2. • The crude drug contains the active constituents, which can be
isolated from these drugs by various methods of extraction and
separation.
• Extraction is defined as the process of isolation of soluble material
from an insoluble residue, which may be liquid or solid, by treatment
with a solvent on the basis of the physical nature of crude drug to be
extracted, i.e. liquid or solid, the extraction process may be liquid—
liquid or solid—liquid extraction.
Introduction
Part-I
3. TYPES OF EXTRACTS
Decoction:
This is the ancient and more popular process of extracting water soluble and heat
stable constituents from crude drugs by boiling in water for about 15 min.
The boiled crude drug—water mixture is then cooled; filtered and sufficient volume
of cold water is passed through the drug to produce the required volume.
Infusion: An infusion is generally a dilute solution of the readily soluble constituents
of crude drugs.
It is nothing but a type of periodic maceration of the drug with either cold or boiling
water.
The infusion is filtered to remove the crude vegetable material and then produced in
a required volume by addition of water
4. Digestion: Digestion is also a type of maceration in which moderate heating is
preferred during extraction.
Heating causes the digestion of drug material and increases the solvent efficiency.
It is preferred for the drugs in which the use of moderately elevated temperature does
not cause the degradation of constituents.
Tinctures: Tinctures are the alcoholic or hydroalcoholic solutions prepared from
crude drugs or from the pure organic or inorganic substances.
Tinctures of crude drugs may contain 10–20 g of drug per 100 ml of tincture.
The methods used for the preparation of tinctures are: maceration and percolation.
Iodine tincture is an example of inorganic pharmaceuticals, belladonna tincture is
prepared by percolation while compound benzoin tincture, sweet orange peel tincture
are prepared by maceration.
5. Liquid Extracts: The liquid extracts are also termed as fluid extracts in some
official books like USP.
It is a liquid preparation of crude drugs which contain ethyl alcohol as a solvent and
preservative.
It may contain active constituents to the extent of 1 g of drug per ml.
Pharmacopoeial liquid extracts are prepared by the percolation or modified
percolation techniques.
Soft Extract: The extracts which are produced as semisolid or liquids of syrupy
consistency are termed as soft extracts.
These extracts are used in the variety of dosage forms ranging from ointments,
suppositories or can be used in the preparation of some other pharmaceuticals.
Glycyrrhiza extract USP comes in the form of soft extract.
6. Dry Extract: Dry extracts are also known as the powdered extracts or dry powders.
The total extracts obtained by using suitable process of extraction, are filtered,
concentrated preferably under vacuum and dried completely.
The tray drying or spray drying is used for making dry extracts. Just like soft extracts, these
powdered extracts can be used for the manufacture of some medicinal preparations.
7. Properties of solvents
Ideal solvent
Cheap
Non-toxic
Stable i.e. chemically and physically inert
Selective ( Required constitutents with
minimum amount)
Eg1: Petroleum ether for removal of fat from
drug
Eg2: Water and ethanol
8. Solvents used in extractions
Ethanol : Alkaloids, alkaloidal salts,
glycosides, tannins, volatile oils,
resins etc
9. Solvents used in extractions
Water : Proteins, Gums, colouring agents , sugars,
tannins, etc
Dissolves enzymes, organic salts and acids
Cannot dissolve fatty substances like waxes, fats,
fixed oils etc
11. INFUSION
• Drugs - Soft in nature, water penetrates to the tissues and the active
constitutents are water soluble
• Apparatus: Beaker or a teapot – Infusion pots
• Infusions are prepared by simply soaking a drug in water for a 15 min.
12. INFUSION
• May be hot or cold, depending on whether decomposition of
ingredients could occur at higher temperatures
• In this method menstruum (solvent) is separated by simple straining
• The marc (solid part) should not be pressed
• Final volume is not made up – Dilution
• Freshly prepared and consumed within 24hrs
13. INFUSION
Eg
1. Infusion of Senna
2. Infusion of quassia
Conc. Infusion – Maceration and percolation
Alcohol- Menstruum or preservative( 20-25%)
Eg: Conc. Compound infusion of chirata
Conc. Compound infusion of gentian
15. DECOCTION
• Process – Water soluble and heat stable constitutents of hard and woody
crude drugs are extracted out
• Water – Used as menstruum, for specified time
• After boiling – liquid is cooled and filtered, menstruum passed through
marc – produce final volume (Uniform product)
• Ratio of drug to water is 1: 4, 1: 16
• Brought down to 1/4th of its original volume by boiling during
process
• Eg Black tea or coffee
16. DIGESTION
• Is a modified form of maceration
• Gentle heat
• Applicable only to those drugs where moderately elevated
temperature is not objectionable
• Solvent action of the menstruum is increased by gentle heat
17. MACERATION
• Simple maceration for organized drugs:
• Organized drugs – Have specific cell structure like roots, leaves,
stems,flowers etc
• Suitable state of subdivision – crushed or cut small drugs are
placed in contact with whole of the menstruum – closed vessel-
2-7days – occasional stirring.
