 Modern methods of extraction

 application of latest techniques like

Spectroscopy, chromatography and
electrophoresis in the isolation,
purification and identification of
crude drugs.
 It is the method of removing active
constituents from a solid or liquid by means of
liquid solvent.
 It can be defined as the process of isolation of
soluble material from am insoluble residue by
treatment with solvent
 In this method the wanted components are
dissolved by the use of selective solvents known
as menstrum & undissolved part is a marc.
 After the extraction unwanted matter is removed.
 Extract: Marc Menstrum

• preparations • Solid • Solvent

of crude residue used for
drugs which obtain after extraction
contain all extraction
the
constituents
which are
soluble in
the solvent.
 Extraction is the process of efficiently dissolving
and separating the desired constituents from the
crude drug with the use of solvent/s
 It is controlled by mass transfer.
 Plant constituents are usually contained inside the
cells. Therefore, The solvent used for extraction
must diffuse into the cell to dissolve the desired
compounds whereupon the solution must pass the
cell wall in the opposite direction and mix with the
surrounding liquid .
 An equilibrium is established between the solute
inside the cells and the solvent surrounding the
fragmented plant tissues
 the choice of solvent depends upon the
charactristics of secondary metabolites like
polarity, pH and thermal stability.
 Be highly selective for the compound to be
extracted.
 Not react with the extracted compound or with other
compounds in the plant material
 Have a low price.
 Be harmless to man and to the environment.
 Be completely volatile.
 Should not mix up with water.
 Should have the big capacity in relation to extract
 The density of solvent should be different from water
density.
 Should have the minimum viscosity.
 Sample size
 Quantity of the exctract required
 Extraction time
 Choice of solvent
 Cost*
 It is necessary to avoid microbial contamination
 Done after collection
 Unless specified, drug should be dried below 60o C
• Less exposure to heat
Shade drying
• Less chances of chemical alteration
• Most efficient & economic
Sun drying
• Use less intense sun light
• Less explored yet
Far infrared drying
• Costly, used for expensive drugs
• Low pressure rapid drying method
Vacuum drying
• For thermolabile compounds

Oven/hot air drying • Used often

 • Maximizes surface are→ increase mass transfer
Size reduction • 30-40 mesh size is optimum

• Maceration, percolation, sox halation, etc

Extraction

• Through muslin cloth, filter paper, filter press

Filtration

• Evaporation of solvent
• Hot air oven, vacuum camber drying, rotary vacuum
Concentration evaporator

• Spray drying, etc

Drying
 maceration= soaking
 The whole / coarsely powdered
crude drug is placed in a stoppered
container with the solvent.
 Allow to stand @RT for a period of
at least 3 days with frequent
agitation until the soluble matter
gets dissolved.
 The mixture then is strained, the
marc is pressed
 The combined liquids are clarified
by filtration or decantation after
standing.
 This method is best suitable for use
in case of the thermolabile drugs.
 Modified maceration: is used for unorganised drug
like gums and resins
› Pressing is avoided
› Fresh menstrum is used to wash marc and make
the volume
 Multiple maceration: to obtain concentrated extract,
total menstrum is divided into 2 parts (double
maceration) or 3 part (triple maceraton) and each
part is used for maceration separately.
 Vacuum maceration: when pressure is reduced to
desired extent, menstrum is allowed to enter→
increases permeabilty of cell walls→ reduced
extraction time
 Kinetic maceration: the vessel is agitated in
constant motion for first 24 hours
› 20-50% more yield usually
 Re-maceration: first maceration is carried with
small amount of menstrum→followed by
remaining amount of solvent
 Circulation maceration: solvent is continously
circulated by pump
 Percolate= I pass through
 It is continuous flow of the
solvent through the bed of crude
drug material to get the extract
 It implies a slow passage of
menstrum under the influence of
gravity through column of drug
powder and during this
movement it goes on extracting
the drug molecules layerwise.
 In percolation the drug is
exhaustively extracted by fresh
menstrum
Moisten the drug with sufficient quantity if menstrum
Allow to stand for 4 hrs in closed vessel
Pack it in percolator
Add suffiecient mensrtum to saturate the drug
When liquid starts dropping, close the outlet
Add sufficient menstrum to form layer above drug
Allow to stand for 24 hrs
Allow to percolate until 3/4th of final extract volum
Press marc and mix the liquid with extract
Adjust final volum
Clarify further if necessary
 Intermittent percolation: 24 hr maceration and 12 hr
maceration are alternated with percolation to effect
extraction
 Re-percolation: drug is divided into 4/5 lots→
percolation followed→ extract is used aas menstrum
for next lot. Same menstrum for every new lot
 Hot percolation: percolation at elevated temperature
increases the efficiency
 Reserved percolation: first part of percolate is
reserved and subsequent percolate is collected
separately. Most of active constituents are in first
percolate. Second percolate contains less active
constituents
 Circulatory percolation: menstrum is
continuously circulated
 Diffusion percolation: menstrum is made to flow
form bottom to top under hydrostatic pressure
 Diacolation: same as diffusion just instead of
hydrostatic pressure, positive pressure of
compressed air is utilized
 It is used for extraction of vitamins, volatile
ingredients and soft ingredients in which the
powdered drug is extracted with hot/cold water
Soak powdered drug in hot/lukewarm water for
specified period with/without stirring

