1. Principles of Separation Techniques in Natural Products Isolation
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Principles of Separation Techniques in Natural Products Isolation
Mohammed N. Sabir
October 2016
11/10/2018

2. Introduction to isolation of natural products (NP)…
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Introduction to isolation of natural products (NP)…
What are NP?
Where they exist?
How to know their activities?
Why they are important?
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3. The systematic method for isolation of NP…
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
The systematic method for isolation of NP…
Identification of the natural source (plant, M.O. etc.).
If plants are involved, you need taxonomic identification of the genus, species and the family as well as the plant part used.
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4. The systematic method for isolation of NP…
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
The systematic method for isolation of NP…
Physicochemical investigation for NP is needed.
The plant crude drug will be subjected for extraction procedure. (you need literature review here)
There are different extraction procedures selected according to the physicochemical preferences of the NP.
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5. The systematic method for isolation of NP…
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
The systematic method for isolation of NP…
The solubility of the bioactive metabolite is crucial for selection of the proper solvent for extraction.
Here comes the concept of like-dissolve-like.
The dielectric constant for the solvent determines its polarity.
The functional groups of the target metabolite determines its polarity and solubility preferences.
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6. What is dielectric constant?
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
What is dielectric constant?
Measures the ability of substances to insulate charges from each others.
It is used to measure the polarity of a solvent.
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7. Dielectric constant of water and some common organic solvents
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Dielectric constant of water and some common organic solvents
Solvent μ
n-Hexane 1.9
Acetic acid 12.9
Acetone
Acetonitrile 37.5
Benzene
Carbon tetrachloride
Chloroform 4.81
Cyclohexane 2.02
Diethylamine 3.6
Diethyl ether 4.33
Ethanol
Ethyl acetate 6.02
Methanol 32.7
Pyridine
Toluene
Water
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8. Why the polarity of a solvent is important?
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Why the polarity of a solvent is important?
How the solvent polarity help in extraction of NP?
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9. The systematic method for isolation of NP…
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
The systematic method for isolation of NP…
Extraction is a process through which a compound or closely related ones (probably isomers) are separated from a mixture.
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10. The systematic method for isolation of NP…
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
The systematic method for isolation of NP…
Techniques for extraction of NP…
Maceration
Soxhelation
Distillation
Percolation or Infusion
Supercritical fluid extraction
Sonication
Decoction
Sublimation
Freeze drying
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11. Important concepts about solvents…
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Important concepts about solvents…
Partition or distribution coefficient
Is the distribution of solute between two immiscible phases.
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12. The systematic method for isolation of NP…
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
The systematic method for isolation of NP…
Another form of extraction is called partitioning (liquid-liquid extraction).
Here, the bioactive substance will be separated according to its partition coefficient to one of two immiscible solvents.
Dunn K.,
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13. Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
The key criteria for choosing proper solvent is the solubility of the solute in the a particular solvent.
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14. The systematic method for isolation of NP…
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
The systematic method for isolation of NP…
In addition to water, the most common organic solvents used for extraction of bioactive metabolites are:-
Methanol
Ethanol
Chloroform
Diethyl ether
Petroleum ether
Ethyl acetate
n-hexane
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15. The systematic method for isolation of NP…
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
The systematic method for isolation of NP…
In some cases mixture of solvents are used as in water with alcohol.
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16. Method of extraction of NP…
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Method of extraction of NP…
Maceration…
When the crude plant material is displaced (soaked) in a solvent for a certain period of time to allow softening of the cellular material and dissolving its bioactive metabolites in the solvent.
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17. Diagrammatic representation for maceration of St. john’s wort
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Diagrammatic representation for maceration of St. john’s wort
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18. No heat or energy required. Easy. Simple.
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Advantages…
No heat or energy required.
Easy.
Simple.
Applicable for a wide range of NP.
No instrumentation required.
Cheap.
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19. Not applicable for crude materials with hard tissues.
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Disadvantages…
Longer time required.
The amount of the extracted material is usually less when compared to other methods.
Not applicable for crude materials with hard tissues.
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20. https://en.wikipedia.org/wiki/Percolation
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Percolation and infusion
The passage of the solvent through the crude material.
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21. Convenient for polar (water soluble) NP. Safe method.
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Advantages…
Easy and quick method.
Cheap.
Convenient for polar (water soluble) NP.
Safe method.
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22. Minimum contact between the solute and the solvent.
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Disadvantages…
Minimum contact between the solute and the solvent.
Not convenient for crude drugs that are rich with fibers.
Not convenient for organic solvents.
