2.4 PURIFICATION
Purification of the biomolecules from the crude extract is an integral task as purified compounds are much more effective than the latter. Advances in biotechnology have opened up numerous possibilities for the purification and large-scale production of many biomolecules (Rodrigues et al., 1999). Since the plant extracts usually occur as a combination of various type of bioactive compounds or phytochemicals with different polarities different levels of chromatographic separations are used - Paper Chromatography, Thin layer Chromatography, Column chromatographic and High Performance Liquid Chromatography. TLC and Column chromatography, HPLC are still widely used because of convenience, availability of variety of stationary phases for separation of phytochemicals. In TLC crude extracts are fractionated into different components. This technique simplifies the process of isolation and identification of bioactive compound (Sasidharan et al., 2011). Seanego and Ndip, (2012) used TLC to isolate bioactive molecules from Garcinia kola seeds; Zeeshan et al., (2012) from leaves of Ageratum houstonianum; Lawrence et al. (2009) from leaves of Aloe vera. Another important technique is the TLC bioautographic method which combines chromatographic separation and in situ activity determination facilitating the localization and target-directed isolation of active constituents in a mixture (Hamburger and Cordell 1987; Rahalison et al., 1991; Shahverdi, 2007). Another important purification technique followed after TLC is the HPLC. It is a versatile, robust, and widely used technique for the isolation of natural products (Cannell, 1998). Currently, this technique is gaining popularity among various analytical techniques as the main choice for fingerprinting (Fan et al., 2006). The biologically active entity is often present only as minor component in the extract and the resolving power of HPLC is ideally suited to the rapid processing of such multicomponent samples on both an analytical and preparative scale. Asghari et al., (2011); Deng et al., (2012); Nazzaro et al., (2012) isolated purified bioactive compounds from cinnamom husks, fruit wastes, chestnut and hazelnut shells using HPLC. There are also other methods employed to detect phytochemicals among which is the diode array detector (DAD) coupled with mass spectrometer (MS) (Tsao and Deng, 2004), liquid chromatography and gas chromatography coupled with mass spectrometry (LC/MS and GC/MS) (Cai et al., 2002). It provides abundant information for structural elucidation of the compounds when tandem mass spectrometry (MS) is applied. Therefore, the combination of HPLC and MS facilitates rapid and accurate identification of chemical compounds (Ye et al., 2007). Anand (2005); Nalina and Rahim (2007); Dwivedi et al., (2010); Sugumaran et al., (2011) used both GC/MS and LC/MS to analyse bioactive molecules from pomegranate waste and betel leaf, including the petiole. Since bioactive compounds occurring in plant material consist of multi-component mixtures, their separation and determination still creates problems. Practically most of them have to be purified by the combination of several chromatographic techniques and various other purification methods to isolate bioactive compound(s).
2.5 STRUCTURAL CHARACTERIZATION
The purified compound needs to be structurally determined. This involves accumulating data from a wide range of spectroscopic techniques, such as UV-visible spectroscopy, Infra Red (IR), Nuclear Magnetic Resonance (NMR) which gives some basic clue regarding the structure of the molecule. Although almost all parts of the electromagnetic spectrum are used for studying matter in organic chemistry, but natural products are concerned with energy absorption from three or four regions - Ultraviolet (UV), Visible, Infrared (IR), radio frequency, and electron beam (Kemp, 1991).
Nature of the compound can be determined making use of UV-visible spectroscopy (Kemp 1991a). IR has proven to be a valuable tool for the characterization and identification of compounds or functional groups (chemical bonds) present in an unknown mixture of M.oleifera and J. curcas extract (Eberhardt et al., 2007). The identification of functional groups in a compound may be detected using IR by analyzing the different bonds present. These bonds (C-C, C=C, C≡C, C-O, C=O, O-H, N-H etc) have different vibrational frequencies, and the presence of these bonds in an organic molecule can be detected by identifying the characteristic frequency as an absorption band in the infrared spectrum (Kemp, 1991b). In addition, IR spectra of pure compounds are usually so unique that they are like a molecular “fingerprintâ€. For most common plant compounds, the spectrum of an unknown compound can be identified by comparison to a library of known compounds. NMR is concerned with the magnetic properties of certain atomic nuclei, notably the nucleus of the hydrogen atom-the proton-and that of the carbon-13, an isotope of carbon. Studying a molecule by NMR spectroscopy enables us to record differences in the magnetic properties of the various magnetic nuclei present, and also to deduce what the positions of these nuclei are within the molecule, and also which atoms are present in neighboring groups. It can measure how many atoms are present in each of these environments (Kemp 1991c; Thitilertdecha 2010; Bouallagui et al., 2012).
