Techniques for extraction and isolation of natural products: a comprehensive review

This is a very simple extraction method with the disadvantage of long extraction time and low extraction efficiency. It could be used for the extraction of thermolabile components.

Ćujić et al. achieved high yields of total phenols and total anthocyanins from chokeberry fruit at an optimized condition with 50% ethanol, a solid–solvent ratio of 1:20 and particle size of 0.75 mm, which suggested that maceration was a simple and effective method for the extraction of phenolic compounds from chokeberry fruit [11]. A study on the extraction of catechin (1, Fig. 1) from Arbutus unedo L. fruits using maceration, microwave-assisted and ultrasound extraction techniques showed that microwave-assisted extraction (MAE) was the most effective, but a lower temperature was applied in maceration with nearly identical extraction yields, which can be translated into economic benefits [12]. Jovanović et al. evaluated the extraction efficiency of polyphenols from Serpylli herba using various extraction techniques (maceration, heat assisted extraction and ultrasonic-assisted extraction). Based on the content of total polyphenols, ultrasonic-assisted extraction produced the highest total flavonoids yield and no statistically significant difference were found between maceration and heat assisted extraction [13]. Cajanus cajan leaves are used in Chinese folk medicine for the treatment of hepatitis, chickenpox and diabetes. Flavonoids are the bioactive compounds. Jin et al. compared extraction rates of orientoside (2), luteolin (3), and total flavonoids from C. cajan leaves by microwave-assisted method, reflux extraction, ultrasound-assisted extraction, and maceration extraction. The extraction efficiency of orientoside, luteolin, and total flavonoids was found to be the lowest in the extract from maceration method [14].

Percolation is more efficient than maceration because it is a continuous process in which the saturated solvent is constantly being replaced by fresh solvent.

Zhang et al. compared the percolation and refluxing extraction methods to extract Undaria pinnatifida. They found that the contents of the major component, fucoxanthin (4, Fig. 2), from the percolation extraction method was higher than that from the refluxing method while there was no significant difference in extract yield between the two methods [15]. Goupi patch is a compound Chinese medicine preparation consisting of 29 Chinese medicines. Fu et al. used the whole alkaloids content determined by acid–base titration as the index and optimized the ethanol percolation method as soaking the medicine with 55% alcohol for 24 h and then percolating with 12 times the amount of 55% alcohol [16]. When using the extracting rate of sinomenine (5) and ephedrine hydrochloride (6) as the index, Gao developed another optimized percolation method: soaking the medicine with 70% ethanol for 24 h and then percolating with 20 times the amount of 70% ethanol. The transfer rates of sinomenine and ephedrine hydrochloride were 78.23 and 76.92%, respectively [17].

The extract from decoction contains a large amount of water-soluble impurities. Decoction cannot be used for the extraction of thermolabile or volatile components.

The ginsenosides (7–31) in ginseng encounter hydrolysis, dehydration, decarboxylation and addition reactions during decocting (Fig. 3) [18]. Zhang et al. investigated the chemical transformation of a famous TCM preparation, Danggui Buxue Tang, an herbal decoction containing Astragali Radix and Angelicae Sinensis Radix. They found that two flavonoid glycosides, calycosin-7-O-β-d-glucoside (32, Fig. 4) and ononin (33), in Astragali Radix, could be hydrolyzed to form calycosin (34) and formononetin (35), respectively, during decocting. The hydrolysis efficiency was strongly affected by pH, temperature, and the amount of herbs [19]. Two compounds of TCM, Sanhuang Xiexin Tang (SXT) and Fuzi Xiexin Tang (FXT), have been used in China for the treatment of diseases such as diabetes for thousands of years. SXT is composed of Rhei Radix et Rhizoma, Scutellariae Radix and Coptidis Rhizoma while FXT is produced by adding another TCM, Aconiti Lateralis Radix Preparata, in SXT. Zhang et al. applied an UPLC-ESI/MS method to monitor 17 active constituents in SXT and FXT decoctions and macerations. The decoction process might enhance the dissolution of some bioactive compounds compared with the maceration process. The contents of 11 constituents [benzoylaconine (36), benzoylhypaconine (37), benzoylmesaconine (38), berberine (39), coptisine (40), palmatine (41), jatrorrhizine (42), aloe-emodin (43) and emodin (44), baicalin (45), wogonoside (46)] in decoctions of SXT and FXT were significantly higher than those in macerations of SXT and FXT. The β-glucuronidase in herbs could catalyze the hydrolysis of the glucuronic acid group from glycosides (baicalin and wogonoside) to transfer into aglycones [baicalein (47) and wogonin (48)]. The high temperature in the decoction process deactivated the activity of the β-glucuronidase and prevented the transformation of glycosides to their aglycones, which led to the discovery of the higher contents of baicalin and wogonoside in decoctions as well as the higher contents of baicalein and wogonin in macerations. The interaction between chemicals from different herbs was also observed. The diester-diterpenoid alkaloids were not detected in the decoction and maceration of FXT, but diester-diterpenoid alkaloid hypaconitine (49) was found in the decoction of the single herb Aconiti Lateralis Radix Preparata. The constituents of the other three herbs in FXT might promote the transformation from diester-diterpenoid alkaloids in Aconiti Lateralis Radix Preparata to other less toxic monoester-diterpenoid alkaloids, which might explain the mechanism of toxicity reduction and efficacy enhancement of TCM by formulation [20].

