GCMS

GGAASS CCHHRROOMMAATTOOGGRRAAPPHHYY--
MMAASSSS SSPPEECCTTRROOMMEETTRRYY ((GGCC--MMSS))
Presented by
APARNA.T UNDER THE GUIDANCE OF
Mr. Ch. DEVADASU M.Pharm.,
Assistant professor
Department of PA & QA
VIGNAN PHARMACY COLLEGE
(Approved by AICTE, PCI &
Affiliated to JNTU-K)
VADLAMUDI, 522213.
VIGNAN PHARMACY COLLEGE 13/9/2014

 IUPAC: chromatography is a physical method of separation in which the
components to be separated are distributed between two phases. One of
which is stationary (stationary phase) while the other (the mobile phase)
moves in a definite direction.
 Elution chromatography is a procedure in which the mobile phase is
continuously passed through or along the chromatographic bed and the
sample is fed into the system as a finite slug. EX: Gas Liquid
Chromatography(GC)
 Gas chromatography is a separation method in which the components
of a sample partition between two phases one of these phases is a
stationary bed with a large surface area, and the other is a gas which
percolates through the stationary bed.
VIGNAN PHARMACY COLLEGE 13/9/2014 2

 The father of modern gas chromatography is Nobel Prize
winner John Porter Martin, who also developed the first
liquid-gas chromatograph. (1950)

In GC the main principle of separation is partition.
1. A gaseous mobile phase flows continuously through
the column which is coated with a liquid stationary
phase.
2. The sample is introduced into the heated injection
port where it is vaporized and carried in to the column.
3. The sample partition between the mobile phase and
stationary phase, and is separated in to individual
components based on relative solubility in liquid
stationary phase at the given temperature.
4. The components of the sample separate from one
another based on their relative vapor pressures and
affinities for the stationary bed.

THE CHROMATOGRAPHIC PPRROOCCEESSSS -- PPAARRTTIITTIIOONNIINNGG
(gas or liquid)
MMOOBBIILLEE PPHHAASSEE
SSTTAATTIIOONNAARRYY PPHHAASSEE
Sample
out
Sample
in
(solid or heavy liquid coated onto a solid or support system)

In the animation below the red molecules are more soluble in the
liquid (or less volatile) than are the green molecules.

Distribution Coefficient
Definition:
Concentration of component A in stationary phase
Concentration of component A in mobile phase
Different affinity of any 2 components to
stationary phase causes the separation.

 G. C is a separation technique.
 Small amounts of sample for example 1 ml of air,1 micro
lit. of the solution either liquids and solids in solution are
injected in to an Instrument .
 The machine is called a Gas chromatograph
 this machine by using injection port, column and detector
generates a written record of analysis .. a series of peaks .
Series of peaks are called a chromatogram.
 Chromatogram is simply a written record of the
analysis performed by the gas chromatograph.

The concept of mass spectrometry was first put forth by
Sir J.J Thomson, English Physicist Who discovered the
electron in 1887.
He got 1906 Nobel Laureate in Physics.
DEMPSTER
Sir J.J Thomson

What does a mass spectrometer do?
Mass –spec or simply MS is a super important technique
Mass spec is easy technique to give you Molecular weight
(from molecular ion (M+)
You can get Molecular formula (Elements present).
Nearly ALL ELEMENTS in the periodic table can be determined by
mass spectrometry.
MS is incredibly valuable in getting structure (from fragments) of Bio
molecules such as peptides and proteins and also
natural products and also organic structures.
It can give information about chemical structures.

THEORY
The mass spectrometer is an instrument which help in separating the individual
atoms or molecules because of difference in their masses.
17
Consider a molecule M, Which is bombarded with a beam of electrons
M + e- M+. +2e-where,
M+. is molecular ion or radical ion
2e- is electron
Now voltage “v” is applied in an electric field then ions are accelerated. In this
condition the energy given to each particle is zV and this is equal to kinetic
energy which is equal to 1/2mv2 .

i.e. potential energy=kinetic energy
zV = 1/2mv2
2zV = mv2
2zV/m= v2
= v
Where V = Velocity of particle
m = mass
z = charge of an electron
V = Acceleration voltage
All the particles posses some energy zV with some kinetic energy
1/2mv2, but m value changes from molecule to molecule with respect velocity
‘v’ also changes. i.e. ½ mv=zV

