Chromatography
Chromatography
• First application by M. S. Tswett 1903
• For the separation of plant pigments. Since the
components had different colors the Greek
chromatos, for color, was used to describe the
process, so it means color writing.
• A physical method of separation in which the
components to be separated are distributed
between two phases:
A stationary phase and a mobile phase that
moves in a definite direction.
• Chromatography is a laboratory technique that
separates components within a mixture by using the
differential affinities of the components for a
mobile medium and for a stationary adsorbing medium
through which they pass.
• Affinity – natural attraction or force between things. (The
smaller the affinity a molecule has for the stationary phase, the
shorter the time spent in a column. )*
• Mobile Medium – phase which move over or through stationary
phase and carries sample along with it, thus resulting in the
separation of its component. it is either liquid or gas. (mobile
phase)
• Stationary Medium – the part of the apparatus that does not
move with the sample (stationary phase)
Illustration of Chromatography
Stationary Phase
Separation
Mobile Phase
Mixture
Components
Components
Affinity to Stationary
Phase
Affinity to Mobile
Phase
• Analyze
Blue
----------------
Insoluble in Mobile Phase
• Identify
Black


Red


Yellow

        
• Purify
• Quantify
Advantages of chromatography
• Can separate complex mixtures with great precision.
Even very similar components, such as proteins that
may only vary by a single amino acid.
• can purify basically any soluble or volatile substance
if the right adsorbent material, carrier fluid, and
operating conditions are employed.
• Exact quantitative analysis is done even from trace
compounds.
• Small material consumption.
• The quantization has a broad linearity range.
• Analyses of several compound can be done during one
run.
• Chromatography is a fast analysis method.
• Well establishes instrumentation with high level
automation is commercially available.
Types of
chromatography
Based on
stationary phase
Based on mobile
phase
Column
Liquid
Planar
Gas
Kinds of Chromatography
1. Liquid Column Chromatography
gel filtration ,ion exchange, affinity, adsorption,
reverse phase, metal binding (column)
2. Gas Liquid Chromatography (column)
3. planar Chromatography (Thin-layer & paper )
LIQUID COLUMN CHROMATOGRAPHY
A sample mixture is passed through a column packed
with solid particles which may or may not be
coated with another liquid.
With the proper solvents, packing conditions, some
components in the sample will travel the column
more slowly than others resulting in the desired
separation.
ION EXCHANGE
•protein interact with stationary phase by chargecharge interaction
•Positively charged proteins adhere to negatively
charged functional groups (carboxylates, sulfates:cation
exchanger)
•Sequential elution, change of pH
SIZE EXCLUTION/GEL
FILTERATION/GEL PERMEATION
 porous beads as stationary phase
 stroke radius; function of molecular mass and
shape
 greater the stroke radius faster will be the
elution
 gel is made
polyacrylamide
of
dextran
agarose
or
AFFINITY
 sensitivity of most proteins towards ligands
 use of resins to attach ligands
 elution by competition with soluble ligand or by
disruption of interation.
Gas liquid chromatography
 separation of volatile mixture
 stationary phase is non-volatile liquid, coated on
an inert solid
 mobile phase is a inert gas (ex. Argon or helium )
or an unreactive gas such as nitrogen.
Planar chromatography
 It is a method for separation and determination of
substances, allowing to carry out qualitative and
quantitative analysis of chemical components in complex
mixtures.
 A separation technique in which the stationary phase
serves as a plane.
 The plane can be either a paper (paper
chromatography) or a layer of solid particles sorbent
(silica gel, cellulose, aluminum oxide, ion exchange resin)
spread on a support such as a glass- or a plastic- plate
(thin layer chromatography).
For qualitative analysis the different mobilities of substances
are used, the distances passed by different substances are
different. The distance between the starting line and the center
of the spot of substance hx, mm characterizes the substance.
Retention factor, RF, provides better way to indentify
substances .
For quantitative determination the intensity of the spot is used:
the bigger the amount of substance in the mixture, the more
intensive is the spot. Also the size of the spot can give
quantitative information – the bigger the spot, the bigger the
content of this compound in the mixture. Intensity of the spots
is evaluated by comparing with the intensities of analyte spots
with known amounts visually or using densitometer.
Principles of Paper Chromatography
• Capillary Action – the movement of liquid within the spaces of
a porous material due to the forces of adhesion, cohesion, and
surface tension. The liquid is able to move up the filter paper
because its attraction to itself is stronger than the force of
gravity.
• Solubility – the degree to which a material (solute) dissolves
into a solvent. Solutes dissolve into solvents that have similar
properties. (Like dissolves like) This allows different solutes
to be separated by different combinations of solvents.
Separation of components depends on both their solubility in
the mobile phase and their differential affinity to the mobile
phase and the stationary phase.
Thin Layer Chromatography (TLC)
• Different compound in sample mixture travel different
distance according to how strongly they interact with the
stationary phase as compared to mobile phase. ( adsorption)
So it depend on : 1. activity of stationary phase.
2. polarity of mobile phase.
3. structure of substrate .
• Principle

