Major Phytoconstituents and their analysis

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Transcript Major Phytoconstituents and their analysis

 Alkaloids
 phenolic compounds
 Terpenoids
 Lipids
 Amino acids
 Carbohydrates
 Alkaloids are difficult to define since they do not
represent a homogenous group of compounds in
chemical, biochemical or physiological terms. But,
they are all organic nitrogenous compounds that
possess basic properties.
 Alkaloids are not rigidly classified, but a useful
approach can be based on the heterocyclic system
from which a group of these compounds is
formally derived. Other approaches use the plant
species from which alkaloids are isolated or
• powdered plant is warmed on a
water bath with 0.1N sulphuric
acid, for a few minutes. Then
cooled and filtered.
• The filtrate is made alkaline by
drop wise addition of ammonia
• Transfer the alkaline solution to a
separatory funnel with chloroform.
Allow to stand and then separate
the chloroform layer (lower layer).
• Repeat this extraction procedure
with chloroform.
• Mix the chloroform layers .
1. General detecting reagents:
 Many alkaloids in quantities as small as 10-100 µg in solution
may form precipitate or turbidity with certain reagents. On
this basis, these alkaloids precipitating reagents are used for
testing the presence or absence of alkaloids in crude extracts
of plant materials, and for testing whether a particular step in
an extraction procedure has exhausted the alkaloid content.
 Under controlled conditions of mixing and slow evaporation, a
drop of an alkaloid solution reacting with a drop of an
appropriate alkaloid-precipitating reagent on a microscope
slide, forms micro-crystals with characteristic shape and
manner of aggregation for particular alkaloid. This is
sometimes used to aid in the identification of the alkaloid.
General detecting reagents:
 the reagents most commonly used for the testing of alkaloids by
precipitation or micro-crystal formation are the following:
Wagner’s reagent: (Iodine potassium iodide) Produces yellowishbrown precipitate, usually flocculent in nature.
Mayer’s reagent: (Potassium mercuric iodide) Produces, with slightly
acidic solution of most alkaloids, yellow or white precipitates that are
amorphous, but after a while frequently become crystalline.
Marma’s reagent: (Cadmium potassium iodide) Produces, with slightly
acidic solution of most alkaloids, yellow or white precipitates that are
amorphous, but after a while become crystalline.
Dragendorff’s reagent: (Bismuth potassium iodide) Produces with
most alkaloids an orange-red precipitate.
Hager’s reagent: (Saturated solution of picric acid) Produces yellow
crystalline precipitate with most alkaloids.
2. Specific colour reagents:
 In some cases, under standardised conditions, the intensity of the
colour formed is in linear proportion to the concentration of the
alkaloids in solution; and may be used in quantitative determination
of these groups of alkaloids.
1. Vitali-Morin reaction:
 Tropane alkaloids: (Ex: Hyoscyamine, atropine and scopolamine
 The alkaloid is mixed with a few drops of fuming nitric acid and
evaporated to dryness on a water bath. Then cooled and 0.5 ml of
5% solution of potassium hydroxide in methanol (freshly prepared)
added to the residue.
A bright purple colour will be formed which changes to red and
subsequently fades to colourless.
2. Specific colour reagents:
Gerrard reaction:
A few mg of the tropane alkaloids react with 2% solution
of mercuric chloride in 50% alcohol to produce the
following colours:
 Atropine: a red colour upon warming
 Hyoscyamine: a red colour upon warming
 Hyoscine : a white precipitate
3. Froehde’s Reagent: (0.5% solution of ammonium
molybdate or molybdic acid in conc.sulphuric acid). gives
pink colour with Opioids .
4. Mandelin’s Reagent: (saturated solution of ammonium
vanadate). Gives brick-red colour with Ephedrine.
5. Marquis Reagent: (cone. sulphuric acid containing one
drop of 40% formaldehyde per ml). Gives purple colour
with most opium derivatives.
Separation of purine alkaloids (e.g: caffeine, theobromine
and theophyllin) avoids the problem of irreversible
column poisoning in ion exchange chromatography.
Stationary phase:
Reversed phase C18 columns.
Mobile phase:
The solvent is 5% glacial acetic acid in
water at a flow rate of 4 ml/ mm.