Filter

Press the marc

Extract again with fresh hot/lukewarm water

 Concetration done by
boiling
 Drug is boiled with
water for minutes to
hours and then filtered
 It is suitable for drugs
that are hard in nature
containing water soluble
constituents and are not
affected by prolonged
heating
 Also called as Sohxlet extraction
 Most preferred method of extraction
 Same quantity of solvent is made to circulate
through extractor of drug by evaporation and
subsequent condensation
 [Link]
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WyXCQgQjRx6BAgBEAQ&url=https%3A%2F%2
[Link]%2Fwiki%2FSoxhlet_extractor
&psig=AOvVaw0jj28p6d67i2sCq2HDxT9V&ust=
1564046887756971
 Water distillation: Clavenger’s
apparatus
 Steam distillation:
 Expression of volatile oils:
› Sponge: volatile oil ia
absorbed on the sponge
after squeezing orange peel
› Ecuelli
› Mechanical method:
mechanical/ hydraulic
pressure is applied by
grater/centrifugation. Apparatus used for Ecuelle
method of extraction
Pc:[Link]
 Enflurage: used for extraction
from delicate plant parts
› Fatty material ( lard or tallow
)is used
› Kept for 24 hours
› The again fresh petals are
loaded
› Continues till fatty material is
saturated→ cold enflurage
› Fat is heated and petals are
stirred→strain→put new
petals/flowers
› Fatty material is dissolved in
suitable lipophilic solvent
evaporate ethanol to have
pure fragrance
› Fat is usually used for soap
making
 Also called as sonication
extraction
 Sound waves of high
frequency pulse of 20 kHz
are genrated in ultrasonic
bath
 Sample with sovent is
kept in suitable solvent.
 The ultrasound increses
the cell
permiabilty→maximum
extraction
 Not suitable for large
scale
 Microwaves are (frequency 300 MHz- 300 GHz) nonioinizing
electromagnetic waves.
 High temperature produced ny microwaves evaporate the
moisure in cell→dehydrates cellulose→ ruptures
cellwall→extraction with solvent
 Rapid extraction within 5-10 min
 Types:
› Solvent extractor
› Solvent free extractor
› Microwave reflux
› Microwave assisted extractor
› Sub-500 W microwave extractor
› Drydist model of milestone
› Monolithic equipment for MAE
› Closed vessel mono model of CEM Co.
PC: Preliminary study: Kinetics of oil extraction from sandalwood by microwave-
assisted hydrodistillation Article
 Also called as CCD
 It is liquid liquid extraction
process based on partiton
coefficient
 The apparatus contains series
of tube
 First tube contains
› Heavy stationary liquid phase
› Mixture to be separated
› Light mobile liquid phase
 The rest of tube contain only
stationary phase initially PC:[Link]
com/webobject/76AE847A-75A5-
4B07-A435-279768396365
 the of the tubes contain only stationary phase

