STK31003 Extraction Dec 2024 PDF

STK31003 Terrestrial Natural Product Chemistry Extraction of Natural Products  EXTRACTION  Extraction is a common technique used in organic chemistry to isolate a target compound.  In the extraction process, a solute is transferred from one phase to another to separ...

STK31003 Terrestrial Natural Product Chemistry Extraction of Natural Products  EXTRACTION  Extraction is a common technique used in organic chemistry to isolate a target compound.  In the extraction process, a solute is transferred from one phase to another to separate it from unreacted starting materials or impurities.  EXTRACTION  In the two main types of extraction, which are liquid- liquid extraction and liquid-solid extraction, the separation is based on solubility. The acid-base extraction is a liquid-liquid extraction that is based on acid-base reactions and a substance will be extracted when reacting with an acid or a base.  If an organic solvent contains a polar compound and a non-polar compound, a liquid-liquid extraction can be performed to extract the polar compound out of the organic solvent.  This extraction process will take place because the polar compound will be more soluble in a polar solvent, like water.  EXTRACTION  Extraction is separating the medicinally active mixture of many naturally active compounds usually contained inside plant materials (tissues) using selective solvents through the standard procedure.  It can also be defined as the treatment of the plant material with solvent, whereby the medicinally active constituents are dissolved and most of the inert matter remains undissolved.  Thus, the purpose of all extraction is to separate the soluble plant metabolites, leaving behind the insoluble cellular materials known as residue.  The Aim For Extraction Two most fundamental question should be asked at the outset of an extraction: What am I trying to isolate? Why am I trying to isolate it?  What am I trying to isolate? There a a number of possible targets of an isolation  An unknown compounds responsible for a particular biological activity  A certain compound known to be produced by a particular organism  A group of compounds within an organism that are all related in some way, such as a common feature  What am I trying to isolate?  All the metabolites produced by one natural product source that are not produced by a different “control” source, e.g. two species of the same genus, or the same organism grown under different conditions  A chemical “dissection” of an organism, in order to characterize all of its interesting metabolites. Such a inventory might be useful for chemical, ecological or chemotaxonomic reasons, among others.  Why am I trying to isolate it? Reason can be:  To purify sufficient amount of compound to characterize it partially or fully  To provide sufficient material to allow for conformation or denial of proposed structures – can be done simply by comparison with standard of known structure; require less material; partially pure compound is adequate. Why am I trying to isolate it?  The generation/production of the maximum amount of a known compound so that it can be used for further work, such as more extensive biological testing  Purity of Compound  For characterizing fully a compound should be suitable for NMR and for this purposes purity of 95 – 100% are required.  If the compound is present at high concentration in the starting material, and there already exist a standard against which to compare it, structure conformation can be done with less pure material and the purification will probably require fewer steps.  Purity of Compound  If a natural product is required for biological testing, it is important to know at least the degree of purity and, preferably the nature of the impurities. The impurity might be responsible for the biological activities.  To generate pharmacological data, very pure compound are required (> 99% pure)  For X-Ray Crystallography studies, extremely pure compounds are required, generally > 99.9% pure ADME - Absorption, distribution, metabolism, and excretion ANDA - Abbreviated New Drug Application BLA - Biologics License Application FDA - Food and Drug Administration NDA - Nondisclosure Agreement IND - Investigational New Drug Pharmacokinetics: The study of what the body does to the drugs Pharmacodynamics: The study of what the drugs does to the body  Phytochemistry  Involve the study of chemical compounds in plants  Isolation  Purification  Structural elucidation  The isolated compounds can show various biological activities and can be used in medicine, agriculture, aquaculture etc.  Phytochemical Screening  Phytochemical screening is a simple method that can be used in the initial stage in order to determine various secondary metabolites that might be present in a particular extract.  Natural products that can be determined based in this methods include: alkaloids, saponin, steroid, cardiac glycoside, phenolic compounds such as flavonoids.  Requirement for Phytochemical Screening  Simple or easy methods  Fast  Minimum apparatus  High sensitivity although the concentration is low  Clear results  Reproducible results  Extract Preparation  Samples is extracted on the day of collection to avoid physiological changes before extraction.  