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PROTOCOL FOR HERBARIUM PREPARATION
Specimen Photographs
Specimen Name……………………………… Taxonomical Classification…………
Kingdom…………. Division…………… Class………………. Order………………. Family……………… Genus………………. Species……………….
Details of Herbarium
Department of Botany…………………
Field Number……………………………….....
Specimen Name…………………………………… Common Name…………………………………. Place of Collection……………………… Month and Time of Collection……………… Description of Specimen………………
Specimen Collect By……………………..................
Date of Collection Herbarium Prepared By: Herbarium Authenticated By:
EXTRACTION AND ITS QUALITY ASSURANCE
Extractionistheseparationofmedicinallyactiveportionsof plant(andanimal)tissuesusingselectivesolventsthrough
standard procedures. The extraction method separates the soluble plant metabolites (chemical constituents) and
leavesbehindthe insoluble cellular marc.The products obtainedfromplantsare relatively complexmixturesof metabolites,inliquidorsemisolidstateor(afterremoving
Types of extraction techniques
thesolvent)indrypowderform,andareintendedfororal
or external use.
Galenicals(namedaftersecondcenturyGreekphysician
Galen)includeclassesofpreparationsknownasdecoctions, infusions,uid extracts, tinctures,semisolid extracts or
powdered extracts.
Thepurpose of standardized extraction procedures forcrude drugs (medicinal plant parts) is toattain
Section 2 Pharmacognosy
299
Extracts and its types
the therapeutically desired portions and to eliminate
unwantedmaterialbytreatmentwithaselectivesolvent knownasmenstruum.Theextractthusobtained,after
standardisation, may be used as medicinal agent as
suchintheformoftincturesoruidextractsorfurther processedtobeincorporatedinanydosageformsuch
as tablets and capsules. These products contain complex
mixtureofmany medicinalplantmetabolites,suchas alkaloids,glycosides,terpenoids,avonoidsandlignans.
In order to be used as a modern drug, an extract may
befurther processed throughvarious techniques of fractionationto isolate individual chemical entities suchas vincristine, vinblastine, hyoscyamine, hyoscine, pilocarpine,forskolinandcodeine.
Thebasicparametersinuencingthe quality of anextract arethe plant parts used as starting material,the solvent usedforextraction, themanufacturingprocess(extraction technology)used with the type of equipment employed, andthe crude-drug: extract ratio. The use of appropriate extractiontechnology, plant material, manufacturing equipment,extractionmethodandsolventandtheadherence togoodmanufacturingpracticescertainlyhelptoproduce
a good quality extract. From laboratory scale to pilot scale, all the conditions and parameters can be modelled using
processsimulationforsuccessfulindustrial-scaleproduction.
Apercolator is a narrow, cone-shapedvessel open at
both ends. The powdered crude drug is moistened with
anappropriateamountofthespecied menstruum and allowedtostandforapproximately4hinawell-closed container,afterwhichthemassispackedandthetopof
the percolator is closed. Additional menstruum is added
toformashallowlayerabovethemass,andthemixture isallowedtomaceratein the closed percolator for24h. Theoutletofthepercolatorthenisopenedandtheliquid
contained therein is allowed to drip slowly. Additional menstruum is added as required, until the percolate
measuresabout three-quarters of the requiredvolume ofthe nished product. The marc is thenpressed and theexpressedliquidisaddedtothepercolate.Sufcient menstruumisaddedtoproducetherequiredvolume,and themixedliquidisclariedby ltration or by standing followedbydecanting.
INFUSION
Freshinfusionsarepreparedbymaceratingthecrudedrug forashortperiodoftimewithcoldorboilingwater.These aredilutesolutionsofthereadilysolubleconstituentsof
crude drugs.
GENERAL METHODS OF EXTRACTION OF MEDICINAL PLANTS
Maceration is a process in which powdered crude drug
(wholeorcoarselypowdered)isplacedwiththesolvent
in a stoppered container and allowed to stand at room
temperatureforaperiodofatleast3dayswithfrequent agitationuntil the soluble matter has dissolved. The
mixture then is strained, the marc (the damp solid material)
ispressed, and the combined liquids are clarifiedby ltrationordecantationafterstanding.