19. MACERATION
• Liquid is strained and marc pressed
• Expressed liquid is added to strained liquid
• Combined liquid – Decanted and filtered
• Final volume is not adjusted
20. MACERATION
• Water or alcohol – used as menstruum
• Closed vessel – Avoids evaporation of the menstruum
• Drug : menstruum ratio is 1:10
• Sufficient time – menstruum penetrates and diffuses the soluble
constitutents out
• Stirring required – disperse the conc. layer of the dissolved
constitutents around solid particles
21. MACERATION
• Filtration – Insoluble cell contents
• Extracts are to be allowed to stand and then filtered – colloidal material –
cloudy solution due to coagulation.
• Eg: Tincture of squill
• Tincture of lemon,
• Tincture of orange etc
22. MACERATION
•Simple maceration for unorganized drugs:
• Drugs have no cellular or tissue structure and are obtained
from plants as their exudates
• Eg: Gums, resins
23. MACERATION
• Placing a weighed amount of drug in contact with 4/5th of the
menstruum in a closed vessel – 2-7 days with occasional shaking.
• After specified period the clear liquid is decanted or filtered.
• Marc is not pressed
• Volume is adjusted by passing more of (remaining 1/5th ) menstruum
through the marc.
24. MACERATION
• Unorganized drugs – Marc left behind forms a compact mass
• Doesn’t retain any appreciable amount of menstruum therefore pressing the
gummy residue is neither practicable nor necessary
• Adjustment to volume by washing the gummy residue with more menstruum
leads to uniform composition
• Eg: tincture of benzoin, tincture of tolu, tincture of myrrh
25. MACERATION
•Repeated maceration – effective
•Active constitutents left behind in the first pressing of marc –
extracted out in the next maceration
•Menstruum is divided in equal parts
26. MACERATION
• Double maceration:
• Maceration is carried out twice and menstruum is divided in two
equal parts ( same quantity)
• Weighed amount of drug – contact with menstruum with
occasional shaking for specified time
• Liquid is strained and marc pressed
27. MACERATION
• Combined the liquids, the 2nd part of menstruum is added to the ,marc –
allowed to stand.
• Clear liquid is strained, marc is pressed
• The 2 liquids are combined filtered and evaporated to get a required
concentration
• Eg: Conc. compound infusion of gentian
28. Maceration
•Triple maceration:
• Marc is not pressed
• Menstruum is divided into 3 equal parts
• Weighed amount of drug – contact in closed vessel – specified
time period – occasional shaking
• Clear liquid is strained and reserved
• Similar 2nd and 3rd maceration
29. Maceration
• The marc is pressed at the end of 3rd maceration
• Expressed liquid and liquids obtained from 2nd and 3rd maceration are
mixed
• 1st maceration – concentrated liquid
• 2nd and 3rd is combined and evaporated
• 90% alcohol is added before making up the final volume – avoid growth of
micro organisms
• Eg: Liquid extract of Senna
30. PERCOLATION
• Simple percolation
• Used in preparation of liquid extracts
• Drug is moistened with sufficient quantity of menstruum – packed in
percolator
• Drug – allowed to be contact – 24h, menstruum is added from top and
percolation is started
• Marc is pressed – expressed liquid is added to percolator
31. PERCOLATION
•Required volume is produced – menstruum
•Clarified, decanted and filtrated
•Eg: Tincture of belladonna,
• Compound tincture of cardamom,
• Strong tincture of ginger
32. PERCOLATION
• Stages:
• Size reduction of the drug:
• Suitable degree of size reduction
• Imbibition : Powdered drug is moistened – menstruum – allowed to stand for
4hrs in a closed vessel.
• This period drug swells up and menstruum penetrates the cell walls.
• After the lapse of time , the moistened drug is passed through a coarse sieve to
remove lumps and mix the dry powder.
33. PERCOLATION
• Percolators : Open and closed percolators
• Open percolators : Cheap and easy to handle
• Menstruum is water or dilute alcohol
• Closed percolators: Menstruum is volatile
• Eg: Alcohol, ethers etc
• Elevated temperate : Steam jacketed percolators used
34. PERCOLATION
• Packing : Moistened drug is evenly
packed in the percolator
• Percolator – conical vessel having a
lid at the top
• False bottom on which filter paper
or cotton wool is placed to support
the column of the drug and helps
to escape the percolate
• Base fixed with tap – percolate is
collected
35. PERCOLATION
• For packing, a piece of cotton wool , fibres of flax hemp or any other suitable
material moistened with menstruum is places on the false bottom of
percolator.
