An Introduction to Chromatography

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An Introduction to Chromatography Powered By Docstoc
					CHROMATOGRAPHY


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                Definition
   Chromatography is a separation process that is
achieved by distributing the components of a mixture
between two phases, a stationary phase and a mobile
                       phase.




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             Chromatography
• Historically , the word of chromatography was
  used by Tswett in 1906
• He described the separation of plant pigments by
  percolating a petroleum-ether extract through a
  glass column packed with powdered calcium
  carbonate .
• Colored zones were produced by the various
  pigments migrating through the column at
  difference rates
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• The technique, as described by Tswett was largely
  ignored for a along time and it was not until the
  late 1930s and early 1940s that Martin and
  Synge(2) introduced liquid-liquid chromatography
  by supporting the stationary phase, in this case
  water, on silica in a packed bed and used it to
  separate some acetyl amino acids. In their paper,
  they recommended replacing the liquid mobile
  phase by a suitable gas, as the transfer of sample
  between the two phases would be faster, and thus
  provide more efficient separations. In this manner,
  the concept of gas chromatography was created


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       Chromatography today :
• Today, chromatography is an extremely versatile
   technique; it can separate gases, and volatile
   substances by GC, involatile chemicals and
   materials of extremely high molecular weight
   (including biopolymers) by LC and if necessary
   very inexpensively by TLC. All three techniques,
  (GC), (LC) and TLC have common features that
   classify them as chromatography systems.

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                                 Classification
                            Chromatography


            GC                                               LC


   GSC             GLC              Column C                             Surface C

                                             Adsorption                             Paper
                                           chromatography
                                                                   TLC          chromatography

                                              Partition
                                           chromatography

                                             Ion exchange
                                           chromatography
         Chromatography
                                               Pair ion
                                           chromatography

    Column             Paper               Ligand exchange
chromatography     chromatography          chromatography

                                            Size Exclusion
                                           chromatography
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        The basic principle :
• Variation in the rate :
   - Which different components of a mixture
  migrate through a stationary phase under
  the influence of a mobile phase
• Rates of migration vary :
  - Because of differences in distribution
  ratios .

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 Mechanism of chromatography
• During a chromatographic separation solute
  molecules are continually moving back and
  forth between the stationary and mobile
  phases .
• The rate of migration of each solute is
  therefore determined by the proportion of
  time in spends in the mobile phase , or in
  other words by its distribution ratio .
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                   Sorption :
• The process whereby a solute is transferred
  from a mobile phase to a stationary phase is
  called sorption .




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        Mechanism of sorption
•   Surface adsorption
•   Partition
•   Ion exchange
•   Exclusion




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         Surface adsorption
• The original method employed by Tswett
  involved surface adsorption where the
  relative polarities of solute and solid
  stationary phase determine the rate of
  movement of that solute through a column
  or across a surface .



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        Adsorption systems :
• Separations in which surface adsorption is
  the predominant sorption process depend
  upon polarity differences between solute
  molecules .
• The approximate order of increasing
  strength of adsorption is :
Paraffines<olefines<ethers<esters<ketones<al
  dehydes<amines<alcohol<acids

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          Adsorption systems :
• During the separation process there is comptetion
  for adsorption sites between solute molecules and
  those of the mobile phase
• Solute and solvent molecules are continually
  being adsorbed and desorbed as the mobile phase
  travels through the system.
• Solute of low polarity spend more time in the
  mobile phase than those that are highly polarity .
• Consequently the components of a mixture are
  eluted in order of increasing polarity .

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                     Partition
• If a liquid is coated on the surface of an
  inert solid support the sorption process is
  one of partition and movement of the solute
  is determined solely by its relative
  solubility in the two phases or by its
  volatility if the mobile phase is a gas .



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            Partition systems :
• In a partition system the stationary phase is a
   liquid coated on to a solid support .
  ( silicagel , cellulose )
• Conditions closely resemble those of counter-
   current distribution so that in the absence of
   adsorption by the solid support .
• Solutes move through the system at rates
   determined by their relative solubilities in
   stationary and mobile phases .