Samples extracted with ethanol or methanol  80% ethanol or methanol is suitable for most extraction  Extract Preparation  ~200g plants part are pulverized and added to 600 ml 80% MeOH and heated slowly for about 1h.  Filter using Buchner or Whatman No.1 filter paper  Concentrate at ~40 oC using rotary evaporater until ~ 1g/mL  Used for phytochemical, microbiological and pharmacological screening.  Alkaloid Screening  Plants extract equivalent to 20g plants evaporated to give conc. syrup.  The samples treated with 10 mL 2M HCl and heated for about 10 min and add about 0.5g clarifying agents.  Final volume ~ 5 mL, divided into 4 parts.  Test tube A use as control  Test tube B add Mayer reagent  Test tube C add Wagner reagent  Test tube D add Dragendorff reagent  Alkaloid Screening  Formation of precipitate or cloudy solution shows positive results for alkaloids.  Quantitative test can be done based on :  not clear cloudy solutions (1+);  clear cloudy solutions (2+)  Precipitate (3+)  Strong precipitate (4+)  White, orange and brown precipitate respectively with Mayer, Dragendorff and Wagner reagents.  Alkaloid Screening  Screening in the field can also be performed  2-4 g samples ground with sand and add 10ml CHCl3 followed by 10 mL CHCl3/NH3 and filter  To filtrate add 20 drops 2M H2SO4 and shake.  Aqueous layer separated and add few drops of Mayer reagent  Formation of white precipitate or cloudy solutions indicate the presence of alkaloids  Saponin Screening  Triterpenoid saponins, steroid saponin, sapogenin.  Saponin can be detected easily because the ability to form honey comb foam; hemolize red blood cells and kill small organism like fish.  Saponin can dissolve easily in 80% MeOH or EtOH  Saponin hydrolisis can be performed by using 2M HCl  Saponin Screening- Foam test  Extracts equivalent to~ 2g plants is used.  Add about 10 ml water  Shake vigorously for 30 sec and leave it for 30 min  Formation of foam like honey comb indicate the presence of saponin  Quantitative determination  Liebermann-Burchadd Test (L-B test)  To determine either steroid or triterpenoid saponins  Plants extracts equivalent to 10g plants is used  10 ml dilute H2SO4 is added and the mixture boiled for 10 min and cooled to room temp.  Extracted with 10 ml CHCl3  Liebermann-Burchadd Test (L-B test)  CHCl3 extract dried using anhydrous sodium sulphate.  Add about 3 drops acetic anhydride and shake to give homogenous solution  Add about 1 drops conc. sulphuric acid and let the sample to mix slowly  Observe the colour changes within 1 h  Blue or greenish blue indicate steroid saponin  Red or purple colour indicate triterpenoid saponin.  Cardiac Glycoside  Various test available  Determine the steroid skeleton – L-B test (Blue or greenish blue)  Determine the unsaturated lactone – Kedde test (red, purple, violet)  Determine deoxy sugars/sugars – Keller Killiani test (red brown)  Positive test with all indicate cardiac glycoside  Cardiac Glycoside Sugar moiety : One to 4 sugars are found to be present in most cardiac glycosides attached to the 3b-OH group. The sugars most common are L-rhamnose, D-glucose, D-digitoxose, D- digitalose, D-digginose, D-sarmentose, L- vallarose, and D-fructose. These sugars predominantly exist in the cardiac glycosides in the b-conformation. The presence of acetyl group on the sugar affects the lipophilic character and the kinetics of the entire glycoside.  Cardiac Glycoside  Unsaturated lactone – Kedde test  0.1 – 0.2 ml plant extract is spotted on Whatman No. 1 paper.  Chromatogram develop in CHCl3  Chromatogram dried and sprayed with Kedde reagent.  Purple/red/violet colour indicate positive results for unsaturated lactone  Cardiac Glycoside  Kedde Reagents: Mix A – 2% 3,5- dinitrobenzoic acid in MeOH; Mix B – 5.7% aqueous NaOH.  Equal volume of A and B are mix prior to use.  Fresh reagents should be used.  Cardiac Glycoside  Deoxy sugar – Keller Killiani test  Plants extract equivalent to 10g plants evaporated to dryness.  Defatted with hexane and hexane discarded  Residue dissolved in 2 ml glacial acetic acid which contain 5% FeCl3 and the mixture transferred in to a test tube  Cardiac Glycoside 1 ml conc sulphuric acid added  Let the solution to mix  Red brown colour indicate positive result for deoxy sugar  Flavonoid Screening  All flavonoids are based on the flavon nucleus  All flavonoids can dissolve easily in MeOH or EtOH  Flavonoids are phenolic compounds and the colour changes when treated with base and observed easily either on chromatogram or in solution.  Flavonoid Screening  Flavonoids are conjugated aromatic compounds and will show strong absorption in the UV or Visible region.  Most flavonoids are attached to sugars  Flavonoids can be determined based on specific colour test, UV, TLC or paper chromatography  Flavonoid Screening  Plants extract equivalent to10 g plants evaporated to dryness using steam bath and than cooled to room temp.  Residues defatted using hexane and extracted several times with hexane  Hexane extract discarded  Residues dissolved in 20 ml 80% MeOH or EtOH and divided into four test tube  Flavonoid Screening  Test tube A as a control  Test tube B – Wilstatter Cyanidin test: Add 0.5 ml conc HCl and 1 cm Mg ribbon (or 0.5 g Mg powder).  