• A 10% of moistened drug is introduced into percolator – pressed lightly with
rod or any other device to give compression.
• More of drug is introduced and pressed till whole of the drug is packed in the
percolator.
36. PERCOLATION
• Maceration:
• After packing – Menstruum is added to saturate the material and top is covered by the
lid
• When the liquid starts to drip at the bottom of the percolator the tap is closed
• The menstruum layer is maintained on top the packing
• Dryness leads to cracking of the column and inefficient percolation
37. PERCOLATION
• Percolator is set aside to macerate the drug for specified period of time (not
less then 24hrs).
• Menstruum penetrates into the tissues and dissolves the active constituents
and gets extracted with small amount of menstruum.
38. PERCOLATION
• After 24hrs of maceration of the drug – lower tap of percolator is
opened and liquid is collected.
• At controlled speed until 3/4th volume of finished product is
obtained.
• Menstruum is added over the drug as bed should not be kept dry.
39. PERCOLATION
• Percolate is tested for complete exhaustion of drug by various
tests.
• Marc is then pressed and expressed liquid is added to already
collected percolate (80-90% of the final volume).
• Liquid is allowed to settle – decant and clarifies by filtration.
40. Soxhlet extraction
Soxhlet extraction is the process of continuous extraction in which the same solvent can be circulated
through the extractor for several times.
This process involves extraction followed by evaporation of the solvent.
The vapours of the solvent are taken to a condenser and the condensed liquid is
returned to the drug for continuous extraction.
Soxhlet apparatus, designed for such continuous extraction, consists of a body of extractor
attached with a side tube and siphon tube as shown in Figure below.
The extractor from the lower side can be attached to distillation flask and the mouth of the
extractor is fixed to a condenser by the standard joints.
42. The crude drug powder is packed in the soxhlet apparatus directly or in a thimble
of filter paper or fine muslin.
Extraction assembly is set up by fixing condenser and a distillation flask.
The vapours pass through the side tube and the condensed liquid gradually increases the
level of liquid in the extractor and in the siphon tube.
A siphon is set up as the liquid reaches the point of return and the contents of the
extraction chamber are transferred to the flask.
The cycle of solvent evaporation and siphoning back can be continued as many times as
possible without changing the solvent so as to get efficient extraction.
Soxhlet extraction is advantageous in a way that less solvent is needed for yielding more
concentrated products.
The extraction can be continued until complete exhaustion of the drug
43. Properties of supercritical fluids
• A supercritical fluid is any substance above its critical temperature
and critical pressure. In the supercritical area there is only one state-
of-the-fluid and it possesses both gas- and liquid-like properties.
• A supercritical fluid exhibits physicochemical properties intermediate
between those of liquids and gases. Characterisitics of a supercritical
fluid are :
• Dense gas
• Solubilities approaching liquid phase
• Diffusivities approaching gas phase.
43
44. Supercritical Fluid Extraction
44
• Manipulating the pressure , the
densities can be altered
• Addition of polar solvents can
modify the properties
Supercritical
CO2(31.1°C, 73.8bar)
45. Properties of supercritical fluids
• Therefore, the properties of gas-like diffusivity, gas-like viscosity, and liquid-like
density combined with pressure-dependent solvating power have provided the
impetus for applying supercritical fluid technology to various problems.
45
• SF combines desirable properties of gases and liquids
• Solubility of liquids
• Penetration power of gases
• Process flexibility: Density of SF and solubility of a solute in it can be changed in a
continuous manner by change of pressure
• Environmental perspective: Innocuous substances such as water and carbon
dioxide can be used as extracting solvents instead of organics
46. Supercritical fluid extraction
• Certain gases behave like a free flowing liquids or supercritical fluids at the
critical point of temperature and pressure.
• Such supercritical fluids have a very high penetration powers and extraction
efficiency.
• This principle was first used in the food packing industries for the deodorization of
the packed food products.
• The gases like carbon dioxide are held as a supercritical fluid at the critical point
of 73.83 bar pressure and 31.06°C temperature.
• At this critical point CO2 behaves as a liquefied gas or free-flowing liquid and
assists the extraction of the phytochemical constituents from the crude drugs.
47. SFE: Enhanced penetration
• Diffusivity of solvent molecules in a SF approach gaseous
state diffusivity
• Solute diffusivity within a SF approaches that shown in
gaseous phase
Solvent
(SF)
Solute
48. • The advantages of CO2 in supercritical fluid extraction are that it is sterile and
bacteriostatic.
• It is noncombustible and nonexplosive. CO2 is harmless to environment and no
waste products are generated during the process, and it is available in large
amount under favourable condition.
• The mixture to be fractionated is passed in the extraction column along the length
of which the heater is located. CO2 is purged through the column.