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  General aspect of chromatography
Liquid chromatography                      Gas chromatography




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              Stationary phase :
• The stationary phase is the part of the chromatographic
  system though which the mobile phase flows where
  distribution of the solutes between the phases occurs. The
  stationary phase may be a solid or a liquid that is
  immobilized or adsorbed on a solid. In general
  immobilization by reaction of a liquid with a solid is used
  in liquid chromatography and absorbtion of a liquid on a
  solid is used in gas chromatography but there are many
  exceptions to both of these generalizations. The stationary
  phase may consist of particles (porous or solid), the walls
  of a tube (eg. capillary) or a fibrous material (eg paper).


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           Stationary phases :
• Almost and polar solid
  can be used
  - Silicagel
  - Alumina




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   Activity of stationary phase :
• Activity is determined by the overall
  polarity and by the number of adsorption
  sites :
• In silicagel the adsorption sites are the
  oxygen atoms and silanol groups ( -S-OH )
  which readily from hydrogen bonds with
  polar molecules .
• Removing water by oven drying

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             mobile phase

 In GC, the mobile is an inert Gas (argon,
 helium, nitrogen, hydrogen)
 In LC, the mobile phase is a liquid (solvent or
 binary solvent mixture such as water, methanol,
 ethanol, acetonitryl etc).
 In Chiral chromatography, the mobile phase is
 gas or liquid.



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             Mobile phase :
• The eluting power of a solvent is
  determined by overall polarity , the polarity
  of the stationary phase and the nature of the
  sample components .




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 chromatography phase normal:
   stationary phase is polar (silica grafting with NH2,
   CN, diol……..)
   mobile phase is a little polar or non polar (hexane,
   dichloromethane, )
   solute : little polar or middle polar
 reverse phase chromatography:
   stationary phase is a polar (silica grafting of alkyl, C8,
   C18 ……)
   mobile phase is polar (water, methanol, acetonitrile,
   ……….)
   solute: a polar or middle polar
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 Chromatographic development :
• A solute progresses through the
  chromatographic system, albeit through
   a column or along a plate, only while it is in
  the mobile phase. This process, whereby the
  substances are moved through the
  chromatographic system, is called
  chromatographic development.

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 Chromatographic development :
• There are three types of chromatographic
  development :
1 - Elution development
2 - Displacement development
3 - Frontal analysis



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  Evaluating column performance
 Separation occurs mainly in column, so the
 accurate resolution is basis on column efficiency
 (the narrowness of peak), peak shape (whether it is
 tails or front) and the column ability to separate
 compounds.
 factors involve in specimen separation:
    Retention time
    Death time or hold up time and average linear velocity
    Retention factor
    Separation factor
    Number of theoretical plate
    Separation (Trennzahl) number
    Peak shape (symmetric or asymmetric)
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                 Retention time (tR)
 Retention time (tR): During sample molecule pass through the
 column, the solute spend a part of time in mobile phase and a
 part of time in stationary phase. This time is called retention
 time.
                  The time which the solute spend
 Adjusted time (tR'):
  in stationary phase

    t’r : adjusted time                     t’r = tr – tm
    t r : retention time
    tm : hold up time or dead
       time
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        Dead time or hold up time
                (tM / t0)
tM represent the time that the un retarded substance
  (mobile phase) spend in the column.
 calculation equation


        t’R’                                     t0 = tR- t’R
               R
        ‫شکل‬



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          Average linear velocity
                    (µ)
 µ is the average speed of mobile phase
  (Gas or liquid), through the column. µ is
  expressed by cm/sec or mL/min.
 Calculating equation:
µ : is linear velocity of mobile phase
L : is the column length                 L
                                                          cm /s
tM : is the retention time of solute µ = tM
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            Retention factor
                  (K)
 K is the ratio of the amount of time that a
 solute spend in stationary and mobile phase.
 K is calculating by equation below:

                    tR - tM                         t’R
                  K= t      =                       tM
                        M




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            Separation factor
                  (α)
 α is a measure of time interval between two
  peaks.
Separation factor calculating by equation below

                      K1
         α =
                      K2


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                     Theoretical plate
                 or column efficiency (N)



