CHM510

CHAPTER 2

GAS CHROMATOGRAPHY

1
 Course Learning Outcomes

Students should be able to:

1. Explain the fundamental concepts and theories of separation

techniques in gas chromatography (GC).

2. Sketch, label the schematic diagrams and discuss the function of

each component in GC.

3. Identify the strength and limitations of GC technique.

4. Suggest and justify the most suitable and efficient separation

technique to be employed for an analysis.

2
 General Chromatography

Chromatography

Gas Liquid

GSC GLC RPC NPC IEC SEC AC

GC : Volatile solutes
(gas has lowest density, can only elute volatile comps with small molecular
weight, Mw)
(Sample MUST be volatile at temperatures BELOW 350oC)

LC : Any mobile phase soluble solutes

3
GC separation is based on :

1. Differences in boiling points of the solutes

(volatilities).

2. Solutes’ interaction with the stationary phase

(polarity).

4
Gas-solid chromatography (GSC)

➢ Based on solid stationary phase (charcoal, molecular

sieves).

➢ Retention of analytes by physical

adsorption/desorption process on solid surfaces of
stationary phase.

5
Gas-solid chromatography (GSC)

➢ Used for the separation of species that are not

retained by GLC (low molecular weight gaseous, eg. air
components, hydrogen sulfide, carbon monoxide &
carbon dioxide, carbon disulfide, nitrogen oxides, rare
gases).

➢ Perform with both packed (molecular sieve) & capillary

column (porous polymer, e.g. PLOT).

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Gas-solid chromatography (GSC)

➢ Limited application :
• Semi-permanent retention of polar molecules.
• Tailing of elution peaks.

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Capillary Column

➢ Porous layer open tubular column (PLOT)

➢ Stationary phases are comprised of a thin layer (usually

<10 µm) of small particles adhered to the surface of the
packing

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Gas-liquid chromatography (GLC)

➢ Known as gas chromatography.

➢ Stationary phase is liquid (high boiling point)

that is immobilized on the surface of solid
support/walls of capillary tubing.

➢ Liquids are used to modify the stationary

phase.

9
 The chromatographic process
- Partitioning
Separation is achieved by partitioning the sample
component between the phases

(gas)
MOBILE PHASE

Sample
Sample
out
in

STATIONARY PHASE
(liquid coated onto a solid support)

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 Schematic diagram of
gas chromatography
Sample is injected & vaporized
onto the head of the column

As each
component
emerges from the
column, it will be
detected

Separation is based on the differences in

migration rates among the analytes in samples

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 Performing GC separations

➢ Sample is injected at the injected port.

➢ Analytes (volatiles) are vaporized & transported through the column by

the flow of gaseous mobile phase (He, N2 or H2).

➢ The column is kept at a control temperature inside an oven, so that the

mixture remains in vapor form to be eluted.

➢ The detector is maintained at a higher temperature than the column so

that all analytes will be gaseous.

➢ From the time the mixtures are injected until they reach the detector,
they are being retained by the liquid stationary phase in the column.

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GC Instrumental components

1. Carrier gas (mobile phase)

2. Columns

3. Sample injection system

4. Detectors

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1. Carrier gas
(mobile phase)

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 Carrier gas (mobile phase)

➢ Function : As a mobile phase.

▪ Transportation of injected sample from the injection

port to the column & subsequent transfer of
separated components to the detector

Mobile phase does not interact with

molecules of the analyte

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➢ Pressure regulators, gauges & flow meters are required to
control the flow rate of the gas.

TANK PRESSURE REGULATOR

Inlet
Outlet Gauge
Gauge

Gas
Shutoff Inlet

Pressure Control

GC gas control Outlet Fitting or H2 Snubber

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➢ Type of gases for carrier gas (mobile phase)

✓ Helium (common)
✓ Nitrogen
✓ Hydrogen
✓ Argon

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➢ Carrier gas :

✓ Does not interact with molecules of the analyte

(chemically inert).

✓ Highly purity gas (99.995% - 99.9995%) & free of

oxygen & moisture (dry).

✓ Contains a molecular sieve to remove water & other

impurities or used in connection with desiccants,
hydrocarbon trap to filter any contaminants from
reaching the column.

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 Advantages & disadvantages of carrier gas

Gas Advantage Disadvantage

Nitrogen Cheap, readily available Long run times
Helium Good compromise, safe, Expensive
compatible with many
detectors

Hydrogen Shorter run times (low Explosive

Mw, higher flow rate can
be used), cheap

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20
 Typical carrier gases

Carrier Gas Symbol Detector Type

He, N2, H2 FID Flame Ionization
N2, Ar/Methane ECD Electron Capture
He, H2, N2, Ar TCD Thermal Conductivity
He, N2, H2 PID Photoionization
He, N2, H2 ELCD Electrolytic Conductivity
He, N2, NPD Nitrogen Phosphorus
He, N2, H2 FPD Flame Photometric
He MS Mass Spectrometer

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➢ The carrier gas flow can be quantified by:

✓ Flow rate (mL/min) (dependent to the column diameter)

✓ Linear velocity (cm/sec) (independent to the column
diameter)

Flow rate (F) is how much mobile phase passed/minute (mL/min).

