gives a miniature explantion of important points in gas chromatography

1. GAS CHROMATOGRAPHY
BY
DEVI MANOZNA.T B.PHARM
I/II M.PHARMACY
(PHARMACEUTICAL ANALYSIS & QC )

2. INTRODUCTION
“Chromatography is a physical method of separation in which
the components to be separated are distributed between two
phases, one of the phases constituting a of large surface
area,
the other being a that percolates through or along the
stationary bed.” (Ettre & Zlatkis, 1967, “The Practice of Gas
Chromatography)
Chromatography dates to 1903 in the work of the Russian
scientist, Mikhail Semenovich Tswett. German graduate
student Fritz Prior developed solid state gas chromatography
in
1947.

3. CHROMATOGRAPHY MEANS……….
It is the collective term for a set of laboratory
techniques for the separation of mixtures. The mixture is dissolved in a
fluid called the mobile phase, which carries it through a structure
holding another material called the stationary phase. The various
constituents of the mixture travel at different speeds, causing them to
separate. The separation is based on differential partitioning between
the mobile and stationary phases. Subtle differences in a
compound's partition coefficient result in differential retention on the
stationary phase and thus changing the separation.
Chromatography may be preparative or analytical. The purpose of
preparative chromatography is to separate the components of a
mixture for more advanced use (and is thus a form of purification).
Analytical chromatography is done normally with smaller amounts of
material and is for measuring the relative proportions of analytes in a
mixture. The two are not mutually exclusive.

4. BASED ON THE PRINCIPLE OF SEPARATION,
IT IS CLASSIFIED AS:
ADSORPTION CHROMATOGRAPHY:
In this the components of mixture are separated based on
their relative affinities towards the stationary phase.
This method is used for a larger quantity of mixtures .
(e.g: TLC,CC,GSC)
PARTITION CHROMATOGRAPHY:
In this the components of mixture are separated based on
the relative solubilities or partition co-efficient. This method
is used for a smaller quantity of mixture. This method is an
accurate method than the GSC. (e.g) GLC, HPLC).

5. TYPES OF CHROMATOGRAPHY

6. Gas Chromatography
Gas Chromatography is a chromatographic technique
in which the mobile phase is a gas.
GC is currently one of the most popular methods for
separating and analyzing compounds. This is due to its
high resolution, low limits of detection, speed,
accuracy and reproducibility.
GC can be applied to the separation of any compound
that is either naturally volatile (i.e., readily goes into
the gas phase) or can be converted to a volatile
derivative. This makes GC useful in the separation of
a number of small organic and inorganic compounds.

7. FATHER OF GC Mr.A.J MARTIN
1903 - Mikhail Tswett separated
plant pigments using paper
chromatography
liquid-solid chromatography
1930’s - Schuftan & Eucken use
vapor as the mobile phase gas solid
chromatography
Archer John Porter Martin, who was
awarded the Nobel Prize for his
work in developing liquid–liquid
(1941) and paper (1944)
chromatography, laid the foundation
for the development of gas
chromatography and he later
produced liquid-gas chromatography
(1950). Erika Cremer laid the
groundwork, and oversaw much of
Prior's work.

8.  In gas chromatography (GC), the sample is vaporized and
injected onto the head of a chromatographic column.
Elution is brought about by the flow of an inert gaseous
mobile phase.
 The mobile phase does not interact with molecule of the
analyte; its only function is to transport the analyte
through the column.
 Gas-liquid chromatography is based upon the partition of
the analyte between a gaseous mobile phase and a liquid
phase immobilized on the surface of an inert solid.

9. Factors which influence the GC separation
Volatility of compound:
Low boiling (volatile)components
will travel faster through the column than high
boiling point components.
Polarity of compounds:
Polar compounds will move more slowly,
especially if the column is polar.
Column temperature:
Raising the column temperature speeds up all
the compounds in a mixture.

10. Column packing polarity:
Usually, all compounds will move
slower on polar columns, but polar compounds
will show a larger effect.
Flow rate of the gas through the column:
Speeding up the carrier gas flow
increases the speed with which all compounds
move through the column.
Length of the column:
The longer the column, the longer it
will take all compounds to elute. Longer columns
are employed to obtain better separation.