Reflux extraction is more efficient than percolation or maceration and requires less extraction time and solvent. It cannot be used for the extraction of thermolabile natural products.

Refluxing with 70% ethanol provided the highest yield of the natural bio-insecticidal, didehydrostemofoline (50, Fig. 5) (0.515% w/w of the extract), from Stemona collinsiae root among the extracts prepared by different extraction methods (sonication, reflux, Soxhlet, maceration and percolation) [21]. Zhang compared the extraction efficiency of active ingredients (baicalin (45, Fig. 4) and puerarin (51) from a TCM compound composing seven herbs with two different methods, decoction and reflux. The reflux method was found to be better than the decoction method and the highest yields of baicalin and puerarin were obtained from the reflux method with 60% ethanol as the extraction solvent [22].

The Soxhlet extraction method integrates the advantages of the reflux extraction and percolation, which utilizes the principle of reflux and siphoning to continuously extract the herb with fresh solvent. The Soxhlet extraction is an automatic continuous extraction method with high extraction efficiency that requires less time and solvent consumption than maceration or percolation. The high temperature and long extraction time in the Soxhlet extraction will increase the possibilities of thermal degradation.

Wei et al. obtained ursolic acid (52, Fig. 6) from the TCM Cynomorium (Cynomorii Herba) with a yield of 38.21 mg/g by Soxhlet extraction [23]. The degradation of catechins in tea was also observed in Soxhlet extraction due to the high extraction temperature applied. The concentrations of both total polyphenols and total alkaloids from the Soxhlet extraction method at 70 °C decreased compared to those from the maceration method applied under 40 °C [24, 27].

Pressurized liquid extraction (PLE) has also been described as accelerated solvent extraction, enhanced solvent extraction, pressurized fluid extraction, accelerated fluid extraction, and high pressure solvent extraction by different research groups. PLE applies high pressure in extraction. High pressure keeps solvents in a liquid state above their boiling point resulting in a high solubility and high diffusion rate of lipid solutes in the solvent, and a high penetration of the solvent in the matrix. PLE dramatically decreased the consumption of extraction time and solvent and had better repeatability compared to other methods.

Pressurized liquid extraction has been successfully applied by the researchers at the University of Macau and other institutes in extracting many types of natural products including saponins, flavonoids and essential oil from TCM [8, 25‐27]. Some researchers believed PLE could not be used to extract thermolabile compounds due to the high extraction temperature, while others believed it could be used for the extraction of thermolabile compounds because of the shorter extraction time used in PLE. Maillard reactions occurred when PLE was used at 200 °C to extract antioxidants from grape pomace [28]. Anthocyanins are thermolabile. Gizir et al. successfully applied PLE to obtain an anthocyanin-rich extract from black carrots because the degradation rate of anthocyanins is time-dependent, and the high-temperature-short-duration PLE extraction conditions could overcome the disadvantage of high temperature employed in the extraction [29].