Average linear velocity (ū) is the distance passed (cm)/sec by mobile phase
in the column (cm/sec).

tM = time at which mobile phase travels through the column.

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Recommended linear velocities & flow rates

Linear velocity (cm/sec) Flow rate (mL/min)

Diameter
(mm)
Helium Hydrogen Helium Hydrogen

0.18 30-45 45-60 0.5-0.7 0.7-0.9

0.25 30-46 45-61 0.9-1.3 1.3-1.8

0.32 30-47 45-62 1.4-2.2 2.2-2.9

0.53 30-48 45-63 4.0-6.0 6.0-7.9

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 Van Deemter Plot for GC
➢ Theories of band broadening in GC can be explained by
the Van Deemter equation.

➢ Van Deemter curve for GC differ using 3 different carrier

gases: N2, He, H2

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 Van Deemter Plot
➢ A plot of plate height vs average linear velocity of mobile
phase.

➢ Determine the optimum mobile phase flow rate to obtain

the minimum H.

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➢ With N2, the optimum flow rate is lower compared to H2.
Gas density : N2 > He > H2 DM : H2 > He > N2
➢ B α diffusion coefficient of analyte in mobile phase, DM
➢ H2 is lighter than N2 (heavier; larger Mw), therefore DM in H2 is higher than
in N2.

➢ Hence, B term is more

significant in H2 at low flow
rate compared to N2. This
increase band broadening,
thus poor resolution.
➢ A highly dense gas gives best
efficiency since diffusivity is
lower, but a low density gas
gives faster speed.

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➢ The slope of the curve at higher flow rate is shallower when using H2 due
to smaller C term (the equilibrium of analyte between the mobile phase
(H2) & stationary phases is more efficient).

➢ H2 & He give better Rs (smaller H) than N2 at high flow rate because

solute diffuse more rapidly through H2 & He than N2.

➢ The more rapidly a solute diffuses between phases, the smaller is the C
term.

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 The mobile phase mass transfer (CMv)

CMv = (fM(k’)dp2/DM)v
dp = particle diameter of packing
DM = mobile phase diffusion coefficient

➢ CMv is less if dp is smaller (hence greater surface area), or

the solute diffusion coefficient in the mobile phase, DM is
larger.
➢ Small particles reduce the distance solute must diffuse in
the mobile phase

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 Sample Problem 2.1
GC, HPLC & SFC are 3 types of separation methods with different mobile
phases. Compare & discuss how each mobile phase affects the B term
of van Deemter in each of the above separation method.

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 Sample Problem 2.2
Which term (A, B or C) is most significant in GC? Explain why & suggest
one approach to minimum the effect.

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 Effect of mobile phase flow rate on H for GC and LC

➢ B/v is important in GC, because of larger diffusion rates in gases (104-

105 times greater than liquids).

➢ The minimum curves relating H to flow rate are broadened in GC.

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2. Column

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 Type of Column

Types of column

Packed column Open tubular (capillary) column

Wall-coated open Support-coated Porous-layer

tubular column open tubular open tubular
(WCOT) column (SCOT) column (PLOT)
(used in GSC)

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Column Dimension

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 Packed column

➢ ~ 2-6 mm I.D. tubing.

➢ 1-5 m length.
➢ Used to separate gases that are
poorly retained.
➢ Provide greater sample capacity.
➢ Disadvantages :
X Broader peak
X Longer tR
X Less resolution

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 Tube made of glass, metal (stainless steel, copper,
aluminium) or teflon with 2-6 mm I.D. & 1-5 m in
length.

Packed with fine particles of solid support

(commonly diatomaceous earth) coated
with a thin layer (0.05-1 µm) liquid
stationary phase.

Solid support :
✓ Small & uniform particles size
✓ Mechanical stability
✓ Porous
Solid support ✓ High surface area
✓ Serve to hold the ✓ Inert
liquid stationary
phase

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 Column Wall
Carrier
Gas

Solid Support
Liquid Phase

➢ Small, uniform particle size decreases multiple path, A term (Eddy

Diffusion) (requiring higher pressures).
➢ Particles size in µm or mesh size – 60-80 mesh (260-170 µm) , 80-100
mesh (170-149 µm)

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➢ Small diameter particle (dp), decrease the time required
for solute equilibration, thus improve column efficiency.

CMv = (fM(k’)dp2/DM)v

➢ However, the smaller the dp, the less space between

particles thus, the more pressure required to force
mobile phase through the column.

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➢ Flow of the mobile phase through a capillary column is
relatively unimpeded when in comparison with a packed
column.

➢ This allow for a faster mobile phase compared to packed

column. Thus reduce B, decreasing band broadening.

B/v = 2ɣDM/v

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➢ Due to the difficulty of packing the tubing
uniformly, these types of columns have a larger diameter
than capillary columns & have a limited range of length.

➢ As a result, packed columns can only achieve about 50%

of the efficiency of a comparable Wall-coated open
tubular column (WCOT) column.

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 Capillary column

➢ Have a very small I.D.(0.1-0.5 mm).

