Mass Spectrometry and Its Applications

Goals:

1) Understanding basic principle of mass spectrometry.

2) Knowing its important applications

3) As a training tool for future research

4) As an analytical skill for chemical analysis.

Grading: One exam for understanding basics of MS & its applications (50) & Research Reports (80). But the final score can not be more than100.

A Few Reference Books• Mass Spectrometry Basics; Chris. G. Herbert and Robert A. W.

Johnstone• Mass Spectrometry: Principles and Applications; Edmond de

Hoffmann & Vincent Stroobant• The Expanding Role of Mass Spectrometry in Biotechnology; Gary

Siuzdak• Biomedical Applications of Mass Spectrometry; Clarence H. Suelter &

J. Throck Watson• Time-of-Flight Mass Spectrometry; Bob J Cotter• Electrospray Ionization Mass Spectrometry; Richard B. Cole• Quadrupole Ion Trap Mass Spectrometry; Raymond E. March & John

F. J. Todd• Liquid Chromatography-Mass Spectrometry:An Introduction; Bob

Ardrey• Protein Sequencing and Identification Using Tandem Mass

Spectrometry; Michael Kinter & Nicholas Sherman• Mass Spectrometry in the Biological Sciences; edited by A. L.

Burlingame & Steven A. Carr• It is not essential to have any of the above books. Hoffmann’s book is

available in local bookstores

Brief Bio-sketch of Tutor • Name: 陳仲瑄• School: 1969,BS; Chemistry Dept. NTU

1974, Ph.D University of Chicago• Working Experience: 1974 ~ 2005 Oak Ridge National

Laboratory; 2005 ~ Now; GRC, Sinica. Adjunct Professor: Vanderbilt University; University of Tennessee; Knoxville; Honorary Professor: Beijing Tsing Hwa University

• Possible Advantages: Experience in many different research fields. Willing to learn from students. Eager to make friends with young people.

• Disadvantages: No much teaching experience; Not at NTU all the time; No Chinese word processing skill; Possible Generation Gap (?) and etc.

陳妍秀 吳尚臻

劉子晨 次家祺

陳映伃 許健明

王彥之 許齡尹

王南絢 李宛俐

陳淑婷 (?) 沈尚昱

李治平 余明龍

林芝毓 許旭辰

趙景豪 沈錦昌

劉已維 藍永翔

李威漢 李孟倫

黃如立

Outline on Mass Spectrometry Course

Chung-Hsuan ChenI. Introduction:

I.a Basic of Mass SpectrometryI.b Major Applications of Mass SpectrometryI.c Vacuum Science and Technology

II. Ionization: II.a Electron IonizationII.b Chemical IonizationII.c PhotoionizationII.d Matrix-assisted Laser Desorption/IonizationII.e Electrospray IonizationII.f Other Ionization methods: FAB, Field Ionization, SIMS etc.II.g Major challenges for Future Development

III. Mass Analysis:III.a Quadrupole III.b Ion TrapIII.c Time-of-flightIII.d Magnetic SectorIII.e FTICRIII.f Major Challenges for Future Development

Outline of MS Course (continued)IV Detection:

IV.a Charge CollectorIV.b Charge Amplifier DetectorIV.c Scientillator DetectorIV.d Other Detectors and Major Barriers to

overome.V. Tandem Mass Spectrometry

V.a Basic of MS/MSV.b Collision induced DissociationV.c Ion-Molecule Interaction

VI Chromatography/Mass Spectrometer CouplingVI.a GC/MSVI.b LC/MSVI.c CE/MS

Outline of MS Course (continued)VII Biomolecule Analysis:

VII.a Oligonucleotide, Protein, Polysaccharide AnalysisVII.b DNA Analysis and SequencingVII.c Proteomic & Metabolomic ApplicationsVII.d Disease Diagnosis

VIII Other Applications:

VIII.a Trace Element AnalysisVIII.b Organic Polymer AnalysisVIII.c Environmental ApplicationVIII.d Material Analysis

IX Future Challenges:

IX.a Ionization MechanismIX.b Mass to Charge Ratio ExtensionIX.c Quantitative MeasurementsIX.d Dynamic Range ExtensionIX.e Cost ReductionIX.f Size Reduction

X. Conclusion:

What can a Mass Spectrometer do?

