Use of C hromatography T echniques to Identify and Isolate

1. Use of Chromatography Techniques to Identify and Isolate Chemical Substances from algae Dictyotapulchella plant. Raquel Bezerra(UFPB)Camila Figueiredo (UFPB), Annaleticia Braz (UFPB),Dr Emidio(UFPB) Jones Abankwa(SUNY Oswego) Introduction Objective 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 liquid-liquid 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 DictyotaPulchellaa 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 Acknowledgements 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 Dictyotapulchella (Dictyotaceae), a Brown Alga, Is Mediated via Inhibition of Calcium Influx in Rats. Marine Drugs. 2011; 9(10):2075-2088. 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.