Application of Chromatography

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Chapter 4 Application of
Chromatography

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• Chromatography is a physic and
  physiochemistry analysis method, it plays a key
  role in the analysis of drugs.

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• Depending on the mechanism of the seperation
  process, chromatographic method can be classified
  as
• (1)Adsorption chromatography
• (2)Partition chromatography
• (3)Ion-exchange chromatography
• (4)Size-exclusion chromatography

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(1)Absorption chromatography

• It is based on the different affinity of
  individual components to the adsorbent(stationary
  phase),so that they can be eluted successively
  with a solvent or gas( mobile phase).

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(2)Partition chromatography

• It is based on the distribution of individual
  components between two phases. The stationary
  phase is coated on or chemically bonded to a
  solid support of large surface area. The mobile
  phase is a liquid or a gas. The supports commonly
  used are silica gel, diatomaceous earth,
  cellulose powder, polymers with suitable
  functional grounds etc.

Source http//elchem.kaist.ac.kr
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(3)Ion-exchange chromatography
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(4)Size-exclusion chromatography

• Size-exclusion chromatography is also known
  as gel permeation chromatography or gel
  filtration chromatography.
• It is based on the different permeability of
  components of different molecular size into a
  support of definite pore size.

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• Chromatography may be calssified into 3 types
  according to the operational method
• (1) column chromatography
• (2) paper chromatography
• (3) thin-layer chromatography

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Thin Layer Chromatography(TLC)
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The use of TLC

• In the past decades, with the development
  of the technology of TLC, it became a most widely
  used qualitative method in the analysis of
  natural products.

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• (1)Simple equipments
  (2)Short time for assay
• (3)Good seperation effect
  (4)Convenience for colouration
• (5)High sensitivity

The advantages of TLC
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• The most widely used one is the adsorption
  TLC, which is based on the two regulations
  occurred in the process of adsorption
• (1) The adsorption should be reversible,
  adsorption and adsorption stripping kept mobile
  equilibrium during the separation
• (2) The absorption ability of each adsorbent
  is varied with the different substances.

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Adsorbent
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• The adsorbent commonly used are silica gel,
  aluminum oxide, polyamide powder etc.

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Preparation of TLC plates
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• Materials for the plate
• Glass
• Plastic slice
• Aluminiun foil

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• Pre-coated plates
• There are two kinds of pre-coated plates
• A TLC plates
• B HPTLC
• Silica gel plates silica gel GF254 plates,
  polyamide membrane

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• Home-made plates
• It could be used if the plates need special
  treatment and chemical modification in order to
  meet the seperation requirements given that the
  quality of the chromatogram is ensured.

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• The most widely used stationary phase are silica
  gel G, silica gel GF254 , silica gel H(no gypsum
  , i. e. CaSO4, ,calcium sulfate ), silica gel
  HF254 , microcrystallite cellulose etc, the
  partical size of which is generally 10-40 µm in
  diameter. Water or solution of carboxymethylcellul
  ose with the concentration of 0.2-0.5 is then
  added appropriately to form a uniform layer of
  definite thickness. The surface of the glass
  plate should be smooth and with no globule after
  cleaning.

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Sample application
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• Micro-liter capillary or other hand-operated,
  semi-automatic and full-automatic sample
  applicators are used generally.

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Development
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• Place the plate loaded with sample into the
  chromatographic chamber, closed the chamber
  tightly. When the mobile phase has moved over the
  prescribed distance(usually 8-15cm for TLC, while
  5-8cm for HPTLC)
• Take it out and dry it in air.

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Visualization and detection
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• The coloured components of the sample could be
  detected directly under visible light.
  Appropriate visualization reagent could also be
  used for colouration in the way of spraying or
  immersion. They could also be visualized by being
  heated and detected directly under the sunlight.
  Fluorescent chromatogram could be detected at
  365nm if the components can emit fluorescence
  itself or when meeting with some reagents.

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• The silica gel plates with fluorescer (e.g.
  Silica gel GF254 plate) could be used if the
  components have no colour but have ultraviolet
  absorption. The chromatogram formed by
  fluorescence inhibitor can be observed on the
  plate at 254nm.

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What's this?
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• Rf value (the shift ratio value) The ratio of
  the distance between the center of the origin
  and that of the spot to the distance between the
  center of origin and the frontal of the mobile,
  i. e.
• (L2)/ (L1)?
• 0.3 ltRflt 0.8

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Figure 4. TLC Chromatogram of Venenum Bufonis
(Chansu) from different places Developing
agentcyclohexane-chloroform-acetone(433) Color
developing agent10 solution of sulfuric acid
in ethanol,observe under 365nm ultra-violate
light
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How much?
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Assay
TLC-UV
TLC- Scan
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Source http//www.sorbfil.com
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The process of thin-layer chromatography combines
UV to determine the content of component in
Chinese medicines.
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Figure 4. HPTLC Chromatogram of Radix Ginseng
from different places
Developing agentChloroform-ethyl
acetate-methanol-water(15402210)

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The new technology of TLC
(overpressured layer chromatography,OPLC)

• OPLC combined the advantages of TLC and HPLC
  ,while the shortage of both of them are avoided.
  The high separation efficiency is ensured by
  allowing the developing process occurred in a
  high pressure and tightly closed environment.
  The separation will not be effected by the
  humidity and temperature.

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OPLC Chromatogram
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Any questions?