Chromatography Chromatography Chromatography • Definition Chromatography

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Chromatography Chromatography Chromatography • Definition Chromatography Powered By Docstoc

• Definition: Chromatography is a separation
  method in which a mixture is applied as a
  narrow initial zone to a stationary, porous
  sorbent and the components are caused to
  undergo differential migration by the flow of
  the mobile phase, a liquid or a gas.
• Michael Tswett (1872-1919), in 1906, published a
  paper describing the separation and isolation of
  green and yellow chloroplast pigments by
  column adsorption chromatography and stated
  that “Chromatography is a method in which the
  components of a mixture are separated on an
  adsorbent column in a flowing system”.
• “Chroma” is Greek for “color.”
• “Graphein” is Greek for “to write.”
• 1903-1906 Tswett invented chromatography with
  use of pure solvent to develop the chromatogram;
  devised nomenclature; used mild adsorbents to
  resolve chloroplast pigments.
• 1930-1932 Karrer, Kuhn, and Strain used
  activated lime, alumina and magnesia absorbents.
• 1935 Holmes and Adams synthesized synthetic
  organic ion exchange resins.
• 1938 Reichstein introduced the liquid or flowing
  chromatogram, thus extending application of
  chromatography to colorless substances.
• 1938 Izmailov and Schraiber discussed the use of
  a thin layer of unbound alumina spread on a
  glass plate.
• 1939 Brown had the first use of circular paper
• 1940-1943 Tiselius devised frontal analysis and
  method of displacement development.
• 1941 Martin and Synge introduced column
  partition chromatography.
• 1944 Consden, Gordon, and Martin first
  described paper partition chromatography.
• 1947-1950 Boyd, Tompkins, Spedding, Rieman,
  and others applied ion-exchange
  chromatography to various analytical problems.
• 1948 Lederer and Linstead applied paper
  chromatography to inorganic compounds.
• 1951Kirchner introduced thin-layer
  chromatography as it is practiced today.
• 1952 James and Martin developed gas
• 1956 Sober and Peterson prepared first ion-
  exchange celluloses.
• 1956 Lathe and Ruthvan used natural and
  modified starch molecular sieves for
  molecular weight estimation.
• 1959 Porath and Flodin introduced cross-
  linked dextran for molecular sieving.
• 1964 J. C. Moore developed Gel permeation
  chromatography as a practical method.
       Theoretical Concept:
        Distribution Ratio
• Chromatography is a separation technique
  where component molecules (solutes) in a
  sample mixture are transported by a mobile
  phase over a stationary phase.
• Attraction of the solute for the stationary
  phase results in retardation of its movement
  through the chromatography system.
        Theoretical Concept:
         Distribution Ratio
• Each component or solute is distributed between
  the two phases with an equilibrium established
  defined by the distribution ratio
• Thus for component S
                     [SS] <=> [SM]
  where [SS] is the concentration of S in a unit
  volume of the stationary phase, and [SM] is the
  concentration of S in a unit volume of the mobile
    Theoretical Concept:
     Distribution Ratio
• The distribution ratio, KS (also called
  as partition ratio or partition
  coefficient), for A, is therefore
              KS = [SS] / [SM]
        Theoretical Concept:
         Distribution Ratio
• Each component separated will have a different value
  for K, reflecting their relative affinities for the
  stationary phase; the generalized form of the
  distribution equation for each component is
                        K = CS / C M
Chromatographic Technique
• Adsorption Chromatography:
  – The stationary phase is a solid on which
    the sample components are adsorbed. The
    mobile phase may be a liquid (liquid-solid
    chromatography) or a gas (gas-solid
  – The components distribute between the
    two phases through a combination of
    sorption and desorption processes.
  Chromatographic Technique