U.V. detector at 254 nm.
4. TLC
Stationary phase:
• Silica gel is the most active stationary phase .
The layer of silica gel is weakly acidic. Salts are thus formed during TLC of
strong bases on silica gel and they remain at the origin when neutral
solvents are employed.
•Alumina is less active.
•Cellulose layers are generally impregnated with formamide. Adsorbent
layers containing an added indicator which fluoresces in short wave UV254
are very satisfactory.
Mobile phase:
• solvents containing ammonia or organic bases like pyridine, piperidine
and diethylamine, in the TLC of strong bases on silica gel.
•Alkaloids of various groups were successfully separated on aluminium
oxide layer with benzene and with benzene-ethanol containing 2.5, 10 or
30% ethanol and also with chloroform and ether.
Detection: Many alkaloids can be seen even in daylight. A
large number yield typical fluorescent colours in UV365.
• The agent most commonly used for detecting alkaloids is
Dragendorff reagent.
•Other reagents used include:
•Iodoplatine spray:( platinic chloride and potassium iodide
in sufficient water). It is a general locating reagent for
nitrogenous bases.
•Antimony trichloride spray: (antimony trichloride in HCl)
•Cerium sulphate in sulphuric acid or in phosphoric acid.
•Ninhydrin reagent for phenyl alkylamines
•Cinnamaldehyde-hydrochloric acid for indole alkaloids
•Van Urk reagent (p-dimethylaminobenzaldehyde-H2SO4
and oxidant) for Ergot alkaloids
•Sulphuric acid for purine alkaloids
5. Examples:
Indole alkaloids
Silica gel
Mob.phase chloroform-96% alcohol (9:1) or
methanol- methyl ethyl ketoneheptane (8.4:33.6:58)
cellulose powder saturated with
20% solution of formamide in
heptane-methyl ethyl ketone (1:1) in
an atmosphere of ammonia
detected in U.V. light or using ferric chloride or trichloroacetic acid
Sarpagine, serpentine, ajmaline, yohimbine, rescinnamine, reserpine and
Opium alkaloids
Silica gel
Mob.phase chloroform-ethanol
Alkaline silica gel
Cellulose layer saturated
with formamide
They were visualised by Dragendorffs reagent
morphine, paracodeine,
codeine, papaverine and
Extraction and separation of Rauwolfia
Alkaloids by T.L.C
 contains the following alkaloids:
 Ajmaline, Serpentine, Reserpine. Which can be separated on
alumina as well as on silica gel plates.
0.3 gm of the Rauwolfia serpentina roots sample is moistened with 4 drops of
25% ammonia solution and subsequently extracted for 10 min, shaking with
7 ml chloroform. The filtrate is evaporated.
Dissolve the residue in 1 ml methanol. A sample of 5-110 μl is applied as spots
on a silica gel plate.
50 mg reserpine in chloroform (50 ml). 1-5 μl are applied
Solvent systems:
Methanol-Chloroform 25:75 or Acetone-Methanol-Glacial acetic acid 70:25:5
UV/ lodoplatinate Reagent/ Dragendorff’s Reagent
Phenolic compounds:
 Phenolic compounds constitutes a wide range of naturally
occurring substances, but there are two main groups: Simple phenolics: which include catechol and resorcinol;
phenolic acids and cinnamic acids (e.g. caffeic acid) and their
lactone derivatives, the coumarins.
 Flavonoids the flavonol glycosides and their aglycons are
generally termed flavonoids. They comprise the widely
occurring water soluble plant pigments, the anthocyanins and
flavones, and a number of related substances, e.g. isoflavones,
catechins and tannins.
Rutin, quercitin & citrus flavonoids are common constituents.
• Acid hydrolysis of the plant
tissue fresh or dried.
• Then phenols are extracted
into ether.
Simple phenols :
One - dimensional
Two- dimensional
Stationary phase:
Stationary phase:
Silica gel
Mobile phase:
• benzene-methanol-acetic acid
• aqueous acetic acid
Mobile phase:
acetic acid-chloroform and ethyl
acetate-benzene .
