GAS
CHROMATOGRAPHY
((GC)
GC)
• Most Valuable for Separation of :
# Volatizible Compounds of relatively
low polarity.
Volatizible Compounds:
- have a high vapor pressure
- can vaporize easily
Basic Principles
• A gaseous mobile phase is used to pass a
mixture of volatile solutes through a
column containing the stationery phase.
• The mobile phase is referred to as a
carrier gas, that carries the volatized
sample molecules through the column.
• Solute separation is base on :
# Relative differences in the solutes’
vapor pressure;
and
# The interaction with the stationery
phase.
• The column effluent carries the
separated solutes to the detector
in the order of their elution.
Ø Solutes are identified:
* Qualitatively by their specific
retention times (compared to a standard).
* Quantitatively by the peak size
(height, area) which is proportional to
the amount of eluted solute.
Types of GC
Stationery
Phase
Separation
Depends on
GSC
GLC***
solid
Liquid
differences
in interaction
on stationery
surface.
solute
partitioning
between gaseous
Mobile phase and
liquid stationery
phase.
Instrumetation
See animation in folder
+GC1.mov
A Basic GC consists of:
• A gas cylinder containing the inert mobile carrier
gas.
• A flow gas controller.
• A column to separate the solutes.
• An injector to introduce the sample or derivatized
sample into the column.
• A column oven to heat the column.
• A temperature controller to control the
temperature of the injector and detector.
• An online detector.
• A
computer to control the system and process
data.
The Carrier Gas Supply
and Flow contoller
•
I- Carrier Gas : should be:
* dry
* inert (so as not to interfere
with the column).
*Highly pure ( impurities harm the
column and decrease the detectors’
performance).
• The inert gas can be :
Helium (He), Nitrogen (N2), or Argon
(Ag).
Ø The choice of the carrier gas depends
upon:
* Type of column.
* Type of detector.
• The Flow Controller:
A constant flow of gas is needed for:
*column efficiency.
* reproducible elution times.
o Systems that provide constant flow
rates vary from simple mechanical
devices to programmable electronic
ones.
o The flow rate depends on the type of
column:
-packed: 10-60ml/min.
-capillary: 1-2ml/min
The Injector
*A micro-syringe needle is used to inject
a 1-10µL aliquot of prepared sample into
the injector septum into a heating
region.
• The volatile analytes and the solvent are
then “flash vaporized” into the column by the
carrier gas.
• To ensure rapid and complete solute
volatilization the temperature of the
injector is maintained at 30-500 higher than
the column temperature.
Column Technology
• The column is packed in an oven.
Main Types of Columns
Packed
*Filled with support
particles:
- uncoated→GSC
-coated with
stationery phase→ GLC
*Dimensions:
# ID: 1-4mm
# Length:~ 1 m.
*made of
stainless steel.
Capillary
*fabricated by coating
the
inner wall of fused silica
tubes with a thin film of
liquid phase.
# ID: 0.1-0.5mm.
# Length: 10-150m.
Fused silica: fragile→
outside walling by Al ↑es
durability.
Capillary Column
Temperature Control
• There should be careful control of the
temperature of the
*column,
*detector and
*injector
Temperature Control
COLUMN
INJECTOR and DETECTOR
OVEN
ELECTRIC HEAT RESISTANCE
DETECTORS
• The detector produces a signal which is
amplified and displayed on the recorder as
a series of peak (Chromatogram).
* The retention times identifies the
component.
(quanlitative)
*The peak size (height, area) is proportional
to the amount of eluted solute.
(quantitation)
TYPES Of DETECTORS
Flame Ionization Detector. (FID)
Photo-ionization Detector. (PID)
Electron Capture Detector. (ECD)
Thermal Conductivity Detector.
(TCD).
• Mass Spetometer.
•
•
•
•
• Column effluent + H2+ AIR
Flame
Thermal ionization of the organic
compounds
+ve ions + ecurrent proportional to the
amount of carbon material delivered to
the detector.
Flame Ionization
Detector. (FID
Fid.mov
Photo-ionization Detector.
(PID)
• A variant of FID.
• Instead of the flame in FID, the
energy for ionization in PID is
provided by
an intense UV lamp.
PID
Electron Capture
Detector. (ECD)
• Responds only to substances capable
of capturing electrons eg. halogens
(Br, I, F, Cl).
● Usually used in analysis of
polychlorinated compounds
eg.pesticides
Electron Capture
Detector. (ECD)
Ecd.mov
• Because not all compounds contain
the functional electron capturing
groups (F, Cl…etc.), derivatization
with reagents containing
polychlorinated or polyflourinated
moieties is a common practice used in
ECD.
MASS SPECTROSCOPY
• After the eluent exits the column
the following sequence of events
occur:
Ø1- Creation of Ionized molecular
fragments usually by a high energy
beam of electrons in the ionization
chamber.
Ø2-Ionized fragments are sucked by a
vacuum system to a mass analyzer or
separator which separates the
ionized fragments according to their
mass/ charge ratio.
• Measurement of the ionized
fragments.
GC-MS
+MS principle.mov
Importance of
GC-MS
• Couples:
◊ the resolving power of GC
with
◊ the outstanding specificity of
MS (only nanograms or pgs of
an analyte are needed)
USES OF GC-MS
• Analysis of drugs
• Qualify reference materials
• Assign certified values to many
clinical analytes.
Sample Preparation
• Sample extraction.
• Solvent extraction.
• Sample derivatisation.
Sample extraction.
• Example: extraction of barbiturates
from serum.
1- acidification
• Serum
(barbiturates)
3-Shake or
Centrifuge
soluble form of
barbiturates
2- Dissolve in organic
solvent
organic layer
(barbiturates)
interferences
(eg. proteins)
Solvent extraction.
• Is also frequently used to ↑ the
concentration of the analyte prior to GC
Sample derivatisation
• Derivatisation may be done in order
to:
Ø Chemically modify non-volatile
compounds to ↑ their volatility for GC. eg.
by acylation or esterification.
Ø ↑ing the sensitivity and specificity of a
particular separation.
Ø ↑ing delectability eg. fluorinating the
analyte for usage with ECD.
Computer
• System Control.
• Data Processing.
Analyte identification
• The retention time, or volume, at which an
unknown solute elutes from the column is
matched to that of a reference (standard
compound).
Analyte Quantification
• A calibration (standard) curve must be
used for measuring analyte
concentration.
External calibration
Internal calibration
External calibration
• Reference (standard) solutions containing
known quantities of same analyte to be
measured are processed in a manner
identical to the samples containing the
analyte.
• A calibration curve of peak height or
(area) versus concentration is constructed
and used to calculate the concentration of
the analyte in the samples.
Internal calibration
• Also called internal standardization.
• A different compound called internal standard, is
added to each reference solution and each
sample.
• By plotting the ratio of the peak height or (area)
of the calibrator to the peak height or (area) of
internal standard versus the concentration of
the calibrator,
a calibration curve that corrects for systematic
losses is constructed. The curve is then used to
compute the concentration of the analyte in
unknown samples
ww.shsu.edu-~chm_tgc-sounds-flashfiles-FID.sw
+GC1,1.mov