Chromatography Chromatography Chromatography • Definition Chromatography

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Chromatography Chromatography Chromatography • Definition Chromatography Powered By Docstoc
					Chromatography
            Chromatography

• 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.
                  History
• 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.”
History
                   History
• 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.
                   History
• 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
  chromatography.
• 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.
                   History
• 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
  chromatography.
                  History
• 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
  phase.
    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
     Classifications
• 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
    chromatography).
  – The components distribute between the
    two phases through a combination of
    sorption and desorption processes.
  Chromatographic Technique
       Classifications

• 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
        Classifications
• 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
       Classifications
• 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
      columns
           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
  bonding.
• 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
     column.
       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
     vent).
     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
  Effect)
  – 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
    vaporization.
  – 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-
    resolved.
  – The solution is to raise the column temperature
    during a chromatographic run, so that for a
    homologous series peaks emerge at regular
    intervals.
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
     long
   – 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
  materials.
• Fused silica tubing has recently
  become the preferred type
  because it produces flexible inert
  columns.
• 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
    polyimide.
    • 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
        methylsiloxane
•   50% Trifluoropropylmethyl-     OV210, DB-210, QF-1
        siloxane
•   50% Cyanopropylphenyidi-       BP225, OV225
        methylsilaxane
•   Polyethylene glycol            BP20, DB-WAX, W20M
•   Cyanopropylsilarylene          BPX70
•   Dimethylsiloxane-carborane     HT5
    copolymer
•   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
       versatile.
 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
       groups.
       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)
       columns.
     • 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
    length.
  – 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
    columns.
 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
    mm.
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
• 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.
                          Detectors
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
              Detectors
• Thermal Conductivity Detector
  – Measures the ability of a substance to
    transport heat from a hot region to a cold
    region.
  – Are simple and universal.
  – Are not sensitive enough for capillary
    columns.
                Detectors
• Flame Ionization

     • Universal organic detector

     • Forms ions when compounds are burned
             Detectors
• Mass Selective Detector
  (Mass Spectrometer)
GC/MS
Selective Ion Monitoring