1. Phenols absorb in the short U.V. and can be detected in wavelength 253 nm as
dark absorbing spots on plates spread with silica gel containing fluorescent
2. Reagents:
• The best specific reagent to detect them is Folin-Ciocalteu ) a mixture
of phosphomolybdate and phosphotungstate(: Phenolic compounds appear as blue
or blue to grey spots after spraying and the plate is exposed to ammonia vapour.
•Vanillin-HCI (lg vanillin in 10 ml con.HCl) and vanillin-H2SO4 (10% vanillin in
Ethanol-conc.H2SO4 2: 1) give a range of pink colours with resorcinol and
phloroglucinol derivatives.
Two dimensional chromatography:
Typical Rf values for some phenolic compounds in four solvents are shown in Table
2. Paper Chromatography
Two- dimensional
One - dimensional
Stationary phase:
Mobile phase:
• formate-formic acid-water
• butanol - 2M NH4OH (1:1)
• water
Mobile phase:
• benzene-acetic acid-water
6:7:3 and sodium formateformic acid-water 10:1:200
in many of the best solvents (e.g. butanol-acetic acidwater) simple phenols tend to cluster together near the
solvent front.
3. Gas Chromatography
GC has not been widely used for phenol
separation, because most phenols have to be
converted to suitable derivatives
(trimethylsilyl [−Si(CH3)3] ethers or acetates)
to make them sufficiently volatile.
1. Flavonoids: natural pigments
paper chromatography
Stationary phase:
 Mobile phase:
• Ethyl acetate saturated with water
• Phenol saturated with water
• m,p-Cresols saturated with water
• Acetic acid-water (15:85)
• Heptane-Butanol-water (29:14:57)
The spots are detected under U.V
light, or by spraying with Na2CO3 or
lead acetate solutions.
Flavonol aglycones,
Flavonol glycosides,
Flavone aglycones,
Flavanone aglycones
Flavanone glycosides
Flavan aglycone.
2. Anthocyanins and Anthocyanidins:
( as chloride salts) can be separated on paper
using Butanol, acetic acid or m-cresol, acetic
acid as the mobile phase.
3. Quinones:
 Anthraquinones:
 can be separated in pure form by eluting
through calcium oxalate or silicic acid
Furthermore, silica gel plates impregnated
with 3.75% tartaric acid solution and
developed with chloroform-methanol (99:1)
give excellent separation of anthraquinones.
 Detection of anthraquinones by their visible
UV. four or five absorption bands are seen of
which three lie between 215-300 nm.
Example: analysis of flavonoids of the
strawberry – Arbutus unedo L. (Ericaceae)
Quercitrin, isoquercitrin, hyperoside and rutin
 (1.0 g) of air-dried, powdered leaves and fruits were refluxed with 10.0 mL
methanol for 30 minutes, filtered, and the filtrate was concentrated under reduced
Flavonoid and phenolic acid standards were prepared as 0.05% solutions in
Thin-layer chromatography (TLC) was performed on 20 x 20 cm TLC plates
coated with 0.25 mm layer of silica gel. After application of the extract and
standard solutions, the plates were developed for 19 cm.
Two chromatography solvents were used: ethyl acetate/formic acid/acetic
acid/water, 100:11:11:26 (V/V) and ethyl acetate/formic acid/water, 8:1:1 (V/V).
Visualization of the flavonoids and phenolic acids was achieved by spraying the
sheets with polyethylene glycol reagent.
Typical intense fluorescence in UV light at = 365 nm was produced immediately
on spraying (flavonoids appeared as orange-yellow bands, whereas phenolic acids
formed blue fluorescent zones).
 Terpenoid, a term which is used to indicate that all
such substances have a common biosynthetic origin.
 Thus, terpenoids are all based on the isoprene
molecule. CH2 = C (CH3) — CH = CH2
Isoprene itself is rare in plants but does occur, the
carbon skeletons of terpenoids is built up from the
union of two or more of these C5 units ( isoprene or
iso-pentane units linked together in various ways and
with different types of ring closures, degrees of
unsaturation and functional groups).
 They range from the essential oil components, the
volatile mono- and sesquiterpenes (C10 and C15),
through the less volatile diterpenes (C20) to the
involatile triterpenoids and sterols (C30) and
carotenoid pigments (C40).
Classification of terpenoids:
Isoprene units
No of carbon
1. Monoterpenes: Typical monoterpene skeletons
include acyclic, monocyclic and bicyclic systems.