Section 2 Pharmacognosy
Percolationis the procedure used most frequently to
extractactiveingredientsinthepreparation of tinctures
300
anduidextracts.
Thegeneral principles andmechanisms involved in maceration,percolationandinfusionfortheextractionof thecrude drugsisgenerallyreferredtoasleaching.The processesofleachingmayinvolvesimplephysicalsolution
or dissolution.
Theextractionproceduresareaffectedbyvariousfactors,
namely:
1. Rateoftransportofsolventintothemass.
2. Rateofsolubilisationofthesolubleconstituentsbythe solvent.
3. Rate of transport ofsolution out of the insoluble
material.
Theextraction of crude drugs ismostly favoured by increasingthesurfaceareaofthematerialtobeextracted anddecreasingtheradialdistancestraversedbetweenthe solids(crudedrugparticle).Masstransfertheorystatesthat themaximumsurfaceareaisobtainedbysizereductions whichentail reduction of material intoindividual cells. However,thisisnotpossibleordesirableinmanycases ofvegetablematerial.
earthenvessel,itshouldnotbenew:Watershouldrstbe boiledinthevessel.Inlarge-scalemanufacture,wooden vats,porcelainjarsormetalvessels are used in place of earthenvessels.Someexamplesofsuchpreparationsare
Karpurasava, Kanakasava, Dasmularista. Some medicinal
preparationsof Ayurveda (likeAsava and Arista) adopt thetechnique offermentation for extracting the active
principles.
DIGESTION
Digestionisaformofmacerationusedwhenmoderately elevatedtemperatureisnotobjectionable.Itisdonewith gentleheatingduringtheprocessofextraction.Thesolvent efciencyofthemenstruumistherebyincreased.
DECOCTION
Decoctionis suitable for extracting water-soluble, heat­stableconstituents.Thecrudedrugisboiledinaspecied volumeofwaterforadenedtime;itisthencooledand strainedorltered.Thisprocessisusedforpreparationof Ayurvedicextractscalled“Quath” or “Kawath”.
Theprocedureforpreparationincludesstartingratioof crudedrugtowaterisxed,e.g.1:4or1:16;thevolume isthenbroughtdowntoone-fourthofitsoriginalvolume
by boiling during the extraction procedure. Then, the
concentrated extract isfiltered and used as suchor processedfurther.
HOT CONTINUOUS EXTRACTION (SOXHLET)
Inthis method, thenely ground crude drug is placed inaporousbagor“thimble”madeofstronglterpaper, whichis placed in chamber of the Soxhletapparatus. Theextractingsolventinaskisheated,anditsvapours
condense in condenser. The condensed extract drips into the thimble containing the crude drug, and extracts it by
contact.When thelevelofliquidinchamberrisestothe topofsiphontube,theliquidcontentsofchambersiphon intoask.
This process is continuous and is carried out until a drop
ofsolvent from the siphon tubedoes not leaveresidue whenevaporated.
Theadvantage is thatlarge amounts of drug can be extractedwithamuchsmaller quantity of solvent. This affectstremendouseconomyintermsoftime,energyand consequentlynancialinputs.Atsmallscale,itisemployed
as a batch process only, but it becomes much more
economicalandviablewhenconvertedintoacontinuous
extraction procedure on medium or large scale.
AQUEOUS ALCOHOLIC EXTRACTION BY FERMENTATION
Theextractionprocedureinvolvessoakingthecrudedrug, inthe form of either a powder ora decoction (Kasaya), foraspeciedperiodoftime,duringwhichitundergoes fermentationandgeneratesalcoholin situ;thisfacilitates theextractionoftheactiveconstituentscontainedinthe plantmaterial.Thealcoholthusgeneratedalsoservesasa preservative.Ifthefermentationistobecarriedoutinan
COUNTER-CURRENT EXTRACTION
Thewet raw material is pulverisedusing toothed disc disintegratorstoproduceneslurry.Inthis process, the materialtobeextractedismovedinonedirection(generally inthe form of ne slurry)within a cylindrical extractor whereit comes in contact withextraction solvent. The furtherthestartingmaterialmoves,themoreconcentrated
the extract becomes. Complete extraction is thus possible
whenthequantitiesofsolventandmaterialandtheirow ratesareoptimised.Theprocessishighlyefcient,requiring littletime and posing no risk from hightemperature. Finally,sufcientlyconcentratedextractcomesoutatone endoftheextractorwhilethemarc(practicallyfreeofvisible solvent)fallsoutfromtheotherend.
