GC-MS

SPEKTROMETRI MASSA
 Merupakan suatu metode analisis instrumental yang digunakan untuk
proses identifikasi serta penentuan struktur dari suatu komponen
sampel/molekul organik berdasarkan perhitungan massa dari molekul
tersebut serta pola fragmentasinya
Kegunaan utama MS:
 menentukan massa molekul senyawa organik
 membantu menentukan rumus molekul senyawa organik (elusidasi
struktur)

 Tidak melibatkan penyerapan atau emisi cahaya

 Spektrometri massa menggunakan perbedaan rasio massa terhadap

muatan (m/z) dari atom atau molekul terionisasi untuk pemisahannya
 SPEKTROMETRI MASSA Ionization sources
Chemical Ionization (CI)
Atmospheric Pressure CI!(APCI)
Electron Impact!(EI)
Electrospray Ionization!(ESI)
Fast Atom Bombardment (FAB)
Field Desorption/Field Ionization (FD/FI)
Matrix Assisted Laser Desorption
Ionization!(MALDI)
Thermospray Ionization (TI)

Analyzers
quadrupoles
Time-of-Flight (TOF)
 IONIZATION
magnetic sectors
producing ions from the sample Fourier transform

 SORTING OF IONS and quadrupole ion traps

separate the ions based on their mass-to-charge ratio

Detectors
 DETECTION OF IONS electron multiplier
measure the quantity of ions of each mass-to-charge ratio Faraday cup
Inlet/Sample introduction system
 The Mass Spectrum
Graph of ion intensity versus mass-to-charge ratio (m/z)

X-axis mass-to-charge ratio (m/z)

Y-axis relative abundance of each m/z
Base peak the most abundant peak the tallest peaks come from
the most stable species
M+ Molecular Ion Peak; m/z corresponds to MW of
singly charged molecule
 Fragmen yang melimpah terbentuk dengan mudah dan mempunyai
tendensi yang rendah untuk terfragmentasi lebih lanjut, atau relatif stabil.

 Fragmen yang paling melimpah dinyatakan mempunyai kelimpahan relatif

100% dan disebut dengan base peak. Kelimpahan fragmen-fragmen yang
lain dinyatakan relatif terhadap base peak
What about peaks at greater m/z than M+?
Two sources:
Isotopic Abundance
 Isotopic Abundance
• Some distinguishing characteristics of certain
elements in a mass spectrum.

Element Distinguishing Characteristic

N Odd M+
S Large M+2 (about 4 %)
Cl M+2 one-third as large as M+
Br M+2 as large as M+
Mass Spectrum with Bromine

Mass Spectrum with Chlorine

 The GC-MS
 A mixture of compounds is separated by gas chromatography,
then identified by mass spectrometry.

GC is used to separate the volatile and thermally stable substitutes in a sample

whereas GC-MS fragments the analyte to be identified on the basis of its mass.
The further addition of mass spectrometer in it leads to GC-MS/MS.
 Spektrometri massa dalam GC/MS
• EI is the most widely used type of ionization in GC/MS. The
other ionization techniques currently commercially available
for GC/MS are called soft ionization: chemical ionization (CI)
and field ionization (FI).

• The four types of m/z analyzers used in GC/MS are the

Quadrupole Mass Filter (most widely used), Time-of-Flight
(rapidly gaining in popularity), and the Magnetic Sector
Analyzers (rarely used in most laboratories today).
 Ion Sources
1. Hard ion sources (Hard
Ionization)
leave excess energy in
molecule - extensive
fragmentation

2. Soft ion sources (Soft

Ionization)
little excess energy in
molecule. They do not (in
the most part) degrade the
molecule during the
ionization process - reduced
fragmentation
 SPEKTROMETRI MASSA
 Ionisasi: Sampel diionisasi agar terbentuk ion positif.
 Metode Ionisasi
EI – electron ionization/electron impact
Prinsip Dasar
 Molekul sampel dalam fase uap dibombardir dengan elektron berenergi
tinggi (70 eV) yang menyebabkan lepasnya satu elektron dari molekul
tersebut untuk membentuk suatu kation radikal (radical cation).

n<π<σ M + e-  M+• + 2e-

 Kation radikal tersebut mengandung semua atom-atom dari molekul asal,

minus satu elektron, dan disebut ion molekul (molecular ion), dan
dinyatakan dengan M+•

 Jika ion molekul tersebut tidak stabil, maka akan mengalami proses
fragmentasi membentuk ion-ion yang lebih kecil.
EI Source
filament
Under high vacuum
70 eV e-

