Gas Chromatography Gas chromatography is a technique used

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Gas Chromatography
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• 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

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• 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.

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• 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

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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.

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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.

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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

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• Three temperature zones can be identified
• Injector temperature, TI, where TI should allow
  flash vaporization of all sample components.
• Column temperature, Tc, which is adjusted as the
  average boiling points of sample components.
• 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

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• 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

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• A carrier gas should have the following
  properties
• Highly pure (gt 99.9)
• Inert so that no reaction with stationary phase
  or instrumental components can take place,
  especially at high temperatures.
• A higher density (larger viscosity) carrier gas
  is preferred.
• Compatible with the detector since some detectors
  require the use of a specific carrier gas.
• A cheap and available carrier gas is an advantage.

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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.

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• 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.

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• 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.

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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.

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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

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• 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.

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Detection Systems

• Several detectors are available for use in GC.
  Each detector has its own characteristics and
  features as well as drawbacks. Properties of an
  ideal detector include

• High sensitivity
• Minimum drift
• Wide dynamic range
• Operational temperatures up to 400 oC.
• Fast response time
• Same response factor for all solutes
• Good reliability (no fooling)
• Nondestructive
• Responds to all solutes (universal)

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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.

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• Note that gases should always be flowing through
  the detector including just before, and few
  minutes after, the operation of the detector.
  Otherwise, the filament will melt. Also, keep
  away any oxygen since oxygen will oxidize the
  filament and results in its destruction.

• Remember that TCD characteristics include
• Rugged
• Wide dynamic range (105)
• Nondestructive
• Insensitive (10-8 g/s)
• Flow rate sensitive

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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.

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• 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.

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• 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

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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.

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• 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

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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 (lt100-300
  mm diameter), which is coated with stationary
  phase. However, glass tubes are also used for
  large-scale separations.

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• 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.

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Capillary/Open Tubular

• Open tubular or capillary columns are finding
  broad applications. These are mainly of two
  types
• Wall-coated open tubular (WCOT) lt1 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.

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• 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.

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• 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

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• Capillary columns advantages compared to packed
  columns
• higher resolution
• shorter analysis times
• greater sensitivity
• Capillary columns disadvantage compared to packed
  columns
• smaller sample capacity

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Solid Support Materials

• The solid support should ideally have the
  following properties
• Large surface area (at least 1 m2/g)
• Has a good mechanical stability
• Thermally stable
• Inert surface in order to simplify retention
  behavior and prevent solute adsorption
• Has a particle size in the range from 100-400 mm

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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

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Weight of liquid stationary phase 100

• Loading
• 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.

Weight of stationary phase plus solid support
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• 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.

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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

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• The polarity of the stationary phase can be
  changed by derivatization with different
  functional groups such as a phenyl group.
  Bleeding of the column is cured by bonding the
  stationary phase to the column or crosslinking
  the stationary phase.

• Liquid Stationary Phases should have the
  following characteristics
• Low volatility
• High decomposition temperature (thermally
  stable)
• Chemically inert (reversible interactions with
  solvent)
• Chemically attached to support (to prevent
  bleeding)
• Appropriate k' and a for good resolution

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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

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• 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

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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

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• 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.

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• The thickness of the stationary phase affects the
  performance of the column as follows
• Increasing thickness of stationary phase allows
  the separation of larger sample sizes.
• Increasing thickness of stationary phase reduces
  efficiency since HS increases.
• Increasing thickness of stationary phase is
  better for separation of highly volatile
  compounds due to increased retention.

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• Much more efficient separations can be achieved
  with capillary columns, as compared to packed
  columns, due to the following reasons
• Very long capillary columns can be used which
  increases efficiency
• Thinner stationary phase films can be used with
  capillary columns
• No eddy diffusion term (multiple paths effect) is
  observed in capillary columns

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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

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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
•  

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• 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

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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
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• 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)

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• 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

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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.

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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.

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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.

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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.

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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.

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• 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.

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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 gtfour
  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.

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• 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.

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• 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

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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.

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Mass Spectrometry
O
C
H
3
Mass Spectrometer
C
N
C
H
3
C
N
C
H
C
C
N
N
O
H
Typical sample isolated compound (1 nanogram)
194
Mass Spectrum
67
109
Abundance
55
82
42
136
165
94
40
60
80
100
120
140
160
200
180
Mass (amu)
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