When all charged particles have been accelerated by an applied voltage, they
enter into a magnetic field “H”. Then attractive force is HzV. And balancing
force of particle is mv2 /r.
Centripetal = Centrifugal
HzV=mv2/r
Hz=mv/r
From the above equation v=
Hz=m / r
By squaring on both sides
H2 z2 = m2 (2zV/m) / r2
H2 z = 2v m/ r2
m/z = H2 r2 / 2v

MASS SPECTRUM
The mass spectrum is the plot of mass to charge ratio of positively charged
ions against their relative abundance. The m/z ratio are taken along the
abscissa, while relative abundance is taken on ordinate.
BASE PEAK:
The most intense peak in the mass spectrum is called the base peak. Base
peak is the highest peak it is assigned a relative intensity of 100%.

MOLECULAR ION PEAK:
The ion formed from a molecule by removal of one electron of lowest
ionization potential is known as molecular ion.
The molecular ion is detected as mass to charge ratio that corresponds to
molecular weight of molecule. The molecular ion peak gives the molecular
weight of compound . The molecular ion peak is highest mass number except
isotope peak. Base peak Molecular ion peak
Fragment ions

FRAGMENT IONS:
The ions produced from the molecular ion by cleavage of bonds are called
fragment ions
They have lower masses and used as building blocks to reconstruct the
molecular structure. Fragmentation of molecular ion cleavage bond occurs in
heterolytic and homolytic cleavage.
METASTABLE IONS:
Mass spectrum of molecule shows sharp peaks at m/z integrals. But some show
diffuse, broad low intensity peaks at non integral m/z values these are called
metastable ions
m1
+ m2
++ neutral fragment

If in the reaction m1
+--------->m2
++ takes place in source then the
daughter ion may be m2
+. But m1
+----->m2
++ if occurs after the source and
before arrival at collector at lower mass than m2+ and is said to be
metastable ion m*.The peak (m*) due to such fragmentation therefore
occurs at lower mass than m2+ and generally broad. The relation between
the m* with that of m1
+ & m2+ can be written as
m*=(m2)2/m1

Relative abundance
108 110
29 –C2 H5
79 81
m/z values
M+2
M+
CH3CH2Br

Gas chromatography–mass spectrometry (GC-MS, or
alternatively HPLC-MS) is an ADVANCED ANALYTICAL
INSTRUMNTAL technique that combines the physical
separation capabilities of GGAASS CCHHRROOMMAATTOOGGRRAAPPHHYY with
the mass analysis capabilities of MASS SPECTROMETER

27
Gas
chromatograp
hy
Mass
spectrometry
GC-MS
Separates
mixture of
components
into individual
Identifies
molecules
based on their
mass
A chemical
analysis technique
combining two
instruments to
provide for
powerful
separation &
identification.

Coupling of GC to
GCM S:
Atmospheric
density
heated (200-
300 ∘C)
Interfaces
MS
High vacuum
(10-6 torr)
heated
The interface b/w the GC&MS is an important
role to play in the overall efficiency of the
instrument.
Both system are heated (200 -300 ∘C) both deal
Ownitlhy coonme pporuonbdlesm in i st hteh avta tphoer asttamtoes. pheric pressure
output of the GC must be reduced to vacuum of
10-5 – 10-6 torr for the MS inlet

Types of interfaces:

Capillary direct interface:
Today most GC-MS systems use capillary columns &
Capillary direct interface:
Today most GC-MS systems use capillary columns &
fused silica tubing permits an inert,high efficiency,direct
transfer between the 2 systems.
fused silica tubing permits an inert,high efficiency,direct
transfer between the 2 systems.
Flow rates is 5ml/min.
Flow rates is 5ml/min.

Jet separator (packed column):
•The separator consist of two glass tubes aligned with a
Small distance between them.
•Carrier gas entering from the GC column is pumped away
by a separate vacuumed system.
•The larger sample molecules maintain their momentum
&pass preferentially in to the second capillary.
•Sample enrichment occurs & the initial atmospheric
pressure is reduced.

Bothe T & P surfaces activity of the glass jet separator must be controlled.

Watson & Biermann effusion
separator:
• It consists of a sintered glass tube .
• The carrier usually Helium, passes preferentially
through the sintered glass tube & the effluent in
concentrated by a factor of up to 100.
• The gas flow rates in the order of 20-60ml/min.

