đại cương về khối phổ

Phương pháp khối phổ (tiếng Anh: Mass spectrometry MS) là một kĩ thuật dùng để đo đạc tỉ lệ khối lượng trên điện tích của ion; dùng thiết bị chuyên dụng là khối phổ kế. Kĩ thuật này có nhiều ứng dụng, bao gồm: Xác định các hợp chất chưa biết bằng cách dựa vào khối lượng của phân tử hợp chất hay từng phần tách riêng của nó Xác định kết cấu chất đồng vị của các thành phần trong hợp chất Xác định cấu trúc của một hợp chất bằng cách quan sát từng phần tách riêng của nó Định lượng lượng hợp chất trong một mẫu dùng các phương pháp khác (phương pháp phổ khối vốn không phải là định lượng) Nghiên cứu cơ sở của hóa học ion thể khí (ngành hóa học về ion và chất trung tính trong chân không) Xác định các thuộc tính vật lí, hóa học hay ngay cả sinh học của hợp chất với nhiều hướng tiếp cận khác nhau. Một khối phổ kế là một thiết bị dùng cho phương pháp phổ khối, cho ra phổ khối lượng của một mẫu để tìm ra thành phần của nó. Có thể ion hóa mẫu và tách các ion của nó với các khối lượng khác nhau và lưu lại thông tin dựa vào việc đo đạc cường độ dòng ion. Một khối phổ kế thông thường gồm 3 phần: phần nguồn ion, phần phân tích khối lượng, và phần đo đạc.

HCMC 2013

(C) HKD 2013

BACKGROUND

uses high energy electrons to break a molecule into fragments.

‰ Separation and analysis of the fragments provides information about:

„ Molecular weight

„ Structure

‰ The impact of a stream of high energy electrons causes the molecule to lose an electron forming a radical cation

„ A species with a positive charge and one unpaired electron

H

-Molecular ion (M + ) m/z = 16

(C) HKD 2013

BACKGROUND

electrons can also break the molecule or the radical cation into fragments.

(not detected by MS) m/z = 29

molecular ion (M+) m/z = 30

+ C H

H H

+ H

H

H C H

H C H H

H C H

H C H H

H C H H

‰ Molecular ion (parent ion):

„ The radical cation corresponding to the mass of the original molecule

mass in the spectrum

„ Some exceptions w/specific isotopes

„ Some molecular ion peaks are absent.

H H

H H

(C) HKD 2013

BACKGROUND

M+base peak

‰ C3H6O and C3H8O have nominal masses of 58 and 60, and can be distinguished by low-resolution MS.

‰ C3H8O and C2H4O2 both have nominal masses of 60.

‰ Distinguish between them by high-resolution MS.

C2H4O2

C3H8O

60.02112

60.05754 60

60

Molecular Formula

Nominal Mass

Precise Mass

) High resolution MS can replace elemental analysis for chemical formula confirmation.

Mass Analzyer Detector

System

Vacuum pumps system

‰ Electron impact (EI): vapor of sample is bombarded with

‰ Laser Desorption & Matrix-Assisted Laser Desorption

( MALDI ): hit the sample with a laser beam.

‰ Electrospray Ionization ( ESI ): a stream of solution passes through a strong electric field (106V/m).

WAYS TO PRODUCE IONS

(C) HKD 2013

BACKGROUND

MALDI FAB

(C) HKD 2013

BACKGROUND

ICP

IONIZATION METHODS

ionization technique, limited to relatively low

MW compounds (<600 amu).

2 Chemical Ionization (CI): ionization with very little fragmentation, still for low MW compounds (<800 amu).

very labile compounds.

4 Spray ionization (SI)

for LC-MS, biomolecules, etc.(C) HKD 2013

BACKGROUND

Electron ionization

energy electrons (typically 70 eV).

fragmentation.

M

Neutral molecule

Molecular ion

M +

-E << 70 eV

+++ + +

Fragment ions

Electron ionization

Advantages

• inexpensive, versatile and reproducible.

• fragmentation gives structural information.

• large databases if EI spectra exist and are searchable.