➢ 10-1000 m long (no packing, can be
longer & narrower).
➢ 0.1-5 µm thick stationary phase
coated on inner walls.
➢ 3 types of columns :
▪ WCOT
▪ SCOT
▪ PLOT

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 Wall-coated open tubular column, WCOT - The inner
wall are directly coated with liquid stationary phase
(no support material).

Porous-layer open tubular column, PLOT -

Stationary phase (solid particles are the active
stationary phase) on inside wall of column

Support-coated open tubular column, SCOT - Liquid

stationary phase coated on solid support attached to
inside wall of column (thin film ~30 µm).

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Tubes / capillary (capillary made of metal, glass, stainless steel or
thin fused silica (SiO2))

Stationary phase

SCOT: the inner wall of the capillary is lined with a thin layer of solid
support onto which the stationary phase has been adsorbed.

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 Fused silica capillary column

➢ One of the most popular types of capillary columns is a special WCOT

column called the fused-silica wall-coated (FSWC) capillary column.

➢ Much thinner (0.1 mm) than the glass capillary columns & are given
strength by the polyimide coating.

➢ Fused silica coated on the outside with a polyimide polymer for

support & protection for the fragile silica capillary, allowing them to
be coiled.

➢ Flexible & can be wound into coils.

➢ They have the advantages of physical strength, flexibility, low

reactivity & smaller sampling size .

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(imparts a brownish color to the columns)

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Compared with packed column, capillary column offers :
✓ Higher Rs with narrow peak
✓ Shorter analysis times due to the higher flow rates applied
✓ Greater sensitivity

Disadvantage :
X Lower sample capacity
100 ng for a 0.25 mm I.D. column with 0.25 µm thick film
5 µg for a 0.53 mm I.D. column with 5 µm thick film

Resolution efficiency (Rs):

WCOT > SCOT > PLOT > packed column

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Properties of GC columns

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➢ Capillary columns tend to have lower H (greater N per given column
length) since there is no A term (no packing).

➢ However, C term is highly dependent on diameter of column.

➢ Narrow ID column (eg. 250 µm) has lower C term compared to wide ID
column (eg. 500 µm)

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 Column temperature

➢ The optimum column temperature is dependent upon the boiling

point of the sample.

➢ A temperature slightly above the average boiling point of the sample

results in an elution time of 2-30 minute.

➢ Minimal temperature give good Rs but increase in elution times.

➢ If sample has a wide boiling range, then temperature programming

can be useful.

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 GC columns

Factor to be considered in selecting the column:

1. Thickness of stationary phase
2. Column diameter & length
3. Stationary phase

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➢ Film thickness primarily affect the retentive character & the
capacity of a column.

➢ Thick films are used with highly volatile analytes, it retain

solutes for a longer time and thus, provide a greater time for
separation to take place.

1. Thick film and narrow bore columns provide good Rs &

sample capacity.

✓ Can be used with most detectors.

✓ tR are longer than thin film.

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2. Thick films and wide bore columns are required with thermal
conductivity and IR detectors.
✓ Have high sample capacity.
✓ Low Rs & long tR.

3. Thin films and narrow bore columns are useful for separating
species of low volatility (high boiling point) in a reasonable
time & more efficient.

Disadvantages :
▪ Low sample capacity
▪ Require high sensitive detector

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 GC column comparisons

Description Thin film narrow bore Thick film narrow bore Thick film wide bore

Inner Diameter 0.10-0.32 mm 0.25-0.32 mm 0.53 mm

(I.D)

Film thickness ~ 0.2 µm ~ 1-2 µm ~ 2-5 µm

Advantages High resolution Good capacity High capacity

Fast separation Good resolution

Disadvantages Low capacity Moderate resolution Low resolution

Long tR for high bp Long tR for high bp
comps comps

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 Sample Problem 2.3
If a GC column is changed from packed to capillary column, explain
the effect of the changes on the H.

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 Stationary Phase

➢ Liquid stationary phase : Non-volatile liquid coated on inside of

column or on fine solid support. The boiling point of the liquid should
be at least 100 ᵒC higher than the maximum operating temperature
of the column.

➢ Ideal properties :

▪ Low volatility
▪ Thermal stability
▪ Chemical inertness
▪ Low viscosity

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➢ Choice :
✓ Depend on the analyte of interest using the principle “like-dissolve-
like”.
✓ polar liquid for polar analytes
✓ nonpolar liquid for nonpolar analytes

➢ The polarity of the stationary phase must be comparable to the polarities of

the analytes, so that there will be interaction between the analytes & the
stationary phase (eg: Non polar analytes when separated using polar
stationary phase (sp) fast elution due to no @ less interaction with sp.

➢ When the match is good, the order of elution is determined by the boiling
point of the analytes.

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➢ Order of elution:
➢ Less retained analytes elute earlier.
➢ Given similar molecular characteristics, more volatile analytes (low
boiling point) elutes earlier. Low molecular masses elute earlier.

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➢ Polar stationary phases contain functional groups e.g., –CN, -CO- and –
OH.

➢ Polyesters phases are highly polar.

➢ Hydrocarbon-type stationary phase and dialkyl siloxanes are non polar.

➢ Analytes:

➢ Polar – alcohols, acids & amines.

