CHROMATOGRAPHY
Lab # 5
introduction
• Chromatography is a laboratory technique for the
separation of mixtures.
• It involves passing a mixture dissolved in a mobile phase
through a stationary phase, which separates the analyte.
• Different chemicals in a mixture have different degrees of
dissolving in a liquid (mobile phase) or sticking to a solid
surface (stationary phase).
Chromatography terms
• The analyte: is the substance to be separated during
chromatography.
• The eluate: is the mobile phase leaving the column.
• The eluent: is the solvent that will carry the analyte.
• The mobile phase: is the phase which moves in a
definite direction, It may be a liquid (LC), a gas (GC).
• The stationary phase: is the substance which is fixed in
place for the chromatography procedure. Examples
include the silica layer in thin layer chromatography.
Principle of chromatography
• Chromatography involves a sample being dissolved in a
mobile phase (which may be a gas or a liquid).The mobile
phase is then forced through an immobile, immiscible
stationary phase.
• The phases are chosen such that components of the
sample have differing solubilities in each phase.
Principle of chromatography
• A component which is quite soluble in the stationary
phase will take longer to travel through it than a
component which is not very soluble in the stationary
phase but very soluble in the mobile phase.
• As a result of these differences in mobilities , sample
components will become separated from each other as
they travel through the stationary phase.
Steps of chromatography
1. Compound is placed on stationary phase.
2. Mobile phase passes through the stationary phase.
3. Mobile phase solubilizes the components.
4. Mobile phase carries the individual components a
certain distance through the stationary phase,
depending on their attraction to both of the phases.
5. The molecules in the mixture that adsorbs the most to
the stationary phase is moving slowest through the
particle bed.
Types of chromatography
1. Column chromatography.
2. Planar chromatography.
a. Paper chromatography.
b. Thin layer chromatography
Column chromatography
• In column chromatography, the stationary phase (solid
adsorbent) is placed in a vertical glass column and the
mixture is added to the top and the mobile phase will
flows down through the column so, the substance with
less ability to adsorb in the stationary phase will be
separated first .
Column chromatography
Column chromatography
Planar chromatography
• A separation technique in which the stationary phase is
present as or on a plane.
• The plane can be a paper, or a layer of solid particles
spread on a support e.g. a glass plate (TLC).
Thin Layer Chromatography
• Thin-layer chromatography (TLC) is a very commonly
used technique in synthetic chemistry for identifying
compounds, determining their purity and following the
progress of a reaction.
• It is performed on a sheet of glass, plastic, or aluminum
foil, which is coated with a thin layer of adsorbent material
(0.25mm), usually silica gel, aluminum oxide, or cellulose.
This layer of adsorbent is known as the stationary phase.
Thin Layer Chromatography
Retardation Factor
• Rf is constant value for a substance (if it meager in the
same condition) so, it can be use to identify the substance
as one of its physical proprieties.
Amino acids
• Proteins consist of amino acids interconnected by peptide
bonds.
• Mixtures of amino acids can be separated on
chromatographic sheet.
• The separated amino acids are visualized using solution
of ninhydrin.
• Purple color develops upon reaction of amino acid with
ninhydrin.
Amino acids
Amino acid
Rf value
alanine
0.38
arginine
0.20
asparagine
0.5
aspartic acid
0.24
Turmeric
• Active constituent:
1. 1-5% orange yellow volatile oil
2. 0.3% of yellow crystalline dye (curcumin)
3. 30-50% starch
4. resin
Separation of Turmeric using TLC
• Procedure:
1. in the TLC, draw 2 horizontal lines (the first one at
1.5cm from the bottom edge and the other one at 1cm
from top edge)
2. draw one spot in the bottom line with around 8mm from
the edge of the plate
3. by a capillary tube add one spot of the sample in the
line of bottom ,wait to dry then repeat this process three
times
4. if the spots not dry use the hotplate to dry it.
Separation of Turmeric using TLC
fill the chamber with 10 ml of solvent ( make sure that
the height of solvent lower than the lower line of the
sheet)
solvent system: ethanol: chloroform : ethanol : acetic acid
(94+5+1)
5. pot the sheet in a chamber, close the it and wait until
the solvent reach the top line of sheet.
6. remove the sheet from the chamber AND move the
plate over ammonia vapour
7. circle the spots and calculate the Rf factor for the
samples
5.
Separation of Turmeric using TLC
desmethoxy curcumin
bisdesmethoxy curcumin
sample
Dist. traveled by solvent
curcumin
Dist. traveled by sub.x
Front of solvent
Result
Thank you