gas chromatography-mass spectrometry (GC-MS) hyphenated technique

ADVANCED SPECTRAL ANALYSIS (MPC201T) UNIT-IV_CHROMATOGRAPHY
Prepared by: - Subham Kumar Vishwakarma (shubhamkumarvishwakarma7@gmail.com), Guided by: - Dr. S Raja, Gitam
University Visakhapatnam (AP)
1
TABLE OF CONETENT
S.
NO.
TOPIC CONTENT
A. GC-MS (Gas Chromatography–mass
spectrometry)
• Principle,
• Instrumentation
• Applications
B. GC-AAS (Gas Chromatography-Atomic
Absorption spectrometry)
C. LC-MS (Liquid Chromatography-Mass
spectrometry)
D. LC-FTIR (Liquid Chromatography-Fourier-
Transform Infrared spectrometry)
E. LC-NMR (Liquid Chromatography-Nuclear
Magnetic Resonance)
F. CE-MS (Capillary Electrophoresis-Mass
Spectroscopy)
G. HPTLC (High Performance Thin Layer
chromatography)
H. Super critical fluid chromatography
I. Ion Chromatography
J. I-EC (Ion - Exclusion Chromatography)
K. Flash chromatography

2
Chromatography
Introduction: -
• Chromatography is derived from the Greek word ‘chroma’ means ‘color’ and ‘graphein’
means writing or recording.
• Chromatography, literally "color writing", was first employed by Russian-Italian scientist
Mikhail Tsvet in 1900, primarily for the separation of plant pigments such as chlorophyll,
carotenes, and xanthophylls.
• Separation of chemical constituents according to affinity for stationary phase and mobile
phase.
• Chromatography is an important biophysical technique that enables the separation,
identification, and purification of the components of a mixture for qualitative and
quantitative analysis.
• Chromatography separates a component of mixture which is dissolved in a substance
called the mobile phase and is carried out by a second substance called the stationary
phase.
Terminology: -
Term Definition
Mobile phase or carrier solvent moving through the column
Stationary phase or adsorbent substance that stays fixed inside the column
Eluent fluid entering the column
Eluate fluid exiting the column (that is collected in flasks)
Elution the process of washing out a compound through a column using a
suitable solvent
Analyte mixture whose individual components have to be separated and
analysed
Instrument Chromatograph
Result Chromatogram
Retention time Time from start of Chromatography to maximum solution of
solute (retention time Vs peak intensity)
Retardation factor (RF) It is the fraction of time that solute spends in mobile phase

3
Principle: -
• Chromatography is usually based on principle of partition of solute between two phases. It
usually consists of a Mobile Phase and a Stationary Phase.
• The Mobile Phase usually refers to the mixture of the substances to be separated dissolved in
a liquid or a gas.
• The Stationary Phase is a porous solid matrix through which the sample contained in the mobile
phase percolates.
OR
• The principle of chromatography is like-dissolve-like or like-prefer-like. The basis of all forms
of chromatography is the partition or distribution coefficient ‘K’ which describes the way in
which a compound distributes itself between two immiscible phases. The Partition coefficient;
is defined as the molar concentration of analyte in the stationary phase divided by the molar
concentration of the analyte in the mobile phase
𝐾 =
Concentration of component in stationary phase
Concentration of component in mobile phase
• The distribution of analytes between phases can be described simply. An analyte is in
equilibrium between the two phases;
Amobile ⇌Astationary
Different types of Chromatography
Technique Stationary
phase
Mobile
phase
Basis of
separation
Notes
Paper
Chromatography
solid
(cellulose)
Liquid polarity of
molecules
Compound spotted directly
on a cellulose paper
Thin layer
chromatography
solid (silica
or alumina)
Liquid Polarity of
molecules
glass is coated with thin layer of silica on
which is spotted the compound
Liquid column
chromatography
solid (silica
or alumina)
Liquid polarity of
molecules
glass column is packed with slurry of silica
Size exclusion
chromatography
solid
(microporous
beads of
silica)
Liquid size of
molecules
small molecules get trapped in
the pores of the stationary phase, while
large molecules flow through the gaps
between the beads and have very small
retention times. So larger molecules come
out first. In this type of chromatography
there isn’t any interaction, physical or
chemical, between the analyte and the
stationary phase.
Ion-exchange
chromatography
Solid
(cationic or
anionic
resin)
Liquid Ionic charge
of
The
molecules
Molecules possessing the opposite charge
as the resin will bind tightly to the resin,
and molecules having the same charge as
the resin will flow through the column and
elute out first.