11. Separations
To detector
Time 1 Time 2 Time 3

12. MODERN GAS CHROMATOGRAPH

13. Schematic diagram of gas
chromatograph

14. INSTRUMENTATION
A. Carrier gas
B. Flow regulator
C. Injector
D. Column
E. Detector
F. Integrator
G. Display system -
printer/monitor
Thermostated
oven
Integrator

15. Working
Principle
The organic compounds are separated due to
differences in their partitioning behavior between
the mobile gas phase and the stationary phase in
the column.
First, a vaporized sample is injected onto the
chromatographic column.
Second, the sample moves through the column through
the flow of inert gas.
Third, the components are recorded as a sequence of
peaks as they leave the column.

16. Chromatographic Separation
In the mobile phase, components of the sample are uniquely
drawn to the stationary phase and thus, enter this phase
at different times.
The parts of the sample are separated within the column.
Compounds used at the stationary phase reach the detector
at unique times and produce a series of peaks along a time
sequence.

17. Chromatographic Analysis
The number of components in a sample is determined by
the number of peaks.
The amount of a given component in a sample is determined
by the area under the peaks.
The identity of components can be determined by the
given retention times.

18. MOBILE PHASE (GAS)

19. CARRIER GAS
 The main purpose of a carrier gas is to carry the
sample through the column. Secondarily, it should be
providing a suitable matrix for the detector to
measure the sample component.
 This is the mobile phase and should be pure gas so as
not to react with the column or analyte. Gas is usually
He, Ar, N2, or H2. Choice will depend on the type of
detector used. He and H2 give better resolution
(smaller plate height) than N2. Pressure is also
important and as expected the system comes with
regulators. Can you find where in GC equations that
are dependent on pressure?

20. POINTS TO REMEMBER FOR CARRIER
GAS
Oxygen and water impurities can chemically attack the
liquid phase of the column and destroy it.
Trace water can absorb the column contaminants and
produce high detector background or ghost peaks.
Trace hydro carbons can cause high detector
background with FID’s and limit detect ability

21. REQUIREMENTS OF A CARRIER GAS
 Inertness
 Suitable for the detector
 High purity(minimum of 99.995%)
 Easily available
 Cheap
 Should not cause the risk of fire
 Should give best column performance

22. SUB CLASSIFICATION OF GAS
CHROMATOGRAPHIC
TECHNIQUES
(depending on stationary phase)
 GSC : gas solid chromatography
In this type the stationary phase used would be a
solid .
 GLC : gas liquid chromatography
In this type the stationary phase used would be
liquid .

23. 1.) Gas-solid chromatography (GSC)
- same material is used as both the stationary phase and
support material
Magnified Pores in activated carbon

24. Solids usually , traditionally run in packed
columns
These solid should have small and uniform particle size
(example : 80/100 mesh range)
SOMECOMMON GC ADSORBENTS :
•Silica gel
•Activated alumina
•Zeolite molecular sieves
•Carbon molecular sieves
•Porous polymers
•Tenax polymers
Some of these solids have been coated on the inside wals
of capillary columns and these are called as SUPPORT
COATED OPEN TUBULAR COLUMNS (SCOT)

25. ADVANTAGES:
- long column lifetimes
- ability to retain and separate some compounds not
easily
resolved by other GC methods
‚ geometrical isomers
‚ permanent gases
DISADVANTAGE:
- very strong retention of low volatility or polar
solutes
- catalytic changes that can occur on GSC supports
- GSC supports have a range of chemical and
physical
environments
‚ different strength retention sites
‚ non-symmetrical peaks
‚ variable retention times

26. 2.) Gas-liquid chromatography (GLC)
- stationary phase is some liquid coated on a solid
support.
- over 400 liquid stationary phases available for GLC
many stationary phases are very similar in terms of
their retention properties
- material range from polymers (polysiloxanes,
polyesters, polyethylene glycols) to fluorocarbons,
molten salts and liquid crystals

27. The Stationary Phase
Desirable properties for the immobilized liquid phase in a
gas-liquid chromatographic column include:
(1) low volatility (ideally, the boiling point of the liquid
should be at 100oC higher than the maximum operating
temperature for the column)
(2) Thermal stability
(3) chemical inertness
(4) solvent characteristics such that k` and  values for
the solutes to be resolved fall within a suitable range.
The retention time for a solute on a column depends
upon its distribution constant which in turn is related
to the chemical nature of the stationary phase.