➢ Medium polarity – ethers, ketones & aldehydes.

➢ Non polar – saturated hydrocarbons.

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Examples of liquid stationary phases :
1. Polydimethyl siloxanes (PDMS):
➢ Non to strong polarity depending on R groups.
➢ Most stable, robust & versatile.

R R
O Si O Si
R R n

➢ For PDMS, R = CH3, liquid are relatively non polar.

➢ In other polysiloxanes, a fraction of methyl groups are replaced by
functional groups such as phenyl (-C6H5), cyanopropyl (-C3H6CN) &
trifluoropropyl (-C3H6CF3).

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➢ The most basic polysiloxane is the 100% methyl substituted.

➢ The % give the amount of substitution of the named group for methyl
groups on the polysiloxane backbone.

➢ E.g., 5% phenyl PDMS (5% phenyl group and 95% methyl group).

➢ The substituents increase the polarity of the liquids to various degrees.

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Examples of liquid stationary phases :

2. Polyethylene glycol (PEG)

HO-CH2-CH2-(O-CH2-CH2)n-OH

➢ Used for the separation of polar analytes.

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 Stationary Phase Common Maximum Common Applications
Trade Name Temperature (C)

General-purpose nonpolar
Polydimethyl siloxane phase; hydrocarbons;
(NON POLAR) HP-1, SE-30 350 polynuclear aromatics; drugs;
steriods; PCBs
5% Phenyl-polydimethyl Fatty acid methyl esters;
siloxane HP-5, SE-52 alkaloids; drugs; halogenated
350
compounds

50% Phenyl- Drugs; steriods; pesticides;

Polydimethylsiloxane OV-17 250 glycols

50% trifluoropropyl- Chlorinated aromatics;

polydimethyl nitroaromatics; alkyl-substituted
OV-210 200
siloxane benzenes

Polyethylene glycol Carbowax Free acid; alcohol; ethers;

(POLAR) 20M 250 essential oils; glycols

50% cyanopropyl - Polyunsaturated fatty acids; free

polydimethyl OV-275 240 acids; alcohols
siloxane

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Typical chromatograms for open tubular columns coated with
(a) PDMS; (b) 5% phenyl methyldimethyl siloxane; (c) 50% phenyl methyldimethyl siloxane; (d) 50%
poly(trifluoropropyl-dimethyl) siloxane; (e) PEG; (f) 50% poly(cyanopropyl-dimethyl) siloxane. (Courtesy of
J&W Scientific)

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Nonpolar versus polar stationary phase

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Bonded & cross-linked stationary phases

➢ Bonded phase column is the column with stationary phase that

attached to the surface of the packing particles by chemical bonds.

➢ Purpose :

▪ Provide a longer-lasting (or more permanent) stationary phase :

that can be rinsed with a solvent when the film becomes
contaminated.

▪ Thermal stability (not disrupted at elevated temperature or

during temp programming).

65
 Column bleeding
➢ Untreated column slowly lose their stationary phase owing to ‘bleeding’ in
which a small amount of immobilized liquid is carried out of the column
during elution.

➢ 2 causes for column bleeding:

▪ High column temperature
is used
▪ Non bonded packing
is used

66
➢ Process :

▪ Bonding: Attaching a monomolecular layer of the stationary phase to

the silica surface of the column by a chemical reaction.

▪ Cross-linking:

▪ treating the stationary phase while it is in the column with a

chemical reagent that creates cross links between the molecules
making up the stationary phase.

▪ Eg. Incorporate a peroxide into the original liquid/initiated by

exposing the coated column to gamma radiation.

67
 Solid support (packing)

➢ Ideal characteristics of solid support :

▪ Strong, porous, high surface area & inert (non-adsorptive).

➢ Example of solid support :

▪ Diatomaceous earth (algae skeletons).

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➢ A problem in GC is the physical adsorption of polar analytes (eg.
alcohols/aromatic hydrocarbons) on the silicate surfaces of column
packings & capillary walls.

➢ Adsorption results :Distorted peaks, which are broadened & exhibit a

tailing peak.

➢ Adsorption is the consequence of silanol groups that form on the

surface of silicates by reaction with moisture.

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➢ The SiOH groups on the support surface have a strong affinity for polar
molecules & tend to retain them by adsorption.

fully hydrolyzed silicate surface structure

➢ NOTE: Operating pH for silica stationary phase = pH 2-8, siloxane bond (Si-O-
Si) to the stationary phase hydrolysed below pH 2 while above pH 8 it will
dissolved

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Example of peak tailing

71
Asymmetric peak shapes

❖ Tailing---occurs when there

are some other sites on
stationary phase—that have
strong interaction with solute

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 Deactivation of support materials

➢ Treatment of siloxane column for the analysis of polar compounds.

➢ To inhibit physical adsorption of polar analyte (alcohols, aromatic

hydrocarbons) on the silicate surfaces of column packings or capillary walls.

➢ Process:

▪ Silanization using dimethylchlorosilane (DMCS) & washing with

methanol. Acid washing prior to silanization may be required to
remove metal oxide impurities.

