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Phương pháp phổ khối lượng (MS) trong phân tích cấu trúc hoá lý

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Tài liệu phân tích dành cho học viên cao học về "Phương pháp phổ khối lượng (MS) trong phân tích cấu trúc hoá lý"

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PHỔ KHỐI LƯỢNG (MS)

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NGUYÊN TẮC PHƯƠNG PHÁP MS

 Đo khối lượng, không phải đo năng lượng

 Phân tử được bắn phá bằng nguồn năng lượng lớn tạo thành ion phân tử hay mảnh ion

 Tỉ lệ khối lượng/điện tích (m/z) của tất cả các ion được ghi nhận bằng cách thay đổi từ trường

 Độ giàu tương đối của mỗi ion tương ứng với mỗi m/z được thể hiện trong phổ MS

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3 Detector: phát hiện và đếm các ion đã tách, tín hiệu điện thu

được tỉ lệ với lượng ion ghi nhận

PHỔ KHỐI LƯỢNG

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MÁY KHỐI PHỔ

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MÁY KHỐI PHỔ

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CÁC PHƯƠNG PHÁP ION HÓA MẪU

Mẫu có thể được ion hóa bằng các phương pháp:

 Electron impact (EI)

 Chemical ionization (CI)

 Atmospheric pressure ionization (API)

 Atmospheric pressure chemical ionization (APCI)

 Electrospray ionization (ESI)

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KHỐI PHỔ BY ELECTRON IMPACT (EI)

Molecular ion,

radical cation

Fragmentation

Charged, detectable

Uncharged, Non detectable

Charged, detectable

Limitations:

 Fast fragmentation of unstable molecules

 risk of loss of molecular peak

 Molecular ions (radical cations) are unstable

 decompose before reaching the detector (during 20 s)

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MASS SPECTROMETRY BY

ELECTRON IMPACT (EI) – EXAMPLE

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MASS SPECTROMETRY BY CHEMICAL IONIZATION (CI)

Formation of [M+H] or [M-H] , more stable than radical cations

Reagent gas

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MASS SPECTROMETRY BY CHEMICAL IONIZATION (CI) – EXAMPLE

Mass spectrum of proline (i) by electron impact (ii) by chemical ionization (i) EI

(ii) CI

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MASS SPECTROMETRY BY CHEMICAL IONIZATION (CI)

 Useful technique when no molecular ion is observed by EI

 Confirmation of the presence of molecular ion when the signal

by EI is too weak

 Common reagent gases are methane, ammonia and isobutane

 There are 2 modes of chemical ionization

 Positive ion chemical ionization (PICI)

 Negative ion chemical ionization (NICI)

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POSITIVE ION CHEMICAL IONIZATION (PICI)

[GH] + M [MH] + G

Methane:

Relevant molecular peaks observed are MH+, [M+CH5]+ and [M+C2H5]+, mainly MH+

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POSITIVE ION CHEMICAL IONIZATION (PICI)

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POSITIVE ION CHEMICAL IONIZATION (PICI) –

CHOICE OF REAGENT GAS

[GH] + M [MH] + G

 These proton transfer reactions are true protonation reactions

by Bronsted acid in the gas phase

 Factors determine the choice of the gas to be used

 Proton affinity (PA): PAM > PAG

 Energy transfer, e.g NH4+ has low energy transfer than CH5+

 Reactivity of reagent gas toward the sample

 Choice of reagent gas affect the extend of fragmentation of the quasi-molecular ion

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POSITIVE ION CHEMICAL IONIZATION (PICI) –

EXAMPLES

Comparison of (a) 70 eV EI spectrum and (b) methane reagent gas CI spectrum of the

amino acid methionine

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POSITIVE ION CHEMICAL IONIZATION (PICI) –

EXAMPLES

Isobutane CI mass spectrum of gastric content in an overdose case

Milne et al Anal Chem 1970, 42, 1815

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NEGATIVE ION CHEMICAL IONIZATION (NICI)

 Electrons are thermalized in a high pressure source by a

reagent gas, e.g methane

 Compounds with electrophilic moieties (halogen, nitro group)

capture the thermal electrons producing abundant negative ions, typically the molecular anion

M + e M

0-2 eV

M + e [M-A] + A

0-15 eV

 NICI is highly selective & sensitive (like ECD)

 Molecular ions observed are usually M  or [MH]

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NEGATIVE ION CHEMICAL IONIZATION (NICI) –

EXAMPLE

EC spectrum of benzo[a]pyrene, isobutane buffer gas, ion source 200  C

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ADVANTAGES OF CHEMICAL IONIZATION

Positive ion chemical ionization:

 Molecular weight can be obtained and confirmed

 Increased sensitivity & selectivity for many compounds

 Selectivity can be affected by choosing appropriate reagent gas

 CI spectra are complemetary to EI spectra

Negative ion chemical ionization:

