Dr. Ashraf M. M.

Mahmoud,
Associate professor
Common Techniques of Chromatography

Thin Layer Chromatography (TLC)

High-Performance Liquid Chromatography (HPLC)

Gas Chromatography (GC)

 Principles of separation in Chromatography
Respons
e

time
 Instrumentation of GC

Principles Behind the Instrument

Mechanisms of Partition to Stationary
Phase
 Gas Chromatography (GC)

The technique is similar to HPLC except that the mobile

liquid phase is replaced by a moving gas phase, and the
injected sample should be volatile (native or derivatized).

What is the principle ?

What is the GC Instrumentation ?

What is the Applications of GC ?

 Instrumentation of GC

Flow meter

Injector Septum Detector GC

Pressure Flow Chart
regulator controller 
Recorder

Oven

Gas
supply Column
 Pressure Flow meter
Flow
Flow
regulator
controller controller
Injector Septum Detector GC
Gas Supply Pressure
regulator  Recorder
chart

Oven

Gas Gas
Column
supply supply

 The mobile phase (carrier gas) should be chemically inert, dry

and free from O2 (helium, argon, nitrogen and hydrogen).

 The carrier gas should be of high purity; impurities in the

carrier gas such as water vapour, air and trace gaseous
hydrocarbons can cause reactions with sample and cause
column deterioration and affect detector performance.

 The gas supply is associated with pressure regulator and

flow controller.
 Flow meter
Flow meter
Flow
Sample Injection System controller
Pressure
Injector Septum
Injector Septum
Detector
Detector GC
chart
regulator  Recorder
Recorder
Carrier
gas

Preparation of the Sample : Oven

Oven

Column
Samples GC must be volatile. Gas
supply
Column

Samples which are non volatile are converted into a volatile

derivative.

The most commonly method is the silylation (reaction of

trimethylsilyl,
- Si(CH3)3 with an active hydrogen atoms in the analyte (carboxylic
acids, amines, imines, alcohols, phenols, and thiols).

Inorganic metals (aluminum, beryllium, and chromium) can be

analyzed by GC via formation of stable, volatile metal chelates with
trifluoroacetylacetone (TFA) and hexafluoroacetylacetone (HFA).
 Flow meter
Injector Septum
Sample Injection System block
Injector Septum Detector

 Recorder
Carrier
gas

Oven

Injection of the Sample :

Column

The injector block; is heated to a temperature that is at least 50 C

above the sample component with the highest boiling point; this
to ensure rapid vaporization of the entire sample.

The injection system is a self-sealing silicone septum.

The sample is injected by a microliter syringe. The needle of the

syringe is inserted through and the sample injected smoothly into
a heated metal block at the head of the column.
 Flow meter

GC Column Injector Septum Detector

Oven
 Recorder
Carrier
gas

The GC column on which the actual Oven

separation of sample components

is effected. Column
Column

Most GC columns are made from high-purity fused silica capillary, the
inner wall of the capillary coated with the stationary phase.

GC columns vary in length from less than 2 m to 50 m or more.

In order to fit into the column oven, they are usually formed as coils.

The control of column’s temperature is critical to attain a good

separation in GC, thus the column is located inside a thermostated
oven to control the temperature.
Choice of GC Columns
 Column Configuration
Packed Columns
• 2 to 4 mm I.D. and 1 to 4 meters long.
• Packed with a suitable adsorbent.
• Mostly used for gas analysis.
• Peak broadening due to zone (eddy)
diffusion resulting from multitude of
pathways a molecule can pass through
column.
Capillary Columns
• 100 mm to 500 mm I.D. and 10 m to 100 m
long
• Stationary phase is coated on the internal
wall of the column as a film 0.2 mm to 1 mm
thick
• Sharper peaks – no eddy diffusion.
• Up to 500,000 theoretical plates – excellent
separations.
• Most popular type of column in use.
 Flow meter
Flow

GC Detector controller
Pressure
Injector Septum Detector GC
chart
regulator  Recorder

The function: Oven

Monitoring the carrier gas as Column

Gas
it emerges from the column.. supply

The requirements of ideal GC detector:

The same as HPLC detectors.

Types of GC detectors:
 Thermal conductivity detector (TCD)
 Electron capture detector (ECD)
 Flame ionization detector (FID)
 Nitrogen phosphorous detectors (NPD)
 GC Detectors
Thermal Conductivity Detector (TCD):
Measures the decrease in the thermal conductivity of the carrier gas
resulted from the non-conductive analyte molecules.

