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11.5 Purification 221
Method Compound Plant References
Column chromatography Geniposidic acid, caffeic acid, chlorogenic acid, ferulic
VLC Ugonin J and K Helminthostachys
VLC,
preparative TLC,
centrifugal planar
chromatography (CPC)
VLC,
Column chromatography,
TLC
VLC,
Column chromatography,
Preparative TLC
VLC,
Column chromatography
VLC,
Column chromatography,
Preparative TLC
VLC,
Column chromatography
VLC,
Column chromatography
acid, quercetin-3-O-sambubioside, rutin, and
isoquercitrin.
8α-(3,4-dihydroxy-2-methylene-butanoyloxy)dehydromelitensin, 8α-hydroxysonchucarpolide,
Cnicin.
Stigmasterol, (6aR,11aR)-medicarpin,
8-O-methylretusin, formononetin, biochanin A,
7,4′-dihydroxy-8-methoxyisoflavone, daidzein, trans
and cis-4-hydroxymellein, and coumestrol
Cepharanone A, cepharanone B (aristolactam BII),
aristolactam AII, piperolactam A, and piperolactam D,
together with sesamin, lupeol, taraxerol, β-sitosterol,
β-sitostenone, and 4-allyl resorcinol
Yangambin, sesamin, syringaresinol, pinoresinol, and
medioresinol
Caulerpin and trioleoylglycerol Spatoglossum asperum [177]
N-isobutyl-(2E,4E)-tetradecadienamide, N-isobutyl(2E,4E,14Z)-eicosatrienamide, N-isobutyl-15-(3′,4′methylene-dioxyphenyl)-2E,4E,12Zpentadecatrienamide, N-p-coumaroyltyramine,
trans-N-p-feruloyltyramine, together with sesamin,
β-sitosterol, β-sitostenone, lupenone, and taraxerone
Daibucarbolines D and E,
pseudovillosine
Eucommia ulmoides [159]
zeylanica
Centaurea virgata [173]
Spatholobus parviflorus [174]
Piper ribesioides [175]
Piper stylosum [176]
Piper lanatum [178]
Neolitsea kedahensis [179]
[172]
chromatographic methods, such as the permanent loss and
deactivation of adsorptive components, elongation of solute
peaks, and contamination. The equipment consists of multiple CCC channels, which connect separate separation columns with parallel flow tubes. As a result, the multichannel
CCC equipment is capable of simultaneously performing
two or more separate chromatographic procedures. In addition, a recently created three-channel CCC apparatus has
been integrated with traditional parallel chromatographic
devices, such as pumps, sample injectors, effluent detectors,
and collectors, in order to establish an efficient CCC fractionation method for NPs. The effectiveness of this method
was proven by separating the ethyl acetate extracts from
three different natural sources: Solidago canadensis, Suillus
placidus, and Trichosanthes kirilowii. The findings indicate
that multichannel CCC has notable benefits in effectively
isolating natural compounds in substantial amounts for
drug development, despite the need for solvent balancing
and the decreased resolution of shorter CCC columns [180].
11.5 Purification
Purification means the elimination of undesired impurities within an organic/natural compound. Throughout
ancient times, numerous compounds have been investigated for their therapeutic or medicinal attributes,
including turmeric, possessing pain-relieving properties
(e.g. morphine), cough-suppressing effects (e.g. noscapine), anti-inflammatory attributes (e.g. sanguinarine and
berberine), and anticancer properties (e.g. vinblastine
and noscapine), among others [181–183].
The isolation and purification of compounds from NPs
constitute a pivotal stage in the identification of molecular
structures, bioactivity assessment, quantity control of NPs,
and subsequent industrial production. Nonetheless, the
isolation and purification of NPs pose challenges due to
their intricate matrices, low content of active compounds,
and susceptibility to thermal degradation. Consequently,
the judicious selection of techniques and methodologies is
222 11 Isolation, Fractionation, and Purification of Natural Products
imperative to achieve the desired compounds with high
yields. In recent decades, various innovative isolation and
purification techniques, such as membrane filtration,
preparative HPLC, CCC, and SFC, among others, have
been introduced and explored. It is noteworthy, however,
that no single technique offers a comprehensive solution
to all separation challenges, emphasizing the importance
of employing a combination of different techniques
for optimal results [184].