2. Sesquiterpenes: occur as acyclic, monocyclic
and bicyclic systems. Farnesol is the most
important of the acyclic sesquiterpenoids.
3. Diterpenes: hydrocarbons, alcohols, ethers and acids are all
known in this group. The only important acyclic member is
the alcohol phytol, which is present in the molecule of
4. Triterpenes:
derived biosynthetically from
the acyclic C30 hydrocarbon, squalene.
 They have relatively complex structures, most being
either alcohols, aldehydes or carboxylic acids.
Triterpenoid alcohols occur both free and as glycosides.
Many of the glycosides are classed as saponins.
 The most important and widely distributed triterpenoids
are the pentacyclic compounds.
 Sterols are triterpenes. Sterols are present in animals
(cholestrol) as well as in plants (phytosterols) and
microorganisms (ergosterol).
Sterolins and Saponins:
 Many terpenoids and steroid alcohols exist in nature, not
as free alcohols, but as glycosides.
 β-sitosterol glycoside is the most widely distributed plant
sterol, so its glycosides are the commonest sterolins.
 The saponins were originally named because of their
soap-like characteristics. They are powerful surface active
agents which cause foaming when shaken with water and
in low concentration often produce haemolysis of red
blood cells.
• No general method of isolation can be applicable
to all of them.
• However, large number are non-polar
compounds and may, therefore, be separated from
polar plant constituents by extraction with solvents
such as benzene or ether. Such extracts would also
contain other types of lipids , waxes, etc. Most of
these may be removed by saponification in
alcoholic alkali followed by extraction with ether.
most terpenoids and steroids will go into the ether
• Glycosidic compounds are extracted from plants
with 70-96% hot ethanol and lipids removed from
this solution by extraction into benzene.
• Saponins, acid hydrolysis ( 4N HCl) should be
carried out to liberate aglycones , then extracted
with benzene.
• The low molecular weight terpenes are usually
separated by steam distillation.
General techniques:
 Almost all triterpenoids and steroids are separated mainly
by TLC and GC (method of choice for characterisation of
volatile terpenoids). Identification can be confirmed by
m.p, MS, I.R and NMR spectroscopy.
 Separations on paper chromatography are occasionally
valuable for distinguishing different glycosidic
triterpenoids. Paper chromatography of the higher
terpenoids and steroids has generally been unsuccessful
because of the non-polar nature of most of the compounds.
 TLC is practically always carried out on a layer of silica
gel, silica gel plates treated with AgNO3 or alumina. The
best adsorbent layer was found to be silicic acid and the
best general purpose solvent 15% ethyl acetate in hexane.
 Locating Reagents:
 More than fifty detection reagents have been listed to be used
in TLC to separate steroids and related compounds.
 The most common reagents are:
1- Carr - Price reagent: 20% antimony chloride in chloroform .
A range of colours are produced, visible in both day-light and
U.V., on heating sprayed plates for 10 min at 100°C.
2- Liebermann - Burchard Reagent:
The plates are sprayed with a mixture of 1 ml of H2SO4, 20
ml acetic anhydride and 50 ml chloroform and then heated at
85°- 90° for 15 min. Giving a blue-green colour with most
steroids and triterpene alcohols. The sensitivity is quite good
(2-5 μg).
3- H2SO4 or dilutions with water and alcohol, with the addition
of aldehyde or 5% nitric acid.
4- Terpene alcohol and oxo-compounds may be
detected with general reagents for alcohol and oxo
5- terpenes with double bonds, sesquiterpenes, and
some triterpenes may be detected by spraying with
antimony trichloride).
6- Pentacyclic triterpenoids give a violet colour when
heated with 2,6-di-tertbutyl-p-cresol in ethanol.
Steroids give no colour or a yellow-green one.
7- further detection reagents are antimony
pentachloride, potassium permanganate solution,
fluorescin solution, bromine vapour and
phosphomolybdic acid.
Techniques for Mono & Sesquiterpenes
1. GC:
 This is the most important technique for the study of
essential oils, since it yields in one operation both qualitative
and quantitative results.
 For the identification of volatile terpenes in any plant
material, it is essential to combine the use of GC with other
procedures and especially with TLC or GC-MS.
