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

8/11/2019 Phương pháp phổ khối lượng (MS) trong phân tích cấu trúc hoá lý

http://slidepdf.com/reader/full/phuong-phap-pho-khoi-luong-ms-trong-phan-tich-cau-truc-hoa 1/51

PHỔ KHỐI LƯỢNG (MS)

WWW.DAYKEMQUYNHON.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON WWW.DAYKEMQUYNHON.UCOZ.COM



NGUYÊN TẮC PHƯƠNG PHÁP MS

Đo khối lượng, không phải đo năng lượng

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

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

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





PHỔ KHỐI LƯỢNG

Khối lượng của ion phân tử cho biết khối lượng phân tử củahợp chất

Khối lượng của các phân mảnh ion cho biết thông tin về cấu

trúc của hợp chất





Máy khối phổ có 3 bộ phận chính:

1. Buồng ion hóa: để hóa hơi và ion hóa các phân tử thành dòngcác hạt tích điện

2. Từ trường: làm chệch hướng dòng ion và tách chúng theo giátrị m/z

3. Detector: phát hiện và đếm các ion đã tách, tín hiệu điện thuđược tỉ lệ với lượng ion ghi nhận

PHỔ KHỐI LƯỢNG WWW.DAYKEMQUYNHON.COM




MÁY KHỐI PHỔ





MÁY KHỐI PHỔ WWW.DAYKEMQUYNHON.COM




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)



A Q O CO



KHỐI PHỔ BYELECTRON IMPACT (EI)

Unknown molecule

with a lone pair of

electron

Electron bombardement( 70 eV)

Deep vacuum

(10-4 N/m2)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)



WWWDAYKEMQUYNHON COM



MASS SPECTROMETRY BY

ELECTRON IMPACT (EI) – EXAMPLE







CHEMICAL IONIZATION (CI)

CnH2n+2 [CnH2n+1] H2

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

cations

Reagent gas







CHEMICAL IONIZATION (CI) – EXAMPLE

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

(i) EI

(ii) CI







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)






POSITIVE ION CHEMICAL IONIZATION (PICI)

[GH] + M [MH] + GMethane:

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

[M+C2H5]+, mainly MH+






POSITIVE ION CHEMICAL IONIZATION (PICI)

Ammonia:

Relevant molecular peaks observed are MH+, [M+NH4]+

Isobutane:

Relevant molecular peaks observed are MH+






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







EXAMPLES

Comparison of (a) 70 eV EI spectrum and (b) methane reagent gas CI spectrum of theamino acid methionine







EXAMPLES

Isobutane CI mass spectrum of gastric content in an overdose case

Milne et al. Anal. Chem. 1970, 42 , 1815






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 [MH]








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






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

Q





ATMOSPHERIC PRESSURE CHEMICAL

IONIZATION (APCI)

Mechanism for positive ion formation:

Primary ion formation

Secondary ion formation

Analyte ion formation

H3O + M [M+H] + H2O

Q





APCI PROCESS IN THE POSITIVE ION

POLARITY MODE

Q







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

APCI ESI

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)





CHOICE OF APCI OR ESI

POLAR

NON-POLAR

BASIC ACIDIC

ESI ()ESI (+)

APCI (+) APCI ()





ESI/APCI COMPARISON – HYDROCORTISONE





NATURAL ABUNDANCE OF STABLE ISOTOPES

Element Majorisotope

Abundance M + 1isotope

Abundance M + 2isotope

Abundance

Hydrogen 1H 99,985 2H 0,015

Carbon 12C 98,89 13C 1,11

Nitrogen 14N 99,64 15N 0,36

Phosphorus 31P 100,00

Oxygen 16O 99,76 17O 0,04 18O 0,20

Sulfur 32S 95.02 33S 0,75 34S 4,4

Chlorine 35Cl 75,77 37Cl 24,23

Bromine 79Br 50.69 81Br 49.31





INTENSITY IN THE MASS SPECTRUM FOR

EACH ISOTOPE

Element Major

isotope

Intensity M + 1

isotope

Intensity M + 2

isotope

Intensity

Hydrogen 1H 100 2H 0,015

Carbon 12C 100 13C 1,1

Nitrogen 14N 100 15N 0,4

Phosphorus 31P 100

Oxygen 16O 100 17O 0,04 18O 0,2

Sulfur 32S 100 33S 0,8 34S 4,21

Chlorine 35Cl 100 37Cl 32,0

Bromine 79Br 100 81Br 97.3





THE MASS SPECTRUM FOR CARBON

Molecule containing 1 cacbon atom:

12C: 100%13C: 1,1%

100

0

Relative

abundance

m/z

12

50

The peak of 13C isotope is too small to observed






Molecule containing 10 cacbon atoms:

Molecule

containing

Only 12C

atoms

9 12C atoms and

1 13C atom

8 12C atoms and

2 13C atom

Probability of

occurrence

(0.9889)10

= 0.8944

(0.9889)9(0.0111)*10

= 0.1004

(0.9889)8(0.0111)2*45

= 0.005071

Intensity 100 11,2 0.6 (not observed)

100

0

Relative

abundance

m/z

120

50

121

The peak of 13C isotope is

about 1/10 the peak of 12C






Molecule containing 100 cacbon atoms:

