3. Contents:
IUPAC Definition of Chromatography
Introduction
Definition
Types of GC
Principle
Instrumentation
Working animation
Troubleshoots
Applications
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4. IUPAC Definition of Chromatography :
“A physical method of separating sample components from a mixture by
selective adsorption or partitioning of the analyte between two phases:
a mobile phase and a stationary phase”.
Stationary Phases
1. Solids (alumina, silica, polymers,
carbon…)
• Adsorption chromatography
2. Liquids (siloxanes, polyethylene
glycols…)
• Partition chromatography
Mobile Phases
1. Liquids (methanol, water…)
•Changing dielectric strength,
temperature, pH
2. Gases (nitrogen, helium, hydrogen,
argon)
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5. INTRODUCTION:
Gas chromatography is the process of separating components
from the given crude drug by using a gaseous mobile phase.
The suggestion that separation of components of a mixture in
the gaseous state could be achieved using a gaseous mobile
phase was first given by Martin and Synge in 1941.
They suggested the use of gas-liquid partition chromatograms
for analytical purposes.
The first description of instrumentation and application was
made by James and Martin in 1952.
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6. DEFINITION:
Gas chromatography is a separation technique based on partitioning
analytes between two immiscible phases: gaseous mobile phase (Carrier
gas) and a stationary solid or immobilized liquid phase (packed or hollow
capillary column).
Used for separation of volatile substances, or substances that can
be made volatile, from one another in a gaseous mixture at high
temperatures.
It involves a sample being vaporized and injected onto the head of the
chromatographic column. The sample is transported through the column by
the flow of inert, gaseous mobile phase. The column itself contains a liquid
stationary phase which is adsorbed onto the surface of an inert solid.
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7. Gas Solid Chromatography(GSC)
The stationary phase is a solid. It is the affinity of solutes towards
adsorption onto the stationary phase which determines, in part, the
retention time. The mobile phase is, of course, a suitable carrier gas. This
gas chromatographic technique is most useful for the separation and
analysis of gases like CH4, CO2, CO, ... etc.
Gas Liquid Chromatography(GLC)
The stationary phase is immobilized liquid with very low volatility while
the mobile phase is a suitable carrier gas. GLC is the most widely used
technique for separation of volatile species.
Types of Gas Chromatography: 7
8. PRINCIPLE:
The sample solution injected into the instrument enters a gas stream
which transports the sample into a separation tube “column”. (Helium
or nitrogen is used as carrier gas.)
The various components are separated inside the column. The
detector measures the quantity of the components that exit the
column.
To measure a sample with an unknown concentration, a standard
sample with known concentration is injected into the instrument.
The standard sample peak, retention time (appearance time) and
area are compared to the test sample to calculate the concentration.
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10. COMPONENTS OF GAS CHROMATOGRAPH
Carrier gas
Flow
meter
Injectors
Column
Oven
Detector• Flame ionization (FID)
• Thermal conductivity (TCD)
• Electron capture (ECD)
• Nitrogen-phosphorus
• Flame photometric (FPD)
• Photo-ionization (PID)
• Atomic emission (AED)
• GC-MS
Recorder
Detector types
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11. CARRIER GAS (Mobile phase):
Sole purpose is to transport solutes (sample molecules) through the column
Commonly used carrier gases are He, N₂, H₂, Ar & CO₂
Should have following properties:
Chemically inert with stationary phase
Of High purity >99.9%
Good thermal conductivity
Compatible with detector
Higher density
Cheap and available
Non flammable
Non toxic
Non polar because stationary phase is polar
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12. Molecular sieves:
A molecular sieve is a material with very small holes of precise
and uniform size. These holes are small enough to block large
molecules and allow small molecules to pass. Many molecular
sieves are used as desiccants.
Glass tubes filled with specific adsorbent materials to remove
impurities ( moisture, hydrocarbon, O2) from gas (mobile phase).
Used to avoid undesirable chemical changes into sample
components and stationary phase, or adverse effects on detector
performance.
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13. Common contaminants and means of removing them are:
Air or oxygen: at levels above about 10 ppm, is removed by a
cartridge containing oxygen trap. Oxygen trap is filled with 500
cc of active oxygen adsorbent that binds covalently with
oxygen
Hydrocarbons:
removed by a cartridge containing activated carbon.
This molecular sieve use activated charcoal in-line trap to
remove gaseous hydrocarbons
Water vapors (moisture): removed with moisture filter.
Molecular sieves: 13
14. CARRIER GAS CYLINDER WITH MOLECULAR SIEVES
Moisture
filter
Hydrocorbon
filter
Indicating Oxygen
filter
Molecular sieves
Two-stage regulator
Tank
On/off valve
14
15. Gas filters required for a GC instrument with Flame Ionization (FID)
detector.
15
16. FLOW METER:
May be a ROTAMETER or a SOAP BUBBLE FLOW METER or a AIR BUBBLE
METER.