Supercritical fluid extraction (SFE) uses supercritical fluid (SF) as the extraction solvent. SF has similar solubility to liquid and similar diffusivity to gas, and can dissolve a wide variety of natural products. Their solvating properties dramatically changed near their critical points due to small pressure and temperature changes. Supercritical carbon dioxide (S-CO₂) was widely used in SFE because of its attractive merits such as low critical temperature (31 °C), selectivity, inertness, low cost, non-toxicity, and capability to extract thermally labile compounds. The low polarity of S-CO₂ makes it ideal for the extraction of non-polar natural products such as lipid and volatile oil. A modifier may be added to S-CO₂ to enhance its solvating properties significantly.

Conde-Hernández extracted the essential oil of rosemary (Rosmarinus officinalis) by S-CO₂ extraction, hydro distillation and steam distillation. He found that both yields of essential oil and antioxidant activity of SFC extract were higher than those from other two methods [30]. S-CO₂ modified with 2% ethanol at 300 bar and 40 °C gave higher extracting selectivity of vinblastine (53, Fig. 7) (an antineoplastic drug) from Catharanthus roseus, which is 92% more efficient for vinblastine extraction compared to traditional extraction methods [31].

Ultrasonic-assisted extraction (UAE), also called ultrasonic extraction or sonication, uses ultrasonic wave energy in the extraction. Ultrasound in the solvent producing cavitation accelerates the dissolution and diffusion of the solute as well as the heat transfer, which improves the extraction efficiency. The other advantage of UAE includes low solvent and energy consumption, and the reduction of extraction temperature and time. UAE is applicable for the extraction of thermolabile and unstable compounds. UAE is commonly employed in the extraction of many types of natural products [32, 33].

Jovanović et al. achieved a higher yield of polyphenols from Thymus serpyllum L. by UAE at an optimized condition (50% ethanol as solvent; 1:30 solid-to-solventratio; 0.3 mm particle size and 15 min time) than maceration and heat-assisted extraction methods [13]. Wu et al. found that there was no statistically significant difference for extracting ginsenosides, including ginsenosides Rg1 (54, Fig. 8) and Rb1 (7, Fig. 3), chikusetsusaponins V (55), IV (56) and IVa (57), and pseudoginsenoside RT1 (58), from the TCM Panacis Japonici Rhizoma between UAE and reflux using 70% aqueous methanol to extract for 30 min [34]. Guo et al. found both the reflux method and UAE had the advantages of time-saving, convenient operation and high extract yield and that UAE is relatively better than reflux methods for TCM Dichroae Radix using the extract yield and content of febrifugine (59) as the indexes [35].

Microwaves generate heat by interacting with polar compounds such as water and some organic components in the plant matrix following the ionic conduction and dipole rotation mechanisms. The transfers of heat and mass are in the same direction in MAE, which generates a synergistic effect to accelerate extraction and improve extraction yield. The application of MAE provides many advantages, such as increasing the extract yield, decreasing the thermal degradation and selective heating of vegetal material. MAE is also regraded as a green technology because it reduces the usage of organic solvent. There are two types of MAE methods: solvent-free extraction (usually for volatile compounds) and solvent extraction (usually for non-volatile compounds) [36, 37].

Chen optimized the conditions for MAE to extract resveratrol (60, Fig. 9) from the TCM Polygoni Cuspidati Rhizoma et Radix (the rhizome and radix of Polygonum cuspidatum) by orthogonal experiment. An extraction yield of 1.76% of resveratrol was obtained from the optimized conditions as follows: extraction time 7 min, 80% ethanol, ratio of liquid to solid 25:1 (ml:g), microwave power 1.5 kw [38]. Benmoussa et al. employed the enhanced solvent-free MAE method for the extraction of essential oils from Foeniculum vulgare Mill. seeds at atmospheric pressure without any addition of solvent or water. The yield and aromatic profile in the enhanced solvent-free MAE extract was similar to those extracted by hydro distillation and cost only one-sixth of the time of hydro distillation [39]. Xiong et al. developed an MAE to extract five main bioactive alkaloids, liensinine (61), neferine (62), isoliensinine (63), dauricine (64), and nuciferin (65), from the TCM Nelumbinis Plumula (lotus plumule, the green embryo of Nelumbo nucifera seeds) using univariate approach experiments and central composite design. The MAE conditions was optimized as follows: 65% methanol as the extraction solvent, microwave power of 200 W and extraction time of 260 s [40, 44].