 Capillary columns, 200 - 300 cm in length and filled with
silicone oil, Diethylene glycol succinate, Carbowax or mineral
oil can be maintained isothermally at low temperatures (60
ºC) or programmed for a temperature range that provides
good resolution. It is important that the support material
should be freed from traces of iron, base or acid, since
terpenes are sensitive to such impurities.
2. TLC:
Stationary phase:
•silica gel is the most widely used adsorbent.
•Terpene alcohols are best separated on paraffin-impregnated plates and 70%
methanol as solvent.
• To separate terpenes according to the number of double bonds, silica gel
plates spread with 2.5% aqueous AgNO3 instead of water.
Ag forms weak bonds with unsaturated molecules causing them to move more
slowly than saturated ones.
Mobile phase:
• benzene, chloroform, benzene-chloroform (1: 1) and benzene-ethyl acetate
(19: 1)
• terpene alcohols: 70% methanol.
•The solvent system to comply with the AgNO3 treated plates is methylene
dichloride-chloroform-ethyl acetate-n-propanol (45:45:45:45).
spraying with 0.2% aqueous KMnO4, 5% antimony chloride in chloroform,
H2SO4 or vanillin. H2SO4 (8 ml ethanol with 0.5 gm vanillin in 2 ml H2SO4); the
plates are heated after spraying at l00°-105°C until full development of colours.
Techniques for Diterpenes
 Diterpenes are separated by GC and TLC using the same
methods as for the lower terpenes.
 However, diterpenes are less volatile than sesquiterpenes and
slightly different GC techniques may be required in some
 TLC on silica gel AgNO3 (10: 1) with petroleum ether as a
solvent has been exploited.
 Detection is by standard procedure (H2SO4, Antimony
chloride reagent or 0.2% KMnO4).
Techniques for Triterpenes
1. GC:
 Non-volatile alcohols can be acetylated, and other
compounds converted to trimethylsilyl esters.
 Liquid phases such as dimethylpolysiloxane, Diethylene
glycol succinate and silicones could be used in the
separation of triterpenoids.
 Concentrations of 1-3% solutions of the above phases
are applied to a solid support.
 High temperatures (220-250°C) are needed and gas flow
rates of 50-100 ml/min.
Polycyclic triterpenes are suitable candidates for chromatographic
systems devised for steroids.
Stationary phase:
•AgNO3-impregnated silica gel layers have proved superior to
untreated silica gel in the resolution of unsaturated triterpenes.
•Highly oxygenated triterpenes have been chromatographed on
Mobile phase:
• AgNO3-impregnated silica gel : chloroform
•Highly oxygenated triterpenes :
petroleum ether-chloroform- acetic acid (40:4: 1)
Previously mentioned detecing reagents.
Techniques for steroids
Liquid Column Chromatography:
 trifluoroethylene polymer is suitable stationary
phase for the reversed phase liquid-liquid partition
chromatography of steroid hormones.
 Mobile phase: water or
 hormones were well separated in a matter of
2. GC:
 Trimethylsilylation has been used most extensively,
because it yields derivatives that are very suitable
for quantitative analysis and mass spectrometry.
 The steroid acetates are stable derivatives, some
produce a response in electron capture detector.
 Methyl ethers are particularly useful for
quantitative analysis and Ketonic steroids are most
frequently chromatographed as their Omethyloximes.
2. GC:
Stationary phase:
•The most widely used stationary phases for steroids have been
dimethylpolysiloxane, they have been supplanted by silicones, which
are characterised by superior stability, higher maximum operating
temperature and wider range of operating temperatures.
•The open capillaries have excellent resolving power, but require
careful preparation and appropriate instrumentation.
Most investigators prefer the flame-ionisation detector .
The electron-capture detector is even more sensitive but more difficult
to use
3. TLC:
Stationary phase:
•Silica gel plates with gypsum binder.
•polyamide plates have been recommended for steroid glucuronides.
•silica gel plates modified with silver nitrate for unsaturated steroids.
Most of the reagents used for detecting steroids contain sulphuric
acid, produces characteristic colours and fluorescence response as
well as permanent black zones.
