CHROMATOGRAPHY
Chromatography- Chromatography is an important biophysical technique that enables the
separation, identification, and purification of the components of a mixture for qualitative and
quantitative analysis A wide range of chromatographic procedures makes use of differences in
size, binding affinities, charge, and other properties to separate materials. It is a powerful
separation tool that is used in all branches of science and is often the only means of separating
components from complex mixtures.
Principle of Chromatography Chromatography is based on the principle where molecules in
mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is
separating from each other while moving with the aid of a mobile phase. 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 the mobile
phase, and leave the system faster.Three components thus form the basis of the
chromatography technique.
Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid
adsorbed on the surface solid support”.
Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
Separated molecules The type of interaction between the stationary phase, mobile phase, and
substances contained in the mixture is the basic component effective on the separation of
molecules from each other.
Applications of Chromatography-
Pharmaceutical sector
To identify and analyze samples for the presence of trace elements or chemicals.
Separation of compounds based on their molecular weight and element composition.
Detects the unknown compounds and purity of mixture.
In drug development.
Chemical industry
In testing water samples and also checks air quality.
HPLC and GC are very much used for detecting various contaminants such as
polychlorinated biphenyl (PCBs) in pesticides and oils.
In various life sciences applications
Food Industry- In food spoilage and additive detection. Determining nutritional quality of food
Molecular Biology Studies
Various hyphenated techniques in chromatography such as EC-LC-MS are applied in the
study of metabolomics and proteomics along with nucleic acid research.
HPLC is used in Protein Separation like Insulin Purification, Plasma Fractionation, and
Enzyme Purification and also in various departments like Fuel Industry, biotechnology, and
biochemical processes.
,1. Column chromatography-
Definition- Column chromatography is a technique in which the substances to be separated are
introduced onto the top of a column packed with an adsorbent, passed through the column at
different rates that depend on the affinity of each substance for the adsorbent and for the
solvent or solvent mixture, and are usually collected in solution as they pass from the column at
different times. It is a solid-liquid technique in which the stationary phase is a solid & the mobile
phase is a liquid or gas.
Principle- In column chromatography the stationary phase is packed into a glass or metal
column. The mixture of analytes is then applied and the mobile phase, commonly referred to as
the eluent, is passed through the column either by use of a pumping system or applied gas
pressure.The stationary phase is either coated onto discrete small particles (the matrix) and
packed into the column or applied as a thin film to the inside wall of the column.As the eluent
flows through the column the analytes separate on the basis of their distribution coefficients and
emerge individually in the eluate as it leaves the column.
Instrumentation of Column Chromatography
A stationary phase:- Chosen to be appropriate for the analytes to be separated.
A column:- In liquid chromatography these are generally 25- 50 cm long and 4mm internal
diameter and made of stainless steel whereas in gas chromatography they are 1-3m long and 2-
4mm internal diameter and made of either glass or stainless steel.They may be either of the
conventional type filled with the stationary phase, or of the microbore type in which the
stationary phase is coated directly on the inside wall of the column.
A mobile phase and delivery system: Chosen to complement the stationary phase and hence
to discriminate between the sample analytes and to deliver a constant rate of flow into column.
An injector system: To deliver test samples to the top of the column in a reproducible manner.
A detector and chart recorder: To give a continuous record of the presence of the analytes in
the eluate as it emerges from the column. Detection is usually based on the measurement of a
physical parameter such as visible or ultraviolet absorption or fluorescence. A peak on the chart
recorder represents each separated analyte.
A fraction collector: For collecting the separated analytes for further biochemical studies.
, Steps in Column Chromatography
A. Preparation of the Column-The column mostly consists of a glass tube packed with a
suitable stationary phase. A glass wool/cotton wool or an asbestos pad is placed at the botton of
the column before packing the stationary phase. After packing, a paper disc kept on the top, so
that the stationary layer is not disturbed during the introduction of sample or mobile phase.
There are two types of preparing the column, they are:
1. Dry packing / dry filling- In this the required quantity of adsorbent is poured as fine dry
powder in the column and the solvent is allowed to flow through the column till equilibrium is
reached.
2. Wet packing / wet filling- In this, the slurry of adsorbent with the mobile phase is prepared
and is poured into the column. It is considered as the ideal technique for packing. Before using
column, it should be washed properly and dried. The column should also be free from impurity
and uniformly filled with the stationary phase.
B. Introduction of the Sample- The sample which is usually a mixture of components is
dissolved in minimum quantity of the mobile phase. The entire sample is introduced into the
column at once and get adsorbed on the top portion of the column. From this zone, individual
sample can be separated by a process of elution.
C. Elution- By elution technique, the individual components are separated out from the column.
It can be achieved by two techniques:
Isocratic elution technique: Same solvent composition or solvent of same polarity is used
throughout the process of separation. Eg. Use of chloroform alone.
Gradient elution technique: Solvents of gradually ↑ polarity or ↑ elution strength are used
during the process of separation eg. initially benzene, then chloroform, then ethyl acetate then
chloroform
D. Detection of Components- If the compounds separated in a column chromatography
procedure are colored, the progress of the separation can simply be monitored visually. If the
compounds to be isolated from column chromatography are colorless. In this case, small
fractions of the eluent are collected sequentially in labelled tubes and the composition of each
fraction is analyzed by TLC.
Applications
Column chromatography is one of the most useful methods for the separation and
purification of both solids and liquids. Its major application includes:
Separation of mixture of compounds.
Removal of impurities or purification process.
Isolation of active constituents.
Isolation of metabolites from biological fluids.
Estimation of drugs in formulation or crude extracts.
Limitations
Time consuming method.
More amounts of solvents are required which may be expensive.
Automation makes the technique more complicated and costly.
