GAS CHROMATOGRAPHYDr. Gangadhar Chatterjee

MBBS, MD (3rd year PGT)

Grant Govt. Medical College, Mumbai, India

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GAS CHROMATOGRAPHY

Separation of gaseous & volatile substances Simple & efficient in regard to separationGC consists of GSC (gas solid chromatography) GLC (gas liquid chromatography)

Gas → Mobile phase

Solid / Liquid → Solid phase

GSC not used because of limited no. of Solid phase

GSC principle is ADSORPTION

GLC principle is PARTITION

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The organic compounds are separated due to differences in their partitioning behavior between the mobile gas phase and the stationary phase in the column.

Principle

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Sample to be separated is converted into vapor

And mixed with gaseous M.P

Component more soluble in the S.P → travels slower

Component less soluble in the S.P → travels faster

Components are separated according to their Partition Co-efficient

Criteria for compounds to be analyzed by G.C

1.VOLATILITY:

2.THERMOSTABILITY:

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The father of modern gas chromatography is Nobel Prize winner John Porter Martin, who also developed the first liquid-gas chromatograph. (1950)

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GC-MS Quadri- TOF

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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.

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. 04/08/2023

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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.

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PRACTICAL REQUIREMENTS

Carrier gas Flow regulators & Flow meters Injection devices Columns Temperature control devices Detectors Recorders & Integrators

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Syringe

Injector

Detector

Carrier Gas Cylinder Column

To Waste or Flow Meter

Flow Controller

Two-Stage Regulator

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Requirements of a carrier gas

Inertness Suitable for the detector High purity Easily available Cheap Should not cause the risk of fire Should give best column performance

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CARRIER GAS

» Hydrogen ( H2 ) better thermal conductivity Disadvantage: it reacts with unsaturated compounds &

inflammable» Helium ( He) excellent thermal conductivity it is expensive

» Nitrogen ( N2) reduced sensitivity it is inexpensive

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Flow regulators & Flow meters deliver the gas with uniform pressure/flow rate flow meters:- Rota meter & Soap bubble flow meter

Rota meter placed before column inlet

it has a glass tube with a float held on to a spring.

the level of the float is determined by the flow rate of carrier gas

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Soap Bubble Meter

◊ Similar to Rota meter & instead of a float, soap bubble formed indicates the flow rate

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THEORETICAL PLATE An imaginary unit of the column where equilibrium has

been established between S.P & M.P It can also be called as a functional unit of the column

HETP – Height Equivalent to a Theoretical Plate Efficiency of a column is expressed by the number of

theoretical plates in the column or HETP If HETP is less, the column is ↑ efficient. If HETP is more, the column is ↓ efficient

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HETP= L (length of the column)

N(no of theoretical plates)

HETP is given by Van Deemter equation

HETP= A + B +Cu

u

A = Eddy diffusion term or multiple path diffusion which arises due to packing of the column

B = Molecular diffusion, depends on flow rate

C = Effect of mass transfer, depends on flow rate

u = Flow rate

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Efficiency ( No. of Theoretical plates)

It can be determined by using the formula

n = 16 Rt2

w2

n = no. of theoretical plates

Rt = retention time

W = peak width at base The no. of theoretical plates is high, the column

is highly efficient For G.C the value of 600/ meter

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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.

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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.

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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. 04/08/2023

Sample preparation

1. The prerequisite in GC separation is that all solutes being separated must be: (a) fairly volatile, and (b) thermally stable.

(c) Usually, the solute should be dissolved in a non-aqueous matrix (H2O changes column behavior).

2. Lack of volatility prevents the direct use of GC for many solute. One way to overcome this difficulty is to derivatize the solutes into more volatile forms.

Cl

Cl

O

OH

O2,4-dichlorophenoxyacetic acid(A cancer suspect agent).

Silylation

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3. Derivatization of a solute can be used for any of the following reasons

(a) To increase the volatility of the solute.

(b) To increase the thermal stability of solute

(c) To improve the response for the solute on certain detectors (e.g., incorporating halogen atoms into a solute so that it can be detected using an electron capture detector).