Techniques for Carotenoids
 The techniques employed are TLC and PC. Column
chromatography is essential for large scale isolation of
 There is no single stationary phase and solvent system
which can be applied universally to all carotenoids. The
choice depends largely on the relative polarities of the
compounds to be separated.
Example: Simultaneous Determination of Free
and Esterified Fatty Alcohols, Phytosterols
and Solanesol in Tobacco Leaves by GC
 Tobacco samples were dried for 3 h at 40 ºC, and then ground to pass
though a 32-mesh screen.
 Extraction Procedure of Free Forms:
1g of ground tobacco was weighed into a conical flask, 30 mL acetone
was added. After ultrasonication for 30 min, the acetone extract was
filtered and evaporated on a vacuum.
• Saponification and Extraction Procedure of Esterified Forms:
The ground tobacco with 20 mL of 2 M KOH in 85% ethanol. The
resulting mixture was heated at 60 ºC with continuous stirring for 1 h
to insure the complete hydrolysis. After the mixture was cooled to room
temperature, 20 mL hexane, 10 mL deionized water and 1.5 g solid
potassium chloride were added.
 Preparation of Trimethylsilyl (TMS) Derivatives:
 freshly prepared pyridine-BSTFA (N,O-bis(trimethylsilyl)
trifluoroacetamide) with 1% TMCS (trimethylchlorosilane) (1:1, v/v)
added, then the mixture was heated at 80 ºC for 60 min. After cooling, the
mixture was diluted to 1 mL with dichloromethane and 1 µL of the solution
was analyzed by GC and GC–MS.
GC-FID Conditions
gas chromatography equipment including automated sample-injection
system, spilt/spiltless injector and flame ionization detection (FID).
Separation was performed on a DB-5 fused silica capillary column (30 m 9
0.25 mm I.D. * 0.25 µm film thickness.
The oven temperature was programmed at 2 ºC min-1 from 210 to 270 ºC
and held for 12 min, then raised to 310 ºC at 10 ºC min-1 and maintained
for another 24 min, giving a total run time of 70 min. Helium was used as
carrier gas and set at 1.2 mL min-1
Other parameters were injection port temperature 270 ºC, detector 300 ºC,
split ratio 5:1 and injection volume 1 µL
 Plant tissues contain lipid mixtures of such complexity
that one analytical method is usually insufficient to
isolate each component, and a combination of two or
more techniques is required. In many cases a
preliminary separation into two or more groups of lipid
classes can greatly facilitate the ultimate resolution of the
mixture into its pure components.
 Lipids have been classified in several different ways but
the most satisfactory system divides them into:
1. saponifiable lipids (complex): yield soaps (salts of fatty
acids) on alkaline hydrolysis.
2. unsaponifiable lipids (simple): do not contain fatty
acids and hence are unsaponifiable (terpenes).
 The saponifiable lipids are classified according to their
1. Fatty acids: almost always have an even number of carbon
atoms, but exceptions do occur. The vast majority of natural
fatty acids have an unbranched carbon chain and differ from
one another in chain length and degree of unsaturation.
2. Simple lipids (fatty acid esters):
 Triglycerides: esters of glycerol with three fatty acid molecules.
it is either: fats (saturated fatty acids) or oils (unsaturated
fatty acids).
 Other fatty acid esters: waxes and cutins
3. Phospholipids or phosphatides: esters of phosphoric acid and
long-chain fatty acids. The simplest phospholipids are
phosphatidic acids esterified with glycerol. They are polar
compounds, soluble in moderately polar solvents, such as
4. Glycolipids: lipids containing sugar units but not phosphorus.
Cetyl palmitate wax
• Lipids are water insoluble organic
compounds that can be extracted from
plant tissues by non-polar solvents, e.g.
chloroform, ether or petroleum ether.
• by passing a solution of lipids in ether
through a silicic acid column.
Phospholipids and glycolipids are
retained by the adsorbent, and these
may be recovered by washing the
column with chloroform-methanol
mixtures. And neutral lipids and free
fatty acids will be eluted.
•Alumina has little use in the
separation of lipids due to the fact that
it catalyses saponification and
autoxidation of glycerides.
1. TLC
Stationary phase:
silica gel or silica gel impregnated with AgNO3 .
Silica gel is established as the most appropriate stationary phase for
the majority of polar lipids.
