Gas Chromatography
1
Gas chromatography is a technique used for
separation of volatile substances, or substances
that can be made volatile, from one another in a
gaseous mixture at high temperatures. A
sample containing the materials to be separated
is injected into the gas chromatograph. A mobile
phase (carrier gas) moves through a column that
contains a wall coated or granular solid coated
stationary phase. As the carrier gas flows
through the column, the components of the
sample come in contact with the stationary
phase. The different components of the sample
have different affinities for the stationary phase,
which results in differential migration of solutes,
thus leading to separation
2
Martin and James introduced this separation
technique in 1952, which is the latest of the
major chromatograhpic techniques. However,
by 1965 over 18000 publications in gas
chromatography (GC) were available in the
literature. This is because optimized
instrumentation was feasible. Gas
chromatography is good only for volatile
compounds or those, which can be made
volatile by suitable derivatization methods or
pyrolysis. Thus, about 20% of chemicals
available can be analyzed directly by GC.
3
Gas chromatography can be used for both
qualitative and quantitative analysis.
Comparison of retention times can be
used to identify materials in the sample by
comparing retention times of peaks in a
sample to retention times for standards.
The same limitations for qualitative
analysis discussed in Chapter 26 also
apply for separations in GC. Quantitative
analysis is accomplished by measurement
of either peak height or peak area
4
Gas - Solid Chromatography
(GSC)
The stationary phase, in this case, is a solid like
silica or alumina. It is the affinity of solutes
towards adsorption onto the stationary phase
which determines, in part, the retention time.
The mobile phase is, of course, a suitable carrier
gas. This gas chromatographic technique is
most useful for the separation and analysis of
gases like CH4, CO2, CO, ... etc. The use of
GSC in practice is considered marginal when
compared to gas liquid chromatography.
5
Gas - Liquid Chromatography
(GLC)
The stationary phase is a liquid with very low
volatility while the mobile phase is a
suitable carrier gas. GLC is the most
widely used technique for separation of
volatile species. The presence of a wide
variety of stationary phases with
contrasting selectivities and easy column
preparation add to the assets of GLC or
simply GC.
6
Instrumentation
It may be wise to introduce instrumental
components before proceeding further in
theoretical background. This will help
clarify many points, which may, otherwise,
seem vague. It should also be noted that a
detector will require special gas cylinders
depending on the detector type utilized.
The column temperature controller is
simply an oven, the temperature of which
can be varied or programmed
7
To Waste or Flow
Meter
Two-Stage
Regulator
Syringe
Detector
Injector
Flow Controller
Carrier Gas
Cylinder
8
Column
Three temperature zones can be identified:
1. Injector temperature, TI, where TI should allow
flash vaporization of all sample components.
2. Column temperature, Tc, which is adjusted as
the average boiling points of sample
components.
3. Detector Temperature, TD, which should
exclude any possible condensation inside the
detector.
Generally, an intuitive equation can be used to
adjust all three zones depending on the
average boiling point of the sample
components. This equation is formulated as:
TI = TD = Tc + 50 oC
9
The Carrier Gas
Unlike liquid chromatography where wide
varieties of mobile phase compositions are
possible, mobile phases in gas
chromatography are very limited. Only
slight changes between carrier gases can
be identified which places real limitations
to chromatographic enhancement by
change or modification of carrier gases
10
A carrier gas should have the following properties:
1. Highly pure (> 99.9%)
2. Inert so that no reaction with stationary phase
or instrumental components can take place,
especially at high temperatures.
3. A higher density (larger viscosity) carrier gas is
preferred.
4. Compatible with the detector since some
detectors require the use of a specific carrier
gas.
5. A cheap and available carrier gas is an
advantage.
11
Longitudinal Diffusion Term
This is an important factor contributing to band
broadening which is a function of the diffusivity
of the solute in the gaseous mobile phase as
well as the molecular diffusion of the carrier gas
itself.
HL = K DM /V
Where; DM is the diffusion coefficient of solute in
the carrier gas. This term can be minimized
when mobile phases of low diffusion, i.e. high
density, are used in conjunction with higher flow
rates.
12
The same van Deemter equation as in LC
can be written for GC where:
H = A + B/V + CV
The optimum carrier gas velocity is given by
the derivative of van Deemter equation
Vopt = { B/C }1/2
However, the obtained velocity is much
greater than that obtained in LC.
13
The carrier gas pressure ranges from 10-50 psi.
