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									    Gas Chromatography




1
Gas chromatography is a technique used for
 separation of volatile substances, or substances
 that can be made volatile, from one another in a
 gaseous mixture at high temperatures. A
 sample containing the materials to be separated
 is injected into the gas chromatograph. A mobile
 phase (carrier gas) moves through a column that
 contains a wall coated or granular solid coated
 stationary phase. As the carrier gas flows
 through the column, the components of the
 sample come in contact with the stationary
 phase. The different components of the sample
 have different affinities for the stationary phase,
 which results in differential migration of solutes,
 thus leading to separation
2
Martin and James introduced this separation
 technique in 1952, which is the latest of the
 major chromatograhpic techniques. However,
 by 1965 over 18000 publications in gas
 chromatography (GC) were available in the
 literature. This is because optimized
 instrumentation was feasible. Gas
 chromatography is good only for volatile
 compounds or those, which can be made
 volatile by suitable derivatization methods or
 pyrolysis. Thus, about 20% of chemicals
 available can be analyzed directly by GC.
3
Gas chromatography can be used for both
 qualitative and quantitative analysis.
 Comparison of retention times can be
 used to identify materials in the sample by
 comparing retention times of peaks in a
 sample to retention times for standards.
 The same limitations for qualitative
 analysis discussed in Chapter 26 also
 apply for separations in GC. Quantitative
 analysis is accomplished by measurement
 of either peak height or peak area

4
    Gas - Solid Chromatography
               (GSC)
The stationary phase, in this case, is a solid like
  silica or alumina. 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. The use of
  GSC in practice is considered marginal when
  compared to gas liquid chromatography.
5
    Gas - Liquid Chromatography
                (GLC)
The stationary phase is a 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. The presence of a wide
 variety of stationary phases with
 contrasting selectivities and easy column
 preparation add to the assets of GLC or
 simply GC.
6
           Instrumentation
It may be wise to introduce instrumental
   components before proceeding further in
   theoretical background. This will help
   clarify many points, which may, otherwise,
   seem vague. It should also be noted that a
   detector will require special gas cylinders
   depending on the detector type utilized.
   The column temperature controller is
   simply an oven, the temperature of which
   can be varied or programmed
7
                                        To Waste or Flow
                                            Meter
    Two-Stage
    Regulator                 Syringe               Detector

                             Injector




                   Flow Controller




     Carrier Gas                        Column
      Cylinder


8
Three temperature zones can be identified:
1. Injector temperature, TI, where TI should allow
    flash vaporization of all sample components.
2. Column temperature, Tc, which is adjusted as
    the average boiling points of sample
    components.
3. Detector Temperature, TD, which should
    exclude any possible condensation inside the
    detector.
Generally, an intuitive equation can be used to
    adjust all three zones depending on the
    average boiling point of the sample
    components. This equation is formulated as:
TI = TD = Tc + 50 oC
9
The Carrier Gas
Unlike liquid chromatography where wide
 varieties of mobile phase compositions are
 possible, mobile phases in gas
 chromatography are very limited. Only
 slight changes between carrier gases can
 be identified which places real limitations
 to chromatographic enhancement by
 change or modification of carrier gases

10
A carrier gas should have the following properties:
1. Highly pure (> 99.9%)
2. Inert so that no reaction with stationary phase
   or instrumental components can take place,
   especially at high temperatures.
3. A higher density (larger viscosity) carrier gas is
   preferred.
4. Compatible with the detector since some
   detectors require the use of a specific carrier
   gas.
5. A cheap and available carrier gas is an
   advantage.
11
     Longitudinal Diffusion Term
This is an important factor contributing to band
  broadening which is a function of the diffusivity
  of the solute in the gaseous mobile phase as
  well as the molecular diffusion of the carrier gas
  itself.

HL = K DM /V
Where; DM is the diffusion coefficient of solute in
  the carrier gas. This term can be minimized
  when mobile phases of low diffusion, i.e. high
  density, are used in conjunction with higher flow
  rates.
12
The same van Deemter equation as in LC
 can be written for GC where:
H = A + B/V + CV
The optimum carrier gas velocity is given by
 the derivative of van Deemter equation
Vopt = { B/C }1/2

However, the obtained velocity is much
 greater than that obtained in LC.

13
The carrier gas pressure ranges from 10-50 psi.
  Higher pressures potentially increase
  compression possibility while very low
  pressures result in large band broadening due
  to diffusion. Depending on the column
  dimensions, flow rates from 1-150 mL/min are
  reported. Conventional analytical columns (1/8”)
  usually use flow rates in the range from 20-50
  mL/min while capillary columns use flow rates
  from 1-5 mL/min depending on the dimensions
  and nature of column. In most cases, a
  selection between helium and nitrogen is made
  as these two gases are the most versatile and
  common carrier gases in GC.
14
                  Injectors
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.

