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Practical High Sensitivity LC-MS

Practical High Sensitivity LC-MS. Fundamentals, Challenges, and Prospects. Gary A. Valaskovic, Ph.D. New Objective, Inc. Main Topics. Anatomy of Electrospray Introduction to Nanospray The Nanobore LC Advantage Flow Splitting and Sample Injection Nanobore LC to MS Interfacing

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Practical High Sensitivity LC-MS

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  1. Practical High Sensitivity LC-MS Fundamentals, Challenges, and Prospects Gary A. Valaskovic, Ph.D. New Objective, Inc.

  2. Main Topics • Anatomy of Electrospray • Introduction to Nanospray • The Nanobore LC Advantage • Flow Splitting and Sample Injection • Nanobore LC to MS Interfacing • Keys to Success

  3. Anatomy of ESI • Adapted from Kebarle & Tnag, Anal. Of Chem., 1993, 64, 972A

  4. Anatomy of ESI

  5. What is Nanospray?

  6. Why Use Nanospray? • ESI-MS (as commonly implemented) is a concentration sensitive detector. There is little or no loss in signal/noise as you reduce the flow rate. You can obtain the same S/N for most compounds from 1 mL/min to 10 nL/min (with the right equipment)! Adapted From Cody, Appl. Elec. Mass. Spec., Pramanik, Ganguly, Gross Eds.

  7. Why Use Nanospray? There are three reasons to use Nanospray: • Sensitivity • Sensitivity • Sensitivity • Nanospray is one of the key technologies for MS-based Proteomics

  8. How Does Nanospray Yield Sensitivity? • Two ways to obtain sensitivity with Nanospray: • Off-line “Static” Nanospray • Extend the analysis time for a given sample • Sum spectra to increase S/N • Complete MS/MS or MSn possible • On-line LC-Nanospray • Analyze a small volume sample (1 µL or much less) • Concentrate your sample into as small a volume as possible

  9. Static Nanospray Methodology • Direct infusion of 0.5 to 5 µL sample • Sample must be “clean” • No pumps - flow is generated by electrostatic “pressure” • Typical Tip ID: 1 - 4µm • Typical flow rate: 10 - 50 nL/min HV MS Inlet Glass needle - 0.7 mm bore Conductive Coating Tip ID 1 - 4 µm Liquid sample 1 - 5 µL

  10. Static Nanospray Extends Analysis Time Adapted From Corey & Pinto, Appl. Elec. Mass. Spec., Pramanik, Ganguly, Gross Eds.

  11. Static Nanospray Limitations • Sensitivity is good, but inferior to LC methods • Typically 10 -100 fmol proteins and peptides • Sample prep is not integral, sample must be clean and concentrated • Typically 100 nM to 10 µM • Limited utility on complex mixtures (OK on single bands but unable to handle “shotgun” methods) • Highly dependent on operator skill • Limited throughput • Automation is possible but $$$

  12. “On-line” Nanospray with Nanobore LC • Integral sample clean-up • On-line injection of 1 - 20 µL • Gradient elution from split flow HPLC pump • Column ID ≤ 100 µm • Typical flow rate: 100 - 500 nL/min Gradient pump @ 200 µL/min MS Inlet In-line filter Column Tip Micro-injection valve (or autosampler) Flow split 1000:1

  13. 4.6 mm 50 µm Why Use Nanospray LC? Elute your sample into the smallest practical volume for the highest S/N!

  14. Why Use Nanobore LC? The Concentration Advantage! Adapted From Tomer & Moseley, Mass. Spec. Rev., 1994, 13, 431

  15. Requirements for LC System • Gradient Operation • Binary required; tertiary, quaternary preferred • Injection • 1 - 20 µL Typical • Accommodate sample trapping • Flow rate ≈ 100 to 1000 nL/min • Typically pre-column flow split from conventional pump

  16. Flow Splitting Methods • Simple “T” Splitter (build) • Inexpensive! Easy to do. Split is non-linear but reproducible. • Balanced Flow Splitter (build or buy) • Good performance, inexpensive • High-Pressure Flow Splitter (buy) • Good performance, $$$ • “Active” Mass Flow Control (buy) • Good performance, $$$

  17. Simple Flow Splitting • Use a simple Tee • Use a small bore (20 - 50 µm ID) tubing to create a flow “calibrator” • Adjust split ratio by adjusting the length of the calibrator • Fine tune by setting the pump flow • Ratios from 1:10 to 1:1000 are readily obtained

  18. Nanospray Source Requirements • Mechanical requirements • XYZ Stage for tip positioning • Tip and spray imaging system • Junction and proximal HV contact • Tip requirements • ID of 10 - 30 µm • Typically fused-silica, 360 µm OD • Uncoated or coated

  19. Objective Lens Tip Holder HV Contact CCD Camera XYZ Stage Injection Valve On-line Nanospray Source www.newobjective.com

  20. Monitor Illuminator Source On-line Nanospray Source

  21. What About Sample Injection? • Gradient elution in reverse phase enables sample stacking: • Large (1 - 20 µL) injection volumes are OK If we ran isocratically, a 75 µm ID column would require a 10 - 20 nL injection volume!

