Phytoconstituents are defined as the chemical compounds that can be found naturally in plants where the word phyto means “plant” in Greek. Isolation and purification of phytoconstituents is the most difficult and complex operation in phytopharmaceutical research. Plant extracts are complex mixtures which contain hundreds or thousands of different constituents. Plant constituents presence in a crude extract make the isolation and measurement of active constituents more difficult. Examples of phytoconstituents are fats, sugars, tannins and so on. Powerful separation techniques with high efficiency and sensitivity are needed. Thus, modern chromatographic techniques such as thin layer chromatography, column chromatography and high-speed counter-current chromatography are used for the isolation and purification of phytoconstituents.
Thin layer chromatography (TLC) is one of the most common and easiest method for purifying components. It is a technique where a solute will undergoes distribution between two phases: stationary phase and mobile phase. Stationary phase will act through adsorption. The absorbent is usually coated on the glass slide or plastic sheet creating thin layer of the stationary phase. The example of precoated plates used is silica gel plates. Mobile phase will be liquid in nature. In addition, the separation can be achieved on the basis of partition or combination of partition and adsorption.
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Separation of the phytoconstituents that present in the plant extract depends on the differences in adsorptive or desorptive behavior in respect of the stationary phase. As example, the photosynthetic pigments can be extracted from kiwi fruit chloroplasts by breaking up the fruit tissue in a very small amount of suitable solvent. The different pigments can be separated by thin layer chromatography by using different solvent mixture.
Firstly, the kiwi fruit is cut into two rectangle, which is about 5mm X 15mm. The two pieces of kiwi fruit is then placed in the microcentrifuge tube that has been added with the extraction solvent . The skin and flesh of the kiwi fruit is squeezed with the help of forceps against the side of the tube. The kiwi fruit pieces also been squeezed between the forceps. The liquid is then slowly develop a green colour. The bottle of chromatography solvent is stand on a level surface. The depth of the solvent in the bottle is measured and recorded without opening the bottle. Then, the pipette tip is used by placing it just below the surface of the top green layer of the solvent. Some of the green liquid drawn into the tip. The tip of the pipette is vertically hold and briefly touch the middle of the pencil line on the TLC strip with the pipette strip. A small drop soaks onto the white coating. When the spot has dried, second drop of the green kiwi extract is placed on the same spot. After that, the strip is lowered carefully into the bottle of chromatography solvent. The strip is removed from the bottle once the solvent has reached approximately 2mm from the end of the strip. Rf (retention factor) value can be calculated by measuring the distance travelled by sample and distance travelled by solvent front to identify the pigments on a chromatogram.
The advantages of using thin layer chromatography for the isolation of biologically active natural products are it is cost-effective and flexible in choosing the stationary and mobile phase. The separation of compounds achieved more rapidly and with less amount of plant sample.
Column chromatography (CC) is useful in the isolation and purification of phytoconstituets. It involves ion exchange, molecular sieves and adsorption phenomenon. It uses the solid-liquid technique. The stationary phase of column chromatography is solid where the solid adsorbent is placed in the column. For example, silica gel is used as stationary phase. The column usually is a vertical glass column. The mobile phase or eluent of column chromatography is either liquid or gas. The mobile phase is added to the top and it will flow down through the column filled with the adsorbent by gravity or external pressure. Example of mobile phase are pet ether, methanol, hexane, benzene, chloroform dichloromethane and water. An equilibrium is achieved between the solute adsorbed on the silica gel and the eluting solvent flows down through the column.
The most commonly used forms of column chromatography are adsorption chromatography, partition chromatography, ion exchange chromatography and gel chromatography. Column chromatography is prepared by using a glass tube with a diameter from 5mm to 50mm and a column bed height of 5cm to 1m with a tap and different filter at the bottom. The filter may be a glass flirt or glass wool plug. This is to prevent the loss of the stationary phase. The column was packed with silica gel by wet packing method. The stationary phase which is the silica gel is poured into the column to form a bed of silica. The extract is then poured on the bed of silica and a layer of cotton is used to cover it. More solvents are poured over it and the column is then eluted gradiently. A solvent used as a mobile phase is known as eluent.
