Chromatography:
Chromatography, originally meaning
the Greek word for "color writing", is a method used
in analytical chemistry for separating and identifying components
of mixtures. Russian botanist Mikhail S. Tswett was the first
to develop a general chromatographic technique (1903). Some
examples of various types of chromatography are:
· paper chromatography
· Thin-layer chromatography ( used in this ISU )
· gel permeation chromatography
· ion chromatography
· countercurrent chromatography
Thin-Layer Chromatography3
Thin-layer chromatography was developed
in 1958 and is very similar to paper chromatography, commonly
used for the analysis of mixtures. The technique is as follows:
- a thin layer of absorbent material such as silica gel, alumna,
or polyamide (probably the best absorbent for all types of flavonoids)
is evenly spread along
3 based on Golier 1997 Interactive
Encyclopedia under "Chromatography"
- a glass plate or plastic film and
then dried.
- A small sample of solution is placed on the thin layer
- The solvent in the solution is then evaporated, leaving only
the mixture to be separated
- The plate is placed in an upright position in a jar
- A chosen solvent is added to the bottom of the container
- As a result the liquid rises along the plate by capillary action
Different components within the sample
rise along the plate at different speeds, therefore separating
the compounds within the mixture. When finished, the resulting
chromatograms can be examined under ultraviolet light to locate
the colour and type of flavonoid produced.
The "retardation factor"
is the "rate at which the pigment migrates"
(Black, p. 5) along the thin-layer plate. The retardation factor
is calculated:
Rf 4 = distance from
application to sample's final position
Distance from application to solvent front
4 Formula taken from Stephanie Black's
Independent study. Can vary slightly between sources and accuracy
is + 5%.
Crystallization:
A crystal is a solid in which the
atoms are arranged in a three dimensional pattern. Crystals
contain valuable knowledge in that they have their own unique
physical properties that reflects its structure chemical composition,
and the nature of bonding among its atoms. Crystallization is
a very common procedure used in the laboratory for the purpose
of separating solid materials in purified forms from a solution
or liquid mixture. Reasons for crystallization include:
· concentration of a liquid solution
· purification of a chemical species (as in this ISU)
· to achieve a change in physical properties
· separation of a chemical species from a mixture
No matter which process is used,
patience is greatly required.
Crystallization Process:
It is generally best to purify the
solution as much as possible before attempting the crystallization
process since impurities generally impede crystallization (eg.
By precipitation or filtering). After it has been purged of
impurities, the solution can be crystallized by one of the following
methods:
· evaporative concentration (used in this ISU)
· reduction of solubility by adding an unfavoured solvent
· cooling
· or other modification which can be improvised according
to the circumstances
Common solvents used in this process
are water, methanol, ethanol, acetic acid, acetone. These five
compounds are powerful solvents due to their polarity which is
used to dissolve polar substances, following the chemists' rule
that "like dissolves like" (Van Hook, p. 193). The
addition of water usually reduces the solubility of alcohols
resulting in solvent pairs of: Methanol-water or ethanol-water.
Crystal growth frequently starts at the wall of the container,
at the surface of a liquid or at a liquid-liquid boundary.
The ability of a substance to crystallize "depends on
its shape and size of its molecules or ions and on the magnitude
and kind of lattice forces"(Van Hook, p. 442). Some favoured
circumstances are:
· the presence of high, permanent dipole moments
· spherical molecules which require little arrangement
to form a lattice versus long shaped molecules
· possessing a low molecular weight as difficulty increases
as weight increases
(Van Hook, p. 442)
Note that when attempting to crystallize a compound by cooling
or evaporation, the solution may become "saturated with
respect to other solutes and separate as gum, oil, or amorphous
solid and contaminated the crystals"(Van Hook, p. 396).
Such contamination is common as may necessitate further crystallization.
Re-crystallization refers to the
"melting, dissolution, or vaporization of an already crystalline
compound to control the shape or size of the crystal or to separate
it from other compound". (Van Hook, p.396) Re-crystallization
may be performed many times until the desired purity is attained.
(Rousseau, p.727-28)(Van Hook, p. 192-556)
Crystallization and Recrystallization route: Fig. 9
Flavonoids:
Flavonoids are compounds which occur
in all parts of higher plant forms: roots, stems, leaves, flowers,
pollen, fruit, seeds, wood and bark.
Such as: anthocyanins flavones glycosides
Chalcones flavonols
Aurones flavonones
They are naturally occurring benzo-y-derivatives
which give foliage their colour as well as many other functions
which are currently being studied. Different plant organs or
tissue within organs can accumulate many different types of flavonoid
derivatives. Flavonoids vary in structure, however they all
share the same basic carbon skeleton:
Fig. 4
(Geissman, p. 1)
Examples of flavonoids include:
Fig. 5
Fig. 6
(Mabry, p. 24, 26)
Much interest have been taken in the last 30 years on flavonoids
due to the wide variety of effects they have on both plants and
animals which could be applicable to medicine as well as many
other areas of science. Some of the varied effects currently
being studied on flavonoid activity include:
· acting as inhibitors of chloroplasts, electron transport
and other metabolic pathways
· chelators
· anti-oxidants
· free radical scavengers
· active mutagenic, carcinogenic, anti-carcinogenic agents
eg. Quercetin, biocharin A
· active cytoxic, antineoplastic, anti-immflamatory,
anti-allergenic properties
· as inhibitors of some aspects of platelet function
(Stafford, p. 234)
In addition, flavonoids have been
shown to act as signals in fungal and bacterial recognition which
suggests that they might be a type of internal chemical messenger.
Derivatives of rutin are being tested for treating vascular
diseases and eupatoretin has shown some forms of anti-tumour
activity.
Flavonoids as Anti-oxidants
It is the acid-base and redox properties
of flavonoids that render them convenient biochemical anti-oxidents.
In humans, the major portion of ingested flavonoids are present
in the gastro-intestinal tract before they are excreted out of
the body as bile. Consequently, it is reasonable to assume that
the action of flavonoids as biological anti-oxidants takes place
during rigorous oxidative processes in digestion. Research has
shown that flavonoids reduce the superoxide radical in the pH
range from 7-10. Albeit low, these reactions "constitute
an efficient inactivation of the super-oxide radical to produce
hydrogen peroxides and the flavonoid radicals"(Jovanovic,
p. 4851). The reaction is as follows:
Fig. 7
(Jovanovic, p. 4851)
The ability of flavonoids to react with the super-oxide radical
seem to depend on their redox properties which are highly sensitive
to any substitution of the B-ring and their charge.
Flavonoid Biosynthesis
Though scientists are still debating
over the actual biosynthesis pathways of flavonoids, there is
general agreement that a cinnamic acid derived from phenylalanine
and 3 acetate units form the flavonoid/ isoflavonoid skeleton
previously mentioned. The next step along the pathway are the
chalcones or flavonones which are considered to be the primary
C15-intermediates. From this junction do the various other
derived flavonoids result.
Fig. 8
(Borz
and Weirman, p. 186)