Thisextractionprocesshassignicantadvantages:
1. Aunitquantityoftheplantmaterialcanbeextracted withmuchsmallervolumeofsolventascomparedto
other methods like maceration, decoction, percolation.
2. CCE is commonly done at room temperature, which
sparesthethermolabileconstituentsfromexposureto
heat which is employed in most other techniques.
3. Asthe pulverisation of the drug is done underwet
conditions, the heat generated during comminution is neutralised by water. This again spares the thermolabile
constituentsfromexposuretoheat.
4. The extraction procedure has been rated to be more
efcientandeffectivethancontinuoushotextraction.
ULTRASOUND EXTRACTION (SONICATION)
The procedureinvolves the use of ultrasoundwith frequencies ranging from20–2000 kHz; this increases thepermeability ofcell walls and produces cavitation. Althoughtheprocessisusefulinsomecases,likeextraction ofrauwolaroot,itslarge-scaleapplicationislimiteddue tothehighercosts.Onedisadvantageoftheprocedureis theoccasionalbutknowndeleteriouseffectofultrasound energy(>20kHz)ontheactiveconstituentsofmedicinal plantsthroughformationoffreeradicalsandconsequently
undesirable changes in the drug molecules.
SUPERCRITICAL FLUID EXTRACTION
Supercriticaluidextraction(SFE)isanalternativesample preparationmethodwithgeneralgoalsofreduceduseof organicsolventsandincreased sample throughput. The factorstoconsiderincludetemperature,pressure,sample volume,analytecollection,modier(cosolvent)addition, flowand pressure control, and restrictors.Generally, cylindricalextractionvesselsareusedforSFEand their performanceisgoodbeyondanydoubt.Thecollectionof theextractedanalytefollowingSFEisanotherimportant step:Signicantanalytelosscanoccurduring this step,
Section 2 Pharmacognosy
301
Extraction of herbal drugs
leadingthe analyst to believe thatthe actual efciency waspoor. There are many advantages to theuse of
CO
asthe extracting uid.Inadditiontoitsfavourable
2
physicalproperties, carbon dioxide is inexpensive, safe andabundant.Butwhilecarbondioxideisthepreferred uidfor SFE, it possesses severalpolarity limitations. Solventpolarityisimportantwhenextractingpolarsolutes andwhenstronganalyte-matrixinteractionsarepresent. Organic solvents are frequently added to the carbon dioxideextractinguidtoalleviatethepolaritylimitations. Oflate, instead of carbon dioxide, argon isbeing used becauseitisinexpensiveandmoreinert.Thecomponent recoveryratesgenerallyincreasewithincreasingpressure ortemperature: The highest recovery rates in case of argonareobtainedat500atmand150°C.Theextraction procedurepossessesdistinctadvantages:(i)Theextraction ofconstituentsatlowtemperature,whichstrictlyavoids damagefrom heat and some organic solvents. (ii) No solventresidues.(iii) Environmental friendlyextraction procedure.Thelargestareaofgrowthinthedevelopment ofSFEhasbeentherapidexpansionofitsapplications.SFE ndsextensiveapplicationintheextractionofpesticides, environmentalsamples, foods and fragrances, essential
oils, polymers and natural products.
PHYTONICS PROCESS
Anew solvent based on hydrouorocarbon-134a and a
new technology to optimise its remarkable properties in the
extractionofplantmaterialsoffersignicantenvironmental advantagesandhealthandsafetybenetsovertraditional processesfor the production of high quality natural fragrantoils,avoursandbiologicalextracts.Advanced
Section 2 Pharmacognosy
PhytonicsLimitedhasdevelopedthispatentedtechnology
termed “phytonics process”. The products mostly extracted
302
bythisprocessarefragrantcomponents ofessentialoils
and biological or phytopharmacological extracts which
canbeuseddirectlywithoutfurtherphysicalorchemical
treatment.