15
                             Syringe


     Septum


                                       Carrier
                                        Gas

     Vaporization
      Chamber




                      To
                    Column

16
     Column Configurations and
              Ovens
The column in chromatography is undoubtedly the
  heart of the technique. A column can either be a
  packed or open tubular. Traditionally, packed
  columns were most common but fast
  developments in open tubular techniques and
  reported advantages in terms of efficiency and
  speed may make open tubular columns the best
  choice in the near future. Packed columns are
  relatively short (~2meters) while open tubular
  columns may be as long as 30-100 meters
17
Packed columns are made of stainless steel or
  glass while open tubular columns are usually
  made of fused silica. The temperature of the
  column is adjusted so that it is close to the
  average boiling point of the sample mixture.
  However, temperature programming is used
  very often to achieve better separations. The
  temperature of the column is assumed to be the
  same as the oven which houses the column.
  The oven temperature should be stable and
  easily changed in order to obtain reproducible
  results.
18
         Detection Systems
Several detectors are      1.   High sensitivity
                           2.   Minimum drift
  available for use in
                           3.   Wide dynamic range
  GC. Each detector        4.   Operational temperatures up
  has its own                   to 400 oC.
  characteristics and      5.   Fast response time
  features as well as      6.   Same response factor for all
                                solutes
  drawbacks.               7.   Good reliability (no fooling)
  Properties of an ideal   8.   Nondestructive
  detector include:        9.   Responds to all solutes
                                (universal)



19
a. Thermal Conductivity Detector
             (TCD)
This is a nondestructive detector which is used for the
  separation and collection of solutes to further perform
  some other experiments on each purely separated
  component. The heart of the detector is a heated
  filament which is cooled by helium carrier gas. Any
  solute passes across the filament will not cool it as much
  as helium does because helium has the highest thermal
  conductivity. This results in an increase in the
  temperature of the filament which is related to
  concentration. The detector is simple, nondestructive,
  and universal but is not very sensitive and is flow rate
  sensitive.


20
21
22
Note that gases should     Remember that TCD
 always be flowing            characteristics
 through the detector         include:
 including just before,    1. Rugged
 and few minutes after,    2. Wide dynamic range
 the operation of the         (105)
 detector. Otherwise,
 the filament will melt.   3. Nondestructive
 Also, keep away any       4. Insensitive (10-8 g/s)
 oxygen since oxygen       5. Flow rate sensitive
 will oxidize the
 filament and results in
 its destruction.

23
     b. Flame Ionization Detector
                 (FID)

This is one of the most sensitive and reliable
 destructive detectors. Separate two gas
 cylinders, one for fuel and the other for O2
 or air are used in the ignition of the flame
 of the FID. The fuel is usually hydrogen
 gas. The flow rate of air and hydrogen
 should be carefully adjusted in order to
 successfully ignite the flame.

24
25
26
The FID detector is a mass sensitive
 detector where solutes are ionized in the
 flame and electrons emitted are attracted
 by a positive electrode, where a current is
 obtained.
The FID detector is not responsive to air,
 water, carbon disulfide. This is an
 extremely important advantage where
 volatile solutes present in water matrix can
 be easily analyzed without any
 pretreatment.
27
Remember that FID characteristics include:
• Rugged
• Sensitive (10-13 g/s)
• Wide dynamic range (107)
• Signal depends on number of carbon atoms in
   organic analytes which is referred to as mass
   sensitive rather than concentration sensitive
• Weakly sensitive to carbonyl, amine, alcohol,
   amine groups
• Not sensitive to non-combustibles – H2O, CO2,
   SO2, NOx
• Destructive
28
 Electron Capture Detector (ECD)

This detector exhibits high intensity for halogen containing
  compounds and thus has found wide applications in the
  detection of pesticides and polychlorinated biphenyls.
  The mechanism of sensing relies on the fact that
  electronegative atoms, like halogens, will capture
  electrons from a b emitter (usually 63Ni). In absence of
  halogenated compounds, a high current signal will be
  recorded due to high ionization of the carrier gas, which
  is N2, while in presence of halogenated compounds the
  signal will decrease due to lower ionization.