• Partition Chromatography
  – The stationary phase of partition
    chromatography is a liquid supported on an
    inert solid.
  – The mobile phase may be a liquid (liquid-liquid
    partition chromatography) or a gas (gas-liquid
    chromatography, GLC).
   Chromatographic Technique
• Ion Exchange and Size Exclusion Chromatography
  – Ion exchange chromatography uses an ion exchange
    resin as the stationary phase. The mechanism of
    separation is based on ion exchange equilibria.
  – In size exclusion chromatography, solvated molecules
    are separated according to their size by their ability to
    penetrate a sieve like structure (the stationary phase).
  Chromatographic Technique
• Affinity Chromatography
  – Affinity chromatography uses highly specific
    interactions between one kind of solute molecule
    and a second molecule covalently attached
    (immobilized) to the stationary phase.
      Gas Chromatography
• History
  –   Metal packed columns
  –   Glass packed columns
  –   Metal capillary columns
  –   Glass capillary columns
  –   Chemically bonded fused-silica capillary
           Gas Chromatography
• Requires the analyte to be thermally stable, reasonably
  volatile, and have a molecular weight of less than ~ 500 amu.
• The mobile phase is an inert gas such as He, H2 or N2.
• The stationary phase is a liquid that is immobilized on the
  surface of a solid support by adsorption or by chemical
• Gas chromatographic separation occurs because of
  differences in the adsorption equilibria between the gaseous
  components of the sample and the stationary phases.
   Gas Chromatography
• Basic components of a GC system
               Sample Injection
• Samples are introduced into
  the injector port via a glass
  syringe with a capacity of 1 –
  10 mL.
• Gas-tight syringes are
  available for injecting gases
  and vapors with Teflon-
  tipped plungers for improved
  sealing of the plunger with
  the syringe barrel against the
  backpressure created by the
  inlet pressure of the injector.
Sample Injection
    Sample Injection Systems
• Features of Injection System
  – Rapid clean switching or injection of the sample into the
    mobile phase with no tailing or dispersion of sample
  – Correct inlet temperatures high enough to vaporize
    instantaneously all components in a sample without
    decomposition and condensation
  – Minimum dead volumes to avoid diffusion of the sample
    in the mobile phase
  – Design of the overall inlet system for good precision
    (better than 1%)
  – No contamination of samples or catalytic effects
  – No loss of retention of sample in the inlet system
  – No septum bleed or leak
      Sample Injection Systems

• Split Injectors
   – Split Injectors are used
     for more concentrated
     samples since only a
     small fraction of the
     injected sample is
     introduced into the
       Sample Injection Systems

