SUBJECT

EXTRACTION
SEPARATION AND PURIFICATION
OIL AND GREASE REMOVAL
CHROMATOGRAPHY
THIN LAYER CHROMATOGRAPHY
GAS CHROMATOGRAPHY
HIGH PRESURE LIQUID CHROMATOGRAPHY

Solid Phase Extrac1on

SPE uses the anity of solutes dissolved or
suspended in a liquid (known as the
mobile phase) for a solid through which the
sample is passed (known as the
staSonary phase) to separate a mixture into
desired and undesired components.

Solid Phase Extrac1on

SEPARATION AND PURIFICATION

Chromatography is a technique in which compounds
to be separated are distributed between a mobile
phase and a sta1onary phase.
In such a system, dierent distribuSons based on
selecSve adsorpSon give rise to separaSon.
There are dierent types of chromatography, such as
paper, thin layer, or column chromatography, each
with its own strengths and weaknesses.

Chromatography
Column chromatography in chemistry is a method
used to purify individual chemical compounds from
mixtures of compounds.
Column chromatography is one of the most useful
methods for the separaSon and puricaSon of both
solids and liquids when carrying out small-scale
experiments. The separaSon can be liquid/solid
(adsorpSon) or liquid/liquid (parSSon) in column
chromatography.

Chromatography

Chromatography
Column chromatography is advantageous over most
other chromatographic techniques because it can be
used in both analy%cal and prepara%ve applicaSons.
It can be used to determine the number of components
of a mixture and as well as the separaSon and
puricaSon of those components.
Many compounds are not visible to the eye when
dissolved in a solvent or adsorbed on a adsorbent.
VisualizaSon processes make these substances visible.
The used techniques for this purpose include UV lights
that cause uorescence or phosphorescence and
chemical reacSons that give colored compounds.

Chromatography
The sta$onary phase or adsorbent in column
chromatography is a solid. The most common
staSonary phase for column chromatography
is

Chromatography
These adsorbents are sold in dierent mesh sizes,
indicated by a number on the bo\le label: silica gel 60
or silica gel 230-400 are a couple of examples.
This number refers to the mesh of the sieve used to size
the silica, specically, the number of holes in the mesh or
sieve through which the crude silica parScle mixture is
passed in the manufacturing process.
If there are more holes per unit area, those holes are
smaller, thus only smaller silica parScles are allowed to
pass the sieve. The larger the mesh size, the smaller the
adsorbent parScles are.

Chromatography
Alumina is quite sensiSve to the amount of water
which is bound to it; the higher its water content,
the less polar sites it has to bind organic compounds,
and thus the less sScky it is.
This sSckiness or acSvity is designated as I, II, or III
with I being the most acSve. Alumina comes in three
forms: acidic, neutral, and basic. The neutral form of
acSvity II or III, 150 mesh, is most commonly
employed.

Chromatography
The mobile phase or eluent is either a pure
solvent or a mixture of dierent solvents

Chromatography
The polarity of the solvent, which is passed
through the column, aects the relaSve rates
at which compounds move through the
column.
Polar solvents can more eecSvely compete
with the polar molecules of a mixture for the
polar sites on the adsorbent surface and will
also be\er solve the polar consStuents.

Chromatography
Consequently, a highly polar solvent will move even
the highly polar molecules rapidly through the
column. If a solvent is too polar, movement becomes
too rapid, and li\le or no separaSon of the
components of a mixture will result.
On the other hand, if a solvent is not polar enough,
no compounds will elute from the column.

Chromatography

Chromatography

Chromatography

Chromatography

Thin Layer Chromatography (TLC)

Thin-layer chromatography (TLC) is a
chromatography technique used to separate
non-volaSle mixtures.
Thin-layer chromatography is performed on a
sheet of glass, plas1c, or aluminium foil,
which is coated with a thin layer of adsorbent
material, usually silica gel, aluminium oxide,
or cellulose.

Thin Layer Chromatography (TLC)

TLC can be divided into three groups,
PreparaSve TLC
AnalyScal TLC
High performance thin layer
chromatography (HPTLC)

Thin Layer Chromatography (TLC)

Prepara1ve TLC uses plates with relaSvely thick
coaSngs (0.5-2.0 mm). Afer separaSon on the
plates, spots containing from 10-1000 mg of
sample can be scraped o the plates and
subjected to further analysis.