Observe the colour change within 10 min  Red (flavone); Crimson (flavonol); Magenta (Flavonone)  Colour usually develop within 1 – 2 min after the addition of acid  Flavonoid Screening  Test Tube C : Bate – Smith and Metcalf test: Add 0. 5 ml conc HCl in test tube C. Red or purple colour that develop immediately after addition of acid indicate the presence of calchone or aurone  Test Tube D: Leucoantocyanidin test – Add 0.5 ml conc HCl in test tube D and heated with steam bath for ~ 15 min. The formation of red or purple colour indicate the presence of leucoantocyanidin  Sample Selections for Phytochemical Studies  Random sampling  Based on traditional uses/ethno- pharmacological uses  Based on specific genera or family (for examples cardenolides found in family such as Crassulaceae, Hyacinthaceae, Iridaceae, Melianthaceae, Ranunculaceae, and Santalaceae; while alkaloids in Ranunculaceae, Leguminosae, Papaveraceae, Menispermaceae, and Loganiaceae).  Based on specific compounds such as alkaloids etc.  EXTRACTION  Sample fresh/dried – dried in open air and ensure no fungal growth.  Grind sample to smaller size to facilitate extraction process  Extract samples – General procedures are available: Normal extraction or Bioassay Guided Isolation; choice of solvents in the initial step: Polar or non polar  Fractionation/Solvent partition  Purification – Various chromatographic procedures: TLC, PC, CC, PTLC, HPLC  Structural elucidation – Spectroscopic Methods  Biological activity testing  Procedure for obtaining the active principles from plants - Bioassay Guided Spectroscopical synthesis Spectroscopical data on-line: data off-line: - LC/UV - UV - LC/MS - MS - LC/NMR structure elucidation - NMR - IR... extraction separation medicinal plants extract(s) fractions pure constituent(s) toxicology bio-assay bio-assay structure modification bio-assay Bioassay Guided Isolation  Isolation & Identification of Phytochemicals CC, PTLC, 1 Cold extraction / soxhlet 2 Fractionation VCC 3 purification HPLC extraction (20-24 hrs) Chromatographic Crude techniques Rhizomes / Extracts/ Fractions fruits/ essential oils leaves 1 Hydrodistillation Phytochemicals (6 – 8 hrs) 5 Bioactivity screening Bioactive Spectroscopic analysis Derivatives phytochemicals Structures Bioactive Derivatives  EXTRACTION  Extraction with organic solvent: Percolation; maceration, extraction with Soxhlet Apparatus  Extraction with water: Infusion; decoction; steam/hydro distillation  FRESH or DRIED SAMPLES  Fresh and dried samples can be used. Ideally, fresh plant tissues should be used for phytochemical analysis.  Alternatively, plants may be dried before extraction. In most reported cases, dried materials are preferred considering their long conservation time compared to fresh samples. Furthermore, fresh specimens are fragile and tend to deteriorate faster than dried ones.  Phytoconstituents such as Essential Oils (EOs) are found in fewer dried samples than in fresh samples.  DRYING METHODS  Drying is the most common method to preserve the plant material from enzymatic degradation, such as hydrolysis of glucoside, etc. It should be dried as quickly as possible in the open room at ambient room temperature with air circulation around the plant material to avoid heat and moisture.  However, they placed in shallow trays with good atmospheric air-up dryness either in the sunshine or in shade depending on nature of the indicated or identified constituents.  Direct sunlight is usually avoided to reduce the possibility of chemical reactions, responsible for forming of the artifact that may result from chemical transformations after exposure to ultraviolet radiation.  DRYING METHODS  Alternatively, plant materials can be dried under optimum temperature conditions between 40 and 50°C, or they can be dried in the oven if needed.  Generally, plant material is dried at temperatures below 30°C to avoid the decomposition of thermolabile compounds.  Plants containing volatile or thermolabile components may be lyophilized (freeze-dried). In freeze-drying the frozen material is placed in an evacuated apparatus with a cold surface maintained at −60 to −80°C. Water vapors from the frozen material then pass rapidly to the cold surface to yield the dry material.  GRINDING Lowering particle sizes increase surface contact between samples and extraction solvents and therefore, increase the yield rate and yield. Grinding resulted in coarse smaller samples, meanwhile, powdered samples gave a more homogenized and smaller particle, leading to better surface contact with solvents used for extraction. Before the extraction, pretreatments such as drying and grinding of plant materials are usually conducted to increase the extraction efficiency.  GRINDING It is essential that the particles are of as uniform size as possible because larger particles take a longer time to complete the extraction process. Smaller particle size is ideal for efficient extraction. Conventional methods are usually used to reduce the particle size of dried plant samples viz. mortar and pestle or electric blenders and mills, etc.  