4
Affinity
chromatography
solid
(agarose or
porous glass
beads on to
which are
immobilized
molecules
like enzymes
and
antibodies)
Liquid Binding
affinity of
the analyte
molecule to
the
molecule
immobilized
on the
stationary
phase
if the molecule is a substrate
for the enzyme, it will bind tightly to the
enzyme and the
unbound analytes will pass through in the
mobile phase, and elute out of the column,
leaving the substrate bound to the enzyme,
which can then be
detached from the stationary phase and
eluted out of the column with an
appropriate solvent
Gas
chromatography
Liquid or
solid
support
Gas
(inert
gas
like
argon
or
helium)
Boiling
point of the
molecules
samples are volatilized and the molecule
with lowest boiling point comes out of the
column first. The molecule with the
highest boiling point comes out of the
column last.

5
INTRODUCTION: -
• Gas Chromatography – Mass Spectrometry (GC-MS) is an analytical method that combines the
features of gas- liquid chromatography and mass spectrometry to identify different substances
with in a test sample.
• Gas chromatography is a technique used to separate drugs that might be present in a sample. The
detector for the GC is the Mass spectrometry detector.
• Mass spectrometer (MS) is an instrument that serves for establishment of the molecular weight
and structure of both inorganic and organic compounds, and the identification and determination
of analytes in complex mixtures.
Principle: -
• GC-MS instrument separates chemical mixtures (the GC component) and identifies the
components at a molecular level (the MS component).
• GC works on the principle that a mixture will separate into individual substances when heated.
The heated gases are carried through a column with an inert gas.
• As the separated substances emerge from the column opening, they flow into MS.
• Mass spectrometry identifies compounds by the mass of the analyte molecule.
OR
✓ The Gas Chromatography/Mass Spectrometry (GC/MS) instrument separates chemical
mixtures (the GC component) and identifies the components at a molecular level (the MS
component). It is one of the most accurate tools for analyzing environmental samples.
✓ The GC works on the principle that a mixture will separate into individual substances when
heated. The sample is injected into the GC inlet where it is vaporized and swept into a
chromatographic column by the carrier gas (helium).
✓ The sample flows through the column and the compounds comprising the mixture of interest
are separated by virtue of their relative interaction with the coating of the column (stationary
phase) and the carrier gas (mobile phase).
✓ The latter part of the column passes through a heated transfer line and ends at the entrance to
ion source where compounds eluting from the column are converted to ions. A beam of
electrons ionize the sample molecules resulting in the formation of molecular ion and smaller
ions with characteristic relative abundances that provide a ‘fingerprint’ for that molecular
structure. The mass analyser separates the ions and is then detected.
Instrumentation: -

6
Fig. Gas chromatography
Fig. Mass spectrometer

7
or
Fig. This schematic shows the major components of an early gas chromatograph-mass spectrometer
The GC-MS is composed of two major building blocks:
• Gas chromatograph
• Mass spectrometer
• The molecules are retained by the capillary column and elute from the column at different
times.
• The Mass spectrometer capture, ionize, accelerate, deflect and detect the ionized molecules
separately by breaking each molecule into ionize fragments and detecting these fragments
using their mass to charge ratio.
GAS CHROMATOGRAPHY-MASS SPECTROMETRY: -
✓ Carrier gas
✓ Flow regulators & Flow Meters
✓ Injection devices
✓ Columns
✓ GC-MS interface
✓ Ionization (Ion source)
✓ Mass analyzer
✓ Detectors
CARRIER GAS: -
Requirements of a carrier gas: -
• Inertness
• Suitable for the detector
• High purity

8
• Easily available
• Cheap
• Should not cause the risk of fire
• Should give best column performance
1. Hydrogen
better thermal conductivity
disadvantage: it reacts with unsaturated compounds & inflammable
2. Helium
excellent thermal conductivity
it is expensive
3. Nitrogen
reduced sensitivity
it is inexpensive
Flow regulators & Flow meters: -
Deliver the gas with uniform pressure/flow rate - Rota meter & Soap bubble flow meter
✓ Rota meter: - placed before column inlet
it has a glass tube with a float held on to a spring. the level of the float is determined by the
flow rate of carrier gas.
✓ Soap Bubble Meter: -Similar to Rota meter & instead of a float, soap bubble formed indicates
the flow rate.
Injection Devices: -
✓ The injection port consists of a septum through which a syringe needle is inserted to inject the
sample.
✓ The sample is injected into a stream of inert gas usually at an elevated temperature by a micro
syringe.
✓ The vapourised sample is carried into a column packed with the stationary phase.
✓ To ensure rapid & complete solute volatilization temp of injector → 30-50 degree Celsius
>column temp
The injection port Is a hollow, heated, glass-lined cylinder: -
✓ The injector is heated so that all components in the sample will be vaporized.
✓ If the temperature is too low, separation is poor and broad spectral peaks should result or no
peak develops at all.