(d) To improve the separation of the solute from other sample components (i.e., changing the structure of a solute will also affect its retention on the column)

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4. Most derivatization reactions can be classified into one of three group: (a) Silylation

(b) Alkylation

(c) Acylation

Most of these reactions are performed using minimal amount of sample and reagents (i.e., 0.1~2.0 mL) are typical carried out at room temperature. Some, however, do require heating to moderate temperatures (60 ~ 100 OC).

5. Silylation

(a) This is the most common type of derivation techniques used in GC.

(b) It involves replacing an active hydrogen on the solute (i.e. R-OH, RCOOH, R-NH2, etc.) with an alkylsilyl group (usually –SiMe3). The result of this reaction is that the solute is converted into a less polar, more volatile and more thermally stable form.

(c) The most common reagent used in silylation is trimethylchlorosilane (TMS). Examples of its use are shown below:

Cl

Cl

O

OH

O ClSi

Me3Cl

Cl

O

SiMe3

O+

ClSi

Me3R OH R OSi

Me3+ + HCl

The resulting Product of this reaction is usually just referred to as a TMS-derivative.

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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.

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Carrier Gas

Syringe

Vaporization Chamber

To Column

Septum

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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.

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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.

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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.

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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.

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A gas chromatography oven, open to show a capillary column

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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 ( Eddy’s diffusion co-efficient)in the van Deemter equation which stands for multiple path effects is zero and the equation for capillary columns becomes

H = B/V + CV

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Capillary columns advantages compared to packed columns

1. higher resolution2. shorter analysis times3. greater sensitivity

Capillary columns disadvantage compared to packed columns

4. smaller sample capacity5. Need better experience

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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 100-400 mm

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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

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Weight of liquid stationary phase * 100% %Loading =

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.

Weight of stationary phase plus solid support

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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.

A thicker film can tolerate a larger sample size.

Film thicknesses in the range from 0.1 – 5 mm are common.

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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:

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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.

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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

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Detection Systems

Characteristics of the Ideal Detector: The ideal detector for gas chromatography has

the following characteristics:

1. Adequate sensitivity

2. Good stability and reproducibility.

3. A linear response to solutes that extends over several orders of magnitude.

4. A temperature range from room temperature to at least 400oC.

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Characteristics of the Ideal Detector

5. A short response time that is independent of flow rate.

6. High reliability and ease of use.

7. Similarity in response toward all solutes or a highly selective response toward one or more classes of solutes.

8. Nondestructive of sample.

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Thermal Conductivity Detectors(TCD)

A very early detector for gas chromatography, and one that still finds wide application, is based upon changes in the thermal conductivity of the gas stream brought about by the presence of analyte molecules.

The sensing element of TCD is an electrically heated element whose temperature at constant electrical power depends upon the thermal conductivity of the surrounding gas.

The heated element may be a fine platinum, gold, or tungsten wire or a semiconducting thermistor.

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Thermal Conductivity Detectors(TCD) The advantage of the thermal conductivity detector

is its simplicity, its large linear dynamic range(~105), its general response to both organic and inorganic species, and its nondestructive character, which permits collection of solutes after detection.

A limitation of the katharometer is its relatively low sensitivity (~10-8 g solute/mL carrier gas).

Other detectors exceed this sensitivity by factors as large as 104 to 107.

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Flame Ionization Detectors (FID)

The flame ionization detector is the most widely

used and generally applicable detector for gas

chromatography.

The effluent from the column is mixed with

hydrogen and air and then ignited electrically.

Most organic compounds, when pyrolyzed at the

temperature of a hydrogen/air flame, produce ions

and electrons that can conduct electricity through

the flame.

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Flame Ionization Detectors (FID)

A potential of a few hundred volts is applied.

The resulting current (~10-12 A) is then measured.

The flame ionization detector exhibits a high sensitivity (~10-13 g/s), large linear response range (~107), and low noise.

A disadvantage of the flame ionization detector is that it is destructive of sample.

