a capillary tube  10  used in LC-ESI-MS includes a tubular body  12  of uniform inner diameter (“I.D.”) and a tapered end  14 , which has an orifice of a diameter “d.” Tube  10  can accommodate a sorbent bed or solid phase  18  that is defined by the wall of the body  12 . A porous retention frit  16  formed of, e.g., silicate, separates the sorbent bed  18  and the tapered end  14 . 
     The capillary tube  10  can have I.D. in the range of from about 5 micron to about 2 mm, e.g., 5 to 75 microns, 10 to 50 microns, or 15 to 25 microns. The diameter d of the orifice of the tapered end  14  can range from a few hundred nanometers to about a few microns, e.g., from 0.5 to 3 microns or 0.5 to 1.5 microns. The frit  16  adheres to the smooth inner wall surface of the tube  10  and is located back from the orifice at a distance “L” which varies from 2 mm to 5 mm. The frit  16  also has a length “l” that varies from about 0.5 mm to 2 mm. The sorbent bed  18  is normally injected under high pressure into the tube  10  in the form of a slurry subsequent to forming the frit  16 . Since the frit is porous, a slurry of sorbent  18  can be easily injected into the tube  10 . 
     The empty capillary tubes used for HPLC columns are those known in the art, and can, for example, be made of ceramic materials, such as borosilicate glass, fused-silica, polyimide coated fused-silica and aluminum coated fused-silica; metals, such as stainless steel, glass-lined stainless steel or silica lined stainless steel; or they can be made of polymeric materials or fused silica-lined polymeric materials. The polymeric material that can be used include fluoropolymers, such as ethylene tetrafluoroethylene (ETFE), fluorinated ethylene propylene (FEP) and polytetrafluoroethylene (PTFE); polyolefins, such as high density linear polyethylene (HDPE), low-density linear polyethylene (LDPE) and polypropylene; polyketones, such as polyetheretherketone (PEEK) and silica-lined PEEK; acrylics, such as polymethylmethacrylate (PMMA), polyamides, such as nylon 6, nylon 11 and nylon 12; and polyimide. Useful capillary tubes for use as capillary columns in accordance with the present disclosure are those of, stainless steel, PEEK and HDPE, and polyimide-coated fused silica. 
     The internal or external shapes of capillary columns can have a variety of regular geometric shapes, such as round, oval, square, rectangular, polygonal, such as pentagonal, hexagonal, and the like; or can have irregular shapes. The term “internal shape” of the capillary columns, as used herein, has the same meaning as the “bore” of a capillary column. Tubes commonly used for these columns have a round internal shape or bore. 
     General Methodology of Making the Chromatographic Columns 
       FIGS. 2   a - 2   e  illustrate an exemplary method to make the new fused silica capillary columns with silicate-based frits and integrated emitter tips. As formed, the columns with integrated emitters can be used in LC-ESI-MS experiments. 
     Forming Porous Frits in Silica Tubing 
     As shown in  FIG. 2   a , a small section  22  (e.g., 1.5 cm) of fused silica is exposed by removing a polyimide coating (show in light grey), for example, via torch heating near one end of a polyimide-coated used silica capillary tube  20 . Next, referring to  FIGS. 2   b  and  2   c , a frit-forming precursor  24  such as a potassium and lithium silicate solution (shown dark grey with a few circles and dots) is drawn up to the fused silica window  22  by capillary action. A soldering iron (not shown) at about 300° C. is brought close to the section  22  and the focused heat induces local polymerization of the precursor  24  to form a porous frit  26  approximately at the leading edge of the silicate solution with a confined length “l” usually in the range of 1 to 2 mm. Any unpolymerized silicate solution is then expelled from the capillary by pressurized rinse with an aqueous solution. 
     In particular embodiments, tetramethylammonium silicate is added to the potassium and lithium silicate solution in an effort to better control the pore size of the frit. Without wishing to be bound by theory, it is believed that the pore size is determined, in part, by the size of the cation in the silicate solution. Tetramethylammonium is a larger cation compared to potassium and lithium cations, therefore the average pore size can be increased by increasing the abundance of tetramethylammonium silicate in the silicate solution. The volume ratio of the tetramethylammonium silicate solution to the potassium and lithium silicate solution is preferably from about 2:1 to 1:3, e.g., about 1:2. For example, a frit-forming precursor solution can be made by mixing a 50 μL tetramethyl ammonium silicate (15-20 wt. %) aqueous solution, a 100 μL Klebofon™ 1524 silicate solution (e.g., of approximately 77.0 wt % water, 2.5 wt % Li 2 O, and 20.5 wt % SiO 2 ), and a 10 μL, formamide. Unexpectedly, besides the benefit of pore-size control, mixing tetramethylammonium silicate and lithium silicate solution also allows creation of remarkably robust frits. As a result, the robust frit is less likely to shed fine particulates that may clog the pores of the frit and/or the orifice when high pressure is applied to the LC column. As an example, the robustness of the frits made as described herein is evidenced by several weeks of repeated use of the LC column with integrated emitter tips of less than 1.5 μm in diameter, at column flow rates of e.g. 3-5 nL/min. 