Chromatography- Chromatography is an important biophysical technique that enables the
separation, identification, and purification of the components of a mixture for qualitative and
quantitative analysis A wide range of chromatographic procedures makes use of differences in
size, binding affinities, charge, and other properties to separate materials. It is a powerful
separation tool that is used in all branches of science and is often the only means of separating
components from complex mixtures.
Principle of Chromatography Chromatography is based on the principle where molecules in
mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is
separating from each other while moving with the aid of a mobile phase. 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 the mobile
phase, and leave the system faster.Three components thus form the basis of the
chromatography technique.
Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid
adsorbed on the surface solid support”.
Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
Separated molecules The type of interaction between the stationary phase, mobile phase, and
substances contained in the mixture is the basic component effective on the separation of
molecules from each other.
Applications of Chromatography-
Pharmaceutical sector
To identify and analyze samples for the presence of trace elements or chemicals.
Separation of compounds based on their molecular weight and element composition.
Detects the unknown compounds and purity of mixture.
In drug development.
Chemical industry
In testing water samples and also checks air quality.
HPLC and GC are very much used for detecting various contaminants such as
polychlorinated biphenyl (PCBs) in pesticides and oils.
In various life sciences applications
Food Industry- In food spoilage and additive detection. Determining nutritional quality of food
Molecular Biology Studies
Various hyphenated techniques in chromatography such as EC-LC-MS are applied in the
study of metabolomics and proteomics along with nucleic acid research.
HPLC is used in Protein Separation like Insulin Purification, Plasma Fractionation, and
Enzyme Purification and also in various departments like Fuel Industry, biotechnology, and
biochemical processes.
,1. Column chromatography-
Definition- Column chromatography is a technique in which the substances to be separated are
introduced onto the top of a column packed with an adsorbent, passed through the column at
different rates that depend on the affinity of each substance for the adsorbent and for the
solvent or solvent mixture, and are usually collected in solution as they pass from the column at
different times. It is a solid-liquid technique in which the stationary phase is a solid & the mobile
phase is a liquid or gas.
Principle- In column chromatography the stationary phase is packed into a glass or metal
column. The mixture of analytes is then applied and the mobile phase, commonly referred to as
the eluent, is passed through the column either by use of a pumping system or applied gas
pressure.The stationary phase is either coated onto discrete small particles (the matrix) and
packed into the column or applied as a thin film to the inside wall of the column.As the eluent
flows through the column the analytes separate on the basis of their distribution coefficients and
emerge individually in the eluate as it leaves the column.
Instrumentation of Column Chromatography
A stationary phase:- Chosen to be appropriate for the analytes to be separated.
A column:- In liquid chromatography these are generally 25- 50 cm long and 4mm internal
diameter and made of stainless steel whereas in gas chromatography they are 1-3m long and 2-
4mm internal diameter and made of either glass or stainless steel.They may be either of the
conventional type filled with the stationary phase, or of the microbore type in which the
stationary phase is coated directly on the inside wall of the column.
A mobile phase and delivery system: Chosen to complement the stationary phase and hence
to discriminate between the sample analytes and to deliver a constant rate of flow into column.
An injector system: To deliver test samples to the top of the column in a reproducible manner.
A detector and chart recorder: To give a continuous record of the presence of the analytes in
the eluate as it emerges from the column. Detection is usually based on the measurement of a
physical parameter such as visible or ultraviolet absorption or fluorescence. A peak on the chart
recorder represents each separated analyte.
A fraction collector: For collecting the separated analytes for further biochemical studies.
, Steps in Column Chromatography
A. Preparation of the Column-The column mostly consists of a glass tube packed with a
suitable stationary phase. A glass wool/cotton wool or an asbestos pad is placed at the botton of
the column before packing the stationary phase. After packing, a paper disc kept on the top, so
that the stationary layer is not disturbed during the introduction of sample or mobile phase.
There are two types of preparing the column, they are:
1. Dry packing / dry filling- In this the required quantity of adsorbent is poured as fine dry
powder in the column and the solvent is allowed to flow through the column till equilibrium is
reached.
2. Wet packing / wet filling- In this, the slurry of adsorbent with the mobile phase is prepared
and is poured into the column. It is considered as the ideal technique for packing. Before using
column, it should be washed properly and dried. The column should also be free from impurity
and uniformly filled with the stationary phase.
B. Introduction of the Sample- The sample which is usually a mixture of components is
dissolved in minimum quantity of the mobile phase. The entire sample is introduced into the
column at once and get adsorbed on the top portion of the column. From this zone, individual
sample can be separated by a process of elution.
C. Elution- By elution technique, the individual components are separated out from the column.
It can be achieved by two techniques:
Isocratic elution technique: Same solvent composition or solvent of same polarity is used
throughout the process of separation. Eg. Use of chloroform alone.
Gradient elution technique: Solvents of gradually ↑ polarity or ↑ elution strength are used
during the process of separation eg. initially benzene, then chloroform, then ethyl acetate then
chloroform
D. Detection of Components- If the compounds separated in a column chromatography
procedure are colored, the progress of the separation can simply be monitored visually. If the
compounds to be isolated from column chromatography are colorless. In this case, small
fractions of the eluent are collected sequentially in labelled tubes and the composition of each
fraction is analyzed by TLC.
Applications
Column chromatography is one of the most useful methods for the separation and
purification of both solids and liquids. Its major application includes:
Separation of mixture of compounds.
Removal of impurities or purification process.
Isolation of active constituents.
Isolation of metabolites from biological fluids.
Estimation of drugs in formulation or crude extracts.
Limitations
Time consuming method.
More amounts of solvents are required which may be expensive.
Automation makes the technique more complicated and costly.
