Column chromatography is a technique used to separate mixtures of chemical compounds based on how they interact with a stationary and mobile phase. The stationary phase is typically a solid adsorbent packed into a glass column, while the mobile phase is a liquid that passes through the column. Compounds separate as they move through the column at different rates depending on their relative affinities for the stationary and mobile phases. Fractions are collected as compounds elute from the column and can be analyzed by thin layer chromatography to identify the purified compounds. Column chromatography is useful for preparative separations and purification of synthetic or natural products.
This presentation contains all the topics related to column chromatography. That includes introduction, principle,apparatus, experimental aspects of column chromatography, application of column chromatography, advantage and disadvantage of column chromatography with reference.
Introduction to chromatography, Definition of Chromatography, Types of column chromatography, Theory of chromatography, Practical considerations in column chromatography , Factors affecting efficiency of a column, Applications.
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 presentation contains all the topics related to column chromatography. That includes introduction, principle,apparatus, experimental aspects of column chromatography, application of column chromatography, advantage and disadvantage of column chromatography with reference.
Introduction to chromatography, Definition of Chromatography, Types of column chromatography, Theory of chromatography, Practical considerations in column chromatography , Factors affecting efficiency of a column, Applications.
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.
High Performance Thin Layer Chromatography (HPTLC) instrumentationMadhuraNewrekar
HPTLC is an advancement of TLC. It is a high performance liquid chromatography with automation compared to Thin Layer Chromatography(TLC).Speed, Efficiency and Accuracy are important advantages. Evaluation time is less due to updated automation in instrumentation.
Steps involved in HPTLC and the materials and instruments required in those steps are described in brief.
COLUMN CHROMATOGRAPHY - SEPERATION OF THE MIXTURE OF COMPONENTS IJN TO INDIVIDUAL COMPONENTS BY USING STATIONARY PHASE AND MOBILE PHASE UPON THE USING OF COLUMN
Paper chromatography is an analytical method used to separate coloured chemicals or substances.It is now primarily used as a teaching tool, having been replaced in the laboratory by other chromatography methods such as thin-layer chromatography (TLC).
It is instrumental analytical technique. it is one of the major type of chromatography technique. its basic principle is adsorption. it has many applications in various fields
Chromatography is a powerful and advanced techniques for separating mixtures. Many types of chromatographic techniques are known, such as paper, thin layer, column chromatography, each with its own strength and weakness.
Chromatography system in general have a stationary phase and a mobile phase.
In column chromatography both phases are placed in a column container, i.e. all the chromatographic operations are carried out using column.
Column chromatography in chemistry is a method using for the Identification, separation and purify individual chemical compounds from mixtures of compounds in the large amount.
Column Chromatography is a separation technique in which components of mixture is separated by using a glass column packed with stationary phase and liquid mobile phase flowing continuously through the column.
It is suitable for the physical separation of gram quantities of material. A solvent acts as the mobile phase while a finely divided solid surface acts as the stationary phase.
Usually a glass tube with a diameter from 1cm to 10cm and a height of 20 cm to 50cm with a tap at the bottom, is used for this purpose.
Depending upon the flow of solvent down, column chromatography may be separated into two categories.
Gravity column chromatography
If the solvent is allowed to flow down the column by Gravity, or downward process, it is known as gravity column chromatography .
Flash chromatography
If the solvent is forced down the column by positive air pressure , it is called flash chromatography.
High Performance Thin Layer Chromatography (HPTLC) instrumentationMadhuraNewrekar
HPTLC is an advancement of TLC. It is a high performance liquid chromatography with automation compared to Thin Layer Chromatography(TLC).Speed, Efficiency and Accuracy are important advantages. Evaluation time is less due to updated automation in instrumentation.
Steps involved in HPTLC and the materials and instruments required in those steps are described in brief.
COLUMN CHROMATOGRAPHY - SEPERATION OF THE MIXTURE OF COMPONENTS IJN TO INDIVIDUAL COMPONENTS BY USING STATIONARY PHASE AND MOBILE PHASE UPON THE USING OF COLUMN
Paper chromatography is an analytical method used to separate coloured chemicals or substances.It is now primarily used as a teaching tool, having been replaced in the laboratory by other chromatography methods such as thin-layer chromatography (TLC).
It is instrumental analytical technique. it is one of the major type of chromatography technique. its basic principle is adsorption. it has many applications in various fields
Chromatography is a powerful and advanced techniques for separating mixtures. Many types of chromatographic techniques are known, such as paper, thin layer, column chromatography, each with its own strength and weakness.
Chromatography system in general have a stationary phase and a mobile phase.