28. The Stationary Phase
To have a reasonable residence time in the column, a
species must show some degree of compatibility
(solubility) with the stationary phase. Here, the
principle of “like dissolves like” applies, where “like”
refers to the polarities of the solute and the
immobilized liquid.
Polar stationary phases contain functional groups such
as –CN,--CO and –OH. Hydrocarbon-type stationary
phase and dialkyl siloxanes are nonpolar, whereas
polyester phases are highly polar. Polar solutes include
alcohols, acids, and amines; solutes of medium polarity
include ethers, ketones, and aldehydes.

30. Water
Carboxylic acids
Amides
Alcohol/amines
Esters/aldehydes/ketone
s
Ethers
Halides
Aromatic hydrocarbons
Olefins
Aliphatic hydrocarbons
Polar
Non-polar
Polarity of Stationary Phase

31. CHOICE OF CARRIER
GAS:
31
Advantages Disadvantages
Hydrogen  Cheap
 Gives the most time efficient
separation
 Still very efficient at high gas
velocities i.e.. 60 cm/ sec
 Can form an explosive mixture
with air
 Some industries in some
countries have regulated
AGAINST the use of hydrogen
 is a reductive gas
Helium  Very inert, will not react with
analytes
 Gives a very time efficient
separation
 Non flammable
 Expensive
 A non-replenishable resource
Nitrogen  Cheap
 Very inert, will not react with
analytes
 Non flammable
 Very slow velocity to achieve
good efficiency
 Narrow range for maximum
efficiency

32. Flow regulators & Flow meters
18-Jan-1532
Flow regulators – To deliver the gas with uniform
pressure or flow rate.
Flow meter – To measure the flow rate of carrier
gas.
TYPES OF FLOW METERS
1. Rota meter .
2. Soap bubble flow meter.

33. 1. ROTAMETER
18-Jan-1533

34. 34 Soap bubble flow meter
Aqueous
solution of
soap or
detergent
 soap bubble meter
 soap bubbles formed
indicates the flow rate.
Glass tube with a inlet tube at
the bottom.
 Rubber bulb-----store soap
solution
 When the bulb is gently
pressed of soap solution is
converted into a bubble by
the pressure of a carrier
gas &travel up

35. 35
inlet tube

37. Sample injection systems

38. Inlets are points of sample introduction
Ideal sample inlets for column type :
1. PACKED COLUMNS :
 Flash vaporizer
 on- column
2. CAPILLRY COLUMNS:
 Split injector
 Splitless injector
 On-column

39. Mode of Injections

40. SPLITLESS INJECTOR
In this case the sample has to be diluted in a volatile solvent
and around 1-5ml is injected in the heated injection port.
Septum perge is essential in split less injection
ADVANTAGES OF SPLITLESS INJECTION
•Improved sensitivity over a slit injector .
DIS –ADVANTAGES OF SPLITLESS INJECTOR
•its time consuming
•Initial temperature and time of opening the split valve needs
to be optimized .
•Not well suited for volatile compounds (boiling points of peaks
of interest to be about 30 degrees centigrade higher than the
solvent)

41. SPLIT INJECTOR
It’s the oldest , simplest and easiest injection
technique.
ADVANTAGES OF SPLIT INJECTION
•High resolution seperations
•Neat samples can be introduced
•Dirty samples can be introduced by putting a deactivated
glass wool plug in the liner to trap non volatile components.
DIS-ADVANTAGES
•Trace analysis is limited.
•Process sometimes discriminates between high molecular
weight solutes so that the sample entering the column is not
represntative of sample injected .

42. OTHER TYPES OF INLETS
•DIRECT INJECTION :
It involves in injecting a small sample into a glass liner where
vapors are carried directly into the column .
•ON-COLUMN INJECTION :
It deals with inserting precisely aligned needle into the
capillary column and making injections inside the column.
•FLASH VAPOURIZER :
It involves in heating the port to a temperature well above the
boiling point to ensure rapid volatilization .
•STATIC HEADSPACE :
it concentrates the vapour over a solid or liquid sample (best
chosen for residual solvent analysis)

43. Sample Injection System
 Column efficiency requires that the sample be of
suitable size and be introduced as a “plug” of vapor;
slow injection of oversized samples causes band
spreading and poor resolution.
 The most common method of sample injection
involves the use of microsyringe to inject a liquid or
gaseous sample through a self-sealing, silicone-
rubber diaphragm or septum into a flash vaporizer
port located at the head of the column (the sample
port is ordinarily about 50oC above the boiling point
of the least volatile component of the sample).