• Once the extraction column is pressurized, drug material gets saturated in the
supercritical fluid which moves along the length of the column.
• The operating conditions, i.e. pressure and temperature, are selected.
49.
50. Factors affecting Choice of an extraction Process
1. Character of a drug
2. Therapeutic value of drug
3. Stability of drug
4. Cost of drug
5. Solvent
6. Concentration of product
50
51. • Once an extract has been generated by a suitable extraction protocol the next step
is to fractionate the extract using a separation method so that a purified biologically
active component can be isolated.
• Possibly the simplest separation method is partitioning, which is widely used as an
initial extract purification.
• Partitioning uses two immiscible solvents to which the extract is added; this
can be sequential by using immiscible organic solvents of increasing polarity.
• Typically, this may take place in two steps: (1) water/light petroleum ether (hexane)
to generate a non-polar fraction in the organic layer; (2) water/dichloromethane or
water/chloroform or water/ethyl acetate to give a medium-polar fraction in the
organic layer.
Part II: Isolation/Separation
52. The remaining aqueous layer will contain polar water-soluble natural products. This is
a soft separation method and relies on the solubility of natural products and not a
physical interaction with another medium (e.g. adsorption on silica gel in thinlayer
chromatography
Sublimation
• As a matter of fact there are very few natural products which have sublimating
nature. In this process the compound if subjected to heating, changes from solid
state to gaseous state directly without passing through a phase of liquid.
• Such compounds from the gaseous state get deposited on the cooler surface in
the form of crystals or cake.
• The process is traditionally used for the separation of camphor from the chips of
wood of Cinnamomum camphora to obtain solid sublimate of camphor.
• Sublimation can also be used for the isolation of caffeine from tea or for the
purification of material present in a crude extract.
53. Fractional Crystallization
• Crystallization is an old but a very important method for the purification of the
compounds from the mixture.
• Crystallization mostly depends upon the inherent character of the compound which
forms crystals at the point of supersaturation in the solvent in which it is soluble.
• Many phytopharmaceuticals and natural products are crystalline compounds which
tend to crystallize even in the mixtures.
• Compounds, such as sugars, glycosides, alkaloids, steroids, triterpenoids,
flavonoids, etc., show the crystalline nature with certain exceptions.
• The processes, such as concentration, slow evaporation, refrigeration are used for
crystallizing the products.
• In case of sugars, osazone formation leads to the crystallization of the derivatives
in the form of various types of crystals enabling the analysis of the sugars.
54. Fractional Distillation
• For the distillation the component should have volatile nature.
• Therefore, fractional distillation is mostly used for the separation of essential oil
components.
• Most of the volatile components are steam volatile and if the process of fractional
distillation is skillfully used, various low-boiling and high-boiling components can be
separated from the total oil.
• This process is largely used for the separation of hydrocarbons from the oxygenated
volatile oil components—the product referred to as terpenless essential oils.
• The components like citral, citronellal and eucalyptol are even now separated by
fractional distillation. It is used in the separation of hydrocyanic acid from plant
material.
56. Chromatography
• Chromatography is a physical method of separation in which the components to
be separated are distributed between two phases, one of which is stationary
(stationary phase) while the other moves in a definite direction (the mobile
phase).
• In chromatography, a solute molecule moves along a small channel (made up by
the stationary phase) the remainder of which is filled with solvent molecules
(mobile phase).
• The solute is subjected to two forces acting in different directions: the impelling
force provided by the solvent’s flow and the retarding force consisting of
interactions with the stationary phase, which are governed by weak intermolecular
forces such as London forces and hydrogen bonds, among others.
57.
58.
59. The principle of chromatography differs according to the stationary and mobile phase
used. According to this the types are:
Adsorption Chromatography: Adsorption chromatography is probably one of the
oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase
that is adsorbed onto the surface of a stationary solid phase. The equilibration
between the mobile and stationary phase accounts for the separation of different
solutes.
Partition Chromatography: This form of chromatography is based on a thin film
formed on the surface of a solid support by a liquid stationary phase. Solute
equilibrates between the mobile phase and the stationary liquid.
Ion Exchange Chromatography: In this type of chromatography, the use of a resin
(the stationary solid phase) is used to covalently attach anions or cations on to it.
Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin
by electrostatic forces.
60. Molecular Exclusion Chromatography:
Also known as gel permeation or gel filtration, this type of chromatography lacks an
attractive interaction between the stationary phase and solute.
The liquid or gaseous phase passes through a porous gel which separates the
molecules according to its size.
The pores are normally small and exclude the larger solute molecules, but allow
smaller molecules to enter the gel, causing them to flow through a larger volume.
This causes the larger molecules to pass through the column at a faster rate than the
smaller ones.