 Highly sensitive & selective ionization technique (NICI > PICI > EI), ideal for analysis of analytes in complex matrices

 Molecular weight can be obtained and comfirmed

 Also complementary to PICI & EI spectra

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ATMOSPHERIC PRESSURE IONIZATION (API)

 Mostly used in HPLC/MS

 There are two API techniques

 Atmospheric pressure chemical ionization (APCI)

 Electrospray ionization (ESI)

MS/MS API-365

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ATMOSPHERIC PRESSURE CHEMICAL

IONIZATION (APCI)

Mechanism for positive ion formation:

 Primary ion formation

 Secondary ion formation

 Analyte ion formation

H3O + M [M+H] + H2O

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APCI PROCESS IN THE POSITIVE ION

POLARITY MODE

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ESI PROCESS IN THE POSITIVE ION

POLARITY MODE

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CHOICE OF APCI OR ESI

 Both APCI & ESI can be used for analysis of medium to quite high polar compounds and may give different sensitivity

Analyte has low molecular mass Analyte has high molecular mass

(commonly biological molecules) Analyte has medium polarity Analyte has high polarity

Analyte has low thermal stability Softer ionization technique (less fragmentation)

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CHOICE OF APCI OR ESI

POLAR

NON-POLAR

ESI () ESI (+)

APCI (+) APCI ()

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ESI/APCI COMPARISON – HYDROCORTISONE

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NATURAL ABUNDANCE OF STABLE ISOTOPES

Chlorine 35 Cl 75,77 37 Cl 24,23 Bromine 79 Br 50.69 81 Br 49.31

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INTENSITY IN THE MASS SPECTRUM FOR

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THE MASS SPECTRUM FOR CARBON

Molecule containing 1 cacbon atom:

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THE MASS SPECTRUM FOR CARBON

Molecule containing 10 cacbon atoms:

Molecule

containing

Only 12 C atoms

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THE MASS SPECTRUM FOR CARBON

Molecule containing 100 cacbon atoms:

Molecule

containing

Only 12 C atoms

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Relative intensity of (M + 2) ion:

Due to the occurrence of a couple of 13C or an 18O atom

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MOLECULES CONTAINING 1 Cl ATOM

Relative intensity of (M + 2) ion:

Mainly due to the contribution of a 37Cl atom

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MOLECULES CONTAINING 1 Br ATOM

Relative intensity of (M + 2) ion:

Mainly due to the contribution of a 81Br atom

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MOLECULES CONTAINING n Cl OR Br ATOMS

There are n + 1 peaks of molecular ion whose relative intensity given by the equation (a + b)n

a, b: relative abundances of two isotopes

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MOLECULES CONTAINING 2 CHLORINE OR

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MOLECULES CONTAINING n Cl AND m Br ATOMS

Relative intensities of molecular ions are given by the equation

(a + b)n (c + d)m

a, b: relative abundances of 35Cl and 37Cl (3, 1)

c, d: relative abundances of 79Br and 81Br (1, 1)

n = 1, m = 1: (a + b)(c + d) = ac + (ad + bc) + bd

= 3 + 4 + 1

IM : IM+2 : IM+4 = 3 : 4 : 1

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SUMMARY OF MOLECULES CONTAINING

n Cl AND m Br ATOMS

I M I M+2 I M+4 I M+6

Cl 2 ClBr

Br 2

Cl 3

Cl 2 Br ClBr 2

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IDENTIFICATION OF MOLECULAR ION

 Molecular ion can be more easily identified by appropriate ionisation techinique

 Ralative intensity of isotopic ions can also help to distinguish molecular ion from impurities

 The mass parity and the nitrogen rule are also utile in the

identification of molecular ion and its fragments

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THE MASS PARITY AND THE NITROGEN RULE

A compound, that contains 2n (n 0) nitrogen atoms, has an

even mass number

A compound, that contains 2n + 1 (n 0) nitrogen atoms, has an odd mass number

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THE MASS PARITY AND THE NITROGEN RULE –

NON-VOLATILE MOLECULES

Molecular ion:

 analyze by ESI-MS, normally by HPLC/ESI-MS

Molecular ion: [M+H]+ (positive mode)

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THE MASS PARITY AND THE NITROGEN RULE –

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THE MASS PARITY AND THE NITROGEN RULE –

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THE MASS PARITY AND THE NITROGEN RULE –

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THE MASS PARITY AND THE NITROGEN RULE –

VOLATILE MOLECULES

Molecular ion:

Analyze by EI-MS, normally by GC/EI-MS

Molecular radical ions: M+

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THE MASS PARITY AND THE NITROGEN RULE –

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THE MASS PARITY AND THE NITROGEN RULE –

80% ions on EI spectrum have odd mass number

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FRAGMENTATION

Fragmentation kinetic:

 depend on the energy transferred to ions

 this energy is higher in EI than in ESI

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