1) A mass spectrometer (MS) is a device which aims to weigh atoms, molecules, cluster, nano-particle, virus, cell and etc. In general, it can only determine mass.

2) Indeed, present mass spectrometers can not even measure mass directly, it can only measure mass-to-charge ratio (M/Z) for a particle in gas phase. For most mass spectrometers, Z is equal to 1 so that mass can be determined.

Essential Parts of a MS• Ionizer: A MS can only measure M/Z of an

ion so that ionization process is necessary. Electron ionization, chemical ionization and photoionization are among examples.

• M/Z Separator: Magnetic Sector, Time-of-Flight, quadrupole, ion trap

• Detector: charged particle detectors such as channeltron, electromultiplier and microchannel plates.

Schematic of Mass Spectrometry

Ionizer

↓Mass-to-charge ratio Analyzer

↓

Detector

Mass Spectrometry Categorization• Based on ionization:

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometryElectrospray ionization (ESI) mass spectrometryInduction-coupled plasma mass spectrometry (ICP-MS)Atmospheric Ionization Mass SpectrometrySecondary Ionization Mass Spectrometry (SIMS)

• Based on M/Z Separation:Quadrupole Mass spectrometryIon trap mass spectrometrymagnetic sector mass spectrometrytime-of-flight mass spectrometryFourier Transform Ion Cyclotron Resonance Mass SpectrometryIon Mobility Mass Spectrometry

Mass Spectrometry Categorization(continued)

• Based on Applications:Environmental Mass SpectrometryBiological Mass SpectrometryCell Mass SpectrometryPortable Mass Spectrometry

• Based on Configuration:Tandem Mass Spectrometry

• Based on Sample Introduction:GCMS; LCMS, Electrophoresis Mass Spectrometry

MS Operation• Nearly all mass spectrometers need to operate

under high vacuum condition with the pressure less than 10 -5 Torr with the only exceptions of an ion trap mass spectrometer (milli-Torr) and an ion mobility mass spectrometer (Torr).

• Never turn on a mass spectrometer without knowing the chamber pressure.

• A tour to major mass spectrometry facilities in Genomic Research Center, Sinica will be arranged.

Mass Ranges for Different Species One atomic mass unit (A.M.U or u) = 1 Dalton (Da) = 1.66 x

10-24 gAtom: < 300 DaSmall Molecule: 2 ~ 2,000 Da.Protein: 500 ~ 10,000,000 DaHuman Chromosome: 109 ~ 1011 DaHuman Genome: 2 x 1012 DaMicrobial Genome: 109 ~ 1010 DaNanoparticle: 104 ~ 1012 DaMicroparticle: 1011 ~ 1022 DaVirus: ~ 109 DaCell: 1013 ~ 1015 Da

Isotope Concern

• Average Mass: a value based on the stoichimetric calculation of various isotope. Cl is considered as 35.45 Daltons

• Monoisotopic mass: Molecular weight based on each individual isotope.

• A mass spectrometer with high enough mass resolution should measure monoisotopic masses not average mass. For example mass spectrum for Cl2 should be obtained with three peaks as 35Cl2, 37Cl2 and 35Cl37Cl.

• Since the natural abundances of each isotope is known, multiple peaks from the same compound can provide additional information for molecule determination.

Key Parameters in MS

• Mass Accuracy: Internal calibration is often needed for high mass accuracy

• Mass Resolution: M/ΔM• Detection Sensitivity: Ieff x Teff x Deff

• Dynamic Range: wish to have 8 orders of magnitude

• Reproducibility: poor reproducibility mostly comes from the ionization process

• Quantitative Measurement: Up to now, MS is not very good for quantitative measurement.