The general procedure of column chromatography are column preparation, load the column, develop the column, collect each fractions and analyze the fractions. The phytoconstituents from the methanolic extract of Limonia acidissima can be isolated using column chromatography. The common name of Limonia acidissima is wood apple due to the hard shell of the fruit. To isolate the natural plant constituents, the plant constituents are first distributed between the stationary phase and mobile phase. The silica is mixed with hexane. The slurry formed is charged into the column in a small portion by touching the sides of the glass without disturbing the stationary phase. After each addition, tap it gently to make sure that the air bubbles are removed and uniform packaging.
Then, the methanolic extract of Limonia acidissima is dissolved in the solvent system and adsorbed on the silica gel. It is let to dry. After drying, the mixture in powder form is loaded at the top of the column and is eluted with n-hexane, followed by hexane-ethyl acetate mixture and ethyl acetate.
Several fractions are collected. As the individual fractions are collected, TLC experiment is carried out to check each fractions. Those fractions that having the same Rf value are mixed together and they are let to evaporate to remove the solvent. The fractions are grouped together based on their homogeneity. HPTLC analysis of MELA and its fractions are then carried out and shows the presence of different phytoconstituents such as carbohydrates, coumarins, glycosides, tannins, flavonoids and triterpenoids.
The advantages of column chromatography are it is less expensive and the stationary phase is easily disposable as it undergoes recycling. It is useful in separating compound mixtures. Other applications of column chromatography are isolate active ingredients and metabolites from biological fluids.
High-speed counter-current chromatography (HSCCC) instrument is one of the type of counter-current chromatography apparatus. It uses liquid-liquid partition chromatographic technique or two-phase solvent system. It consists of stationary phase and mobile phase in where the liquid used for both of these phases should be immiscible solvents. Non-reactive solvents should be used to ensure an effective and successful HSCCC separations. It does not require solid support. Thus, this reduces the risk of irreversible adsorption on solid support and there will be no loss of substrate by binding to the column. Moreover, HSCCC also known as the hydrodynamic counter-current chromatography column.
High-speed counter-current chromatography consists of a multilayer coil separation column. The column consists of helically coiled inert Teflon tubing which rotates on its own axis. One end of the tube is known as “head”, which is the higher-pressure end. The other end of the tube is known as “tail”, which is the lower-pressure end. Elution will occur in “head-to-tail” mode when lower phase is chosen as the mobile phase is HSCCC. However, in opposite case, elution occur in “tail-to-head” mode. It also consists a gear which is assembly arranged so that the helical coils can revolve around a central axis to produce planetary motion. HSCCC operates based on the principle of partition of the solutes between two immiscible liquid phase. Partition of the solutes between two immiscible liquid phases is based on the differences in capacity factor and distribution coefficient of the analyte. The chemical constituents with more partition coefficient in mobile phase will be eluted first while the chemical constituents with higher partition coefficient in stationary phase will be eluted later. Centrifugal force is used to retain the liquid in stationary phase while the liquid in mobile phase is pumped through the column. This gives high yield and purity. When two liquid phases enclosed in the coil, the planetary motion produce the countercurrent movement. This causes the complete separation of two phases as it relies on the Archimedes’ screw force in the helical coil.
