Chromatography(gc ms & lc ms)

• Chromatography is the science which is studies
the seperation of molecules based on
differences in their structure and/or
composition.
• Chromatography was first developed and
defined by the Russion Botonist Mikhail
Tswett in 1903. He produced a colourful
seperation of plant pigments using a column of
calcium carbonate(chalk).

 Charged molecules or molecular fragments are
generated in a high vacuum or immediately prior to a
sample entering a high vacuum using a variety of
methods for ion production.
 magnetic fields to enable the determination of their
molecular weight (m/z).The history of mass
spectroscopy (MS) started in 1912 when Thomson obtd.

 Combining the two processes reduces the possibility of error, as it is extremely
unlikely that two different molecules will behave in the same way in both a gas
chromatograph and a mass spectrometer.
 Therefore, when an identifying mass spectrum appears at a characteristic retention
time in a GC-MS analysis, it typically lends to increased certainty that the analyte
of interest is in the sample.

 Gas Chromatography was first described in 1952 by James
and martin with the separation of a mixture of small
carboxylic acids.
 The power of GC was substantially enlarged by the
introduction of open capillary columns in 1958 by Golay.
 The introduction of the fused-silica capillary column in
1976 by Dandeneau and Zerner can be considered as a
breakthrough in the development of GC.
 The use of a mass spectrometer as the detector in gas
chromatography was developed during the 1950s by
Roland Gohlke and Fred McLafferty.

• It is an instrumental method for the seperation and
identification of chemical compounds.
• Gas chromatography is a chromatographic
technique that can be used to separate volatile
organic compounds.
• The organic compounds are seperates due to
differences in their partitioning behaviour between
the mobile phase and the stationary phase in the
column.

 The sample solution is injected into the GC inlet
where it is vaporized and swept onto a
chromatographic column by the carrier gas (usually
helium).
 The sample flows through the column and the
compounds comprising the mixture of interest are
separated by 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.

 The GC-MS is composed of two
major building blocks: the gas
chromatograph and the mass
spectrometer.
 Carrier Gas, N2 or He, 1-2 mL/min
 Injector
 Oven
 Column
 Detector

 Inert
 Helium (hydrogen/ nitrogen).
 Choice dictated by detector, cost, availability
 Pressure regulated for constant inlet pressure(
below 0.3MPa).
 Flow controlled for constant flow rate
~20ml/min for packed column
~1ml/min for open capillary column
 Chromatographic grade gases (high purity)

State
 Organic compounds must be in solution for injection into the
gas chromatograph.
 The solvent must be volatile and organic.
Amount
 Depending on the ionization method, analytical sensitivities of
1 to 100 pg per component are routine.
Preparation
 Sample preparation can range from simply dissolving some of
the sample in a suitable solvent .

Purge and Trap
(Aqueous and Soils / Volatiles Preparation)
Courtesy of Environmental Conservation

Sonication
(Soils, Solids / Semivolatiles)
Courtesy of Environmental Conservation

Solid Phase Extraction
(Aqueous / Semivolatiles)
Courtesy of Stanford
Cartridge
Sample

Typical GC setup –
courtesy of Waters,
Inc.

 Capillary Columns
 Packed Columns

 Made of the highest purity fused silica obtained
with an external polyimide coating
 Length-10 to 100 m -depends on application.
 For fast analysis shorter column are applied e.g.-
for heat sensitive and for high boiling compound.
 Large columns are required for high resolution
seperation.

 A more polar stationary phase is applied for the analysis
of more polar compounds.
 So least polar column- CP-Sil 5 and CP-Sil 8 applied.
 For high resolution more polar stationary phase have to
be applied.
 In GC-MS low bleed columns are applied.

 These columns, less commonly used today, have
diameter of 1.6 to 9.5mm and a length of
between 1–3m.
 Manufactured from steel or glass, the internal
wall of the tube is treated to avoid catalytic
effects with the sample.
 They can withstand a carrier gas flow rate
within the range 10–40 mL/min.
 They contain an inert and stable porous support
on which the stationary phase can be
impregnated or bounded (between 3 and 20 per
cent).

 Two capillary tubes aligned with a small
space between them. (1 mm)
 A vacuum is created between the two tubes
using a rotary pump.
 The GC effluent enters the vacuum region,
those molecules which continue in the same
direction enter the second capillary tube and
continue to the ion source.

