Use of Chromatography Techniques to Identify and Isolate
Chemical Substances from algae Dictyota pulchella plant.
Raquel Bezerra(UFPB)Camila Figueiredo (UFPB), Annaleticia Braz (UFPB),Dr Emidio(UFPB)
Jones Abankwa(SUNY Oswego)
Objective
Introduction
Chromatography involves a series of processes for separating sample extract dissolved in a mobile phase(can be
gas, liquid, supercritical fluid) and then forced through an immobile, immiscible stationary phase(can also be solid or
liquid). Chromatography facilitate in the handling of isolation problem present in a complex mixture. In order to apply
a technique of chromatography, to a natural plant, the plant has to be change into an extract and broken down into
fractional components using solvents with different polarities.
We studying how different techniques of chromatography are utilize to isolate substance and also identify their
chemical structure through the analysis of data from mass spectra, nuclear magnetic resonance of 1H and 13c
NMR, IR and ultraviolet.
Theory and Methods
Marine algae are known for their rich sources of diverse biologically active compounds with great pharmaceutical potentials. It can be derived from
compounds which shows biological activities such as anticoagulant anti-viral, antioxidant, and ant-cancer. Marine algae have different families with
the Dictyotascae family being one of them. This genus dictyota is represented by more than 40 species and is the richest in the Dictyotacae family. It
is also one of the most abundant seaweeds in tropical marine habitats.
Chromatography is particularly for the isolation of small materials that have high essential value. It can separate a mixture into its individual
component and can provide a quantitative estimate of each constituent. Separation of two sample extract involve their different distribution between
two non-miscible phases(stationary and mobile phase). Different distribution of substance between mobile and stationary phase results in different
migration velocities.
The first step involve in changing the plant into extract is by collecting the plant and grinding and drying it in a mechanical mil and afer extraction.
Methnol or ehanol are commonly used in plant extractin, however with the algae plant organic solvent like dichloromethane and methanol are used
for th extraction. After the process of percolation, the extract is put in a rotavapor and is concentrated to remove all the ethanol used during
percolation. Apart from concentration, the rotavapor helps protect the extract and eliminates unwanted emission of vapors during the process of
concentration. Now the concentrated extract is stored in a refrigerator to avoid fungi infection.
After obtaining the extract, it may be subjected to partition using organic solvents of increasing degree of polarity. This kind of solvent can be a liquidliquid partition in which the extract is dissolved in a hydro-alcoholic solution and then placed in a separator funnel where solvents, beginning with
lower polarity to the more polar.(Hexane, dichloromethane, ethyl acetate, methanol) passed through the funnel. After being concentrated individually,
result in four phases(hexane, dichloromethane, ethyl acetate, methanol) are collected. To get an idea of the compounds present in each phase both
NMR spectrum of 1 H and 13 C and analytical thin-layer chromatography(CCDA/TLC) are needed to be done to choose the best phase which can
provide us with this information. With this reason in mind, we subject all the phases to chromatographic process for isolation of the compounds
present in the extract. Column chromatography is then used for this isolation process. Column chromatography is used when the phases pass
through a column glass packed with silica gel. The fractions obtained from the column chromatography were monitored with CCDA/TLC. For the
CCDA, the fractions which appeared as one spot are considered pure and are routed to obtain the spectra.
Figure 1. This diagram depicts the
setup used isolate and identify
compounds in the plant
Figure 2. This diagram depicts
the plant Dictyota Pulchellaa
Figure 3. This diagram depicts
Column Chromatography
Results
We observed that by using a light UV spectra
and CCDA, we are able to see how substances
present in the plant extract are isolated and
structured. All the substances present in this
extract are considered as pure because they
appear as one spot. This is the only information
required to send a substance to the spectra for
identification and it’s significant.
Figure 4. Shows a light UV of a substance that has
been isolated by using CCDA.
Conclusion
References
Although chromatography appear to help in the process of
isolation and identification of substances in plant, it requires
different mixtures of solvents which sometimes becomes
difficult for one to achieve pure isolated substances.
Queiroz T.M., Machado N.T., Furtado F.F., Oliveira-Filho A.A., Alustau
M.C., Figueiredo C.S., Miranda G.E.C., Barbosa-Filho J.M., Braga V.A.,
Medeiros I.A. Vasorelaxation, Induced by Dictyota pulchella
(Dictyotaceae), a Brown Alga, Is Mediated via Inhibition of Calcium
Influx in Rats. Marine Drugs. 2011; 9(10):2075-2088.
Acknowledgements