61. Affinity Chromatography:
This is the most selective type of chromatography employed.
It utilizes the specific interaction between one kind of solute molecule and a second
molecule that is immobilized on a stationary phase.
For example, the immobilized molecule may be an antibody to some specific protein.
When solute containing a mixture of proteins is passed by this molecule, only the
specific protein is reacted to this antibody, binding it to the stationary phase.
This protein is later extracted by changing the ionic strength or pH.
62. Retention
The retention is nothing but a measure of the speed at which a compound moves in a
chromatographic system.
In continuous development systems like HPLC or GC, where the compounds are eluted with the
eluent, the retention is usually measured as the retention time Rt, the time between injection and
detection.
In interrupted development systems like TLC, PC the retention is measured as the retention factor Rf,
the run length of the compound divided by the run length of the eluent front.
63. Thin Layer Chromatography
The thin layer chromatography is a widely used, fast technique for the qualitative analysis of a mixture
of compounds.
The stationary phase consists of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat
carrier like a glass plate, a thick aluminium foil, or a plastic sheet.
TLC has certain advantages over PC.
Fractionations can be effected more rapidly with smaller quantities of the mixture.
The separated spots are usually more compact and more clearly demarcated from one another, and
the nature of the film is often such that drastic reagents, such as concentrated sulphuric acid, which
would destroy a paper chromatogram, can be used for the location of separated substances.
TLC plates are made by mixing the adsorbent with a small amount of inert binder like calcium sulfate
(gypsum) and water, spreading the thick slurry on the carrier, drying the plate, and activation of the
adsorbent by heating in an oven.
66. Several methods exist to make colourless spots visible.
Often a small amount of a fluorescent dye is added to the adsorbent that allows the
visualization of UV absorbing spots under a black light (UV254).
Even UV light without fluorescent dye could scan the compounds, both in long (365
nm) and short (254 nm) wavelength ultraviolet light.
Iodine vapors are a general unspecific colour reagent.
67. The result of the TLC analyses is more often monitored after the chemical
development of the plate.
The plate is quickly immersed or carefully sprayed with chemical reagents followed
by air drying at room temperature or heating at a certain temperature in an oven or
on a hot surface.
Development reagents can be unspecific or specific for a certain class of natural
products.
Examples of the most common developing reagents used in phytochemistry are
shown in table (next slide).
68.
69. There are a number of advantages to TLC
Cost-effective compared with instrumental methods and requires little training or
knowledge of chromatography.
Easy scale-up from analytical to preparative mode with quick isolation of milligram to
gram amounts of product.
Flexibility of choice of mobile and stationary phases.
A separation may be readily optimized to ‘zero in’ on one component and methods may be
quickly developed.
Practically any separation can be achieved with the correct mobile and stationary phases.
A large number of samples may be analysed or separated simultaneously.
70. The major disadvantages of TLC are that:
Loading and speed are poor compared with instrumental chromatography.
There is poor detection and control of elution compared with high-performance liquid
chromatography.
71. PREPARATIVE THIN-LAYER CHROMATOGRAPHY
• This method employs glass or aluminium plates that are pre-coated with sorbent (e.g. silica gel)
of varying thickness dependent on the amount of material to be loaded onto the plates.
• The coating of preparative plates may be 1–2 mm thick, that is up to 10 times thicker than
analytical plates.
• The compound mixture is loaded at 1–2 cm from the bottom edge of the plate as either a spot or a
continuous band.
• The plate is then lowered into a tank containing a predetermined solvent that will migrate up the
plate and separate the compound mixture according to the polarity of the components.
• It is rapid and cheap and has been the method of choice for separating lipophilic compounds.
• Preparative plates are available from suppliers as pre-coated plates of 1–2 mm thickness in silica,
alumina or C18.
• However, homemade plates offer greater flexibility by allowing the incorporation of modifying
agents into the sorbents (e.g. silver nitrate for separation of olefinic compounds – known as
argentation TLC), use of other sorbents (ion exchange, polyamide, cellulose) and the addition of
indicators and binders.
73. • The sample is dissolved in a small volume of solvent and applied as a thin line 2
cm from the bottom of the plate and dried.
• The plate is then eluted in a suitable solvent and UV-active compounds are
visualized at 254 or 366 nm.
• Natural products that are not UV-active will need development using a suitable
spray reagent such as vanillin-sulphuric acid, Dragendorff’s reagent etc.
• The bands containing pure natural product are scraped off the plate and the
natural product is desorbed from the sorbent.
• This desorption may be carried out by placing the compound-rich sorbent into a
sintered glass funnel and washing with a suitable solvent followed by collection
and concentration of the filtrate.
• The purified ‘band’ should then be assessed for purity by analytical TLC.
• Disadvantages of preparative TLC are poor loadings and speeds that compare
unfavorably to flash chromatography.