Disadvantages

• fragmentation at expense of molecular ion.

• sample must be relatively volatile.

(C) HKD 2013

BACKGROUND Chemical ionization (CI)

exchange, electron capture, adduct formation, etc.

‰ Common CI reagents: methane, ammonia, isobutane, hydrogen, methanol.

thermoneutral ion-molecule reactions will occur.

‰ choice of reagent allows tuning of ionization.

Positive Chemical ionization (CI)

„ Uses reagent gas (methane, isobutane or ammonia)

„ Soft ionization method Æ only little fragmentations

„ Produces positive ions

„ Detects pseudo molecular ions Æ molecular weight information can be obtained

„ Lower ion source temperature will produce more pseudo molecular ions Be careful that lower ion source temperature can cause more contamination.

Secondary reagent ions

„ Uses reagent gas (methane, isobutane or ammonia)

„ Soft ionization method Æ only little fragmentations

„ Produces negative ions

„ Higher reagent gas pressure will produce more low energy electrons, and in turn, more molecular ions.

„ Lowering the ion source temperature can produce more resonance capture reaction and increase molecular ions.

Analytes

(C) HKD 2013

MASS ANALYSER – MAGNETIC SECTOR

m/z = B(C) HKD 20132r2/2V

BACKGROUND

9 Classical mass spectra

9 Very high reproducibility

9 Best quantitative performance of all MS analyzers

9 High resolution

9 High sensitivity

9 10,000 Mass Range

9 Requires Skilled Operator

9 Usually larger and higher cost than other mass analyzers

9 Difficult to interface to ESI

9 Low resolution MS/MS without multiple analyzers

MASS ANALYSER – MAGNETIC SECTOR

MASS ANALYSER – QUADRUPOLE

z 䠖charge

k 䠖constant

V :voltage applied to rods

We can select mass

(C) HKD 2013

MASS ANALYSER – QUADRUPOLE

9 Relatively small and low-cost systems

9 Able to separate ions at lower vacuum levels (10-2 to 10-3Pa)

9 Quadrupoles are now capable of routinely analyzing up to a m/q ratio

of 3000, which is useful in electrospary ionization of biomolecules

MASS ANALYSER – ION TRAP

MASS ANALYSER – ION TRAP

9 Poor quantitation.

9 Less sensitive

9 Non-classical spectrum

9 Electrodes tend to be contaminated

9 Limited column flow rate

MASS ANALYSER – ION TRAP

(C) HKD 2013

BACKGROUND

MASS ANALYSER – Time-Of-Flight

m/z = 2eUt2/L2

MASS ANALYSER – Time-Of-Flight

resolution = 2dt/t(C) HKD 2013

BACKGROUND

9 Good for kinetic studies of fast reactions and for use with gaschromatography to analyze peaks from chromatograph

9 High ion transmission

9 Can register molecular ions that decompose in the flight tube

9 Extremely high mass range (>1MDa)

9 Fastest scanning

9 Requires pulsed ionization method or ion beam switching (duty cycle

is a factor)

9 Low resolution (4000)

9 Limited precursor-ion selectivity for most MS/MS experiments

MASS ANALYSER – Time-Of-Flight

Fourier Transform Ion Cyclotron Resonance

Fourier Transform Ion Cyclotron Resonance

(FT ICR) analyzers

 = qB/m = v/r

‰ The decay over time of the image current resulting after applying ashort radio-frequency sweep is transformed from the time domain into afrequency domain signal by a Fourier transform

(C) HKD 2013

BACKGROUND

9 The highest recorded mass resolution of all mass spectrometers(>500,000)

9 Very good accuracy (<1ppm)

9 Well-suited for use with pulsed ionization methods such as MALDI

9 Non-destructive ion detection; ion remeasurement

9 Stable mass calibration in superconducting magnet FTICR systems

Fourier Transform Ion Cyclotron Resonance

(FT ICR) analyzers

9 Expensive.

9 Requires superconducting magnet

9 Subject to space charge effects and ion molecule reactions

9 Artifacts such as harmonics and sidebands are present in the massspectra

9 Many parameters (excitation, trapping, detection conditions) comprisethe experiment sequence that defines the quality of the massspectrum

9 Generally low-energy CID, spectrum depends on collision energy,collision gas, and other parameters

Fourier Transform Ion Cyclotron Resonance

(FT ICR) analyzers

(C) HKD 2013

BACKGROUND

‰ Only cations are detected.