11.5.1 Importance and Goals of Purification
The term “natural products” typically denotes chemical
substances discovered in nature exhibiting distinctive
pharmacological or biological activities. Encompassing a
broad spectrum, NPs include alkaloids, terpenoids, flavones, lignans, coumarins, and other diverse compounds.
In the contemporary context of cataloged biodiversity, NPs
serve as a prolific source of chemical, structural, and bioactive diversity. They constitute principal starting materials
for industries engaged in pharmaceuticals, cosmetics, flavors, and dietary supplements [185].
The isolation of compounds from natural sources constitutes a paramount, challenging, and time-intensive phase
in NP research and production. Commencing with the
extraction process, subsequent separation processes and
techniques aim to achieve high purity of individual compounds or compound groups for in-depth studies, including molecule structure identification, bioactivity testing,
quality control of natural sources, or further industrial production [186].
The primary objective of purification and isolation is the
separation of a single compound or group of compounds
from inert constituents and undesirable compounds. While
NPs exhibit significant potential as pharmaceutical candidates, their application is constrained by impurities, given
the coexistence of the desired product with numerous
other chemical entities. Therefore, a meticulous purification strategy must be devised for the target product.
Furthermore, active components within NPs commonly
exhibit characteristics such as low content, coexistence
with homologues and structural analogs, and thermolability. Additionally, the inherent complexity of NP matrices
makes the isolation and purification of specific components laborious and challenging. Consequently, the selection of a suitable strategy, utilizing appropriate techniques
and operational conditions, becomes imperative to achieve
high yields of the target compound(s) [187, 188].
It is important to monitor the activity of NPs, including
extracts, fractions, and purified compounds, through a
minimum of three purification steps. This is crucial for
establishing a correlation between chemical purity and
biological activity. The continued development of isolation and purification technologies holds significant
importance for advancing research on NPs. The evolution of modern separation techniques facilitates the
investigation and application of an increasing number of
natural compounds in pharmaceuticals, cosmetics, flavors, and dietary supplements.
While preisolation or enrichment techniques can concentrate and enhance the target compounds in extracts, resulting extracts remain intricate and may comprise various
chemical compound classes. To obtain bioactive compounds with high purity, appropriate isolation and purification techniques are essential. In recent decades, several
potent purification technologies, including prep-HPLC,
CCC, and SFC, among others, have been developed.
However, each technology possesses unique characteristics
and is optimally suited for specific applications, with none
universally suitable for the separation of all NPs [188].
Addressing the complex task of isolating and purifying
NPs frequently requires the application of a mix of diverse
purification technologies. This approach is customized to
the specific properties of the components in the extract and
the features of the desired compounds. The subsequent
sections briefly present the principles and features of different purification technologies and offer examples of their
applications in purifying NPs.
11.5.2 Crystallization, Distillation, and Sublimation
11.5.2.1 Crystallization
Crystallization is a phenomenon wherein the solute spontaneously precipitates from a solution to create a distinct
phase. The fundamental principles underlying the crystallization process are solubility and saturation. In a supersaturated solution, minute solute particles undergo
precipitation, initiating the formation of the crystal
nucleus. Subsequently, the solute diffuses onto the nucleus
surface, facilitating its continuous growth into a fully
developed crystal. Integrated crystallization processes
encompass three essential steps:
i) the establishment of a supersaturated solution;
ii) nucleation; and
iii) crystal growth: the creation of solute crystals is
prompted by the solution being in a state of
supersaturation.
The preparation of a supersaturated solution typically
involves seven methods: evaporation, cooling, chemical
reactions, salting-out, isoelectric point, composite, and
azeotropic distillation. Each of these techniques contributes to achieving conditions where the concentration of
11.5 Purification 223
the solute exceeds its normal saturation point, initiating
the crystallization process [189–191].
11.5.2.2 Distillation
Distillation serves as a purification method relying on evaporation and condensation. In a broader sense, distillation
involves separating substances from a liquid mixture by
selectively evaporating and condensing them. Chemists
commonly use this technique in laboratories to purify and
identify organic liquids. Since various compounds have different boiling points, distillation allows the separation of
components in a mixture. There are three primary methods
for purifying organic compounds, and the choice of the most
suitable method depends on the mixture’s properties [192].
