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Group 6
Jualo, Zaldhy O.
Salubre, Jomar C. .
Arandilla, Aila Marie C.
Edillion, Shaina D.
Griño, Alliza Joy M.
Lanoza, Joanna Melanie M.
Mabunga, Abegail P.

1. Basic concepts of chromatography.

Chromatography differs from other methods of separation in that a wide variety of

materials, equipment, and techniques can be used. Which are characterized by a
distribution of the molecules to be separated between two phases, one stationary and the
other mobile phase molecules with a high tendency to stay in the stationary phase will
move through the system at a lower velocity than will those which favor the mobile phase.
The shape, rigidity and particle size distribution profile of the gel matrix are important

2. Different types of chromatography.


Paper Paper chromatography was introduced YES NO
Chromatography in 1944. In paper chromatography, the
stationary phase and the mobile phase
are both liquid. Paper generally serves
as a support for the liquid stationary
phase. The dissolved sample is applied
as a small spot or streak one half inch
or more from the edge of a strip or
square of filter paper (usually
cellulose), which is then allowed to dry.
The dry strip is suspended in a closed
container in which the atmosphere is
saturated with the developing solvent
(mobile phase), and the paper
chromatogram is developed. The end
closer to the sample is placed in contact
with the solvent, which then travels up
or down the paper by capillary action,
separating the sample components in
the process. When the solvent front has
traveled the length of the paper, the
strip is removed from the developing
chamber and the separated zones are
detected by an appropriate method.
Thin Layer Thin-layer chromatography (TLC), first YES NO
Chromatography described in 1938, has largely replaced
paper chromatography because it is
faster, more sensitive, and more
reproducible. The resolution in TLC is
greater than in paper chromatography
because the particles on the plate are
smaller and more regular than paper
fibers. There are several distinct
advantages to TLC: high sample
throughput, low cost, the possibility to
analyze several samples and standards
simultaneously, minimal sample
preparation, and that a plate may be
stored for later identification and
Gas Gas chromatography is a column YES YES
Chromatography chromatography technique, in which
the mobile phase is gas and the
stationary phase is either an
immobilized liquid or a solid packed in a
closed tube. Gas chromatography is
used to separate thermally stable
volatile components of a mixture. Gas
chromatography, specifically gas–liquid
chromatography, involves vaporizing a
sample and injecting it onto the head of
the column. Under a controlled
temperature gradient, the sample is
transported through the column by the
flow of an inert, gaseous mobile phase.
Volatiles are then separated based on
several properties, including boiling
point, molecular size, and polarity
Column Column chromatography is the most YES NO
Chromatography useful method of separating
compounds in a mixture. Fractionation
of solutes occurs as a result of
differential migration through a closed
tube of stationary phase, and analytes
can be monitored while the separation
is in progress. In column liquid
chromatography, the mobile phase is
liquid, and the stationary phase can be
either solid or liquid supported by an
inert solid.
Ion Exchange Ion exchange is a NO YES
Chromatography separation/purification process
occurring naturally, for example, in soils
and is utilized in water softeners and
deionizers. Three types of separation
may be achieved: (1) ionic from
nonionic, (2) cationic from anionic, and
(3) mixtures of similarly charged
species. In the first two cases, one
substance binds to the ion-exchange
medium, whereas the other substance
does not. Batch extraction methods can
be used for these two separations;
however, chromatography is needed
for the third category. Ion-exchange
chromatography may be viewed as a
type of adsorption chromatography in
which interactions between solute and
stationary phase are primarily
electrostatic in nature.
Gel Filtration Gel filtration chromatography, also YES YES
Chromatography known as molecular exclusion, gel
permeation (GPC), and size-exclusion
chromatography (SEC), is probably the
easiest mode of chromatography to
perform and to understand. It is widely
used in the biological sciences for the
resolution of macromolecules, such as
proteins and carbohydrates, and also is
used for the fractionation and
characterization of synthetic polymers.
Affinity Affinity chromatography is unique in YES YES
Chromatography that separation is based on the specific,
reversible interaction between a solute
molecule and a ligand immobilized on
the chromatographic stationary phase.
While discussed here as a separate
type of chromatography, affinity
chromatography could be viewed as
the ultimate extension of adsorption
chromatography. Affinity
chromatography usually involves
immobilized biological materials as the
stationary phase. These ligands can be
antibodies, enzyme inhibitors, lectins,
or other molecules that selectively and
reversibly bind to complementary
analyte molecules in the sample.
High Performance High performance liquid YES NO
Liquid chromatography is basically a highly
Chromatography improved form of column
chromatography. Instead of a solvent
being allowed to drip through a column
under gravity, it is forced through under
high pressures of up to 400
atmospheres. That makes it much
faster. It also allows you to use a very
much smaller particle size for the
column packing material which gives a
much greater surface area for
interactions between the stationary
phase and the molecules flowing past
it. This allows a much better separation
of the components of the mixture.

