1. Detectors of HPLC

2. HPLC Bulk Property Detectors
Refractive Index Detector

3. HPLC Bulk Property Detectors
Refractive Index Detector
Plus:
1) Measures a bulk property
2) Nearly Universal (different RI than mobile phase)
3) Comparable response for different analytes
4) Detects species with no chromophores
Minus:
1) Temperature dependent
2) Poor sensitivity (LOD ≈ 100 ng)
3) No gradient elution

4. HPLC Bulk Property Detectors
Evaporative Light Scattering Detector
Solid state laser

5. HPLC Bulk Property Detectors
Evaporative Light Scattering Detector
Plus:
1) Measures a bulk property
2) Nearly Universal (must be non-volatile)
3) Does not detect liquids (gradients are ok)
4) Detects species with no chromophores
Minus:
1) Signal not linear with concentration
2) Fair sensitivity (LOD ≈ 1 ng)
3) No salts or buffers in mobile phase

6. HPLC Specific Property Detectors
Conventional UV-Vis Absorption Detector
Photodiode 1
Photodiode 2
Hg Pen
Lamp
254 nm

7. HPLC Specific Property Detectors
Multi-wavelength UV-Vis Absorption Detector
Deuterium
Lamp
Photodiode
Array

8. HPLC Specific Property Detectors
Multi-wavelength UV-Vis Absorption Detector

9. UV-Vis detection of sugars in juice

10. HPLC Specific Property Detectors
Multi-wavelength UV-Vis Absorption Detector
Plus:
1) Measures a specific property
2) Nearly Universal (must have chromophore)
3) Most common of all detectors (~75%)
4) Potential to provide qualitative info.
5) Simple, robust
Minus:
1) Fair sensitivity (LOD ≈ 1 ng)
2) Expensive with PDA
3) Misses some important analytes

11. HPLC Specific Property Detectors
Fluorescence Detector

12. HPLC Specific Property Detectors
Fluorescence Detector

14. HPLC Specific Property Detectors
Fluorescence Detector
Plus:
1) Measures a specific property
2) Highly Selective (must fluoresce)
3) Second most common of all detectors (~15%)
4) High sensitivity (LOD ≈ 0.01 ng)
5) Can interrogate very small volumes
Minus:
1) Not Universal
2) Limited Applications

15. HPLC Specific Property Detectors
Electrochemical Detector

16. HPLC Specific Property Detectors
Electrochemical Detector
Plus:
1) Measures a specific property
2) Highly Selective (depends on reduction potential)
3) High sensitivity (LOD ≈ 0.01 ng)
Minus:
1) Not Universal
2) Must have electrolyte in mobile phase
3) Mobile phase must be aqueous
4) Gradients not possible

17. What is Mass Spectrometry?
IUPAC Definition: The branch of science dealing with all aspects of mass spectrometers and the results obtained with these instruments.
Applications:
identification
Quantification
Molecular structure
higher-order structure (H/D exchange, cross-link)
tissue imaging

18. Mass Definitions
Molecular masses are measured in Daltons (Da) or mass units (u).
One Dalton = 1/12 of the mass of a 12C atom.
Monoisotopic mass = sum of the exact masses of the most abundant isotope of each element present, i.e., 1H= , 12C= , 16O= , etc.
This is the most accurately defined molecular mass and is preferred if a measurement of it can be determined.
Average mass = sum of the abundant averaged masses (“atomic weights”) of the constituent atoms of a given molecule.
The result is a weighted average over all of the naturally occurring isotopes present in the compound. This is the common chemical molecular weight that is used for stoichiometric calculations (H=1.0080, C=12.011, O=15.994, etc.). The average mass cannot be determined as accurately as the monoisotopic mass because of variations in natural isotopic abundances.
The mass to charge ratio (m/z). A quantity formed by dividing the mass (in u) of an ion by its charge number; unit: Thomson or Th. .

