Analytical Instrumentation

Gas Chromatography
What?
 Chromatography is defined as physical method of separation, in which mixture of
Analytes is separated using two phases, one is stationary phase and other is mobile
phase which percolates through the stationary phase.
 The separation occurs because of difference in affinity occurs between Analytes and
stationary phase
Components
 Based on this approach three components
form the basis of the chromatography
technique.

• Stationary phase: This phase is always

composed of a “solid” phase or “a layer of a
liquid adsorbed on the surface a solid
support”.

• Mobile phase: This phase is always

composed of “liquid” or a “gaseous
component.”

• Supporting medium: A solid surface on

which the stationary phase is bound or coated
Basic terminologies
 Adsorption: Interaction of solute molecules with the surface of the
stationary phase
 Eluent: The mobile phase
 Elution: Motion of the mobile phase through the stationary phase
 Elution time: The time taken for a solute to pass through the system.
A solute with a short elution time travels through the stationary
phase rapidly, i.e. it elutes fast
 Stationary phase: The part of the chromatography system that is
fixed in place
 Normal phase: stationary phase is POLAR thus solutes interact
strongly and run slowly (mobile phase is non polar)
 Reverse phase: mobile phase where POLAR solutes run fast i.e.
reverse order

Types of chromatography
 Gas chromatography (GC)

 A physical separation method involves the

distribution of component between two
phases: stationary phase and mobile phase
 stationary phase - solid <(10%)
-liquid >(90%)
 mobile phase - gas
Advantages

➢ Short Analysis Time

➢ Choice of Stationary

➢ Wide Choice of Detectors

➢ Ease of Operation
Woking principle
 When analytes are introduced into the column, the
molecules distribute between the stationary and
Detector mobile phases
 The molecules in the mobile are carried down the
column
Detector  Those in the stationary phase are temporarily
immobile and do not move down the column
 All molecules of the same compound travel through
the column at nearly the same rate and appear as
Detector a band of molecules (sample band)
 Sample band of compound which is less compound
soluble’ in the stationary phase moves faster,
Detector because more of the molecules spend more time in
the mobile phase (carrier gas)
Migration rates of compounds in
column

 Compound chemical structure

 Stationary phase chemical structure

 Column temperature
Basic definitions
 Retention Time (tR): A measure of the amount of time an analyte spends in the column

 Dead time (tm): it is time a non retained compound spends in the mobile phase which is also the
time the non retained compound spends in the column.

 Adjusted retention time: it is a time that a compound spends in the stationary phase. It is the
time difference between the dead time and retention time for a compound.

Tr = tR -tm
Cont.

 Capacity factor/retention factor/ Partition ratio: It is the ratio of mass of

the compound in the stationary phase relative to mass of the compound in
mobile phase

 Phase ratio (𝜷): it relates the column diameter and film thickness of
stationary phase
𝑟
𝜷= 2𝑑𝑓
Cont.
 Distribution constant (KD): Ratio of analyte concentration in the stationary and mobile phase

 Selectivity/ separation factor (𝜶): A measure of the time or distance between the maxima of
two peaks. α = 1 means the two peaks have the same retention and co –elute

 Linear velocity (u): It is the velocity by which the carrier gas or mobile phase travel through the
column.
𝑳
u=
𝒕𝒎
Resolution (R)
 A measure of overlap between two peaks; the higher the resolution, the less the overlap
 Separation (α) is only the distance between two peak maxima; resolution takes both maxima α
and the width of the peaks into account
Schematic of GC
 Basic GC Injector Structure

 Sample is introduced to the column through

GC injector, by using
 Syringe injection
 Autosampler injection
 Valve injection
Types of GC injectors

 Four types of injectors:

 Split and splitless injector
 Direct injector
 Programmed Temperature Vaporization injector
 Packed Injector

