detectors of hplc. hplc bulk property detectors refractive index detector

$: Detectors of HPLC. HPLC Bulk Property Detectors Refractive Index Detector$
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


Evaporative Light Scattering Detector

Solid state laser


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 Specific Property Detectors

Conventional UV-Vis Absorption Detector

Hg PenLamp

254 nm

Photodiode 2

Photodiode 1


Multi-wavelength UV-Vis Absorption Detector

DeuteriumLamp

PhotodiodeArray

UV-Vis detection of sugars in juice



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


Fluorescence Detector


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


Electrochemical Detector


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

17

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

18

.

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

20

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.)

29

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

31

-

+-

++

++

--

--

--

-

--

--

-

-

- ++ +

+ +

+

++

+

++

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

m/z

Disadvantages No Fragment Library Wide variety of

matrices Quantitation Difficult


(>100,000 amu) Thermally Labile

Compounds (R.T.) Easy to Operate

34

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

+

+

+++

+++ +

++

+ +++

+

37

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


(>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

45

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

47

•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

49

+

+

+

+

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

54

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

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

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

65

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

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

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

detectors of hplc. hplc bulk property detectors refractive index detector

Documents

detectors of hplc slide

juice slide

tissue imaging slide

average mass

fair sensitivity lod

high sensitivity lod

mass units

monoisotopic mass