  22. Injection Strategies • On-column Injection (Pressure Bomb) • High sensitivity • Zero sample loss or waste • Time consuming (manual) • “Micro” Injection Valve • 0.1 - 5 µL • Easy to use • Sample Trapping • Faster injection of large volumes (5 - 20 µL) • Trap protects columns for increased lifetime • Some peptides lost during injection and analysis

  23. To Column Pressure Bomb Gas In Sample Vial Bomb Injection

  24. Fused Silica Column Sample Trapping • Trap Cartridge/Column • 100 - 500 µm ID • 1 - 25 mm in length • Typically C18 or SCX • Loading rate 1 - 20 µL/min • Enable hundreds/thousands of injections on an analytical column

  25. Sample Trapping Load Injection Loop

  26. Sample Trapping Load Sample Trap & Wash

  27. Sample Trapping Elute into Column

  28. How Do We Interface? • Liquid sheath for make-up flow (The Early Days) • Generally not used, compromised sensitivity • “Direct Connect” interface with fused-silica tip • No “make-up” or sheath liquid • Reasonable sensitivity • Plumbing can be a challenge • Integration of LC column with emitter • Highest sensitivity • Robust interface • Greater ease of use

  29. HV ZDV Metal Union Tip 5 - 30 µm HV Union PEEK or Teflon Distal Coating Direct Connect InterfaceJunction Contact

  30. SIC, 653.5 m/z SIC, 653.5 75 µm ID, C18 Distal Coated 10 µm PicoTip™ Water/CH3CN/Formic Acid 45 Minute gradient Micromass Q-TOF Base Peak, RIC Performance BenchmarkTryptic Digest of BSA - 125 fmol Data courtesy Art Moseley, GlaxoSmithKline

  31. Direct Connect InterfaceCommon Problems • Poor peak shape • Difficult post-column plumbing, requiring a “perfect” connection • Impractical with columns smaller than ≈75 µm • Clogged tips and columns • Difficult to distinguish point of plug - is it the column or the tip? • Air bubbles in line • Out-gassing, leaks, electrolysis, etc.

  32. 75 µm ID, C18 Frit Tip: 8 - 15 µm PicoFrit™ Packed Tip Performance Pack the LC column directly into the tip! “Zero” post column volume Emmett & Caprioli, J. Am. Soc. Mass. Spec. 1994, 5, 605-613

  33. HV Pt electrode PEEK “T” Packed C18 PicoFrit™ Packed Tip Approach • Junction style HV contact for robustness (arc immunity) • Junction can be far behind tip (10 cm or more) • Pre-column volume does not hurt chromatography

  34. PicoFrit™ ApproachAnalytical Advantages • Tip size optimal for column flow rate • Typically 8 -15 µm for 75 µm ID column • HV contact on inlet side of column • Minimal contribution to band broadening w/sample stacking • Eliminates air bubbles (high pressure side of column) • Robust and easy to use • Economical • Concurrent fabrication of tip and column

  35. Packed Tip AppraochAnalytical Advantages • Optimal sensitivity and resolution • Spray directly from column • Virtually zero post-column volume • Virtually eliminates tip clogging • Robust lifetime • 500+ injections/column with sample trapping • Easy to use • Fewer connections to make

  36. PicoFrit™ ColumnsPerformance Benchmark Data courtesy James P. Murphy III, Ph.D.

  37. PicoFrit™ ColumnsPerformance Benchmark

  38. Keys to Success

  39. Minimize Particle Contamination

  40. Minimize Particle Contamination Contaminated Column Head Clean Column Head • Mobile Phase Stocks • Change Stocks Regularly (weekly or better) • Use bottled water, preferrably distilled in glass • Avoid “ultrpure” meg-ohm water from in-house systems • These can contain high levels of carbon particulates Poor quality water is the primary cause of clogged columns!

  41. Minimize Particle Contamination • Fittings and Unions • Use PEEK or FEP adapter sleeves • Don’t over tighten fittings • Avoid graphitized ferrules (common in GC) • Discard contaminated fittings OUCH!

  42. Minimize Particle Contamination • Injection valves • Avoid “scribing” surface of rotor with fused-silica • Inspect surfaces often • Pump components • Inspect/replace seals, fittings, check valves and filters Watch out!

  43. Measuring Column Flow Rate • Let a droplet collect at tip for 5-10 minutes (ESI is off) • Collect the droplet by capillary action • Measure the volume and calculate flow rate

  44. Source Tuning: Go For the Best Spray 850V Stream and Plume 50% ACN, 0.1% Formic Acid 500 nL/min, 15 µm Picofrit™ tip, LCQ™ Deca XP Inlet

  45. 1150V Stream and Plume Source Tuning: Go For the Best Spray 850V Stream and Plume 50% ACN, 0.1% Formic Acid 500 nL/min, 15 µm Picofrit™ tip, LCQ™ Deca XP Inlet

  46. 1450V Good Plume Source Tuning: Go For the Best Spray 850V Stream and Plume 50% ACN, 0.1% Formic Acid 500 nL/min, 15 µm Picofrit™ tip, LCQ™ Deca XP Inlet

  47. 1850V Optimal Plume Source Tuning: Go For the Best Spray 850V Stream and Plume 50% ACN, 0.1% Formic Acid 500 nL/min, 15 µm Picofrit™ tip, LCQ™ Deca XP Inlet

  48. 2050V Split Plume Source Tuning: Go For the Best Spray 850V Stream and Plume 50% ACN, 0.1% Formic Acid 500 nL/min, 15 µm Picofrit™ tip, LCQ™ Deca XP Inlet

  49. Spray Morphology: Composition 5% ACN 50% ACN 95% ACN 1700V 1900V 2100V 2300V 2500V 30 µm Tip @ 500 nL/min 0.1% Formic Acid 3100V

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