45. Sampling place

46. 46

47. COLUMNS

48. GC Columns
Capillary columnsPacked columns
•Typically a glass or
stainless steel coil.
•1-5 total length and 5
mm inner diameter.
• Filled with the st. ph.
or a packing coated
with the st.ph.
•Thin fused-silica.
•Typically 10-100 m in
length and 250 mm
inner diameter.
•St. ph. coated on the
inner surface.
•Provide much higher
separation eff.
•But more easily
overloaded by too
much sample.

49. GAS CHROMATOGRAPHIC COLUMNS
Open tubular Columns
Open tubular, or capillary, columns are of two basic types
,namely
 WALL—COATED OPEN TUBULAR (WCOT) :
These are simple capillary
tubes coated with a thin layer of the stationary
phase. In support-coated open tubular columns,
the inner surface of the capillary is lined with a
thin film (~30 m) of a support material, such as
diatomaceous earth. This type of column holds
several times as much stationary phase as does a
wall-coated column and thus has a greater sample
capacity.

50.  SUPPORT-COATED OPEN TUBULAR (SCOT) :
These are surface coated open
tubular columns. These are no longer available in fused
silica
 PLOT :
These are the porous layer open
tubular column which is less than 5% of all gas
chromatographic use these days

51. Packed Columns
Packed columns are fabricated from glass, metal
(stainless steel, copper, aluminum), or Teflon tubes
that typically have lengths of 2 to 3 m and inside
diameters of 2 to 4 mm. These tubes are densely
packed with a uniform, finely divided packing
material, or solid support, that is coated with a
thin layer (0.05 to m) of the stationary liquid
phase. In order to fit in a thermostating oven, the
tubes are formed as coils having diameters of
roughly 15 cm.

52. 3.SCOT COLUMNS (SUPPORT COATED
OPEN TUBULAR COLUMN)
 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

54. capillary
PACKED
• Packed
• Capillary

55. Solid Support Materials
The most widely used support material is
prepared from naturally occurring diatomaceous
earth, which is made up of the skeletons of
thousands of species of single-celled plants
(diatoms) that inhabited ancient lakes and seas.
Such plants received their nutrients and
disposed of their wastes via molecular diffusion
through their pores. As a consequence, their
remains are well-suited as support materials
because gas chromatography is also based upon
the same kind of molecular diffusion.

56. COLUMNS:
Columns in GC are two types
1) packed column
2) capillary column
Packed column
(capillary column)
Open tubular column
56

57. Liquid phase
Solid support coated
with liquid phase
Porous Adsorbent
Porous Layer Open
Tubular
(PLOT)
Wall-coated Open
Tubular
(WCOT)
Support-coated Open
Tubular
(SCOT)
Types of open tubular column:
57

58. Typical gc dual column

59. The oven
Inside here
Column
59

60. TEMPERATURE CONTROL DEVICES
 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.
 Two types of operations:
 Isothermal programming:- this method is
generally used for all compounds
 Linear programming:- this method is efficient
for separation of complex mixtures

61. Temperature Control
• Isothermal • Gradient
0
40
80
120
160
200
240
0 10 20 30 40 50 60
Time (min)
Temp(degC)
Instrumentation - Oven

62. Column Ovens
 Column temperature is an important variable that must
be controlled to a few tenths of a degree for precise
work. Thus, the column is ordinarily housed in a
thermostated oven. The optimum column temperature
depends upon the boiling point of the sample and the
degree of separation required.
 Roughly, a temperature equal to or slightly above the
average boiling point of a sample results in a
reasonable elution time (2 to 30 min). For samples with
a broad boiling range, it is often desirable to employ
temperature programming, whereby the column
temperature is increased either continuously or in
steps as the separation proceeds.

63. The thermostated oven serves to control the temperature of
the column within a few tenths of a degree to conduct precise
work. The oven can be operated in two manners: isothermal
programming or temperature programming. In isothermal
programming, the temperature of the column is held constant
throughout the entire separation. The optimum column
temperature for isothermal operation is about the middle
point of the boiling range of the sample. However, isothermal
programming works best only if the boiling point range of the
sample is narrow. If a low isothermal column temperature is
used with a wide boiling point range, the low boiling
fractions are well resolved but the high boiling fractions are
slow to elute with extensive band broadening. If the
temperature is increased closer to the boiling points of the
higher boiling components, the higher boiling components elute
as sharp peaks but the lower boiling components elute so
quickly there is no separation.

