Chromatography is a family of analytical chemistry techniques used to separate mixtures based on differences in how components interact with two phases - a stationary phase and a mobile phase. The document discusses the history and definition of chromatography, as well as several types including liquid chromatography, gas chromatography, paper chromatography, thin layer chromatography. It provides examples of chromatography uses in fields like petroleum engineering, forensics, and environmental testing. The key aspects and processes of each technique are described.

1. ‫مح‬‫مم‬ ‫مد‬‫سكشن‬ ‫عبدالسالم‬ ‫محمود‬ ‫دوح‬4
‫م‬‫ــــــ‬‫وه‬ ‫خــالد‬ ‫حمد‬‫ـــ‬‫الشريـــف‬ ‫بة‬‫سكشن‬4
‫محـمد‬ ‫الفتاح‬ ‫عبد‬ ‫جميل‬ ‫محمود‬‫سكشن‬5
‫أح‬ ‫إبراهــــيم‬ ‫حمدي‬ ‫بيومي‬‫ـــ‬‫مد‬‫سكشن‬2
Suez University
Faculty of Petroleum and Mining Engineering
Petroleum Engineering Department
Chromatography
Dr.Hamdy El-Kady

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Chromatography
Definition and its History
It is a family of analytical chemistry techniques for the separation of mixtures. It also could
be defined as a physical method of separation that distributes components to separate between
two phases, one stationary (stationary phase), the other (the mobile phase) moving in a definite
direction. It was the Russian botanist Mikhail Tsvet (Mikhail Semyonovich Tsvet) who
invented the first chromatography technique in 1901.He continued to work with
chromatography in the first decade of the 20th century, primarily for the separation of
plant pigments such as chlorophyll, carotenes, and xanthophylls. Since these components
have different colors (green, orange, and yellow, respectively) they gave the technique its
name. New types of chromatography developed during the 1930s and 1940s made the
technique useful for many separation processes.
Keep in Mind :
The separation of molecules depends on differences of
1- size 2- shape
3-Mass 4- Charges
5- Solubility 6- Adsorption
Uses of chromatography & Advantages of using chromatography
- Government laboratories used to check:
1- for approved dyes in food.
2- that vegetables contained tiny amounts of pesticides and herbicides.
- it is also used in the field of petroleum engineering.
- Its main advantages are:
1- Require very minute amount for identification.
2- Can be used to identify substances that cannot be easily melted or distilled.

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Types of Chromatography:
1- Liquid chromatography: is used in the world to test water samples to look for pollution
in lakes and rivers. It is used to analyze metal ions and organic compounds in solutions.
Liquid chromatography uses liquids which may incorporate hydrophilic, insoluble
molecules.
2- Partition chromatography e.g. paper chromatography: is one of the most common types
of chromatography. It uses a strip of paper as the stationary phase. Capillary action is
used to pull the solvents up through the paper and separate the solutes.
3- Gas Chromatography: is used in airports to detect bombs and is used is forensics in
many different ways. It is used to analyze fibers on a person's body and also analyze
blood found at a crime scene. In gas chromatography helium is used to move a gaseous
mixture through a column of absorbent material. Thin layer chromatography (TLC).
4- Thin-layer Chromatography: uses an absorbent material on flat glass or plastic plates.
This is a simple and rapid method to check the purity of an organic compound. It is
used to detect pesticide or insecticide residues in food. Thin-layer chromatography is
also used in forensics to analyze the dye composition of fibers.
Keep in Mind :
All types of chromatography involve interaction between:
1- The mixture to be separated. 2- The stationary phase.
3- The mobile phase.
Liquid chromatography
Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid.
It can be carried out either in a column or a plane. Present day liquid chromatography that
generally utilizes very small packing particles and a relatively high pressure is referred to
as high performance liquid chromatography (HPLC).
In HPLC the sample is forced by a liquid at high pressure (the mobile phase) through a
column that is packed with a stationary phase composed of irregularly or spherically shaped
particles, a porous monolithic layer, or a porous membrane. HPLC is historically divided into
two different sub-classes based on the polarity of the mobile and stationary phases. Methods
in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile
phase, silica as the stationary phase) are termed normal phase liquid chromatography
(NPLC) and the opposite (e.g., water-methanol mixture as the mobile phase and C18

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(octadecylsilyl) as the stationary phase) is termed reversed phase liquid chromatography
(RPLC).
Paper chromatography
Paper chromatography is an analytical
method used to separate colored
chemicals or substances. It is primarily
used as a teaching tool, having been
replaced by other chromatography
methods, such as thin-layer
chromatography. A paper
chromatography variant, two-
dimensional chromatography involves
using two solvents and rotating the paper
90° in between. This is useful for
separating complex mixtures of
compounds having similar polarity, for
example, amino acids. The setup has
three components. The mobile phase is a
FIG- Preparative HPLC apparatus