• Split Injectors
   – Split Vent: A small
     volume of the carrier
     gas flows into the
     column (1-4 ml/min)
     while a much higher
     volume (10-100
     ml/min) flows out of
     the split line (split
     Sample Injection Systems
• Split Injectors
   – Split Ratio: The split ratio is the ratio of the carrier
     gas flow in the column and out of the split vent.
      • Typical split ratios range from 1:100 to 1:1000.
      • Lower split ratios introduce more sample into the column.
      • Using a 1:50 split ratio introduces approximately 1/50 (or 2%)
        of the sample into the column while a 1:100 split ratio
        introduces about 1/100 (or 1%) of the sample into the column.
     Sample Injection Systems
• Splitless Injectors
   – Splitless injection is
     suitable for trace level
     determinations in trace
     analysis where the
     analytes may be in ppm
     (µg/ml) concentration.
      Sample Injection Systems
• Splitless Injectors
   – Upon sample
     vaporization, the vapors
     are mixed with the
     carrier gas.
   – At 15-60 seconds after
     the injection, the injector
     automatically enters the
     "purge on" mode.
      Sample Injection Systems
• Cold Trapping (Solvent
  – One requirement of
    splitless injections is that
    the initial column
    temperature be at least
    10oC below the boiling
    point of the sample solvent.
  – Since the column
    temperature is below the
    solvent boiling point, the
    sample solvent condenses at
    the front of the column.
      Sample Injection Systems
• On-column Injector
  – On-column injectors
    deposit the sample directly
    into the column without
    utilizing sample
  – The biggest drawback to
    on-column injections is
    column contamination.
              GC Column Ovens
• Column temperature is an important variable that
  must be controlled to a few tenths of a degrees for
  precise work.
• The column is ordinarily housed in a
  thermostatically controlled oven.
• Desirable characteristics of the chromatograph
  oven are:
   – Rapid temperature response to follow accurately the
     temperature program profile
   – Low thermal mass for fast cool-down at the conclusion
     of the analysis.
            GC Column Ovens
• Oven temperature programming
  – An isothermal GC run does not yield a
    satisfactorily separated mixture of analytes.
  – If the column temperature is high enough to give
    satisfactory peaks for the less volatile compounds,
    the low-boiling constituents will be less well-
  – The solution is to raise the column temperature
    during a chromatographic run, so that for a
    homologous series peaks emerge at regular
GC Column Ovens
  Chromatographic Columns
• Packed Columns: Stationary phase is a
  liquid that is coated onto a solid support.
   – The column may be made of glass or metal and
     typically 2 - 6 mm i.d. and 1 - 3 m in length.
   – Advantages: more concentrated samples may be
     analyzed; more solvent may be injected; can be
     used in preparative applications.
   – Disadvantage: analyte separation is less
     favorable; unsuitable for trace analysis.
     Chromatographic Columns
• Capillary Columns: stationary phase is a liquid
  that is bond to the inner surface of the column.
   – The column may be 0.2 - 0.7 mm i.d. and 10 - 100 m
   – Advantages: high resolution chromatography;
     multitude of liquid phases; inert surface upon which to
     coat the liquid phase.
   – Disadvantages: small sample size; long
     chromatographic runs; “quirky” behavior depending
     upon chromatographic conditions.
               Capillary Columns
• Fused silica, glass and stainless
  steel are the primary tubing
• Fused silica tubing has recently
  become the preferred type
  because it produces flexible inert
• Metal columns are generally
  avoided since catalyzed reactions
  with the analytes may occur.
• Capillary columns are
  constructed of three parts - fused
  silica tubing, polyimide coating
  and stationary phase
        Glass Capillary Columns
• Wall coated open tubular
  (WCOT) capillary columns
  are the most commonly used
  GC columns.
• Current column technology
  uses the surface properties of
  pure silica tubing to
  immobilize the stationary
  phase producing extremely
  stable inert columns.
  Glass Capillary Columns
• Fused Silica Tubing
   – The fused silica used to manufacture capillary
     columns is synthetic quartz typically containing
     less than 1 ppm metallic impurities.
• Silylation Process
   – Silanol groups (Si-OH) on the tubing surface are
     reacted with a silane type of reagent. Typically, a
     methyl or phenyl-methyl silyl surface is created
     for most columns
     Glass Capillary Columns
• Polyimide Coating
  – Immediately after the drawing process, the
    outer surface of the tubing is coated with
    • This fills any flaws in the tubing.
    • It also provides a strong, waterproof barrier.
       Glass Capillary Columns
• Stationary Phases
  – The suitability of a stationary phase for a particular
    application depends on the selectivity and the degree
    to which polar compounds are retarded relative to
    what their retardation would be on a completely
    non-polar stationary phase.
  – A method to select the appropriate stationary phase
    for analysis of a sample mixture is to consider the
    polar characteristics of the analytes and select a
    stationary phase of similar polarity.
            Glass Capillary Columns
                 Liquid Phases
•   Cross-linked stationary phase Commercial equivalent
•   Dimethylsiloxane               BP1, DB1, HP1, SE30,OV1, CPSil
•   55% Diphenyidimethylsiloxane BP5, DB5, HP2, SE54
•   14% Cyanopropylphenyidi-       BP10, DB1701, OV1701
•   50% Trifluoropropylmethyl-     OV210, DB-210, QF-1
•   50% Cyanopropylphenyidi-       BP225, OV225
•   Polyethylene glycol            BP20, DB-WAX, W20M
•   Cyanopropylsilarylene          BPX70
•   Dimethylsiloxane-carborane     HT5
•   Dimethylsilarylene             BPX5
 Glass Capillary Columns
• Stationary Phases
  – Polysiloxanes
     • Polysiloxanes are the most common stationary
       phases. They are available in the greatest
       variety and are the most stable, robust and
 Glass Capillary Columns
• Stationary Phases
  – Polysiloxanes
     • The most basic polysiloxane is the 100 %
       methyl substituted such as DB-1 or HP-1.
     • When other groups are present, the amount is
       indicated as the percent of the total number of
       Glass Capillary Columns
• Stationary Phases
  – Polysiloxanes
     • A low-bleed phase is available which incorporates
       phenyl or phenyl type groups into the backbone of the
       siloxane polymer.