 The carrier gas molecules are more easily
diverted from the linear path by collisions.
 The analyte molecules are much larger and
carry more momentum.
 The surface of the separator must be inactive
and a reasonably even temperature.

packed columns
capillary columns

The insides of the GC-MS, with the column of the
gas chromatograph in the oven on the right.

• The gas chromatograph utilizes a capillary column
which depends on the column's dimensions (length,
diameter, film thickness) as well as the phase properties.
• The difference in the chemical properties between
different molecules in a mixture will separate the
molecules as the sample travels the length of the column.
• The molecules take different amounts of time (called the
retention time) to come out of (elute from) the gas
chromatograph, and this allows the mass spectrometer
downstream to capture, ionize, accelerate, deflect, and
detect the ionized molecules separately.
• The mass spectrometer does this by breaking each
molecule into ionized fragments and detecting these
fragments using their mass to charge ratio.

 Electron impact ionisation
 Chemical Ionisation

 The physics behind mass spectrometry is that a charged
particle passing through a magnetic field is deflected along a
circular path on a radius that is proportional to the mass to
charge ratio, m/e.

• Used to confirm molecular weight.
• Known as a “soft” ionisation technique.
• Differs from EI in that molecules are ionised
by interaction or collision with ions of a
reagent gas rather that with electrons.
• Common reagent gases used are Methane ,
Isobutane and Ammonia.
• Reagent gas is pumped directly into ionisation
chamber and electrons from Filament ionise
the reagent gas.

 The most common type of mass spectrometer (MS)
associated with a gas chromatograph (GC) is the
quadrupole mass spectrometer, sometimes referred
to by the Hewlett-Packard (now Agilent) trade name
"Mass Selective Detector" (MSD).
 Another relatively common detector is the ion trap
mass spectrometer.
 Other detectors may be encountered such as time of
flight (TOF), tandem quadrupoles (MS-MS).

• A mass spectrometer is typically utilized in one of two ways:
• Full Scan or Selective Ion Monitoring (SIM).
• The typical GC/MS instrument is capable of performing both
functions either individually or concomitantly, depending on
the setup the particular instrument.

GC/MS Data Processing
Important to Review Peak Integration
Chromatogra
m
(maximum
information content)
Final Report
GC/MS

Environmental Monitoring and
Cleanup
GC-MS is becoming the tool of choice for
tracking organic pollutants in the
environment. The cost of GC-MS
equipment has decreased significantly, and
the reliability has increased at the same
time, which has contributed to its increased
adoption in environmental studies. There
are some compounds for which GC-MS is
not sufficiently sensitive, including certain
pesticides and herbicides, but for most
organic analysis of environmental samples,
including many major classes of pesticides,
it is very sensitive and effective.

• GC-MS can analyze the particles from a human body in order
to help link a criminal to a crime. The analysis of fire debris
using GC-MS is well established, and there is even an
established American Society for Testing Materials (ASTM)
standard for fire debris analysis.
• GCMS/MS is especially useful here as samples often contain
very complex matrices and results, used in court, need to be
highly accurate.

Law Enforcement
GC-MS is increasingly used for detection of illegal narcotics, and may
eventually supplant drug-sniffing dogs. It is also commonly used in forensic
toxicology to find drugs and/or poisons in biological specimens of suspects,
victims, or the deceased.
Food, Beverage and Perfume Analysis
Foods and beverages contain numerous aromatic compounds, some naturally
present in the raw materials and some forming during processing. GC-MS is
extensively used for the analysis of these compounds which include esters, fatty
acids, alcohols, aldehydes, terpenes etc. It is also used to detect and measure
contaminants from spoilage or adulteration which may be harmful and which is
often controlled by governmental agencies, for example pesticides.

Astrochemistry
• Several GC-MS have left earth. Two
were brought to Mars by the Viking
program.Venera 11 and 12 and Pioneer
Venus analysed the atmosphere of Venus
with GC-MS.
• The Huygens probe of the Cassini-
Huygens mission landed one GC-MS on
Saturn's largest moon, Titan.The material
in the comet 67P/Churyumov-
Gerasimenko will be analysed by the
Rosetta mission with a chiral GC-MS in
2014.

• It is the combination of liquid
chromatography and the mass
spectrometry.
• In LC-MS we are removing the detector
from the column of LC and fitting the
column to interface of MS.
• In the most of the cases the interface used
in LC-MS are ionization source.