Have following properties:
Deliver the gas with uniform pressure/ flow rate.
Maintains the gas pressure and flow of gas per minute.
Adjusts the gas pressure at 10--50 psi.
Adjusts the gas flow
• in case of packed column at 25—150 ml/min
• in case of open tubullar column at 1—25 ml/min
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17. SAMPLE INJECTION SYSTEM:
FUNCTION:
Vaporize the sample
Introduce the gaseous sample onto column
Gaseous, liquid and solid samples are introduced rapidly into the flowing
mobile phase at the top of the column through an injection port using a
micro-syringe, micro pipette, valve or other device.
Type of injector depends upon the physical state of the sample
Temperature of the sample port maintained about 50ﹾC higher than the
boiling point of least volatile component
Vaporize sample without decomposing it
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18. INJECTOR:
The injection port Is a hollow, heated, glass-lined cylinder
The injector is heated so that all components in the sample
will be vaporized.
If the temperature is too low, separation is poor and broad
spectral peaks should result or no peak develops at all.
If the injection temperature is too high, the specimen may
decompose or change its structure.
The temperature of the sample port is usually about 50°C
higher than the boiling point of the least volatile component
of the sample.
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20. GC Injection Techniques:
Vaporizing:
The liquid sample is evaporated prior to be transferred to the
separation column.
Split injection
Split-less injection
Programmed temperature vaporization (PTV) injection.
Non-vaporizing:
The liquid sample evaporates into the separation column (or a
precolumn)
On-column injection
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21. SEPTA:
Ensure optimal performance of your GC
instrument with bleed and temperature.
Made of low-bleed silicone, have
excellent mechanical properties, are
ideal for demanding GC and GC-MS
applications, and may be used reliably
up to 400 °C.
Septum must be replaced at least after
200 injections.
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22. Split Injector (vaporizing-injector)
The split vent is open, very small
part of the sample go into the
column.
Use when analyzing high
concentration or neat samples.
Yields the sharpest peaks if the
split gas is properly mixed.
Standard for capillary columns.
split vent
open
22
23. Splitless Injector (vaporizing-injector)
The split vent is closed, most of the
sample go into the column.
When analyzing low concentration or
diluted samples.
Splitless times of ~ 1 minute are
typical.
Standard for capillary columns.
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24. On-column injection:
Sample aliquots are directly introduced
onto the analytical column ( 0.2-0.5 ) at
low temperatures (60– 80°C).
On column injection is favored for an
analyte that can be thermally degradated
at the elevated heated split or split-less
mode (around 200 C).
These injection mode require careful
awareness to attain a good reproducibility.
A liner of the wider volume is favorable for
this injection.
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25. Column:
The column is where the
chromatographic separation of the
sample occurs.
Several types of columns are available
for different chromatographic
applications:
It is coated with a stationary phase
which greatly influences the separation
of the compounds.
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26. Columns:
Gas chromatography columns are of two designs:
packed and capillary.
Packed columns are typically a glass or stainless steel coil (typically
1-5 m total length and 5 mm inner diameter) that is filled with the
stationary phase, or a packing coated with the stationary phase.
Capillary columns are a thin fused-silica (purified silicate glass)
capillary (typically 10-100 m in length and 250 mm inner diameter)
that has the stationary phase coated on the inner surface.
Capillary columns provide much higher separation efficiency than
packed columns but are more easily overloaded by too much
sample.
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27. Packed columns:
A glass or stainless steel coil
Solid particles either porous or non-
porous coated with thin (1 μm) film of
liquid
3 - 6 mm Internal diameter;
1 - 5 m length
Large sample capacity and used for
preparative work
Alumina, silica gel, zeolite and porous
polymers are used as adsorbent
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28. Capillary or open tubular column:
Made by stainless-steel tube, glass, copper, cupronickel or fused silica
material
Kept in a suitable thermostat for maintaining a constant temperature.
Inner wall of the tube is coated with liquid or solid stationary phase
10—100m in length (normally 30—50m) and have internal diameter
near to 0.3mm (varies from 0.1—0.5mm) with internal wall thickness
0.1mm (immobilized liquid stationary phase)
Flexible and can be shaped into coils
Give better resolution, larger theoretical plate number, greater
sensitivity, smaller sample capacity
28
29. Types of Capillary column:
Wall coated open tubular column (WCOT)
Support coated open tubular column
(SCOT)
Porous layer open tubular column (PLOT)
Fused silica open tubular column (FSOT)
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30. Wall coated open tubular column (WCOT):
Consist of a capillary tube
Walls are coated with liquid stationary phase.
Made by extending the inner wall of columns by substances
such as fused silica.
