detectors of hplc. hplc bulk property detectors refractive index detector
TRANSCRIPT
Detectors of HPLC
HPLC Bulk Property Detectors
Refractive Index Detector
Refractive Index Detector
Plus:1) Measures a bulk property2) Nearly Universal (different RI than mobile phase)3) Comparable response for different analytes4) Detects species with no chromophores
Minus:1) Temperature dependent2) Poor sensitivity (LOD ≈ 100 ng)3) No gradient elution
HPLC Bulk Property Detectors
Evaporative Light Scattering Detector
Solid state laser
HPLC Bulk Property Detectors
Plus:1) Measures a bulk property2) 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 concentration2) Fair sensitivity (LOD ≈ 1 ng)3) No salts or buffers in mobile phase
Evaporative Light Scattering Detector
HPLC Bulk Property Detectors
HPLC Specific Property Detectors
Conventional UV-Vis Absorption Detector
Hg PenLamp
254 nm
Photodiode 2
Photodiode 1
HPLC Specific Property Detectors
Multi-wavelength UV-Vis Absorption Detector
DeuteriumLamp
PhotodiodeArray
HPLC Specific Property Detectors
Multi-wavelength UV-Vis Absorption Detector
UV-Vis detection of sugars in juice
HPLC Specific Property Detectors
Multi-wavelength UV-Vis Absorption Detector
Plus:1) Measures a specific property2) 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 PDA3) Misses some important analytes
HPLC Specific Property Detectors
Fluorescence Detector
HPLC Specific Property Detectors
Fluorescence Detector
HPLC Specific Property Detectors
Plus:1) Measures a specific property2) 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 Universal2) Limited Applications
Fluorescence Detector
HPLC Specific Property Detectors
Electrochemical Detector
HPLC Specific Property Detectors
Plus:1) Measures a specific property2) Highly Selective (depends on reduction potential)3) High sensitivity (LOD ≈ 0.01 ng)
Minus:1) Not Universal2) Must have electrolyte in mobile phase3) Mobile phase must be aqueous4) Gradients not possible
Electrochemical Detector
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IUPAC Definition: The branch of science dealing with all aspects of mass spectrometers and the results obtained with these instruments.
Applications:
1. identification
2. Quantification
3. Molecular structure
4. higher-order structure (H/D exchange, cross-link)
5. tissue imaging
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.
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.
Ionization Techniques and
Mass Analyzers
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Data System
Mass spectrum
out
Inlet System
Mass Analyzer
Ion Source
Detector
Vacuum envelope
Sample in
Inlet systems: •GC•HPLC •Direct injection
Ion sources:
Mass analyzers:
•CI•EI•ESI•APCI•MALDI
•Time-of-flight (TOF)•Quadrupole•Ion trap•FT-ICR•Orbitrap
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)
Disadvantages Parent Identification Need Volatile Sample Need Thermal
Stability No Interface to LC Low Mass Compounds
(<1000 amu) Solids Probe Requires
Skilled Operator
Advantages Well-Established Fragmentation
Libraries No Supression Insoluble Samples Interface to GC Non-Polar 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 Interface to GC Insoluble Samples
Disadvantages No Fragment Library Solubility in Matrix
(MNBA, Glycerol) Quantitation Difficult Needs Highly Skilled
Operator Relatively Low
Sensitivity
Advantages Parent Ion High Mass Compounds
(10,000 amu) Thermally Labile
Compounds (R.T.)
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Sinapinic acid -cyano-4-hydroxycinnamic acid (CHCA) 2,5-dihydroxybenzoic acid (DHB)HO
COOH
OHCH3O
CH3O
HO CH=CH-COOH HO CH=C-COOH
CN
•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)
30
±20 kV
Sample and matrix, crystallized on stage
Desorbed sample ions and neutrals
Pulsed laser (337 nm)
3.5 ns
Sample stage
Mass analyzer
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-
+-
++
++
--
--
--
-
--
--
-
-
- ++ +
+ +
+
++
+
++
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
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m/z
Disadvantages No Fragment Library Wide variety of
matrices Quantitation Difficult
Advantages Parent Ion High Mass Compounds
(>100,000 amu) Thermally Labile
Compounds (R.T.) Easy to Operate
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Sample in solution flows into capillary
tube
Nitrogen flowing in outer tubeaids
nebulization
Tube held at +1- 5 kV
0 kV
35
36
++
++
+
+
++
+ +++
++
+ +++ +
++
+++
+++
++
+
+
++
++
+
+++
+++
+++
MH+
[M+2H]2+
[M+3H]3+
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.
+
+
+
++++++
++++
Needle at High Voltage
+
+
+++
+++ +
++
+ +++
+
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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 saltNew Objective, Inc.