 [Link]
 Also called as DCC/ DCCC
 Is combination of liquid- liquid extraction and column
chromatography
 A liquid stationary phase is held in a collection of vertical
glass columns connected in series.
 The mobile phase passes through the columns in the form of
droplets.
 The DCCC apparatus may be run with the lower phase
stationary and the upper phase being introduced to the
bottom of each column.
 Or it may be run with the upper phase stationary and the
lower phase being introduced from the top of the column.
 The mobile phase is pumped at a rate that will allow droplets
to form that maximize the mass transfer of a compound
between the upper and lower phases.
 Compounds that are more soluble in the upper phase will
travel quickly through the column, while compounds that
are more soluble in the stationary phase will linger.
 Separation occurs depending upon partition coefficient of
compounds between the two phases.
 The process uses high temperature and pressure→
the extraction taking less time and requiring less
solvent, and possibly also giving better analyte
recovery
 The elevated temperature is employed to increase
extraction efficiency of the analyte of interest
 The elevated pressure (100-200 bars) is used to
keep the solvent in a liquid state as the temperature
is increased above its boiling point and also
increases permeability of solvent.
 The temperatue and pressure is below supercritical
point
 The CO2 is not used as solvent
 Requires minutes to complete extraction process
 PC: [Link]
 At critical point, substance exists in vapour-liquid
equilibrium
 SCF offer liquid like density, gas like viscocity,
gas like copressibilty and high diffusivity than
liquid
 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 system must contain a pump for the CO2, a pressure cell to contain the sample, a
means of maintaining pressure in the system and a collecting vessel.
 The liquid is pumped to a heating zone, where it is heated to supercritical conditions.
 It then passes into the extraction vessel, where it rapidly diffuses into the solid matrix
and dissolves the material to be extracted.
 The dissolved material is swept from the extraction cell into a separator at lower
pressure, and the extracted material settles out.
 The CO2 can then be cooled, re-compressed and recycled, or discharged to
atmosphere.

PC: [Link]
 SPE is a sample preparation process by which
compounds that are dissolved or suspended in a
liquid mixture are separated from other compounds
in the mixture according to their physical and
chemical properties.
 It uses the affinity of solutes dissolved or suspended
in a liquid (known as the mobile phase) for a solid
through which the sample is passed (known as
the stationary phase) to separate a mixture into
desired and undesired components.
 The syringe barrel/ small glass/ plastic column
called cartidge is packed with stationary phase and
sample solution is gently forced with vacuum
 PC:[Link]
 Depends upon the physical and chemical
properties of compounds to be separated and
also upon the proportion of the component in the
extract
 Methods like fractional crystallization, fractional
distillation, fractional liberation, etc
 Chemical derivatization can also be employed
based on groups or moieties present in the
compound and chemical reactions.
 It is an important method for the purification of
compounds from mixture.
 In fractional crystallization the compound is
mixed with a solvent, heated, and then gradually
cooled so that, as each of its constituent
components crystallizes, it can be removed in its
pure form from the solution.
 Many natural products are crystalline in nature
even in mixture, process such as concentration,
slow evaporation, refrigeration are used for
crystallization
 Fractional distillation is
a process by which
components in a
chemical mixture are
separated into different parts
(called fractions) according
to their different boiling
points.
 This method is used for the
separation of the
components from volatile
mixtures
 Largely using in the
separation of hydrocarbons
from oxygenated volatile oil
eg citral, eucalyptol
 In this process the groups of compounds having the
tendency of precipitation from the solution.
 This process is often used in separation of cinchona
alkaloids, morphine etc.
 Some groups of compounds lend themselves to fractional
liberation from a mixture , ex : a mixture of alkaloid salts in
aqueous solution when treated with aliquots of alkali , will
give first the weakest base in the free state followed by base
liberation in ascending order of basicity .
 If the mixture is shaken with an organic solvent after each
addition , then a fractionated series of bases will be obtained.
 A similar scheme can be used for organic acids soluble in
water – immiscible solvents ; in this case, starting with a
mixture of the acid salts , it is possible to fractionally liberate
the acids by addition of mineral acids .
 It is a laboratory technique for the separation of a
mixture of phytoconstituents.
 Chromatography=from Greek chroma "color and
graphein "to write"
 The mixture is dissolved in a fluid called
the mobile phase, which carries it through a
structure holding another material called
the stationary phase.
 The separation is based on differential
partitioning between the mobile and stationary
phases.
 Chromatography usually consists of mobile phase and
stationary phase.
 The mobile phase = to the mixture of substances to be
separated + a liquid or a gas.
 The stationary phase = a porous solid matrix through
which the mobile phase along with sample percolates.
 The interaction between the mobile phase and the
stationary phase results in the separation of the
compounds from the mixture depending upon its
partition coefficient between mobile phase and
stationary phase.
 Subtle differences in a compound's partition
coefficient result in differential retention on the stationary
 Due to this various constituents of the mixture travel at
different speeds along with mobile phase, causing them
to separate.
 Chromatography may be preparative or
analytical.
 The purpose of preparative chromatography is to
separate the components of a mixture for later
use, and is thus a form of purification.
 Analytical chromatography is done normally with
smaller amounts of material and is for
establishing the presence or measuring the
relative proportions of analytes in a mixture.
 Classification of chromatography