Pulsed electric field extraction significantly increases the extraction yield and decreased the extraction time because it can increase mass transfer during extraction by destroying membrane structures. The effectiveness of PEF treatment depends on several parameters including field strength, specific energy input, pulse number and treatment temperature. PEF extraction is a non-thermal method and minimizes the degradation of the thermolabile compounds.

Hou et al. obtained the highest yield of the ginsenosides (12.69 mg/g) by PEF using the conditions of 20 kV/cm electric field intensity, 6000 Hz frequency, 70% ethanol–water solution, and 150 l/h velocity. The yield of the ginsenosides of the PEF extraction method is higher than those of MAE, heat reflux extraction, UAE and PLE. The entire PEF extraction process took less than 1 s and much less than the other tested methods [41]. In a study of antioxidants extracted from Norway spruce bark, Bouras found that much higher phenolic content (eight times) and antioxidant activity (30 times) were achieved after the PEF treatment compared to untreated samples [42].

The structure of the cell membrane and cell wall, micelles formed by macromolecules such polysaccharides and protein, and the coagulation and denaturation of proteins at high temperatures during extraction are the main barriers to the extraction of natural products. The extraction efficiency will be enhanced by EAE due to the hydrolytic action of the enzymes on the components of the cell wall and membrane and the macromolecules inside the cell which facilitate the release of the natural product. Cellulose, α-amylase and pectinase are generally employed in EAE.

Polysaccharide is one of the bioactive ingredients in the TCM Astragali Radix. Chen et al. studied the EAE of polysaccharide from the radix of Astragalus membranaceus using various enzymes and found that glucose oxidase offered better performance in extracting polysaccharide than the other seven enzymes tested (amyloglucosidase, hemicellulase, bacterial amylase, fungal amylase, pectinase, cellulose and vinozyme). The polysaccharide yield under the optimized EAE condition using glucose oxidase increased more than 250% compared with that from non-enzyme treated method [43]. The extraction yield of chlorogenic acid (66, Fig. 10) from Eucommia ulmoides leaves was greatly improved when using cellulase and ionic liquids [44]. Strati el al. found that carotenoid and lycopene (67) extraction yields from tomato waste were increased by the use of pectinase and cellulase enzymes. Compared to the non-enzyme treated solvent extraction method, sixfold and tenfold higher yields of the two target compounds were obtained in samples treated with cellulase and pectinase, respectively [45].

Hydro distillation (HD) and steam distillation (SD) are commonly used methods for the extraction of volatile oil. Some natural compounds encounter decomposition in HD and SD.

The chemical composition and antibacterial activity of the primary essential oil and secondary essential oil from Mentha citrata were significantly affected by distillation methods. Both primary essential oil and secondary essential oil yields by HD were higher than those by SD [46, 50]. Yahya and Yunus found that the extraction time did affect the quality of the essential patchouli oil extracted. When the extraction time increased, the contents of some components decreased or increased [47].

Adsorption column chromatography is widely used for the separation of natural products, especially in the initial separation stage, due to its simplicity, high capacity and low cost of adsorbents such as silica gel and macroporous resins. The separation is based on the differences between the adsorption affinities of the natural products for the surface of the adsorbents. The selection of adsorbents (stationary phase) as well as the mobile phase is crucial to achieve good separation of natural products, maximize the recovery of target compounds and avoid the irreversible adsorption of target compounds onto the adsorbents.

Silica gel is the most widely used adsorbent in phytochemical investigation. It was estimated that nearly 90% of phytochemical separation (preparative scale) was based on silica gel. Silica gel is a polar absorbent with silanol groups. Molecules are retained by the silica gel through hydrogen bonds and dipole–dipole interactions. Thus, polar natural products are retained longer in silica gel columns than nonpolar ones. Sometimes, certain polar natural products might undergo irreversible chemisorption. The deactivation of silica gel by adding water before use or using a water-containing mobile phase will weaken the adsorption. Severe tailing may occur when separating alkaloids on silica gel, and the addition of a small amount of ammonia or organic amines such as triethylamine may reduce the tailing. Twelve alkaloids belonging to the methyl chanofruticosinate group including six new alkaloids, prunifolines A–F (68–73, Fig. 11), were obtained from the leaf of Kopsia arborea by initial silica gel column chromatography using gradient MeOH–CHCl₃ as the mobile phase followed by centrifugal TLC using ammonia saturated Et₂O–hexane or EtOAc/hexane systems as the eluent [48].

