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Chromatography

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24 views6 pages

Chromatography

Brb

Uploaded by

Ahmed Ali
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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Chromatography

Definition and Principle


Chromatography is a technique used to separate different components of a
mixture based on their relative distribution between two phases, a stationary
phase and a mobile phase.
The basic components in any chromatographic technique are:
1. The mobile phase (gas or liquid), which carries the complex mixture
(sample).
2. The stationary phase (solid or liquid), through which the mobile phase
flows.
3. The column holding the stationary phase.
4. The separated components (eluate).
A substance which is more distributed in mobile phase will migrate more than
the one which is more distributed in stationary phase. This will lead to their
separation.

Advantages of Chromatography
1) Can separate very complex mixtures (drugs, plastics, flavorings, foods,
pesticides, tissue extracts, fuels, air samples, water samples, ...)
2) Very small sample sizes
3) Separated components can be collected individually
4) Analyses can be highly accurate and precise

Basic forms of Chromatography


Planar and column are the two basic forms of chromatography.
1. In planar chromatography, the stationary phase is coated on a sheet of
paper (paper chromatography) or is bound to a solid surface [thin-layer
chromatography (TLC)].
2. In column chromatography, the stationary phase may be a pure silica
or polymer, or it may be coated onto, or chemically bonded to, support
particles. The stationary phase may be “packed” into a tube, or it may be
coated onto the inner surface of the tube. Column chromatography
includes both gas chromatography (GC) and liquid chromatography
(LC); with classification dependent on whether the mobile phase is a gas
or a liquid.

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Separation Mechanisms
Chromatographic separations are classified by the chemical or physical
mechanisms used to separate the solutes. These include
1. Ion-exchange
2. Adsorption
3. Partition
4. Affinity
5. Size-exclusion mechanisms.

Ion-Exchange
Ion-exchange chromatography relies on the exchange of ions between charged
groups bound to a stationary phase and ions of opposite charge in the mobile
phase. Depending on the charge of the stationary phase, components binding
to the column may be cations (positively charged) or anions (negatively
charged).
Ion-exchange chromatography has a number of clinical applications,
including:
1. Analyses of amino acids and hemoglobins.
2. It is also used to analyze small inorganic and organic ions with
conductivity detection, a technique termed ion chromatography.
3. Water purification is an important preparative application of ion-
exchange chromatography.

Adsorption
Adsorption chromatography, also known as liquid-solid Chromatography. The
basis of separation by adsorption chromatography is the difference between
adsorption (The accumulation of gases, liquids, or solutes on the surface of a
solid or liquid.) and desorption to remove an absorbate or adsorbate from an
absorbent or adsorbent) of solutes at the surface of a solid particle. i.e. is based
on the competition between the sample and the mobile phase for
adsorptive sites on the solid stationary phase.
Electrostatic, hydrogen-bonding, and dispersive (Van der Waals) interactions
are the physical forces that control this type of chromatography. Liquid-solid
chromatography is not widely used in clinical laboratories because of technical

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problems with the preparation of a stationary phase that has homogeneous
distribution of absorption sites.

Partition Chromatography
Partition chromatography is also referred to as liquid – liquid Chromatography.
The differential distribution of solutes between two immiscible liquids is the
basis for separation by partition chromatography , one of the immiscible liquids
serves as the stationary phase. Partition chromatography is applicable to any
substance that may be distributed between two liquid phases.

Affinity Chromatography
Specific molecular interactions are used as the basis for affinity
chromatography. Examples include:
1. Interactions of antibody with antigen,
2. Enzyme with substrate,
3. Aptamer ( a short segment of DNA, RNA, or peptide that binds to a
specific molecular target (such as a protein) with ligand (A ligand is an
ion or molecule that binds to a central metal atom to form a complex)
4. Receptor with ligand, and
5. Lectin (carbohydrate-binding proteins,) with sugar.