On the basis of
On the basis of On the basis of
interaction of solute
chromatographic physical state of
to the stationary
bed shape mobile phase
phase

Adsorption Two Three Liquid

Chromatography Dimensional Dimensional Chromatography

Partition Thin Layer Column Gas

Chromatography Chromatography Chromatography Chromatography

Super Critical
Ion Exchange Paper
Fluid
Chromatography Chromatography
Chromatography

Size Exclusion
Chromatography
 Chromatograph - equipment that enables a
sophisticated separation
› EX. Gas chromatography or Liquid
chromatography
 Eluent - Fluid entering column/ solvent that carries
the analyte.
 Eluate - Mobile phase leaving the column.
 Stationary phase - Immobilized phase
› Immobilized on the support particles or on the
inner wall of the column tubing.
› Examples : Silica layer - Thin Layer
Chromatography
 Mobile phase - Moves in a definite direction. Liquid
(LC), Gas (GC). The mobile phase moves through
the chromatography column (the stationary phase)
where the sample interacts with the stationary
phase and is separated.
 Retention time: Time takes for a particular analyte to
pass through the system (from the column inlet to
the detector) under set conditions.
 Sample (Analyte) :Substance analyzed in
chromatography.
 Solvent: Any substance capable of solubilizing
another substance.
 It is the visual output of the chromatograph
 Column chromatography involves the following:

