GAS CHROMATOGRAPHYLECTURE 8

PRINCIPLES

In Gas Chromatography, the components of a vaporized sample are separated as a result of being partitioned between a mobile gaseous phase and a liquid or a solid a liquid or a solid stationary phasestationary phase held in the column.

A sample is being injected at the inlet/injector and vaporized into the chromatographic column.

The sample is transported through the column by the flow of inert gaseous mobile phase.

As the sample passes through the column, they are separated and detected electronically by detector.

PRINCIPLES

Gas Chromatography

Gas is called “carrier gas”. Typical carrier gas: helium or

nitrogen. Pressure from a compressed gas

cylinder containing the carrier gas is sufficient to create the flow through the column.

There are two types.Gas-liquid chromatography (GLC)

mobile phase – gasstationary phase - liquid

Gas-solid chromatography (GSC)mobile phase – gasstationary phase - solid

Shortened to Gas Chromatography

Gas Chromatography

INSTRUMENTATION

A. Carrier gas

B. Flow regulator

C. Injector

D. Column

E. Detector

F. Integrator

G. Display system -printer/monitor

Thermostated oven

Integrator

INSTRUMENTATION

Injection port and detector must be kept warmer than the column,

1. To promote rapid vaporization of the injected sample.

2. To prevent sample condensation in the detector.

INSTRUMENTATION

Samples must be…..

Volatile Thermally stable. When injected onto the head of a

chromatographic column and vaporized.

Mobile phase transports the analytes (sample) through column.

Mobile phase can not interact with the molecules of the analyte.

Referred as carrier gas.

Mobile phase

A. Carrier gas

Must be chemically inert. Most common carrier gas is

Helium(He) Some specific detectors are using

Nitrogen gas(N2), Hydrogen gas(H2), Carbon dioxide gas(CO2) and Argon.

The carrier gas should not contain traces of water or oxygen. Both are harmful to the stationary phase.

B. Flow regulator

The function of flow regulator is to control the flow rate of the carrier gas using the pressure regulators, gauges and flow meters.

The pressure at the head of the column is stabilized mechanically OR through the use of an electronic

device.

C. Injectors Functions

1. An inlet for the sample.2. To vaporize and mix the sample with

the carrier gas before the sample enters the head of the column.

Temperature is set about 50°C higher than boiling point of the least volatile component of the sample.

Modes of injection and characteristics of injectors vary depending on type of column used whether split/splitless.

Mode of Injections

Column efficiency requires sample to be……1. Of a suitable size2. Introduced as a “plug” of vapor

Band broadening and poor resolution are caused by…….1. Slow injection.2. Oversized sample.

D. Sample Injection System

Sample introduction usually……1. In the form of neat liquid or

solution.2. Introduced in a small volumes.a. 1 μL - 20 μL for packed column.b. 1 x 10-3 μL for capillary column.

D. Sample Injection System

Examples Direct injection using

microsyringe Loop injectors Auto samplers Headspace

D. Sample Injection System

D. Sample Injection System

DIRECT INJECTION USING

MICROSYRINGE

LOOP INJECTORS

D. Sample Injection System

HeadspaceA headspace sample is 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.

D. Sample Injection System

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

S = the sample phaseThe sample phase contains the compound(s) of interest. It is usually in the form of a liquid or solid in combination with a dilution solvent or a matrix modifier.

Once the 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 as depicted by the arrows. The sample is then taken from the headspace.

E. Oven

Must have sufficient space to hold the column.

Can be heated to the desired temperature for analysis.

Atmosphere inside the oven is constantly agitated by forced ventilation which has small thermal inertia.

Reproducible of retention time,tR which require control of the column temperature within a few tenths of a degree.

Optimum temperature depends on the boiling points of the sample components.

A temperature that is roughly ≥ the average boiling point of the sample results in a reasonable elution period.

Samples with broad boiling range, necessary to employ temperature programming.

E. Oven

Definition:A technique in which the column temperature is increased either continuously or in steps as the separation proceeds.

In general, optimum resolution is associated with minimal temperature.

Low temperature, result in longer elution times hence slower analysis.

Temperature Programming

Using Temperature programming, low boiling point constituents are separated initially at temperatures that provide resolution.

As separation proceeds, column temperature is increased so that the higher boiling point constituents come off the column with good resolution and at reasonable lengths of time.

Temperature Programming

A technique in which the column temperature is constantly maintained throughout the

separation.

Isothermal Elution

Isothermal at 1500C

Temperature programmed:

500C to 2500C at 80C/min

F. Columns

Two types of columns1.Packed column 2.Capillary column

Packed column:1-5m in length, 2-4mm

i.d

Capillary Column:10-100m in length, very small

i.d

Less commonly used Made of glass or steel Length: 1 to 5 m Internal diameter: 2 to 4 mm

These column is densely packed with uniform, finely divided solid support, coated with thin layer (0.05 to1μm) of stationary liquid phase.

Accommodate larger samples.

Cross-sectional view of packed

column

1. Packed Column

Carrier gas flow between 10 – 40 mL/min. Not well adapted for trace analysis. Contain an inert & stable porous support

on which the stationary phase can be impregnated(coated) or bound.

Advantages:

1.Large sample size2.Ease & convenience of use

1. Packed Column

Widely used in GC analysis Also known as open tubular column Length: 10 – 100 m Coiled around a light weight of metallic

support. Types of capillary column

I. FSOT (Fused Silica Wall Coated) - i.d. 0.1 - 0.3 mm

II. WCOT (Wall Coated) - i.d. 0.25 – 0.75 mm

III.SCOT (Support Coated) - i.d. 0.5 mm

2. Capillary Column

Advantages:1.High resolution2.Short analysis time3.High sensitivity

2. Capillary Column

Properties and characteristics of GC Column

G. Stationary PhaseDesirable properties for the immobilized liquid stationary phase:Low volatility (ideally the boiling point of the liquid at least 1000C higher than the maximum operating temperature for the column)Thermal stability.Chemical inertness.Solvent characteristics such as k and α values for the solutes to be resolved fall within a suitable range.