Alumina (aluminum oxide) is a strong polar adsorbent used in the separation of natural products especially in the separation of alkaloids. The strong positive field of Al³⁺ and the basic sites in alumina affecting easily polarized compounds lead to the adsorption on alumina that is different from that on silica gel. The application of alumina in the separation of natural products has decreased significantly in recent years because it can catalyze dehydration, decomposition or isomerization during separation. Zhang and Su reported a chromatographic protocol using basic alumina to separate taxol (74, Fig. 11) from the extract of Taxus cuspidate callus cultures and found the recovery of taxol was more than 160%. They found that the increase of taxol came from the isomerization of 7-epi-taxol (75) catalyzed by alumina. It was also found that a small amount of taxol could be decomposed to baccatin III (76) and 10-deacetylbaccatin III (77) in the alumina column [49]. Further investigation into the separation of taxol on acidic, neutral and basic alumina indicated that the Lewis souci and the basic activity cores on the surface of alumina induced the isomerization of 7-epi-taxol to taxol [50].

The structures of polyamides used in chromatography contain both acryl and amide groups. Hydrophobic and/or hydrogen bond interaction will occur in polyamide column chromatography depending on the composition of the mobile phase. When polar solvents such as aqueous solvents are used as the mobile phase, the polyamides act as the non-polar stationary phase and the chromatography behavior is similar to reversed-phase chromatography. In the contrast, the polyamides act as the polar stationary phase and the chromatography behavior is similar to normal phase chromatography. Polyamide column chromatography is a conventional tool for the separation of natural polyphenols including anthraquinones, phenolic acids and flavonoids, whose mechanisms are ascribed to hydrogen bond formation between polyamide absorbents, mobile phase and target compounds. Gao et al. studied the chromatography behavior of polyphenols including phenolic acids and flavonoids on polyamide column. It was found that the polyamide functioned as a hydrogen bond acceptor, and the numbers of phenolic hydroxyls and their positions in the molecule affected the strength of adsorption [51]. In addition to polyphenols, the separation of other types of natural products by polyamide column chromatography were also reported. The total saponins of Kuqingcha can be enriched by polyamide column chromatography, which significantly reduced the systolic pressure of SHR rat [52]. Using a mixture of dichloromethane and methanol in a gradient as the eluent, the seven major isoquinoline alkaloids in Coptidis Rhizoma including berberine (39), coptisine (40), palmatine (41), jatrorrhizine (42), columbamine (78), groenlandicine (79) (Fig. 4), and magnoflorine (80, Fig. 11) were separated in one-step polyamide column chromatography [53].

Adsorptive macroporous resins are polymer adsorbents with macroporous structures but without ion exchange groups that can selectively adsorb almost any type of natural products. They have been widely used either as a standalone system, or as part of a pretreatment process for removing impurities or enriching target compounds due to their advantages, which include high adsorptive capacity, relatively low cost, easy regeneration and easy scale-up. The adsorptive mechanisms of adsorptive macroporous resins include electrostatic forces, hydrogen bonding, complex formation and size-sieving actions between the resins and the natural products in solution. Surface area, pore diameter and polarity are the key factors affecting the capacity of the resins [54]. 20(S)-protopanaxatriol saponins (PTS) (81) and 20(S)-protopanaxadiol saponins (PDS) (82, Fig. 11) are known as two major bioactive components in the root of Panax notoginseng. PTS and PDS were successfully separated with 30 and 80% (v/v) aqueous ethanol solutions from the D101 macroporous resin column, respectively. The chromatography behaviors of PDS and PTS were close to reversed-phase chromatography when comparing the chromatographic profiles of macroporous resin column chromatography to the HPLC chromatogram on a Zorbax SB-C₁₈ column [55]. Recently, Meng et al. obtained the total saponins of Panacis Japonici Rhizoma (PJRS) using D101 macroporous resin. The contents of the four major saponins, chikusetsusaponins V (55), IV (56) and IVa (57), and pseudoginsenoside RT1 (58) (Fig. 8), in the obtained PJRS was more than 73%. The PJRS served as the standard reference for quality control of Panacis Japonici Rhizoma [56]. Some researchers assumed that the principal adsorptive mechanism between macroporous resins and polyphenols was associated with the hydrogen bonding formation between the oxygen atom of the ether bond of the resin and the hydrogen atom of phenolic hydroxyl group of the phenol. The hydrogen bonding interaction force was significantly affected by the pH value of the solution [57, 58].

