A review of Chromatography: principles, Classification, Applications.
By: Mohamed Ahmed Sayed
October 24, 2021
Department of chemistry, Helwan University
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Abstract ....................................................................................... Error! Bookmark not defined.
Introduction and some terminologies ............................................. Error! Bookmark not defined.
Classification ................................................................................ Error! Bookmark not defined.
Principle of chromatography ......................................................... Error! Bookmark not defined.
Planar chromatography ................................................................ Error! Bookmark not defined.
Paper chromatography (PC) ...................................................... Error! Bookmark not defined.
er chromatography (TLC)lay-Thin ............................................ Error! Bookmark not defined.
Column chromatography .............................................................. Error! Bookmark not defined.
atography (HPLC)Liquid chrom ............................................... Error! Bookmark not defined.
Applications of liquid chromatography ...................................... Error! Bookmark not defined.
Conclusion ................................................................................... Error! Bookmark not defined.
References .................................................................................... Error! Bookmark not defined.
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Chromatography is a separation process used to separate components in a mixture. The
components of the mixture are dispersed in a liquid solution known as the mobile phase., which
holds it through a structure containing another substance known as the stationary phase.
Component separation requires differential partitioning between the mobile and stationary
phases. The analytical goal of chromatography is to determine the qualitative and quantitative
chemical makeup of a sample, and its primary purpose is to purify and extract one or more
components of a sample. This paper will discuss the history and basics of what chromatography
is meant and the main principles of how we can run it. besides, we will mention and focus on
an application for each chromatographic type such as planar, TLC, gas, liquid separation
Keywords: chromatographic separation technique; thin layer chromatography; High-
performance liquid chromatography; elution time; applications.
Introduction and some terminologies
Chromatography means color-writing and the more specific definition is, it is a physical
process of separation at which a mixture of compounds can be separated and isolated, purified
into different molecules that depend on different distribution rates depending on 1. Solubility
2. Affinity (if polar or non-polar molecules) 3. Interaction with fixed material (the stationary
phase, which we will define later), the components in the mixture are dispersed between two
phases, the stationary phase, and the mobile phase, that moves at various speeds in a specified
It is known that Michael tswett, the Russian botanist in 1901 observe that chlorophyll
pigments are separated into different colored components when he uses a column containing
CaCO3 and moves its mixture on it .so, he is named the founder and father of chromatography,
Archer John Porter Martin and Richard Laurence Millington in 1952 won Nobel Prize in
Chemistry for their work and efforts in developed many- based separation techniques like
partition (liquid-liquid chromatography). [3,4]
Any chromatographic separation technique must contain the three main parts as
follows: 1. Sample 2. Mobile phase 3. Stationary phase. stationary phase: it is the solid
substance at which the mixture of the components will be separated and isolated, its nature is
a solid or a liquid only. Mobile phase: it is a solid or liquid substance that carries a mixture
composed of a sample to be purified, isolated, separated at the surface of the stationary phase.
There are two types of chromatographic separation techniques, the first is Normal
phase liquid chromatography (NPLC) in which the stationary phase is polar.in contrast, the
mobile phase is non-polar, the second is reversed-phase liquid chromatography (RPLC) in
which the stationary phase is non-polar, and the mobile phase is polar. To perform a good
separation, we should choose the suitable parameter between the stationary and mobile
The main purpose of chromatography is in between primitive that depend on separate
and isolate only the mixture sample rather than determine the concentration of the purified
sample, and the analytical that determine the chemical composition of a sample and its
We can classify and summarize the chromatographic method technique into three
different ways as the following:
1) Depend on the shape of the stationary phase. e.g.- planar and column chromatography.
2) Depend on the physical state of both stationary and mobile phase. e.g.-gas and liquid
3) Depend on the interaction between stationary and mobile phase. e.g.- affinity, ion
exchange, partition, adsorption, size exclusion chromatography.
Figure (1) shows the classification of the chromatographic method.
Principle of chromatography 
molecules in a mixture are fixed on the surface of the stationary face, and the mobile
phase will be injected to pass on the solid phase carrying the mixture to be separated. Molecular
features linked to adsorption (liquid-solid), partition (liquid-solid), and affinity or variations
among their molecular weights are the most important factors effective on this separation
process. For these differences, some components in the mixture take a long time on the
stationary phase and move slowly through the chromatographic system, while others leave the
Figure (1). A graphical diagram shows the classification of chromatography according to three
different parameters to form many and vary techniques.
Figure (2). illustrates the chromatographic separation process internal the column technique
until it reaches the detector.
Figure (2) shows the main procedure for the chromatographic separation method.
Here, the mobile phase is a liquid solution that moves through the stationary phase that
may be liquid or cellulosic (paper chromatography) or solid containing silica gel or alumina
(thin layer chromatography) by gravity or capillary action. 
Paper chromatography (PC)
The stationary phase and the mobile phase are both liquids (partition chromatography),
the polar adsorbed water in the paper act as the stationary phase in a 2D plate. The dissolving
sample is placed as a small spot one-half inch from the edge of filter paper and left to dry. The
dry spot will be held at the front end in a closed chamber saturated with atmosphere, and the
end closer to the sample contacts the solvent, which moves up or down by the capillary action
(depending on the mode of action whether ascending means moves up along the paper or
descending that moves down due to high viscosity of thus mobile phase). when the mobile
phase mixture reaches the final length of the paper, we removed the un-colored spots that are
separated in the paper and measure each separated zone by an appropriate method called
Retention factor or rate flow (Rf). 