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1. Silanization using
DMCS

2. Washing with
methanol

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 Kovats Retention Index (RI)
➢ Retention index relates the retention time of a solute to the
retention time of linear alkanes

75
76
3. Sample injection
system

77
 Injection port / inlets
(sample introduction)
➢ The inlet is a piece of hardware attached to the column head.

➢ To flash/ instantly evaporate the sample & introduce it into the column.

➢ Tinj > 50 oC higher than the boiling point of least volatile component of
the sample (250°-275° C is adequate) to ensure fast & complete
vaporization.

➢ The injection port temperature must be high enough to completely

vaporize the sample.

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➢ Injection using a microsyringe (gases are injected by a gas-tight syringe)
through a rubber septum.

▪ Septum must be stable at injection temperature & replaced regularly

to maintain seal (lifetime : ~20 manual injection or ~100 auto sampler
injections).

▪ Decomposed sample, non-volatile components & septum debris

accumulate in the glass liner (must be replaced periodically).

▪ The septum purge outlet prevents septum bleed components from

entering the column.

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➢ Column efficiency requires :

▪ Introduced onto the column as a "plug" of vapour.

▪ Sample volume should not be too large due to it will cause broad peak
due to slow vaporization of sample.

➢ Slow injection of large samples causes band broadening & loss of

resolution.

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Packed column
➢ Volume injected :
▪ Liquid samples – 0.1-10 µL
▪ Gas samples – 1-10 mL

➢ All the vaporized sample from the injector enters onto the
column.

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Capillary columns
➢ Volume ~10-3 µL.

➢ The size of the capillary column limits the amount of analyte that can be
injected, otherwise, overloading occurs.

➢ Alternative to get the amount of analytes

that are injected onto the column smaller
(the remainder going to waste) without
having to dilute concentrated samples is
the split/splitless capillary GC injector.

82
Recommended procedure for sample loading into syringe

1. Fill the syringe with sample without air bubble.

2. Retract the syringe plunger, pulling ~2 µL of air into the syringe.

This give air peak, which can be used to indicate the dead time of
the column & prevents loss of sample due to vaporization.

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 Injector types

1) Split injector
2) Splitless injector
3) On-column injector

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85
Septum Nut Septum

Split/Splitless Inlet Weldment

Viton O-Ring
Liners

Gold-Plated Seal (Splitless)

86
 Splitless Packing Split Packing

Liner
Ground
4 inches long. Ground
Portion
Portion
6 mm O.D. / 4 mm I.D.
6 mm O.D. / 2 mm I.D. O-Rings O-Rings

Quartz Wool Quartz Wool

Packing Loosely Packed Loosely Packed
Silanized glass wool
Quartz Wool
recommended. Tightly Packed
Top portion loosely packed.
Dimple Dimple
Bottom portion tightly
packed.

4-mm i.d. 2-mm i.d. 4-mm i.d.

Wide-Bore Liner Narrow-Bore Liner Wide-Bore Liner
(P/N N612-1001) (P/N N612-1002) (P/N N612-1001)

87
 1) Split Injection (sample splitter)

➢ Used when analytes are in high concentration (> 0.1%) and

would overload the column.

➢ Splitting of sample allows deposit of small of a µL (0.2 – 2 %)

in the column – introduces only a small amount of sample into
the column.

➢ The rest is sent to waste.

➢ Produces narrow peak widths and high reproducibility.

88
➢ Large part of sample vented out via the split valve will:
▪ Prevents overloading of the column.
▪ Produces narrow & sharp peaks.

➢ After sample vented out, split outlet is closed.

➢ Split ratio: the proportion of sample vented out from the column (typically
write 20:1 to 100:1).

▪ E.g. 50:1, that is, for every 50 units of gaseous sample that are vented
out to waste, 1 unit goes on the column.

▪ Higher ratio – Too much sample vented out, small amount of sample
enter the column, hence small peak & not reproducible.

▪ Too low – Poor peak shape, column overload.

89
 (a) Split ratio : 1/25 (b) Split ratio: 1/75

Effects of a low split ratio. Effects of a high split ratio.

All of the peak heights were All of the peak heights were
increased due to the greater reduced due to the smaller
amount of the sample amount of the sample
introduced into the column. introduced into the column.

90
 2) Splitless Injection (split vent is closed)

➢ Most of the sample is introduces on the column. Sample spends a large

amount of time in the injector.

➢ Used for low concentration samples (dilute samples) or trace analysis

(less than 0.01% of the sample) of high boiling compounds in low boiling
solvents.

➢ Relatively small samples (< 10 µL).

➢ Split vent opened 30-90 seconds after injection, removes bulk of the
solvent (large solvent peak may overlap with the analyte peak), but
leaves most of the sample condensed at the top of the column.

91
➢ Purpose :

▪ To reduce/remove amount of solvent into the column while

ensuring all the analytes to enter the column.

▪ Thus, reduce solvent peak and separate the analyte from the
solvent.

▪ Low conc. (e.g. sample extracted using SPME) : ensure all the
samples will be transferred into the column.

92
➢ Requires a low initial column temperature to dissolve & refocus the solutes
at the start of the column & then temperature programming.

➢ Transfer times are slower and peaks are broader when compared to split
injection.