64. THE EFFECT OF COLUMN TEMPERATURE
ON THE SHAPE OF THE PEAKS.

65. DETECTION SYSTEMS
Characteristics of the Ideal Detector: The ideal
detector for gas chromatography has the following
characteristics:
1. Adequate sensitivity
2. Good stability and reproducibility.
3. A linear response to solutes that extends over
several orders of magnitude.
4. A temperature range from room temperature to at
least 400oC.

66. Characteristics of the Ideal Detector
5. A short response time that is independent of
flow rate.
6. High reliability and ease of use.
7. Similarity in response toward all solutes or a
highly selective response toward one or more
classes of solutes.
8. Nondestructive of sample.

68. Few mostly used detectors
1) FID ( flame ionization detector )
2) TCD ( thermal conductivity detector )
3) ECD ( electron capture detector )
4) FPD ( flame photometric detector )
5) HID ( helium ionization detector )
6) NPD ( nitrogen-phosphorus detector )
7) PID ( photo ionization detector )
8) TID ( thermionic ionization detector )
9) CCD ( catalytic combustion detector )
10) NPD/DELCD ( combination NPD and dry electrolytic conductivity
detector ) 11) FID/DELCD ( combination FID and dry electrolytic
conductivity detector )
12) FID/FPD ( combination FID and FPD )
13) Dual FPD ( dual wavelength FPD for both sulfur and phosphorus )
14) FID dual FPD ( dual FPD plus FID combination )

69. 1. Flame Ionization Detector
(FID)

70.  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.
How does FID works?

71. 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.

72.  Disadvantages
 Weakly sensitive to carbonyl, amine, alcohol
& amine groups.
 Not sensitive to non-combustibles analyte
such as H2O, CO2, SO2, NOx.
 Destructive method.

73. 2. Thermal Conductivity
Detector (TCD)

74. 2. TCD
 A universal detector.
 Has a moderate sensitivity.
 Less satisfactory with carrier gas whose
conductivities closely resemble those of most sample
components.

75.  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.
How does TCD works?

76.  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.
How does TCD works?

77. 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.

78. 3. Electron Capture Detector
(ECD)

79.  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 decreases markedly.
How does ECD works?

80. 4.) Electron Capture Detector (ECD)
- radiation-based detector
- selective for compounds containing
electronegative atoms, such as halogens
Process
- based on the capture of electrons by
electronegative atoms in a molecule
- electrons are produced by ionization of the
carrier gas with a radioactive source
‚ 3H or 63Ni
- in absence of solute, steady stream of
these electrons is produced
- electrons go to collector electrode where
they produce a current
- compounds with electronegative atoms
capture electrons, reducing current

81.  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.

82. 3.) Nitrogen-Phosphorus Detector (NPD)
- used for detecting nitrogen- or phosphorus containing compounds
- also known as alkali flame ionization detector or thermionic detector
Process
- same basic principal as FID
- measures production of ions when a solute
is burned in a flame
- ions are collected at an electrode to
create a current
- contains a small amount of alkali metal
vapor in the flame
- enhances the formation of ions from
nitrogen- and phosphorus- containing compounds
Alkali Bead

83. NITROGEN PHOSPHORUS
DETECTOR

84. 3.) Nitrogen-Phosphorus Detector (NPD)
advantages:
- useful for environmental testing
‚ detection of organophosphate pesticides
- useful for drug analysis
‚ determination of amine-containing or basic
drugs
- Like FID, does not detect common mobile phase
impurities
or carrier gases
- limit of detection: NPD is 500x better than FID in
detecting
nitrogen and phosphorus- containing compounds
- NPD more sensitive to other heterocompounds, such as
sulfur-, halogen-, and arsenic- containing molecules
disadvantage:
- destructive detector
- NPD is less sensitive to organic compounds compared to
FID

85. Atomic Emission Detectors (AED)
The atomic emission detector is available
commercially. In this device the eluent is
introduced into a microwave-energized helium
plasma that is coupled to a diode array optical
emission spectrometer. The plasma is sufficiently
energetic to atomize all of the elements in a
sample and to excite their characteristic atomic
emission spectra.

87. Thermionic Detectors (TID)
The thermionic detector is selective toward organic
compounds containing phosphorus and nitrogen. Its
response to a phosphorus atom is approximately ten
times greater than to a nitrogen atom and 104 to 106
larger than a carbon atom. Compared with the flame
ionization detector, the thermionic detector is
approximately 500 times more sensitive to phosphorus-
containing compounds and 50 times more sensitive to
nitrogen bearing species. These properties make
thermionic detection particularly useful for detecting
and determining the many phosphorus-containing
pesticides.

