Gas chromatography is a technique used to separate components of a vaporized sample. It works by partitioning the components between a mobile gaseous phase and a stationary phase within a column. The sample is injected and vaporized, then transported through the column by the mobile phase gas. As the components pass through the column they are separated and detected. Common detectors include the flame ionization detector (FID), thermal conductivity detector (TCD), and electron capture detector (ECD). The FID responds to organic compounds, the TCD is universal but less sensitive, and the ECD selectively detects halogen-containing compounds.
Gas chromatography is one of the widely used chromatographic techniques, which use inert gas as the mobile phase.
In gas chromatography the components of a sample, after vaporization, are separated by being partitioned between gaseous mobile phase and solid or liquid stationary phase.
The inert gas does not interfere with the analyte but transport the components through the column and facilitate the separation.
The mobile phase is comprised of an inert gas such as helium, argon or nitrogen.
The stationary phase consists of a packed column in which the packing or solid support, or a liquid coat act as stationary phase.
The main principles involved are adsorption and partition for gas solid chromatography and gas liquid chromatography, respectively.
Principle and application of ptgc and isothermal programmingAthira39
Gas chromatography is the separation of gaseous and volatile substances which is achieved by employing gas as a mobile phase and moving it through a column containing stationary phase which could be a liquid or solid.
Two methods of temperature control are used during gas chromatography:
Isothermal operation and;
Temperature programming
Gas chromatography and its instrumentationArgha Sen
Gas chromatography is an unique technology which helps us in separating volatile analytes. Its is an easy and reproduciple method for detecting residual solvents found in APIs.
Gas chromatography is one of the widely used chromatographic techniques, which use inert gas as the mobile phase.
In gas chromatography the components of a sample, after vaporization, are separated by being partitioned between gaseous mobile phase and solid or liquid stationary phase.
The inert gas does not interfere with the analyte but transport the components through the column and facilitate the separation.
The mobile phase is comprised of an inert gas such as helium, argon or nitrogen.
The stationary phase consists of a packed column in which the packing or solid support, or a liquid coat act as stationary phase.
The main principles involved are adsorption and partition for gas solid chromatography and gas liquid chromatography, respectively.
Principle and application of ptgc and isothermal programmingAthira39
Gas chromatography is the separation of gaseous and volatile substances which is achieved by employing gas as a mobile phase and moving it through a column containing stationary phase which could be a liquid or solid.
Two methods of temperature control are used during gas chromatography:
Isothermal operation and;
Temperature programming
Gas chromatography and its instrumentationArgha Sen
Gas chromatography is an unique technology which helps us in separating volatile analytes. Its is an easy and reproduciple method for detecting residual solvents found in APIs.
in this slides contains principle and types of detectors used in Gas Chromatography.
Presented by: J.Vinay Krishna. (Department of industrial pharmacy),
RIPER, anantapur.
In this slide contains principle, instrumentation, methodology, and application of gel chromatography.
Presented by: SATHEES CHANDRA (Department of pharmaceutical analysis).
RIPER, anantapur
High Performance Thin Layer Chromatography (HPTLC) instrumentationMadhuraNewrekar
HPTLC is an advancement of TLC. It is a high performance liquid chromatography with automation compared to Thin Layer Chromatography(TLC).Speed, Efficiency and Accuracy are important advantages. Evaluation time is less due to updated automation in instrumentation.
Steps involved in HPTLC and the materials and instruments required in those steps are described in brief.
in this slides contains principle and types of detectors used in Gas Chromatography.
Presented by: J.Vinay Krishna. (Department of industrial pharmacy),
RIPER, anantapur.
In this slide contains principle, instrumentation, methodology, and application of gel chromatography.
Presented by: SATHEES CHANDRA (Department of pharmaceutical analysis).
RIPER, anantapur
High Performance Thin Layer Chromatography (HPTLC) instrumentationMadhuraNewrekar
HPTLC is an advancement of TLC. It is a high performance liquid chromatography with automation compared to Thin Layer Chromatography(TLC).Speed, Efficiency and Accuracy are important advantages. Evaluation time is less due to updated automation in instrumentation.
Steps involved in HPTLC and the materials and instruments required in those steps are described in brief.
Gas chromatography is widely used techniques for separation of gaseous and volatile substances which are difficult to separate and analyze It is simple and inexpensive method , generally efficient in regard to separation.
A separation technique in which the mobile phase is a gas. Gas chromatography is always carried out in a column.
Separating mixtures of gases or volatile materials based primarily on their physical properties.