EXTRACTION TECHNIQUES Extraction is separating the medicinally active mixture of many naturally active compounds usually contained inside plant materials (tissues) using selective solvents through the standard procedure. It can also be defined as the treatment of the plant material with solvent, whereby the medicinally active constituents are dissolved and most of the inert matter remains undissolved. Thus, the purpose of all extraction is to separate the soluble plant metabolites, leaving behind the insoluble cellular materials known as residue.  EXTRACTION TECHNIQUES The obtained product is a relatively complex mixture of metabolites, in liquid or semisolid state or (after removing water) in dried powder form. Extraction is based on the difference in solubility between the solute, other compounds in the matrix, and the solvent used to stabilize.  EXTRACTION TECHNIQUES  In general, there are three common type of extractions: liquid/solid, liquid/liquid and acid/base.  The extraction of these active compounds needs appropriate extraction methods that consider the plant parts used as starting material, the solvent used, extraction time, particle size and the stirring during extraction.  Extraction methods include solvent extraction, distillation method, pressing, and others. Solvent extraction is the most widely used method.  EXTRACTION TECHNIQUES  The solvent used, the plant part used as starting material and the extraction procedure are three basic parameters reported that influence the quality of an extract. Proper extraction procure is the first step towards isolating and identifying the specific compounds in crude herbal material. It plays a significant and crucial role in the outcome.  Successful extraction begins with careful selection and preparation of plant sample and thorough review of the appropriate literature for indications of which protocols are suitable for a particular class of compounds or plant species.  EXTRACTION TECHNIQUES  For instance, if the components are volatile or prone to degradation, they can first be frozen and homogenized with liquid nitrogen. The extraction, in most cases, involves soaking the plant material in solvent for some specific time.  Reported properties on an excellent extraction solvent include low toxicity and ease of evaporation at low heat.  MACERATION  This process is conducted by soaking the plant materials (coarse or powered) in a closed stoppered container in a solvent allowed to stand at room temperature for 2–3 days with frequent stirring to obtain plant extracts. A sealed extractor is used to avoid solvent evaporation at atmospheric pressure.  The process is intended to soften and break the plant’s cell walls to release the soluble phytoconstituents. The mixture is then pressed or strained by filtration or decantation after a specific time.  MACERATION  Maceration is the simplest and widely used procedure. The extraction procedure in this stationary process works on principle of molecular diffusion, which is a time-consuming process.  Maceration ensures dispersal of the concentrated solution accumulation around the particles’ surface and brings fresh solvent to the surface of particles for further extraction.  PERCOLATION  It is conducted by passing the solvent through the plant material at a controlled and moderate rate (e.g. 5–7 drops per min) until the extraction is complete before evaporation.  The concentrated plant extracts are commonly collected at the bottom of the vessel.  To obtain a significant amount of extract, successive percolations can be performed by refilling the percolator with fresh solvent and pooling all extracts together.  PERCOLATION  This procedure is mostly used to extract active compounds in the preparation of tinctures and fluid extracts.  Its major disadvantage is that large volumes of solvents are required, and the procedure can be time- consuming.  PERCOLATION  Percolation: Most widespread methods for plants extraction.  Conical glassware container with a tap at the base used to set the rate of solvent elution  Hot/cold solvents  Very fine or very large size samples not suitable  MACERATION  Maceration is also commonly used  Powdered materials in any shaped glass or stainless steel containers can be used  Solvent added until it moisture the samples  Shaking occasionally by mechanical or magnetic stirrer will facilitate extraction  INFUSION  Infusion is a simple chemical process used to extract plant material that is volatile and dissolves readily or release its active ingredients easily in solvents.  Infusion and decoction use the same principle as maceration; both involve soaking the plant material in boiled or cold water which is then allowed to steep in the liquid. The maceration time for infusion is, however shorter. The liquid may then be separated and concentrated under a vacuum using a rotary evaporator.  Infusion finds its application in tea preparation and consumption prescribed in psychophysical asthenia, diarrhea, bronchitis, asthma, etc. In various country, the infusion of the bark of Prunus africana is taken orally to increase the ease of urination and reduce inflammation and cholesterol deposits.  