     The robustness of the frits is further evidenced by the fact that the new columns with integrated electrospray emitters made by the methods disclosed herein have been tested to withstand ultra high pressures, e.g., over 5000, 7500, 9000, and 9500 psi. 
     Packing the Chromatographic Column 
     After the frit  26  is formed, a slurry formed of sorbent  28  or particles of specified diameter (e.g., Monitor 3 μm diameter, 120 Å pore size C18 particles) and a mobile phase (e.g., an organic solvent) is injected under pressure from the column end opposite to where the silicate entered the column, as illustrated in  FIG. 2   d . In some embodiments, pressure packing the LC resin  28  can also be used to simultaneously expel the unpolymerized silicate. More detail of packing chromatographic columns is described in Yang, U.S. Pat. No. 4,483,773. 
     Forming Single-Micron Electrospray Emitters 
     After the column  20  is packed with sorbent bed  28 , the column can be dried with pressurized gas, such as helium, for a few minutes and an integrated emitter tip of sub- or single micron diameter, e.g., 0.5-1.5 μm can be formed using a laser-based pipette puller, such as the P-2000 of Sutter Instruments Company, Calif. Referring now to  FIG. 2   e , the packed capillary  20  is placed into a laser-based pipette puller and a small diameter orifice emitter tip is then pulled 2-5 mm from the frit  26 . The orifice diameters of these emitters can range from less than 3 microns down to 2.5, 2.0, 1.5, 1.0, or even less than 1 micron. 
     Storing the Packed Columns with Integrated Emitters 
     For convenient handling and storage, the column can be sheathed in a TEFLON® tubing (e.g., 0.062 inch O.D.×0.0115 inch I.D.) that is counter-bored (0.038 inch I.D.) at one end using a pin vise drill to provide a protective cover for the tip when not in use. For extended storage, the column may be placed into a conical style tube (TEFLON® sleeve oriented downward) containing 5 mL of dilute acetic acid. Without wishing to be bound by theory, it is believed that this procedure keeps the emitter tip bathed in liquid such that trace quantities of nonvolatile salts that may be present in the HPLC solvents do not precipitate on the tip. Alternately, the column may be dried by expelling all liquids with pressurized helium. 
     EXAMPLES 
     The following examples are illustrative, but not limiting. 
     Starting Materials for Making the Chromatographic Column Assemblies 
     Fused-silica tubing is available, e.g., from Polymicro Technologies (Phoenix, Ariz.). Various polyetheretherketone (“PEEK”) nuts, ferrules, and other fittings are available, e.g., from Valco Instrument Co. (Houston, Tex.) and Upchurch Scientific (Oak Harbor, Wash.). TEFLON® tubing was purchased from Zeus Industrial Products (Orangeburg, S.C.). Bulk resins for reversed phase chromatography (Monitor 5 μm diameter, 120 Å pore size C18 particles; Monitor 3 μm diameter, 120 Å pore size C18 particles) can be obtained, e.g., front Column Engineering (Ontario, CA). Other resins can be used interchangeably depending on the specific use of the chromatographic columns as is known in this field. 
     Frit-forming compounds or precursors such as potassium and lithium silicate solutions can be obtained commercially, e.g., from PQ Corporation (Valley Forge, Pa.). Formamide, tetramethylammonium silicate, glacial acetic acid are available, e.g., from Sigma-Aldrich (St. Louis, Mo.). HPLC grade acetonitrile can be purchased from Fisher Scientific Intl. (Hampton, N.J.). 
     Example 1 
     Forming Silicate-Based Frits in Analytical Columns 
     Analytical columns (“AC”) were constructed from 360 μm O.D.×50 μm I.D. and 360 μm O.D.×25 μm I.D. fused silica capillary tubing. A 2.5 cm section of polyimide was removed approximately 3 cm from one end of the fused silica tubing. Silicate-based frits were cast in situ following the general protocol described above. 50 μL of tetramethylammonium silicate (Sigma-Aldrich, part#438669) was added to a microcentrifuge tube, followed by the addition of 100 μL of lithium silicate (Klebofon™ 1524 from PQ Corporation, Valley Forge, Pa.). This solution was briefly stirred, with subsequent addition of 10 μL of formamide (Sigma-Aldrich, part# F5786) followed by another brief stirring at room temperature. This solution was allowed to migrate approximately 1 cm into one end of the fused silica tubing by capillary action. 