In column chromatography both phases are placed in a column container, i.e. all the chromatographic operations are carried out using column.
Column chromatography in chemistry is a method using for the Identification, separation and purify individual chemical compounds from mixtures of compounds in the large amount.
Column Chromatography is a separation technique in which components of mixture is separated by using a glass column packed with stationary phase and liquid mobile phase flowing continuously through the column.
It is suitable for the physical separation of gram quantities of material. A solvent acts as the mobile phase while a finely divided solid surface acts as the stationary phase.
Usually a glass tube with a diameter from 1cm to 10cm and a height of 20 cm to 50cm with a tap at the bottom, is used for this purpose.
Depending upon the flow of solvent down, column chromatography may be separated into two categories.
Gravity column chromatography
If the solvent is allowed to flow down the column by Gravity, or downward process, it is known as gravity column chromatography .
Flash chromatography
If the solvent is forced down the column by positive air pressure , it is called flash chromatography.
Introduction
Definition
History
Types of chromatography
Principle of column chromatography
Types of column chromatography
Process of column chromatography
Requirement
Procedure
Precautions
Applications
Advantage of Column chromatography
Disadvantage of Column chromatography
Conclusion
References
The present slide gives us an insight into the different aspects of application of columnn chromatrography the principle behind it and at the same time its recent advances.
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2. COLUMN CHROMATOGRAPHY
• Column chromatography in chemistry, analysis,
pharmacognosy, etc is a method used to separate/purify
individual chemical compounds from mixtures of compounds.
• It is often used for preparative applications on scales from
micrograms up to kilograms.
• This is a solid-liquid technique in which the stationary phase is a
solid & mobile phase is a liquid.
• Column chromatography is an extremely valuable technique for
purification of synthetic or natural products.
• Compounds are separated by Column Chromatography through
the same mechanism as TLC
3. COLUMN CHROMATOGRAPHY
• Column Chromatography was developed by the
Russian-Italian botanis, Mikhail Semyonovich Tswett,
in 1906 used adsorption columns in his investigations
of plant pigments.
4. PRINCIPLE
Adsorption
• Mixture of components dissolved in the
Mobile Phase is introduced into the column.
• Components moves depending upon their
relative affinities.
• Relative affinities: It is defined as an
equilibrium constant representing the ratio of
the equilibrium activities of a component in
two different phases.
5. A chemist in the 1950s using column chromatography.
The Erlenmeyer receptacles are on the floor.
6.
7.
8. COLUMN CHROMATOGRAPHY
• Adsorption column chromatography, the adsorbent,
packed in a glass column, and a solvent, the mobile
phase, that moves slowly through the packed column.
A solvent used as a mobile phase is called an eluent.
9. COLUMN CHROMATOGRAPHY
• A compound attracted more strongly by
the mobile phase will move rapidly
through the column, and elute from, or
come off, the column dissolved in the
eluent.
• In contrast, a compound more strongly
attracted to the stationary phase will
move slowly through the column.
10. Experimental aspects of column
chromatography:
• Adsorbents: The usual adsorbents
employed in column
chromatography are silica, alumina,
calcium carbonate, calcium
phosphate, magnesia, starch, etc.,
• Alumina is generally suitable for
chromatography of less polar
compounds(Non-polar solvents).
Silica gel gives good results with
compounds containing polar
functional groups.
11. COLUMN CHROMATOGRAPHY
• Adsorbent in C.C should meet following criteria
◘ Particles should be spherical in shape & uniform in size
◘ Mechanical stability must be high
◘ They shouldn’t react chemically
◘ It should be useful for separating for wide variety of
compounds
◘ It should be freely available & inexpensive
(The particle size of the commercially available grade is
in the range 50 – 200 µm.)
12. Selection of Stationary Phase
• Success of chromatography depends upon proper selection
of Stationary Phase, it depends on the following:
1. Removal of impurities
2. No. of components to be separated
3. Length of the column used
4. Affinity differences b/w components
5. Quantity of adsorbent used
13. Mobile Phase
• They act as solvent, developer & eluent.
The function of a mobile phase are:
• As developing agent
• To introduce the mixture into the column – as solvent
• To developing agent
• To remove pure components out of the column – as
eluent
14. • The choice of the solvent is depend on the solubility
characteristics of the mixture. The solvents should also
have sufficiently low boiling points to permit ready
recovery of eluted material.
• However, polarity as seen the most important factor in
adsorption chromatography.
• Different mobile phases used: (in increasing order of
polarity)
• Petroleum ether, carbon tetrachloride, cyclohexane,
ether, acetone, benzene, toluene, esters, water, etc
• It can be used in either pure form or as mixture of
solvents
15.
