Chromatography

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Chromatography
Dr.A.DINESH KARTHIK HEAD, P G RESEARCH DEPT. OF
CHEMISTRY SHANMUGA INDUSTRIES ARTS SCIENCE
COLLEGE, TIRUVANNAMALAI-606603. dineshkarthik2008
_at_gmail.com.
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Chromatography

• Is a technique used to separate and identify the
  components of a mixture.
• Works by allowing the molecules present in the
  mixture to distribute themselves between a
  stationary and a mobile medium.
• Molecules that spend most of their time in the
  mobile phase are carried along faster.

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Introduction Invention of Chromatography Chromatog
raphy Paper Chromatography Thin Layer
Chromatography (TLC) Liquid Chromatography
(LC) High Pressure Liquid Chromatography
(HPLC) Ion Chromatography Gas Chromatography
(GC) Applications of chromatography Conclusion
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Invention of Chromatography
Mikhail Tswett invented chromatography in 1901
during his research on plant pigments. He used
the technique to separate various plant pigments
such as chlorophylls, xanthophylls and
carotenoids.
Mikhail Tswett Russian Botanist (1872-1919)
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Chromatography (Greek chroma color and
graphein writing ) Tswett named this new
technique chromatography based on the fact that
it separated the components of a solution by
color.
Common Types of Chromatography
Tswetts technique is based on Liquid
Chromatography. There are now several common
chromatographic methods. These include Paper
Chromatography Thin Layer Chromatography
(TLC) Liquid Chromatography (LC) High Pressure
Liquid Chromatography (HPLC) Ion
Chromatography Gas Chromatography (GC)
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Thin Layer Chromatography
Here the mobile phase is a liquid
Flowing past a thin layer of powder on a solid
support.
Substances that are less attracted to the solid
or are more soluble in the liquid move faster.
And so move further up the plate by the time that
the process has been stopped by taking the plate
out of the liqiud. - larger Rf
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THIN LAYER CHROMATOGRAPHY
Stationary Phase ---------gt Silica
Gel Mobile Phase -------------gt Solvent
(developing)
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THIN LAYER CHROMATOGRAPHY
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Paper and Thin Layer Chromatography
The solvent moves up paper by capillary
action, carrying mixture components at different
rates.
solvent front
Later
solvent
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Rf distance moved by substance distance
moved by solvent front
For substances that are very soluble in the
liquid Rf will be close to ....
1
For substances that are rather insoluble in the
liquid Rf will be close to ....
0
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Comparing Chromatography to the Flow of a
River...
Light leaf
Water flow
Heavy stone
Base
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How Does Chromatography Work?
In all chromatographic separations, the sample is
transported in a mobile phase. The mobile phase
can be a gas, a liquid, or a supercritical
fluid. The mobile phase is then forced through a
stationary phase held in a column or on a solid
surface. The stationary phase needs to be
something that does not react with the mobile
phase or the sample. The sample then has the
opportunity to interact with the stationary phase
as it moves past it. Samples that interact
greatly, then appear to move more slowly.
Samples that interact weakly, then appear to move
more quickly. Because of this difference in
rates, the samples can then be separated into
their components.
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Mobile Phase / Stationary Phase

• A site in which a moving phase (mobile phase) and
  a non-moving phase (stationary phase) make
  contact via an interface that is set up.
• The affinity with the mobile phase and stationary
  phase varies with the solute. ? Separation occurs
  due to differences in the speed of motion.

Mobile phase
Weak
Strong
Stationary phase
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Gas Liquid Chromatography
Here the mobile phase is an unreactive gas ( eg
Nitrogen) flowing through a tube.
And the stationary phase is an involatile liquid
held on particles of a solid support.
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Column Chromatography and Planar Chromatography
Separation column
Paper or a substrate coated with particles
Packing material
Paper Chromatography Thin Layer Chromatography
(TLC)
Column Chromatography
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Separation Process and Chromatogram for Column
Chromatography

Output concentration
Chromatogram
Time
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Chromatogram
tR
tR Retention time
Peak
Intensity of detector signal
t0
t0 Non-retention time
h
A Peak area
A
h Peak height
Time
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Ion Exchange Chromatography
R
N
R
Anion exchange
R




SO3-
Cation exchange






Electrostatic interaction (Coulomb force)
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Stationary Phase Used in Ion Exchange Mode