MS Applications (non-biomedical)

• Pollutant Analysis

• Trace Metal Analysis

• Explosive Analysis

• Illegal Drug Detection

• Alcohol Analysis

• Organic Chemical Analysis

• Inorganic Chemical Analysis

MS for Biomedical Applications

• Proteomic Analysis• DNA sequencing• DNA fingerprinting for Forensic Applications• Biomolecule structure analysis• Polysaccharide Analysis• Metabolomic Analysis & Pharmacological

Applications• Disease Diagnosis

Basic Vacuum Technology for MS

• Gas Phase Collision Kinetics:Gas Phase collision can destroy existing ions and /or produce new ions to make MS analysis difficult. Electronics can be unstable when gas pressure is too high.

• Vacuum Technology:“Good” vacuum condition is generally required for the operation of a mass spectrometer. Basic knowledge on vacuum technology becomes essential.

Vacuum Category

Gas Phase Collision Kinetics

• Collision frequency (ν) to a surface is proportional to gas number density and mean velocity of gas molecules.

ν= ¼ nvave = n (kT/2πm)1/2

At room temperature (300 K) and 1 atmosphere of air, the collision frequency is ~ 2.4 x 1023 /cm2 sec which is equivalent to the gas pumping speed of 12 liter per second per square centimeter. The design of outlet pumping flange should allow the maximum use of the pumping speed of the pump.

Leak & Outgas Concern

• IF there is a leak of 0.01 mm at 1 atmosphere, the leak rate is about

1.2 x10-3 liter-Atm/sec. If the vacuum is at 10-5 Torr, it needs the pumping speed of ~100,000 liter/sec which is impossible. Thus, no leaks are allowed for any high vacuum and ultra-high vacuum chamber. Although outgas rate is usually much smaller, any material with high outgas rate can not be allowed in the ultra-high vacuum chamber either.

Differential Pumping

• For certain mass spectrometers, carrier gas needs to be leaked into the chamber to carry the sample for analysis, differential pumping becomes essential.

• Addition of differential pumping often adds the size, cost and alignment difficulty.

Mean Free Path

Mean Free Path (L) can be estimated as

L = vaver/ fcollision = kT/(20.5 πσ2 P) ~ 0.005/P

P: Torr, T: 300K, Ion interaction cross section: 0.7 nm. For large biomolecular ion,σ can be much larger. In general, L is preferred to be longer than 1 m, pressure needs to be at least 5 x 10 -5 Torr.

Vacuum Pump

• Rough Pump (From atmosphere to milli-Torr):a) Mechanical Rotatory Pump: 20 to 250 liter/sec; Advantage: rugged, convenient and relatively inexpensive; Disadvantage: Oil contaminationb) Cryosorption Pump (Liquid nitrogen & activated charcoal) Advantage:oil free. Disadvantage: Liquid nitrogen is needed and activated charcoal needs to be replaced. c) All high vacuum pumps need to be backed by a rough pump to pump the chamber down to milli-Torr region.

Vacuum Pump (continued 1)

• Diffusion Pump:Range: 10-3 ~ 10-9 TorrOil jet to carry residual gas down for pumpingSpeed: 50 ~ 600 liters/ secAdvantages: Fast Pumping speedDisadvantages: Heating, cold trap and cooling are often required. If electricity is discontinued, it can become a disaster for the entire experiment.

* Diffusion pump is gradually replaced by ion pump and turbo pump.

Vacuum Pump ( continued)• Ionization Pump:

(a) An electronic discharge is set up between anode and cathode plates of which the cathode is fabricated of titanium or tantalum material.(b) The discharge is confined by a magnetic field, which causes the emitted electrons to travel in a long spiral path, under acceleration of 2000 ~ 5000 volts.© Electron collision with residual gas molecules to produce ionization.(d) Ions colliding with cathode to produce clean surface for further reactions to produce oxide or nitride to remove the residual gas.(e) Ion pump only works efficiently under ultra-high vacuum.(f) Ion pump is not efficient in pumping rare gas atoms such as He and Ar.

Vacuum Pump (continued)• Sublimation Pump:

(a) Titanium is vaporized to deposit on the wall. Such deposit of fresh titanium have a great chemical affinity for all of active gases, including hydrogen,nitrogen and oxygen.