Moleculecontaining Only12

Catoms 9912

C atoms and1 13C atom 9812

C atoms and2 13C atom

Probability of

occurrence

(0.9889)100

= 0.3275

(0.9889)99(0.0111)*100

= 0.3676

(0.9889)98(0.0111)2

*4950 = 0.2043

Intensity 89.1 100 55.6

100

0

Relative

abundance

m/z

121

50

120

122

Intensity of isotope increases with

the number of carbon atom in the moleculeWWW.FACEBOOK.COM/DAYKEM.QUYNHON WWW.DAYKEMQUYNHON.UCOZ.COM




MOLECULES CONTAINING C, H, N, O, F, P, I

CaHbNcOdFePf Ig

Relative intensity of (M + 1) ion:

IM + 1

IM=

Ex: C6H

5NO

2

1.11*6 + 0.36*1

7 %

% IM + 1 100% 1,11a + 0.36c (%)

% IM + 1

Due to the important contribution of a 13C and an 15N atom


O C S CO G C OWWW.DAYKEMQUYNHON.COM




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

IM + 2

IM (1.11 % * a)2

/ 2 + 0.20 % * d

(1.11 * a)2 / 200 + 0.20 * d (%)

Ex: (CH3)3PO

(1.11*3)2 / 200 + 0.20 * 4

0.85 %

MOLECULES CONTAINING C, H, N, O, F, P, I

CaHbNcOdFePf Ig

% IM + 2 = 100%

% IM + 2

% IM + 2


MOLECULES CONTAINING 1 Cl ATOMWWW.DAYKEMQUYNHON.COM



MOLECULES CONTAINING 1 Cl ATOM


Mainly due to the contribution of a 37Cl atom

32.0 * 1 (%)

Ex: C6H5Cl

100

0

Relative

abundance

m/z

M

50M + 2

IM+2 1/3 IM

% IM + 2


MOLECULES CONTAINING 1 B ATOMWWW.DAYKEMQUYNHON.COM



MOLECULES CONTAINING 1 Br ATOM


Mainly due to the contribution of a 81Br atom

= 97.3 * 1 (%)

Ex: C6H5Cl

100

0

Relative

abundance

m/z

M

50

M + 2

IM+2 IM

% IM + 2




MOLECULES CONTAINING 2 CHLORINE ORWWW.DAYKEMQUYNHON.COM



MOLECULES CONTAINING 2 CHLORINE OR

BROMINE ATOMS

RCl2

(a + b)2 = a2 + 2ab + b2

RBr 2

(3 + 1)2 = 32 + 2*3*1 + 12

= 9 + 6 + 1

IM : IM+2 : IM+4 = 9 : 6 : 1

100

0

Relativeabundance

m/z

M

50

M + 2

M + 4

(1 + 1)2 = 12 + 2*1*1 + 12

= 1 + 2 + 1

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

100

0 m/z

M50

M + 2

M + 4


MOLECULES CONTAINING Cl AND B ATOMSWWW.DAYKEMQUYNHON.COM



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




IDENTIFICATION OF MOLECULAR IONWWW.DAYKEMQUYNHON.COM



IDENTIFICATION OF MOLECULAR ION

Molecular ion can be more easily identified by appropriateionisation 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


THE MASS PARITY AND THE NITROGEN RULEWWW.DAYKEMQUYNHON.COM



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

Molecule M

EtOH 46

H2NNH2 32Et3N 101

Ex:





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)

or [M-H]- (negative mode)

In positive mode:

(M+H)+ even oddM odd even

Nitrogen number 2n+1 2n







Fragment ions:

[M+H] [F+H] + M’ (not observed)

(M+H)+ odd

M even

[F+H]+ odd

Nitrogen number 2nM’ even

Nitrogen number 2m

(i)


THE MASS PARITY AND THE NITROGEN RULE –WWW.DAYKEMQUYNHON.COM





Fragment ions:


(M+H)+ even

M odd

[F+H]+ odd

Nitrogen number 2nM’ odd

Nitrogen number 2m+1

(ii)







Fragment ions:


(M+H)+ even

M odd

[F+H]+ even

Nitrogen number 2n+1M’ even

Nitrogen number 2m

(ii)







THE MASS PARITY AND THE NITROGEN RULE –VOLATILE MOLECULES

Fragment ions:Fragmentation

M+ even

F+ even

Nitrogen number 2n

M’ even

Nitrogen number 2m

(1)

(2)

(1)





THE MASS PARITY AND THE NITROGEN RULE –VOLATILE MOLECULES

Fragment ions:Fragmentation

M+ odd

F+ odd

Nitrogen number 2n+1

R odd

Nitrogen number 2m+1

(1)

(2)

(2)

80% ions on EI spectrum have odd mass numberWWW.FACEBOOK.COM/DAYKEM.QUYNHON WWW.DAYKEMQUYNHON.UCOZ.COM

FRAGMENTATIONWWW.DAYKEMQUYNHON.COM



FRAGMENTATION

Fragmentation kinetic:

depend on the energy transferred to ions

this energy is higher in EI than in ESI

Fast fragmentation in EI

Low fragmentation in ESI MS/MS

EI ESI

Fragmentation Fragmentation

MS


EXAMPLE OF FRAGMENTATIONWWW.DAYKEMQUYNHON.COM



EXAMPLE OF FRAGMENTATION

The fragmentation is often induced by the charge

m/z = 132

Fragmentation

m/z = 57 m/z = 76

Documents

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