Simple distillation under atmospheric pressure is appropriate for organic liquids with low boiling points, generally
below 150 °C. This is crucial to prevent decomposition and
overcome associated challenges that arise when heating
organic compounds beyond this temperature. The suitability of simple distillation is also contingent on the organic
liquid being relatively pure, with no more than 10% liquid
contaminants. Additionally, it is effective when the organic
liquid possesses a nonvolatile component, such as a solid
contaminant like a polymer, or when it is contaminated by
a liquid with a boiling point differing by at least 70 °C.
Conversely, fractional distillation under atmospheric
pressure is employed for separating mixtures of liquids
with boiling points that are separated by less than 70 °C.
This method is especially apt for achieving the separation
of components with closer boiling points, allowing for a
more refined purification process [193].
Reduced pressure distillation under vacuum becomes
necessary when the boiling point of a compound or solvent
is excessively high, exceeding 150 °C under atmospheric
pressure. This situation makes it challenging to distill the
compound or solvent without significant decomposition.
Additionally, reduced pressure distillation is employed
when the compound undergoes decomposition upon heating at atmospheric pressure. By conducting distillation
under reduced pressure (vacuum), the boiling point of the
compound is effectively lowered, reducing the risk of thermal decomposition and allowing for the distillation of
high-boiling compounds under more favorable conditions.
This technique is particularly useful when working with
heat-sensitive compounds that may degrade at higher
temperatures [194].
11.5.2.3 Sublimation
The verb form associated with the process of sublimation is
“sublime,” or alternatively, “sublimate,” albeit the latter
usage is less common. Furthermore, “sublimate” can also
denote the resulting product obtained through the process
of sublimation. Conversely, the opposite of sublimation is
referred to as deposition, wherein a substance undergoes a
transition directly from a gaseous to a solid state without
an intervening liquid phase [195].
Sublimation refers to the phase transition wherein a substance undergoes a direct transformation from its solid
state to the gaseous state, bypassing the intermediary liquid phase. This phenomenon is notably observed in the
water cycle when snow and ice undergo sublimation,
changing into water vapor in the atmosphere without first
melting into liquid water. While all solids have the potential to sublime, most do so at extremely low rates that are
scarcely detectable. Under normal pressures, the majority
of chemical molecules and elements exist in three distinct
states across varying temperatures, with the transformation from a solid to a gaseous state often necessitating an
intermediary liquid phase. It is crucial to underscore that
the pressure denoted in this context pertains to the partial
pressure of the substance, distinct from the overall pressure, such as atmospheric pressure, encompassing the
entire system. Consequently, any solid material has the
potential to undergo sublimation, provided its vapor pressure surpasses the concurrent partial pressure of the corresponding substance in the surroundings. In certain cases,
substances sublime at appreciable rates, as observed, for
instance, in water ice just below 0 °C [195, 196].
Certain substances, like carbon and arsenic, are more
likely to undergo sublimation directly from a solid to a gas
rather than evaporating from a liquid. This is because their
triple point in the phase diagram occurs at a high pressure,
which is the lowest pressure at which the substance can
exist as a liquid.
Sublimation happens when heat is absorbed, giving molecules enough energy to break free from their neighboring
molecules and turn into vapor. This process requires extra
energy, so it’s called an endothermic change. The enthalpy
of sublimation can be figured out by adding together the
enthalpy of fusion and the enthalpy of vaporization.
Gronbach and colleagues employed this method to discover 52 phytochemicals in sea buckthorn fruit powder sublimates. They identified even more markers in sublimates
from different sea buckthorn extracts. When compared with
sublimates from three other fruit powders, sea buckthorn
showed 27 distinct phytochemicals. Specific markers were
also present in the sublimates of dry extracts, with most
exclusive to the sublimates. Significantly, many of these sublimated compounds had not been previously documented in
sea buckthorn literature. Sublimation, in this context, provides a new approach for identifying previously unknown
plant constituents. The researchers claim to be the first to
224 11 Isolation, Fractionation, and Purification of Natural Products
showcase the effectiveness of sublimation for identifying
plants and their extracts, suggesting potential applications in
detecting food fraud. Further research in analytical chemistry is necessary, and future studies could investigate sublimation with various plant materials [197–199].
11.5.3 Advanced Purification Techniques
The first step toward reaching the final stage involves making sure that the purification process is complete.