     Next, a variable temperature soldering iron was set to 375° C. and used to induce polymerization of the silicate solution beginning at the opposite end of the silicate plug. By slowly rotating the fused silica capillary and positioning the soldering iron immediately adjacent to the distal end of the silicate plug, gradual polymerization was easily visualized and frits of 2-5 mm in length were readily cast in 5-20 seconds. Immediately after frit formation unpolymerized silicate was ejected from the capillary by a pressurized rinse with an aqueous solution of 0.1% acetic acid. Excess fused silica tubing was trimmed using a tile scribe so that the frit was positioned at the very end of the fused silica capillary.  FIGS. 3   a - 3   c  are optical images that show discrete steps in this process for construction of 50 μm I.D. analytical columns. First, the silicate solution was allowed to migrate to four fifths the length of the exposed window ( FIG. 3   a ). Next, polymerization was induced using the soldering iron as described above, with care taken to form frits of 1-2 mm in length ( FIG. 3   b ). After ejection of excess silicate solution the frits were re-heated with the soldering iron at 400° C. for several seconds ( FIG. 3   c ). The re-heating can provide extra mechanical stability to the silicate-based frits.  FIG. 3   e  is a scanning electron micrograph that shows a cross-section of the capillary tube (25 μm I.D.) filled with the mesoporous frit as formed. 
     Example 2 
     Packing the Analytical Columns 
     To pack the columns described in Example 1, a dense slurry of reversed phase resin was made in 1 mL, of acetonitrile and pressure packed behind the frit at pressures of approximately 500-1500 psi. A bed length of 6-8 cm was achieved in less than 10 minutes (5 μm diameter beads used for the 50 μm I.D. AC and 3 μm diameter beads used in the 25 μm I.D. AC). 
     Example 3 
     Forming Single and Sub-Micron Electrospray Emitters on Analytical Columns 
     The packed ACs in Example 3 were dried with pressurized helium for 5 minutes before an integrated emitter tip of 0.75-1.5 μm diameter was formed 2-4 mm beyond the frit using a laser-based pipette puller (P-2000, Sutter Instruments Company, Calif., program settings: Heat=500, Fil=0, Vel=5, Del=130, Pull=25). A Nikon I.C.-66 microscope (Micro Optics, Fresh Meadows, N.Y.) was used at 1000× or 1500× magnification for visual confirmation of tip diameter.  FIG. 3   d  shows a high resolution electron micrograph image of a sub-micron integrated emitter tip as formed in this procedure. 
     Example 4 
     Using AC in Liquid Chromatography Mass Spectrometry Analysis 
     A Waters® NanoAcquity (Waters Corp., Milford, Mass.) and an Agilent 1100 series binary pump (Agilent Technologies, Santa Clara, Calif.) were used to generate solvent gradients for online LC-MS experiments. Samples were manually pressure loaded onto a precolumn (a fritted 360 μm O.D.×100 μm I.D. or 360 μm O.D.×50 μm I.D. fused silica capillary tubing packed with 5 μm diameter beads), which was then inserted into one outlet arm of a PEEK Y-style connector. The inlet and second cadet of the PEEK Y were connected to the HPLC effluent and waste lines, respectively. The base HPLC flow rate was set at 200 μL/min, and an adjustable restrictor was inserted into the waste line to provide variable backpressure (and hence flow) through the AC made in Example 3. 
     Typically 60-80 bar backpressure measured at the binary pump provided flow rates of 2-10 nL/min through the PC and AC. Final flow rate for each experiment was estimated from measurement of the displaced volume spanning the PC and AC, and the observed delay time for breakthrough of the organic gradient (resulting in sharp rise of the total ion chromatogram baseline) during LC-MS analysis. An Upchurch micro union with 9 nL internal swept volume was used to connect the pre- and analytical columns. The emitter tip position with respect to the mass spectrometer inlet was controlled by a NanoESI Source (Proxeon Biosystems A/S, Odense, Denmark). HPLC solvent A was 0.2 M acetic acid and solvent B was 70% acetonitrile/0.2 M acetic acid. The solvent gradient was ramped 0%-100% B in 50 min. Mass spectrometry data acquisition was performed in data-dependent mode on a hybrid linear ion trap-Orbitrap instrument (ThermoFinnigan, San Jose, Calif.) under the following conditions: MS scan, 350≦m/z≦1500; top 8 most abundant MS/MS scans with exclusion list and 3 Da width isolation window; 1.5 kVolt ESI voltage; 200° C. capillary temperature. 