 1. Adsorption/retention of substance on
stationary phase
 2. Separation of adsorbed substance using
mobile phase.
 3. Recovery of individual components by
continuous flow of mobile phase.
 Stationary phase: silica gel, alumina,
 Paper chromatography is a technique that involves placing a
small dot or line of sample solution onto a strip of
chromatography paper.
 The paper is placed in a jar containing a shallow layer of solvent
and sealed.
 As the solvent rises through the paper, it meets the sample
mixture, which starts to travel up the paper with the solvent.
 This paper is made of cellulose, a polar substance, and the
compounds within the mixture travel farther if they are non-
polar.
 More polar substances bond with the cellulose paper more
quickly, and therefore do not travel as far.
 Retention factor : Rƒ = zero, - Solute remains in the stationary
phase and thus it is immobile.
 Rƒ = 1 - Solute has no affinity for the stationary phase and
travels with the solvent front.
 Sometimes,it is rather difficult to separate a complex
mixture of substances by a single run with one
solvent system. In such a case, a second run is
carried out by a different solvent system, in a
direction perpendicular to the first run. This is
referred to as two dimensional chromatography.
 Thin layer chromatography (TLC) is a widely
employed laboratory technique and is similar to
paper chromatography.
 However, instead of using a stationary phase of
paper, it involves a stationary phase of a thin layer
of adsorbent like silica gel, alumina, or cellulose .
 Compared to paper, it has the advantage of faster
runs, better separations, and the choice between
different adsorbents.
 The mobile phase runs upward along the plate due
to capillary action and the components get
separated depending upon their polarity towards
stationary phase
 Gas chromatography (GC), also sometimes known as Gas-Liquid
chromatography, (GLC), is a separation technique in which the mobile
phase is a gas.
 It is the method of choice for the separation of volatile substances or
the volatile derivatives of certain non-volatile substances.
 Stationary phase is an inert solid material ( kieselgurh/ firebrick)
impregnated with a non-volatile liquid (silicon/PEG)
 This is packed in narrow column and maintained at high temperature
around 2000C
 A sample is rapidly heated and vaporized at the injection port. The
sample is transported through the column by a mobile phase
consisting of an inert gas (Ar/He/N).
 Sample components are separated based partion coefficeint between
mobile phase and staionary phase .
 The higher a component's affinity for the stationary phase, the slower
it comes off the column. The components are then detected and
represented as peaks on a chromatogram.
 It is commonly used for quantitative estimation of lipids, drugs and
vitamins
 Also called as high pressure liquid chromatography
 It relies on pumps to pass a pressurized (5000-1000 psi)
liquid solvent ie mobile phase containing the sample
mixture through a column filled with a solid adsorbent
material.
 Stationary phase-immobilized thin layer liquid on micro
glass or plastic beads tightly packed on narrow column
 Mobile phase- solvent system passed under high
pressure through column
 The eluents can be detected by detectors
 It can be applied inn the form of adsorption, ion
exchange, partition or molecular sieve chromatography
 Due to rapidity in detection its is used for detection of
amino acids, peptides, carbohydrates, proteins, lipids,
nucleic acids, vitamins, hormones, drugs, etc
 It retains analyte molecules based on it’s ionic
interaction
 It can be further divided into:
› Cation exchange chromatography- retains
cations on negatively charged stationary
phase with negatively charged functional
groups and is used when the molecule of
interest is positively charged.
› Inion exchange- retains anions on positively
charged stationary phase with positively
charged functional groups is used when the
molecule of interest is negatively charged.
 When amino acid mixture is passed through the cation
exchange chromatgraph, individual amino acid can be
eluted using buffers of different pH
 A change in pH affects the charge on the particular
molecules and, therefore, alters binding.
 Bound proteins are eluted out by utilizing a gradient of
linearly increasing salt concentration, usually NaCl.
 With increasing ionic strength of the buffer, the salt ions
will compete with the desired proteins in order to bind to
charged groups on the surface of the medium.
 This will cause desired proteins to be eluted out of the
column.
 Proteins that have a low net charge will be eluted out
first as the salt concentration increases causing the ionic
strength to increase.
 Proteins with high net charge will need a higher ionic
strength for them to be eluted out of the column.
 Size-exclusion chromatography (SEC) is also known as gel permeation
chromatography (GPC) or gel filtration chromatography and separates
molecules according to their size, shape & molecular weight.
 It is also referred to as molecular sieving or molecular exclusion
chromatography.
 The chromatography column is packed with fine, porous beads which are
composed of dextran polymers (Sephadex), agarose (Sepharose), or
polyacrylamide (Sephacryl or BioGel P). The pore sizes of these beads
are used to estimate the dimensions of macromolecules
 Smaller molecules are able to enter the pores of the media and,
therefore, molecules are trapped and removed from the flow of the mobile
phase.
 Molecules that are larger than the average pore size of the packing are
excluded and thus suffer essentially no retention→ the first to be eluted.
This is how the molecules are separated.
 It is also useful for determining the tertiary structure and quaternary
structure of purified proteins, especially since it can be carried out under
native solution conditions.
PC: wikipedia
 Spectroscopy is the study of the interaction between matter and
electromagnetic radiation
 Traditionally, spectroscopy involved the visible spectrum of light, but
X-ray, gamma, and UV spectroscopy also are valuable analytical
techniques.
 Spectroscopy can involve any interaction between light and matter,
including absorption, emission, scattering, etc.
 When a beam of electromagnetic radiation passes through a sample,
the photons interact with the sample.
 They may be absorbed, reflected, refracted, etc.
 Absorbed radiation affects the electrons and chemical bonds in a
sample.
 In some cases, the absorbed radiation leads to the emission of lower-
energy photons.
 Spectroscopy looks at how the incident radiation affects the sample.
 Emitted and absorbed spectra can be used to gain information about
the material.
 is absorption spectroscopy or reflectance spectroscopy
in part of the ultraviolet (10-400nm) and the full,
adjacent visible spectral regions (400-800 nm)
 Absorption of the ultra-violet radiations results in the
excitation of the electrons from the ground state to
higher energy state.
 UV spectroscopy obeys the Beer-Lambert law, which states
that: when a beam of monochromatic light is passed through a
solution of an absorbing substance, the rate of decrease of
intensity of radiation with thickness of the absorbing solution is
proportional to the incident radiation as well as the
concentration of the solution.