Throughout the isolation process, continuous analysis
would have been carried out, and an appropriate analytical
system should already be set up. However, since no further
purification work is anticipated, conducting additional
analysis at this point may be helpful in accurately determining contamination levels. If an isolated peak is observed
on one (preferably more than one) HPLC system, it indicates a single component in the sample being analyzed.
Similarly, if a single spot is seen on different TLC systems,
it suggests a single component. However, to ensure confidence in this conclusion, it’s important to confirm that the
systems used effectively separate and detect all components [125].
It is recommended to use a gradient system for eluting
compounds with a broad range of polarities and detecting
at a low wavelength. This is especially useful for compounds without a characteristic chromophore, as they may
still show some absorbance at the end. Using two different
systems helps reduce the risk of impurities going undetected due to coelution.
Although TLC may not have the same resolving power as
HPLC, its advantage lies in not requiring compounds to
have a chromophore for detection. Almost all compounds
can be visualized through staining. Specific and sensitive
stains can be applied to identify suspected contaminants.
Although nuclear magnetic resonance (NMR) and mass
spectrometry are potent tools for detecting and quantifying
contaminants, it is preferable to ascertain in advance that
impurities will not significantly interfere with the structure
elucidation process [194, 200].
Notably, the development of hyphenated techniques
involving HPLC, such as LC/UV, LC/MS, LC/MSn, and
LC/NMR, represents a significant addition. These techniques are indispensable for the early detection and identification of compounds in crude plant extracts. The
integration of various instrumental and ancillary equipment has further expanded the capabilities of chromatography in phytochemical research and has become an
essential tool for scientists working in the fields of natural
product isolation, characterization, and analysis [193,
201–204].
11.5.3.1 Flash Chromatography
The concept of flash chromatography was introduced in
1978, promising rapid separation of significant amounts of
compounds within a significantly shorter time frame (15
minutes compared to two to three hours). While this technique is widely utilized by organic chemists, it has seen
limited evolution over several decades and remains the
preferred method for the purification of organic compounds. Some minor modifications have been suggested to
enhance accessibility to this technique in teaching laboratories [205].
Introducing flash chromatography systems in chromatography modules provides availability to a diverse range of
prepacked cartridges featuring various sorbent weights,
particle diameters, and phase characteristics, including
normal, reverse, grafted, and chiral phases. Correlating
sorbent weights with HPLC column length and/or diameter allows for understanding the increase in active site
number in both scenarios. This approach also familiarizes
with HPLC column selection. The diverse range of cartridges allows the study of parameters beyond those typically explored in LC laboratories, which often focus on the
impact of mobile phase composition on chromatographic
separation [206].
The introduction of automated flash chromatography
systems like Reveleris (Buchi), Isolera (Biotage), or
Puriflash (Interchim) has significantly revolutionized compound purification techniques. These advancements have
not only transformed methods in organic synthesis but
have also opened up new possibilities in the realm of education, specifically within analytical chemistry. These
automated systems are increasingly popular in companies
and academic research laboratories, serving as a complement or replacement for traditional CC. The use of prepacked silica cartridges in these systems enhances safety by
eliminating the need for researchers to handle hazardous
silica gel.
Moreover, some flash chromatography systems come with
dual detectors, including a UV detector and an evaporative
light-scattering detector (ELSD). This capability allows to
examine how the choice of detection mode affects chromatograms obtained in a single run. While factors like the weight
of silica (column loading), particle sizes, and detector type
have been known. This limitation arises from the infrequency
of owning traditional HPLC columns with different lengths
and silica particle sizes. Additionally, most HPLC instruments typically possess a single detector, ranging from the
common UV–vis detector to the advanced (and expensive)
tandem mass spectrometer [207–210].
Antonio and colleagues presented an effective method
for purifying molecules from intricate extracts, a crucial
11.5 Purification 225
aspect in natural product research. Their comprehensive
approach centered on using mass spectrometry to guide
the isolation of antifungal compounds. Initially, they used
off-line HPLC antifungal activity-based profiling and
HPLC-PDA-MS profiling to identify and locate compounds
of interest on a minor scale. Then, they transferred the analytical gradient to the flash chromatographic level. Finally,
they isolated the targeted bioactive molecules with highresolution flash chromatographic columns (15 µm spherical particles) connected to a single quadrupole mass
spectrometer through a splitter system. This innovative isolation strategy proved to be successful in the large-scale
purification of antifungal components from the liverwort
Chiloscyphus polyanthos. The team demonstrated that this
rational methodology has significant potential for efficiently purifying bioactive compounds on a larger scale,
eliminating the need to repeat a given bioassay at each isolation step. Seven sesquiterpene lactones were isolated
through this approach, with five of them exhibiting bioactivity. Additionally, one compound was identified as a new
discovery. The absolute configuration of some compounds
was established for the first time using electronic circular
dichroism spectroscopy [211].