Higher pressures potentially increase
compression possibility while very low
pressures result in large band broadening due
to diffusion. Depending on the column
dimensions, flow rates from 1-150 mL/min are
reported. Conventional analytical columns (1/8”)
usually use flow rates in the range from 20-50
mL/min while capillary columns use flow rates
from 1-5 mL/min depending on the dimensions
and nature of column. In most cases, a
selection between helium and nitrogen is made
as these two gases are the most versatile and
common carrier gases in GC.
14
Injectors
Septum type injectors are the most common.
These are composed of a glass tube where
vaporization of the sample takes place. The
sample is introduced into the injector through a
self-sealing silicone rubber septum. The carrier
gas flows through the injector carrying vaporized
solutes. The temperature of the injector should
be adjusted so that flash vaporization of all
solutes occurs. If the temperature of the injector
is not high enough (at least 50 degrees above
highest boiling component), band broadening
will take place.
15
Syringe
Septum
Carrier
Gas
Vaporization
Chamber
To
Column
16
Column Configurations and
Ovens
The column in chromatography is undoubtedly the
heart of the technique. A column can either be a
packed or open tubular. Traditionally, packed
columns were most common but fast
developments in open tubular techniques and
reported advantages in terms of efficiency and
speed may make open tubular columns the best
choice in the near future. Packed columns are
relatively short (~2meters) while open tubular
columns may be as long as 30-100 meters
17
Packed columns are made of stainless steel or
glass while open tubular columns are usually
made of fused silica. The temperature of the
column is adjusted so that it is close to the
average boiling point of the sample mixture.
However, temperature programming is used
very often to achieve better separations. The
temperature of the column is assumed to be the
same as the oven which houses the column.
The oven temperature should be stable and
easily changed in order to obtain reproducible
results.
18
Detection Systems
Several detectors are
available for use in
GC. Each detector
has its own
characteristics and
features as well as
drawbacks.
Properties of an ideal
detector include:
19
1.
2.
3.
4.
5.
6.
7.
8.
9.
High sensitivity
Minimum drift
Wide dynamic range
Operational temperatures up
to 400 oC.
Fast response time
Same response factor for all
solutes
Good reliability (no fooling)
Nondestructive
Responds to all solutes
(universal)
a. Thermal Conductivity Detector
(TCD)
This is a nondestructive detector which is used for the
separation and collection of solutes to further perform
some other experiments on each purely separated
component. The heart of the detector is a heated
filament which is cooled by helium carrier gas. Any
solute passes across the filament will not cool it as much
as helium does because helium has the highest thermal
conductivity. This results in an increase in the
temperature of the filament which is related to
concentration. The detector is simple, nondestructive,
and universal but is not very sensitive and is flow rate
sensitive.
20
21
22
Note that gases should
always be flowing
through the detector
including just before,
and few minutes after,
the operation of the
detector. Otherwise,
the filament will melt.
Also, keep away any
oxygen since oxygen
will oxidize the
filament and results in
its destruction.
23
Remember that TCD
characteristics
include:
1. Rugged
2. Wide dynamic range
(105)
3. Nondestructive
4. Insensitive (10-8 g/s)
5. Flow rate sensitive
b. Flame Ionization Detector
(FID)
This is one of the most sensitive and reliable
destructive detectors. Separate two gas
cylinders, one for fuel and the other for O2
or air are used in the ignition of the flame
of the FID. The fuel is usually hydrogen
gas. The flow rate of air and hydrogen
should be carefully adjusted in order to
successfully ignite the flame.
24
25
26
The FID detector is a mass sensitive
detector where solutes are ionized in the
flame and electrons emitted are attracted
by a positive electrode, where a current is
obtained.
The FID detector is not responsive to air,
water, carbon disulfide. This is an
extremely important advantage where
volatile solutes present in water matrix can
be easily analyzed without any
pretreatment.
27
Remember that FID characteristics include:
• Rugged
• Sensitive (10-13 g/s)
• Wide dynamic range (107)
• Signal depends on number of carbon atoms in
organic analytes which is referred to as mass
sensitive rather than concentration sensitive
• Weakly sensitive to carbonyl, amine, alcohol,
amine groups
• Not sensitive to non-combustibles – H2O, CO2,
SO2, NOx
• Destructive
28
Electron Capture Detector (ECD)
This detector exhibits high intensity for halogen containing
compounds and thus has found wide applications in the
detection of pesticides and polychlorinated biphenyls.
The mechanism of sensing relies on the fact that
electronegative atoms, like halogens, will capture
electrons from a b emitter (usually 63Ni). In absence of
halogenated compounds, a high current signal will be
recorded due to high ionization of the carrier gas, which
is N2, while in presence of halogenated compounds the
signal will decrease due to lower ionization.