Porous layer open tubular column (PLOT):
30
31. Support coated open tubular column (SCOT)
The inner wall of the capillary is lined
with a thin layer of support material
Support material may be
diatomaceous earth
Stationary phase adsorbed on
support layer
Less efficient than packed columns
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33. Fused silica open tubular column (FSOT):
A new type of WCOT column
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34. Column temperature and oven:
The column is enclosed in an insulated and
thermostatically-controlled oven with a heater and
circulating fan to maintain a uniform temperature from
ambient to about 400ºC
Column temperature must be high enough to provide
sufficient vapor pressure for components of the sample to
be eluted in suitable time.
Temperature of the oven ranges from 5ºC--400ºC and
may decrease up to with -25ºC with cryogenic cooling
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36. Detectors:
Numerous types of detectors available
Requirements: -
• Sensitive to the analytes of interest
• Compatible with the column, carrier gas, solvent, etc.
• Useful linear range
• GC Detectors have it’s own temperature control
• Measures response as a voltage or a current
• Short response time independent of flow rate
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37. Detectors:
Some of the most commonly used detectors are
described below:
Thermal conductivity detector (TCD)
Flame ionization detector (FID)
Electron capture detector (ECD)
Flame photometric detector (FPD)
Atomic emission detector (AED)
GC-MS
Nitrogen phosphorous detector (NPD)
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38. Thermal conductivity detector (TCD)
Known as Katharometer
Depends upon the thermal conductivity of the mobile phase
passing around a tungsten-rhenium filament
Senses changes in thermal conductivity of the column effluent
and compares it to a reference flow of carrier gas
Simple, non-destructive and employed for packed column
Used for detection of organic and inorganic species
Sensitivity is low
Not suitable for capillary gas chromatography
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40. Flame ionization detector (FID):
Most frequently used detector in GC
Its operation is based on detection of ions from during combustion of
organic compounds in a hydrogen flame
Carbon dioxide and carbon monoxide are not detectable by FID
FID compatible carrier gases include nitrogen, helium and argon
Insensitive to non-hydrocarbons
Low detection limits
Gives 100 times better detection than TCD
Destructive technique to detect the components
40
42. Electron capture detector (ECD):
Working principle is electron capturing by the sample or
analyte
Sensitive to halogens, chlorinated insecticides, carbonyl
peroxides, nitro-compounds and organo-metallics
β-emitter nickle-63 is used to emit β-particles
Concentration of the analyte is proportional to degree of
electron capture
Sensitivity decreases with moisture
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44. Flame photometric detector (FPD):
FDP uses a photomultiplier tube to detect spectral lines
of the samples
Examples (phosphorous, halogens, Sulphur, metals etc.)
as these are burned in hydrogen-air flame
Excited sample elements emit radiation of specific
wavelengths in the flame which are filtered and
measured by a photomultiplier tube
Analyze environmental samples
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45. Atomic emission detector (AED):
Based on atomic emission of sample
Elements (N, S, P, Br, Cl, F, O C, Si, etc.) generate atomic emission spectra
and detected by a series of photomultiplier tubes or photo diode-array
photometer
Figure. Schematic of atomic emission detector.
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46. GC-MS:
Gas Chromatography-Mass Spectrometry (GC-MS) is very powerful
analytical tool in which gas chromatograph is attached with mass
spectrometer.
Separation of sample components is based on their retention time
The separated components of sample exit from the GC column, enters
the ionization chamber of MS.
Sample components are ionized and these ionized fragments are
separated, accelerated and detected using their mass to charge ratio
(m/e).
This is the best detector which identifies the exact molecular mass of
every component.
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50. Troubleshooting categories:
1. Baseline disturbances.
2. Irregular peak shapes or sizes.
3. Retention time shifts.
4. Loss of separation or resolution.
5. Quantitation difficulties.
6. Rapid column deteriorations.
7. Ghost peaks.
8. Broad solvent fronts.
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51. Troubleshooting Tools:
1. An electronic leak detector
2. A flow meter
3. An accurate thermometer
4. A reliable analytical column
5. New syringes
6. Spare septa and high temperature septa
7. Spare ferrules
8. Detector cleaning solutions
9. Spare recorder and electrometer cables
10. Instrument manuals
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52. Applications of GC:
Used for separation of hydrocarbons and refinery gases in petroleum industries
Determines minute quantities of herbicides, fertilizers and pesticides in
vegetables, fruits and animal tissues
Analyze environmental hazards substances
Analyze and separate aromas of flowers, beverages ingredients, contents of
food and flavor
Extensively used in pharmaceutical industries to check intermediates, purity of
samples and drugs assay
Important technique for forensic and clinical analysis, toxicological cases, fatty
acids, steroids, biological specimens and body secretions
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53. Ensure quality of various industrial and agricultural products
Study reaction mechanism
Analyze detergents, soap, rubber products, resins, plastics,
binders, coatings & plasticizers and polymers
Used in the separation of radioactive products
High degree of resolution of GLC allows purity of a sample to be
checked
Applications of GC: 53