 The plate theory needs to assume that the solute, during its
 passage through the column, is always in equilibrium with the
 mobile and stationary phases. But the equilibrium between the
 solute and phases never actually occurs. So to obtain this
 equilibrum, the column must divided in number of cell or plat.
 Every plat has a specific size and solute spend limite time in each
 plat.
  so in the existing of small plats, the solute will spend little time in
 each plat and it will elute fast.
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          Equations of theoretical plat
                  calculation
N, is the theoretical plat
                                                                      2
tR , is retention time of solute
                                                                 tR
                                                    N= 16
                                                                 wb
wb , is the peak width at the
Base, In unit of time.                                                2
                                                                 tR
                                                   N= 5.54       wh
wh , is peak width at the half
Of height in unit of time.


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Column Resolution :




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Mechanism of separation




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             Dispersion interaction
 It is the main interaction for all PSX and PEG stationary phase.
 More volatile compound (low boiling point), elute first.
 Effective for the solute with 30 difference in boiling point.




                                                               Clark’s p 428
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             Dipole interaction
 dipole interaction of PEG and cyanopropyl, trifluoropropyl
 substituted PSXs enable these phase to separate solute
 molecule, which has different dipole site.
 this interaction is used for pesticide, halocarbons and
 drugs.




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                    Hydrogen bond
 functional group that show strong hydrogen bond with
 stationary phase,such as alcohols, carboxylic acid, amines ,
 aldehydes, esters and ketones, are less effective to separate.
 hydrocarbons, halocarbons and ethers has produce weak
 hydrogen bonds.




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Mechanism of separation in
  liquid chromatography




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     Mechanism of separation in
     adsorption chromatography

 in sorption chromatography the stationary
 phase is solid and mobile phase is liquid
 and the analyte is adsorbed by stationary
 phase .




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  Mechanism of separation in ion-exchange
            chromatography

       Sol+/-M + X+/-S                     Sol+/-S + X+/-M

 in cation exchange chromatography
   Sol+M + X-S          Sol+S + X-M
   SO -3 for strong acids and CO -2 for weak acid
 in Ion exchange chromatography
   Sol-M + X+S         Sol-S + X+M
   NR+3 for strong bases and NHR+2 for weak bases

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Mechanism of separation in affinity
        chromatography




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         Mechanism of separation in size-exclusion
                    chromatography
 are too severe, the incoming resin can be returned to the supplier as unaccep
Size Separation Mechanism




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Mechanism of separation in partition
        chromatography




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     Mechanism of separation in Gas
           chromatography
separation performing by adsorption and disorption
  mechanism.
 separation according to difference between the
  boiling point of substance
 separation according to dispersion of analyte in
  Liquid and Gas phase
 separation of polar substance by using polar
  stationary phase and vice versa (packed column)
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             Peak shape & asymmetry

 Symmetric peak (needle shape peak), show the efficacy
 of column and enough theoretical plats.
 tailing peak, show the affinity of solute to the stationary
 phase.
 front peak (shark’s fin peak), show the column overload




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 polysiloxanes




 polyethylene glycol




 cyclodextrin
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Different form of columns

         Packed column




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                                           Co-eluting
Normal peak   Boarding peak                  peaks             Tailing peak




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• partition chromatography
• Chromatography in which separation is based
  mainly on differences between
  the solubility of the sample components in the
  stationary phase (gas
  chromatography), or on differences between the
  solubilities of the components
  in the mobile and stationary phases (liquid
  chromatography)

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                   Detectors
 Mass or MS-MS spectrometer
 Flam ionization detector (FI D)
 Electron capture detector (ECD)
 Nitrogen Phosphor detector (NPD)
 Atomic emission detector (AE D)
 Infra-red detector (IR)


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Liquid chromatography column
                         Silica column             Monolithic column




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Gas chromatography column




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    Separation (Trennzahl) number
                 (TZ)
 TZ, is the measurement of how many resolved peaks
   can be accommodate between adjusted number of
   homologous series.
 Calculating equation:
TZ: separation number
∆ tR : the difference in
 retention time between A & B               ∆ tR
                                   TZ =             -1
wh(A) and wh(B) are peak widths at      wh(A) + wh(A)
the half height.

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