Thin Layer Chromatography (TLC)

Analy1cal TLC uses plates usually 20 x 20 cm in size
with a 0.25 mm coaSng of parScles with diameters
from 12-20 mm and are developed with the solvent
front moving from 10-12 cm. These plates are
commonly used for qualita1ve purposes.

Thin Layer Chromatography (TLC)

High performance thin layer chromatography (HPTLC)
can produce fast, high-resoluSon analysis with
quanStaSve accuracy and precision close to column
HPLC.
It uses thinner layers of staSonary phase and smaller
sample volumes, thus reducing the loss of resoluSon
due to diusion.

Thin Layer Chromatography (TLC)

The equilibrium between the free (completely
dissolved in the liquid or gaseous mobile
phase) and adsorbed (stuck on the surface of
the solid staSonary phase) states depends on
three factors:
the polarity and size of the molecule
the polarity of the staSonary phase
the polarity of the solvent

Thin Layer Chromatography (TLC)

The polarity of the molecules is determined by their
structures. By selecSng dierent staSonary and
mobile phases one can change the equilibrium
between the free and absorbed states.
It is important to understand chromatography at this
molecular level because this allows one to choose
mobile and staSonary phases that will separate just
about any mixture of molecules.

Thin Layer Chromatography (TLC)

The staSonary phase is
typically alumina
(Al2O3.xH2O)n or silica gel
(SiO2.xH2O)n.
The covalent network of
these absorbents create
very polar materials.

Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC)

Common Mobile Phases listed by Increasing Polarity.

Thin Layer Chromatography (TLC)

SpoGng the TLC Plate
Once the sample is prepared, a spohng capillary must
be used to add the sample to the plate.
The spohng capillaries must be extremely small.

Thin Layer Chromatography (TLC)

TLC Plate ready to be spo\ed

Thin Layer Chromatography (TLC)

Development
The bo\le is lled with a small amount of the mobile
phase and capped with a cork. In addiSon, a piece of
lter paper is put in the bo\le to help create an
atmosphere saturated with solvent.

Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC)

Visualiza1on
Some organic compounds are colored. If you
are fortunate enough to be separaSng organic
molecules that are colored such as dyes, inks or
indicators, then visualizing the separated spots
is easy.
However, since most organic compounds are
colourless, this rst method does not always
work.

Thin Layer Chromatography (TLC)

In most cases observing the separated spots by UV
light works well. TLC plates normally contain a
uorescent indicator which makes the TLC plate glow
green under UV light of wavelength 254 nm.
Compounds that absorb UV light will quench the
green uorescence yielding dark purple or bluish
spots on the plate. Simply put the plate under a UV
lamp, and the compounds become visible to the
naked eye.

Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC)

Visualiza1on reagent
DetecSon of organic compounds with spraying
of reagent into TLC plate

Thin Layer Chromatography (TLC)

Visualiza1on reagent
2,4-Dinitrophenylhydrazine
For detec$on of aldehydes and ketones
Spray plate with soluSon of 0.4 g 2,4-DNPH in
100ml 2N hydrochloric acid, add 1ml ethanol
Results: Yellow-red spots will be seen.

Thin Layer Chromatography (TLC)

Visualiza1on reagent
Bromocresol green
For detec$on of organic acids
Dip chromatogram in a soluSon of 0.1g
bromocresol green in 500ml ethanol and 5ml
0.1M NaOH
Results: Acids yield yellow spots on a blue
background.

Thin Layer Chromatography (TLC)

Visualiza1on reagent
Vanillin / potassium hydroxide for detecSon of amines and
amino acids
Phosphomolybdic acid for detecSon of Fa\y acid methyl
esters, keto acids, lipids, steroids, and sulphated bile acids.
KMnO4 for detecSon diol, C=C, reacSve methylene, phenol,
thiol, phosphine
Cerium-ammonium-molybdate, CAM Universal
Anisaldehyde For detecSon of phenols, sugars, steroids, and
terpenes
Iodopla1nate For detecSng alkaloids, amines, and organic
nitrogen compounds.

Thin Layer Chromatography (TLC)

Rf Values
In addiSon to qualitaSve results, TLC can also
provide a chromatographic measurement
known as an Rf value. The Rf value is the
retardaSon factor or the raSo-to-front
value expressed as a decimal fracSon.

Thin Layer Chromatography (TLC)

Rf Values
EssenSally it describes the
distance traveled by the
individual components. If two
spots travel the same distance or
have the same Rf value then it
might be concluded that the two
components are the same
molecule.
For Rf value comparisons to be
valid; however, TLC plates must
be run under the same exact
condi1ons.

THE END