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TECHNIQUES TO SEPERATE AND CHARACTERS OF BIOACTIVE COMPOUNDS

 Separation techniques:

  • Thin layer chromatography.
  • Gas chromatography.
  • High performance liquid chromatography.
  • Column chromatography.

Other techniques:

  • Fractional distillation.
  • Crystallization.
  • solvent Partitioning.

Thin layer chromatography:

  • Preparation: A thin layer of adsorbent material (like silica gel) is coated onto a solid support (usually glass, plastic, or aluminum).

  • Spotting: A small amount of the mixture you want to separate is applied as a spot near the bottom of the TLC plate.

  • Development: The TLC plate is placed in a container with a solvent (or a mixture of solvents) called the mobile phase. The solvent travels up the plate by capillary action.

  • Separation: As the solvent moves, it carries the components of the mixture with it.

  •  Visualization: Once the solvent has traveled a certain distance, the plate is removed from the container. The separated compounds may be visible, or they may need to be visualized using UV light, chemical sprays, or other methods.

Column chromatography:

  • Column Preparation: A vertical column is packed with a solid material called the stationary phase. This material can be silica gel, alumina, or other substances.

  • Sample Loading: The mixture of compounds to be separated is dissolved in a small amount of liquid (the mobile phase) and carefully loaded onto the top of the column.

  • Elution: More mobile phase is added to the top of the column, and it flows down through the stationary phase.

  • Separation: As the mobile phase travels through the column, the different compounds in the mixture interact with the stationary phase.

  • Collection: As the separated compounds elute (come out) from the bottom of the column, they are collected in separate fractions.

High performance liquid chromatography:

  • Mobile Phase: A liquid solvent (or a mixture of solvents) called the mobile phase is pumped under high pressure through the system.

  • Sample Injection: The sample containing the mixture of compounds to be analyzed is injected into the mobile phase stream.

  • Column: The mobile phase carries the sample through a column packed with a stationary phase. This stationary phase can be a solid material (like silica gel) or a liquid coated onto a solid support.

  • Separation: As the mixture travels through the column, the different components interact with the stationary phase.

  • Detection: As the separated compounds elute (come out) from the column, they pass through a detector.

  •  Data Analysis: The detector signal is recorded and displayed as a chromatogram.

Gas chromatography:

  • Vaporization: The sample containing the mixture of compounds is injected into a heated injection port, where it is vaporized.

  • Carrier Gas: An inert gas, usually helium or nitrogen, called the carrier gas, carries the vaporized sample through a chromatographic column.

  • Column: The column is a long, narrow tube packed with a stationary phase. This stationary phase can be a solid adsorbent, or a liquid coated onto a solid support.

  • Separation: As the vaporized sample travels through the column, the different compounds interact with the stationary phase. 

  • Detection: As the separated compounds elute (come out) from the column, they pass through a detector.

  •  Data Analysis: The detector signal is recorded and displayed as a chromatogram. 

 Nuclear Magnetic Resonance (NMR) Spectroscopy:

It shows the properties of atomic nuclei to provide information about the connectivity and arrangement of atoms within a molecule.
  • Connectivity: Which atoms are bonded to which other atoms.
  • Structure: The 3D arrangement of atoms in space.
  • Functional groups: The presence of specific groups of atoms (like -OH, -COOH).
Eg: 1H NMR (for hydrogen atoms) and 13C NMR (for carbon atoms) are the most common.


Mass Spectrometry (MS):

 MS measures the mass-to-charge ratio of ions. It provides information about the molecular weight of a compound and its fragmentation pattern
  • Molecular weight: The size of the molecule.
  • Elemental composition: The types and numbers of atoms present.
  • Structure: By analyzing how the molecule fragments, you can get clues about its structure.
Eg: GC or HPLC (GC-MS, LC-MS) to identify and characterize phytochemicals.

Infrared (IR) Spectroscopy:

IR spectroscopy measures the absorption of infrared light by a molecule. Different functional groups absorb IR light at different frequencies.

  • Functional groups: The presence of specific groups of atoms (like -OH, -COOH, C=O).

Ultraviolet-Visible (UV-Vis) Spectroscopy:

UV-Vis spectroscopy measures the absorption of ultraviolet and visible light by a molecule.

  • Chromophores: The presence of specific groups of atoms that absorb UV-Vis light (like conjugated double bonds).
  • Quantification: It can be used to determine the concentration of a phytochemical.
  1. X-ray crystallography: Provides a very detailed 3D structure of a molecule, but requires the compound to be in crystalline form.
  2. Raman spectroscopy: Provides complementary information to IR spectroscopy, particularly for certain types of bonds.

FT-IR:

FT-IR spectroscopy provides a "fingerprint" of a molecule.
  • O-H stretches: Indicates the presence of alcohols or carboxylic acids.
  • C=O stretches: Indicates the presence of carbonyl groups (like in ketones, aldehydes, or esters).
  • C-H stretches: Indicates the presence of alkanes or aromatic rings.
  1. Identifying functional groups: It's a quick and easy way to identify the types of chemical bonds present in a molecule.
  2. Identifying unknown compounds: By comparing the FT-IR spectrum of an unknown compound to a library of known spectra, you can often identify the compound.
  3. Monitoring reactions: FT-IR can be used to monitor the progress of a chemical reaction by observing the disappearance of reactants and the appearance of products.
  4. Analyzing complex mixtures: It can be used to analyze complex mixtures of compounds, such as those found in plant extracts.


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