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23. Extraction by sonication…
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Extraction by sonication…
Is the use of Ultrasonic source as an aid for extraction. The U/S waves with cause cellular lysis (disruption) and softening which in turn facilitates the movement of the solvent through the crude plant material.
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24. Extraction by sonication… A source of Ultrasound is needed.
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Ultrasonic extraction from herbs
Extraction by sonication…
A source of Ultrasound is needed.
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25. Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
When sonicating liquids at high intensities, the sound waves that propagate into the liquid media result in alternating high-pressure (compression) and low-pressure (rarefaction) cycles, with rates depending on the frequency. During the low-pressure cycle, high-intensity ultrasonic waves create small vacuum bubbles or voids in the liquid. When the bubbles attain a volume at which they can no longer absorb energy, they collapse violently during a high-pressure cycle. This phenomenon is termed cavitation.
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26. Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
During the implosion very high temperatures (approx. 5,000K) and pressures (approx. 2,000atm) are reached locally. The implosion of the cavitation bubble also results in liquid jets of up to 280m/s velocity. The resulting shear forces break the cell envelope mechanically and improve material transfer. Ultrasound can have either destructive or constructive effects to cells depending on the sonication parameters employed.
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27. Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
U/S can lead to a 1permeabilization of cell membranes to ions, and it can 2reduce the selectivity of the cell membranes significantly.
3The mechanical activity of the ultrasound supports the diffusion of solvents into the tissue.
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28. This process is also 5economic and 6environmental friendly.
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
As ultrasound breaks the cell wall mechanically by the cavitation shear forces, it facilitates the transfer from the cell into the solvent.
The particle size reduction by the ultrasonic cavitation 4increases the surface area in contact between the solid and the liquid phase.
This process is also 5economic and 6environmental friendly.
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29. Synergies of Ultrasound with Temperature and Pressure
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Synergies of Ultrasound with Temperature and Pressure
Ultrasonication is often more effective when combined with other anti-microbial methods, such as: thermo-sonication, i.e. heat and ultrasound mano-sonication, i.e. pressure and ultrasound mano-thermo-sonication, i.e. pressure, heat and ultrasound.
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30. Not convenient for glycosides.
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Disadvantages…
Not convenient for glycosides.
Not suitable for compounds that are affected by increase of energy of the medium (ex. Compounds with double bonds).
High energy may cause inter- and intramolecular bonding.
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31. Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Conclusion…
In sonication, there should be a control over the intensity of the energy provided and the time of the extraction to get maximum amount of the intact solute.
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32. Supercritical fluid extraction (SFE)…
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Supercritical fluid extraction (SFE)…
Supercritical Fluid Extraction (SFE) is the process of separating one component (the extractant) from another (the matrix) using supercritical fluids as the extracting solvent. Extraction is usually from a solid matrix, but can also be from liquids. SFE can be used as a sample preparation step for analytical purposes, or on a larger scale to either strip unwanted material from a product (e.g. decaffeination) or collect a desired product (e.g. essential oils). These essential oils can include limonene and other straight solvents. Carbon dioxide (CO2) is the most used supercritical fluid, sometimes modified by co-solvents such as ethanol or methanol. Extraction conditions for supercritical carbon dioxide are above the critical temperature of 31 °C and critical pressure of 74 bar. Addition of modifiers may slightly alter this. The discussion below will mainly refer to extraction with CO2, except where specified.
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33. Supercritical fluid extraction (SFE)…
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Supercritical fluid extraction (SFE)…
A supercritical fluid (SCF) is any substance at a temperature and pressure above its critical point, where distinct liquid and gas phases do not exist. It can effuse through solids like a gas, and dissolve materials like a liquid. In addition, close to the critical point, small changes in pressure or temperature result in large changes in density, allowing many properties of a supercritical fluid to be "fine-tuned". Supercritical fluids are suitable as a substitute for organic solvents in a range of industrial and laboratory processes. Carbon dioxide and water are the most commonly used supercritical fluids, being used for decaffeination and power generation, respectively.
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34. Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
In general terms, supercritical fluids have properties between those of a gas and a liquid. In Table 1, the critical properties are shown for some components, which are commonly used as supercritical fluids.
Table 1. Critical properties of various solvents (Reid et al., 1987)
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35. Table 1. Critical properties of various solvents (Reid et al., 1987)
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Table 1. Critical properties of various solvents (Reid et al., 1987)
Solvent
Molecular wt.
Critical temp.