➢ Two techniques can be used to get a narrow band (refocus the solutes) at
the head of the column: solvent focusing and analyte focusing.

➢ The difference between the two methods is the initial temperature of the
column oven.

93
1) Solvent focusing, the initial oven temperature is low enough (~20 °C
below the boiling point of solvent) to allow the solvent to recondense at
the head of the column. This forms a zone of liquid solvent that
traps/focus all of the analytes at the head of the column.

2) Analyte focusing requires an initial oven temperature (60-80 °C below

the boiling point of the earliest eluting compound) that allows the
solvent to move through the column as a vapor immediately after
injection. Analytes that have a significantly higher boiling point than the
solvent are recondensed at the head of the column because of the lower
oven temperature.

94
The Splitless Process

➢ The sample is injected & vaporized.

➢ Column temperature is kept at relatively low temp, lower (by at least 10 oC)
than the boiling point of the solvent.

➢ Volatile solvents will condense at the top of the column & solute will
dissolve & refocus in the solvent (solvent trapping).

➢ Less volatile solute will re-condense on top of the colder column & refocus
too (cold trapping).

➢ Then, split vent will be opened to remove bulk of the solvent. (Note: large
solvent peak may tailing and will overlap with the analyte peak).

95
➢ Sample that is concentrated at the top of the column and then will
introduce into the column giving a sharp peak.

➢ This approach requires a low initial column temperature to dissolve and

refocus the solutes at the start of the column and then temperature
programming is applied to elute the analytes.

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 toluene
toluene

Overlapping of analyte peak with the

solvent peak

➢ Advantages :
✓ Good for trace analysis (less than 100 ppm).
✓ Good for high boiling point compounds.
✓ Easily automated.

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98
 3) On column injection

➢ Used for samples that easily decompose above their boiling point
or thermally labile compounds.

➢ Can handle dilute/concentrated solutions & relatively large/small

volumes.

➢ Best for quantitative analysis.

➢ Widely used in packed column, less in capillary column.

➢ Entire sample reaches the detector

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➢ Sample is directly injected into the column as a liquid without
vaporization with the aid of a syringe with a long, narrow needle
whereby the injector is maintained at low temperature.

➢ Analyte will later vaporize by temperature programming of the

column.

➢ Advantages:

▪ Trace analysis : enables quantitative insertion of sample into

column.

▪ Labile components not stressed thermally.

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101
4. Detectors

102
 Detectors

➢ A specific detector responds to a single chemical

compound.

➢ A selective detector responds to a range of compounds

with a common physical or chemical property.

➢ A non-selective / universal detector responds to all

compounds except the carrier gas.

➢ A non-destructive detector does not destroy sample.

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 Flame Ionization Detector
(FID)
➢ Analytes containing carbon burn in hydrogen-oxygen flame and produce
ions.
➢ CH + O → CHO+ + e-

➢ CHO+ ions are collected on a cathode and

the current they produce results in the
signal (increase the current).

➢ The more CH-groups a molecule contains,

the larger is the detector response.

➢ Gases are not an organic compound and

therefore cannot be detected by FID.

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➢ Useful :
✓ Responds to the number of carbon atoms entering the detector per
unit time, it is mass-sensitive, rather than a concentration-sensitive.
✓ General detector for the analysis of organic compounds or
hydrocarbons.
✓ Insensitive to nonhydrocarbons (H2, He, N2, O2, CO, CO2, H2O, NH3,
NO, H2S).
✓ High sensitivity.
✓ Large linear response range.
✓ Low noise.
✓ Robust.
✓ Easy to use.

➢ Disadvantage : destructive detector, it destroys the sample.

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 Electron-Capture Detector
(ECD)
➢ The sample eluate from a column is passed over a
radioactive β-emitter like nickel-63.
➢ An electron from the emitter causes ionization of
the carrier gas (often N2) and will produce a burst
of electrons.
➢ In the absence of organic species, a background
current between a pair of electrodes results from
this ionization process.
➢ When analytes that can capture electrons e.g with
halogens, peroxides & nitro groups enter the
detector, electrons from the background current
are captured, therefore the current is suddenly
decreased.
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➢ Advantages:

✓ Non-destructive.

✓ Sensitive.

✓ Selective to organic molecules or analytes which contain

electronegative functional groups that can capture electrons e.g.
halogens, peroxides, quinones & nitro groups.

✓ Insensitive to amines, alcohols, ketones & hydrocarbons.

✓ Widely used detectors for environmental samples such as chlorinated

pesticides & polychlorinated biphenyls.

➢ Disadvantage : involve radioactive component.

ECD cannot be used with inert gas like He due to no interaction of electrons
with the carrier gas molecules to form a background current.

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 Thermal Conductivity Detector
(TCD)

➢ The TCD works by measuring the change in

carrier gas thermal conductivity caused by the
presence of the sample, which has a different
thermal conductivity from that of the carrier
gas.

➢ Their design is relatively simple & consists of

an electrically heated source that is
maintained at constant power.

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➢ The temperature of the source depends upon the thermal conductivities
of the surrounding gases.

