Tài liệu phân tích dành cho học viên cao học về "Phương pháp phổ khối lượng (MS) trong phân tích cấu trúc hoá lý"
Trang 1PHỔ KHỐI LƯỢNG (MS)
Trang 2NGUYÊN 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
Trang 43 Detector: phát hiện và đếm các ion đã tách, tín hiệu điện thu
được tỉ lệ với lượng ion ghi nhận
PHỔ KHỐI LƯỢNG
Trang 5MÁY KHỐI PHỔ
Trang 6MÁY KHỐI PHỔ
Trang 7CÁC 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)
Trang 8KHỐI 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)
Trang 9MASS SPECTROMETRY BY
ELECTRON IMPACT (EI) – EXAMPLE
Trang 10MASS SPECTROMETRY BY CHEMICAL IONIZATION (CI)
Formation of [M+H] or [M-H] , more stable than radical cations
Reagent gas
Trang 11MASS SPECTROMETRY BY CHEMICAL IONIZATION (CI) – EXAMPLE
Mass spectrum of proline (i) by electron impact (ii) by chemical ionization (i) EI
(ii) CI
Trang 12MASS 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)
Trang 13POSITIVE ION CHEMICAL IONIZATION (PICI)
[GH] + M [MH] + G
Methane:
Relevant molecular peaks observed are MH+, [M+CH5]+ and [M+C2H5]+, mainly MH+
Trang 14POSITIVE ION CHEMICAL IONIZATION (PICI)
Trang 15POSITIVE 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
Trang 16POSITIVE ION CHEMICAL IONIZATION (PICI) –
EXAMPLES
Comparison of (a) 70 eV EI spectrum and (b) methane reagent gas CI spectrum of the
amino acid methionine
Trang 17POSITIVE ION CHEMICAL IONIZATION (PICI) –
EXAMPLES
Isobutane CI mass spectrum of gastric content in an overdose case
Milne et al Anal Chem 1970, 42, 1815
Trang 18NEGATIVE 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 [MH]
Trang 19NEGATIVE ION CHEMICAL IONIZATION (NICI) –
EXAMPLE
EC spectrum of benzo[a]pyrene, isobutane buffer gas, ion source 200 C
Trang 20ADVANTAGES 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
Trang 21ATMOSPHERIC 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
Trang 22ATMOSPHERIC PRESSURE CHEMICAL
IONIZATION (APCI)
Mechanism for positive ion formation:
Primary ion formation
Secondary ion formation
Analyte ion formation
H3O + M [M+H] + H2O
Trang 23APCI PROCESS IN THE POSITIVE ION
POLARITY MODE
Trang 24ESI PROCESS IN THE POSITIVE ION
POLARITY MODE
Trang 25CHOICE 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)
Trang 26CHOICE OF APCI OR ESI
POLAR
NON-POLAR
ESI () ESI (+)
APCI (+) APCI ()
Trang 27ESI/APCI COMPARISON – HYDROCORTISONE
Trang 28NATURAL ABUNDANCE OF STABLE ISOTOPES
Chlorine 35 Cl 75,77 37 Cl 24,23 Bromine 79 Br 50.69 81 Br 49.31
Trang 29INTENSITY IN THE MASS SPECTRUM FOR
Trang 30THE MASS SPECTRUM FOR CARBON
Molecule containing 1 cacbon atom:
Trang 31THE MASS SPECTRUM FOR CARBON
Molecule containing 10 cacbon atoms:
Molecule
containing
Only 12 C atoms
Trang 32THE MASS SPECTRUM FOR CARBON
Molecule containing 100 cacbon atoms:
Molecule
containing
Only 12 C atoms
Trang 34Relative intensity of (M + 2) ion:
Due to the occurrence of a couple of 13C or an 18O atom
Trang 35MOLECULES CONTAINING 1 Cl ATOM
Relative intensity of (M + 2) ion:
Mainly due to the contribution of a 37Cl atom
Trang 36MOLECULES CONTAINING 1 Br ATOM
Relative intensity of (M + 2) ion:
Mainly due to the contribution of a 81Br atom
Trang 37MOLECULES 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
Trang 38MOLECULES CONTAINING 2 CHLORINE OR
Trang 39MOLECULES 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
Trang 40SUMMARY 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
Trang 41IDENTIFICATION 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
Trang 42THE 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
Trang 43THE 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)
Trang 44THE MASS PARITY AND THE NITROGEN RULE –
Trang 45THE MASS PARITY AND THE NITROGEN RULE –
Trang 46THE MASS PARITY AND THE NITROGEN RULE –
Trang 47THE MASS PARITY AND THE NITROGEN RULE –
VOLATILE MOLECULES
Molecular ion:
Analyze by EI-MS, normally by GC/EI-MS
Molecular radical ions: M+
Trang 48THE MASS PARITY AND THE NITROGEN RULE –
Trang 49THE MASS PARITY AND THE NITROGEN RULE –
80% ions on EI spectrum have odd mass number
Trang 50FRAGMENTATION
Fragmentation kinetic:
depend on the energy transferred to ions
this energy is higher in EI than in ESI