It converts the components of a sample into ions by
bombardment with electrons, ions, molecules.
IONIZER;
CH3OH + 1e CH3OH+ + 2e
molecular ion or radical ion
The gas molecules exiting the GC are bombarded by a
high energy electron beam.

 An electron which sticks a molecule may impart enough
energy to remove another electron from that molecule.
 The charged molecule is known as molecular ion.
 The molecular ion can causes that ion to break into
smaller pieces.
CH3OH+ CH2OH+ + H-VIGNAN
PHARMACY COLLEGE 13/9/2014 35

 The most common form of ionization is EI.
 Electrons are produced by tungsten filament.
 These electrons accelerated towards the ion source chamber.
 The electrons require an energy equal to the voltage B/W the
filament & ion source chamber.
 70 ev is commonly used.
 A proportion of electron beam will strike the electron trap
producing trap current.

 A permanent magnet is positioned across the ion chamber to
produce a magnetic flux in parallel to the electron beam.
 A (+)ve ion repelle voltage & (-)ve ion excitation voltage
works to gather to produce an electric field in the source
chamber.
 Such that ions leaves through ion exit slit.
 The ions are directed through the various focusing &
centering lenses are focused on to the source exit slit.

 In CI a reagent gas methane or ammonia or isobutene
are introduced into the mass spectrometer.
 The reagent gas will interact with the electron to produce
radical electrons.
EG;
R + e R+ + 2e

 It is a good for organic compounds.
 Usually produces (M + H)+ (M+ CH3)+ adducts.
 Adducts are not always abundant.
ISOBUTANE
 Usually produces (M + H)+ ,(M+C4H9)+ adducts & some
fragmentation.
 Adducts are more abundant than for methane CI .

 Fragmentation is absent.
 Polar compounds produces ( M+NH4)+ adducts.
 Basic compounds produces (M+H)+ adducts.
 Non polar, non basic compounds are not ionized.

44
Ionization
method
electron
impact
Chemical
ionization
Typical analyses Relatively small
volatile
Relatively small
volatile
Sample
introduction
GC (or) liquid/solid
probe
GC/Liquid /solid
probe
Mass range 1-1000 Dolton's 1-1000 Daltons
Method highlights Hard method
versatile
provides structure
information
Soft method
molecular ion peak
(M+H)+

45
Negative ion chemical ionization(NICI):
In NICI a r eagent gas is used & the electrons collide with it so
that their energies are reduced to 10Ev.
Molecules with a high affinity for electrons are able to
capture these low energy thermal electrons.
This is known as NICI but it does not involved in the
formation of a chemical adduct.

46
AB+ e
AB-Resonance
electron capture
A- +B
Dissociative
electron capture.

47
They deflects ions down a curved tubes in a magnetic fields based on
their kinetic energy determined by the mass, charge and velocity. The
magnetic field is scanned to measure different ions.

 In a quadrupole mass analyser a set of four rods are arranged parallel to
the direction. Here a DC current and radio frequency RF is applied to
generate oscillating electrostatic field in between the rods. Based on this
only m/z is been determined and stable oscillation takes place. And ion
travels in quadrupole axis with cork screw type of trajectory.
48

 TOF mass analyser is based on simple idea that the velocities of
two ions are created by uniform electromagnetic force applied to
all the ions at same time, causing them to accelerate down a flight
tube.
 Lighter ions travels faster and strike the detector first so that the
m/z ratio of ions is detected.
49

The ion trap mass analyser operates by similar principles where it
consists of circular ring electrode
Plus two end caps that form a chamber. Here AC or DC power
along RF potential is applied between the cups and the ring
electrode.
There the ions entering into the chamber are trapped by
electromagnetic fields and they oscillates in concentric
trajectories. This process is called resonant ejection.

DATA HANDLING
All the mass spectrometers now employ computer control of same functions
and also use a computerised display and output.
The amount of data generated even by a fairly modest mass spectrometer is
very large indeed, a single run may store data for upto 100 fragments from
each type of molecule and if, GCMS analyses is being performed, a complete
mass spectrum is generated and stored every sec for upto 90 min

54
LIMITATIONS

•Only Compounds with Vapour Pressure exceeding about 1010 torr
can be analyzed b as chromatography –mass spectrometry.
•Certain isomeric compounds cannot be distinguished by mass
spectrometry (EG : naphthalene vs. azulene).