Retention factor =
Retention factor is a qualitatively determination and identifier to the new separated
components, and it is a standard value in a range of 0,1, when the value of Rf is close as possible
to 0, it refers to the good interaction that occurs between the sample components and the
stationary phase due to high polarity of both stationary and mobile phase. On the other hand,
when the value of Rf within range equals 1, this means that a week or low interaction occurs
between the stationary phase and sample components. Therefore, the mobile phase is a polar
substance, and the stationary phase is a non-polar one.
Distance moved by solute
Distance moved by solvent
In the '50s, some studies have used this method to purify amino acids compounds [9-
13], as well as drug purification [14,15], plant extract purification, and separation for abscise
acid , and isolation of gram-positive bacteria cell wall teichoic acids .
In conclusion, the process is useful due to it is rapid and uses only a small amount of material.
The technique's disadvantages, on the other hand, include its extensive and time-
consuming procedures, as well as its low resolving power and reproducibility [18,19].
Because the two techniques operate on the same principle, paper chromatography is
sometimes replaced with Thin Layer Chromatography (TLC), When samples are run on the
same paper chromatography as unknowns, moreover, paper chromatography is especially
successful at identifying unknown compounds.
Thin-layer chromatography (TLC)
In thin-layer chromatography, the mobile phase is liquid while the stationary phase is
solid and interacts with a high surface area to form solid-liquid adsorption. Capillary action
propels the mobile phase upward through the stationary phase (thin plate soaked with the
solution). This upward motion rate is affected by the polarity of the substance, solid phase, and
solvent. Thus, we can use a colored chemical substance that will accept the non-developing
thin plate color to be developed and appear at chromatogram and identify as a separated peak
each one, the most common substance is ninhydrin and blacklight visualization technique. 
Thin-layer chromatography can purify macromolecules such as amino acids, active
ingredients, preservatives in drugs and drug preparations and contribute to synthetic-
manufacturing processes, aromatic amines on silica gel layers, the biological source for active
substances and their metabolites e.g., urinary constituents such as steroids, amino acids,
porphyrins, and bile acids. Also, it separates a complex drug component, determines pesticides
by using cationic and non-ionic surfactant-mediated systems as mobile phases .
The column is a three-dimensional shape model that may be packed or open tubular in
geometrical structure. in packed, the stationary phase is especially filled and occupy the wall
and spaces of all the column. But, in the open tubular, the stationary phase is with the column
There are two main abroad groups classified the column chromatography as the
following: 1. Liquid chromatography (HPLC).2. Gas Chromatography (GC).
The classification depends on the polarity for each phase can be 1. Normal Phase
Chromatography (NPLC).2. Reversed-Phase Chromatography (RPLC).
The classification depends on many other practical factors for each phase can be 1.
Isothermal (constant temperature).2. isocratic (constant mobile phase). 
Liquid chromatography (HPLC)
It is called high-performance liquid chromatography or high-pressure liquid
chromatography, HPLC is primarily based on the use of a column that contains packing
material. (Stationary phase), a pump that drives the mobile phase(s) across the column, and a
detector that displays the molecule retention durations. The retention time is governed by the
interactions between the stationary phase, the molecules being analyzed, and the solvent(s)
utilized . The sample is usually added to the mobile phase stream and is slowed by chemical
or physical interactions with the stationary phase. Gradient elution changes the mobile phase
composition during the analysis. The gradient separates analyte mixtures based on the analyte's
affinity for the mobile phase. The nature of the stationary phase and the sample influence the
choice of mobile phase, additives, and gradient. 
There are many types of HPLC, we will focus on only Normal phase-HPLC, Reversed-
HPLC, size exclusion-HPLC. In normal phase-HPLC, A polar stationary phase and a non-polar
mobile phase are used. The polar stationary phase interacted with and held the polar analyte.
Adsorption strengths raise as analyte polarity increases, and the interaction between the polar
analyte and the polar stationary phase prolongs elution time or retention factor. On the other
hand, Reversed-HPLC has a non-polar stationary phase and a moderately polar aqueous mobile
phase. it works on the principle of hydrophobic interactions, which are caused by repulsive
forces between a polar solvent, a comparatively non-polar analyte, and a non-polar stationary
phase. besides, size exclusion-HPLC or gel permeation chromatography can separate based on
particle size, it determines the tertiary and quaternary structures of proteins and amino acids.
Also, the molecular weight of polysaccharides. 
Applications of liquid chromatography
In Pharmaceutical applications [24-27], 1-evaluate of pharmaceutical product shelf-
life.2-Identify active constituents in dosage forms. 3- develop pharmaceutical quality control
In Environmental applications [27-31],1- Identify diphenhydramine in deposited
samples.2- Pollutant biomonitoring.
In Clinical [32,33,34,22], 1- Estimation of bilirubin and bilivirdin levels in blood
plasma in the presence of hepatic diseases.2- Detection of endogenous neuropeptides in brain
It can be concluded from the entire review that each type of chromatographic separation
technique has its great effective, sensitive, major work application in industry and clinical and
most human being fields. Chromatography techniques improve chemical and instrumentation
productivity by giving more information due to increased resolution, speed, and sensitivity.
The time spent refining new methods can be significantly reduced.
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