16. COLUMN CHARACTERISTICS
• The main function of all the columns is to support
the stationary phase.
• The material of the column is mostly good quality
neutral glass since it shouldn’t be affected by
solvents. An ordinary burette can also be used as
column for separation.
• Column dimensions - length & diameter ratio
(10:1,30:1 or 100:1)
• Various accessories are attached to the top and
bottom of the column for maintenance of the
elution process.
17. PREPARATION OF THE COLUMN
• It consists of a glass tube with bottom portion of the
column – packed with glass wool/cotton wool or may
contain asbestos pad,
» Above which adsorbent is packed
» After packing a paper disc kept on the top, so that the
adsorbent layer is not disturbed during the introduction of
sample or mobile phase.
18. Packing techniques in Column
Chromatography
• There are two types of preparing the column, they are:
i. Dry packing / dry filling
ii. Wet packing / wet filling
• The column should be free from impurity, before using
column, it should be washed properly and dry it.
• Before filling column with stationary phase, cotton/glass
wool is kept
• It should be uniformly filled
19. Dry Packing Technique
Adsorbent is packed in the column in dry form
Filled up to 3/4th of the actual height of the column
Fill the solvent, till equilibrium is reached
DEMERIT: Air bubbles are entrapped b/w Mobile Phase &
Stationary Phase→ cracks appear in the adsorbent layer.
• After filling tapping can be done to remove void spaces.
21. COLUMN CHROMATOGRAPHY
Wet Packing Technique
» ideal & common technique
The material is slurred with solvent and generally added to
the column in portions.
◊ Allow to settle S.P uniformly & avoid the cracking in the
column of adsorbent.
» Solid settle down while the solvent remain upward.
» This solvent is removed then again cotton plug is placed.
it’s usually faster and seems to use up less liquid
22. 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.
25. COLUMN CHROMATOGRAPHY
Development technique ( Elution)
• By elution technique, the individual components are
separated out from the column.
• The two techniques are:
(i) Isocratic elution technique : In this elution technique, same
solvent composition or solvent of same polarity is used
throughout the process of separation.
• Example: chloroform only
26. COLUMN CHROMATOGRAPHY
(ii) Gradient elution techniques:
(Gradient – Gradually)
Solvents of gradually ↑ polarity or ↑ elution strength are used
during the process of separation.
E.g. Initially benzene, then chloroform, then ethyl acetate then
methenol
27. COLUMN CHROMATOGRAPHY
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 colourless. In this case, small fractions of the eluents are
collected sequentially in labelled tubes and the composition
of each fraction is analyzed by TLC.
28. COLUMN CHROMATOGRAPHY
• Eluting the sample: Components a, b, and c separate as
column progresses.
• Fractions can be collected in test tubes, vials, beakers, or
flasks.
30. FACTORS AFFECTING COLUMN EFFICIENCY
1. Dimension of the column: column efficiency has been
improved by increasing length/width ratio of the column.
2. Particle size of column packing: separation to be improved
by decreasing the particle size of the adsorbent.
3. Uniformity of packing: Non uniform packing results in
irregular movement of solutes through column & less
uniform zone formation, (i.e. band broadning or tailing)
4. Temperature of the column: The speed of the elution
increases at higher temperatures.
5. Quality of solvents: Solvents should be of low viscosity (to
give efficient resolution) & h igh volatility (to get rapid
recovery of the substances)
6. Continuity of flow: Discontinuous flow disturbs resolution
7. Condition of adsorbent: Deactivation of adsorbent decreases
separation.
31. APPLICATIONS
►Separation of mixture of compounds
►Purification process
►Isolation of active constituents
►Estimation of drugs in formulation
►Isolation of active constituents
►Determination of primary and secondary glycosides in
digitalis leaf.
► separation of diastereomers
32. COLUMN CHROMATOGRAPHY
Advantages of C.C
» Any type of mixture can be separated
» Any quantity of mixture can be separated
» Wider choice of Mobile Phase
» Automation is possible
Disadvantages of C.C
» Time consuming
» more amount of Mobile Phase are required
» Automation makes the techniques more complicated &
expensive
33. TLC vs. CC
• Same type of separation technique (liquid-solid separations)
• TLC is much simpler (it doesn’t require as much preparation
as CCdoes with all of the packing material, solvent and eluent),
• Cheaper (even though CC is cheap as well when compared
to HPLC)
• Takes less time to finish.
• Column requires more amount of solvents
• Column Chromatography is that it’s not really reproducible.
• Column Chromatography is better because columns are
longer than a TLC plate. Also, TLC (as opposed to column
chromatography) takes place in an open system which means it
can be affected by humidity or temperature