Extraction moves compounds from one liquid to another, so that they can be more easily manipulated or concentrated. It also enables the selective removal of components in a mixture. It is essential that you know whether the aqueous layer is above or below the organic layer in the separatory funnel, as it dictates which layer is kept and which is eventually discarded.
Two immiscible solvents will stack atop one another based on differences in density. The solvent should also be volatile so that it can be removed from the solute easily. The water-immiscible organic solvent generally possesses a non-polar or low polarity. It is also critical to know the densities of the solvents to determine the identities of the top and bottom layers.
Most organic liquids have a lower density than water, with the exception of chlorinated organic solvents, and will settle to the bottom of the separatory funnel. Acid-base extraction is a type of liquid-liquid extraction that separates organic compounds based on their acid-base properties. If a solute is an acid or base, its charge changes as the pH is changed. Generally, most organic compounds are neutral, and therefore more soluble in organic solvents than they are in water.
However, if the organic compound becomes ionic, then it becomes more soluble in water. This is useful in extracting an organic acid or base compound from an organic phase to an aqueous phase. Acid-base extraction harnesses this property by transforming the solute into its water-soluble salt form, thereby changing its solubility. The solubility of the organic compound and its salt must be dramatically different in order for the technique to be effective.
For example, consider a mixture containing an organic carboxylic acid, an amine, and a neutral compound. Carboxylic acids consisting of six carbons or more are insoluble in water and entirely soluble in organic solvents. However, their conjugate bases an ionic compound are water-soluble and insoluble in organic solvents. An amine consisting of at least seven carbons is insoluble in water but soluble in organic solvents.
The conjugate acid of that amine an ionic compound is water-soluble and insoluble in organic solvents. When reacted with a base, the carboxylic acid is neutralized to its salt form. The other compounds in the mixture remain neutral. Once the carboxylic acid is transformed into a salt, it will partition to the aqueous phase, while the neutral compounds remain in the organic phase.
Acid-base extraction is also used to separate two weak acids or two weak bases with a significant difference in their pK a. In the case of the acids, the relatively stronger acid, having a small pKa value, is neutralized to a salt using a weak base.
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. Zhang compared the extraction efficiency of active ingredients baicalin 45 , Fig. 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. The degradation of catechins in tea was also observed in Soxhlet extraction due to the high extraction temperature applied. 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 , 26 , 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.
Anthocyanins are thermolabile. Gizir et al. 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.
The low polarity of S-CO 2 makes it ideal for the extraction of non-polar natural products such as lipid and volatile oil. A modifier may be added to S-CO 2 to enhance its solvating properties significantly. He found that both yields of essential oil and antioxidant activity of SFC extract were higher than those from other two methods [ 30 ].
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 ]. Wu et al. Guo et al. 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. An extraction yield of 1. Benmoussa et al. 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. 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. 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.
Chen et al. The extraction yield of chlorogenic acid 66 , Fig. Strati el al. 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 ]. The components in the extract from above methods are complex and contain a variety of natural products that require further separation and purification to obtain the active fraction or pure natural products.
The separation depends on the physical or chemical difference of the individual natural product. Chromatography, especially column chromatography, is the main method used to obtain pure natural products from a complex mixture.
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. 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.
Alumina aluminum oxide is a strong polar adsorbent used in the separation of natural products especially in the separation of alkaloids. 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. 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 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. 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. 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.
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 ]. Recently, Meng et al. 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. 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 , 60 , 61 ]. Wang et al. A pair of isomers, brasiliensic acid 83 , Fig. Some research groups also applied silver nitrate in the two-phase system in high-speed counter-current chromatography HSCCC to improve the separation.
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. 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.
A novel polyacrylamide-based silica stationary phase was synthesized by Cai et al. 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. 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. The high system pressure in CPC prevents the improvement of the resolution by increasing the length of the column. 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. Tang et al. Li et al. 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. In MF, the semipermeable membrane allows smaller molecules to pass through and retains the larger molecules. MF of natural products could be characterized as microfiltration, ultrafiltration, and nanofiltration based on the pore size of the membrane applied. Place the solution to be extracted in the separatory funnel. As the organic solvent and water are not miscible with each other, you should be able to see the two layers organic and aqueous layers clearly.
You should also have two beakers ready, one labeled "organic layer" and the other labeled "aqueous layer". To remove all inorganic substances from the organic layer, shake the separatory funnel to increase the contact between these substances and the water.
The proper way to hold a separatory funnel is to grasp the funnel so that the stopper is in the palm of one hand the stopcock is held with the other. This way leaks are prevented and any pressure built up inside the funnel will not pop the stopper off. It is imperative to VENT the separatory funnel of any gas pressure. After a few shakes, hold the funnel upside down with the funnel stem pointed away from nearby people, and open the stopcock to release any pressure.
Close the funnel and shake the funnel a little more vigorously and vent again as necessary. This "shake and vent" method can be repeated two or three times as needed. When finished, the funnel can be returned to the ring stand and the layers allowed to separate. To determine which layer is which, one can simply add distilled water to the funnel.
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