Pharmacognosy

CONTENT
CHROMATOGRAPHY_______________________________________________________
TYPES OF CHROMATOGRAPHY___________________________________________
Column Chromatography_________________________________________________
1. STATIONARY PHASE:___________________________________________________________
Adsorbents:______________________________________________________________________
The Solvent_____________________________________________________________________
Analysis of Column Eluents____________________________________________________
PREPARATION OF THE COLUMN:__________________________________________________
PACKING OF THE COLUMN:_______________________________________________________
1. Dry PACKING TECHNIQUE:_____________________________________________________________
2. WET PACKING TECHNIQUE:___________________________________________________________

DETECTION OF COMPONENTS:____________________________________________________
FACTORS AFFECTING COLUMN EFFICIENCY:______________________________________
APPLICATIONS___________________________________________________________________
ADVANTAGES OF C.C.:____________________________________________________________
DISADVANTAGES OF C.C.:_________________________________________________________

Pharmacognosy

CHROMATOGRAPHY

Chromatography is a separation technique used to separate and identify the

components of a mixture.
Chromatography works by allowing the molecules present in the mixture to
distribute themselves between a stationary and mobile phase.

TYPES OF CHROMATOGRAPHY
1. Solid liquid Chromatography
Stationary Phase----Solid, Mobile Phase------Liquid
Tech: C.C., TLC, FCC, HPLC
2. Liquid liquid Chromatography
Stationary Phase----Liquid, Mobile Phase------Liquid
Tech: Paper Chromatography
3. Gas - Liquid Chromatography
Gas - Solid Chromatography
Stationary Phase----Solid / liquid, Mobile Phase------Gas
Tech: Gas Chromatography

Column Chromatography

Column chromatography is generally used as a purification technique:

it isolates desired compounds from a mixture.
The mixture to be analyzed by column chromatrography is placed
inside the top of the column.
The liquid solvent (the eluent) is passed through the column by
gravity or by the application of air pressure.
An equilibrium is established between the solute adsorbed on the
adsorbent and the eluting solvent flowing down through the column.
Because the different components in the mixture have different
interactions with the stationary and mobile phases, they will be carried
along with the mobile phase to varying degrees.
A weakly adsorbed compound will spend more time in the solvent, and will therefore be
eluted first.

1. STATIONARY PHASE:
CRITERIA:
Spherical in shape
Mechanical stability must be high.

Pharmacognosy
They should not react chemically.
It should be useful for separating for wide variety of compounds.
It shud be freely available and inexpensive.
Adsorbents:
Typical adsorbents used in C.C. are Alumina (aluminum oxide), Activated
charcoal, Magnesium silicate, Silica gel (silicon dioxide)., Inorganic carbonates,
starch, cellulose, sucrose.
Silica gel is slightly acidic while alumina may be acidic, neutral or basic. For
greatest effectiveness the particles of solid adsorbent should be of uniform size
and large surface area.
The strength of adsorption depends upon the compounds involved. Since the
adsorbents are polar, the more polar compounds are adsorbed more strongly.
Thus, non-polar compounds are eluted first.

The Solvent

The polarity of the solvent which is passed through the column affects
the relative rates at which compounds move through the column.

Polar solvents can more effectively compete with the polar molecules
of a mixture for the polar sites on the adsorbent surface and will also
better solvate the polar constituents.

Consequently, a highly polar solvent will move even highly polar

molecules rapidly through the column. If a solvent is too polar,
movement becomes too rapid, and little or no separation of the
components of a mixture will result.

If a solvent is not polar enough, no compounds will elute from the

column.

Often a series of increasingly polar solvent systems are used to elute a

column. A less-polar solvent is first used to elute a less-polar
compound. Once the less-polar compound is off the column, a morepolar solvent is added to the column to elute the more-polar
compound

Thin-Layer Chromatography (TLC) is generally used to determine the

system for a column chromatography separation.

Pharmacognosy
Analysis of Column Eluents

If the compounds separated in a column chromatography procedure

are colored, the progress of the separation can simply be monitored
visually.

More commonly, the compounds to be isolated from column

chromatography are colorless. In this case, small fractions of the
eluent are collected sequentially in labeled tubes and the composition
of each fraction is analyzed by TLC.

PREPARATION OF THE COLUMN:

Bottom portion of the column packed with glass wool/ cotton wool or may
contain asbestos pad.
Above which adsorbent is packed.
After packing a paper disc kept on the top
PACKING OF THE COLUMN:
1. Dry PACKING TECHNIQUE:
Adsorbent is packed in the column in dry form
Fill the solvent till equilibrium is reached.
2. WET PACKING TECHNIQUE:
Ideal and common technique.
For the wet method, a slurry is prepared of the eluent with the stationary phase powder and
then carefully poured into the column.

Adsorbent + M.P. in a beaker and poured into column.

S.P. settles uniformly and no crack in the column of adsorbent.
Slurry is a suspension of the adsorbent in a solvent which the sample will
also be dissolved or suspended with such as silica gel + hexane.