Critical pressure
Critical density
g/mol K
MPa (atm)
g/cm3
Carbon dioxide (CO2)
44.01
304.1
7.38 (72.8)
0.469
Water (H2O)
18.015
( )
0.322
Methane (CH4)
16.04
190.4
4.60 (45.4)
0.162
Ethane (C2H6)
30.07
305.3
4.87 (48.1)
0.203
Propane (C3H8)
44.09
369.8
4.25 (41.9)
0.217
Ethylene (C2H4)
28.05
282.4
5.04 (49.7)
0.215
Propylene (C3H6)
42.08
364.9
0.232
Methanol (CH3OH)
32.04
512.6
8.09 (79.8)
0.272
Ethanol (C2H5OH)
46.07
513.9
6.14 (60.6)
0.276
Acetone (C3H6O)
58.08
508.1
4.70 (46.4)
0.278
Nitrous oxide (N2O)
44.013
306.57
7.35 (72.5)
0.452
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36. Comparison of Gases, Supercritical Fluids and Liquids
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Table 2 shows density, diffusivity and viscosity for typical liquids, gases and supercritical fluids.
Table 1. Critical properties of various solvents (Reid et al., 1987)
Comparison of Gases, Supercritical Fluids and Liquids
Density (kg/m3)
Viscosity (µPa∙s)
Diffusivity (mm²/s)
Gases 1 10
1–10
Supercritical Fluids
100–1000
50–100
0.01–0.1
Liquids
1000
500–1000
0.001
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37. Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
In addition, there is no surface tension in a supercritical fluid, as there is no liquid/gas phase boundary. By changing the pressure and temperature of the fluid, the properties can be "tuned" to be more liquid- or more gas-like. One of the most important properties is the solubility of material in the fluid. Solubility in a supercritical fluid tends to increase with density of the fluid (at constant temperature). Since density increases with pressure, solubility tends to increase with pressure. The relationship with temperature is a little more complicated. At constant density, solubility will increase with temperature. However, close to the critical point, the density can drop sharply with a slight increase in temperature. Therefore, close to the critical temperature, solubility often drops with increasing temperature, then rises again.[3]
All supercritical fluids are completely miscible with each other so for a mixture a single phase can be guaranteed if the critical point of the mixture is exceeded
Table 1. Critical properties of various solvents (Reid et al., 1987)
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38. Figure 1. Carbon dioxide pressure-temperature phase diagram
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Figure 1. Carbon dioxide pressure-temperature phase diagram
Table 1. Critical properties of various solvents (Reid et al., 1987)
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39. Table 1. Critical properties of various solvents (Reid et al., 1987)
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Advantages[edit]
Selectivity[edit]
The properties of a supercritical fluid can be altered by varying the pressure and temperature, allowing selective extraction. For example, volatile oils can be extracted from a plant with low pressures (100 bar), whereas liquid extraction would also remove lipids. Lipids can be removed using pure CO2 at higher pressures, and then phospholipids can be removed by adding ethanol to the solvent.[1] The same principle can be used to extract polyphenols and unsaturated fatty acids separately from wine wastes.[2]
Speed[edit]
Extraction is a diffusion-based process, in which the solvent is required to diffuse into the matrix and the extracted material to diffuse out of the matrix into the solvent. Diffusivities are much faster in supercritical fluids than in liquids, and therefore extraction can occur faster. In addition, due to the lack of surface tension and negligible viscosities compared to liquids, the solvent can penetrate more into the matrix inaccessible to liquids. An extraction using an organic liquid may take several hours, whereas supercritical fluid extraction can be completed in 10 to 60 minutes.[3]
Table 1. Critical properties of various solvents (Reid et al., 1987)
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40. Table 1. Critical properties of various solvents (Reid et al., 1987)
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Limitations[edit]
The requirement for high pressures increases the cost compared to conventional liquid extraction, so SFE will only be used where there are significant advantages. Carbon dioxide itself is non-polar, and has somewhat limited dissolving power, so cannot always be used as a solvent on its own, particularly for polar solutes. The use of modifiers increases the range of materials which can be extracted. Food grade modifiers such as ethanol can often be used, and can also help in the collection of the extracted material, but reduces some of the benefits of using a solvent which is gaseous at room temperature.
Table 1. Critical properties of various solvents (Reid et al., 1987)
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41. Figure 1. Schematic diagram of SFE apparatus
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Table 1. Critical properties of various solvents (Reid et al., 1987)
Figure 1. Schematic diagram of SFE apparatus
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42. Rf-value = Spot Dist./Solv. Dist.
Pharmacognosy I – Third Lecture 4- Principles of Separation Techniques
Gravity (Flash) Column Chromatography
Thin Layer Chromatography Techniques (TLC)
Solvent Front
Rf-value = Spot Dist./Solv. Dist.
Spot Dist.
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