DECOCTION The process involves boiling the plant material in water to obtain plant extracts. Heat is transferred through convection and conduction, and the choice of solvents will determine the type of compound extracted from the plant material. The sample is boiled in a specified volume of water for a defined time (15 to 60 minutes.) It is then cooled, strained, filtered, and added enough water through the drug to obtain the desired volume. This method is suitable for extracting thermostable (that does not modify with temperature) and water soluble compounds.  INFUSION & DECOCTION  Infusion and decoction are simple methods for extraction with water  Infusion – Boiling or cold water is added to the sample  Decoction – The samples boiled for ~ 15 min in water  Useful for water soluble compounds  STEAM & HYDRODISTILLATION Steam and hydrodistillation methods are usually used to extract volatile compounds, including essential oil, insoluble in water, from various aromatic and medicinal plants. This is conducted by boiling the plant materials in water to obtain EOs after vapor condensation. Steam distillation occurs at a temperature lower than the boiling point of the ingredients. The method is useful for thermo-sensitive bioactive compounds e.g., natural aromatic compounds.  STEAM & HYDRODISTILLATION  The heat leads to breakage in the sample’s pores and then enables the release of the target compound from a matrix. As Raoult’s law states that while mixing two immiscible liquids, the boiling point will be reduced.  Therefore, in the mixture of volatile compounds having a boiling point between 150 and 300°C and water having a boiling point at about 100°C (at atmospheric pressure), the mixture evaporation will be getting closer to that of the water  STEAM & HYDRODISTILLATION There are similarities between the hydrodistillation and the steam distillation principles. In brief, plant material is immersed in water or a proper solvent followed by heating to boiling under atmospheric pressure in the distilling apparatus. In a condenser, EOs vapors and water undergo a liquefaction process, and EOS are then separates from water/solvent after collection of the condensate in the decanter. The distillation time depends on the plant material being processed. STEAM & HYDRODISTILLATION  SOXHLET EXTRACTION  In this method, finely ground sample is placed in a porous bag or “thimble” made from a strong filter paper or cellulose, set in the thimble chamber of the Soxhlet apparatus.  Extraction solvents are heated in a round bottom flask, vaporized into the sample thimble, condensed in the condenser, and dripped back. When the liquid content reaches the siphon arm, the liquid content is emptied into the bottom flask again, and the process is continued.  The disadvantages include a large amount of solvent is required. This method is unsuitable for thermolabile compounds as prolonged exposure (long extraction time) to heat may lead to their degradation. SOXHLET EXTRACTION  MICROWAVE-ASSISTED EXTRACTION (MAE)  Microwaves are part of the electromagnetic spectrum of light [300 MHz; (λ = 1 m) up to 300 GHz ; (λ = 1 mm)]. These waves are made up of two perpendicular oscillating fields which are used as energy and information carriers.  In this extraction process, the use of microwave energy results in faster heating. Due to the exposure of each molecule to the microwave field, its direct effects include, thermal gradients reduction, volume generation due to heat, equipment size reduction, because of the higher process rates, and thus increase in productivity, through better usage of the same equipment process volume.  MAE is a feasible green solvent extraction procedure as it uses water or alcohol at elevated temperature and controlled pressure conditions.  MICROWAVE-ASSISTED EXTRACTION (MAE) This procedure has demonstrated various benefits like ease to handle and understand steadiness. Many studies reported that MAE has higher yields and is significantly faster than conventional methods for extracting active substances from plant materials. MAE can be presented as a potential alternative to the traditional solid-liquid extraction techniques.  MICROWAVE-ASSISTED EXTRACTION (MAE) A few of the potential advantages are as follow: a lesser amount of solvent is required (few milliliters of solvent can be used); shorter extraction time, from few seconds to few minutes (15–20 min); improved extraction yield; favorable for thermolabile constituents; during extraction, it provides a stirring, by which the mass transfer phenomenon is improved MICROWAVE-ASSISTED EXTRACTION (MAE) Supercritical Fluid Extraction (SFE)  SFE is used for separating components from the matrix with the application of supercritical fluids as the extracting solvent.  Using CO2 as the extracting fluid has many advantages. Besides, its lower boiling point (31°C) and its critical pressure (74 bar). Moreover, carbon dioxide is abundant in nature, safe and inexpensive.  