Separation principles Use the principle of “like dissolve like”

where like refers to the polarity of the analyte and the immobilized liquid stationary phase.

Polarity of organic functional group in increasing order Aliphatic hydrocarbons<olefins<aromatic

hydrocarbons<halides<ethers< esters/ aldehydes/ketones<alcohols/amines< amides<carboxylic acids<water

G. Stationary Phase

Polarity of the stationary phase should match that of sample components.

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

G. Stationary Phase

The choice of stationary phase should match that of sample components.

Non polar Stationary phase Polar Stationary phase

WaterCarboxylic acidsAmidesAlcohol/aminesEsters/aldehydes/

ketonesEthersHalidesAromatic hydrocarbonsOlefinsAliphatic hydrocarbons

Polar

Non-polar

Polarity of Stationary Phase

Aliphatic hydrocarbons < esters/aldehydes/ketones < alcohols/amines < water

Pentane, Hexane

Heptane, Octane

Acetone, 3-pentanone

Methyl ethyl ketone

Propanol, Butanol

Pentanol

PolarNon-polar

G. Stationary Phase applications

HO C

H

H

C O

H

H

C C OH

H

H H

Hn

Polyethylene glycol (PEG)Use for separating polar species

Si

R

R

R

O Si O

R

R

Si R

R

Rn

Polydimethyl siloxane, the R groups are all CH3. (Non-polar)

Many liquid statationary phase are based on polysiloxanes or polyethylene glycol (PEG)

G. Stationary Phase

H. Detectors

Some detectors are universal. They are sensitive to almost every

compound that elutes from the column. Most detectors are selective.

They are sensitive to a particular type of compound. Give response that is dependent on the concentration of analyte in the carrier gas.

Yield(produce) simple chromatogram.

Characteristics of ideal detector1. High reliability & ease to use.2. Similarity response toward all

solutes or alternatively a high predictable & selective response toward one or more classes of solute.

3. Detector should be nondestructive.

H. Detectors

Characteristics of ideal detector4. Adequate sensitivity.5. Good stability and reproducibility.6. Linear response to solutes that

extends over several orders of magnitude.

7. Temperature range (from room temperature to at least 400 0C)

H. Detectors

Several types of detectors.

1. Flame Ionization Detector (FID)2. Thermal Conductivity Detector

(TCD)3. Electron Captured Detector

(ECD)

H. Detectors

1. Flame Ionization Detector (FID)

Effluent from the column is passes through a small burner fed H2 and air.

Combustion of the organic compounds flowing through the flame creates charged particles (ionic intermediates are responsible for generating a small current between the two electrodes).

The burner, held at ground potential acts as one of the electrodes.

The second electrode called as a collector, is kept at a positive voltage & collects the current that is generated.

Signal amplified by electrometer that generate measurable voltage.

How does FID works?

Advantages Rugged Sensitive (10-13 g/s) Wide dynamic range (107) Signal depends on number of C atoms

in organic analyte - mass sensitive not concentration sensitive.

1. FID

Disadvantages Weakly sensitive to carbonyl, amine,

alcohol & amine groups. Not sensitive to non-combustibles

analyte such as H2O, CO2, SO2, NOx. Destructive method.

1. FID

2. Thermal Conductivity Detector (TCD)

A universal detector. Has a moderate sensitivity. Less satisfactory with carrier gas

whose conductivities closely resemble those of most sample components.

2. TCD

Consists of an electrically heated source whose temperature at constant electric power depends on the thermal conductivity of the surrounding gas.

The electrical resistance of this element (fine platinum, gold or tungsten wire or thermistor) depends on the thermal conductivity of the gas.

Operating principles relies on the thermal conductivity of the gaseous mixture.

The thermal conductivity affects the resistance of the thermistor as a function of temperature.

How does TCD works?

Twin detectors are normally used One located ahead of sample injection chamber and the other immediately beyond the column or alternatively, the gas stream can be split.

When the solutes elutes from the column there is a change in the composition of the mobile phase thus in the thermal conductivity.

this results in a deviation from thermal equilibrium, causing a variation in the resistance of one the filament.

this variation is proportional to the concentration of the analyte, provided its concentration in the mobile phase is low.

How does TCD works?

Advantages Simple Large linear dynamic range Responds to both organic and

inorganic species Nondestructive; permits collection

of solutes after detection.

Disadvantage Relatively low sensitivity.

2. TCD

3. Electron Capture Detector (ECD)

Sample elute from a column is passed over a radioactive β emitter, usually nickel-63.

An electron from the emitter causes ionization of carrier gas (often N2) and the production of a burst of electrons.

In the absence of organic species, a constant standing of current.

In the presence of organic molecules containing electronegative functional groups that tend to capture electrons, the current decreases markedly.

How does ECD works?

Most widely used for environmental samples

Advantages Selectively responds to halogen-containing

organic compounds such as pesticides and polychlorinated biphenyls.

Highly sensitive towards halogens, peroxides, quinones and nitro groups.

Disadvantages Insensitive to functional groups such as

amines, alcohols and hydrocarbons.

3. ECD

Other detectors Nitrogen-Phosphorous Detector

(NPD) Flame Photometry Detector (FPD) Mass spectrometer (GC-MS)

H. Detectors

Detector

Principle of operation

Principle class of compound detected

FID Ionization of solute molecules

in a flame

Organics

TCD Thermal conductivity

Any samples

ECD Current Compounds containing electronegative

elements

H. Detectors