88. Element-selective detectors
18-Jan-1588
It includes :
 Thermionic ionization detector (TID).
 Give selective response to phosphorus and /or
nitrogen containing compounds.
 Use- For determination of pesticides like malathion
and parathion.
 Flame photometric detector
 For the determination of compounds containing
sulphur and phosphorus.

89. RECORDERS & INTEGRATORS
18-Jan-1589
RECORDERS
Used to record the responses obtained from detectors
after amplification.
Retention time can be found out.
INTEGRATORS
Record the individual peaks with retention time , height
and width of peaks , peak area , percentage of area
etc:-

90. Analysis of Output
 Less than ideal spectral peaks may indicate less than ideal
analytical procedures or equipment. The technician can
readily observe whether the output exhibits unsatisfactory
results. Ideally, the spectral peaks should be symmetrical,
narrow, separate (not overlapping), and made with smooth
lines. GC evidence may be suspect if the peaks are broad,
overlapping, or unevenly formed. If a poorly shaped peak
contains a steep front and a long, drawn-out tail, this may
indicate traces of water in the specimen.
 The GC technician should inject the specimen into the
septum rapidly and smoothly to attain good separation of the
components in a specimen. If the technician injects the
specimen too slowly, the peak may be broad or overlap. A
twin peak may result from the technician hesitating during the
injection. A smoothly performed injection, without abrupt
changes, should result in a smoothly formed peak. A twin
peak may also indicate that the technician injected two
specimens consecutively.

91. GENERAL CHROMATOGRAM

93. CHROMATOGRAM

94. CHROMATOGRAM

95. ADVANTAGES OF G.C
 Very high resolution power, complex mixtures can be
resolved into its components by this method.
 Very high sensitivity with TCD, detect down to 100 ppm
 It is a micro method, small sample size is required
 Fast analysis is possible, gas as moving phase- rapid
equilibrium
 Relatively good precision & accuracy
 Qualitative & quantitative analysis is possible

96. DIS ADVANTAGES
Response Factor
The size of a spectral peak is proportional to the amount of the
substance that reaches the detector in the GC instrument. No
detector responds equally to different compounds. Results using one
detector will probably differ from results obtained using another
detector. Therefore, comparing analytical results to tabulated
experimental data using a different detector does not provide a reliable
identification of the specimen.
A "response factor" must be calculated for each substance with a
particular detector. A response factor is obtained experimentally by
analyzing a known quantity of the substance into the GC instrument and
measuring the area of the relevant peak. The experimental conditions
(temperature, pressure, carrier gas flow rate) must be identical to
those used to analyze the specimen. The response factor equals the
area of the spectral peak divided by the weight or volume of the
substance injected. If the technician applies the proper technique, of
running a standard sample before and after running the specimen,
determining a response factor is not necessary.

97. Worn Septum
An injection port septum should last between 100 and
200 injections. Higher injection port temperatures
shorten the septum's lifespan. A leaking septum
adversely affects the GC instrument's sensitivity.
If a portion of the specimen leaks back out of the
septum, the amount of the specimen is not
recorded. This event makes any eventual quantitative
result erroneous. If air should leak into the injection
port through a worn septum, the oxygen and water
contained in air may skew the results. Any oxygen may
react with the specimen components. If this happens,
the GC instrument will provide results indicating the
presence of this unintended reaction product, instead of
the original compounds present in the specimen vial. Any
water in the column adversely affects the GC
instrument's ability to separate components.

98. Injection Port Temperature
The temperature of the GC injection port must be high
enough to vaporize a liquid specimen instantaneously. 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. If this occurs, the GC
results will indicate the presence of compounds that were not
in the original specimen.
Residual Impurities
Ideally, all components of a specimen elute completely
from the GC column. If any substance remains inside the
column, the substance may elute during subsequent analyses
with other specimens. This may result in an unexpected peak
in the output. The peak produced should be broad.