➢ The source is usually a thin wire made of platinum or gold. The

resistance within the wire depends upon temperature, which is
dependent upon the thermal conductivity of the gas.

➢ TCDs usually employ two detectors, one of which is used as the

reference for the carrier gas & the other which monitors the thermal
conductivity of the carrier gas & sample mixture.

➢ Carrier gases such as helium and hydrogen has very high thermal
conductivities so the addition of even a small amount of sample is
readily detected.

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➢ Advantages:

✓ Universal, not selective (broad application to inorganic & organic

compounds).

✓ Non-destructive.

➢ Disadvantage : Not sensitive.

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 GC detectors & their detection limits
Symbol Detector Type Selectivity Detection limit
Most organic cmpds that ionize in
FID Flame Ionization 100 pg
flame

Halides, nitrates, nitriles, peroxides,

50 fg
ECD Electron Capture anhydrides, organometallics

Thermal
TCD Universal 1 ng
Conductivity

Aliphatics, aromatics, ketones, esters,

PID Photoionization aldehydes, amines, heterocyclics, 2 pg
organosulphurs, some organometallics

Nitrogen
NPD Nitrogen, phosphorus 10 pg
Phosphorus
Sulphur, phosphorus, tin, boron,
Flame
FPD arsenic, germanium, selenium, 100 pg
Photometric
chromium

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 Detection techniques & their applications
Detector Used for
Mass spectrometer (MS) Identification of unknown compounds
Flame Ionization Detector Compounds containing Carbon
(FID)

Thermal Conductivity Universal detection for gases without

Detector (TCD) Carbon

Nitrogen Phosphorous Selective detection of Nitrogen and

Detector (NPD) Phosphorous containing compounds

Electron Capture Detector Selective detection of halogen containing

(ECD) compounds

Atomic Emission Detector Selective detection of elemental

(AED) composition

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 Interfacing GC with
spectrometric detectors

➢ Mass spectrometry (MS)

➢ Fourier Transform Infrared Spectroscopy (FTIR)

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 Mass Spectrometry Detector

➢ Mass spectrometer: Detector which fragments the molecules of eluting

compounds. The individual fragments are than detected.

➢ GC-MS composed of two blocks: GC & Mass spectrometry (MS)

➢ Molecules elute from the GC & allows the MS to capture, ionize,

accelerate, deflect & detect the ionized molecules separately.

➢ The MS does this by breaking each molecule into ionized fragments and
detecting these fragments using their mass to charge ratio.

➢ Analysis of the mass spectra of each analyte and comparison of spectra

from MS libraries to experimental spectra accomplishes the identification
of analysis.

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➢ Advantages of MS:

▪ The MS is used to identify

comps based on their
structure, meaning that it will
provide the structural
information of each compound
match from the library of mass
spectra of known compounds
stored on the computer & thus
can identify the compound.

▪ Measures the mass-to-charge-

ratio (m/z) of charged analytes.

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116
Method Development for GC

Method development is the process of

determining what conditions are adequate
and/or ideal for the analysis required.

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 Areas to optimize

➢ Injector

➢ Carrier gas

✓ Optimal range of velocities based on van Deemter curve.

✓ Too low or high velocity results in loss of Rs.

✓ Balance Rs vs analysis time.

➢ Column Temperature

✓ The optimum column temperature is dependent upon the boiling point

of the analyte in sample.

✓ Minimal temperatures give good Rs, but increase the elution times.

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Factors which affect GC separations

➢ Efficient separation of compounds in GC is dependent on

the compounds traveling through the column at different
rates.
➢ The rate at which a compound travels through a particular
GC system depends on :
a) Volatility of compound : Low boiling (volatile)
components will travel faster through the column than
high boiling components. Boiling points of the
different components is the main factor in GC
separation.

119
b) Polarity of compounds : Polar compounds will move slowly
if the column is polar. Differences in polarity of the
compounds is only important if you are separating a mixture
of compounds which have widely different polarities.

c) Column temperature : Raising the column temperature

speeds up the elution of all the compounds in a mixture.
Hence, decrease the tR & sharpen the peak. Separation
based on isothermal temperature or temperature
programming.

120
d) Column polarity : Usually, all compounds will move slower
on polar columns, but polar compounds will show a larger
effect.

d) Flow rate of the gas through the column : Speeding up the

carrier gas flow increases the speed with which all
compounds move through the column.

d) Length of the column : The longer the column, the longer

it will take all compounds to elute. Longer columns are
employed to obtain better separation (higher N).

121
➢ Factors to be considered in selecting the column :

✓ Polarity of stationary phase

✓ Column length & diameter
✓ Thickness of stationary phase

122
Column Length vs. resolution

123
Column Diameter vs. resolution

124
 The heights of the later eluting peaks are
ISOTHERMAL reduced & the peak widths increased
because they are more affected by the lower
Constant column temperature temperature program used.
throughout the analysis.

Oven temperature :100 oC

TEMPERATURE PROGRAMMING
Oven temperature:
Column temperature is 60oC for 1 min
increased either continuously or 60-180oC at 20o/min
in steps as the analysis
proceeds.