Silver nitrate is another useful solid support in the separation of natural products. Those natural products containing the π electrons reversibly interact with silver ions to form polar complexes. The greater the number of double bonds or aromaticity of the natural product, the stronger the complexation forms. Silver nitrate is typically impregnated on silica gel (SNIS) or alumina for separation. Several research groups reported the separation of fatty acids on SNIS [59‐61]. Wang et al. reported the isolation of zingiberene from ginger oleoresin by SNIS column chromatography [62]. A pair of isomers, brasiliensic acid (83, Fig. 11) and isobrasiliensic acid (84), were separated from Calophyllum brasiliense by Lemos et al. on an SNIS column [63, 69]. Some research groups also applied silver nitrate in the two-phase system in high-speed counter-current chromatography (HSCCC) to improve the separation. Xanthochymol (85) and guttiferone E (86) are a pair of π bond benzophenone isomers from Garcinia xanthochymus by AgNO₃-HSCCC. The elution order of the π bond isomers in this AgNO₃-HSCCC separation is internal π bond (earlier) < terminal, which is identical to that observed from SNIS column chromatography [64].

Partition chromatography (PC) follows the liquid–liquid extraction principle based on the relative solubility in two different immiscible liquids. In the early stage, one liquid phase was coated to a solid matrix (silica gel, carbon, cellulose, etc.) as the stationary phase and another liquid phase was employed as the mobile phase. The disadvantage of an easily removed stationary phase and unrepeatable results has led to this kind of PC being rarely used today. The bonded-phase, in which the liquid stationary phase is chemically bound to the inert support, which is used as the stationary phase overcomes those drawbacks. Commercially available alkyl such as C8 and C18, aryl, cyano and amino substituted silanes are often used as bonded phases, which are widely used to separate a variety of natural products, especially in the final purification step.

Three PTS (notoginsenoside R1 (87) (Fig. 11), ginsenosides Rg1 (55) (Fig. 8) and Re (88) (Fig. 11)) and two PDS [ginsenosides Rb1 (7) and Rd (9)] (Fig. 3) were well separated in a C18 column using the EtOH–H₂O system as the mobile phase [65]. A novel polyacrylamide-based silica stationary phase was synthesized by Cai et al. and was successfully applied in the separation of galactooligosaccharides and saponins of Paris polyphylla with EtOH–H₂O as the mobile phase [66].