29
30
Remember the following facts about ECD:
1. Electrons from a b-source ionize the carrier gas
  (nitrogen)
2. Organic molecules containing electronegative
  atoms capture electrons and decrease current
3. Simple and reliable
4. Sensitive (10-15 g/s) to electronegative groups
  (halogens)
5. Largely non-destructive
6. Insensitive to amines, alcohols and
  hydrocarbons
7. Limited dynamic range (102)
8. Mass sensitive detector

31
 Gas Chromatographic Columns
     and Stationary Phases

Packed Columns
These columns are fabricated from glass, stainless
  steel, copper, or other suitable tubes. Stainless
  steel is the most common tubing used with
  internal diameters from 1-4 mm. The column is
  packed with finely divided particles (<100-300
  mm diameter), which is coated with stationary
  phase. However, glass tubes are also used for
  large-scale separations.

32
Several types of tubing were used ranging from
  copper, stainless steel, aluminum and glass.
  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. These find excellent and wide
  use because of easy packing and good routine
  separation characteristics. Column length can
  be from few feet for packed columns to more
  than 100 ft for capillary columns.

33
      Capillary/Open Tubular
Open tubular or capillary 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
These are used for fast and efficient separations but are
   good only for small samples. The most frequently used
   capillary column, nowadays, is the fused silica open
   tubular column (FSOT), which is a WCOT column.



34
The external surface of the fused silica columns is
  coated with a polyimide film to increase their
  strength. The most frequently used internal
  diameters occur in the range from 260-320
  micrometer. However, other larger diameters are
  known where a 530 micrometer fused silica
  open tubular column was recently made and is
  called a megapore column, to distinguish it from
  other capillary columns. Megapore columns
  tolerate a larger sample size.


35
36
37
38
39
It should be noted that since capillary
   columns are not packed with any solid
   support, but rather a very thin film of
   stationary phase which adheres to the
   internal surface of the tubing, the A term in
   the van Deemter equation which stands
   for multiple path effects is zero and the
   equation for capillary columns becomes
H = B/V + CV

40
Capillary columns advantages compared to
   packed columns
1. higher resolution
2. shorter analysis times
3. greater sensitivity

Capillary columns disadvantage compared
   to packed columns
1. smaller sample capacity
41
     Solid Support Materials
The solid support should ideally have the
   following properties:
1. Large surface area (at least 1 m2/g)
2. Has a good mechanical stability
3. Thermally stable
4. Inert surface in order to simplify retention
   behavior and prevent solute adsorption
5. Has a particle size in the range from 100-
   400 mm
42
     Selection of Stationary Phases
General properties of a good liquid stationary
 phase are easy to guess where inertness
 towards solutes is essential. Very low volatility
 liquids that have good absolute and differential
 solubilities for analytes are required for
 successful separations. An additional factor that
 influences the performance of a stationary
 phase is its thermal stability where a stationary
 phase should be thermally stable in order to
 obtain reproducible results. Nonvolatile liquids
 assure minimum bleeding of the stationary
 phase

43
                Weight of liquid stationary phase * 100%
% Loading =
              Weight of stationary phase plus solid support

Increasing percent loading would allow for
  increased sample capacity and cover any
  active sites on the solid support. These
  two advantages are very important,
  however increasing the thickness of
  stationary phase will affect the C term in
  the van Deemter equation by increasing
  HS, and therefore Ht.
44
Generally, the film thickness primarily affects the
 retention character and the sample capacity of a
 column. Thick films are used with highly volatile
 analytes, because such films retain solutes for a
 longer time and thus provide a greater time for
 separation to take place. Thin films are useful for
 separating species of low volatility in a
 reasonable time. On the other hand, a thicker
 film can tolerate a larger sample size. Film
 thicknesses in the range from 0.1 – 5 mm are
 common.

45
     Liquid Stationary Phases
In general, the polarity of the stationary
  phase should match that of the sample
  constituents ("like" dissolves "like"). Most
  stationary phases are based on
  polydimethylsiloxane or polyethylene
  glycol (PEG) backbones:



46
The polarity of the        Liquid Stationary Phases
  stationary phase can        should have the following
                              characteristics:
  be changed by
  derivatization with      • Low volatility
  different functional     • High decomposition
                              temperature (thermally
  groups such as a            stable)
  phenyl group.            • Chemically inert
  Bleeding of the             (reversible interactions
  column is cured by          with solvent)
  bonding the stationary   • Chemically attached to
  phase to the column;        support (to prevent
  or crosslinking the         bleeding)
  stationary phase.        • Appropriate k' and a for
                              good resolution