To mass
analyzer

GC column

anode
repeller Acceleration
slits
 EI process

• M + e- M+*

f1 f2 f4
f3
This is a remarkably reproducible process. M will
fragment in the same pattern every time using a
70 eV electron beam
 Metode Ionisasi
EI – electron ionization/electron impact

Advantages of EI:
• high ion currents - sensitive
• fragmentation aids identification
• can be applied to all volatile analytes
Disadvantages of EI:
• weak or absent M+ peak inhibits determination of MW
• molecules must be vaporized (MW < 103 Da)
• molecules must be thermally stable during vaporization
 Metode Ionisasi
CI – chemical ionization
Prinsip Dasar
 Proses ionisasi menggunakan reagen gas (mis: metan, isobutan atau ammonia)
yang diionkan secara electron impact.

 Energi ionisasi lebih kecil dibanding EI (metode alternatif untuk volatile analytes),
sehingga fragmentasinya lebih kecil dan kelimpahan relatif M+• tinggi.

 BM molekul sampel
diperoleh dari
protonasi molekul
sample, dan harga m/z
yang diperoleh adalah
satu unit lebih besar
dibanding BM yang
sebenarnya.
 Metode Ionisasi
CI – chemical ionization
Keunggulan:
• Memberikan informasi berat molekul melalui molekul ion seperti ion
[M + H]+,bahkan ketika EI tidak akan menghasilkan ion molekuler.
• Spektrum massanya sederhana, pola fragmentasi berkurang
dibandingkan dengan EI

Kekurangan:
• Sampel harus secara termal mudah menguap dan stabil
• Fragmentasinya kurang dari EI, pola fragmen tidak cukup informatif
• Hasilnya tergantung pada jenis reagen gas, tekanan pereaksi gas
atau waktu reaksi,dan sifat sampel.
 Mass Analyzer
Quadropole
Terdapat empat buah
pipa dari logam yang
diberi arus DC dan Rf
(Radio Frekuensi)

Ion dari ion source akan

melalui bagian tengah
quadrupole

Pergerakan akan
bergantung medan
listrik quadrupole

Resolusi rendah
 Mass Analyzer
Triple Quadropole

A triple quadrupole (QQQ-MS-MS). The middle quadrupole is used as a collision

chamber. It is operated in the radiofrequency voltage mode only, where it will transmit
all masses. Middle quadrupole is where fragmentation of the sample occurs in the
presence of an inert gas like Ar, He, or N2. A characteristic daughter ion is produced as
a result of the collisions of the inert gas with the analyte. Upon exiting the collision
cell, the fragmented ions then travel onto the second quadrupole mass filter, Q3,
where m/z selection can occur again.
 Mass Analyzer
Triple Quadropole
 Mass Analyzer
Magnetic Sector
Analyzers
The ions enter the flight
tube and are deflected by
the magnetic field, B.
depending on their mass to
charge ratio.

m/z = (B2r2)/2V

This equation shows that the m/z ratio of the ions

that reach the detector can be varied by changing
either the magnetic field (B) or the potential
difference of the ion accelerating plates (V).
 Mass Analyzer
Time of Flight (TOF)
Separates ions based on
flight time

Ions accelerated by an
electric field

Lighter ions reach the

detector first

m/z = (2V/L2)t2
can be solved for charge-
mass ratio A simplified schematic of a time of flight spectrometer
and the principle of the ion reflector (reflectron)
 Ion Detector
Types of detector

The detector’s purpose is to translate the ion arrival into an

electric signal measured by the electronic system of the mass
spectrometer. These signals are produced by ions striking a
surface to initiate the first release of electrons or photons.

 The Faraday Cup

 The Electron Multiplier
 The Photomultiplier
 Ion Detector
The Electron Multiplier

Electron multipliers with discrete dynodes. In this device, positive ions strike a
conversion cathode liberating electrons which are then accelerated and ‘multiplied’ via
a series of up to twenty dynodes . This type of detector is extremely sensitive, having a
gain of up to 108. Aluminium-based dynodes have improved performances of the
traditional materials (Cu/Be alloys)
 SPEKTROMETRI MASSA
Sample is first Vaporized before
passing through an ion chamber
Then bombarded with a stream
of high-energy electrons.
This results in the formation
of a molecular ion as well as
a number of fragments
These ions are then fed into an electric field where they are accelerated and
passed through a narrow slit, producing a narrow beam of positive ions.
This beam is then subjected to a magnetic field causing the ions to be deflected a
particular distance depending on the mass to charge ratio
The ions deflected are detected by a particular instrument, which then sends
messages to a recorder resulting in the formation of a mass spectrum.
Data for the GC-MS is displayed in several ways
1. One is a total-ion chromatogram, which sums the
total ion abundances in each spectrum and plots
them as a function of time.