DETECTION OF COMPONENTS:
Colored components----- Visually
Colourless components--------Different properties which can be used are
UV / Visible detector, Flourescence detector, RI detector, Flame Ionization
Detector,..
FACTORS AFFECTING COLUMN EFFICIENCY:
Nature of the solvent
Dimensions of columns
Particle size of column packing
Temperature of the column

Pharmacognosy
APPLICATIONS
Separation of mixture if the components
Purification process
Isolation of metabolites
Estimation of drugs in formulation.
ADVANTAGES OF C.C.:
Any type of mixture can be separated
Any quantity of mixture can be separated
Wider choice of M.P.
Automation is possible

DISADVANTAGES OF C.C.:
Time consuming
Increased amounts of M.P. is required.
Automation makes the technique more complicated and expensive.

PLANT PIGMENTS
The plant life around us is full of wonderfully rich and vibrant colors. From red
to violet, the entire color spectrum can be found in plant materials. These colors
come
from a surprisingly small number of plant pigments. The three primary groups of
plant
pigments are anthocyanins, carotenoids, and chlorophyll. Others include porphyrins,
betalains, flavonoids etc. Each of these absorbs light at
different parts of the visible light spectrum and has a different function in the plant.
The light that is absorbed may be used by the plant to power chemical
reactions, while the reflected wavelengths of light determine the color the
pigment will appear to the eye.

The primary function of pigments in plants is photosynthesis, which uses the

green pigment chlorophyll along with several red and yellow pigments that
help to capture as much light energy as possible.

Pharmacognosy
Chlorophyll is the primary pigment in plants; it is a chlorin that absorbs yellow
and blue wavelengths of light while reflecting green. It is the presence and
relative abundance of chlorophyll that gives plants their green color. T he energy
of sunlight is captured by chlorophyll pigments to make food during the process of
photosynthesis.
There are 3 kinds of Chlorophyll:

Chlorophyll "a": mainly responsible for photosynthesis. Found in all plants, algae and
cyanobacteria carrying out photosynthesis.

Chlorophyll "b": found only in green algae and in plants.

Chlorophyll "c": found only in photosynthetic kind of organisms found in aquatic ecosystem
e.g. Chromista and dinoflagellates.

All chlorophylls serve as the primary means plants use to intercept light in order
to fuel photosynthesis.

Carotenoids are red, orange, or yellow tetraterpenoids. They function as

accessory pigments in plants, helping to fuel photosynthesis by gathering
wavelengths of light not readily absorbed by chlorophyll. The most familiar
carotenoids are carotene (an orange pigment found in carrots), lutein (a yellow
pigment found in fruits and vegetables), and lycopene (the red pigment
responsible for the color of tomatoes). Carotenoids have been shown to act as
antioxidants and to promote healthy eyesight in humans.
Flavonoids are widely distributed plant pigments. They are water soluble and
commonly occur in vacuoles, membrane-enclosed structures within cells which
also store water and nutrients. Anthocyanins (literally "flower blue") are
water-soluble flavonoid pigments that appear red to blue, according to pH. They
occur in all tissues of higher plants, providing color in leaves, plant stem, roots,
flowers, and fruits, though not always in sufficient quantities to be noticeable.
Anthocyanins are most visible in the petals of flowers, where they may make up
as much as 30% of the dry weight of the tissue.They are also responsible for the
purple color seen on the underside of tropical shade plants such as Tradescantia
zebrina; in these plants, the anthocyanin catches light that has passed through
the leaf and reflects it back towards regions bearing chlorophyll, in order to
maximize the use of available light.

Betalains are red or yellow pigments. Like anthocyanins they are water-soluble,
but unlike anthocyanins they are indole-derived compounds synthesized from
tyrosine. This class of pigments is found only in the Caryophyllales (including
cactus and amaranth), and never co-occur in plants with anthocyanins.

Pharmacognosy
Betalains are responsible for the deep red color of beets, and are used
commercially as food-coloring agents.

Paper Flower (Bougainvillea glabra), also known as lesser bougainvillea, is a

species of bougainvillea

The genus Bougainvillea, in the

Nyctinaginacea (Four-oclock)
family of plants, has 14 species, with three that are horticultural important.
Bougainvillea spectabilis Willd. Bougainvillea glabra Choisy Bougainvillea
peruviana
Family
:

Nyctaginace
ae

Paper Flower (Bougainvillea glabra), also known as lesser bougainvillea, is a

species of bougainvillea
They are thorny, woody vines growing anywhere from 1 to 12 meters tall,
scrambling over other plants with their spiky thorns. The thorns are tipped with
a black, waxy substance. They are evergreen where rainfall occurs all year, or
deciduous if there is a dry season. The leaves are alternate, simple ovateacuminate, 413 cm long and 26 cm broad. The actual flower of the plant is
small and generally white, but each cluster of three flowers is surrounded by
three or six bracts with the bright colours associated with the plant, including
pink, magenta, purple, red, orange, white, or yellow. Bougainvillea glabra is
sometimes referred to as "paper flower" because the bracts are thin and papery.
The fruit is a narrow five-lobed achene. Bougainvilleas are popular ornamental
plants in most areas with warm climates