47
      Bonded and Crosslinked
        Stationary Phases
The purpose of bonding and cross-linking is to
  prevent bleeding and provide a stable stationary
  phase. With use at high temperatures, stationary
  phases that are not chemically bonded or
  crosslinked slowly lose their stationary phase
  due to bleeding in which a small amount of the
  physically immobilized liquid is carried out of the
  column during the elution process. Crosslinking
  is carried out in situ after a column is coated with
  one of the polymers
48
49
In summary, stationary phases are usually
  bonded and/or crosslinked and the following
  remarks are usually helpful:
1. Bonding occurs through covalent linking of
  stationary phase to support
2. Crosslinking occurs through polymerization
  reactions to join individual stationary phase
  molecules
3. Nonpolar stationary phases are best for
  nonpolar analytes where nonpolar analytes are
  retained preferentially
4. Polar stationary phases are best for polar
  analytes where polar analytes are retained
  preferentially
50
     Gas-liquid chromatography
               (GLC)
Packed columns are fabricated from glass, metal,
  or Teflon with 1 to 3 m length and 2 to 4 mm in
  internal diameter. The column is packed with a
  solid support (100-400 mm particle diameter
  made from diatomaceous earth) that has been
  coated with a thin layer (0.1-5 mm) of the
  stationary liquid phase. Efficiency increases with
  decreasing particle size as predicted from van
  Deemter equation. The retention is based on
  absorption of analyte (partition into the liquid
  stationary phase) where solutes must have
  differential solubility in the stationary phase

51
Open tubular capillary columns, either WCOT,
 SCOT are routinely used. In WCOT the capillary
 is coated with a thin film (0.1-0.25 mm) of the
 liquid stationary phase while in SCOT a thin film
 of solid support material is first affixed to the
 inner surface of the column then the support is
 coated with the stationary phase. WCOT
 columns are most widely used. Capillary
 columns are typically made from fused silica
 (FSOT) and are 15 to 100 m long with 0.10 to
 0.5 mm i.d.

52
The thickness of the stationary phase affects the
   performance of the column as follows:

1. Increasing thickness of stationary phase allows
   the separation of larger sample sizes.
2. Increasing thickness of stationary phase
   reduces efficiency since HS increases.
3. Increasing thickness of stationary phase is
   better for separation of highly volatile
   compounds due to increased retention.
                                                 •
53
Much more efficient separations can be
   achieved with capillary columns, as
   compared to packed columns, due to the
   following reasons:
1. Very long capillary columns can be used
   which increases efficiency
2. Thinner stationary phase films can be
   used with capillary columns
3. No eddy diffusion term (multiple paths
   effect) is observed in capillary columns
54
55
     Temperature Programming
Gas chromatographs are usually capable of
 performing what is known as temperature
 programming gas chromatography (TPGC). The
 temperature of the column is changed according
 to a preset temperature isotherm. TPGC is a
 very important procedure, which is used for the
 attainment of excellent looking chromatograms
 in the least time possible. For example, assume
 a chromatogram obtained using isothermal GC
 at 80 oC, as shown below:
56
57
58
59
60
 The General Elution Problem
Look at the chromatogram below in which six
  components are to be separated by an elution
  process using isothermal conditions at for example
  120 oC:




61
It is clear from the figure that the separation is
   optimized for the elution of the first two
   components. However, the last two
   components have very long retention and
   appear as broad peaks. Using isothermal
   conditions at high temperature (say for
   example 200oC) can optimize the elution of
   the last two compounds but, unfortunately,
   results in bad resolution of the earlier eluting
   compounds as shown in the figure below
   where the first two components are coeluted
   while the resolution of the second two
   components becomes too bad:
62
63
One can also optimize the separation of the
middle too components by adjusting the
isothermal conditions (for example at say 160
oC). In this case, a chromatogram like the one

below can be obtained:




64
However, in chromatographic separations we
 are interested in fully separating all
 components in an acceptable resolution.
 Therefore, it is not acceptable to optimize the
 separation for a single component while
 disregarding the others. The solution of this
 problem can be achieved by consecutive
 optimization of individual components as the
 separation proceeds. In this case,
 temperature should be changed during the
 separation process. This is called
 temperature programming gas
 chromatography (TPGC)
65
First, a temperature suitable for the
  separation of the first eluting component is
  selected, and then the temperature is
  increased so that the second component
  is separated and so on. The change in
  temperature can be linear, parabolic, step,
  or any other formula. The chromatographic
  separation where the temperature is
  changed during the elution process is
  called TPGC. A separation like the one
  below can be obtained:
66
67
     Temperature Zones in GC
Three temperature zones should be adjusted
  before a GC separation can be done. The
  injector temperature should be such that fast
  evaporation of all sample components is
  achieved. The temperature of the injector is
  always more than that of the column, which
  depends on the operational mode of the
  separation. The detector temperature should be
  kept at some level so as to prevent any solute
  condensation in the vicinity of the detector body.
68
Gas-solid chromatography (GSC)
Gas-solid chromatography is based upon
  adsorption of gaseous substances on solid
  surfaces. Distribution coefficients are generally
  much larger than those for gas-liquid
  chromatography. Consequently, gas-solid
  chromatography is useful for the separation of
  species that are not retained by gas-liquid
  columns, such as the components of air,
  hydrogen sulfide, carbon disulfide, nitrogen
  oxides, and rare gases. Gas-solid
  chromatography is performed with both packed
  and open tubular columns.