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Electron-Capture Detectors(ECD) The electron-capture detector has become one of the most widely

used detectors for environmental samples because this detector selectivity detects halogen containing compounds, such as pesticides and polychlorinated biphenyls (PCB)

The effluent from the column is passed over a emitter, usually Nickel-63.

An electron from the emitter causes ionization of the carrier gas and the production of a burst of electrons.

In the absence of organic species, a constant standing current between a pair of electrodes results from this ionization process.

The current decreases markedly, however, in the presence of those organic molecules that tend to capture electrons.

Electron-Capture Detectors(ECD) The electron-capture detector is selective in its

response being highly sensitive to molecules containing electronegative functional groups such as halogens, peroxides, quinones, and nitro groups.

It is insensitive to functional groups such as amines, alcohols, and hydrocarbons.

An important application of the electron-capture detector has been for the detection and determination of chlorinated insecticides.

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Thermionic Detectors (TID) The thermionic detector is selective toward

organic compounds containing phosphorus and nitrogen.

Its response to a phosphorus atom is approximately ten times greater than to a nitrogen atom and 104 to 106 larger than a carbon atom.

Compared with the flame ionization detector, the thermionic detector is approximately 500 times more sensitive to phosphorus-containing compounds and 50 times more sensitive to nitrogen bearing species.

These properties make thermionic detection particularly useful for detecting and determining the many phosphorus-containing pesticides.04/08/2023 71

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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 same level so as to prevent any solute condensation in the vicinity of the detector body.

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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.

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Interfacing GC with other Methods Chromatographic methods (including GC) use retention

times as markers for qualitative analysis.

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. 04/08/2023

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Mass SpectrometryAnalytical method to measure the molecular

or atomic weight of samples

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Different elements can be uniquely identified by their masses

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MS Principles

Different compounds can be uniquely identified by their masses

CH3CH2OH

NOH

HO

-CH2-

-CH2CH-NH2

COOH

HO

HO

Butorphanol L-dopa Ethanol

MW = 327.1 MW = 197.2 MW = 46.1

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Mass Spectrometry

For small organic molecules the MW can be determined to within 5 ppm or 0.0005% which is sufficiently accurate to confirm the molecular formula from mass alone

For large biomolecules the MW can be determined within 0.01% (i.e. within 5 Da for a 50 kD protein)

Recall 1 dalton = 1 atomic mass unit (1 amu)

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MS Principles• Find a way to “charge” an atom or molecule

(ionization)

• Place charged atom or molecule in a magnetic field or subject it to an electric field and measure its speed or radius of curvature relative to its mass-to-charge ratio (mass analyzer)

• Detect ions using microchannel plate or electron multiplier tube

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Mass Spectrometer Schematic

InletIon

SourceMassFilter Detector

DataSystem

High Vacuum SystemRough pumpsRotary pumpsTurbo pumpsDiffusion pumps

VaporHPLCGCSolids probe

MALDIESIFABEI/CI

TOFQuadrupoleIon TrapMag. SectorFTMS

Microch plateElectron Mult.

PC’sUNIXMac

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Mass Spec Principles

Ionizer

Sample

+_

Mass Analyzer Detector

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ion trajectory not in register

(too heavy)

IonSource

Detector

ion trajectory not in register

(too light)

ion trajectory in register

S

N

Magnetic Sector Mass Analyzer

Electromagnet

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Typical Mass Spectrum• Characterized by sharp, narrow peaks

• X-axis position indicates the m/z ratio of a given ion (for singly charged ions this corresponds to the mass of the ion

• Height of peak indicates the relative abundance of a given ion (not reliable for quantitation)

• Peak intensity indicates the ion’s ability to desorb or “fly” (some fly better than others)

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194

67 109

5582

42

16513694

40 60 80 100 120 140 160 180 200

Ab

un

dan

ce

Mass (amu)

Mass Spectrum

NC

CNH

CO

C

O

N

N

C H

C 3H

C3H

MassSpectrometer

A Typical Mass Spectrum

Typical sample: isolatedcompound (~1 nanogram)

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