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this parathion of the bioactive compounds, several isolation procedures, namely, thin layer
chromatography, ash chromatography, high-performance liquid chromatography, column
chromatography, and Sephadex chromatography have to be employed to attain the desired
bioactive compounds with the greatest purity. The puried complexes were then taken
for quality characterization which involves structural as well as biological determination
(Sasidharan et al., 2011).
FIGURE 9.1 Process flowchart showing the process of purification and utilization of bioactive compounds.
9.6.1 QUALITATIVE AND QUANTITATIVE TECHNIQUES/CHROMATOGRAPHIC OR NONCHROMATOGRAPHIC TECHNIQUES
TLC is an easy, fast, low-cost technique that provides researchers with rapid answers
about the number of constituents present in a mixture. Furthermore, TLC is also
employed to confirm the characteristics of the desired compounds present in a mixture
by comparing the retardation factor (Rf) of a compound to the Rf of an identified
compound. Further tests in the identification of compounds include spraying of phytochemical screening reagents that lead to the alteration in visual properties which depend
on the phytochemical’s presence in the plant extract. Alternatively, the plate is observed
in UV light. It has also been utilized to confirm the purity as well as identification of the
isolated compounds (Sasidharan et al., 2011).
In comparison to other chromatographic techniques, such as paper chromatography,
the distinct advantages of TLC are exibility, speed, and sensitivity of the process. TLC
is generally an adsorption chromatography in which samples are separated on the basis
of interactions among thin layers of adsorbent linked to the plate (Ingle et al., 2017).
⏎
204
The different adsorbents utilized for the separation of different compounds are listed in
Figure 9.2.
FIGURE 9.2 Adsorbents used for separation of different bioactive compounds in TLC
⏎
HPLC is a useful, robust, and extensively employed practice for isolating natural bioactive
compounds. This method is now obtaining wide approval amidst different analytical procedures and has become the key option for fingerprinting, quantitative analysis, and quality
control (Fan et al., 2006). Usually, natural plant foods are often isolated after evaluating
moderately crude extracts in biological assays to entirely indicate the bioactive ingredients.
Biologically active moieties in natural sources are usually found only as minor components
in extracts and their solution of HPLC is preferably suitable for instant treating of such
multicomponent samples on both analytical as well as preparative scales. The purification/
refining of desirable constituents from natural sources by the use of HPLC is the way
of separating or extracting bioactive compounds of interest from other compounds with
similar structures or impurities. All compounds must exhibit a specific peak under defined
chromatographic specifications. On the basis of the resultant compound of interest to be
isolated, and how close the related components are, the chromatograph can pick conditions viz., appropriate mobile phase, flow rate, appropriate detector , and column to achieve
optimal separation (Sasidharan et al., 2011).
HPLC could be combined with simple detectors applied to record chromatographic
marks, proling, or quantitative reasons. Some of the detectors used with HPLC are ultraviolet detectors, ELSD (evaporative light-scattering detector), electron capture detectors or
detectors for hyphenated systems that produce multidimensional information for certication and online transcription, for example, MS, UV diode array (DAD), nuclear magnetic
resonance (NMR) (Wolfender, 2009).
Because of the simplicity of usage and inexpensive nature, HPLC-UV is utilized by
different pharmacopeias for the purpose of quantifying specic desired bioactive compounds
205
in the quality control of foods from natural sources, medicines, and herbal supplements.
Apart from the HPLC-UV, spectral data from the HPLC-DAD (Diode-Array Detection)
could be used to verify a number of chromatograms over an extensive wavelength range,
allowing qualitative as well as quantitative exploration of ngerprint chromatogram peaks
(Zhang et al., 2011). Additionally, ELSD (evaporative light scattering detector) is another
detector utilized for liquid chromatography. It is mainly used to detect components with
weak chromophores, for instance, aglycon or the glycosidic forms of terpenes, saponins,
and a few alkaloids; however, it is commonly used along with some other techniques like
MS and UV–Vis (Adnani et al., 2012).
FTIR has been shown to be an effective tool for characterizing and identifying bioactive
compounds or functional units (chemical bonds) present in unknown mixtures of plant
extracts. It aids in identifying as well as determining the structure of molecules. Moreover, the FTIR spectrum of pure compounds is generally so distinctive that they resemble
molecular “fingerprints.” Usually, for the commonly extracted compounds from plant
sources, the spectra of unknown compounds can be recognized by comparing them with
libraries of identified compounds (Hazra et al., 2007). FTIR has a broad range of applications, from examining small particles and compounds to the assessment of cells and tissues
(Berthomieu and Hienerwadel, 2009).
The preparation of FTIR samples for analysis can be done in many ways. As for a
sample that is liquid in nature, the simplest way is to keep a drop of the sample among
two plates of NaCl. Apparently, the drops form a thin lm layer among the plates. Besides,
the samples that are solid in nature can be ground with potassium bromide (KBr) besides
formerly attened into ne pellets for analysis. Alternatively, solid samples can also be
mixed in a solvent for example methylene chloride, and then after a few drops of the
solution are applied onto a single HATR (high attenuated total reectivity) plate, and the
spectrum is noted as a percentage of transmittance. So the peaks formed at a particular
wave number are specied by the linkage group, and the function by reference is specied
in the manual of the Varian FTIR instrument (Ingle et al., 2017).