29
30
Remember the following facts about ECD:
1. Electrons from a b-source ionize the carrier gas
(nitrogen)
2. Organic molecules containing electronegative
atoms capture electrons and decrease current
3. Simple and reliable
4. Sensitive (10-15 g/s) to electronegative groups
(halogens)
5. Largely non-destructive
6. Insensitive to amines, alcohols and
hydrocarbons
7. Limited dynamic range (102)
8. Mass sensitive detector
31
Gas Chromatographic Columns
and Stationary Phases
Packed Columns
These columns are fabricated from glass, stainless
steel, copper, or other suitable tubes. Stainless
steel is the most common tubing used with
internal diameters from 1-4 mm. The column is
packed with finely divided particles (<100-300
mm diameter), which is coated with stationary
phase. However, glass tubes are also used for
large-scale separations.
32
Several types of tubing were used ranging from
copper, stainless steel, aluminum and glass.
Stainless steel is the most widely used because
it is most inert and easy to work with. The
column diameters currently in use are ordinarily
1/16" to 1/4" 0.D. Columns exceeding 1/8" are
usually used for preparative work while the 1/8"
or narrower columns have excellent working
properties and yield excellent results in the
analytical range. These find excellent and wide
use because of easy packing and good routine
separation characteristics. Column length can
be from few feet for packed columns to more
than 100 ft for capillary columns.
33
Capillary/Open Tubular
Open tubular or capillary columns are finding broad
applications. These are mainly of two types:
• Wall-coated open tubular (WCOT) <1 mm thick liquid
coating on inside of silica tube
• Support-coated open tubular (SCOT) 30 mm thick coating
of liquid coated support on inside of silica tube
These are used for fast and efficient separations but are
good only for small samples. The most frequently used
capillary column, nowadays, is the fused silica open
tubular column (FSOT), which is a WCOT column.
34
The external surface of the fused silica columns is
coated with a polyimide film to increase their
strength. The most frequently used internal
diameters occur in the range from 260-320
micrometer. However, other larger diameters are
known where a 530 micrometer fused silica
open tubular column was recently made and is
called a megapore column, to distinguish it from
other capillary columns. Megapore columns
tolerate a larger sample size.
35
36
37
38
39
It should be noted that since capillary
columns are not packed with any solid
support, but rather a very thin film of
stationary phase which adheres to the
internal surface of the tubing, the A term in
the van Deemter equation which stands
for multiple path effects is zero and the
equation for capillary columns becomes
H = B/V + CV
40
Capillary columns advantages compared to
packed columns
1. higher resolution
2. shorter analysis times
3. greater sensitivity
Capillary columns disadvantage compared
to packed columns
1. smaller sample capacity
41
Solid Support Materials
The solid support should ideally have the
following properties:
1. Large surface area (at least 1 m2/g)
2. Has a good mechanical stability
3. Thermally stable
4. Inert surface in order to simplify retention
behavior and prevent solute adsorption
5. Has a particle size in the range from 100400 mm
42
Selection of Stationary Phases
General properties of a good liquid stationary
phase are easy to guess where inertness
towards solutes is essential. Very low volatility
liquids that have good absolute and differential
solubilities for analytes are required for
successful separations. An additional factor that
influences the performance of a stationary
phase is its thermal stability where a stationary
phase should be thermally stable in order to
obtain reproducible results. Nonvolatile liquids
assure minimum bleeding of the stationary
phase
43
% Loading =
Weight of liquid stationary phase * 100%
Weight of stationary phase plus solid support
Increasing percent loading would allow for
increased sample capacity and cover any
active sites on the solid support. These
two advantages are very important,
however increasing the thickness of
stationary phase will affect the C term in
the van Deemter equation by increasing
HS, and therefore Ht.
44
Generally, the film thickness primarily affects the
retention character and the sample capacity of a
column. Thick films are used with highly volatile
analytes, because such films retain solutes for a
longer time and thus provide a greater time for
separation to take place. Thin films are useful for
separating species of low volatility in a
reasonable time. On the other hand, a thicker
film can tolerate a larger sample size. Film
thicknesses in the range from 0.1 – 5 mm are
common.
45
Liquid Stationary Phases
In general, the polarity of the stationary
phase should match that of the sample
constituents ("like" dissolves "like"). Most
stationary phases are based on
polydimethylsiloxane or polyethylene
glycol (PEG) backbones:
46
The polarity of the
stationary phase can
be changed by
derivatization with
different functional
groups such as a
phenyl group.