Increase in temperature
✓ Decrease retention time
✓ Sharpens peak

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 Temperature Program Method

➢ Analysis of mixtures with a broad boiling point range

causes a group of early eluting comps (low bp.)
separated & late eluting comps (high bp.) will have very
long tR at low isothermal temperature.

126
➢ When column temperature increases, the early eluting
compounds (low boiling point) will overlapped & late eluting
compounds (high boiling point) separated.

127
➢ In the analysis of a broad boiling point range, isothermal
analysis may not produce an efficient separation. The temp
programming able to solve general separation problem such
as long elution times that cause band broadening which lead
to poor Rs.

➢ Thus, use temperature programming with initial low

temperature to separate low boiling point compounds & the
temperature is increased at a rate (either increased
continuously or in steps) to reduce the tR for the high boiling
point compounds.

128
➢ A temperature program allows comps that elute early in the
analysis to separate adequately, while shortening the time
it takes for late eluting comps to pass through the column.

129
Solution to general elution problem

➢ If we could change the separation conditions (ie.

capacity factor, k’) as the separation proceeds, we could
optimize conditions for separation of early peaks in the
first part of the procedure.

➢ Then change conditions to gradually optimize conditions

for late eluting components, so that they are adequately
separated in a reasonable period of time.

➢ How do we change K as separation proceeds?

➢ Gas Chromatography- temperature programming

130
Increase column length

k’A = [(tR)A – tM]/tM

Increase Rs, increase tR

α = (tR)B – tM/(tR)A – tM

131
➢ The most effective way to alter Rs is to change the
selectivity or the capacity factor of the column.

➢ The effect of increasing the efficiency of the column by

increasing the column length, will increase N.

➢ If increased resolution is required, a column with a higher

capacity factor is often the best choice.

➢ However, increasing the capacity factor will increase the

analysis time, so a compromise must be reached between
resolution and analysis time.

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 Overloaded column

➢ Too much solute has been applied to the column.

➢ As the conc. of solute increases, the solute becomes

more & more soluble in the stationary phase (“like
dissolve like”).

➢ There is so much solute in the stationary phase that the

stationary phase begins to resemble solute.

133
➢ The front of an overloaded peak has
gradually increasing concentration.

➢ As the concentration increases, the

band become overloaded.

Asymmetric peak
➢ The solute is so soluble in the
overloaded zone that little solute trail
behind the peak.

➢ The band emerges gradually from the

column but ends abruptly.

134
Sample Preparation for GC

a) Derivatization

b) Pyrolysis

c) Headspace

135
 a) Chemical Derivatization

➢ Derivatization : A process of chemically modifying a comp

to produce a new comp which has properties that are
suitable for an analysis.
➢ Derivatization used when;
▪ analytes are not sufficiently volatile (high molecular
weight),
▪ tailing peak (adsorption to the stationary phase/polar
comp (poor volatility due to intermolecular interactions
caused by hydrogen bonding)
▪ thermally unstable (decompose).

136
➢ Chemical derivatization can : (Advantages)
✓ Increase the volatility & decrease the polarity of
compounds.
✓ Reduce thermal degradation of samples by increasing
their thermal stability.
✓ Reduce tailing.
✓ Increase specific detector response by incorporating
functional groups which lead to higher detector signals,
e.g. CF3 groups (addition of halogen atoms) for ECD.

137
➢ Common derivatization methods can be classified into 4
groups depending on the type of reaction applied :

▪ Silylation
▪ Acylation
▪ Alkylation
▪ Esterification

138
 b) Pyrolysis

➢ Is a method of chemical analysis in which the sample is

heated to decomposition to produce smaller molecules
that are separated by GC & detected using mass
spectrometry.

➢ Pyrolysis is the thermal decomposition of materials in an

inert atmosphere or a vacuum.

➢ The sample is put into direct contact with a platinum wire,

or placed in a quartz sample tube, and rapidly heated to
600–1000 °C.

139
➢ Large molecules cleave at their weakest points and
produce smaller, more volatile fragments. These fragments
can be separated by gas chromatography.

➢ Pyrolysis gas chromatography is useful for the

identification of non-volatile compounds and synthetic
polymeric media, such as acrylics or alkyds, synthetic
varnishes and in environmental samples, e.g. fossils..

140
 c) Headspace

➢ Normally prepared in a vial containing the sample, the

dilution solvent, a matrix modifier and the headspace.

➢ Volatile components from complex sample mixtures

can be extracted from non-volatile sample
components and isolated in the headspace or gas
portion of a sample vial.

➢ A sample of the gas in the headspace is injected into a

GC system for separation of all of the volatile
components.

141
▪ G = Gas phase (headspace); The gas phase is
commonly referred to as the headspace and lies
above the condensed sample phase.

▪ S = Sample phase; Sample phase contains the

compounds of interest. It usually in the form of
a liquid or solid in combination with a dilution
solvent or a matrix modifier.

Sample phase is introduced into the vial and

the vial is sealed, volatile components
diffuse into the gas phase until the
headspace has reached a state of
equilibrium. The sample is then taken from
the headspace.

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THANK YOU FOR YOUR ATTENTION
THANK YOU
Q & A SESSION
CHM 510
ANALYTICAL SEPARATION METHODS

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