HPLC Bulk Property Detectors Refractive Index Detector
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
HPLC Bulk Property Detectors Evaporative Light Scattering Detector Solid state laser
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
HPLC Specific Property Detectors Conventional UV-Vis Absorption Detector Photodiode 1 Photodiode 2 Hg Pen Lamp 254 nm
HPLC Specific Property Detectors Multi-wavelength UV-Vis Absorption Detector Deuterium Lamp Photodiode Array
HPLC Specific Property Detectors Multi-wavelength UV-Vis Absorption Detector
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
HPLC Specific Property Detectors Fluorescence Detector
HPLC Specific Property Detectors Fluorescence Detector
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
HPLC Specific Property Detectors Electrochemical Detector
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
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
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=1.007825, 12C=12.000000, 16O=15.994915, 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. .
Mass Spectrometry Ionization Techniques and Mass Analyzers
Mass Spectrometer Schematic Vacuum envelope Sample in InletSystem 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
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)
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
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
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
Matrix-Assisted Laser Desorption/Ionization (MALDI) OH CH3O CN HO CH=CH-COOH HO COOH CH=C-COOH HO CH3O a-cyano-4-hydroxycinnamic acid (CHCA) 2,5-dihydroxybenzoic acid (DHB) Sinapinic acid • 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.
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
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
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
Electrospray Ionization 0 kV Sample in solution flows into capillary tube Tube held at +1- 5 kV Nitrogen flowing in outer tubeaids nebulization
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.
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.
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
Ionization Methods • Electrospray • 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
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
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)
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
Quadrupole Mass Analyzer/Filter -U - V coswt + U + V coswt • 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
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
Quadrupole Ion Trap 3D Trap End caps Ions out to detector He gas 1x10-3 Torr Insulated spacer Ring electrode (~V) 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)
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
Time-of-Flight (TOF) Mass Analyzer or Source Drift region (flight tube) + + detector + + V • Ions formed in pulses. • Measures time for ions to reach the detector.
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