Bleeding of the
column is cured by
bonding the stationary
phase to the column;
or crosslinking the
stationary phase.
47
Liquid Stationary Phases
should have the following
characteristics:
• Low volatility
• High decomposition
temperature (thermally
stable)
• Chemically inert
(reversible interactions
with solvent)
• Chemically attached to
support (to prevent
bleeding)
• Appropriate k' and a for
good resolution
Bonded and Crosslinked
Stationary Phases
The purpose of bonding and cross-linking is to
prevent bleeding and provide a stable stationary
phase. With use at high temperatures, stationary
phases that are not chemically bonded or
crosslinked slowly lose their stationary phase
due to bleeding in which a small amount of the
physically immobilized liquid is carried out of the
column during the elution process. Crosslinking
is carried out in situ after a column is coated with
one of the polymers
48
49
In summary, stationary phases are usually
bonded and/or crosslinked and the following
remarks are usually helpful:
1. Bonding occurs through covalent linking of
stationary phase to support
2. Crosslinking occurs through polymerization
reactions to join individual stationary phase
molecules
3. Nonpolar stationary phases are best for
nonpolar analytes where nonpolar analytes are
retained preferentially
4. Polar stationary phases are best for polar
analytes where polar analytes are retained
preferentially
50
Gas-liquid chromatography
(GLC)
Packed columns are fabricated from glass, metal,
or Teflon with 1 to 3 m length and 2 to 4 mm in
internal diameter. The column is packed with a
solid support (100-400 mm particle diameter
made from diatomaceous earth) that has been
coated with a thin layer (0.1-5 mm) of the
stationary liquid phase. Efficiency increases with
decreasing particle size as predicted from van
Deemter equation. The retention is based on
absorption of analyte (partition into the liquid
stationary phase) where solutes must have
differential solubility in the stationary phase
51
Open tubular capillary columns, either WCOT,
SCOT are routinely used. In WCOT the capillary
is coated with a thin film (0.1-0.25 mm) of the
liquid stationary phase while in SCOT a thin film
of solid support material is first affixed to the
inner surface of the column then the support is
coated with the stationary phase. WCOT
columns are most widely used. Capillary
columns are typically made from fused silica
(FSOT) and are 15 to 100 m long with 0.10 to
0.5 mm i.d.
52
The thickness of the stationary phase affects the
performance of the column as follows:
1. Increasing thickness of stationary phase allows
the separation of larger sample sizes.
2. Increasing thickness of stationary phase
reduces efficiency since HS increases.
3. Increasing thickness of stationary phase is
better for separation of highly volatile
compounds due to increased retention.
•
53
Much more efficient separations can be
achieved with capillary columns, as
compared to packed columns, due to the
following reasons:
1. Very long capillary columns can be used
which increases efficiency
2. Thinner stationary phase films can be
used with capillary columns
3. No eddy diffusion term (multiple paths
effect) is observed in capillary columns
54
55
Temperature Programming
Gas chromatographs are usually capable of
performing what is known as temperature
programming gas chromatography (TPGC). The
temperature of the column is changed according
to a preset temperature isotherm. TPGC is a
very important procedure, which is used for the
attainment of excellent looking chromatograms
in the least time possible. For example, assume
a chromatogram obtained using isothermal GC
at 80 oC, as shown below:
56
57
58
59
60
The General Elution Problem
Look at the chromatogram below in which six
components are to be separated by an elution
process using isothermal conditions at for example
120 oC:
61
It is clear from the figure that the separation is
optimized for the elution of the first two
components. However, the last two
components have very long retention and
appear as broad peaks. Using isothermal
conditions at high temperature (say for
example 200oC) can optimize the elution of
the last two compounds but, unfortunately,
results in bad resolution of the earlier eluting
compounds as shown in the figure below
where the first two components are coeluted
while the resolution of the second two
components becomes too bad:
62
63
One can also optimize the separation of the
middle too components by adjusting the
isothermal conditions (for example at say 160
oC). In this case, a chromatogram like the one
below can be obtained:
64
However, in chromatographic separations we
are interested in fully separating all
components in an acceptable resolution.