9
✓ If the injection temperature is too high, the specimen may decompose or change its structure.
✓ The temperature of the sample port is usually about 50°C higher than the boiling point of the
least volatile component of the sample.
Injector Types
1. Split/ Splitless Injector
2. On-Column Injector
3. High Oven Temperature On-Column Injector
4. Large Volume On-Column Injector
5. Packed Column Injector
6. Purged Packed Injector
7. Programmable Temperature Vaporizing Injector
1. Split mode
✓ The split vent is open, part of the sample goes into the column.
✓ When analyzing high concentration or neat samples.
✓ Yields the sharpest peaks if the split gas is properly mixed.
✓ Standard for capillary columns.
Split-less mode
✓ The split vent is closed, most of the sample go into the column.
✓ When analyzing low concentration or diluted samples.
✓ Splitless times of ~ 1 minute are typical.
✓ Standard for capillary columns.

10
2. Large Volume on column injector: -
✓ LV-SL injection overcomes the limitation of the maximum sample volume to 1-2 µL of
classical splitless injection by exploiting the Concurrent Solvent Recondensation technique
(CSR).
✓ CSR technique allows injection of large volumes by combining a restricted evaporation rate
with an accelerated sample transfer granted by the pressure surge generated by solvent
evaporation and by the quick solvent recondensation in a precolumn.
LV-SL has the following advantages: -
• It is simpler because it allows injections of up to 50 µL in a conventional split/split-less injector
without any special tuning of operating parameters;
• It is robust versus sample by-products or contaminants and extremely suitable for food
matrices.
COLUMNS: -
✓ Column is one of the important parts of GC which decides the separation efficiency. Columns
are made up of glass and stainless steel.
✓ Is where the chromatographic separation of the sample occurs.
✓ Several types of columns are available for different chromatographic applications.
✓ The heart of the system.
✓ It is coated with a stationary phase which greatly influences the separation of the compounds.
✓
Classification of columns: -
Depending on its use:
✓ Analytical column
✓ Preparative column
Depending on its nature Column Types: -
✓ Packed column: - Are available in packed manner commercially and hence are called as
Packed column. Different columns ranging from non-polar to polar are available.

11
✓ Open tubular/ Capillary Column: - They are made up of long capillary tubing of 30-90
meters in length and have diameter of 0.025 to 0.075 cm. These are made up of stainless steel
and are in the form of a coil. The inner wall of the capillary is coated with stationary phase
liquid in the form of a thin film.
GC-MS interface: -
There are four types of interfaces available:
✓ JET SEPREATOR
✓ PERMSELECTIVE MEMBERANE
✓ MOLECULAR EFFUSION
✓ DIRECT INTRODUCTION
JET SEPARATOR: -
In these separators, the GC flow is introduced into an evacuated chamber through a restricted
capillary. Light particle dispersed away.
Permselective membrane interface: -
It is made of a silicone-rubber membrane that transmits organic non-polar molecules and acts as a
barrier for (non-organic) carrier gases.

12
The molecular effusion /Watson-Biemann interface: -
It is based on the molecular filtering of the gas effluent by means of a porous glass fritted tube.
DIRECT INTRODUCTION: -
In this method capillary column is directly inserted into MS ionisation chamber.
IONIZATION
1. Electron Impact (EI)
2. Chemical Ionization (CI)
3. Field Desorption (FD)
4. Fast Atom Bombardment (FAB)
5. Laser Desorption (LD)
6. Electrospray Ionization (ESI)
7. Matrix Assisted Laser Desorption (MALD)

13
A. Electron Ionization: -
✓ Historically, ions for mass analysis were produced by electron ionization, formerly called
electron-impact ionization.
✓ In this process, the sample is brought to a temperature high enough to produce a molecular
vapor, which is then ionized by bombarding the resulting molecules with a beam of
energetic electrons.
✓ Electrons are emitted from a heated tungsten or rhenium filament and accelerated by
applying approximately 70 V between the filament and the anode.
✓ As shown in the figure, the paths of the electrons and molecules are at right angles and
intersect near the center of the source, where collision and ionization occur.
✓ The primary product is singly charged positive ions formed when the energetic electrons
approach molecules closely enough to cause them to lose electrons by electrostatic
repulsion. Electron ionization is not very efficient, and only about one molecule in a million
undergoes the primary reaction.
M + e-
M'+
+2e-
Here, M represents the analyte molecule, and M#1 is its molecular ion. The positive ions produced
by electron ionization are attracted through the slit in the first accelerating plate by a small potential
difference (typically 5 V) that is applied between this plate and the repellers they enter the mass
analyzer.