(b) Sublimation pump has very high pumping speed under ultra-high vacuum.

© Sublimation pump does not work well for rare gas

Vacuum Pump (continued)

• Turbomolecular Pump:(a) This pump makes use of a multiple-stage high-speed rotator consisting of a series of flat disks rotating between fixed disks which accelerate gas molecules by a collision method and utilize the centrifugal effect to compress these gas molecules. Such pumps operate at a very high rotating speed (>= 24000 rpm).(b) Pumping speed is a couple of hundred liters per second.© Advantages: no oil and no heating. Suitable for ultra-high vacuum(d) Disadvantages: Expensive but privices have come down. Most modern and expensive mass spectrometers are using turbo pump.

Turbomolecular Pump (continued)

Most turbomolecular pumps employ multiple stages consisting of rotor,stator pairs mounted in series. Gas captured by the upper stages is pushed into the lower stages and successively compressed to the level of the fore-vacuum pressure. As the gas molecules enter through the inlet, the rotor,which has a number of angled blades, impacts on the molecules. Thus the mechanical energy of the blades is transferred to the gas molecules. With this newly acquired momentum, the gas molecules enter into the gas transfer holes in the stator. This leads them to the next stage where they again collide with the rotor surface, and this process is continued, finally leading them outwards through the exhaust. Turbomolecular pump is less efficient for very light molecules such as hydrogen and helium

Vacuum Pump (continued)

• Getter Pump:

Similar to Sublimation pump except only getter process. This pump can be used for pumping without any external electricity. The pumping speed is very low. Nevertheless, it can be used for environmental mass spectrometers in field use.

Vacuum Gauge

• Thermal Couple Gauge:

Pressure Range: 1 Torr ~ 0.001 Torr

• Ionization Gauge:

Pressure Range: 10-3 ~ 10-12 Torr

• Mechanical Pressure Gauge: >1 Torr

• MKS Baratron: Precision Pressure Gauge

Mechanical Pressure Gauge

Most pressure gauges are diaphragm seal nowadays

Thermal Couple Gauge

The thermal conductivity for air is nearly constant down to 1 Torr. Then it begins to change linearly with pressure down to 1 mTorr. T/C gauge is based on thermal conductivity. It is a gas species dependent gauge

Ionization Gauge

"Ionization Gauge is based on the ionization measurement. Thus it is gas species dependent.

Leak Detector• Helium Leak Detector:

It is a simple mass spectrometer for measure helium. External helium spray is needed.

• Portable or Hand-held leak detectors: Based on conductivity or discharge such as SF6 as a carrier gas instead of He.

Sealing Materials

• Rubber Gasket (Viton O-Ring):

High Vacuum

* Copper Gasket: Ultra-high vacuum

Flow of Gas• Viscous Flow (short mean free path):

C (conductance) = 5.68 (a4/L)(P2 + P1)a: radius of a tube; L: Tube length

P1: Initial Pressure; P2: Final Pressure• Molecular Flow (long mean free path)

C = 2.64 KA (T/M)1/2; A: area of tube; K is a function of tube length and geometry

* Transition Flow: Difficult to estimate

Conductance Estimate

Assuming a pipe connecting a pump to a chamber to be evacuated:

1/S = 1/Cpump + 1/Cpipe

If Cpump = Cpipe, Conductance is reduced by half. When a small pipe is connected between a chamber, a large pump will not help much. Try your best not to use long and thin pipe for vacuum system.

Pumping Down Time

• Viscous Flow:

t (sec) = V/S Ln (P1 / P2);V: Volume of the system, S: Net pumping speed,P1: initial pressure,P2 : Final Pressure.

* Molecular Flow: Controlled by outgas & leak. t (hour) = Q/S; Q is overall ourgas & leak rate; S is pumping speed at final pressure.

Summary

• Basic Concept of Mass Spectrometry:Ionization Mass Analyzer Detection

• Potential Applications:Chemical Analysis; Disease Diagnosis;Genomic, Proteomic and Metabolomic Analysis

• Vacuum Technology:Gas Kinetics & Vacuum Instrumentation