74. Column Chromatography
• Column chromatography utilizes a vertical glass
column filled with some form of solid support with the
sample to be separated placed on top of this support.
• The rest of the column is filled with a solvent which,
under the influence of gravity, moves the sample
through the column.
• Solvent mixture is eluted according to increasing
polarity (0-100%) eg: n-hexane: ethylacetate;
Dichloromethane:methanol
https://www.youtube.com/watch?v=fF1gXUvyGb4
https://www.youtube.com/watch?v=YyHjsTqUgKY
75. Flash Column Chromatography
• This is a fast, simple, widely used preparative separation
technique, where the stationary bed is packed in a long, narrow
glass lube.
• The flow rate of the mobile phase of the system can be
accelerated either by applying pressure on the top of the column
or by applying suction from the lower end of the column to
decrease the time that the compounds spend in the column or to
increase the flow rate of the mobile phase.
• Typically, silica is used as the stationary phase but other
stationary beds such as reverse phase silica or cellulose are also
used depending on the nature of the compounds to be
separated.
• The particles size should be smaller than that of the column
chromatography.
https://www.youtube.com/watch?v=nbLlH0CT6IA
Not required
77. Using this technique, milligrams to tens of grams can be separated.
The bioactive extract can be dissolved in solvent and loaded onto the column directly; solvent is then
pumped through the column and fractions are collected, resulting in a rapid separation of extract
components.
This is a rapid method; 10 g of extract can be fractionated into 12 fractions of increasing polarity in 30
min using a step gradient solvent system.
There are a number of benefits to this, particularly that speed minimizes contact with reactive
sorbents (e.g. silica) and that hazardous sorbents such as silica, which when free may cause
silicosis, are contained in the cartridges.
Additionally, the cartridges may be re-used, reducing the cost of the bioassay guided process.
The high flow-rates employed by this technique (20–250 ml/min) retain ‘band-like’ movement of the
components through the column, resulting in a high resolution.
Compounds eluting from the column may be detected by TLC (of fractions) or the eluant may be
passed through a UV detector so that compounds that absorb UV light can be detected as they elute
from the column.
78. GEL CHROMATOGRAPHY
• This technique employs a cross-linked dextran (sugar polymer) that, when added to a suitable
solvent (e.g. chloroform or ethyl acetate), swells to form a gel matrix.
• The gel contains pores of a finite size that allow small molecules (< 500 Da) to be retained in the
matrix; larger molecules (> 500 Da) are excluded and move quickly through the gel.
• This gel is loaded into a column and the extract is added to the top of the column. Large
molecules are the first to elute, followed by molecules of a smaller size.
• This is an excellent method for separating out chlorophylls, fatty acids, glycerides and other large
molecules that may interfere with the biological assay.
• Different sorts of gels are available, which may be used in organic solvents (e.g. LH-20) or
aqueous preparations such as salts and buffers (e.g. G-25).
• Therefore, both non-polar and polar natural products can be fractionated using this technique.
79. • Not only are compounds fractionated according to size, but also a small amount of adsorption
chromatography occurs, as the dextran from which the gel is made contains hydroxyl groups that
interact with natural products, facilitating some separation according to polarity.
• A further benefit of this technique is that many different gels are available with a variety of pore
sizes that can be used to separate compounds from 500 to 250,000 Da.
• This is the method of choice for large molecules, in particular proteins, polypeptides,
carbohydrates, tannins and glycosides, especially saponin and triterpene glycosides.
https://www.youtube.com/watch?v=qrUaZWUM9uw
80. High-Performance Liquid Chromatography
• This is a versatile natural
product isolation technique
which is similar to flash
chromatography; however, high
pressure (up to 4,000–5,000
psi) is applied to the system to
move the mobile phase through
the smaller particle sized (2–10
μm) stationary phase bed.
• The column is stainless steel to
withstand the high pressure.
• It employs relatively narrow
columns about 5 mm diameter
for analytical work.
• Reversed phase packing material is
produced by the bonding of
81. High-Performance Liquid Chromatography
• In the commercial material there appears to be a considerable proportion of residual silanol OH groups, and
this would lead to both adsorption and partition effects during separation. the eluting compounds can be
detected using their different physical and structural properties by connecting a detector (UV/visible UV/VIS,
refractive index RI).
• Furthermore, the eluting compounds can be connected to a spectrophotometer (NMR, MS) to study the
spectral characteristics of compounds eluting through the HPLC system. The separation principle of HPLC is
based on the distribution of the analyte (sample) between a mobile phase (eluent) and a stationary phase
(packing material of the column).
• Depending on the chemical structure of the analyte, the molecules are retarded while passing the stationary
phase.
• The specific intermolecular interactions between the molecules of a sample and the packing material define
their time “on-column”.
