3. Learning Objectives
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BME 1201: Biochemistry
1. Basics of chromatography
2. Types of chromatography
3. Uses of chromatography
4. Chromatography
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▪ Chromatography is a combination of two words:
Chromo – Meaning color
Graphy – representation of something on paper
BME 1201: Biochemistry
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BME 1201: Biochemistry
▪ It is a physical separation method in which the components of a mixture are
separated by differences in their distribution between two phases, one of which
is stationary (stationary phase) while the other (mobile phase) moves through
it in a definite direction. The substances must interact with the stationary phase
to be retained and separated by it.
Chromatography - Definition
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BME 1201: Biochemistry
▪ Three components form the basis of the chromatography technique: stationary phase,
mobile phase and separated molecules.
▪ The type of interaction between stationary phase, mobile phase, and substances contained
in the mixture is the basic component effective on separation of molecules from each
other.
▪ The factors effective on this separation process include molecular characteristics related to
adsorption (liquid-solid), partition (liquid-solid), and affinity or differences among their
molecular weights. Because of these differences, some components of the mixture stay
longer in the stationary phase, and they move slowly in the chromatography system, while
others pass rapidly into mobile phase, and leave the system faster.
Basics of Chromatography
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BME 1201: Biochemistry
Column Chromatography
▪ It is the most powerful methods for purifying proteins, which
takes advantage of differences in protein charge, size, binding
affinity, and other properties. A porous solid material with
appropriate chemical properties (the stationary phase) is held in
a column, and a buffered solution (the mobile phase) percolates
through it. The protein-containing solution, layered on the top
of the column, percolates through the solid matrix as an ever-
expanding band within the larger mobile phase. Individual
proteins migrate faster or more slowly through the column
depending on their properties.
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BME 1201: Biochemistry
Ion-exchange Chromatography
▪ It is based on electrostatic interactions between charged
protein groups, and solid support material (column matrix).
Matrix has an ion load opposite to that of the protein to be
separated, and the affinity of the protein to the column is
achieved with ionic ties. Proteins are separated from the
column either by changing pH, concentration of ion salts or
ionic strength of the buffer solution. Positively charged ion-
exchange matrices are called anion-exchange matrices, and
adsorb negatively charged proteins. While matrices bound
with negatively charged groups are known as cation-exchange
matrices, and adsorb positively charged proteins.
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BME 1201: Biochemistry
Affinity Chromatography
▪ It is based on binding affinity. The beads in the column have a
covalently attached ligand (a group or molecule that binds to a
macromolecule such as a protein). When a protein mixture is added to
the column, any protein with affinity for this ligand binds to the
beads, and its migration through the matrix is retarded. For example,
if the biological function of a protein involves binding to ATP, then
attaching ATP to the beads in the column creates an affinity matrix
that can help purify the protein. As the protein solution moves through
the column, ATP-binding proteins (including the protein of interest)
bind to the matrix. After proteins that do not bind are washed through
the column.
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BME 1201: Biochemistry
▪ It is an important biophysical technique that enables the separation,
identification, and purification of the components of a mixture for qualitative
and quantitative analysis. Proteins can be purified based on characteristics
such as size and shape, total charge, hydrophobic groups present on the
surface, and binding capacity with the stationary phase.
▪ Separate and purify biomolecules.
▪ Quantify the amount of specific compounds.
▪ Characterize properties of biomolecules.
▪ Identify unknown compounds.
▪ Study interactions between biomolecules.
▪ Ensure quality in biopharmaceuticals.
Uses of Chromatography
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BME 1201: Biochemistry
Chromatography methods are very effective on separation, and identification of small
molecules as amino acids, carbohydrates, and fatty acids, proteins and nucleic acids.
▪ Affinity chromatography (i.e. ion-exchange chromatography) are more effective in
the separation of macromolecules as nucleic acids, and proteins.
▪ Paper chromatography is used in the separation of proteins, and in studies related to
protein synthesis; gas-liquid chromatography is utilized in the separation of alcohol,
lipid, and amino groups, and observation of enzymatic interactions,
▪ Agarose-gel chromatography is used for the purification of RNA, DNA particles, and
viruses
Uses of Chromatography in Biochemistry
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BME 1201: Biochemistry
Reference Books
1. Lehninger Principles of Biochemistry, 6th Edition by David L. Nelson and
Michael M. Cox.
2. Separation techniques: Chromatography by Ozlem Coskun