High-speed counter-current chromatography (HSCCC) is suitable for the preparative isolation and purification of anthocyanins from the purple sweet potato (Ipomoea batatas). Anthocyanins are colour pigments that can be found in plants and under the classification of flavonoids. Before HSCCC separation, anthocyanin crude extract is prepared. The root of the purple sweet potato are immersed and mashed in the ethanol acidified with 0.5% hydrochloric acid and filtered out. The residues are then washed with 0.5% hydrochloric acid in ethanol for few times. The filtrates are dried using an evaporator and are ready for HSCCC separation. Two-phase solvent system composed of methyl tert-butyl ether/n-butanol/acetonitrile/water/ trifluoroacetic acid (1:4:1:5:0.01, v/v) are used. Each set of the solvent system is added to a separatory funnel and equilibrated thoroughly at room temperature. The column is filled with stationary phase from head to tail. Then, the apparatus will start to rotate at 880rpm. 200mg of the anthocyanin crude extract that had been prepared earlier is dissolved in 4ml of mobile phase. The mobile phase is pumped into the column with a flow rate of 1.5ml per minutes. Injection into the column is done through the injection valve. The UV detector with a wavelength of 520nm is used to monitor the effluent from the outlet of column. The anthocyanin fractions are collected and is analyzed by HPLC for the final purity control. As a result of this operation, four acylated anthocyanins are determined.
Other phytoconstituents that can be isolated from HSCCC include purification of flavonoids from mulberry leaves using two phase solvent system composed of ethyl acetate/n-butanol/water (4:1:5, v/v/v) and isolation and purification of isoflavones from Pueraria lobata using two phase solvent system composed of ethyl acetate/n-butanol/water (2:1:3, v/v/v).
The advantages of HSCCC is simple, fast and highly reproducible. Besides, it does not require solid carriers. Thus, abnormal tailing of chromatographic peaks will not happen due to the absence of solid carriers. Moreover, it saves times due to the rapid process. It is highly sensitive and significant for qualitative and quantitative analysis such as food analysis, environmental analysis, plant analysis and so on.
[bookmark: bau1] In 2016, Neeraj Varma found that phytoconstituents are chemical compounds that can be found in plants . Phytoconstituents responsible for few role such as colour and other organoleptic properties. Wide range of technologies are available to extract the active components and essential oil from the plants. In 2019, Weisheng Feng and others had found that modern technologies and methods of the extraction, isolation and structural identification have been developed. This is to increase the speed of extraction and analysis of phytochemicals. In 2016, Krishnananda P Ingle and others had highlighted on the extraction methods and the analysis of phytoconstituents through application of chromatographic techniques. These include column chromatography, thin layer chromatography and so on.
In 2020, Mahin Basha highlighted that two processes occur simultaneously in any chromatographic separation to ensure the success of the separation of analytes from each other by affecting the behavior of each analyte. The first process involve basic mechanisms such as adsorption, ion exchange, molecular exclusion and partition while the second process involve diffusion process. In 2016, Ozlem Coskun found that chromatography enables separation, identification and purification of components for qualitative and quantitative analysis. The chromatographic technique that based on stationary bed are column and thin layer chromatography.
In 2019, Sandra Šegan and others found out that thin layer chromatography not only is the simplest to perform but also known as the respectable analytical method in different phases of drug discovery and development process. This include identification of bioactive compounds from different natural sources and their isolation and purification. In 2019, Muhammad Sajid Hamid Akash and Kanwal Rehman found out that thin layer chromatography (TLC) is used to separate the components present in mixture using thin stationary phase. Separation of the components is determined by the competition between the adsorption of solute on stationary phase.
In 2015, John Schollar and Dean Madden had reported that extraction of photosynthetic pigments from kiwi fruit chloroplasts can be done by breaking up the tissues in suitable solvent.TLC is then applied to separate these different pigments by using different solvent mixture. By calculating the Rf values, pigments include xanthophyll, chlorophyll A, chlorophyll B, phaeophytin and carotene are identified. Moreover, Isabelle A. Kagan and Michael D. Flythe in 2014 found that TLC can be applied to many different types of plant extracts and aerobic or anaerobic species as well as fungi. They can be used as test organisms if the conditions of the culture are modified to fit the growth requirements of species.
