CLS 572 Fall 2020-21

Gas Chromatography

American University of Science and Technology

Nina Esipenko, Ph.D.

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 Principle
• A gaseous mobile phase (carrier gas) flows under pressure
through a heated tube/column packed with liquid stationary
phase coated onto a solid support or pure solid support.
• The sample is loaded onto the head of the column via a heated
injection port where it is evaporated.

• The column is placed in an oven that is programmed at a given

temperature (temp. is held constant or rises gradually)

• The components of a sample are separated based on:

The length of time they spend in the column
 Partitioning with the stationary phase
 The nature of the stationary phase
 Temperature
• The mobile phase does not interact with the analyte molecules;
its only function is to transport the analyte through the column
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 Keypoins

Application

 Drug characterization
• Quantification of drugs

• Characterization of some raw materials used in synthesis of drug

molecules

• Measurement of drugs and their metabolites in biological fluids

 Impurities determination limit test for solvent residuals and

other volatile impurities in drug substances

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 Keypoins
Strengths:
 Capable of the same quantitative accuracy and precision as HPLC
 Much greater separating power than HPLC when used with
capillary columns
 Determines compounds which lack chromophores
 The mobile phase does not vary and does not require disposal
(even if helium is used as a carrier gas, it is cheaper than organic
solvents used in HPLC)
Limitations:
 Only thermally stable (volatile) compounds can be analyzed
 Quantitative sample introduction is difficult because of the small
volumes of sample injected
Aqueous solutions of salts cannot be injected into the instrument
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Instrumentation

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 Instrumentation
 Injection of the sample may be made manually or using an auto
sampler through resealable rubber septum

 The sample is evaporated in the heated injection port area and

condenses on the head of the column

 Column: Capillary or packed column, while the mobile phase is a

gas: N2(g), He(g) or Ar(g)

 Column enclosed in an oven (temp. between ambient to 400oC)

 Detector: flame ionization detector (FID); thermal conductivity

detector (TCD); MS

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Injector functions: Besides its role as an inlet for the sample, it
must vaporize, mix with the carrier gas and bring about the sample at
the head of the column.
Depending on a column type there are Direct vaporization injector
two types of injectors:
 Direct vaporization injector
 Split/splitless injector

Syringes:
The volumes injected in GC are
routinely in the range of 0.5 - 2 µl
vaporization
chamber

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 Split/splitless injector
For capillary columns able to handle only a small capacity of sample, even
the smallest volume that it is possible to inject with a micro-syringe, can
saturate the column. Special injectors are used which can operate in two
modes,with or without flow splitting (also called split or splitless).

 When working with capillary

columns, this type of injector is
used for very diluted samples in
the splitless mode

 Split mode: The split ratio

typically varies between 1 : 20 and
1 : 500. Only the smallest fraction
of sample will penetrate into the
column

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 GC Oven
 GC ovens incorporate a fan which ensures uniform heat
distribution throughout the oven
Oven can be programmed to either produce a constant
temperature – isothermal conditions or a gradual increase in
temperature
 The advantage of temperature programs are that materials of
widely differing volatilities can be separated in a reasonable time

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 Final temperature
(final hold time)

Injection
temperature
Ramp rate
oC/min

Sample Initial temperature

condensation hold time

Injection temperature:
 250oC is sufficient injector temperature for nearly all samples.
 For volatile samples an injector temperature of 150-200oC is recommended
 For high boiling samples such as steroids, triglycerides or surfactants, an injector
temperature of 275-300oCis recommended.
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Solvent focusing is a technique used to get narrow peaks without doing a split
injection (when making on-column or splitless injections)

Attaining narrow peaks without splitting the sample is important because it allows for
greater sensitivity without sacrificing resolution. By correctly setting the GC
parameters, solvent focusing can be used to obtain narrow and symmetrical peaks for
the majority of analyses

during injections vaporized sample condenses on a cool column.