„ Radicals are “invisible” in MS.

depends on the mass to charge ratio (m/z).

„ Most cations formed have a charge of +1 so the amount of deflection observed is usually dependent on the mass of the ion.

Mass, as m/z Z is the charge, and for doubly charged ions (often seen in

macromolecules), masses show up at half their proper value

High mass

[M+H]+(CI)

Or M•+ (EI)

“molecular ion”

Unit mass spacing

Fragment Ions Derived from molecular ion

or higher weight fragments

In CI, adduct ions, [M+reagent gas] +

M + e - o M + + 2e

-Molecule High Energy

Electron

Molecular Ion (Radical Cation)

‰ A partial MS of dopamine showing all peaks with intensity equal to or greater than 0.5% of base peak.

higher than M+

higher than M+

S

2) Cleavage of 2 s bond (rearrangements)

C H 2

C H 2

Z H

+ .

3) Cleavage of Complex rearrangements(C) HKD 2013

1 Intensity of M.+ is Larger for linear chain than for branched compound

2 Intensity of M.+ decrease with Increasing M.W (fatty acid is

an exception)

3 Cleavage is favored at branching

 reflecting the Increased stability of the ion

M .+ is absent with heavy branching

Fragmentation occur at branching: largest fragment loss

Branched alkanes

Molecular ion is stronger than

in previous sample

(C) HKD 2013

Illustration of first 3 rules

(Branched alkane with Smaller MW)

Molecular ion smaller than linear alkane

Cleavage at branching is favored

43

Loss of Largest substituent Favored

Rule1: intensity of M .+

is smaller with branching

(C) HKD 2013

FRAGMENTATION PATTERNS

4 Aromatic Rings, Double bond, Cyclic structures stabilize M.+

5 Double bond favor Allylic Cleavage

 Resonance – Stabilized Cation

(C) HKD 2013

FRAGMENTATION PATTERNS

RULES

6 a) Saturated Rings lose a Alkyl Chain (case of branching)

b) Unsaturated Rings  Retro-Diels-Alder

9 Cleavage of small neutral molecules (CO2, CO, olefins, H2O ….) results often from rearrangement.

CH2

CH2H

CH2

O C Y

- CH2=CH2 x

CH2

O C Y

forming the most stable carbocations.

(C) HKD 2013

FRAGMENTATION PATTERNS

SUMMARY

‰ Alkenes:

ƒ Fragmentation typically forms

resonance stabilized allylic carbocations

m/z 41, 55, 69

Rule 4: Double Bond Stabilize M+

Rule 5: Double Bond favor

Allylic cleavage

CH2 +CH CH Et

EtMe

-Et

EtMe

CH2 CH CH +

EtMe

ƒ Fragment at the benzylic carbon, forming a

resonance stabilized benzylic carbocation

(which rearranges to the tropylium ion)

M+

C H

H C

H Br

H

C

H H

or

Aromatics may also have a peak at m/z = 77 for the benzene ring.

9 1o alcohol usually has prominent peak at

m/z = 31 corresponding to H2C=OH+

nitrogens are present).

ƒ D-cleavage forming oxonium ion

ƒ Loss of alkyl group forming oxonium ion

carbocation

M+ = 134

C C C H H

H

H H

9 Loss of OR’: peak at M+ - OR’

9 Loss of R’: peak at M+ - R’

identify possible molecular formulas for an unknown hydrocarbon, CnHm.

remainder in step 2)

‰ Example: The formula for a hydrocarbon with M+ =106 can be found:

ƒ Add the heteroatoms to the formula.

peak at m/z = 102 has a strong peak at

1739 cm-1 in its IR spectrum Determine its molecular formula.