sprinkled in the TLC and heated to around 200 °C until colors are produced.
Various color-producing reagents are used, most notably including anisaldehyde,
ninhydrin, and phenylmercury acetate (PMA).
D. Ion Exchange Column Chromatography
Many natural substances contain acidic, basic, or positive dissociating functional
groups. Ion exchange is a method that uses the state in which compounds with
dissociating groups are dissolved in solution. The ion exchange method is a form of
separation that uses polymers combining various kinds of cation and anion, taking
advantage of differences in the substances’ ion affinity. It is typically used for
protein, nucleic acid, amino acid, and peptide isolation. Exchangers can be classifier
broadly by polymer type into ion exchange resin, ion exchange cellulose, and
dextran (Mercer 1974; Peterson and Sober 1956).
The stationary phase in ion exchange chromatography is charged ion exchange
resin, which comes in a large variety of types. Ion exchange resin capacity is
determined by the absolute value of the ions bonding to it. In anion exchange resin,
a positively charged functional group attaches to a supporter through a covalent
bond, and anion in the solute are drawn to the charged location. A positive ion
exchange resin possesses a negatively charge site that bonds with cation in the
solute. Different functional groups, such as carboxymethyl (CM) and diethylaminoethyl (DAEA), attach to different supporters, making them suitable ion
exchange resins for chromatography.
Substances used for supporters include polystylene, polyacrylate, cellulose,
Sephacel, dextran, agarose, and toyopearl. In the mobile phase, the eluant is typically an aqueous solution, and pH, salt concentration and properties, and the solvent’s viscosity and dielectric constant are important considerations. Separated
substances must be charged, or at least ionized.
The process of ion exchange chromatography for protein refinement is as follows (Fig. 10.7). A protein solution passes through a stationary phase column
including ion exchange resin. Cation exchange resin bonds negatively charged
carboxymethyl groups to a cellulose supporter, giving carboxymethyl-cellulose.
Cation proteins bond with the exchange resin through electrostatic force, an
adhesive force resulting from the net positive change in the pH entering the column.
Once the protein has bonded, it is washed with a buffer solution that increases pH or
ionic strength. These changes in the extraction solution weaken the bonds, causing
the proteins to separate first from the exchange resin. Because of the significant
changes to conditions in the bonding stage, solidly bonded molecules are subsequently separated out.
Protein and amino acid separation is an example of one important application of
ion exchange chromatography. This chromatography is also put to effective use in
separation of cation and anion. Halide ions can be separated in a Dowex-2 column
with 1 M sodium nitrate (NaNO 3 , adjust to NaOH) at pH 10.4 as an eluent,
emerging in the order F
− > Cl
− > Br
− > I
− . Alkaline metal ions can be separated
with 0.7 M hydrochloric acid (HCl) in a Dowex-50 or Amberlite IR 120 column,
10.2 Researching Natural Marine Substances
359
Various color-producing reagents are used, most notably including anisaldehyde,
ninhydrin, and phenylmercury acetate (PMA).
D. Ion Exchange Column Chromatography
Many natural substances contain acidic, basic, or positive dissociating functional
groups. Ion exchange is a method that uses the state in which compounds with
dissociating groups are dissolved in solution. The ion exchange method is a form of
separation that uses polymers combining various kinds of cation and anion, taking
advantage of differences in the substances’ ion affinity. It is typically used for
protein, nucleic acid, amino acid, and peptide isolation. Exchangers can be classifier
broadly by polymer type into ion exchange resin, ion exchange cellulose, and
dextran (Mercer 1974; Peterson and Sober 1956).
The stationary phase in ion exchange chromatography is charged ion exchange
resin, which comes in a large variety of types. Ion exchange resin capacity is
determined by the absolute value of the ions bonding to it. In anion exchange resin,
a positively charged functional group attaches to a supporter through a covalent
bond, and anion in the solute are drawn to the charged location. A positive ion
exchange resin possesses a negatively charge site that bonds with cation in the
solute. Different functional groups, such as carboxymethyl (CM) and diethylaminoethyl (DAEA), attach to different supporters, making them suitable ion
exchange resins for chromatography.
Substances used for supporters include polystylene, polyacrylate, cellulose,
Sephacel, dextran, agarose, and toyopearl. In the mobile phase, the eluant is typically an aqueous solution, and pH, salt concentration and properties, and the solvent’s viscosity and dielectric constant are important considerations. Separated
substances must be charged, or at least ionized.
The process of ion exchange chromatography for protein refinement is as follows (Fig. 10.7). A protein solution passes through a stationary phase column
including ion exchange resin. Cation exchange resin bonds negatively charged
carboxymethyl groups to a cellulose supporter, giving carboxymethyl-cellulose.
Cation proteins bond with the exchange resin through electrostatic force, an
adhesive force resulting from the net positive change in the pH entering the column.
Once the protein has bonded, it is washed with a buffer solution that increases pH or
ionic strength. These changes in the extraction solution weaken the bonds, causing
the proteins to separate first from the exchange resin. Because of the significant
changes to conditions in the bonding stage, solidly bonded molecules are subsequently separated out.
Protein and amino acid separation is an example of one important application of
ion exchange chromatography. This chromatography is also put to effective use in
separation of cation and anion. Halide ions can be separated in a Dowex-2 column
with 1 M sodium nitrate (NaNO 3 , adjust to NaOH) at pH 10.4 as an eluent,
emerging in the order F
− > Cl
− > Br
− > I
− . Alkaline metal ions can be separated
with 0.7 M hydrochloric acid (HCl) in a Dowex-50 or Amberlite IR 120 column,
10.2 Researching Natural Marine Substances
359