99. Carrier Gas
If the GC instrument uses hydrogen for the carrier gas, the
technician must consider whether the hydrogen may react with any of
the compounds present in the specimen. If the hydrogen does react,
a broad peak will result. When using a thermal conductivity detector,
care should be taken as a false peak may occur if the carrier gas's
thermal conductivity is in the range of the thermal conductivity of any
compound in the specimen. An unstable carrier gas flow rate may
produce a drifting baseline and false broad peaks. A carrier gas
should be pure. Regular changing of the gas filter should prevent
significant impurities.
Crucial Factors
GC analysis is highly reliable if the instrument is properly
maintained, the technician follows proper procedures, and the
interpretation of the results is competent. While some factors rarely
affect GC analysis, some factors are absolutely essential for the use
of reliable GC evidence. In all cases a technician must process a
standard sample containing a verified composition identical to the
presumed contents of the collected specimen. This standard sample

100. Any output from the collected specimen that does not match the
standard sample is inconclusive. If tabulated reference data
exists for the relevant conditions, the specimen data must match
the reference data. If advance notice of GC testing is available,
an adverse party should observe the procedure. If a retained
consultant or the knowledgeable attorney observes the
technician's use of the GC instrument, important information can
be recorded. The technician's preparation of the specimen and
the subsequent injection can be observed for errors or
malfunctioning equipment. The observer should record the
instrument's make, model, serial number, injection temperature,
column temperature, carrier gas flow rates and pressure, identify
the type of detector used, and observe any manipulation of the
data by use of a computer. Ensure that the technician properly
starts measuring the time at injection and records the time of
elution. Any discrepancy in the time will produce an erroneous
retention time. If the procedure can not be observed, the adverse
party should seek all pertinent information (experimental

102. Headspace gas chromatography analysis
 Headspace GC (HSGC) analysis employs a specialized
sampling and sample introduction technique, making
use of the equilibrium established between the
volatile components of a liquid or solid phase and the
gaseous / vapor phase in a sealed sample container.
Aliquots of the gaseous phase are sampled for
analysis.

103. Pyrolysis gas chromatography
 Pyrolysis GC (PGC) is used principally for the
identification of non-volatile materials, such as
plastics, natural and synthetic polymers, drugs and
some microbiological materials. The thermal
dissociation and fragmentation of the sample
produces a chromatogram which is a fingerprint for
that sample. The small molecules produced in the
pyrolysis reaction are frequently identified using a
GC-MS system and information on molecular structure
for identification is also obtained.

104. Food analysis
 Analysis of foods is concerned with the assay of
lipids, proteins, carbohydrates, preservatives,
flavours, colorants and texture modifiers, and also
vitamins, steroids, drugs and pesticide residues and
trace elements. Most of the components are non-
volatile and although HPLC is now used routinely for
much food analysis, GC is still frequently used. For
examples, derivatization of lipids and fatty acid to
their methyl esters(FAMEs), of proteins by acid
hydrolysis followed by esterification (N-propyl
esters) and of carbohydrates by silylation to produce
volatile samples suitable for GC analysis.

105. Food and Cancer
 Chemicals that can cause cancer have a wide variety
of molecular structures and include hydrocarbons,
amines, certain drugs, some metals and even some
substances occurring naturally in plants and molds. In
this way, many nitrosamines have carcinogenic
properties and these are produced in a number of
ways such as cigarette smoke. GC can be used to
identify these nitro-compounds in trace quantities.

106. Drugs
 There are still numerous GC applications involving both
quantitative and qualitative identification of the
active components and possible contaminants,
adulterants or characteristic features which may
indicate the source of the particular sample. Forensic
analysis frequently users GC to characterize drugs of
abuse, in some cases the characteristic
chromatographic fingerprint gives an indication of the
source of manufacture of the sample or worldwide
source of a vegetable material such as cannabis.
 Analytical procedures, chromatographic methods and
retention data are published for over 600 drugs,
poisons and metabolites. These data are extremely
useful for forensic work and in hospital pathology
laboratories to assist the identification of drugs.

107. Metal chelates and inorganic materials
 Although inorganic compounds are generally non-
volatile, GC analysis can be achieved by converting the
metal species into volatile derivatives. Only some
metal hydrides and chlorides have sufficient volatility
for GC.
 Organometallics other than chelates, which can be
analyzed directly, include boranes, silanes, germanes,
organotin and lead compounds.

108. Environmental analysis
 Environmental pollution is an age-old trademark of
man and in recent years as technology has progressed,
populations have increased and standards of living
have improved. So the demands on the environment
have increased, with all the attendant problems for
the ecosystems. Combustion of fossil fuel, disposal of
waste materials and products, treatment of crops
with pesticides and herbicides have all contributed to
the problems. Technological developments have
enabled man to study these problems and realize that
even trace quantities of pollutants can gave
detrimental effects on health and on the stability of
the environment. There is a vast amount of literature
on the use of GC for studying a wide variety of these
problems.