Split Injection
Split injection involves injecting a liquid sample into a heated
injection port, vaporizing the sample in the injection inlet, and
splitting the vaporized sample into two parts, so that a small fraction
of the vaporized sample enters the column, and the major portion is
vented to waste.
 Split injection was first developed for open tubular columns. It has
been one of the most commonly used methods for many applications,
since it offers many practical benefits for the analysis of
concentrated samples with little risk of band broadening. It is easy to
automate, and it is compatible with both isothermal and
temperature-programmed operation.
 The classical split injector is a flash vaporization device (see Figure.
 The device consists of a heated vaporizing chamber, which is usually
made from a stainless steel tube lined with a removable glass or
quartz liner.
 The carrier gas at a constant pressure enters behind the glass liner
and is therefore preheated. The flow is divided into two different
routes: one to purge the septum and the other, a high flow, to enter
the vaporization chamber, where carrier gas is mixed with the
vaporized sample.
SPLIT RATIO (SR) is the parameter that
 The mixed stream flows by the column inlet and leaves through the
split valve exit. The split ratio, which is controlled by a needle valve,
determines the amount of sample that goes
is the ratio of the split flow to the column flow rate into the capillary column
 Splitless injection
 Splitless injection is usually used for the analysis of trace
level samples.
 The design of splitless injectors differs from that of split
injectors only in the addition of the solenoid valve
downstream from the vent.
 During the splitless period, most of the sample and solvent
enter the column.
 The residual vapor in the injector, however, may cause
peak tailing, especially for the solvent peak.
 To avoid this problem, the inlet is switched back to the
split mode after a nearly complete sample transfer, so that
all remaining solvent and vapors are purged out of the split
vent.
 In this case, the splitless period must be optimized for
each particular sample matrix, since the sample transfer
efficiency will be low if the purge is turned on too quickly.
 Factors having an effect on the splitless efficiency include
the starting oven temperature, the solvent boiling point,
and the polarity of the solvent
Liquid and gas sample injection

 Liquid sample volume:

0.1-10 μL
 Sample injected into
hotzone of the column
for quick vaporization

• Gas sample volume: 0.1-10 mL

• Sample injected into hotzone of
the column for quick
vaporization
GAS SUPPLY SYSTEM

 In GC, a supply of carrier gas is required as the

mobile phase to transfer the samples from the
injector, through the column, and into the detector.
 The commonly used carrier gases are helium (He)
and nitrogen (N2) There is an increasing use of
hydrogen (H2) as carrier gas due to its high
separation efficiency.
 Argon (Ar) and carbon dioxide (CO2) are also used as
carrier gas, but not commonly.
 The carrier gas is required to be inert, dry, and pure.
 Selection of Carrier Gases
 The choice of carrier gas requires consideration of the
separation problem to be solved, the detector used, and
the purity of the gases.
 A further consideration in the selection of carrier gas is its
availability and its cost.
 The selection of the best carrier gas is important, because
it will determine the column separation processes
 The efficiency of the column separation, referred to as the
height equivalent to a theoretical plate (HETP or H),
depends on the solute diffusivity in the carrier gas.
 The relation of H and the mobile phase linear velocity (µ) in
packed columns was described by van Deemter
 In the van Deemter plot, the minimum of the curve (Hmin)
corresponds to the maximum efficiency of the separation.
 Figure shows the van Deemter plots for the common carrier
gases, namely, nitrogen, helium, and hydrogen, using a c
About GC efficiency
 The chromatographic column has discrete sections, which
they named theoretical plates.
 Each plate represents the theoretical distance required for
one adsorption-desorption step of sample components
between the stationary and mobile phase (see figure
below).
 The number of theoretical plates is often used to establish
the efficiency of a column.
 No. of plates separation Efficiency

 The plate height is often called ‘height

equivalent to a theoretical plate’ (HETP)
 GC Columns

 The column, where the actual chromatographic

separation occurs, is described as the heart of
the chromatographic system.
 Two general types of gas chromatographic
columns are most commonly used:
 packed,
 open tubular or capillary.
 Packed columns were developed first, but
today, the majority of GC is carried out on
capillary columns.
 The principal difference between the two kinds
of column is reflected in their plate numbers.
 Packed column
 Packed columns consist of metal (stainless steel, copper,
or aluminum) or glass tubing filled with solid material,
either uncoated adsorbent (GSC) or a solid support coated
with a stationary liquid phase (GLC).
 The selection of tubing material is dictated by the
particular analytical use.
 Glass columns can be used at high temperatures, when
metal tubing would catalyze the decomposition of the
sample.
 In GLC columns, the solid support is needed to hold the
liquid stationary phase.
 The material should consist of inert, uniformly spherical
particles having a large surface area per unit volume
(particle size between 80 and 120 mesh) to minimize the
void volume and to supply a specific surface area for
interaction with the analytes.
 In addition, it should be mechanically strong over a wide
temperature range.
 The most frequently used solid supports for GLC are
diatomaceous earths, either kieselguhr, the most
commonly used adsorbents are activated charcoal, silica
gel, alumina, and glass beads.
Capillary or open tubular columns
 Early columns were constructed of stainless steel, aluminum,
copper, plastic, or glass.
 Fused silica tubing with a polyimide coating is currently
preferred.
 There are three main types of open tubular columns: WCOT,
SCOT, and PLOT
 WCOT capillary columns are the most commonly used in GC.
 They are prepared by coating the inner column walls with the
liquid stationary phase as a thin film.
 Currently, the most commonly used column material is fused
silica, which is manufactured from synthetic quartz with very low
(less than 1 ppm) metallic impurities.
 In SCOT columns, the liquid is located on a porous support as a
thin layer.
 Compared with WCOT, SCOT columns have higher sample capacity
but lower efficiency.
 PLOT columns are similar to SCOT columns in that a porous
material is deposited on the inner column wall
 Typical gas chromatograms of light oil
obtained from packed and capillary columns.
Detectors
 Major two types/ families:
 First family (concentration-dependent detectors):
Detector those give response proportional to
concentration of sample (mole fraction) but
independent of rate of gas flow rate. Example:
Thermal conductivity and electron capture detector
 Second family(mass flow–dependent detectors):
Response of the detector depends upon rate at which
sample is delivered to sensing element, but
independent of sample concentration. Example:
Flame ionization, flame photometric detectors.
First family