2. Another is the mass spectrum at a particular time in

the chromatogram to identify the particular
component that was eluted at that time.
Gas Chromatography/Mass Spectrometry (GC/MS)

Sample introduced into GC inlet

vaporized at 250 °C , swept onto the
column by He carrier gas &
separated on column. Sample
components emerge from column,
flowing into the capillary column
interface connecting the GC column
and the MS.
Qualitative Methods of GC/MS Analysis: Library Search

 Library search is an easy and useful tool for identifying a

chromatographic peak.
 Since a mass spectrum pattern relates to the chemical
structure of the compound, a mass spectrum acts as a
fingerprint in chemical analysis.
 A database of mass spectra is called "mass spectral library".
 We can identify an unknown component by comparing its
measured spectrum to reference spectra registered in mass
spectral libraries.
 Qualitative Methods of GC/MS Analysis: Library Search
An unknown spectrum is compared to reference spectra registered in
spectral libraries and the similarity is estimated for each reference.
 Qualitative Methods of GC/MS Analysis: Library Search

 A conventional library search neglects retention parameters, and the process

includes only comparison of spectra.
 Some chemical components such as isomers have similar spectra, which may
confuse chemists, but show chromatographic peaks at different retention
times.
 The combined use of retention indices when searching libraries makes
identification easier and more reliable.
 To perform this search, spectral libraries with retention indices are required.
 Using the same column and same analysis method described in the library is
recommended to get good results
 Mass Spectral Libraries
Many mass spectral libraries are commercially available. Some of them are for general
use while others are for specific application fields. In general, the data are obtained using
70eV EI. Additionally, users' personal libraries can be made for some GCMS application
software.

FFNSC (Flavor and Fragrance Natural and Synthetic Compounds) Library

This is a library for 3000 flavor and fragrance compounds.

Drug Library
This library contains spectra for 8650 compounds, including drugs, poisons, pesticides and
environmental pollutants.

GC/MS Metabolite Mass Spectral Database

This database contains mass spectral library and method files that specify analytical
conditions and data analysis parameters for amino acids, fatty acids, and organic acids.
 Mass Spectral Libraries
NIST Library
The main library contains spectra for 242,466 general compounds, and 33,782 of these
spectra are registered in a sub-library (2014 edition).

Wiley Library
This library contains spectra for 638,000 general compounds (The 10th edition).

Pesticide Library 3rd edition

This is a library containing mass spectra for 578 compounds measured using the electron
ionization (EI) method and 383 compounds measured using the negative chemical
ionization (NCI) method. Highly reliable identification is possible using the mass spectra of
the EI and NCI modes together. The library also includes a method for analyzing pesticide
residues in food and tap water.
 The Mass Spectrum
Graph of ion intensity versus mass-to-charge ratio (m/z)

X-axis mass-to-charge ratio (m/z)

Y-axis relative abundance of each m/z
Base peak the most abundant peak the tallest peaks come from
the most stable species
M+ Molecular Ion Peak; m/z corresponds to MW of
singly charged molecule
 Glossary
• Molecular ion - The ion obtained by the loss of one
electron from the molecule (m+)
• Base peak - The most intense peak in the MS, assigned
100% intensity
• Radical cation - positively charged species with an odd
number of electrons
• Fragment ions - Lighter cations (and radical cations)
formed by the decomposition of the molecular
ion. These often correspond to stable carbcations.
• m/z - mass to charge ratio
 High-Resolution Mass Spectrometry
 High-resolution results with at least three or four significant figures beyond
the decimal point. Example :

The exact mass of certain nuclides is shown below

 Summary: acquiring a mass spectrum

Ionization Mass Sorting (filtering) Detection

Ion Ion
Source Mass Analyzer Detector
Form ions
Sort Ions by Mass (m/z) Detect ions
(charged molecules)

100

75

Inlet • Solid 50

• Liquid 25

• Vapor 0
1330 1340 1350

Mass Spectrum
Mass Spectrum with Bromine

Mass Spectrum with Chlorine

 Mass Spectra of Alkanes
 The lowest energy electron is going to be in a sigma bond.

