Detector Signal
CHROMATOGRAPHY
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time or volume
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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
GSC
LC
GLC
Column C
Adsorption
chromatography
Surface C
TLC
Partition
chromatography
Ion exchange
chromatography
Chromatography
Column
chromatography
Pair ion
chromatography
Paper
chromatography
Ligand exchange
chromatography
Size Exclusion
chromatography
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Paper
chromatography
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
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•
•
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 countercurrent 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
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 Peak shape
(symmetric
or asymmetric)
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.
 Adjusted time (tR'):
The time which the solute spend
in stationary phase
t’r = tr – tm
t’r : adjusted time
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’
‫شکل‬
R
t0 = tR- t’R
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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
tM : is the retention time of solute µ = tM
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cm /s
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
K= t
=
M
t’R
tM
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Separation factor
(α)
 α is a measure of time interval between two
peaks.
Separation factor calculating by equation below
α =
K1
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
tR , is retention time of solute
N= 16
wb , is the peak width at the
Base, In unit of time.
N= 5.54
wh , is peak width at the half
Of height in unit of time.
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tR
wb
tR
wh
2
2
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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Normal peak
Boarding peak
Co-eluting
peaks
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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
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Monolithic column
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
∆
t
R
retention time between A & B
-1
TZ =
wh(A) and wh(B) are peak widths at
wh(A) + wh(A)
the half height.
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