 The expression of Beer-Lambert law is-

A = log (I0/I) = Ecl

Where, A = absorbance
I0 = intensity of light incident upon sample cell
I = intensity of light leaving sample cell
C = molar concentration of solute
L = length of sample cell (cm.)
E = molar absorptivity
 Chromophore- Chromophore is defined as any isolated
covalently bonded group that shows a characteristic
absorption in the ultraviolet or visible region (200-800 nm).
 Chromophores can be divided into two groups-
› a) Chromophores which contain p electrons and which
undergo pie to pie* transitions.
Eg: Ethylenes and acetylenes
› b) Chromophores which contain both p and nonbonding
electrons. They undergo two types of transitions; pie to
pie* and nonbonding to pie*.
Eg: Carbonyl, nitriles, azo compounds, nitro compounds
 Auxochromes- An auxochrome can be defined as any group
which does not itself act as a chromophore but whose
presence brings about a shift of the absorption band towards
the longer wavelength of the spectrum.
 Eg: –OH,-OR,-NH2,-NHR, -SH etc
 a) Bathochromic effect- This type of shift is also known as red
shift. Bathochromic shift is an effect by virtue of which the
absorption maximum is shifted towards the longer wavelength
due to the presence of an auxochrome or change in solvents.
The nonbonding to pie* transition of carbonyl compounds
observes bathochromic or red shift.
b) Hypsochromic shift- This effect is also known as blue shift.
Hypsochromic shift is an effect by virtue of which absorption
maximum is shifted towards the shorter wavelength. Generally it
is caused due to the removal of conjugation or by changing the
polarity of the solvents.
c) Hyperchromic effect- Hyperchromic shift is an effect by virtue
of which absorption maximum increases. The introduction of an
auxochrome in the compound generally results in the
hyperchromic effect.
d) Hypochromic effect- Hyperchromic effect is defined as the
effect by virtue of intensity of absorption maximum decreases.
Hyperchromic effect occurs due to the distortion of the geometry
of the molecule with an introduction of new group.
 IR spectroscopy (which is short for infrared
spectroscopy) deals with the infrared region of the
electromagnetic spectrum, i.e. light having a longer
wavelength and a lower frequency than visible light.
 The IR spectroscopy concept can generally be analyzed
in three ways: by measuring reflection, emission, and
absorption.
 The major use of infrared spectroscopy is to determine
the functional groups of molecules, relevant to both
organic and inorganic chemistry
 IR radiation does not have enough energy to induce
electronic transitions as seen with UV.
 Absorption of IR is restricted to compounds with small energy
differences in the possible vibrational and rotational states
 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.
 What is a vibration in a molecule?
› “Any change in shape of the molecule-
stretching of bonds, bending of bonds, or
internal rotation around single bonds”.

 Why we study the molecular vibration?

› Because whenever the interaction b/w
electromagnetic waves & matter occur so
change appears in these vibrations.
 FUNDAMENTAL VIBRATIONS
› Vibrations which appear as band in the
spectra.
 NON- FUNDAMENTAL VIBRATIONS
› Vibrations which appears as a result of
fundamental vibrations of molecule.
› [Link]
opy
 OVER TONES: These are observed at twice the
frequency of strong band. Ex: carbonyl group.
 COMBINATION TONES: Weak bands that
appear occasionally at frequencies that are
sum/difference of 2 or more fundamental bands.
 FERMI RESONANCE: Interaction b/w
fundamental vibration & overtones or
combination tones. Ex:CO2
 In IR, the region below 1500 cm-1 is rich in many
absorption bands and the region is known as
fingerprint region.
 Here the number of bending vibrations are
usually more than the number of stretching
vibrations.
 In this region, small difference in the structure
and constitution of a molecule results significant
changes in the absorption bands.
 Many compounds show unique absorption bands
in this region and which is very useful for the
identification of the compound.
 Solid run in solution : Dissolve solid sample in
non-aqueous solvent (which should be IR
inactive) and place a drop of this solution in alkali
metal disc and allow to evaporate, leaving a thin
film which is then mounted on a sepectrometer. ◦
E.g. of solvents – acetone, cyclohexane,
chloroform, carbon tetrachloride etc.
 Mull technique :Finely powdered sample +
mulling agent (Nujol) and make a thick paste
(mull). Transfer the mull to the mull plates and
the plates are squeezed together to adjust the
thickness it is then mounted in spectrometer.
 [Link] pellet technique :Finely powdered
sample is mixed with about 100 times its weight
of KBr in a vibrating ball mill and the mixture is
then pressed under very high pressure in an
evacuable die to form a small pellet( 1-2mm thick
and 1cm in diameter).
 4. Solid films: Here amorphous solid is dissolved
in volatile solvents and this solution is poured on
a rock salt plate (NaCl or KBr), then the solvent
is evaporated by gentle heating.
 Sampling of liquids :Liquid sample can be
sandwiched between two alkali halide plates (NaCl ,
KBr , CaF2).
 Sampling of gases : Here gases sample is
introduced into a glass cell made up of NaCl.
› Very few organic compounds can be examined as
gases. ◦ E.g.: 1,4-dioxane.
 Sampling of solutions :Here 1-5% of solution is
placed in a solution cell made up of metal halides
and a second cell containing the pure solvent act as
a reference.
› Important solvents used are:-chloroform , CCl4,
Carbon disulphide etc.
 Mass spectrometry is a powerful analytical
technique used to quantify known materials, to
identify unknown compounds within a sample,
and to elucidate the structure and chemical
properties of different molecules.
 The complete process involves the conversion of
the sample into gaseous ions, with or without
fragmentation, which are then characterized by their
mass to charge ratios (m/z) and relative
abundances.
 A mass spectrum is the plot of relative abundance of
ions against their mass/charge ratio.
 The basic aspect of organic mass spectrometry
consist of bombarding the vapour of an organic
compound with a beam of energetic electron
accelarated from a filament to an energy of 70
eV to form positively charged ions (molecular
ions).
 The additional energy of the elecrons is
dissipated in breaking the bonds in the molecular
ion, which undergoes fragmentation to yield
several neutral or positively charged species.
 The ions are then separated according to their
mass to charge ration by virtue of magnet and
are detected by detector
 NMR is the most powerful tool available for organic
structure determination.
 It is used to study a wide variety of nuclei:
› 1H
› 13C