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solution that travels up the stationary phase, due to capillary action. The mobile
phase is generally an alcohol solvent mixture, while the stationary phase is a strip
of chromatography paper, also called a chromatogram. A chromatographic
method is called adsorption chromatography if the stationary phase is solid.
Rƒ value, solutes, and solvents
The retardation factor (Rƒ) may be defined as the ratio of the distance traveled by
the substance to the distance traveled by the solvent. Rƒ values are usually
expressed as a fraction of two decimal places. If Rƒ value of a solution is zero, the
solute remains in the stationary phase and thus it is immobile. If Rƒ value = 1 then
the solute has no affinity for the stationary phase and travels with the solvent
front. To calculate the Rƒ value, take the distance traveled by the substance
divided by the distance traveled by the solvent (as mentioned earlier in terms of
ratios). For example, if a compound travels 9.9 cm and the solvent front travels
12.7 cm, (9.9/12.7) the Rƒ value = 0.779 or 0.78. Rƒ value depends on temperature
and the solvent used in experiment, so several solvents offer several Rƒ values for
the same mixture of compound. A solvent in chromatography is the liquid the
paper is placed in, and the solute is the ink which is being separated.
Types of Paper Chromatography
1. Descending Paper Chromatography-In this type, development of the
chromatogram is done by allowing the solvent to travel down the paper. Here,
mobile phase is placed in solvent holder at the top. The spot is kept at the top and
above solvent flow down the paper from above.
2. Ascending Paper Chromatography-Here the solvent travels up the
chromatographic paper. Both Descending and Ascending Paper Chromatography
are used for the separation of organic and inorganic substances.
3. Ascending-Descending Paper Chromatography-It is the hybrid of both of the
above techniques. The upper part of Ascending Chromatography can be folded
over a rod in order to allow the paper to become Descending after crossing the
rod.
4. Radial Paper Chromatography-It is also called Circular Chromatography.
Here a circular filter paper is taken and the sample is deposited at the center of
the paper. After drying the spot, the filter paper is tied horizontally on a Petri dish
containing solvent, so that the wick of the paper is dipped in the solvent. The

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solvent rises through the wick and the components are separated into concentric
circles.
5. Two-Dimensional Paper Chromatography-In this technique a square or
rectangular paper is used. Here the sample is applied to one of the corners and
development is performed at right angle to the direction of the first run.
Gas Chromatography
Gas chromatography is one of the most widely used techniques for analyzing
hydrocarbon mixtures. Some of the advantages of chromatography are the range
of measurement (from ppm levels up to 100 %), the detection of a wide range of
components, and the repeatability of the measurements. Chromatography is used
in the laboratory, in permanently installed online systems, and in the field with
portable systems. No matter the location, style, or brand, all gas chromatographs
are composed of the same functional components: the sample handling system, the
chromatograph oven, and the controller electronics.

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Thin-layer chromatography
Thin-layer chromatography (TLC) is a chromatography technique used to separate non-
volatile mixtures. Thin-layer chromatography is performed on a sheet of glass, plastic, or
aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel,
aluminium oxide (alumina), or cellulose. This layer of adsorbent is known as the stationary
phase.
After the sample has been applied on the plate,
a solvent or solvent mixture (known as the
mobile phase) is drawn up the plate via
capillary action. Because different analytes
ascend the TLC plate at different rates,
separation is achieved. The mobile phase has
different properties from the stationary phase.
For example, with silica gel, a very polar
substance, non-polar mobile phases such as
heptane are used. The mobile phase may be a
mixture, allowing chemists to fine-tune the
bulk properties of the mobile phase.
After the experiment, the spots are visualized.
Often this can be done simply by projecting
ultraviolet light onto the sheet; the sheets are
treated with a phosphor, and dark spots
appear on the sheet where compounds absorb
the light impinging on a certain area.
Chemical processes can also be used to
visualize spots; anisaldehyde, for example,
forms colored adducts with many compounds,
and sulfuric acid will char most organic
compounds, leaving a dark spot on the sheet.
To quantify the results, the distance traveled
by the substance being considered is divided
by the total distance traveled by the mobile
phase. (The mobile phase must not be allowed to reach the end of the stationary phase.) This
ratio is called the retention factor or Rf. In general,a substance whose structure resembles the
stationary phase will have low Rf, while one that has a similar structure to the mobile phase
will have high retention factor. Retention factors are characteristic, but will change depending
on the exact condition of the mobile and stationary phase. For this reason, chemists usually
apply a sample of a known compound to the sheet before running the experiment.

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Thin-layer chromatography can be used to monitor the progress of a reaction, identify
compounds present in a given mixture, and determine the purity of a substance. Specific
examples of these applications include: analyzing ceramides and fatty acids, detection of
pesticides or insecticides in food and water, analyzing the dye composition of fibers in
forensics, assaying the radiochemical purity of radiopharmaceuticals, or identification of
medicinal plants and their constituents .
A number of enhancements can be made to the original method to automate the different
steps, to increase the resolution achieved with TLC and to allow more accurate quantitative
analysis. This method is referred to as HPTLC, or "high-performance TLC". HPTLC
typically uses thinner layers of stationary phase and smaller sample volumes, thus reducing
the loss of resolution due to diffusion.