69
          Molecular Sieves
Molecular sieves are metal aluminum silicate ion
 exchangers, whose pore size depends upon the
 kind of cation present, like sodium in sodium
 aluminum silicate molecular sieves. The sieves
 are classified according to the maximum
 diameter of molecules that can enter the pores.
 Commercial molecular sieves come in pore
 sizes of 4, 5, 10, and 13 angstroms. Molecular
 sieves can be used to separate small molecules
 from large ones.
70
         Porous Polymers
Porous polymer beads of uniform size are
 manufactured from styrene crosslinked
 with divinylbenzene. The pore size of
 these beads is uniform and is controlled by
 the amount of crosslinking. Porous
 polymers have found considerable use in
 the separation of gaseous species such as
 hydrogen sulfide, oxides of nitrogen,
 water, carbon dioxide, methanol, etc.
71
      Quantitative Analysis
GC is an excellent quantitative technique
 where peak height or area is proportional
 to analyte concentration. Thus the GC can
 be calibrated with several standards and a
 calibration curve is obtained, then the
 concentration of the unknown analyte can
 be determined using the peak area or
 height. The detector response factor for
 each analyte should be considered for
 accurate quantitative analysis.
72
Gas chromatographs are widely used as criteria
 for establishing the purity of organic compounds.
 Contaminants, if present, are revealed by the
 appearance of additional peaks. Qualitative
 Analysis is usually done by comparison with
 retention times of standards, which are very
 reproducible in GC, provided good injection
 practices are followed. Injection should be done
 with a suitable Hamilton type syringe through the
 heated septum injector till all needle disappears,
 then the needle is drawn back as steadily and
 fast as possible. This is important for
 reproducible attainment of retention times.
73
        The Retention Index
The retention index, RI, was first proposed by
  Kovats in 1958 as a parameter for identifying
  solutes from chromatograms. The retention
  index for any given solute can be derived from a
  chromatogram of a mixture of that solute with at
  least two normal alkanes (chain length >four
  carbons) having retention times that bracket that
  of the solute. That is, normal alkanes are the
  standards upon which the retention index scale
  is based.
74
By definition, the retention index for a normal
  alkane is equal to 100 times the number of
  carbons in the compound regardless of the
  column packing, the temperature, or other
  chromatographic conditions. The retention index
  system has the advantage of being based upon
  readily available reference materials that cover a
  wide boiling range. The retention index of a
  compound is constant for a certain stationary
  phase but can be totally different for other
  stationary phases.

75
In finding the retention index, a plot of the
  number of carbons of standard alkanes
  against the logarithm of the adjusted
  retention time is first constructed. The
  value of the logarithm of the adjusted
  retention time of the unknown is then
  calculated and the retention index is
  obtained from the plot.
The adjusted retention time, tR’, is defined
  as:
tR’ = tR - tM
76
77
     Interfacing GC with other
              Methods
As mentioned previously, chromatographic
  methods (including GC) use retention times as
  markers for qualitative analysis. However, this
  characteristic does not absolutely confirm the
  existence of a specific analyte as many analytes
  may have very similar stationary phases. GC, as
  other chromatographic techniques, can confirm
  the absence of a solute rather than its existence.
  When GC is coupled with structural detection
  methods, it serves as a powerful tool for
  identifying the components of complex mixtures.
  A popular combination is GC/MS.

78
                                   Mass Spectrometry
                     O             C H3

     H3C            C              N
           N             C
                                          C H                                         Mass
           C             C                                                        Spectrometer
       O            N              N
                    H

        Typical sample: isolated
       compound (~1 nanogram)


                                                                                             194
                                                  Mass Spectrum




                                   67                    109




                         55
                                            82

                42




                                                 94                  136      165


               40             60          80      100          120    140   160       180        200
79                                                    Mass (amu)
80

								
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