GC is an analytical method for separating and detecting bioactive constituents found in
product mixtures to examine the occurrence or nonoccurrence and/or quantity. For successful
characterization using GC analysis, these components must be volatile, typically have
molecular weights below 1250 Da, and should be thermally stable so that these components
do not decompose in the GC system. In this technique, the compounds are distributed
between the gaseous and liquid phases. Usually, the flow is the gas phase; however, the
liquid phase is stationary. Gas phase chromatography encompasses evaporating a sample
and injecting the sample into the top of a chromatographic column. For this reason, the
206
shipment of such samples is done through the column by nonreactive mobile phase flow.
Usually column comprises a stationary phase in liquid form that is adsorbed on the exterior
surface of the inert solid (Ingle et al., 2017).
GC is a commonly applied method used in various industries, such as quality control,
manufacturing, chemical, and phytochemical as well as in pharmaceuticals. The four key
indicators of a successful GC approach are selectivity, efciency, sensitivity, and speed.
Efciency is the capacity of a method to isolate analytes into sharp, compact bands, meaning
more analytes can be separated in a single run. In other words, as efciency increases,
so does peak capacity. Selectivity is the capacity of an approach to distinguish among
analytes and is largely manifest in the order in which the analytes exit the GC column.
Further sensitivity is the capacity of an isolation method to identify very minute analyte
concentrations. Usually among the four indicators of the efcient GC system, efciency
and selectivity are considered to be the parameters of major interest as these indicate if the
analyte could be separated efciently or not (Shellie, 2013).
MS is an essential analytical instrument in the fields of chemistry, pharmacology,
biochemistry , drugs, and various interlinked scientific fields. MS is an important analytical
method to identify unknown components, quantify identified compounds, and explicate the
chemical organization as well as attributes of molecules. Using MS, the molecular mass
of the sample can be examined (Ingle et al., 2017). MS is used to examine combinatory
archives, sequence biomolecules, and study single cells and matter from outer space.
Structural interpretation of unidentified compounds, ecological as well as forensic analysis,
and quality control of pharmaceuticals, foods, and polymers are all based primarily on MS.
Mass spectrometers work by transforming analyte particles into a charged (ionized) state
and analyzing the ions and fragmentations generated through the ionization process based
on their (m/z) (mass-to-charge) ratio (Pitt, 2009).
The GC-MS technique for the characterization of plant-based extracts is not only used
for analyzing the quantities of different bioactive substances contained in herbs/natural
sources used in the pharmaceutical, cosmetic, and food industries, but it can also be an
interesting tool for forensic and environmental applications (Uma et al., 2009). This
technique combines two analytical procedures into one method to analyze mixtures of
components. GC helps to isolate the components of a mixture, while mass spectrometry
examines each component individually. Combining GC and MS (GC-MS) was realized
by commercial instruments in the 1950s and has been existing since the 1970s. GC-MS is
comparatively inexpensive and consistent. GC-MS systems are promptly used in different
fields such as biotechnology and biochemistry research labs and are essential in numerous
207
fields where the analysis and unambiguous identification of complex mixtures is mandatory (Pitt, 2009).
LC-MS is usually a conventional method with the advancement of electrospray ionization
(ESI) delivering a simple as well as powerful user interface. It can be utilized for an extensive variety of biomolecules, and the usage of parallel MS and internal stable isotope ideals
permits the development of very complex and precise assays, although some methods need
to be optimized to minimize ion inhibition processes (Pitt, 2009).
NMR spectroscopy is the examination of molecules by documenting the interaction of
their nuclei with radiofrequency electromagnetic radiation in an effective magnetic field.
NMR is generally employed for examining structural attributes. Its intrinsic sensitivity is
rewarded by the provided deep structural facts (such as chirality, conformation, inclusion
phenomena). There are different categories of NMR techniques, namely solid-state
NMR, solution-state NMR, high-resolution magic angle spinning NMR, comprehensive
multiphase NMR, hyphenated NMR, low-field NMR, and magnetic resonance imaging
(Simpson et al., 2018).
9.7 CONCLUSIONS
The ongoing requirement to extract plant bioactive components from natural sources
stimulates research into practical extraction techniques that will provide more yield over
conventional techniques without any degradation of the biological properties of bioactive molecules. The emergence of the majority of nonconventional extraction procedures
is largely due to advances in chromatography and environmental consciousness as less
usage of solvent. However, since most of these approaches are based on diverse mechanisms and extraction enhancement is the result of various combined processes starting
from natural material selection, pretreatments, and solvent selection to understanding
every component of the nonconventional extraction process is essential. The isolation
and identification of bioactive chemicals still present challenges since they are multicomponent combinations found in plant material which are having different polarities
and are associated with each other. During extraction, the selection of solvents based on
polarity plays an important role in improving the extraction yield of bioactive molecules
from natural sources. To isolate the bioactive compound(s), practically the majority of
them must be purified using a variety of chromatographic techniques combined with
other purification techniques that require in-depth knowledge regarding the behavior of
208
the obtained bioactive molecule for the selection of an appropriate technique so that the
biological properties of the material can be retained to the maximum for their wide food
and industrial applications.
KEYWORDS
• bioactive molecules
• extraction methods
• identification
• natural sources
• quantification techniques
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