Chromatographic Procedures
1. Thin-layer chromatography
2. High – performance liquid chromatography
3. Gas chromatography

Thin-Layer Chromatography
Thin-layer chromatography (TLC) is a variant of column chromatography. A
thin layer of sorbent, such as alumina, silica gel, cellulose, or cross-linked
dextran, is uniformly coated on a glass or plastic plate. Each sample to be
analyzed is applied as a spot near one edge of the plate.

High-Performance Liquid Chromatography


Modern liquid chromatography uses pressure for fast separations. High
performance liquid chromatography (HPLC) is basically a highly improved
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form of column liquid 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.
All chromatographic separations, including HPLC operate under the same
basic principle; separation of a sample into its constituent parts because of the
difference in the relative affinities of different molecules for the mobile phase
and the stationary phase used in the separation.

Figure Components of HPLC

Gas Chromatography
Principle of gas chromatography: The sample solution injected into the
instrument enters a gas stream which transports the sample into a separation
tube known as the "column." (Helium or nitrogen is used as the so-called
carrier gas.) The various components are separated inside the column. The
detector measures the quantity of the components that exit the column.

Advantages of Gas Chromatography


1) High separation efficiency and analysis speed.
2) Small sample consumption and high detection sensitivity.

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3) Gas chromatography has good selectivity and can be used to analyze
azeotropic mixture.
4) Wide range of applications.

Disadvantages of Gas Chromatography


It can only be used to analyze volatile substances.

Mass Spectrometry
Mass spectrometry (MS) is an analytical technique that is used to measure the
mass-to-charge ratio of ions. The results are typically presented as a mass
spectrum, a plot of intensity as a function of the mass-to-charge ratio.

What is a mass spectrometer used for?


mass spectrometry is an analytical tool useful for measuring the mass-to-
charge ratio (m/z) of one or more molecules present in a sample. These
measurements can often be used to calculate the exact molecular weight of the
sample components as well.

What is a basic principle of mass spectrometry?


"The basic principle of mass spectrometry (MS) is to generate ions from either
inorganic or organic compounds by any suitable method, to separate these ions
by their mass-to-charge ratio (m/z) and to detect them qualitatively and
quantitatively by their respective m/z and abundance.

Basic Principle of mass spectrometry


What are the four stages of a mass spectrometry? There are four stages in a
mass spectrometer which we need to consider, these are: (1) ionization (2)
acceleration (3) deflection and (4) detection.

How does mass spectrometry identify compounds?


Most of the ions formed in a mass spectrometer have a single charge, so the
m/z valueis equivalent to mass itself. Modern mass spectrometers easily
distinguish (resolve) ions differing by only a single atomic mass unit (AMU),

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and thus provide completely accurate values for the molecular mass of a
compound.

Mass Spectrometry –MSU chemistry


Who uses mass spectrometry?
Specific applications of mass spectrometry include drug testing and discovery,
food contamination detection, pesticide residue analysis, isotope ratio
determination, protein identification, and carbon dating.
Since mass spectrometers can work both in directed and non-directed fashion
as well as in positive or negative mode, there are almost no molecules it cannot
detect. Thus, even if you work with a synthetic drug, a modified protein or a
hard to solubilize lipid, MS can help you to quantify it.

What is nitrogen rule in mass spectrometry?


The nitrogen rule states that a molecule that has no or even number of nitrogen
atoms has an even nominal mass, whereas a molecule that has an add number
of nitrogen atoms has an add nominal mass.

Nitrogen Rule –Chemistry Libre Texts


What are the three major components of a mass spectrometer?
A mass spectrometer consists of three components: (1) an ion source (2) a mass
analyzer and (3) a detector. The ionizer converts a portion of the sample into
ions.

Which radiation is used in mass spectroscopy?


A 336 nm radiation from nitrogen laser is most commonly used. The laser helps
introducing energy into molecular system in such a way preventing thermal
decomposition.

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