Electron Capture Detector (ECD):

Measures the decrease in the detector current which occurs when
an analyte molecule that tend to capture electrons appears in the
gas stream.

Flame Ionization Detector (FID):

Measures the increase in the electrical signal as a result of
ionization of the analyte molecules by the action of the detector flame.

Nitrogen Phosphorous Detectors (NPD):

Measures the increase in the current resulted from combustion of
nitrogen- and phosphorous-containing compounds in the detector
flame.
 GC Detectors

Characteristics of GC Detectors :

Type Selectivity Sensitivity Temp. limit

(ng) (C)
TCD All organic compounds 5-20 450

ECD Halogens, nitrates, and 0.1-100 350

conjugated carbonyles

FID Compounds with C-H 0.1-10 400

bonds
NPD Compounds containing 0.001-0.01 300
nitrogen and phosphorous
 Flow meter
Flow
controller
Injector Septum Detector GC
GC Recorder Pressure
regulator  Recorder
chart

Oven

Gas Column
supply

Sample injected

Peaks correspond to
individual components
Applications of GC ? Peaks correspond to
individual components

Qualitative Analysis

Quantitative Analysis
Separation of Mixture Components: Authentic

Identification of Compounds: Unknown

Retention time comparsion

Pyrolysis gas chromatography

It is used for the identification of non-volatile materials (plastics,
natural and synthetic polymers, and some microbiological materials.

It is based on the fingerprint chromatogram for the sample, which

results from its thermal dissociation and fragmentation.
Quantitative Analysis Calibration curve

External Standard Method

Peak hight
Concentration

0 10 0 10 0 10 0 10 0 10 0 10 0 10

100 ng/mL 75 ng/mL 50 ng/mL 25 ng/mL 10 ng/mL 5 ng/mL Unknown

Quantitative Analysis Calibration curve

Peak hight ratio

Internal Standard Method

Concentration

Internal
Standard

Compound

0 10 0 10 0 10 0 10 0 10 0 10 0 10

100 ng/mL 75 ng/mL 50 ng/mL 25 ng/mL 10 ng/mL 5 ng/mL Unknown

Aspects of GC Applications:

Food Analysis
Analysis of foods is concerned with confirming the presence
and determination the quantities of the analytes (lipids,
proteins, carbohydrates, preservatives, flavours, colorants,
and also vitamins, steroids, and pesticide residues).

Drug Analysis
GC is widely applied to identification of the active
components, possible impurities as well as the metabolites.
Environmental Analysis
The environmental contaminants; e.g. dichlorodiphenyltrichloro-
ethane (DDT) and the polychlorinated biphenyls (PCBs) are present
in the environment at very low concentrations and are found among
many of other compounds.

GC, with its high sensitivity and high separating power, is mostly
used in the analysis of environmental samples.

Forensic Analysis
In forensic cases, very little sample is available, and the
concentration of the sample components may be very low.

GC is a useful due to its high sensitivity and separation efficiency.

 Peak Base: An interpolation of the base line between the
extremities of the peak. .
 Peak Area: The area enclosed by the peak and the peak
base.
 Peak Height: The distance from the peak maximum to the
peak base.
 Peak Width (tW): The magnitude of the peak base
intercepted by the tangents to the inflection points of the
peak.
 Retention time (tR): The time between sample injection
and the appearance of a solute peak at the detector.
 Dead time (t0): The time for mobile phase to pass through
a column.
 Adjusted Retention Time (tR') is the time solute spent in
the stationary phase and equals to tR' = [ tR- t0].
 Adjusted Retention Time (tR') tR' = [ tR- t0].
 The Capacity Factor (k') is used to describe the migration rates of
solutes on columns, It is defined as:
For solute A, kA' = (tR,A – t0)/t0 ; = t'R,A/t0

 The Selectivity Factor () of a column for the two solutes A and B
in a mixture is defined as
= k'B/k'A

 Chromatoaraohic column efficiency is determined by

1- Plate height (Height Equivarent to Theoretical Plate (HETP)= L/N
2- Number of Theoretical Plates(N) =16 (tR/tW)2
L = length of column

 Resolution of the column (Rs) = (tR,B – tR,A)/0.5 (tw,A+tW,B)

Rs = √N/4 x [α-1]/ α x [k'B/(k'B+1)]
Column efficiency Selectivity Capacity
Resolution and Selectivity
Can One Have Too Much Resolution

RS = 2.5
Separating Efficiency – Peak Asymmetry