 In the concentration-dependent detector, the signal yi of

a sample component i is proportional to the concentration
ci in the carrier gas at any time during the elution:
Cont.
 In the concentration-dependent detector, the effective detector volume
should be small in comparison with the volume of carrier gas in which the
solute is diluted; otherwise, an increase in the peak occurs.
 Sensitivity S can be defined as the change of measured detector signal yi
resulting from the change of the concentration of the eluted analyte ci

 The sensitivity depends on the design and properties of the analyte. The
minimum detectability of a detector, DL, can be calculated from the following
equation:
Second family

 In this type of detector, the peak area A of the same

amount of analyte Qi is not changed at higher or lower gas
flow; in this case, the signal yi is dependent only on the
properties of the eluted sample
Thermal conductivity
 Thermal conductivity is defined as the ability of a
material to conduct heat from its one side to the
other. It is represented with thermal conductivity
coefficient k. Smaller k indicates that the material
has stronger heat insulation and preservation.
 Thermal conductivity detector
 A thermal conductivity detector (TCD) is a detector used in
gas chromatography (GC) to analyze gases and small
hydrocarbon molecules. It is also known as a katharometer.
 It sense passively some physical property of gas without
reacting with it chemically.
 TCD works by having two parallel tubes both containing gas
and heating coils. The gases are examined by comparing the
heat loss rate from the heating coils into the gas.
 Normally one tube holds a reference gas and the sample to be
tested is passed through the other.
 Initially Wheatstone bridge circuits is balanced as He is
flowing through all chambers.
 Using this principle, a TCD senses the changes in the thermal
conductivity of the column effluent and compares it to a
reference flow of carrier gas.
 Most compounds have a thermal conductivity much less than
that of the common carrier gases of hydrogen or helium.
 Therefore, when an analyte elutes from the column, the
thermal conductivity of the effluent is reduced and a
detectable signal is produced.
 Electron capture detectors(ECD)
 In the electron capture detector (ECD), the column
effluent passes over a beta-emitter, such as nickel-63
or tritium.
 The electrons from the emitter bombard the carrier
gas (nitrogen), resulting in ions and a burst of
electrons.
 In the absence of an analyte, the ionization process
yields a constant standing current.
 When organic molecules that contain electronegative
functional groups, such as halogens and phosphorus
groups, pass by the detector, they capture the
electrons and reduce the current between the
electrodes.
 The loss of electron stream is related to the quantity
of analyte in the carrier gas or makeup gas. The ECD
can be classified into a constant potential (direct
current [dc]) mode and a pulsed potential mode.
Cont.
 The sensitivity of the ECD increases in the order of
F<Cl<Br
 By chemical derivatization, the applicability of ECD can
be expanded for the trace analysis of species that capture
electrons only weakly or not at all
 For analysis using a packed column, nitrogen or argon–
methane can be used as carrier gas, and no make-up gas is
needed, whereas in a capillary column, helium or
hydrogen is preferred as carrier gas, with nitrogen or
argon– methane as the make-up gas (25–30 mL/min)
 The applications of ECD illustrate the advantages of a
special detector that is highly sensitive toward molecules
that contain electronegative functional groups, such as
halogens, peroxides, quinines, or nitro groups
 Flame ionization detector (FID)
 It is well known that the flame ionization detector (FID) is the most
commonly used detector in GC.
 The FID consists of a hydrogen/air flame and a collector electrode plate.
The effluent from the column passes through the flame, which oxidizes
the organic molecules and produces ions.
 A collector electrode attracts the negative ions to the electrometer
amplifier, producing an analog signal, which is connected to the data
system
 FID is sensitive to all compounds that contain C–C or C–H and considerably
less sensitive or insensitive to certain functional groups of organic
compounds, such as alcohol, amine, carbonyl, and halogen. In addition,
the detector is also insensitive toward noncombustible gases such as H2O,
CO2, SO2, and NO.
Cont.
 For operating FID, the hydrogen flow usually ranges between 20 and 30
mL/min, and the airflow is about 120–200 mL/min (for packed columns),
or the air:hydrogen ratio should be about 10:1.
 For capillary columns, the column flow rate may be less than 1 mL/min.
 To achieve maximum sensitivity for FID, it is recommended to use make-
up gas, which assists in pushing the sample to the flame and therefore,
enhances the sensitivity. FID is capable of measuring 10−12 g carbon/s.
