Organic Chemistry Laboratory

Isolation of beta Carotene through Column Chromatography

Isolation of natural products through column chromatography, concentration of extracts and
thin layer chromatography

Introduction
Cashew nut shell liquid is a by-product of the cashew industry. The shell of the nut contains a dark reddish
brown viscous liquid. It is called the cashew nut shell liquid. The CSNL content of the raw nut varies
between 20 and 25 percent. Cashew nut shell liquid (CNSL) consists of unsaturated phenolic compounds,
anacardic acid (90%) and cardol (10%) with minor amounts of 2-methyl cardol. CNSL finds a wide array of
applications such as friction linings, paints & varnishes, laminating resins, rubber compounding resins,
cashew cements, polyurethane based polymers, surfactants, foundry chemicals and intermediates for chemical
industry.
OH O
OH

OH

HO
Figure 1. Structure of anacardic acid.

Figure 2. Structure of cardol.

Procedure
Sample preparation
Peel carrot sample and chop finely. Using 100 mL of petroleum ether, extract the oil in the carrot sample by
soaking and stirring in a beaker for 5 minutes. Collect the liquid extract. Do a second extraction to the sample
by adding 50 mL of petroleum ether. Combine the extracts.
Concentrate the liquid extract by evaporating the solvent with the aid of heat (Bunsen burner, water bath or a
hot plate). Maintain a 2-5 mL volume of the concentrated extract.
Packing the column
While doing the first part, you can start working on your column set-up. Clean and dry (or final wash with
petroleum ether) your column apparatus. Place a small amount of glass wool at the inner most part of the
column. Ask the instructor how to do this and the approximate amount of glass wool. Pour small amount (5
mL) of ether through the column to check the rate of flow. At this point, the flow of the solvent should be
fast.
In a beaker, weigh 20-25 grams of silica. Do not inhale or blow the silica particles. Add enough amount of
petroleum to form slurry. Slurry enough in a way that right after stirring the solution, no silica particles should
be suspending at the bottom of the beaker. Pour this slurry into the column, quickly but carefully with the aid
of a stirring rod. Let the solvent flow until it reaches the same level as the silica. You can collect the solvent
in a clean container. This solvent is clean and can be reused. You may place small amount of clean and dry
sand on top of the silica layer. If the flow of the solvent is very slow, you may aid with a vacuum by
connecting the column to a filter flask which is connected to the vacuum source.

Isolation of beta Carotene using Column Chromatography C.A. Malapit, 2010.

Chromatography
Just when the solvent reaches the level of the silica, stop the vacuum and immediately add the sample using a
medicine dropper. The sample should be added in a span of no longer than a minute. Allow the sample to be
completely absorbed into the silica before adding petroleum ether. To test of the sample is completely
absorbed into the silica, add few drops of ether into the column. If the sample is being transferred or extracted
by the solvent, then it is not yet completely absorbed. Do this until the solvent being added remains colorless.
Flush the column with ether and start the vacuum source. Observe the separation of colors (band of pigments)
in the column. Note colors and number of pigments observed. Beta carotene is an orange-yellow pigment that
is of highest amount in the extract. Your goal is to collect as many as possible of this pigment free from other
pigments in the extract. Collect the fractions of the said extract using a test tube. Once you have collected the
beta carotene fraction, you can now stop your column from flushing.
Combine all the fractions collected containing beta carotene only. To know which fractions contains beta
carotene only, perform TLC chromatography using petroleum ether as the mobile phase. For each TLC plate,
three fractions can be developed at the same time. You do not have to analyze all fractions through TLC.
Example, you can analyze only fractions 1, 3, 5, 7, 9 and so on.
Concentrate the combined fractions containing beta carotene only with the aid of heat. Determine the mass of
the beta carotene extracted and the % beta carotene in the sample.

References:

Kubo, I., Muroi, H., & Himejima, M., Structure - Antibacterial activity relationships of anacardic acids.
Journal of Agricultural food Chemicals, 41, 1016-1019, 1993.
Primary Information Services. India. Available online at http://www.primaryinfo.com, search for: cashew nut
shell liquid.

Isolation of beta Carotene using Column Chromatography C.A. Malapit, 2010.