109.  Every year many new substances are synthesized that
differ radically from the natural products that exist
in biosystems. The Environmental Protection Agency is
empowered to control water pollution and the
production, use and disposal of toxic chemicals.
It follows that detailed studies must be made of their
effect on the environment and their method of
movement through the ecosystem. Many of the
compounds are not biodegradable and will thus
progressively pollute the environment. There are a
number of tragic examples of which DDT
(dichlorodiphenyltrichloroethane) and the PCBs
(polychlorinated biphenyls) are well known instances.
The materials of interest are present in environmental
samples at very low concentrations and are often to
be found among a myriad of other compounds from
which they must be separated and identified. It
follows that GC, with its inherent high sensitivity and
high separating power, is one of the more commonly
used techniques in the analysis of environmental
samples.

115. Derivatisation of sample
Treat sample to improve the process of
separation by column or detection by
detector.
They are 2 types
Precolumn Derivatisation
Components are converted to volatile &
thermo stable derivative.
Conditions - Pre column derivatisation
Component ↓ volatile
Compounds are thermo labile

116. Derivatisation
Post column Derivatisation
 Improve response shown by detector
 Components ionization / affinity towards
electrons is increased
Pretreatment of solid support
To overcome tailing
Generally doing separation of non polar
components like esters, ethers…
Techniques: 1. use more polar liquid S.P
2. Increasing amt. of liquid phase
3.Pretreatment of solid support to remove
active sites.

136. Kovat’s retention index
Kovat’s retention index (also known as Kovat’s
index, retention
index; plural retention indices) is a concept used in gas
Chromatography to convert retention times into system-
independent
constants.
The index is named after the Hungarian-born Swiss
chemist Ervin
Kováts who outlined this concept during the 1950s while
performing
research into the composition of the essential oils .
The retention index of a certain chemical compound is
its retention
time normalised to the retention times of adjacently
eluting n-alkanes

137. While retention times vary with the individual
chromatographic system
(e.g. with regards to column length, film thickness,
diameter, carrier gas , Velocity and pressure, and void
time), the derived retention indices are quite
independent of these parameters and allow comparing
values measured by different analytical
laboratories under varying conditions.
Tables of retention indices can help identify
components by comparing experimentally found
retention indices with known values

138. The method takes advantage of the linear relationship between
the values of and the number of carbon atoms in
a molecule. The
value of Kovat’s index is usually represented by I in
mathematical
expressions. Its applicability is restricted to organic compounds.
For
isothermal chromatography, the Kovat’s index is given by the
equation
Where:
Kovat’s retention index,
the number of carbon atoms in the smaller n-
alkane,

139. For temperature programmed chromatography, the Kovat’s
index
is given by the equation
Where:
Kovat’s retention index
the number of carbon atoms in the smaller n-
alkane,
the number of carbon atoms in the larger n-
alkane,
the retention time.

140. PARAMETERS USED IN GC
 Retention time (Rt)
 Retention volume (Vr)
 Separation factor (S)
 Resolution (R)
 Theoretical Plate

141. Parameters used in GC
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
(or) 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 ˣ Flow rate

142. Separation factor (S) :
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 & S 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
R= 2d
W1+W2

143. •Requires only very small samples with little preparation
•Good at separating complex mixtures into components
•Results are rapidly obtained (1 to 100 minutes)
•Very high precision
•Only instrument with the sensitivity to detect volatile
organic mixtures of low concentrations
•Equipment is not very complex (sophisticated oven) •
•Fast analysis,Typically minutes (even sec.) Can be automated
• Small samples (µl or µg needed)
• High resolution Record: N ~ 1.3 x 106
•• Reliable, relatively simple and cheap (~ $20,000)
• Non-destructive and Allows on-line coupling, e.g. to MS
• Sensitive detectors (easy ppm, often ppb)
• Highly accurate quantification (1-5% RSD)
ADVANTAGES OF GAS CHROMATOGRAPHY

144. DISADVANTAGES OF GC
• Limited to volatile samples
– T of column limited to ~ 380 °C
– Need Pvap of analyte ~ 60 torr at that T
– Analytes should have b.p. below 500 °C
• Not suitable for thermally labile samples
• Some samples may require intensive preparation
– Samples must be soluble and not react with the
column
• Requires spectroscopy (usually MS) to confirm
the peak identity

145. ADVANCES IN GC
- MINI GC