MOBILE PHASE
COLUMN
ION SOURCE MASS ANALYSER ION CLLECTION
SYSTEM
VACCUM
SYSTEM
DATA HANDLING
SYSTEM
EFFUENT

To reduce separation time to achieve fast analysis. (
From hours to minutes)
HOW?
The solutes must move faster through stationary phase MEANS
By increasing the mobility of the liquid mobile phase.

The MIGRATION VELOCITY of the liquid mobile phase should be
HIGH.
We can achieve faster migration velocities of liquid mobile
phase by-
1.Applying vacuum at the other end of the chromatographic column
2.Applying high pressure on the liquid mobile phase.
Application of higher pressure on liquid mobile phase
seems to be an easier and more practical way for achieving
fast analysis.

 Sample preparation generally consists of concentrating
the analyte and removing compounds that can cause
background ion or suppress ionization.
 Example of sample preparation include:-
 (1) on –column concentration to increase analyte
concentration.
 (2) desalting to reduce the sodium and potassium
adduct formation that commonly occurs in electro
spray.
 (3) filtration to separate a low molecular-weight
drug from proteins in plasma, milk, or tissue.

• Column type:-
• Specialized mode:-
• The use of di-functional or tri-functional silanes
to create bonded groups with two or three
attachement points leading to phases with higher
stability in low or higher pH and lower bleed for
LCMS
• Most widely used columns for LCMS are:-
(1) fast LC column.
the use of short column. (15-50mm)
(2) Micro LC column.
the use of large column. ( 20-150mm)

• Glass is normally the choice for chromatographic
column material due to its chemical inertness towards
stationary phase, mobile phase and solutes being
separated.
• We could not increase the pressure of a glass column?
• Hence glass will not be able to withstand high
pressures of liquid mobile phase because glass has
relatively small mechanical strength.

• Which IDEAL MATERIAL should be selected in place of
glass?
• STAINLESS STEEL due to its chemical inertness &
mechanical strength.

48
Previous condition :- Liquid mobile phase moving
across the stationary phase in a glass column under
atmospheric pressure.
Present condition :- Liquid mobile phase with high
migration velocity moving across the stationary
phase in a stainless steel column under high
pressure..
Will the column separation efficiency of the
latter be good?
NO

With the increase in the liquid mobile
phase velocity under high pressures,
solute molecules will not get enough
time to interact with the stationary phase
and separation efficiency will be reduced
and we will not be able to achieve good
separation of solutes.
49

How can we achieve good column separation
efficiency?
50
1. By reducing the Particle size of the stationary phase
support.
2. By reducing the width of chromatographic column.

We use Narrow bore (3mm, id) SS columns filled
with small particles of stationary phase support (5µ)
will give us high separation efficiency with low
analytical time when liquid mobile phase moves
through the column under high pressures of the
order of 500 –5000 psi.
51

Can the physically coated stationary phases used
in GC be used in narrow bore SS columns through
which liquid mobile phase moves under high
pressures?
52
Obviously… NO

Because under high pressures of liquid mobile phase,
physically coated liquid stationary phases will be
removed physically or by dissolution in liquid mobile
phase.
We need physically&chemically stable stationary phase.
53

Chemically treated inert (not strictly) particles have
free silinol group at their surface.
54
Silica
particle
CCC
CCC
Si - OH
Si - OH
Si - OH
Free silinol
groups

Early attempts were then made to chemically bonded long chain
aliphatic alcohols with the free silinol groups present on the
surface of silica particles.
55
Si - OH + HO - R
- H2O
Si - O - R

However these chemically bonded phases with
Si - O - C bonds are stable only in acidic media.
56

We cannot restrict our separation work always in acidic
media
57
In basic media Si– O- C bond undergoes hydrolysis.
Si - O - R basic medium Si - OH + R - OH

We therefore would like to use chemical bonding
with ‘ Si ’ of free silinol groups which will be
hydrolytically stable over a wide acidic to basic
pH range.
58

Attempts were then made to carry out chemical
reactions with substituted silane ( Si H4) having
suitable long chain of hydrocarbons attached to it.
59

60
Si - OH
1 2 18
+ Cl – Si – C – C C - H
CH3
CH3
H
H
H
H
H
H
Substituted siliane
1 2 18
– Si – C – C C - H
CH3
CH3
H
H
H
H
H
H
Si - O
-H Cl

This chemically bonded phase ( Si-O-Si bond )
is therefore mechanically strong and hydrolytically
stable over a wide pH range (2-9).
61

62
When the liquid mobile phase is moving
under high pressures through
chromatographic columns, introduction of
a sample on to the column will require
some specially designed injection system.