While carbon dioxide is the preferred fluid for SFE, it possesses several polarity limitations. When extracting polar solutes and when strong analyte- matrix interactions are present solvent polarity is crucial. Carbon dioxide fluid is usually mixed with organic solvents to alleviate the polarity limitations  Supercritical Fluid Extraction (SFE) The SFE extraction procedure possesses distinct advantages:  the extraction of constituents is carried out at a low temperature, strictly avoiding damage from heat and some organic solvents. SFE offers gentle treatment for heat- sensitive material;  fragrances and aroma remain unchanged;  CO2 is an inexpensive solvent;  No solvent residues are left behind;  possibility of direct coupling with analytical chromatographic techniques such as gas chromatography (GC) or supercritical fluid chromatography (SFC);  environmentally friendly extraction procedure. CO2 as the solvent does not cause environmental problems and is physiologically harmless, germicidal, and non-flammable. Supercritical Fluid Extraction (SFE) GENERAL EXTRACTION PROCEDURE GENERAL EXTRACTION PROCEDURE  Solvent Choice  MeOH or 80% EtOH are always used  Alcohol solvent efficiently penetrate cell membrane permitting the extraction of high amount of endocellular components  Chloroform or other non polar solvents may wash out mostly extracellular materials  Aqueous alcohol solvent possess the optimum solubility characteristic  Water seldom used alone – can cause emulsion  Diethyl ether rarely used because of the volatility, flammability, toxicity and tendency to form explosive peroxide  Thin Layer Chromatography  Various coating materials – Silica gel the most common  Various spray reagents  Choice of various solvent system  TLC  TLC plates are made by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate (gypsum) and water.  This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic, and the resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C.  The thickness of the adsorbent layer is typically around 0.1–0.25 mm for analytical purposes and around 1–2 mm for preparative TLC (PTLC). Every type of chromatography contains a mobile phase and a stationary phase.  TLC  The process is similar to paper chromatography with the advantage of faster runs, better separations, and the choice between different stationary phases.  Because of its simplicity and speed TLC is often used for monitoring chemical reactions and for the qualitative analysis of reaction products.  TLC  A small spot of solution containing the sample is applied to a plate, about one centimeter from the base.  The plate is then dipped in to a suitable solvents, such as chloroform or ethanol and placed in a sealed container.  The solvents moves up the plate by capillary action and meets the sample mixture, which is dissolved and is carried up the plate by the solvent.  Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the stationary phase.  TLC  Results also vary depending on the solvent used. For example, if the solvent were a 90:10 mixture of hexane to ethyl acetate, then the solvent would be mostly nonpolar.  This means that when analyzing the TLC, the nonpolar parts will have moved further up the plate. The polar compounds, in contrast, will not have moved as much.  The reverse is true when using a solvent that is more polar than non-polar (10:90 hexane to ethyl acetate). With these solvents, the polar compounds will move higher up the plate, while the non-polar compounds will not move as much.  TLC  The appropriate solvent in context of Thin Layer Chromatography will be one which differs from the stationary phase material in polarity.  If polar solvent is used to dissolve the sample and spot is applied over polar stationary phase TLC, the sample spot will grow radially due to capillary action, which is not advisable as one spot may mix with the other.  Hence, to restrict the radial growth of sample- spot, the solvent used for dissolving samples in order to apply them on plates should be as non- polar or semi-polar as possible when the stationary phase is polar, and vice-versa.  TLC  As the chemicals being separated may be colorless, several methods exist to visualize the spots:  Often a small amount of a fluorescent compound, usually Manganese-activated Zinc Silicate, is added to the adsorbent that allows the visualization of spots under a UV light (UV254). The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence.  Iodine vapors are a general unspecific color reagent Specific color reagents exist into which the TLC plate is dipped or which are sprayed onto the plate  TLC  Once visible, the Rf value of each spot can be determined by dividing the distance traveled by the product by the total distance traveled by the solvent (the solvent front).  These values depend on the solvent used, and the type of TLC plate, and are not physical constants. Chromatogram of 10 essential oils coloured with vanillin reagent  Column Chromatography  ~ 1 g extracts to 100 – 500g adsorbent  Slurry packing – easiest, adsorbent plus solvents and stir to give slurry and add into the column  Dry packing – Add the adsorbent into the column than add solvent slowly; air bubbles  Sample application – Dissolve sample in minimum solvent and add slowly into the column or used dried samples. Dried samples dissolved in small amount of MeOH or EtOAc and add x10 times SiG. Solvent removed and samples can be applied into the column. CC SLURRY PACKING  CC METHODS  Fill the column about one third with solvent (step B).  In a beaker, measure out the required amount of silica or alumina.  