 C - C bonds weaker than C - H bonds

 The radical cation of an alkane will fragment to yield a radical and a cation.

 In straight chain alkanes, an alkyl group is lost from one end of the molecule,
and then successsive losses of 14 mass unit (CH2) are observed.

 fragmentation most likely at a branch

 positive charge remains with fragment with most branching

 More stable carbocations will be more abundant. Order of stability of

carbocations :

primary < secondary < tertiary

Mass Spectra of Alkanes
Fragmentation of Hexane
 Mass Spectra of Alkanes
More stable carbocations will be more abundant.
 Mass Spectra of Alkenes
 π electron is removed.
 Cleavage is favored at second bond away from double bond.
 Alkenes fragment to give Resonance-stabilized allylic carbocations.
 Mass Spectra of Alcohols
 One of the lone pair electrons
gets removed.

 Cleavage of the hydroxyl carbon

– alpha carbon bond (yielding a
resonance stabilized cation).

 Alcohols usually lose a water

molecule (Dehydration), yielding
an alkene radical cation.

 M+ may not be visible.

 Mass Spectra of Amines and Ethers
 Both amines and ethers undergo a similar type of fragmentation reaction.

 If the fragmentation occurs next to a nitrogen or oxygen, then electron

rearrangement may occur to form either iminium ion or oxonium ion.

 Additional stabilized cations

may be formed from a-
cleavage (breaking the carbon-
carbon bond, adjacent to the carbon

bearing a specified functional group).

Mass Spectra of Amines and Ethers
 Mass Spectra of Carbonyl

 Compounds containing carbonyl

groups may fragment next to the
carbonyl. Electron delocalization
occurs to yield an acylium ion

The McLafferty Rearrangement

• Occurs in carbonyl only if the g

(gamma) carbon contains a hydrogen.

• In the mass spectrum, peaks from the

alkene and enol would occurs.
Mass Spectra of Carbonyl
Mass Spectra of Carbonyl
Mass Spectra of Carbonyl
 LC-MS
LC-MS systems combine the outstanding
separation resolution of liquid
chromatography with the outstanding
qualitative capabilities of mass
spectrometry. The mass spectra
obtained from these scan
measurements provides molecular mass
and structural information for eluted
components, which supplements the
qualitative information based on
retention times obtained using other LC
detectors (Figure 1).
 Ionization Method (LC-MS)
ESI - Electrospray Ionization (liquid)
• Generates ions directly from solution.
• A spray of charged droplets is created by the application of a high
potential difference.
• This method can produce multiply charged ions that increase with
increasing molecular weight.
Sample Inlet Nozzle Partial
Pressure = 1 atm vacuum
Inner tube diam. = 100 um (Lower Voltage)

N2 MH+
+
++++ + ++ + +
++ + +
++
++ + +
Sample in solution
++
+
+ ++ +
+
++
+ +
+ +
MH2+
++ ++ + ++ +
N2 gas ++ + +

MH3+

High voltage applied Charged droplets

to metal sheath (~4 kV)
 Ionization Method (LC-MS)

Evaporation of Ions in Electrospray Ionization (ESI)

 Ionization Method (LC-MS)
ESI – electrospray ionization

A schematic of the mechanism of ion formation in atmospheric pressure ionization by electrospray.

The analyte solution flow passes through the electrospray needle that has a potential in the range from
2.5 to 4 kV with respect to the counter electrode. The charged droplets have a surface charge of the
same polarity to the charge on the needle. The droplets (1) shrink until they reach the point that the
surface tension can no longer sustain the charge (‘Coulombic explosion’) (2) and the droplet is ripped
apart, expelling analyte molecules carrying several charges (3). The presence of nitrogen improves the
concentration process (4).
 Ionization Method (LC-MS)
Advantages of ESI:
• Greatest chance of observing molecular ion
• Very labile analytes can be ionized
• Molecule need not be volatile
• Easily coupled with LC/HPLC

Disadvantages of ESI:
• ESI is very sensitive to contaminants
• Distribution of multiple charge states can make spectra of
mixtures hard to interpret
 EI and ESI

• Obtaining molecular mass information is difficult using EI, which requires using a
complementary analytical method such as chemical ionization (CI).
• In contrast, using ESI, which is a soft ionization method, provides a simple spectrum
with a protonated molecule detected at m/z 377 and a sodium adduct ion detected at
m/z 399, and almost no fragment ions.
• However, because use of API tends not to produce fragment ions, it seems difficult to
obtain structural information of functional groups and others by analyzing the
fragment ions.