Advanced Organic Chemistry Lab-1 Course Code: 2597 SPRING 2020 UNIT 4: Column Chromatography Objectives of Activity

ADVANCED ORGANIC CHEMISTRY LAB-1
COURSE CODE: 2597

SPRING 2020
UNIT 4: Column Chromatography

Objectives of Activity:
1-To learn the basic Principle of Column Chromatography.
2-To learn in the application of Column Chromatography for
purification of Organic compounds in lab and medicines in
pharmaceutical industry.
3- To learn how the technique of column Chromatography.
Activity 1: Read the theory written below to learn the basic principle
of Column chromatography, its applications.
Column chromatography in chemistry is a chromatography method

used to isolate a single chemical compound from a mixture.
Chromatography is able to separate substances based on differential
adsorption of compounds to the adsorbent; compounds move through
the column at different rates, allowing them to be separated into
fractions. The technique is widely applicable, as many different
adsorbents (normal phase, reversed phase, or otherwise) can be used
with a wide range of solvents. The technique can be used on scales from
micrograms up to kilograms. The main advantage of column
chromatography is the relatively low cost and disposability of the
stationary phase used in the process. The latter prevents cross-
contamination and stationary phase degradation due to recycling.
Column chromatography can be done using gravity to move the
solvent, or using compressed gas to push the solvent through the
column.
A thin-layer chromatograph can show how a mixture of compounds

will behave when purified by column chromatography. The separation
is first optimised using thin-layer chromatography before performing
column chromatography.
Column preparation
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A column is prepared by packing a solid absorbent into a cylindrical
glass or plastic tube. The size will depend on the amount of compound
being isolated. The base of the tube contains a filter, either a cotton or
glass wool plug, or glass frit to hold the solid phase in place. A solvent
reservoir may be attached at the top of the column.
Two methods are generally used to prepare a column: the dry method
and the wet method. For the dry method, the column is first filled with
dry stationary phase powder, followed by the addition of mobile phase,
which is flushed through the column until it is completely wet, and
from this point is never allowed to run dry.
For the wet method, a slurry is prepared of the eluent with the
stationary phase powder and then carefully poured into the column. The
top of the silica should be flat, and the top of the silica can be protected
by a layer of sand. Eluent is slowly passed through the column to
advance the organic material.
The individual components are retained by the stationary phase

differently and separate from each other while they are running at
different speeds through the column with the eluent. At the end of the
column they elute one at a time. During the entire chromatography
process the eluent is collected in a series of fractions. Fractions can be
collected automatically by means of fraction collectors. The
productivity of chromatography can be increased by running several
columns at a time. In this case multi stream collectors are used. The
composition of the eluent flow can be monitored and each fraction is
analysed for dissolved compounds, e.g. by analytical chromatography,
UV absorption spectra, or fluorescence. Coloured compounds (or
fluorescent compounds with the aid of a UV lamp) can be seen through
the glass wall as moving bands.
Stationary phase: The stationary phase or adsorbent in column
chromatography is a solid. The most common stationary phase for
column chromatography is silica gel, the next most common being
alumina. Cellulose powder has often been used in the past. The
stationary phases are usually finely ground powders or gels and/or are
microporous for an increased surface. There is an important ratio
2|Page
between the stationary phase weight and the dry weight of the analyte
mixture that can be applied onto the column. For silica column
chromatography, this ratio lies within 20:1 to 100:1, depending on how
close to each other the analyte components are being eluted.
Mobile phase (eluent): The mobile phase or eluent is a solvent or a
mixture of solvents used to move the compounds through the column.
It is chosen so that the retention factor value of the compound of
interest is roughly around 0.2 - 0.3 in order to minimize the time and
the amount of eluent to run the chromatography. The eluent has also
been chosen so that the different compounds can be separated
effectively. The eluent is optimized in small scale pre-tests, often using
thin layer chromatography (TLC) with the same stationary phase.
There is an optimum flow rate for each particular separation. A faster
flow rate of the eluent minimizes the time required to run a column and
thereby minimizes diffusion, resulting in a better separation. However,
the maximum flow rate is limited because a finite time is required for
the analyte to equilibrate between the stationary phase and mobile
phase. A simple laboratory column runs by gravity flow. The flow rate
of such a column can be increased by extending the fresh eluent filled
column above the top of the stationary phase or decreased by the tap
controls. Faster flow rates can be achieved by using a pump or by using
compressed gas (e.g. air, nitrogen, or argon) to push the solvent through
the column (flash column chromatography).
3|Page
Photographic sequence of a column chromatography (for a coloured
mixture)
The particle size of the stationary phase is generally finer in flash
column chromatography than in gravity column chromatography. For
example, one of the most widely used silica gel grades in the former
technique is mesh 230 – 400 (40 – 63 µm), while the latter technique
typically requires mesh 70 – 230 (63 – 200 µm) silica gel
Activity 2: Watch the videos to learn how to do Column

Chromatography
https://www.youtube.com/watch?v=B_QyhG2-VBI
https://www.youtube.com/watch?v=ItTtnVKcqDw
Activity 3: Separation of beta carotenes by column chromatography

Watch the video to learn how pure compound (Beta carotene from carrots) can
be purified by column chromatography:
https://www.youtube.com/watch?v=Gexf_PNPefU (Extraction of beta-carotene from carrots)

https://www.youtube.com/watch?v=ci2uu9Cuf5s (Purification by Column Chromatography)
Activity 4: Answer the following Questions
Q-1 Explain the basic Principle of Column Chromatography.
Q-2 Name at least four materials used as adsorbents in Column

Chromatography.
4|Page
Q-3 How many components were present in carrot extract?
Q-4 At which number carotenes were eluted from the column?
Q-5 Write down the composition of eluent (solvent system) used to

elute the carotenes from the column.
Q-6 How do you know that you got pure carotene as a result of column
chromatography?
5|Page

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ADVANCED ORGANIC CHEMISTRY LAB-1

COURSE CODE: 2597

UNIT 4: Column Chromatography

Column chromatography in chemistry is a chromatography method

A thin-layer chromatograph can show how a mixture of compounds

The individual components are retained by the stationary phase

Activity 2: Watch the videos to learn how to do Column

Activity 3: Separation of beta carotenes by column chromatography

https://www.youtube.com/watch?v=Gexf_PNPefU (Extraction of beta-carotene from carrots)

Activity 4: Answer the following Questions

Q-1 Explain the basic Principle of Column Chromatography.

Q-2 Name at least four materials used as adsorbents in Column

Q-4 At which number carotenes were eluted from the column?

Q-5 Write down the composition of eluent (solvent system) used to

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