Glc

HAMZA GILL
HASANAT AHMED
HAMZA IKRAM
DANIYAL KHAN SURI
ZEESHAN ALI BHATTI

Consist of
 Carrier Gas cylinder with pressure regulator
 Sample Injection
 Column Oven
 Open Tubular Columns and Packed Columns
 Detection Systems
 Flow meter
Amplifier
Recorder

 A carrier gas should have the following properties:
 It should be highly pure (> 99.9%).
 It should be inert ,compatible with the detector, cheap and available.
 Carrier gas must be dry, free of oxygen and chemically inert mobile-
phase employed in gas chromatography.

Commonly used gases include nitrogen, helium, argon, and carbon dioxide.
The carrier gas pressure ranges from 10-50 psi.
Helium is most commonly used because it is safe
Flow rates should be from 1-150 ml/min.
Conventional analytical columns usually use flow rates in the range from 20-
50 ml/min while capillary columns use flow rates from 1-5 ml/min.

Pressure regulator is used to control the amount of gas to be passed
to column.
Flow rates should be from 1-150 ml/min.
Conventional analytical columns usually use flow rates in the range
from 20-50 ml/min while capillary columns use flow rates from 1-5
ml/min.

A sample port is necessary for introducing the sample at the head
of the column.
A calibrated micro-syringe is used to deliver a sample volume in
the range of a few microliters through a rubber septum and into
the vaporization chamber.

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.

The column in chromatography is undoubtedly the heart of the
technique.
They have following major types.
 Packed columns
 Open tubular columns

These columns are fabricated from glass, stainless steel, copper, or
other suitable tubes.
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.
Column length can be from few feet for packed columns to more
than 100 ft. for capillary columns.
Disadvantage: Glass is prone to breakage.

Open tubular or capillary or GOLAY 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.

 Improved version of Golay / Capillary columns, have small
sample capacity.
 Made by depositing a micron size porous layer of supporting
material on the inner wall of the capillary column.
 Then coated with a thin film of liquid phase.
Disadvantage: Small sample capacity

The most frequently used capillary column, nowadays, is the fused
silica open tubular column (FSOT), which is a WCOT column.
The external surface of the fused silica columns is coated with a
polyimide film to increase their strength.

 Before introduction of the sample.
Column is attached to instrument & desired flow rate by flow
regulators.
Set desired temp.
Conditioning is achieved by passing carrier gas for 24 hours.

Preheaters: convert sample into its vapor form, present along with
injecting devices
Thermostatically controlled oven: temperature maintenance in a
column is highly essential for efficient separation.
Isothermal programming: also called non-linear
Linear programming: this method is efficient for separation of
complex mixtures

 The temperature control system must be sensitive to 0.01 C and should of
resistance i.e., Pt. or Kovaror tungsten.
 The filament within the cell form a Wheatstone bridge that detects the
difference in thermal conductivity b/w the stream of carrier gas that contain the
sample components and the reference system of pure carrier gas before the
injection point.
 The difference in thermal conductivity generates a signal that is amplified to
drive a recorder pen to a proportional height on a strip chart recorder.

• FLAME IONIZATION DETECTOR (FID)
• NITROGEN PHOSPHORUS DETECTOR (NPD)
• ELECTRON CAPTURE DETECTOR (ECD)
• THERMAL CONDUCTIVITY DETECTOR (TCD)
• FLAME PHOTOMETRIC DETECTOR (FPD)
• PHOTO IONIZATION DETECTOR (PID)
• ELECTROLYTIC CONDUCTIVITY DETECTOR (ELCD)
• MASS SPECTROMETER (MS)

• As solutes elute from the column, they interact with the detector. The detector converts this
interaction into an electronic signal that is sent to the data system. The magnitude of the signal is
plotted versus time (from the time of injection) and a chromatogram is generated.
• Some detectors respond to any solute eluting from the column while others respond only to solutes
with specific structures, functional groups or atoms
• Detectors that exhibit enhanced response to specific types of solutes are called selective detectors.

FLAME IONIZATION DETECTOR (FID):
• Mechanism: Compounds are burned in a hydrogen-air flame. Carbon containing
compounds produce ions that are attracted to the collector. The number of ions
hitting the collector is measured and a signal is generated.
Selectivity: Compounds with C-H bonds. A poor response for some non-hydrogen
containing organics (e.g., hexachlorobenzene ).
Sensitivity: 0.1-10 ng
Linear range: 105-107
Gases: Combustion - hydrogen and air; Makeup - helium or nitrogen
Temperature: 250-300°C,and 400-450°C for high temperature analyses.

NITROGEN PHOSPHORUS DETECTOR (NPD):
• Mechanism: Compounds are burned in a plasma surrounding a rubidium bead supplied with
hydrogen and air. Nitrogen and phosphorous containing compounds produce ions that are attracted
to the collector. The number of ions hitting the collector is measured and a signal is generated.
Selectivity: Nitrogen and phosphorous containing compounds
Sensitivity: 1-10 pg
Linear range: 104-10-6
Gases: Combustion - hydrogen and air; Makeup - helium
Temperature: 250-300°C

• ELECTRON CAPTURE DETECTOR (ECD):
Mechanism: Electrons are supplied from a 63Ni foil lining the detector cell. A current is generated in
the cell. Electronegative compounds capture electrons resulting in a reduction in the current. The
amount of current loss is indirectly measured and a signal is generated.
Selectivity: Halogens, nitrates and conjugated carbonyls
Sensitivity: 0.1-10 pg (halogenated compounds); 1-100 pg
(nitrates); 0.1-1 ng (carbonyls)
Gases: Nitrogen or argon/methane