Different compounds in sample mixture
travel
different distances according to how
strongly
they
interact with the stationary
phase as compared to
the mobile phase.

The specific Retention factor (Rf) of each
chemical
can be used to aid in the
identification of an unknown
substance.
Measuring Rf
Procedure:
• Instruments, chemicals and glassware:
• Eluent. [Mix n-butanol, acetic acid (purity 98 – 100 %) and
distilled water in volume ratio 5:1:5. Stir for 10 minutes, then
let the layers separate. Use upper layer as eluent].
• Developing Solution: Dissolve 0.3 g of ninhydrin in 100 ml nbutanol. Add 3 ml of glacial acetic acid.
• 0.02 M solutions of different amino acids (e.g. leucine,
methionine, alanine and serine) in H2Odd.
• Chromatographic paper
• Elution chamber, Glass capillaries for spotting the samples.
• Drying oven at ~ 60° C.
1. Rubber gloves must be used during this work to avoid
contamination of chromatographic paper with amino acids
from skin, and for protecting skin from solvents and
ninhydrin while working with the sprayer or sprayed
paper.
2. While the paper is being prepared for chromatographic
analysis it should be kept on a piece of filter paper.
3. Mark the starting line to the paper - 8-9 mm from the
edge of the plate - with graphite pencil (very slight line!).
Also mark the locations where the samples will be
spotted. The distance between neighboring spots should
be about 8 mm and the spots should be at least 5 mm
away from the paper’s edge. Usually the spot of unknown
substance is applied to the center of the starting line.
4.
Before applying samples to the paper and filling the elution
chamber fit the length of chromatographic paper with the
height of elution chamber.
5.
The spots of individual amino acids and sample solutions are
applied to the chromatographic paper. Use separate clean and
dry glass capillary for each solution. Dip the capillary into
solution – some solution is drawn into the capillary. With the
filled capillary touch the prepared location on chromatographic
paper. The spot on the paper should not be bigger than 2-3
mm. (You can exercise spotting on a sheet of filter paper.)
6.
After application of samples let the spots dry. Meanwhile
measure with a graduated test-tube 5 ml of eluent into the
elution chamber. Cover the chamber with lids and let the
chamber atmosphere saturate with eluent vapors for at least
10 min.
7. Elution is stopped when the solvent front has
traveled up the plate until 7-10 mm from the lid.
8. Remove the paper from elution chamber and place it
on a sheet of filter paper. After 2-3 minutes mark
the eluent front with pencil and dry the paper in
oven.
9. When the paper is dry, take it into the fume hood and
spray it with solution of ninhydrin until the paper is
slightly damp. Chromatographic paper and the paper
supporting it should lie at 45° angle while spraying.
The chromatographic paper is again put in the drying
oven (60 C) for 15 min to speed up the reactions.
8. Remove chromatographic paper from drying in the
oven, draw the contours and centers of the
chromatographic bands. Calculate RF values by the
method described above.
9. 11. Compare retention of standard substances and
components in sample and determine which amino
acids were present in the sample.
Result
• Determination of amino acids using thin layer
chromatography
1. Adding fluorescence indicators to the sorbent layer
during the process of preparation of the plates or
spraying the plates with fluorescent solutions and
then observing under ultraviolet lamp.
2. I2 vapor as indicator .
3. Ninhydrin spray.
Ninhydrin spray
Development of Ruhemann’s purple from ninhydrin and amino acid.