19. Ionization Techniques and Mass Analyzers
Mass Spectrometry
Ionization Techniques
and
Mass Analyzers

20. Mass Spectrometer Schematic
Vacuum envelope
Sample in
Inlet System
Ion Source
Mass Analyzer
Detector GC
HPLC
Direct injection
Inlet systems:
Data System CI EI
ESI
APCI
MALDI
Ion sources:
Mass spectrum out
Mass analyzers:
Time-of-flight (TOF)
Quadrupole
Ion trap
FT-ICR
Orbitrap

21. Ionization Techniques
Gas-Phase Methods
Electron Impact (EI)
Chemical Ionization (CI)
Desorption Methods
Matrix-Assisted Laser Desorption Ionization (MALDI)
Fast Atom Bombardment (FAB)
Spray Methods
Electrospray (ESI)
Atmospheric Pressure Chemical Ionization (APCI)

22. Electron Impact

23. Advantages
Well-Established
Fragmentation Libraries
No Supression
Insoluble Samples
Interface to GC
Non-Polar Samples
Disadvantages
Parent Identification
Need Volatile Sample
Need Thermal Stability
No Interface to LC
Low Mass Compounds (<1000 amu)
Solids Probe Requires Skilled Operator

24. Chemical Ionization

25. Chemical Ionization Advantages Parent Ion Interface to GC
Insoluble Samples
Disadvantages
No Fragment Library
Need Volatile Sample
Need Thermal Stability
Quantitation Difficult
Low Mass Compounds (<1000 amu)
Solids Probe Requires Skilled Operator

26. FAB

27. Advantages
Parent Ion
High Mass Compounds (10,000 amu)
Thermally Labile Compounds (R.T.)
Disadvantages
No Fragment Library
Solubility in Matrix (MNBA, Glycerol)
Quantitation Difficult
Needs Highly Skilled Operator
Relatively Low Sensitivity

28. MALDI

29. Matrix-Assisted Laser Desorption/Ionization (MALDI)
Analyte is dissolved in solution with excess matrix (>104).
Sample/matrix mixture is dried on a target and placed in the MS vacuum.
Requirements for a satisfactory matrix:
It must co-crystallize with typical analyte molecules
It must absorb radiation at the wavelength of the laser (usually 337 nm)
To transfer protons to the analyte it should be acidic
Typical successful matrices for UV MALDI are aromatic carboxylic acids.
Sinapinic acid
a-cyano-4-hydroxycinnamic acid (CHCA)
2,5-dihydroxybenzoic acid (DHB) HO
COOH OH
CH3O
CH=CH-COOH
CH=C-COOH CN

30. Matrix-assisted laser desorption ionization (MALDI)
Pulsed laser (337 nm)
3.5 ns
Sample stage
Desorbed sample ions and neutrals
Mass analyzer
Sample and matrix, crystallized on stage
±20 kV

31. MALDI Ionization Mechanism
1. Laser pulse produces matrix neutrals, + and - ions, and sample neutrals: M --> M*, MH+, (M-H)- (M= Matrix) - +
2. Sample molecules are ionized by gas-phase proton transfer:
MH+ + A --> AH+ + M (A=Analyte)
(M-H)- + A --> (A-H)- + M

32. MALDI Mass Spectrum of Protein Tryptic Digest
m/z

33. MALDI Advantages Parent Ion High Mass Compounds (>100,000 amu)
Thermally Labile Compounds (R.T.)
Easy to Operate
Disadvantages
No Fragment Library
Wide variety of matrices
Quantitation Difficult

34. Electrospray Ionization
0 kV
Sample in solution flows into capillary tube
Tube held at kV
Nitrogen flowing in outer tube aids nebulization

35. Electrospray ionization

36. Electrospray Ion Formation
Needle at High Voltage
MH+ + + + + + + + + + + + + + + + + + + + + + + + + + +
[M+3H]3+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
[M+2H]2+
Droplets formed in electric field have excess positive ions.
Evaporation of neutrals concentrates charge.
Droplets break into smaller droplets.
Eventually one molecule + n protons is left.

37. Nanospray Online analysis ~ 20 µm tip ID Interface with nanoLC
Flow rate: ~300nL/min
Offline analysis (static infusion)
~ 2 µm tip ID
Flow rate: ~40nL/min
Requires pure sample free from salt
New Objective, Inc.