• Hence, different constituents of a sample are eluted at different times. Thereby, the separation of the
sample ingredients is achieved.
84. Gas Chromatography
Gas chromatography is the most widely used chromatographic technique to analyse volatile
compounds where those compounds are carried by an inert gas like nitrogen, helium or argon through
a heated (50–350°C) stationary bed (silica supported with bonded polar or nonpolar phase).
The choice of stationary phase is governed by the temperature at which the column is to operate and
the nature of the material to be fractionated; it should be nonvolatile at the operating temperature
and should not react with either the stationary and mobile phases or the solutes.
85. Some commonly used, stationary phase materials are nonpolar compound like silicone oils, greases,
high-boiling point paraffins, such as mineral oil, squalene, moderately polar compounds high-boiling
point alcohols and their esters and strongly polar compounds polypropylene glycols and their esters.
There are two general types of column, packed and capillary.
Packed columns contain a finely divided, inert, solid support material (commonly based on
diatomaceous earth) coated with liquid stationary phase.
Capillary columns have an internal diameter of a few tenths of a millimeter.
They can be one of two types; wall-coated open tubular (WCOT) or support-coated open tubular
(SCOT).
Wall-coated columns consist of a capillary tube whose walls are coated with liquid stationary phase.
In support-coated columns, the inner wall of the capillary is lined with a thin layer of support material
such as diatomaceous earth, onto which the stationary phase has been adsorbed. SCOT columns are
generally less efficient than WCOT columns.
Both types of capillary column are more efficient than packed columns.
86. Either a flame ionization detector (FID) or electron capture detector (BCD) detects the
compounds eluting from the column producing a signal which can transform into a peak or a
chromatogram.
The technique is very sensitive, and low concentrations of sample (less than nanograms)
can be analysed.
88. Ultraviolet-Visible Absorption Spectroscopy
Different organic molecules with certain functional groups (chromophores) that contain valence
electrons of low energy can absorb ultraviolet (UV) or visible (VIS) radiation at different wavelengths.
Hence the absorption spectrum of a certain molecule will show a number of absorption bands
corresponding to structural groups within the molecule.
Most commonly used solvent is 95% ethanol, because it solubilizes most classes of compounds.
Other solvents used are water, petroleum, hexane, ether and methanol.
The absorption is recorded using a detector, such as photo diode array (PDA).
This phenomenon could be used to identify the functional groups of a certain molecule or when a
PDA detector is connected to a HPLC system; it could be used to monitor the separation or purity of a
certain mixture or a compound/s that contained different chromophores.
Selection of the detection wavelength of a compound determines the nature of the chromophore
within the molecule.
89. UV spectra of caffeine
UV spectra of green tea extracts
Not required
90. While the compounds which possess chromophores are detected between 200–700 nm
(visible),
others which do not possess chromophores are detected between 200–400 nm (UV).
Strengths
• An easy-to-use, cheap and robust method offering good precision for making quantitative
measurements of drugs in formulations.
• Routine method for determining some of the physico-chemical properties of drugs, which
need to be known for the purposes of formulation.
Limitations
• Only moderately selective. The selectivity of the method depends on the chromophore of
the individual drugs.
• Not readily applicable to the analysis of mixtures
91. Infrared Spectroscopy
This is done by IR spectrophotometer and the plant compounds used is either in liquid, e.g.
chloroform, as a mull with nujol oil or in the solid state, mixed with potassium bromide to form a
thin disc.
The term ‘infra red’ covers the range of the electromagnetic spectrum between 0.78 and 1,000 μm.
In the context of infra red spectroscopy, wavelength is measured in wavenumbers, which have the
unit cm-1.
Electromagnetic radiation ranging between 400 cm-1 and 4000 cm-1 (2500 and 20 000 nm) is passed
through a sample and is absorbed by the bonds of the molecules in the sample causing them to
stretch or bend.
The wavelength of the radiation absorbed is characteristic of the bond absorbing it.
92. The most useful IR region lies between 4,000 and 670 cm-1. IR radiation does not have enough
energy to induce electronic transitions seen with UV.
For a molecule to absorb IR, the vibrations or rotations within a molecule must cause a net change
in the dipole moment of the molecule.
The alternating electrical field of the radiation interacts with fluctuations in the dipole moment of
the molecule.
If the frequency of the radiation matches the vibrational frequency of the molecule, then radiation
will be absorbed causing a change in the amplitude of molecular vibration.
The intensity with which a bond absorbs radiation depends on its dipole moment.
Thus the order of intensity of absorption for the following C–X bonds is:
93. IR spectrum is the most simplest and reliable tool because many functional groups can
be identified by their characteristic vibration frequencies.
It has a role in structural elucidation when new compounds are identified in plants.