 Elucidation of the structure of organic & biological
molecules.
 Impurity profiling of pharmaceuticals.
 Identification of components in thin layer & paper
chromatograms.
 Identification of drugs of abuse & metabolites of drugs of abuse
in blood, urine & saliva.
 Testing for the presence of the drugs in blood in race horses &
in Olympic athletic (in forensic GC-MS).
 Analyzer of aerosol particles.
 Determination of pesticide residues in food.
 Polymer characterization (pyrolysis methods combined GCMS).
 Drug monitoring & toxicology studies.

The TIC GC chromatogram for Psoralen and the EI
spectrum obtained from the chromatographic peak

EI CHLOROQUIN
NICI OF CHLOROQUIN

Impurities can arise from
•Manufacturing process
•Degradation of drug substance
If impurities are greater than 0.1% concentration in drug substance
the name and structure of impurity is to be submitted to regulatory agencies.
USFDA CONSIDERATIONS:
IMPURITIES SHOULD BE LESS THAN 1.0%
Presence of impurities must be illustrated with GC-MS/LC-MS chromatograms

1-naphthol

 GC-MS is becoming the tool of choice for tracking organic
pollutants in the environment.

 GC-MS can analyze the particles from a human body in order
to help link a criminal to a crime.
 GC-MS especially useful here as samples often contain very
complex matrices &results used in court.

 GC-MS used for detection of illegal narcotics & may
eventually supplant drug-sniffing dogs.
 It’s also commonly used in forensic toxicology to find drugs
&poisons in biological specimens of victims .

 GC-Ms is main tool used in sports anti doping
laboratories to test athletes urine samples for prohibited
performance enhancing drugs.
EG : anabolic steroids.

 Food & beverage contain numerous aromatic compounds ,
some naturally present in the raw materials &some forming
during process.
 GC-MS is extensively used for the analysis of these
compounds which include ester, fatty acids , alcohols,
aldehydes, terpenes etc……

 GC-MS 2 were brought to mars by the Viking program.
 Venera11&12 pioneer Venus analyzed the atmosphere of
Venus with GC-MS.
 The material in the comet 67p will be analyzed by the rosetla
mission with a chiral GC-MS in 2014.

 In born errors of metabolism are now detectable by new born
screening tests, especially the testing using GC-MS .
 It can determine compounds in urine even in minor
concentration.
 The measurement of c13-c12 ratio with an isotope ratio mass
spectrometer.
13/9/2014 75
VIGNAN PHARMACY COLLEGE

 GC-MS is one of the best analytical tool iinn aannaallyyssiinngg tthhee
mmoosstt ooff tthhee ccoommppoouunnddss. GGCC--MMSS aass aann aannaallyyttiiccaall mmeetthhoodd
ooff ooppttiioonn iiss iittss iinnccrreeaasseedd sseennssiittiivviittyy && rreelliiaabbiilliittyy eevveenn iinn
vveerryy ssmmaallll qquuaannttiittiieess ((nngg)).

 M.Mcnair,M.Miller, basic gas chromatography- 2nd
Edition. (Pg.No 156-169)
 David G.Watson, Pharmaceutical analysis. (Pg.No 202-
206).
 AH Beckett, J.B Stenlake, Pharmaceutical chemistry 4th
Edition-Part two (Pg.No 474-477).
 Skoog, Holler, Lrouch, Instrumental-Analysis.
 (Pg.No 606-629).

 Gurdeep R.Chatwal, K.Anand, Instrumental methods of
chemical analysis (Pg.No 2.272,2.673)
 Pavia,Lampman, Kriz, (Pg.No 401-415).
 B.K Sharma, Instrumental methods of chemical analysis
(Pg.No 844-938,180-224)
 Dr.S.Ravi Sankar text book of pharmaceutical analysis
3rd Edition (Pg.No 8.1,17.1)

AACCKKNNOOWWLLEEDDGGEEMMEENNTTS
I sincerely thank my guide
Mr. Ch. DEVADASU sir
for his constant guidance & support.
I thank our respected Principal
Dr. P.SRINIVASA BABU
& seminar Committee for giving me
this opportunity.

GCMS

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