 The FID temperature should be maintained hot enough (recommended at
least 200oC) so that condensation does not occur in the system
 The magnitude of the variation in current would be directly proportional
to the number of ions or electrons formed in the flame gases, which in
turn would be proportional to the carbon content of the organic
molecules in the vapour
 There are certain limitations to the use of FID. These are:
 • The FID does not respond to inert gases and inorganic compounds.
 • The emerging components get destroyed in the flame.
 • The response to sample weight has to be separately determined for
each component.
Differential flame-ionisation detector
 Two columns are used in this arrangement.
 If the same packed column as the sample-side one is
employed on the reference-side, the carrier gas is
fed through the reference-side column at the same
speed as on the sample-side
 And if the reference-side column is mounted very
near the sample-side one, under these conditions,
the amount of liquid phase flowing from the
reference-side column can be considered of the same
as that of the liquid phase flowing from the sample-
side.
 On applying the voltages to the electrodes, the
signals produced are shown in Figure indicating that
the sample-side and reference-side signals offset
each other, producing the base lint as straight.
 Nitrogen Phosphorus Detector
 The nitrogen phosphorus detector (NPD) is similar in design to
the FID. This detector is also known as the thermoionic emission
detector or FTD. An electrically heated (up to 800°C)
thermoionic bead is positioned between the jet orifice and the
collector.
 Generally, the bead consists of heated silica doped with an alkali
metal, usually a rubidium or cesium salt.
 Nitrogen- and/or phosphorus-containing molecules will collide
with the hot bead and undergo a catalytic surface reaction,
producing ions, and the ions will be attracted to the collector
electrode, amplified, and output to the data system. A diagram
of the NPD detector is shown in Figure
 This detector is commonly used for analyzing pesticides.
 Compared with FID, this detector is about 500 times more
sensitive for phosphorus-containing molecules and about 50
times more sensitive for nitrogen-bearing compounds.
 NPD is a highly sensitive but also specific detector for nitrogen
and phosphorus
 Flame Photometric Detector
 The flame photometric detector (FPD)
monitors the intensity of the light emitted
from a sample component that has been
excited in a flame.
 The components eluted from the GC column
are decomposed and then excited to a
higher electronic state in a hydrogen-rich
flame.
 FPD is selective for S- and P-containing
molecules. FPD uses a chemiluminescent
reaction in a hydrogen/air flame.
 In the FPD detector, the effluent passes into
a low-temperature H2–air flame, which A combustion chamber to house the flame
converts phosphorus and sulfur to emitting • Gas lines for hydrogen (fuel) and air (oxidant),
• An exhaust chimney to remove combustion products.
species. • Thermal (bandpass) filter to isolate only the visible and UV
radiation emitted by the flame.
Without this, the infrared radiation emitted by the flame’s
combustion reactions would heatup the PMT and increase its
background signal.
Cont.
 The emitting species for S-compounds is
excited S2, which has a maximum at
about 394 nm, whereas for P-compounds,
the emitter is excited HPO, which has a
maximum about 512–526 nm (doublet).
 The emitted visible and UV bands are
filtered using a 526 nm band-pass filter
(for P-compounds) or a 394 nm filter (for
S-compounds), and their intensity is
recorded photometrically. Sulfur
compounds can be detected down to
about 200 ppb, whereas phosphorus can
be detected down to 20 ppb.
 It should be mentioned that the intensity
of light emitted is not linear with the
concentration but approximately
proportional to the square of the sulfur
atom concentration that is emitted by
the excited particles is separated into
individual lines by a photodiode array.
 Methods for measurement of Peak area
 The output from the pre-amplifier after the detector is brought to resistance R1 of
the integrator.
 As soon as the absolute values of the two voltages differ, charging of the integrating
capacitor begins.
 The output of OP2 would be a voltage increasing linearly with time, the slope of
which will correspond to the difference between the signal voltage and the ES .
 As soon as the integrator output voltage reaches or slightly exceeds the voltage value
applied to input E1 , the comparator (OP3 ) output jumps to the saturation voltage
and the relay (Re ) is closed. The integrating capacitor is thus discharged.
 This process can be repeated and the circuit can be used for conversion of an analog
signal into a digital signal. As each relay closure is recorded by a counter, the number
of pulses registered in the counter gives a measure of the area under the peak.