In 2019, Muhammad Sajid Hamid Akash and Kanwal Rehman highlighted column chromatography is a type of adsorption chromatography which is largely used to separate the individual components present in mixture. This technique used on small and large scale for the isolation and purification of the components. In 2015, Devika and Justine Koilpillai found that the ethanol extraction of the dried flower sample (Tagetes erecta Linn.) was subjected to column chromatography separation. Approximately 185 fractions were separated and proved that the sample contains tannins, carbohydrate, alkaloids, saponins, phenols, coumarins, terpenoids and quinines. In 2016, Buddhadeb Mallik and others found that the individual methods for sample loading or elution of columns are different depends on the specific technique used. The general experimental procedure that uses tubular columns is called as column chromatography. Besides, in 2010, M. Mohtasheemul Hasan and others reported that the phytoconstituents can be isolated from the chloroform and butanol extracts of the male flower inflorescence of Phoenix sylvestris Roxb by using column chromatography . Five compounds had been successfully isolated.
In 2018, Bilal Muhammad Khan and Yang Liu reported that HSCCC utilizes a liquid stationary phase. It was important to choose an appropriate solvent system to ensure the efficiency and speed of the separation. In 2009, Neha Senti and others highlighted the major applications of HSCCC are extraction of drugs from plants and isolation and purification of active material, separation of rare earth elements and so on . Separation of alkaloids, flavonoids, dipeptides and proteins and so on shows the versatile and dynamic nature of the technique. In 2019, Pian Zhang and others reported the extracts from mulberry leaves were separated and purified via HSCCC and HPLC was used to detect the product. Two phase solvent system composed of ethyl acetate/n-butanol/water (4:1:5, v/v/v) was selected and showed that the mobile phase flow rate affected the separation efficiency. When mobile phase flow rate decreased, separation efficiency increased.
In 2015, Yanhua Qin and others had showed that HSCCC is a suitable method to separate compounds from Trollius chinensis Bunge. Two steps are involved where hexane/ethyl acetate/ethanol/water (3:7:3:7, v/v) solvent system was selected in the first step while hexane/ ethyl acetate/methanol/water (1:2:1:2, 1:4:1:4, 1:9:1:9, v/v) solvent systems was used in the second step. Eleven compounds that had been successfully separated are vanillic acid, orientin, vitexin, veratic acid, 3,4- dihydroxyphenylethanol, 2”-O-(3”’, 4’’’-dimethoxybenzoyl) orientin, 2’’-O-feruloylorientin, 2’’-O-feruloylvitexin, 2’’-O-(2’’’-methylbutyryl) vitexin, 2’’-O-(2’’’-methylbutyryl) isoswertiajaponin and 2’’-O-(2’’’-methylbutyryl) isoswertisin.
As a conclusion, phytoconstituents play an important role in daily life. From scientific and industrial point of view, commercial utilization of medicinal phytoconstituents is significant. Physical and chemical method are used to isolate and purify the phytoconstituents. Recent method of isolation and purification of phytoconstituents are thin layer chromatography, column chromatography and high-speed counter-current chromatography. These chromatographic techniques are the chemical method. Besides these techniques, new technologies have been successfully developed in recent years to make the isolation and purification of phytoconstituents more effective.
Thin layer chromatography (TLC) is one of the simplest and widely used chromatographic techniques. TLC is important in the field of phytochemistry. Photosynthetic pigments from kiwi fruit chloroplasts can be extracted and these different pigments are separated using TLC. Five pigments are identified such as xanthophyll, chlorophyll A, chlorophyll B, phaeophytin and carotene.
Column chromatography (CC) is another chromatographic technique used for the isolation and purification of phytoconstituents. It uses the solid-liquid techniques and have two phases: stationary phase (silica gel) and mobile phase (suitable solvent). The phytoconstituents from the methanolic extract of Limonia acidissima are isolated using column chromatography.
High-speed counter current chromatography (HSCCC) is the improved technique of counter current chromatography. Both of the stationary phase and mobile phase use immiscible solvent. It consists of a multilayer coil separation column and relies on the Archimedes’ screw force in the helical coil to obtain complete separation. It operates based on the principle of partition of the solutes between two immiscible liquid phases. Centrifugal force is used to retain the liquid in stationary phase while the liquid in mobile phase is pumped through the column. HSCCC is used for the preparative isolation and purification of anthocyanins from the purple sweet potato (Ipomoea batatas).
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