     • The phenyl group strengthens and stiffens the polymer
       backbone which inhibits stationary phase degradation
       at higher temperatures.
       Glass Capillary Columns
• Stationary Phases
  – Polyethylene Glycols
     • Stationary phases with “wax" or “FFAP" in their name are
       some type of polyethylene glycol.
     • They are less stable, less robust and have lower temperature
       limits than most polysiloxanes.
     • With typical use, they exhibit shorter lifetimes and are more
       susceptible to damage upon over heating or exposure to oxygen.
     Glass Capillary Columns
• Stationary Phases
  – Gas-solid Stationary Phase
     • Gas-solid stationary phases are comprised of a thin
       layer (usually <10 mm) of small particles adhered
       to the surface of the tubing.
     • They are called porous layer open tubular (PLOT)
     • Various derivatives of styrene, aluminum oxides
       and molecular sieves are the most common PLOT
       column stationary phases.
       Glass Capillary Columns
• Stationary Phases
  – Gas-solid Stationary Phase
     • PLOT columns are very retentive.
     • Hydrocarbon and sulfur gases, noble and permanent
       gases, and low boiling point solvents are some of the more
       common compounds separated with PLOT columns.
         Chromatographic Columns
Cross-section of GC columns:
   –   (a) 1/8 in. packed column
   –   (b) thin film WCOT column
   –   (c) thick film WCOT column
   –   (d) 1/16 in. micropacked column
   –   (e) PLOT column
   –   (f) SCOT column)
  Capillary Column Dimensions
• Column Length
  – Resolution is a function of the square root of column
  – Shorter column lengths are intended for samples
    containing a relatively small number of compounds
    especially if they are not very similar in structure,
    polarity or volatility.
  – Most analyses are performed with intermediate
    column lengths (20 - 30 meters).
  – Increased retention will be obtained with longer
 Capillary Column Dimensions
• Column Diameter
  – The internal diameter will have a direct impact upon the
    efficiency, retention characteristics and sample capacity of
    a column.
  – Smaller diameter columns are more efficient than larger
    diameter columns.
  – As column diameter decreases, the retention of a given
    solute will increase providing no other changes to the
    chromatographic system have been made.
  – Larger diameter columns have greater sample capacities.
  – Typical column diameters range from 0.18 mm to 0.53
Column Diameter

• Effect of column diameter on retention
  A: DB-5, 30 m x 0.25 mm I.D., 0.25 µm
  B: DB-5, 30 m x 0.32 mm I.D., 0.25 µm
     Capillary Column Dimensions
• Film Thickness
  – Increasing film thickness will cause
    a substantial increase in the
    retention of a solute.
  – Thin film columns are useful for
    the analysis of low volatility or high
    boiling samples.
  – Film thickness runs from 0.10 mm
    to 5.00 mm.
                                      Effect of film thickness on retention
                                       A: DB-5, 30 m x 0.32 mm I.D., 0.25 µm
                                       B: DB-5, 30 m x 0.32 mm I.D., 1.0 µm
 Properties of the Capillary Column
• Bonded and Cross-linked Stationary Phases
  – Cross-linked stationary phases have the individual
    polymer chains linked via covalent bonds.
  – Bonded stationary phases are covalently bonded to the
    surface of the tubing.
  – Columns with bonded and cross-linked stationary
    phases can be solvent rinsed to remove contaminants.
  – Most polysiloxanes and polyethylene glycol stationary
    phases are bonded and cross-linked.
          Properties of the Capillary
• Column Bleed
  – Column bleed is the continuous elution of the compounds produced
    from normal degradation of the stationary phase and increases with
    higher temperatures.
  – On average, polar stationary phases have higher column bleed, and
    significant bleed occurs at lower temperatures.
  – Column bleed increases as a column is used. Exposing the column to
    oxygen (air) and/or consistently using the column at or near its
    upper temperature limit for prolonged periods accelerates the onset
    of higher column bleed.
Detector               Detection Limit         Linear Range
Thermal conductivity   400 pg/mL               >105
Flame ionization       2 pg/s                  >107
Electron capture       5 fg/s                  104
Flame photometric      < 1 pg/s (phosphorus)   >104
Nitrogen-phosphorus    100 fg/s                105
FTIR                   200 pg to 40 ng         104
Mass spectrometric     25 fg to 100 pg         105
• Thermal Conductivity Detector
  – Measures the ability of a substance to
    transport heat from a hot region to a cold
  – Are simple and universal.
  – Are not sensitive enough for capillary
• Flame Ionization

     • Universal organic detector

     • Forms ions when compounds are burned
• Mass Selective Detector
  (Mass Spectrometer)
Selective Ion Monitoring