14
B. Chemical Ionization (CI): -
✓ In chemical ionization–mass spectrometry (CI-MS), the sample molecules are combined
with a stream of ionized reagent gas that is present in great excess relative to the sample.
✓ When the sample molecules collide with the pre-ionized reagent gas, some of the sample
molecules are ionized by various mechanisms, including proton transfer, electron transfer,
and adduct formation. Almost any readily available gas or highly volatile liquid can be used
as a reagent gas for CI-MS.
✓ Common ionizing reagents for CI-MS include methane, ammonia, isobutane, and
methanol.

15
Mass Analyzers: -
✓ To separate the ions produced in the ion source acc. to their mass/charge ratio.
✓ Ideally mass analyzer should be capable of distinguishing small mass differences.
✓ It should also allow passage of a sufficient number of ions to yield radially measurable ion
current.
1. The Magnetic Sector Mass Analyzer: -
The kinetic energy of an accelerated ion is equal to
1
2
𝑚𝑣2
= ȥ𝑉 ……………………(1)
where m is the mass of the ion, v is the velocity of the ion, z is the charge on the ion, and V is the
potential difference of the ion-accelerating plates. In the magnetic sector mass analyzer (Fig. 3.11),
the ions are passed between the poles of a magnet. In the presence of a magnetic field, a charged
particle describes a curved flight path. The equation that yields the radius of curvature of this path is
r =
𝑚𝑣
ȥ𝐵
……………………(2)
where r is the radius of curvature of the path, and B is the strength of the magnetic field. If these two
equations are combined to eliminate the velocity term, the result is
𝑚
𝑧
=
𝐵2𝑟2
2𝑉
……………………(3)
As can be seen from Equation 3, the greater the value of m/z, the larger the radius of the curved path.
The analyzer tube of the instrument is constructed to have a fixed radius of curvature. A particle with
the correct m/z ratio can negotiate the curved analyzer tube and reach the detector.
2. Double-Focusing Mass Analyzers: - For many applications, much higher resolution is needed
and can be achieved through modifications of this basic magnetic sector design. In fact, magnetic

16
sector analyzers are used today only in double focusing mass spectrometers. The particles leaving
the ionization chamber do not all have precisely the same velocity, so the beam of ions passes
through an electric field region before or after the magnetic sector (Fig. 3.12). In the presence of
an electric field, the particles all travel at the same velocity. The particles describe a curved path
in each of these regions, and the resolution of the mass analyzer improves—by a factor of 10 or
more over the magnetic sector alone.
3. Quadrupole Mass Analyzers: - In a quadrupole mass analyzer (Fig. 3.13), a set of four solid
rods is arranged parallel to the direction of the ion beam. The rods should be hyperbolic in cross
section, although cylindrical rods may be used. A direct-current (DC) voltage and a radiofrequency
(RF) is applied to the rods, generating an oscillating electrostatic field in the region between the
rods. Depending on the ratio of the RF amplitude to the DC voltage, ions acquire an oscillation in
this electrostatic field. Ions of an incorrect m/z ratio (too small or too large) undergo an unstable
oscillation. The amplitude of the oscillation continues to increase until the particle strikes one of
the rods. Ions of the correct mass-to-charge ratio undergo a stable oscillation of constant amplitude
and travel down the quadrupole axis with a “corkscrew”-type trajectory. These ions do not strike
the quadrupole rods but pass through the analyzer to reach the detector. Like the magnetic sector
analyzer, the quadrupole can be scanned from high to low values of m/z. A quadrupole mass
analyzer is found in most “benchtop” GC-MS systems and typically has a m/z range from 0 to
1000, although quadrupole analyzers are available on some LC-MS systems with m/z ranges that
approach 2000. Quadrupole mass spectrometers are low-resolution instruments (R~3000)
incapable of providing exact elemental composition of the sample but their relatively low cost
makes them popular for many applications. An additional drawback is their relatively slow
acquisition rate due to their scanning nature.

17
4. Time-of-Flight Mass Analyzers (TOF): -
✓ TOF Analyzers separate ions by time without the use of an electric or magnetic field.
✓ Analyzation is based on the kinetic energy and velocity of the ions.
✓ Separates ions based on flight time in drift tube.
✓ Positive ions are produced in pulses and accelerated in an electric field (at the same
frequency), all particles have the same kinetic energy.
K.E. = zV = 1/2 mv2
Solving for velocity (v)
v = (2zV/m)1/2
The transit time (t) through the drift tube is L/v where L is the length of the drift tube (usually 1‐
3 meters).
t =L (m/z)1/2
(1/ 2V)1/2

18
Application of GC-MS
• Petrochemical and hydrocarbons analysis
• Geochemical research
• Forensic (arson, explosives, drugs, unknowns)
• Environmental analysis
• Pesticide analysis and Food safety
• Pharmaceutical and drug analysis
• Clinical toxicology
• Food and fragrance
• High end research GC-MS
• Service and institution GC-MS analysis
…………………………………. ………………………………….

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