In a separate flask or beaker, measure solvent approximately one and a half times the volume of silica.  Add the silica to the solvent, a little at a time, while swirling. Use a Pasteur pipette or glass rod to mix the slurry.  Pour or pipette some of the slurry into the column. Allow the solvent to drain to prevent overflowing (step C).  Tap the column gently to encourage bubbles to rise and the silica to settle (step D).  Continue to transfer the slurry to the column until all the silica or alumina is added.  Rinse the inside of the column by pipetting solvent down the inside edge.  Drain the solvent until the solvent level is just even with the surface of the stationary phase (step E).  CC DRY PACKING Fill the column with solvent, allowing some to run through the sand and cotton wool to remove air bubbles (step B). Place a dry funnel in the top and gently pour the silica or alumina (stationary phase) into the solvent. Allow the solvent to drain to prevent overflowing (step C). Let the stationary phase settle and gently tap the column (see box below) so that the silica or alumina will pack tightly into the column (step D). Drain the solvent until the solvent level is just even with the surface of the phase (step E). CC  CC  Column chromatography is frequently used by organic chemists to purify liquids (and solids.) An impure sample is loaded onto a column of adsorbent, such as silica gel or alumina. An organic solvent or a mixture of solvents (the eluent) flows down through the column.  Components of the sample separate from each other by partitioning between the stationary packing material (silica or alumina) and the mobile eluent.  Molecules with different polarity partition to different extents, and therefore move through the column at different rates.  The eluent is collected in fractions. Fractions are typically analyzed by TLC to see if separation of the components was successful. Components a, b, and c separate as column progresses. Fractions can be collected in test tubes, vials, beakers, or Erlenmeyer flasks.  CC Analyzing the fractions Analyze the fractions by thin-layer chromatography to determine a) if the fraction contains more than one component and b) if fractions can be combined without affecting the purity of those fractions. intial TLC TLC of fractions  CC Other Comments  The success of your separation will be dependant on how well you pack and load the column. It is important to have level sand and silica. It is also important to carefully and evenly add your sample to the packed column.  Do not allow the silica to dry out as the column progresses. Cracks will form within the silica column if it dries, and compounds can fall down the cracks instead of partitioning between mobile and stationary phases.  CC  Compounds pass through sand quickly and do not stick to it. Sand is used at the bottom of the column to help ensure a level silica gel line.  Sand is used at the top of the column to aid even loading of the sample. Sample diffuses evenly through the sand. Once the pinch clamp is removed from the bottom of the column, sample loads evenly onto the silica. Without sand, the sample would be added directly to the silica and would stick where ever it is added, not evenly across the surface of the silica.  Preparative thin layer chromatography (PTLC) is used to separate and isolate amounts of material larger than are normal for analytical TLC. The quantities processed range from 10 mg to greater than 1 gram.  In preparative TLC, materials to be separated are often applied as long streaks, rather than spots, in the sample application zone.  After development, specific components may be recovered by scraping the sorbent layer from the plate in the region of interest and eluting the separated material from the sorbent using a strong solvent.  The material that is recovered from the layer may require further purification by other chromatographic methods, or the purity may be adequate for identification and structure determination by elemental analysis or spectrometry, for use in biological activity or chemical synthesis studies, or for use as standard reference material for comparison with unknown samples.  PTLC  Used for separation after CC  Silica Gel  Thickness 1 – 2 mm  Scrap out the bands  Dissolved separately  Filter  Filtrate evaporate to dryness  Check with TLC/GC/HPLC for purity Alkaloid Extraction – Acid Base  Alkaloid are basic  Reaction with acid form salts  Neutral alkaloids dissolve in organic solvents  Alkaloid salts dissolve in acid  Use this characteristic to extract alkaloids from other impurities Alkaloid Extraction Alkaloid Extraction – Acid Base  Sample ground to smaller size to facilitate extraction.  