11.5.3.2 Preparative HPLC
In the initial stages of synthetic chemistry, the creation of
compounds has traditionally comprised two primary
phases: first, the synthesis of the compound, followed by
its subsequent purification. Traditional purification techniques such as crystallization, extraction, and distillation
were commonly employed. In the 1950s and 1960s, the first
instruments for preparative CC emerged, typically consisting of a column and an eluent reservoir set up above it.
Manually applying the sample to the column head and
connecting it to the eluent reservoir enabled the flow
through the hydrostatic pressure of the eluent. In the
1970s, the initial preparative HPLC systems were created to
improve throughput and separation capabilities. These systems employed high-pressure pumps to produce flow, benefiting from improved packing materials with smaller
particle sizes in the columns. Merrifield’s innovation of
solid-phase synthesis for peptides in 1963 brought a new
method to synthesis and purification. Attaching the
C-terminal amino acid to an insoluble polymeric support
resin allowed for the high-concentration application of
reaction compounds, streamlining purification through a
simple filtering process.
The pharmaceutical industry adopted the solid-phase
approach for combinatorial synthesis to supply highthroughput screening assays. However, despite this method,
the purified compounds removed from resin beads were
often insufficiently pure for direct use in assays. To address
this, high-throughput purification systems were needed to
avoid bottlenecks in the drug discovery process. Traditional
methods like distillation or extraction lacked the necessary
level of automation required for high-throughput synthesis.
Preparative HPLC emerged as the method that fulfilled the
requirements for automated and user-friendly purification
of large numbers of compounds [212].
In the initial stages, users assembled early systems by
combining components from different suppliers and ran
them with self-developed software. Presently, the market
provides fully automated purification systems from various
vendors. Although analytical HPLC is now a widely
adopted tool in the pharmaceutical industry, ongoing
advancements continue in preparative HPLC. Trends in
this domain involve the introduction of high-throughput
purification systems capable of processing a multitude of
compounds daily. Additionally, there is a rise in walk-up
systems, where a system administrator oversees the setup,
allowing users to independently purify their samples.
In the field of natural product chemistry, the conventional process typically revolves around extracting beneficial compounds from raw natural product extracts. Given
that the structure of these active compounds is often
unknown, it becomes impractical to collect fractions based
on mass. As a result, the preferred methods involve collecting fractions based on time or the appearance of peaks.
Due to the intricate composition of crude extracts, multiple
purification steps and activity testing are often required
sequentially until the active compound is obtained in a
pure form, facilitating the elucidation of its structure.
Furthermore, natural product extracts are utilized to create
compound libraries with high diversity, where numerous
compounds are isolated through consecutive purification
steps using time- and peak-based fraction collection
[213–216].
Minzou and the research team reported a hyphenated
strategy that involved the off-line coupling of 1,1′-diphenyl–
2-picrylhydrazyl-high-performance liquid chromatography
(DPPH-HPLC), high-speed countercurrent chromatography (HSCCC), and preparative high-performance liquid
chromatography (Prep-HPLC). This innovative approach
was employed for the screening and separation of antioxidants from the ethyl acetate fraction of Polygonum multiflo-
rum roots. In the initial stage of the experiment,
DPPH-HPLC was utilized for targeted guidance. This process allowed for the identification of 12 compounds with
potential antioxidant properties. Subsequently, high-speed
countercurrent chromatography and preparative HPLC
were employed to efficiently isolate these compounds. The
structures of the isolated compounds were identified using
226 11 Isolation, Fractionation, and Purification of Natural Products
analytical techniques such as UV spectroscopy, mass spectrometry, and 1H NMR spectroscopy. The hyphenated strategy provided a systematic and effective approach for the
identification and isolation of antioxidants from Polygonum
multiflorum roots [217].
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