• Base Material
• Resin is often used.
• Silica gel is also used.
• Cation Exchange Column
• Strong cation exchange (SCX) -SO3-
• Week cation exchange (WCX) -COO-
• Anion Exchange Column
• Strong anion exchange (SAX) -NR3
• Week anion exchange (WAX) -NHR2

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Dependence of Exchange Capacity of Ion Exchanger
on pH of Eluent
Strongly acidic cation exchanger
Strongly basic anion exchanger
Exchange capacity
Exchange capacity
Weakly acidic cation exchanger
Weakly basic anion exchanger
0
7
14
0
7
14
pH
pH
Cation exchange mode
Anion exchange mode
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Ion Exclusion Chromatography
H
H
H
Depending on the level of dissociation, some weak
acid ions can enter the pore.
Strong acid ions are repelled by charge and
cannot enter the pore.
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Relationship between Retention Time and Salt
Concentration of Eluent in Ion Exchange Mode
Resin
Resin
Resin
The exchange groups are in equilibrium with
anions in the eluent.
An eluent ion is driven awayand a solute ion is
adsorbed.
The solute ion is driven away by an eluent ion
and is adsorbed by the next exchange group.
Solute ions and eluent ions compete for ion
exchange groups.
If the salt concentration of the eluent
increases, the solutes are eluted sooner.
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• Gas Chromatography (GC)
•  
• Gas chromatography is a chromatographic technique
  that can be used to separate volatile organic
  compounds.
• It consists of
• a flowing mobile phase
• an injection port
• a separation column (the stationary phase)
• an oven
• a detector.

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In the animation below the red molecules are more
soluble in the liquid (or less volatile) than are
the green molecules.
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Principle
The organic compounds are separated due to
differences in their partitioning behavior
between the mobile gas phase and the stationary
phase in the column.
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• Mobile phases are generally inert gases such as
  helium, argon, or nitrogen.
• The injection port consists of a rubber septum
  through which a syringe needle is inserted to
  inject the sample.
• The injection port is maintained at a higher
  temperature than the boiling point of the least
  volatile component in the sample mixture.

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• Since the partitioning behavior is dependent on
  temperature, the separation column is usually
  contained in a thermostat-controlled oven.
• Separating components with a wide range of
  boiling points is accomplished by starting at a
  low oven temperature and increasing the
  temperature over time to elute the high-boiling
  point components.
• The more volatile (Low Boiling Point / Higher
  Vapor Pressure) compounds arrive at the end of
  the column first and pass into the detector

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Components of a Gas Chromatograph
Gas Supply (usually N2 or He) Sample Injector
(syringe / septum) Column 1/8 or 1/4 x 6-50
tubing packed with small uniform size, inert
support coated with thin film of nonvolatile
liquid Detector TC - thermal conductivity FID
- flame ionization detector
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Injection port
Recorder
Oven
Detector
Column
Nitrogen cylinder
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GC Theory
Analyte
To detector
Column packing
Time
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Separations
To detector
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GC Columns
Packed columns
Capillary columns

• Thin fused-silica.
• Typically 10-100 m in length and 250 mm inner
  diameter.
• St. ph. coated on the inner surface.
• Provide much higher separation eff.
• But more easily overloaded by too much sample.

• Typically a glass or stainless steel coil.
• 1-5 total length and 5 mm inner diameter.
• Filled with the st. ph. or a packing coated with
  the st.ph.

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Filling the Column
1. Add about 1 tsp of dry/sifted Tide to
fill pipet within 1/4 of top
(Tide has been sifted and dried, so keep lid
closed on container.)
2. Tap column with a pencil to settle the
powder.
3. Use a plug of fiberfill to hold Tide in
place
Do not compact Tide into column. Do not leave
any dead space at head of column.
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A gas chromatography oven, open to show a
capillary column
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GC Detectors After the components of a mixture
are separated using gas chromatography, they must
be detected as they exit the GC
column. Thermal-conduc. (TCD) and flame
ionization (FID) detectors are the two most
common detectors on commercial GCs.