3. Samples that undergo chromatography.

Column chromatography is used for the purification of biomolecules samples.

Since proteins have different characteristic features as size, shape, net charge, stationary
phase used, and binding capacity, each one of these characteristic components can be
purified using chromatographic methods. Ion – exchange chromatography is based on
electrostatic interactions between charged protein groups, and solid support material
(matrix) samples. Gel – permeation (molecular sieve) chromatography is to use dextran
containing samples to separate macromolecules based on their differences in molecular
sizes. Affinity chromatography is used for the purification of enzymes, hormones,
antibodies, nucleic acids, and specific proteins. Paper chromatography support material
consists of a layer of cellulose highly saturated with water; it is a “liquid-liquid”
chromatography. Thin-layer chromatography is a “solid-liquid adsorption”
chromatography. In this method, stationary phase is a solid adsorbent substance coated
on glass plates. Gas chromatography is a “gas-liquid” chromatography. In this method,
the stationary phase is a column which is placed in the device and contains a liquid
stationary phase which is adsorbed onto the surface of an inert solid. Dye- ligand
chromatography was based on the demonstration of the ability of many enzymes to bind
purine nucleotides for Cibacron Blue F3GA dye. Hydrophobic interaction chromatography
(HIC) is based on hydrophobic interactions between side chains bound to
chromatography matrix. Pseudoaffinity chromatography are some compounds such as
anthraquinone dyes, and azo-dyes that can be used as ligands because of their affinity
especially for dehydrogenases, kinases, transferases, and reductases. High-pressure
liquid chromatography (HPLC) is possible to perform structural, and functional analysis,
and purification of many molecules within a short time. This technique yields perfect
results in the separation, and identification of amino acids, carbohydrates, lipids, nucleic
acids, proteins, steroids, and other biologically active molecules.

4. What type of chromatography is used for assay of Aspirin?

The type of chromatographic method that was developed for the determination of
aspirin (acetylsalicylic acid) in drug preparations, tissues, and in solutions is Gas
chromatography. Gas chromatography is a term used to describe the group of analytical
separation techniques used to analyze volatile substances in the gas phase. In gas
chromatography, the components of a sample are dissolved in a solvent and vaporized
in order to separate the analytes by distributing the sample between two phases: a
stationary phase and a mobile phase. The mobile phase is a chemically inert gas that
serves to carry the molecules of the analyte through the heated column. Gas
chromatography is one of the sole forms of chromatography that does not utilize the
mobile phase for interacting with the analyte. The stationary phase is either a solid
adsorbent, termed gas-solid chromatography (GSC), or a liquid on an inert support,
termed gas-liquid chromatography (GLC).


Al-anbaqi, R. (n.d.). Biotechnology & Genetic Engineering. Retrieved April 24, 2019 from

Chemistry Libretexts. (n.d.). Gas Chromatography. Retrieved April 24, 2019 from

Crippen, R.C. & Freimuth, H. C. (1964). Determination of Aspirin by Gas

Chromatography. Retrieved April 24, 2019 from

Ismael, B. & Nielsen, S.S. (2010). Basic principles of chromatography. DOI 10.1007/978-
1-4419-1478-1_27. Springer Science+Business Media, LLC 2010