Not required
95. Mass Spectroscopy
In mass spectrometry, the sample in gas or liquid or solid state is introduced to the spectrometer
followed by ionization, mass analysis, and ion detection/data analysis.
We could get the exact molecular weights of the compounds in microgram amounts of sample.
Volatilization of the sample (liquid or solid state) is done either prior to ionization or along with the
ionization.
A mass spectrometer works by generating charged molecules or molecular fragments either in a
high vacuum or immediately prior to the sample entering the high-vacuum region.
The ionized molecules have to be generated in the gas phase.
Once the molecules are charged and in the gas phase, they can be manipulated by the application
of either electric or magnetic fields to enable the determination of their molecular weight and the
molecular weight of any fragments which are produced by the molecule breaking up.
Thus mass spectrometry can be divided into two sections ion generation and ion separation.
96. The various ionization techniques commonly used are: chemical ionization (CI), electron spray
ionization (El) and desorption ionization techniques.
Then, the ion and its fragments are accelerated by electrical and magnetic fields and the ions are
separated in the basis of their mass/ charge (m/z) ratio and detected.
The data produced by the molecular mass measurements or the fragmentation data enable us to
elucidate the possible chemical structure of the molecule.
Electrospray ionization (ESI)
Thus ESI is now the most widely applied method of ionisation because of its ready
compatibility with high-pressure liquid chromatography (HPLC).
The ionization takes place under atmospheric pressure.
97. The eluent from a HPLC system passes through a quartz or metal needle to which a high
electrical potential, is applied.
If a positive potential is applied, then the negative ions in the eluent are stripped away by
being attracted to the needle thus leaving positively charged solvent droplets which spray
out of the capillary
98. Under the influence of flow of nitrogen gas, the droplets evaporate and, as they do so,
break up due to internal charge–charge repulsion.
In the end gas phase ions are produced which are attracted into the mass spectrometer by
an opposite charge applied to a heated capillary, which has to operate under high vacuum.
In order to maintain high vacuum in the instrument two pumping stages are used, an
intermediate stage immediately after the heated capillary and a high vacuum stage in the
ion separation stage.
99. Strengths
1] The best method for getting rapid identification of trace impurities, which should ideally be carried out using
chromatographic separation in conjunction with high resolution mass spectrometry so that elemental
compositions can be determined
2] The method of choice for monitoring drugs and their metabolites in biological fluids because of its high
sensitivity and selectivity.
Not required
100. Limitations
1] Mass spectrometry is not currently used in routine quality control (QC) but is placed in
a research and development (R&D) environment, where it is used to solve specific
problems arising from routine processes or in process development.
2] The instrumentation is expensive and requires support by highly trained personnel and
regular maintenance.
101. Nuclear Magnetic Resonance Spectroscopy
The nuclear magnetic resonance is a complex but powerful tool for providing information about the
structure of a molecule in a solution.
Proton NMR spectroscopy provides a means of determining the structure of an organic compound by measuring
the magnetic moments of its hydrogen atom.
also known as magnetic dipole moment, is the measure of the object's tendency to align with a
magnetic field
102. Subatomic particles (electrons, protons and neutrons) can be imagined as spinning on their axes. In many atoms (such
as 12C) these spins are paired against each other, such that the nucleus of the atom has no overall spin. However, in some
atoms (such as 1H and 13C) the nucleus does possess an overall spin.
103.
104.
105.
106.
107. The sample of the substance is placed in solution, in an inert solvent between
the poles of a powerful magnet and the protons undergo different chemical
shifts according to their molecular environments within the molecule.
Proton NMR spectra of most organic compounds are characterized by chemical
shifts (ppm).
Every proton accepts the energy according to its radiofrequency. When it is
irradiated with electromagnetic radiation in the radiofrequency of protons in the
compound, the protons resonate and provide a signal (peak) indicating presence
of hydrogen (s) in the structure.
Thus, hydrogen atoms in different environments absorb photons of different
energies.
108. The NMR spectrum of the protons in a molecule is obtained by plotting the amount of
energy absorbed by the spinning nuclei versus the frequency of the RF radiation applied to
the molecule.
This spectrum provides information about the chemical environment of the spinning proton
and can be used to deduce the atomic bonding patterns in the molecule.
The proton NMR cannot give information on the nature of the carbon skeleton of a molecule
but 13C NMR could help to solve it.
Simple NMR spectra are recorded in solution, and solvent protons must not be allowed to
interfere.
Deuterated (deuterium = 2H, often symbolized as D) solvents especially for use in NMR are
preferred, e.g. deuterated water, D2O, deuterated acetone, (CD3)2CO, deuterated methanol,
CD3OD, deuterated dimethyl sulfoxide, (CD3)2SO, and deuterated chloroform, CDCl3.