63
The most popular and commonly used
injector system, is the syringe –loop
injector type.
It is a fixed volume universal injector
which allows introduction of micro
litres of samples on to the
chromatographic column.

64
Thus so far with we have achieved the
following attributes with LC
 Fast Analysis
 Small Sample Size
 Versatility
 Efficiency
 Non-destructive

• It is difficult to interface a liquid
chromatography to a mass-spectrometer
cause of the necessity to remove the solvent.
• The commnly used interface are:-
(1) Electrospray ionization (ESI)
(2) Thermospray ionization (TSI)
(3) Atmospheric pressure chemical ionization
(APCI)
(4) Atmospheric pressure
photoionization(APPI)
(5) Partical beam ionization.

69
As soon as the solutes are eluted they should be
detected and quantitated with sensitive and
specific or universal detectors.

70
With specific and universal detectors we have achieved the
following attributes:
High sensitivity
High reliability and accuracy

71
Thus in place of glass column liquid
chromatography now we have
1 4 5
2
3
1- Pumping system
2- Universal injector
3- Column
4- Detector
5- Recorder

 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.
Types of mass analyzer:-
 (1) Quadrapole mass filter.
 (2) time of flight
 (3) Ion trap
 (4) Fourier transform ion cyclotron
resonance (FT-ICR or FT-MS)

74
1.Pumping system capable of giving
pulseless flow of liquid mobile phase under
high pressures of the order of 500 to
5000psi.
*This makes fast analysis.

75
2.Universal injector which allows to introduce
samples in microlitres.
*This reduces sample size

76
3.Narrow bore SS columns with suitable chemically
bonded phases with small particle size (3-5µ), desired
polarity and pore size and hydrolytically stable over a
wide acidic to basic pH range.
*This gives high separation efficiency , high
versatility in separating solutes with diverse
nature.

77
4.Sensitive and Specific or Universal detector
*This gives high sensitivity , reliability and
accuracy of detection and quantitation of
solutes.

78
5.Computing system allows to record and
compute chromatographic results.

79
All the above factors add
“High Performance” to normal Liquid
Chromatography.
It is therefore known as
“High Performance Liquid Chromatography”
abbreviated as HPLC.

 Molecular weight determination
 Determining the molecular weight of green
fluorescent proteins
 Structural determination e.g. structural
determination of ginsenoside.
 Pharmaceutical application e.g. identification
of bile acids metabolites.
 Biochemical application e.g. rapid protein
identification using capillary lc/ms/ms.

Food application e.g. identification of aflatoxin
in food determination of vitamin D3 in poultry
feed supplement using MS3
Environmental application e.g. detection of
phenyl urea herbicides, detection of low level
of carbaryl in food.

GC-MS LC-MS
 Volatile or stable
compounds can be used
as samples.
 All gases are
determined.
 Mobile phase used is
gas.
 inner wall of the column
is lined with a thin layer
of support material such
as diatomaceous earth.
 Non-volatile compounds
can be used as samples.
 Can be used for all salts &
macromolecules.
 Mobile phase used is
liquid.
 a column that is packed
with a stationary phase
composed of irregularly or
spherically shaped
particles, a porous
monolithic layer, or a
porous membrane

1. Principles and Intrumentation of gas chromatography-mass
spectroscopy by W. M. A. Niessen, hyphen MassSpec
Counsultancy,Leiden,The Netherlands.
2. gas chromatography-mass spectroscopy from Wikipedia ,The free
encyclopedia.
3. Gas chromatography by U. A. Devkate Sir.
4. Encyclo pedia of Chromatography – Jack Cazes
5. Gas chromatography by Ian A[1]. Fowlis 2nd Edition
6. hollas_J.M._ modern _Spectroscopy
7. Handbook of Instrumental Technique for Analytical Chemistry by
Frank Settle.
8. Moderm Instrumentation Methods and Techniques by Francis Rouessac
and Annick Rouessac.
9. Instrumental methods of Analysis by Willard,Merritt,Dean,Settle,7th

10. W. Paul & H. Steinwedel; Zeitschrift für
Naturforschung, 8A; 1953, p448.
11. W. Paul; Agewandte Chemie - International
Edition, 29; 1990, p739.
12. G. C. Stafford et al.; International Journal of
Mass Spectrometry and Ion Processes, 60; 1984,
p85 and Analytical Chemistry, 59; 1987, p1677.

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