Mobile phase:
• triglycerides: mixture of chloroform, methanol and water, to which
acetic acid or ammonia may be added to take advantage of the
different amphoteric characteristics of the various lipid groups.
• Phospholipids and glycolipids : chloroform-methanol-acetic acidwater (85:15:10:3-7).
•Neutral lipids: Petroleum ether-diethyl ether-acetic acid 90:10:1
2. GC:
 Gas chromatography is the most significant analytical
method and is the method of choice in the analysis of
almost all single-chain aliphatic compounds.
 fatty acids are first converted into their more volatile
methyl esters. Among the many reagents used for the
methylation diazomethane, 2, 2- dimethoxypropane, and a
mixture of methanol with boron trifluoride. The latter has
the advantage of not introducing unwanted products due to
secondary reactions.
 Many stationary non-polar phases have been used: such as
polyethylene glycol succinate, which allow the separation of
unsaturated acids on the basis of their degree of
 Amino acids can be defined as carboxylic acids
having at least one amino function present in the
 With the exceptions of proline and hydroxyproline,
each of which has an endocylic N, all members of
the group share a general formula.
 In the solid state or in solution, when the amino
acid is near neutrality, the carboxyl and amine
groups carry charges due to the gain or loss of
protons. The properties of the amino acids makes
separation easy using techniques such as
electrophoresis or ion-exchange chromatography.
General formula
The sample of plant extract is
evaporated under reduced pressure,
and the amino acids recovered by
extraction with 0.5% HCl in 95%
1. Ion-exchange Chromatography
 In acidic solution the carboxyl group is protonated and
thus uncharged, but the amine group is still positive. In
basic solution the amine group loses its extra proton,
becoming neutral, but the carboxyl group retain its charge.
 In ion-exchange chromatography of amino acids accurate
values in amino acid analysis can only be expected if the
sample is handled adequately.
 The sample to be applied on a cation exchanger must be
acidified to about pH 2 in order to ensure proper
adsorption of the amino acid on top of the column. Proteins
and fatty materials tend to clog the resin particles and
produce high back pressure, hence their removal is
Ion-exchange Chromatography
 For the ion-exchange chromatography of amino acids,
strong acid ion-exchangers of the sulphonated
polystyrene type are used.
 The only exceptions are the strongly acidic amino
acids, namely sulphonic acids and o-sulphate esters of
hydroxy amino acids, which are separated on a
strongly basic resin.
 For a given ion-exchange resin, the elution rate of
amino acids is largely determined by the pH and the
composition of the buffer. It is necessary to control the
pH to ±0.01 and the cation concentration to ± 1 mmol/
litre. Any increase in pH, ionic strength or
temperature, will speed up the elution.
Ion-exchange Chromatography
 Sodium citrate buffers are most widely used. The addition
of a small percentage of methanol, ethanol, propanol or
benzyl alcohol to the buffer accelerates the elution rate of
many amino acids, partly because hydrophobic interactions
are reduced.
 Other buffers include volatile pyridinium formate, acetate
and tartrate.
2. TLC
Stationary phase:
Silica gel is commonly used, although
cellulose has found frequent use.
Mobile phase:
A polar solvent system is used because of the polar character
of amino acids. The mobile phase must contain water and
partition chromatography occurs rather than adsorption.
The use of thin-layer and ion-exchange chromatography together is
very useful in the separation of amino acids, e.g. the use of diethyl
amino ethyl cellulose as the stationary phase. In another technique,
involving the use of TLC and electrophoresis together, the sample is
applied to one corner of the plate and run in one direction by
electrophoresis, then in the other direction of conventional TLC.