Defatting process by extraction with less polar solvent such as petroleum ether or hexane or cyclohexane. Filter and discard the solvents  Residues moisture with 10% ammonia or chloroform and add organic solvent such as chloroform to cover the plant materials. Alkaloid Extraction – Acid Base  Occasional shaking will facilitate extraction  Filter and add new solvent  Combine filtrate and concentrate to 1/3 volume  Transfer the filtrate into a separating funnel  Add 1 – 2% mineral acid. 30 ml acid/100 ml extract. Shake slowly. Remove aqueous layer. Reside re-extract with acid Alkaloid Extraction – Acid Base  Combined the acidic fraction. This fractions might contain some impurities  The combined acid fraction extracted with chloroform and the chloroform extract discarded  To the aqueous extract add chloroform and 10% ammonia/sodium carbonate until the solution become alkaline and shake slowly Alkaloid Extraction – Acid Base  Aqueous layer discarded after testing negative with Mayer reagent  The chloroform extract which contain the alkaloids can be dried to give crude alkaloids.  The crude alkaloids can be used for isolation and purification.  Extract with hexane to remove Can add alkali if fatty components extraction of the defatted materials is done with less polar solvent such as chloroform To change alkaloid salt to free bases treat with base such as Na2CO3/NH4OH  Flavonoid  Polyphenolic compounds  Bonded to sugars to give glycosidic bond which enhance the solubility in polar solvents such as alcohol, DMSO, water etc  Flavonoid usually extracted in ethanol, methanol or butanol  Sugar bonded to flavonoid will enhance the solubility in water. Combination between water and alcohol are usually employed.  Flavonoid  Plants materials ground to smaller size.  The samples extracted with MeOH:water; (9:1) in the initial stage and with MeOH:water (1:1) later stage  Sample filtered, the filtrate combined and concentrated to 1/3 volume  Sample transferred into separating funnel and chloroform added to remove non-polar component. The chloroform layer discarded  Aqueous extracts concentrated to dryness and used for further isolation and purification  Flavonoid  For antocyanin fresh materials are used and extracted with 1% HCl in MeOH.  PC, TLC and CC are normally used for the purification processes  In PC solvent such as n-BuOH:HOAc:Water (4:1:5) and t-BuOH:HOAc:water (3:1:1) is suitable for glycoside; for flavon, flavonol HOAc:water:HCl (30:10:3); for antocyanidin HCOOH:HOAC:water (5:3:2) etc  Flavonoid  For CC mixture of various solvent such as CHCl3:MeOH:methyl ethyl ketone: acetone (40:20:5:1) or Benzene:petrol:methyl ethyl ketone:MeOH (60:26:3.5:3.5) can give best separation  Glycoside can be separated in a mixture of MeOH: H2O in suitable ratio  Extraction of Carotenoids  Depending on the polarity, different solvents are used in the extraction process.  For non-polar carotenoids, the most commonly used solvents are hexane, petroleum ether, and tetrahydrofuran.  For polar carotenoids, on the other hand, acetone, ethanol, or ethyl acetate are most commonly used  Extraction of Carotenoids from Pumpkin Peel and Pulp Bioassay Guided Isolation  High Throughput Screening  The key principle of high throughput experimentation is parallelization.  This means that rather than carrying out single experiments one after another, you can run several tests simultaneously.  It is a valuable solution to conducting more cost-effective R&D.  High Throughput Screening  HTS is identification of one or more positive candidates extracted from a pool of possible candidates based on specific criteria.  It is a drug discovery process widely used by pharmaceutical industry.  It allows automation to quickly assay the biological activity of a large numbers of compounds.  High Throughput Screening  High throughput screening (HTS) is the use of automated equipment to rapidly test thousands to millions of samples for biological activity at the model organism, cellular, pathway, or molecular level.  The major advantages of an HTS assay are high sensitivity (single molecule detection), high speed (automation), minimization of sample (microtiter plate assay).  High Throughput Screening  HTS is a translational research technology used for identifying effectors (compounds, peptides, and biologics) that modulate target-specific biochemical or cell-based assays.  In the context of small molecule drug discovery, HTS involves performing assays in the presence of large collections of compounds.  The compounds identified from HTS are referred to as “hits” once the actives are reconfirmed for activity and specificity in downstream assays.  The method uses automation for dispensing compounds and assay reagents and for reading detection signals. Advances in automation, liquid handing, and signal detection, as well as large data computing and analysis, have greatly facilitated screening of mid- to large (103 – >106) compound collections in HTS.  High Throughput Screening  In its most common form, HTS is an experimental process in which 103–106 small molecule compounds of known structure are screened in parallel. Other substances, such as chemical mixtures, natural product extracts, oligonucleotides, and antibodies, may also be screened.  Because HTS typically aims to screen 100,000 or more samples per day, relatively simple and automation- compatible assay designs, robotic-assisted sample handling, and automated data processing are critical.  HTS is commonly used in pharmaceutical and biotechnology companies to identify compounds (called ‘hits’) with pharmacological or biological activity. These are used as starting points for medicinal chemical optimization during pharmacological probe or drug discovery and development.