     Mascot Daemon was used to extract and format MS/MS data for subsequent search against human IPI protein database (Mascot version 2.0.00, Matrix Science, Inc., London, United Kingdom). The search parameters allowed 2 missed cleavages for trypsin and a fixed modification of +57 corresponding to carboxyamidation of cysteine. A mass tolerance was 1.2 Da for precursors and 0.35 Da for fragment ions was specified. Only those peptide sequence identifications common between analyses performed with the large and small column assemblies, and with score ≧20 were exported from Mascot for further analysis. Selected ion chromatograms for 100 of these precursors were generated and then compared in terms of maximum precursor intensity and chromatographic peak width, measured base-to-base. Finally, manual comparison of the corresponding MS/MS spectra was performed to confirm comparison of identical precursors. 
     The chromatographic columns with single and sub-micron integrated electrospray emitters and methods of making such columns allow improved ionization efficiency and thus improved LC-MS performances. Emitter tips in this sub-micron size regimen readily support flow rates below 10 nL/min, and offer the potential for improved detection limits as a result of increased. More advantages are demonstrated below. 
     Referring particularly to  FIG. 4 , a plot of mass spectral peak intensity as a function of column size (red: 360 μm OD×50 μm ID: blue: 360 μm OD×25 μm ID) and column flow rate (listed above each column size, nL/min) for 400 common peptides detected across these conditions is shown. The yellow line shows the average peptide intensity observed across these column and flow rate conditions. Clearly the observed peptide intensity shows a strong and inverse correlation to the combination of column size and flow rate. For example, the average peptide intensity using 25 micron ID column tested at flow rate of 0.75 nL/min is at least ten-fold higher than that using 50 micron ID column at flow rate of 200 nL/min. In this example, both columns have emitters of 0.75-1 μm in orifice diameter. 
     Referring now to  FIG. 5 , chromatographic peak width (seconds) is plotted as a function of column flow rate (nL/min). Individual points are those peptides detected with large (360×50) and small (360×25) column sets. Mean chromatographic peak widths are indicated by the red (360×50) and blue (360×25). The small column set provides superior chromatographic performance at all flow rates used in this study. Furthermore, at flow rates below approximately 3 nL/min., chromatographic peak widths begin to broaden for both large and small column sets. In spite of this peak broadening, the new columns still provide a significant performance improvement. 
     Referring to  FIG. 6 , the number of peptides identified by MS/MS, with Mascot score ≧40, is plotted as a function of column size (red: 360 μm OD×50 μm I.D.; blue; 360 μm OD×25 μm ID) and column flow rate (listed above each column size, nL/min). Clearly the number of peptides identified per LC-MS analysis shows a strong and inverse correlation to the combination of column size and flow rate. Interestingly, for the large column set, longitudinal band broadening reduces the number of peptides identified at the lowest flow rate. In the small columns, equivalent volumetric flow yields four-times higher linear velocity, hence negating the deleterious effects of longitudinal diffusion. As a result the total number of peptides identified using the small columns continues to increase as volumetric flow decreases. 
       FIG. 7  illustrates the reproducibility of peptide identifications observed for LC-MS/MS analyses across the three lowest flow rates used for the small (left) and large (right) column sets. Here, the small column set has an I.D. of 25 μm and an orifice diameter of about 0.75-1 μm while the large column set has an I.D. of 50 μm and an orifice diameter of about 0.75-1 μm μm. Taking the left Venn diagram (using 25 μm I.D. column and about 0.75-1 μm diameter orifice) for example, the largest circle indicates that 2206 species (e.g., peptides) were detected at a flow rate 0.7 nL/min, and the intermediate circle indicates that 1367 species were detected at a flow rate of 3 nL/min. The overlap of the two circles indicate the number of common species that were both detected at the two different flow rates, which in this case is 1045 common peptides. Similarly, the overlap of the three circles indicates the number of common species that were detected at three different flow rates. Generally, the higher the number of common species is, the more reproducible of peptide identification is. The Venn diagrams clearly show a more than two-fold improvement (670 vs. 325) in the number of common peptides observed, therefore clear demonstrate that smaller column set has higher reproducibility of peptide identifications. 
     OTHER EMBODIMENTS 
     A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.