38. ESI

39. ESI Advantages Parent Ion High Mass Compounds (>100,000 amu)
Thermally Labile Compounds (<0º C)
Easy to Operate
Interface to HPLC
Zeptomole sensitivity with nanospray
Disadvantages
No Fragmentation
Need Polar Sample
Need Solubility in Polar Solvent (MeOH, ACN, H2O, Acetone are best)
Sensitive to Salts
Supression

40. Electrospray MALDI Ionization Methods Online LC/MS possible
Poor for mixtures without LC
Quantitation possible
Good for MW <600
Generate highly charged ions
MALDI
Very long sample lifetime; repeated measurements possible
Good for mixtures
Matrix peaks can interfere at MW <600
Salt tolerant
Low maintenance
Generate ions with few charges

41. APCI

42. APCI Advantages Parent Ion Insensitive to Salts Interface to HPLC
Can use Normal Phase Solvents
Handles High Flow Rates
Disadvantages
Need Volatile Sample
Need Thermal Stability

43. Mass Analyzers Double Focusing Magnetic Sector Quadrupole Mass Filter
Quadrupole Ion Trap
Linear Time-of-Flight (TOF)
Reflectron TOF
Fourier Transform Ion Cyclotron Resonance (FT-ICR-MS)

44. Double-Focusing Magnetic Sector
Advantages
Very High Resolution (60,000)
High Accuracy (<5 ppm)
10,000 Mass Range
Disadvantages
Very Expensive
Requires Skilled Operator
Difficult to Interface to ESI
Low resolution MS/MS without multiple analyzers

45. Quadrupole Mass Analyzer/Filter
+ U + V cos wt
-U - V cos wt
Uses a combination of RF and DC voltages to operate as a mass filter.
Mass analyzer.
Mass selection device
Ion transport device (RF-only collision cell).
Mass scan and stability diagram
Randall E. Pedder
Extrel Application Note

46. Quadrupole Mass Filter
Advantages
Inexpensive
Easily Interfaced to Many Ionization Methods
Disadvantages
Low Resolution (<4000)
Low Accuracy (>100ppm)
MS/MS requires multiple analyzers
Low Mass Range (<4000)
Slow Scanning

47. Quadrupole Ion Trap 3D Trap
End caps
Ions out
to detector
Ring electrode (~V)
Insulated spacer
He gas
1x10-3 Torr
Ions in
(from ESI)
Uses a combination of DC and RF fields to trap ions
Ions are sequentially ejected by scanning the RF voltage
Linear Trap
Essentially a quadrupole with end-caps
Advantage: Larger ion storage capacity, leading to better dynamic range
Raymond E. March, JOURNAL OF MASS SPECTROMETRY, VOL. 32, 351È369 (1997)

48. Quadrupole Ion Trap Advantages Inexpensive
Easily Interfaced to Many Ionization Methods
MS/MS in one analyzer
Disadvantages
Low Resolution (<4000)
Low Accuracy (>100ppm)
Space Charging Causes Mass Shifts
Low Mass Range (<4000)
Slow Scanning

49. Time-of-Flight (TOF) Mass Analyzer
Source
Drift region (flight tube) + +
detector + + V
Ions formed in pulses.
Measures time for ions to reach the detector. or

50. Linear Time-of-Flight (TOF)
Advantages
Extremely High Mass Range (>1 MDa)
Fast Scanning
Disadvantages
Low Resolution (4000)
Low Accuracy (>200ppm)
MS/MS not possible

51. Reflectron Time-of-Flight (TOF)

52. Linear and Reflector TOF Analyzers
Reflector compensates for initial variation in kinetic energy, improving resolving power and mass accuracy.

53. Reflectron Time-of-Flight (TOF)
Advantages
High Resolution (>20,000 in some models)
High Accuracy (<5ppm)
10,000 Mass Range
Fast Scanning
Disadvantages
Low Resolution for MS/MS (PSD)

54. Fourier Transform Ion Cyclotron Resonance (FT-ICR)
Ions trapped and
measured in ultrahigh vacuum
inside a superconducting magnet.
Excite + R C
Detect + B0
A.G. Marshall

55. FT-ICR-MS Advantages Extremely High Resolution (>500,000)
Very Good Accuracy (<1 ppm)
MS/MS in one analyzer
Disadvantages
Expensive
Requires Superconducting Magnet
Slow MS/MS

56. Electron Multiplier Multi-Channel Plate (MCP)
From Detector Technolgy:
B. Brehm et al., Meas. Sci. Technol. 6 (1995)

57. Differential Amplifier
Fourier Transform Ion Detection
Differential Amplifier FT
100
150
200
250
Frequency (kHz) 7+ 8+
10+
11+
12+ 9+
600
1000
1400
1800
m/z
Calibration 80
240
400
Time (ms)
Image
Current
Bovine
Ubiquitin
1072
1071
A.G. Marshall