• The others are
• Atomic-emmision detector (AED)
• Chemiluminescence detector
• Electron-capture detector (ECD)
• Flame-photometric detector (FPD)
• Mass spectrometer (MS)
• Photoionization detector (PID)

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GC Detectors Contd The requirements of a GC
detector depend on the separation
application. E.g. An analysis may require a
detector selective for chlorine containing
molecules. Another analysis might require a
detector that is non- destructive so that the
analyte can be recovered for further
spectroscopic analysis. You can not use FID in
that case because it destroys the sample totally.
TCD on the other hand is non-destructive.
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TCD Detector A TCD detector consists of an
electrically-heated wire.The temperature of the
sensing element depends on the thermal
conductivity of the gas flowing around it.
Changes in thermal conductivity, such as when
organic molecules displace some of the carrier
gas, cause a temperature rise in the element
which is sensed as a change in resistance. The
TCD is not as sensitive as other detectors but it
is non-specific and non-destructive.
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ECD Detector Uses a radiactive Beta emitter
(electrons) to ionize some of the carrier gas and
produces a current between a biased pair of
electrodes. When an org. mol. that contains
electornegative functional gr., such as halojens,
phosphorous and nitro groups, pass by the
detector, they capture some of the electrons and
reduce the current.
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• FID Detector
• Consists of a hydrogen/air flame and a collector
  plate.
• The eff. from the GC column passes through the
  flame, shich breaks down org. mol. and produces
  ions.
• The ions are collected on a biased electrode and
  produce an elec. sig.
• Extremely sensitive, large dynamic range.

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• MS Detector
• Uses the difference in mass-to-charge ratio (m/e)
  of ionized atoms or molecules to separate them
  from each other.
• Molecules have distinctive fragmentation patterns
  that provide structural information to identify
  structural components.
• The general operation of a mass spectrometer is
• create gas-phase ions
• separate the ions in space or time based on their
  mass to charge ratio
• Measure the quantity of ions of each
  mass-to-charge ratio.

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MS Detector Contd The ion separation power of an
MS is described by the resolution R
m/Dm Where m is the ion mass and Dm is the
difference in mass between two resolvable peaks
in a mass spectrum. E.g., an MS with a resolution
of 1000 can resolve an ion with a m/e of 100.0
from an ion with an m/e of 100.1.
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GAS LIQUID CHROMATOGRAPHY
Principles Partition of molecules between gas
(mobile phase) and liquid (stationary phase).
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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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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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Gas Chromatography Application

• Separation and analysis of organic compounds
• Testing purity of compounds
• Determine relative amounts of components in
  mixture
• Compound identification
• Isolation of pure compounds (microscale work)
• Similar to column chromatography, but differs in
  3 ways
• Partitioning process carried out between Moving
  Gas Phase and Stationary Liquid Phase
• Temperature of gas can be controlled
• Concentration of compound in gas phase is a
  function of the vapor pressure only.
• GC also known as Vapor-Phase Chromatography (VPC)
  and Gas-Liquid Partition Chromatography (GLPC)

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SEMI- QUANTITATIVE ANALYSIS OF FATTY ACIDS
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TENTATIVE IDENTIFICATION OF UNKNOWN COMPOUNDS
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GLC ADVANTAGES
1. Very good separation 2. Time (analysis is
short) 3. Small sample is needed - ml 4. Good
detection system 5. Quantitatively analyzed
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Food and Cancer

• Chemicals that can cause cancer have a wide
  variety of molecular structures and include
  hydrocarbons, amines, certain drugs, some metals
  and even some substances occurring naturally in
  plants and molds.
• In this way, many nitrosamines have carcinogenic
  properties and these are produced in a number of
  ways such as cigarette smoke.
• GC can be used to identify these nitro-compounds
  in trace quantities.

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Drugs

• There are still numerous GC applications
  involving both quantitative and qualitative
  identification of the active components and
  possible contaminants, adulterants or
  characteristic features which may indicate the
  source of the particular sample.
• Forensic analysis frequently users GC to
  characterize drugs of abuse, in some cases the
  characteristic chromatographic fingerprint gives
  an indication of the source of manufacture of the
  sample or worldwide source of a vegetable
  material such as cannabis.
• Analytical procedures, chromatographic methods
  and retention data are published for over 600
  drugs, poisons and metabolites. These data are
  extremely useful for forensic work and in
  hospital pathology laboratories to assist the
  identification of drugs.

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DISADVANTAGES OF GAS CHROMATOGRAPHY
Material has to be volatilized at 250C without
decomposition.
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Dr.A.DINESH KARTHIKdineshkarthik2008_at_gmail.com