Because the volume of a gas is much larger than that of a liquid, when the sample
condenses, it is focused into a small area on the column.

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There are several parameters in a GC system which affect a sample’s rate of
condensation:

1. initial oven temperature (The lower the initial oven temperature, the greater
the rate of sample condensation)
A good starting point is 50 ºC below the boiling point of the earliest eluting analyte.
This temperature should be held for the duration of the splitless hold time to
ensure that the entire sample is focused onto the column.

2. sample components (The higher the boiling point of the sample components,
the greater the rate of condensation)
when possible, solvent focusing is best achieved with a solvent that has the
greatest difference between the solvent’s boiling point and the initial column
temperature. For example, if the initial temperature is 30 ºC, ethyl acetate (boiling
point. 77.1 ºC) will have a greater focusing effect than dichloromethane (boiling
point 39 ºC).

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Solvent focusing was done on a hydrocarbon test mix using hexane as the solvent
(boiling point 69 ºC). A) Initial GC temp. of 140 ºC. B) Initial GC temp. of 60 ºC.

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Other factors which affect resolution in GC:

 Initial temperature hold time:

Increasing the initial hold time often improves the resolution of the earlier
eluting peaks;
The resolution of later eluting peaks is minimally affected with a change in
the initial hold time.

Lowering the initial temperature and increasing the initial hold time
can be combined to improve the resolution of earlier eluting peaks

 Changing the ramp rate

Better resolution of later eluting peaks often occurs when decreasing the ramp
rate. Only change the ramp rate by about 5 ºC/min each time.

Multiple ramp rates can be used to affect smaller regions of the chromatogram. For
example, if 5 ºC/min was good for the earlier portion of the chromatogram and 15
ºC/min was better for a later portion, both ramp rates can be used within a single
program

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Unknown sample temperature programming strategy:

 start with an initial temperature of 50oC

 ramp rate of 10oC/min

 final temperature equal to the isothermal temperature limit of the column

 final hold time of approximately 30 minutes. The long final hold time is
used to ensure all of the solutes elute from the column.

The program can be stopped several minutes after the last solute has
eluted from the column. This may occur before the final temperature is
reached

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17
Isothermal

vs

gradient

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 GC Column

There are two column types, which differ in their performance:

packed columns and capillary columns

For packed columns the stationary phase is deposited or bonded

by chemical reaction onto a porous support.

For capillary columns a thin layer of stationary phase is deposited

onto, or bound to the inner surface of the column.

capillary columns

packed columns
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 GC Column
Packed columns

 less commonly used today, used for routine analysis

 diameters of 1/8 or 1/4 inch (3.18 and 6.35 mm) and a length of
between 1–3 m
 manufactured from steel or glass, the internal wall of the tube is
treated to avoid catalytic effects with the sample.
 contain an inert and stable porous support on which the
stationary phase can be impregnated or bounded (packed with
solid stationary phase or solid support coated with liquid stationary
phase)
 large choice of stationary phases available
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 Capillary columns

Polyamide coating
Fused silica
Stationary phase

Wall-coated open tubular (WCOT)

Porous Layer Open Tubular (PLOT)
Support coated open tubular (SCOT)

 Capillary columns are made of the highest purity fused silica

obtained by the combustion of tetrachlorosilane SiCl4
The internal diameter of the tube varies from 100 to 530 µm, its
thickness is 50 µm and the length is of 12 to 100 m.
 Capillary columns are rendered flexible by the application of a
polyimide outer coating, a thermally stable polymer Tmax=370 C or a
thin aluminium film. 21
 Selectivity of stationary phase
• Phases: polar and non-polar
For example, alkanes (organic) elute quicker in polar phases; In
contrast, polar analytes elute slower in polar phases
Retention Time: time spent by the analyte in the stationary phase
of the column
Between analyte and Stationary phase:
– As interaction increases, retention time (rt) increases
– As interaction decreases, rt decreases

• Polarity of a stationary phase is measured by the McReynold’s

constant. The higher the constant the more polar the phase.