• THERMAL CONDUCTIVITY DETECTOR (TCD):
• Mechanism: A detector cell contains a heated filament with an applied
current. As carrier gas containing solutes passes through the cell, a change
in the filament current occurs. The current change is compared against the
current in a reference cell. The difference is measured and a signal is
generated.
Selectivity: All compounds except for the carrier gas
Sensitivity: 5-20 ng
Gases: Makeup - same as the carrier gas

• FLAME PHOTOMETRIC DETECTOR (FPD):
Mechanism: Compounds are burned in a hydrogen-air flame. Sulfur and phosphorous
containing compounds produce light emitting species (sulfur at 394 nm and phosphorous at
526 nm). A monochromatic filter allows only one of the wavelengths to pass. A photomultiplier
tube is used to measure the amount of light and a signal is generated. A different filter is
required for each detection mode.
Selectivity: Sulfur or phosphorous containing compounds. Only one at a time.
Sensitivity: 10-100 pg (sulfur); 1-10 pg (phosphorous)
Linear range: Non-linear (sulfur); 103-105 (phosphorous)
Gases: Combustion - hydrogen and air; Makeup - nitrogen

PHOTO IONIZATION DETECTOR (PID):
Mechanism: Compounds eluting into a cell are bombarded with high energy
photons emitted from a lamp. Compounds with ionization potentials below
the photon energy are ionized. The resulting ions are attracted to an
electrode, measured, and a signal is generated.
Selectivity: Depends on lamp energy. Usually used for aromatics and olefins
(10 eV lamp).
Sensitivity: 25-50 pg (aromatics); 50-200 pg (olefins)
Gases: Makeup - same as the carrier gas
Temperature: 200°C

• ELECTROLYTIC CONDUCTIVITY DETECTOR (ELCD):
Mechanism: Compounds are mixed with a reaction gas and passed through a high temperature
reaction tube. Specific reaction products are created which mix with a solvent and pass
through an electrolytic conductivity cell. The change in the electrolytic conductivity of the
solvent is measured and a signal is generated. Reaction tube temperature and solvent
determine which types of compounds are detected.
Selectivity: Halogens, sulfur or nitrogen containing compounds. Only one at a time.
Sensitivity: 5-10 pg (halogens); 10-20 pg (S); 10-20 pg (N)
Linear range: 105-106 (halogens); 104-105 (N); 103.5-104(S)
Gases: Hydrogen (halogens and nitrogen); air (sulfur)
Temperature: 800-1000°C (halogens), 850-925°C (N), 750-825°C (S)

MASS SPECTROMETER (MS):
Mechanism: The detector is maintained under vacuum. Compounds are
bombarded with electrons (EI) or gas molecules (CI). Compounds fragment
into characteristic charged ions or fragments. The resulting ions are focused
and accelerated into a mass filter. The mass filter selectively allows all ions of
a specific mass to pass through to the electron multiplier. All of the ions of the
specific mass are detected. The mass filter then allows the next mass to pass
through while excluding all others. The mass filter scans stepwise through the
designated range . A mass spectrum is obtained for each scan which plots the
various ion masses versus their abundance or number.
Selectivity: Any compound that produces fragments within the selected mass
range. May be an inclusive range of masses (full scan) or only select ions
(SIM).
Sensitivity: 1-10 ng (full scan); 1-10 pg (SIM)
Gases: None
Temperature: 250-300°C (transfer line), 150-250°C (source

 The eluent (carrier gas) is introduced from a gas cylinder outside
the machine. It's called the carrier because that's exactly what it
does—carry the sample we're studying through the machine. In gas
chromatography, the carrier gas is the mobile phase.
 The rate of flow of the carrier is carefully controlled to give the
clearest separation of the components in the sample.
 The carrier enters the machine through an inlet port/splitter.

 The sample being measured is injected into the carrier gas using
a syringe and instantly vaporizes (turns into gas form).
 The gases that make up the sample separate out as they move
along the column (orange), which contains the stationary phase
(typically, it's a thin coating on the inside wall of the column). The
column is a very thin (capillary) tube, sometimes as much as 30–
60m (100-200ft) long, coiled and entirely contained inside an oven
(blue) that keeps it at a high enough temperature to ensure that the
sample remains in gas form. The temperature of the oven can be
carefully controlled.

As the sample separates out and its constituent gases travel along
the column at different speeds, a detector senses and records them.
Various different detectors can be used, including flame ionization
detectors, thermal conductivity detectors, and mass spectrometers
(usually separate machines).
The data analyzer/recorder attached to the machine draws
a chromatogram (chart) with peaks corresponding to the relative
amounts of the different chemicals in the sample.

Retention time (Rt.)
It is the difference in time b/w the point of injection & appearance
of peak maxima.
Rt. measured in minutes or seconds
It is the time required for 50% of a component to be eluted from a
column

Retention volume (Vr)
 It is the volume of carrier gas which is required to elute 50% of the
component from the column.
Retention volume = Retention time × Flow rate

Separation factor (S)
The separation factor (α) is the ability of the chromatographic
system to 'chemically' distinguish between sample components.
Ratio of partition co-efficient of the two components to be
separated. If more difference in partition co-efficient b/w two
compounds, the peaks are far apart & Separation is more.
If partition co-efficient of two compounds are similar, then peaks
are closer.

Resolution (R)
 The true separation of 2 consecutive peaks on a chromatogram is
measured by resolution. It is the measure of both column & solvent
efficiencies.

Where tr1 and tr2 and w1 and w2 are the times and widths of peaks.

 Chromatographic peak should be symmetrical about its center.
 If peak is not symmetrical- shows Fronting or Tailing
Fronting:
 Due to saturation of S.P & can be avoided by using less quantity of
sample.

Tailing:
Due to more active adsorption sites & can also be occur due to flow
path disruption.

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