› 15N

› 19F

› 31P

Atomic weight Atomic number Spin number I

Odd Odd/even ½,3/2,5/2… NMR

spectra
Even Even 1 or 0
Even Odd 1, 2, 3…
 A nucleus with an odd atomic number or an odd
mass number has a nuclear spin.
 The spinning charged nucleus generates a
magnetic field.
 When placed in an external field, spinning
protons act like bar magnets.
 The magnetic fields of
the spinning nuclei will
align either with the
external field, or
against the field.
 A photon with the right
amount of energy can
be absorbed and
cause the spinning
proton to flip.
 Energy difference is
proportional to the
magnetic field strength.
 This energy is provided
through radio waves
 If all protons absorbed the same amount of
energy in a given magnetic field, not much
information could be obtained.
 But protons are surrounded by electrons that
shield them from the external field.
 Circulating electrons create an induced magnetic
field that opposes the external magnetic field.
 Magnetic field strength must be increased for a
shielded proton to flip at the same frequency.
 Depending on their chemical environment,
protons in a molecule are shielded by different
amounts.

=>
 The number of signals shows how many
different kinds of protons are present.
 The location of the signals shows how shielded
or deshielded the proton is.
 The intensity of the signal shows the number of
protons of that type.
 Signal splitting shows the number of protons on
adjacent atoms.
 TMS is added to the sample.
 Since silicon is less electronegative than carbon,
TMS protons are highly shielded. Signal defined
as zero.
 Organic protons absorb downfield (to the left) of
the TMS signal.
 Measured in parts per million.
 Ratio of shift downfield from TMS (Hz) to total
spectrometer frequency (Hz).
 Same value for 60, 100, or 300 MHz machine.
 Called the delta scale.
 More electronegative atoms deshield more
and give larger shift values.
 Effect decreases with distance.
 Additional electronegative atoms cause
increase in chemical shift.
 Electrophoresis is the migration of charged particles or
molecules in a medium under the influence of an applied
electric field.
 Comprehensive term that refers to the migration of
charged particle of any size in liquid medium under the
influence of an electric field.
 Depending on kind of charge the molecule carry, they
move towards either
› To cathode
› Or toAnode
 An ampholyte become positively charged in acidic
condition and migrate to cathode, in alkaline condition
they become negatively charge and migrate to anode.
 Commonly used for isolation of amino acids and
proteins
 The rate of migration of an ion in electrical field
depend on factors,
› Net charge of molecule
› Size and shape of particle
› Strength of electrical field
› Properties of supporting medium
› Temperature of operation