3. Paper Chromatography
One - dimensional
Stationary phase:
Mobile phase:
Phenol-water (pH 5)
• water-saturated phenol and
collidine (most widely used)
•butanol-acetic acid- water (9:1:1)
•By using several solvents
buffered at a chosen pH between
1and 12, it is possible to separate
each amino acid from all the
others by one-dimensional
Two- dimensional
Mobile phase:
1. Methanol:water: pyridine (4:1:0.2) (1st)
Butanol: methyl ethyl ketone: water:
diethylamine (2:2: 1:0.2) (2nd)
2. Methanol:water:pyridine (8:2:0.4) (1st)
Tert. butanol: methyl ethyl ketone: water:
diethylamine (4:4:2:0.4) (2nd)
3. Butanol:acetic acid:water (4:1:5) (1st) mcresol-phenol: pH 9.3 borate buffer (1:1)
4. Butanol: acetic acid: water (4: 1:5) (1st) picoline: acetic acid: water (75:2:23) (2nd).
Detection: The most widely used reagent for the detection of amino acids is
ninhydrin reagent which is not actually specific for amino acids, but reacts
with any primary amine, to produce reddish to bluish-purple colours
(yellow for prolines).
 Carbohydrates occupy a central position in plant metabolism
and are key compounds in the biochemistry of green plants.
Ultimately, all other constituents can be derived from them.
 Carbohydrates are aldehydic or ketonic, polyhydroxy
compounds classified into two broad groups: sugars and
 Variations of the basic carbohydrate structure are also
encountered these include:
(a) Amino sugars, in which one of the hydroxyl groups is replaced
with an amino group, e.g. glucosamine, galactosamine.
(b) Uronic acids, in which the terminal alcohol group is converted o
a carboxylic group, e.g. D-glucuronic acid, D-galacturonic acid.
(c) Desoxy sugars, in which a hydroxyl group is replaced simply
with a hydrogen, e.g. desoxyribose.
(d) Sugar phosphates, in which conventional carbohydrates are
esterified with phosphoric acid.
Classification of the Carbohydrates
•extracting the fresh tissue with
95% ethanol. then concentration
and filtration . The filtrate is then
washed with petroleum ether to
remove lipids.
•Polysaccharides, as well as
glycosides, can be hydrolysed with
H2SO4 . In the case of plant
glycosides, the aglycone must also
be removed by extracting with
ether or ethyl acetate.
1. paper chromatography
Stationary phase:
Mobile phase:
1. Butanol-acetic acid-water
2. Butanol-ethanol-water
3. Butanol-benzene-pyridine-Water
4. Phenol saturated with water
Resorcinol-H2SO4 or Aniline hydrogen phthalate, which is prepared
by dissolving aniline (9.2 ml) and phthalic acid (16 g) in n-butanol
(490 ml), ether (490 ml) and water (20 ml). Different colours appear
after heating the paper with a hair dryer.
After locating the sugar complexes it is advisable to measure their
absorption spectra to confirm their identity.
2. TLC
Stationary phase:
Cellulose or silica gel
Mobile phase:
• Due to the high polarity of carbohydrates, they display very high
water solubility and low solubility in less polar, organic solvents. In
general polar solvents are used. Water in the solvent system saturates
the stationary phase, so the type of separation is based on partition.
• The separations achieved on paper can be obtained by TLC on a
microcrystalline cellulose using the same solvents.
• Silica gel can be used for the separation of sugars using n-butanolacetic acid-ether-water 9 : 6 : 3 : 1
Spray reagents: Aniline hydrogen phthalate or 0.2%
naphthoresorcinol in butanol containing 10% phosphoric acid.
On heating the plate for 10 min at 100°C ketoses give pink, pentoses
green, and hexoses blue colours.
3. GC
 Since GC is a more sensitive technique it can detect 0.5 µg of
sugar compared to 5 µg on paper.
 Trimethylsilyl ether derivatives of the sugars are used instead of
the sugars.
 Chromatography can be carried out on a silonised column
Oven temperature 180°C and Inlet pressure of 15 psi.
4. Paper Electrophoresis:
 This important technique requires charged complexes
and it is more useful for such oligosaccharide and
monosaccharide derivatives than for the parent
 This technique is used to distinguish carbohydrates
with acidic or basic substituents (glucuronic acid,
sugar phosphates, amino sugars) from neutral
 In the standard procedure for electrophoresis of
common sugars, 0.05M sodium borate buffer (pH 9-10)
is used to produce the charged complex and at voltages
of 25-30 V/cm, 90 mm is required for separation.
5. HPLC :
 Carbohydrate analysis columns have been
developed which, when utilised with H2O-CH3CN
35:65, give excellent separation of a wide range of
 The use of both an absorbance detector and a
differential refractometer allows the monitoring
using U.V. at 254nm.