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 Stationary phase
Liquid stationary phases: (nonvolatile (boiling point > 100 oC),
thermally stable liquids)
Most common:
 Polydimethyl siloxane (OV-1, SE-30, Tmax=350 oC; non-polar;
separation of separation of steroids hydrocarbons)
 Polyethylene glycol (Carbowax 20M, Tmax=250 oC; polar;
separation of alcohols, ethers, glycols, free acids)

 50% phenyl-polydimethyl siloxane (OV-17, Tmax=250 oC; separation

of drugs, steroids, pesticides, glycols)
etc.

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 Stationary phase
For packed columns, for which impregnation techniques are very simple,
over 100 stationary phases are avalible.
For bonded phase capillary columns the choice of stationary phase is
limited because the generation of the film at the surface of the column
requires a different principle than impregnation.
Two families: the polysiloxanes and the polyethylene glycols.

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 Detectors
Flame ionization detector (FID) most common
• Analyte burned in flames producing ions (using H2(g))
• Measures the voltage of the electric signal produced by the ions
thus allowing quantitation.
• Detects carbon and hydrogen containing compounds
• Detects as low as 0.1 – 1 ng
Thermal conductivity detector (TCD)
• Cheap and robust
• Used mainly for compounds that do not contain hydrocarbon (low
molecular weight gases)
• Low sensitivity for hydrocarbons
Mass Spectrometry (MS):
• Very sensitive
• Down to trace molecules
• Expensive and not very quantitative
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 FID detector
The Flame ionization detector (FID) is the most widely used detector for GC
analysis because of its large detection sensitivity (10 6-107orders of magnitude from
picograms to micrograms of sample), linear response and applicability to carbon
chemistry.

The FID works by counting the number of carbon ions

formed from the combustion of molecules in the
hydrogen/air flame. For this reason the FID is
considered a carbon detector.

The number of carbon ions formed, usually on the order

of one ion per 106 molecules, determines the sensitivity
of the FID to a given molecule.
CHO+ ions are widely accepted as the ions formed in this
process.
The response of the FID increases linearly with the number of carbons in linear
alkanes because the number of CHO+ ions also increase proportionally.
Thus, FID insensitive to the detection of small molecular weight molecules such as
H2O, CO2, SO2, NOx

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The gas flow issuing from the column passes through the flame of a small burner fed
by a mixture of hydrogen and air.
The detector destroys the organic compound present whose combustion results in the
release of ions and charged particles responsible for the passage of a very weak
current 10−12 A between two electrodes (pd of 100 to 300 V).
One end of the burner, held at ground potential, acts as a polarization electrode while
the second electrode, called the collector, surrounds the flame rather like a collar.

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 TCD detector
 The TCD is a universal detector responding to compounds with a
different ability to transfer heat from the carrier gas.

 Compare to FID detector it is a nondestructive type of detector

 The TCD is particularly useful for the analysis of small

molecular weight molecules in gaseous mixtures
• TCD is useful for the analysis of permanent gases such as CO 2
• TCD are used medically in lung function testing equipment and
in gas chromatography
• Detection of hydrogen gas and monitoring of hydrogen purity

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Heat sensing resistors are connected to form a wheatstone bridge, so that one or
two cells from a bridge.
A carrier gas, helium, flows across the resistors that are heated by applying a
current. The gas cools the resistor at a constant rate as long as the gas mixture does
not change.
Two gas sources enter the detector, a reference gas and the column effluent.
At the beginning of an analysis, the bridge is equilibrated, but as compounds enter
the cell, a change in resistance and disequilibration of the bridge is recorded.

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Rxi-5Sil column: low polarity phase; Crossbond® 1,4 bis(dimethylsiloxy)
phenylene dimethyl polysiloxane

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Determination of solvent residues in pharmaceuticals remaining from
manufacturing process

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E-cigarette exhaling vapors analysis

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