Disadvantages No Fragmentation Need Polar Sample Need Solubility in
Polar Solvent (MeOH, ACN, H2O, Acetone are best)
Sensitive to Salts Supression
Advantages Parent Ion High Mass Compounds
(>100,000 amu) Thermally Labile
Compounds (<0º C) Easy to Operate Interface to HPLC Zeptomole sensitivity
with nanospray
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
Electrospray•Online LC/MS possible
•Poor for mixtures without LC
•Quantitation possible
•Good for MW <600
•Generate highly charged ions
Ionization MethodsIonization Methods
Disadvantages Need Volatile Sample Need Thermal
Stability
Advantages Parent Ion Insensitive to Salts Interface to HPLC Can use Normal Phase
Solvents Handles High Flow
Rates
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)
Disadvantages Very Expensive Requires Skilled
Operator Difficult to Interface to
ESI Low resolution MS/MS
without multiple analyzers
Advantages Very High Resolution
(60,000) High Accuracy (<5
ppm) 10,000 Mass Range
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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).
+ U + V cos t
-U - V cos t
Randall E. Pedder Extrel Application Note
Mass scan and stability diagram
Disadvantages Low Resolution
(<4000) Low Accuracy
(>100ppm) MS/MS requires
multiple analyzers Low Mass Range
(<4000) Slow Scanning
Advantages Inexpensive Easily Interfaced to
Many Ionization Methods
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•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
Ions in(from ESI)
3D TrapEnd caps
Ions outto detector
Ring electrode (~V)
Insulated spacer
He gas1x10-3 Torr
Raymond E. March, JOURNAL OF MASS SPECTROMETRY, VOL. 32, 351È369 (1997)
Disadvantages Low Resolution
(<4000) Low Accuracy
(>100ppm) Space Charging
Causes Mass Shifts Low Mass Range
(<4000) Slow Scanning
Advantages Inexpensive Easily Interfaced to
Many Ionization Methods
MS/MS in one analyzer
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+
+
+
+
Source Drift region (flight tube)
dete
ctor
V
•Ions formed in pulses.
•Measures time for ions to reach the detector.
2
22
L
Vtzm or zmt
Disadvantages Low Resolution (4000) Low Accuracy
(>200ppm) MS/MS not possible
Advantages Extremely High Mass
Range (>1 MDa) Fast Scanning
52
Reflector compensates for initial variation in kinetic energy, improving resolving power and mass accuracy.
Disadvantages Low Resolution for
MS/MS (PSD)
Advantages High Resolution
(>20,000 in some models)
High Accuracy (<5ppm)
10,000 Mass Range Fast Scanning
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B0
Detect++
++
+++
+
+
R C
Excite
+++ + ++
+++
Fourier Transform Ion Cyclotron Resonance (FT-ICR)
•Ions trapped and measured in ultrahigh vacuum inside a superconducting magnet.
A.G. Marshall
zm
1
Disadvantages Expensive Requires
Superconducting Magnet
Slow MS/MS
Advantages Extremely High
Resolution (>500,000) Very Good Accuracy
(<1 ppm) MS/MS in one analyzer
56From Detector Technolgy: http://www.detechinc.com/B. Brehm et al., Meas. Sci. Technol. 6 (1995) 953-958.
Multi-Channel Plate (MCP)
57
Differential Amplifier
FT
100 150 200 250Frequency (kHz)
7+
8+
10+
11+
12+
9+
600 1000 1400 1800
12+
11+
10+
9+
8+
7+
m/z
Calibration
0
80 240 400Time (ms)
ImageCurrent
BovineUbiquitin
10721071
Fourier Transform Ion Detection
A.G. Marshall
58Alexander Makarov, Anal. Chem. 2000, 72, 1156-1162
Orbitrap
TOF•Simultaneous excitation
FTICR•Confined ion trajectory•Image current detection•Fourier transform data conversion
Unique to Orbitrap•3D electric field trapping•No need for magnet•Easy access•Final detection device
59From Alexander Makarov’s 2008 ASMS Award Address
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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.
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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)
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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
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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
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250 l/s turbopump
250 l/s turbopump
250 l/s turbopump
Collision gas
2nd source (accelerates
fragment ions)
Ion gate
Multiplier detectors
Reflectron
Floating collision cellN2 laser
Retarding lens
Beam 1 lens & x/y deflectors
Beam 2 lens, metastable
suppressor & x/y deflectors
MALDI-TOF/TOF (4700 Proteomics Analyzer)
• 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. 2005 ;402 :79-108
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5x10-7
Torr
Liner
Ion mirror
Ion detector
PusherPuller
Q1 q2
MS1 mass selection CID
Ion focus & transfer
q0
N2
ESIspray tip
Roughing pump
Turbopump
Highvacuum
Highvacuum
Highvacuum
Collision gas
MS2 mass analysis
Ion formation
Hybrid Instrument: QqTOF Mass Spectrometer (QSTAR)
Chernushevich, Loboda and Thomson, J. Mass Spectrom. 2001; 36: 849–865
66
Linear Ion Trap (LTQ) Ion Cyclotron
Linear Ion Trap – FT-ICR (LTQ-FT)
John E. P. Syka, et al, Journal of Proteome Research 2004, 3, 621-62
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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
Alexander Makarov, Stevan Horning, et al, Anal. Chem. 2006, 78, 2113-2120
G. C. McAlister, et al, J Proteome Res. 2008, 7, 3127-36
70J. Schwartz, et al, JASMS2002v13p659
Linear quadrupole ion trap (LTQ) video clip
File_146.exe