Therefore, it is not acceptable to optimize the
separation for a single component while
disregarding the others. The solution of this
problem can be achieved by consecutive
optimization of individual components as the
separation proceeds. In this case,
temperature should be changed during the
separation process. This is called
temperature programming gas
chromatography (TPGC)
65
First, a temperature suitable for the
separation of the first eluting component is
selected, and then the temperature is
increased so that the second component
is separated and so on. The change in
temperature can be linear, parabolic, step,
or any other formula. The chromatographic
separation where the temperature is
changed during the elution process is
called TPGC. A separation like the one
below can be obtained:
66
67
Temperature Zones in GC
Three temperature zones should be adjusted
before a GC separation can be done. The
injector temperature should be such that fast
evaporation of all sample components is
achieved. The temperature of the injector is
always more than that of the column, which
depends on the operational mode of the
separation. The detector temperature should be
kept at some level so as to prevent any solute
condensation in the vicinity of the detector body.
68
Gas-solid chromatography
(GSC)
Gas-solid chromatography is based upon
adsorption of gaseous substances on solid
surfaces. Distribution coefficients are generally
much larger than those for gas-liquid
chromatography. Consequently, gas-solid
chromatography is useful for the separation of
species that are not retained by gas-liquid
columns, such as the components of air,
hydrogen sulfide, carbon disulfide, nitrogen
oxides, and rare gases. Gas-solid
chromatography is performed with both packed
and open tubular columns.
69
Molecular Sieves
Molecular sieves are metal aluminum silicate ion
exchangers, whose pore size depends upon the
kind of cation present, like sodium in sodium
aluminum silicate molecular sieves. The sieves
are classified according to the maximum
diameter of molecules that can enter the pores.
Commercial molecular sieves come in pore
sizes of 4, 5, 10, and 13 angstroms. Molecular
sieves can be used to separate small molecules
from large ones.
70
Porous Polymers
Porous polymer beads of uniform size are
manufactured from styrene crosslinked
with divinylbenzene. The pore size of
these beads is uniform and is controlled by
the amount of crosslinking. Porous
polymers have found considerable use in
the separation of gaseous species such as
hydrogen sulfide, oxides of nitrogen,
water, carbon dioxide, methanol, etc.
71
Quantitative Analysis
GC is an excellent quantitative technique
where peak height or area is proportional
to analyte concentration. Thus the GC can
be calibrated with several standards and a
calibration curve is obtained, then the
concentration of the unknown analyte can
be determined using the peak area or
height. The detector response factor for
each analyte should be considered for
accurate quantitative analysis.
72
Gas chromatographs are widely used as criteria
for establishing the purity of organic compounds.
Contaminants, if present, are revealed by the
appearance of additional peaks. Qualitative
Analysis is usually done by comparison with
retention times of standards, which are very
reproducible in GC, provided good injection
practices are followed. Injection should be done
with a suitable Hamilton type syringe through the
heated septum injector till all needle disappears,
then the needle is drawn back as steadily and
fast as possible. This is important for
reproducible attainment of retention times.
73
The Retention Index
The retention index, RI, was first proposed by
Kovats in 1958 as a parameter for identifying
solutes from chromatograms. The retention
index for any given solute can be derived from a
chromatogram of a mixture of that solute with at
least two normal alkanes (chain length >four
carbons) having retention times that bracket that
of the solute. That is, normal alkanes are the
standards upon which the retention index scale
is based.
74
By definition, the retention index for a normal
alkane is equal to 100 times the number of
carbons in the compound regardless of the
column packing, the temperature, or other
chromatographic conditions. The retention index
system has the advantage of being based upon
readily available reference materials that cover a
wide boiling range. The retention index of a
compound is constant for a certain stationary
phase but can be totally different for other
stationary phases.
75
In finding the retention index, a plot of the
number of carbons of standard alkanes
against the logarithm of the adjusted
retention time is first constructed. The
value of the logarithm of the adjusted
retention time of the unknown is then
calculated and the retention index is
obtained from the plot.
The adjusted retention time, tR’, is defined
as:
tR ’ = t R - tM
76
77
Interfacing GC with other
Methods
As mentioned previously, chromatographic
methods (including GC) use retention times as
markers for qualitative analysis. However, this
characteristic does not absolutely confirm the
existence of a specific analyte as many analytes
may have very similar stationary phases. GC, as
other chromatographic techniques, can confirm
the absence of a solute rather than its existence.
When GC is coupled with structural detection
methods, it serves as a powerful tool for
identifying the components of complex mixtures.
A popular combination is GC/MS.
78
Mass Spectrometry
O
H3C
C
N
C H3
N
C
C H
C
C
N
N
H
O
Mass
Spectrometer
Typical sample: isolated
compound (~1 nanogram)
194
Mass Spectrum
67
109
55
82
42
136
94
79
40
60
80
100
120
Mass (amu)
140
165
160
180
200
80