Counter-current chromatography (CCC) is kind of PC that holds the liquid stationary phase by gravity or centrifugal force. CCC has rarely been used in early stages due to its poor stationary retention, long separation time and labor intensive process. CCC was significantly improved in the 1980s, however, when modern CCC, including HSCCC and centrifugal partition chromatography (CPC), were developed. The hydrodynamic CCC systems such as HSCCC have a planetary rotation movement around two rotating axes with no rotating seals, which offers a low pressure drop process. Hydrostatic CCC, e.g., centrifugal partition chromatography, uses only one rotating axis and has a series of interconnecting chambers to trap the stationary phase which offers a higher retention of the stationary phase and a higher system pressure than that of HSCCC. The high system pressure in CPC prevents the improvement of the resolution by increasing the length of the column. High performance CCC (HPCCC) represents a new generation of hydrodynamic CCC and works in the same way as HSCCC, but with a much higher g-level. The HPCCC instruments generate more than 240 g, while early HSCCC equipment gave g-levels of less than 80 g. HPCCC shortens the separation time to less than an hour compared to several hours in previous HSCCC and can achieve at least ten times the throughput of an HSCCC instrument [67]. Compared to the conventional column separation method using a solid stationary phase, both hydrostatic and hydrodynamic CCC systems offer some advantages including the elimination of irreversible adsorption and peak tailing, high loading capacity, high sample recovery, minimal risk of sample denaturation and low solvent consumption. The limitation of CCC is that it only separates the compounds in a relatively narrow polarity window. Over the past 20 years, HSCCC, HPCCC and CPC attracted great attention in separation science and have been widely used in the separation of natural products. Tang et al. developed an HSCCC method using a two-phase solvent system comprising ethyl acetate–n-butanol–ethanol–water (4:2:1.5:8.5, v/v/v/v) to separate six flavone C-glycosides (89‐94, Fig. 12), including two novel compounds from Lophatherum gracile [68]. HSCCC, HPCCC and CPC have also been successfully applied in the separation of volatile oil, which is difficult to separate via conventional column chromatography. Six volatile compounds (curdione (95), curcumol (96), germacrone (97), curzerene (98), 1,8-cineole (99) and β-elemene (100)) were isolated by CPC from the essential oil of Curcuma wenyujin using a nonaqueous two-phase solvent system consisting of petroleum ether–acetonitrile–acetone (4:3:1 v/v/v) [69]. Four major sesquiterpenoids (ar-turmerone (101), α-turmerone (102), β-turmerone (103), and E-atlantone (104)) with similar structures were separated from the essential oil of Curcuma longa in a single HSCCC run using a two-phase solvent system composed of n-heptane–ethyl acetate–acetonitrile–water (9.5/0.5/9/1, v/v) and each compound achieved over 98% purity [70]. Linalool (105), terpinen-4-ol (106), α-terpineol (107), p-anisaldehyde (108), anethole (109) and foeniculin (110) were successfully isolated from the essential oil of Pimpinella anisum by HPCCC using a stepwise gradient elution [71]. Li et al. developed a CPC method for the separation of patchouli alcohol (111) with a nonaqueous ether–acetonitrile (1:1, v/v) solvent system. More than 2 g of patchouli alcohol with over 98% purity were isolated from 12.5 g of essential oil over a 240 ml column [72]. The large volume (several liters) column has been adopted in commercial hydrostatic CCC and hydrodynamic CCC equipment for pilot/industrial scale separation. Few reports could be obtained due to commercial confidentiality. It is difficult to judge whether hydrostatic or hydrodynamic CCC is better for industrial applications. Users might select different types of CCC instrument for different purposes. When the stationary phase is poorly retained in hydrodynamic CCC due to high viscosity and small density differences between the mobile and stationary phases, the hydrostatic CCC is more practical than hydrodynamic CCC because the retention of the stationary phase of hydrostatic CCC is less sensitive to the physical properties of liquid systems and will have a higher retention of the stationary phase. When the stationary phase is well retained in hydrodynamic CCC, higher separation efficiency will be obtained from hydrodynamic CCC than from hydrostatic CCC with the same liquid system and similar column volumes because hydrostatic CCC has relatively low partition efficiency due to a limited degree of mixing, and the hydrodynamic system provides efficient mixing to yield a high partition efficiency.

The separation of natural products by membrane filtration (MF) or gel filtration chromatography (GFC) is based on their molecular sizes.

Ion-exchange chromatography (IEC) separates molecules based on the differences in their net surface charge. Some natural products, such as alkaloids and organic acids possessing a functional group capable of ionization, might be separated by IEC. The charged molecules could be caught and released by ion-exchange resin by changing the ionic strength of the mobile phase (e.g., changing pH or salt concentration). Cation ion-exchange resins were used for the separation of alkaloids, while the anion ion-exchange resins were used for the separation of natural organic acids and phenols.

The positively charged anthocyanins were separated from the neutral polyphenolic compounds in the XAD-7 treated Actinidia melanandra fruit (kiwifruit) extract using Dowex 50WX8 cation ion-exchange resin [81]. Feng and Zhao used semi-preparative chromatography to separate (−)epigallocatechin-gallate [121, Fig. 15)] and (−)epicatechin-gallate (122) in tea crude extract with polysaccharide-based weakly acidic gel CM-Sephadex C-25 [82]. A new alkaloid, fumonisin B₆ (123), along with a known alkaloid, fumonisin B₂ (124), was isolated by IEC over Strata X-C mixed-mode RP-cation-exchange resin followed by reverse-phase chromatography from the fungus Aspergillus niger NRRL 326 cultures extract [83].