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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

Practical High Sensitivity LC-MS

Fundamentals, Challenges, and Prospects

Gary A. Valaskovic, Ph.D.

New Objective, Inc.

main topics
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
anatomy of esi
Anatomy of ESI
  • Adapted from Kebarle & Tnag, Anal. Of Chem., 1993, 64, 972A
why use nanospray
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.

why use nanospray8
Why Use Nanospray?

There are three reasons to use Nanospray:

  • Sensitivity
  • Sensitivity
  • Sensitivity
  • Nanospray is one of the key technologies for MS-based Proteomics
how does nanospray yield sensitivity
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
static nanospray methodology
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

static nanospray extends analysis time
Static Nanospray Extends Analysis Time

Adapted From Corey & Pinto, Appl. Elec. Mass. Spec., Pramanik, Ganguly, Gross Eds.

static nanospray limitations
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 $$$
on line nanospray with nanobore lc
“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

why use nanospray lc

4.6 mm

50 µm

Why Use Nanospray LC?

Elute your sample into the smallest practical volume for the highest S/N!

why use nanobore lc
Why Use Nanobore LC?

The

Concentration Advantage!

Adapted From Tomer & Moseley, Mass. Spec. Rev., 1994, 13, 431

requirements for lc system
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
flow splitting methods
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, $$$
  • “Active” Mass Flow Control (buy)
    • Good performance, $$$
simple flow splitting
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
nanospray source requirements
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
on line nanospray source

Objective Lens

Tip Holder

HV Contact

CCD Camera

XYZ Stage

Injection Valve

On-line Nanospray Source

www.newobjective.com

what about sample injection
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!

injection strategies
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
bomb injection

To Column

Pressure Bomb

Gas In

Sample Vial

Bomb Injection
sample trapping

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
sample trapping26
Sample Trapping

Load Injection Loop

sample trapping27
Sample Trapping

Load Sample Trap & Wash

sample trapping28
Sample Trapping

Elute into Column

how do we interface
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
direct connect interface junction contact

HV

ZDV Metal Union

Tip

5 - 30 µm

HV

Union

PEEK or Teflon

Distal Coating

Direct Connect InterfaceJunction Contact
performance benchmark tryptic digest of bsa 125 fmol

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

direct connect interface common problems
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.
picofrit packed tip performance

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

picofrit packed tip approach

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
picofrit approach analytical advantages
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
packed tip appraoch analytical advantages
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
picofrit columns performance benchmark
PicoFrit™ ColumnsPerformance Benchmark

Data courtesy James P. Murphy III, Ph.D.

minimize particle contamination41
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!

minimize particle contamination42
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!

minimize particle contamination43
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!

measuring column flow rate
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
source tuning go for the best spray
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

source tuning go for the best spray46

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

source tuning go for the best spray47

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

source tuning go for the best spray48

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

source tuning go for the best spray49

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

spray morphology composition
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

source tuning challenges
Source Tuning: Challenges
  • Spray characteristics are sensitive to:
    • Emitter size, shape, distance
    • Flow rate
    • Voltage
    • Mobile phase composition
      • Optimal results require a changing voltage!
  • Bottom line: Tune your spray under “eluting conditions”
performance benchmarks
Performance Benchmarks
  • Cell mapping project at McGill University
  • Daniel Boismenu,
  • Montréal Network for Pharmaco-Proteomics and Structural Genomics
  • Exhaustive proteomic analysis of cell organelles
  • Determine elation between protein function and location

Total of 1350 1-D lanes for cell map:

93 slices per lane

Total of 125,550 slices

1 hour of HPLC-MS/MS per gel slice

5231 days of instrument time = 14 years

performance benchmarks robustness
Performance BenchmarksRobustness

Injection #31: Plasma membrane challenged with insulin.In gel digestion of slice no 30 of 64

75 µm x 10 cm C18 PicoFrit™ column, with 300 µm x 1 mm C18 Trap Cartridge on Micromass Q-TOF

Data courtesy Daniel Boismenu, McGill University

performance benchmarks robustness54
Performance BenchmarksRobustness

Injection #881: Smooth endoplasmic reticulum, aqueous phase.In gel digestion of slice no 45 of 92(Over 1 month of continuous, 24 hr, 7 days/week operation)

… and still going!

Data courtesy Daniel Boismenu, McGill University

keys to success with nanobore lc ms
Keys to Success with Nanobore LC-MS
  • Clean mobile phase
    • Minimize particulate contamination
    • Use multiple high quality in-line filters
  • Know your flow rate
    • Monitor through column flow periodically
  • Use the right injection scheme for your samples
  • Throughput vs. sensitivity
  • Minimize (or eliminate) post-column plumbing
    • Use special care with post-column connections
    • Use a tip-column (PicoFrit™) format
  • Optimize electrospray conditions
    • Stabilize spray with voltage
    • Maximize S/N with emitter position
    • Match tip size to flow rate