Introduction and principle of glc, hplc
columns of hplc
columns of glc
detectors of glc
detectors of hplc
chromatography
classification of chromatography
gas liquid chromatography
high performance liquid chromatography
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2. 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 stationary phase held in the column.
3. PRINCIPLES
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.
4. 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.
5. Gas Chromatography
There are two types.
Gas-liquid chromatography (GLC)
mobile phase – gas Shortened to Gas
Chromatography
stationary phase - liquid
Gas-solid chromatography (GSC)
mobile phase – gas
stationary phase - solid
6. INSTRUMENTATION
A. Carrier gas
B. Flow regulator
C. Injector
Thermostated
D. Column oven
E. Detector
Integrator
F. Integrator
G. Display system
-printer/monitor
8. 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.
9. Samples must be…..
Volatile
Thermally stable.
When injected onto the head of a
chromatographic column and
vaporized.
10. Mobile phase
Mobile phase transports the analytes
(sample) through column.
Mobile phase can not interact with the
molecules of the analyte.
Referred as carrier gas.
11. 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.
12. 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.
13. 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.
15. D. Sample Injection System
Column efficiency requires sample to
be……
1. Of a suitable size
2. Introduced as a “plug” of vapor
Band broadening and poor resolution
are caused by…….
1. Slow injection.
2. Oversized sample.
16. 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.
17. D. Sample Injection System
Examples
Direct injection using microsyringe
Loop injectors
Auto samplers
Headspace
19. D. Sample Injection System
LOOP INJECTORS
DIRECT INJECTION
USING MICROSYRINGE
20. D. Sample Injection System
Headspace
A 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.
21. 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 phase
The 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.
22. 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.
23. E. Oven
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.
24. Temperature Programming
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.
25. 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.
26. Isothermal Elution
A technique in which the column
temperature is constantly maintained
throughout the separation.
28. F. Columns
Two types of columns
1.Packed column
2.Capillary column
Capillary Column:
Packed column:
10-100m in length, very small i.d
1-5m in length, 2-4mm i.d
29. 1. Packed Column
Less commonly used
Made of glass or steel
Length: 1 to 5 m
Cross-sectional view
Internal diameter: 2 to 4 mm of packed column
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.
30. 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 size
2. Ease & convenience of use
31. 2. Capillary 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
32. 2. Capillary Column
Advantages:
1. High resolution
2. Short analysis time
3. High sensitivity
34. G. Stationary Phase
Desirable 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.
35. G. Stationary Phase
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
36. 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.
37. The choice of stationary phase should match
that of sample components.
Non polar Stationary phase Polar Stationary phase
38. Polarity of Stationary Phase
Water
Polar Carboxylic acids
Amides
Alcohol/amines
Esters/aldehydes/ketones
Ethers
Halides
Aromatic hydrocarbons
Olefins
Non-polar
Aliphatic hydrocarbons
42. G. Stationary Phase
Many liquid statationary phase are based on
polysiloxanes or polyethylene glycol (PEG)
H H H H
HO C C O C C OH Polyethylene glycol (PEG)
Use for separating polar species
H H H H
n
R R R
R Si O Si O Si R
Polydimethyl siloxane, the R
groups are all CH3. (Non-polar)
R R R
n
43. 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.
44. H. Detectors
Characteristics of ideal detector
1. 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.
45. H. Detectors
Characteristics of ideal detector
4. 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)
46. H. Detectors
Several types of detectors.
1. Flame Ionization Detector (FID)
2. Thermal Conductivity Detector (TCD)
3. Electron Captured Detector (ECD)
48. How does FID works?
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.
49. 1. FID
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.
50. 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.
52. 2. TCD
A universal detector.
Has a moderate sensitivity.
Less satisfactory with carrier gas whose
conductivities closely resemble those
of most sample components.
53. How does TCD works?
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.
54. 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.
55. 2. TCD
Advantages
Simple
Large linear dynamic range
Responds to both organic and
inorganic species
Nondestructive; permits collection of
solutes after detection.
Disadvantage
Relatively low sensitivity.
57. How does ECD works?
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
58. 3. ECD
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.
59. H. Detectors
Other detectors
Nitrogen-Phosphorous Detector
(NPD)
Flame Photometry Detector (FPD)
Mass spectrometer (GC-MS)
60. H. Detectors
Detector Principle of Principle class of
operation compound detected
FID Ionization of solute Organics
molecules in a
flame
TCD Thermal Any samples
conductivity
ECD Current Compounds containing
electronegative
elements