58. Orbitrap FTICR Confined ion trajectory Image current detection
Fourier transform data conversion
TOF
Simultaneous excitation
Unique to Orbitrap
3D electric field trapping
No need for magnet
Easy access
Final detection device
Alexander Makarov, Anal. Chem. 2000, 72,

59. Image Current Detection in Orbitrap
From Alexander Makarov’s 2008 ASMS Award Address

60. Comparison of Analyzer Types
Ion Trap/
Quadrupole
TOF
OrbiTrap
FT-ICR
Sensitivity
+++
++* to +++
++* +*
Mass Accuracy
+** ++
+++**
Resolving Power
++++**
Dynamic Range
+ to +++**
++**
Upper m/z +
++++
*Sensitivity lowered due to losing ions on way to analyzer, rather than inherent sensitivity.
**Can be improved by scanning narrower mass range or slower.

61. Hybrid/Tandem Instruments
Combine (1) ion selection, (2) ion dissociation, and (3) mass analyzer devices
Quadrupoles and ion traps good for selective isolation of precursor ions and for fragmentation (required for MSMS - Topic of Lecture 2)
Reflectron TOF, FT-ICR, and OrbiTrap have higher mass accuracy and resolving power (high mass accuracy is good for identification – Lecture 3)

62. Ion Isolation Quadupole Continuous ion beam Quadrupole ion trap
Pulsed-mode operation; space charge issue
SWIFT in FTICR
Ultrahigh selectivity; only works well in ICR traps
TOF
Only implemented on TOF/TOF

63. Ion Dissociation
Collision Induced Dissociation (CID or Collision Activated Dissociation (CAD)
ion traps: off-resonance excitation
rf-only multi-poles: higher kinetic energy (HCD) and cascaded CID
TOF/TOF: single collision
Electron capture dissociation (ECD) and Electron transfer dissociation (ETD)
ECD: FTICR, reagent: electron
ETD: ion traps, reagent: free radical anion
Other important factors to consider: how product ions are collected and detected

64. MALDI-TOF/TOF (4700 Proteomics Analyzer)
250 l/s turbopump
Collision gas
2nd source (accelerates fragment ions)
Ion gate
Multiplier detectors
Reflectron
Floating collision cell
N2 laser
Retarding lens
Beam 1 lens & x/y deflectors
Beam 2 lens, metastable suppressor & x/y deflectors
High performance TOF analysis for MS1 and MS2 give high resolving power and good mass accuracy.
High accelerating voltage allows high energy CID, giving a wider range of fragment ions and facilitating side-chain cleavages that distinguish isomeric amino acids Ile and Leu.
Marvin L. Vestal and Jennifer M. Campbell, Methods Enzymol ;402 :79-108

65. Hybrid Instrument: QqTOF Mass Spectrometer (QSTAR)
5x10-7
Torr
Liner
Ion mirror
Ion detector
Pusher
Puller Q1 q2
MS1 mass selection
CID
Ion focus & transfer q0 N2
ESI
spray tip
Roughing pump
Turbo
pump
High
vacuum
Collision gas
MS2 mass analysis
Ion formation
Chernushevich, Loboda and Thomson, J. Mass Spectrom. 2001; 36: 849–865

66. Linear Ion Trap – FT-ICR (LTQ-FT)
Linear Ion Trap (LTQ)
Ion Cyclotron
John E. P. Syka, et al, Journal of Proteome Research 2004, 3,

67. Data Dependent Acquisition
Data Dependent Scans
MSMS based on intensity ranking of precursor ions
Dynamic Exclusion
Precursor m/z of previous MSMS are memorized and no MSMS done on them during a defined time period
Automatic Gain Control (AGC, unique to ion trap)
Control how many ions are scanned – to achieve signal/noise ratio and to minimize space charge effect

68. Scan Sequence of LTQFT
Thermo Application Note: 30046

69. Linear Ion Trap - Orbitrap - ETD
Alexander Makarov, Stevan Horning, et al, Anal. Chem. 2006, 78,
G. C. McAlister, et al, J Proteome Res. 2008, 7,

70. Linear quadrupole ion trap (LTQ) video clip
J. Schwartz, et al, JASMS2002v13p659