Factors affecting choice of extraction procedure
Extract and factors affecting procedures
TECHNIQUES FOR DETECTION OF PHYTOCHEMICAL GROUPS IN EXTRACTS
Carbohydrates
Molisch’s test:5 ml of sugar solutionwith 2 dropsof
Molisch’sreagent(10gα-naptholin100mlof95%alcohol) mixandaddconcentratedsulphuricacidthroughsidesof theslopingtest-tube,apurpleringformingattheinterface betweentheacidandtestlayer.Thereactionformsform ve/member/oxygencontainingrings,knownasfurfural, furtherreacts with Molisch reagent to form coloured
compounds.
Benedict’s testassaysforthepresenceofreducingsugars.
Add1mlofthesolutiontobetestedto5mlofBenedict’s solution(solutionofcoppersulphate,sodiumcarbonate,
and sodium citrate, pH 10.5), and shake test tube. Place
thetubeina boiling water bathandheatfor3minutes. Removethe test tube from theheat and allow them to cool.Formationofagreen,red,oryellowprecipitateisa positivetestforreducingsugars.
Barfoed’s testisatestformonosaccharides.Add1mlofthe
solutiontobetestedto3mloffreshlypreparedBarfoed’s
reagent (copper acetate in dilute acetic acid, pH 4.6). Place
testtubesintoaboilingwaterbathandheatfor3minutes. Coolit,formationofagreen,red,oryellowprecipitateis apositivetestforreducingmonosaccharides.
Note:Donotheatthetubeslonger than 3 minutes, as a positivetestcanbeobtainedwithdisaccharidesiftheyare
heated long enough.
Alkaloids
1. Mayer reagent: Solution I—dissolve1.36 g HgC
l2
in
60mlwater.SolutionII—dissolve5gKIin10mlwater.
Procedure—combine the two solutions and dilute with
waterto100ml.Addafewdropstoanacidiedextract
solution (diluted HCl or H
),and if alkaloids are
2SO4
present, a white to yellowish precipitate will appear. Care should be taken not to agitate the test system,
becausetheprecipitatemayberedissolved.
2. Dragendorff reagent: SolutionI—dissolve8gbismuth
subnitrate [Bi (NO
. H2O]in30%w/vHNO3. Solution
3)3
II—dissolve27.2 g KI in 50ml water. Procedure— combinethe solutions and let stand for 24 h,lter,
and dilute to 100 ml with deionised water. In acid
solutions,anorange-brownishprecipitatewillappear. Thealkaloids may be recovered bytreatment with
Na
and subsequent extraction with diethyl ether.
2CO3
Thisreactionmayalsobeperformedonalterpaper oronaTLCplatebyaddingadropofthereagentonto aspotofthesample.
3. Wagner reagent: Solution—dissolve1.27gI
(sublimed)
2
and2gKIin20mlwater,andmake-upwithwaterto 100mL. Procedure—a brown precipitate in acidic solutionssuggeststhepresenceofalkaloids.
4. Ammonium reineckate: Solution—Add 0.2 g
hydroxylaminetoasaturatedsolutionof4%ammonium
reineckate {NH
[Cr (NH3)2(SCN) 4].H2O},andacidify
4
with dilute HCl. Procedure—when added to extracts,
apinkprecipitatewillappearifalkaloidsarepresent.
The precipitate is soluble in 50% acetone, which may
alsobeusedforcompoundrecrystallisation.
Glycosides
Borntrager’s test for anthraquinone glycosides: Boil
thepowderwithdil.HCl,lter,cool,shakewithorganic solvent,separateorganiclayer,shake with NH
OH, the
4
aqueous layer becomes rose pink or cherry red.
ModiedBorntrager’stest: Incase of dianthrone, e.g.
sennosoides—Boilthepowderwithalc.KOH,lter.Add dil.HCltotheltrateandextractwithether.Oxidisewith
H
. Add NH4OH to the ethereal extract and shake, a rose
2O2
red colour is produced in the aqueous layer.
Keller Killiani test for cardiac glycosides:0.1 mg of
glycosideis dissolved ina mixture of1 ml of5% ferric sulphateand99mlofglacialaceticacidandto this 1–2 dropofconc.H
isadded,itgivesbluecolourwithin
2SO4
2–3minconstitutesapositivetest.
Kedde’s test for cardiac glycosides:SamplewithKedde’s
reagent(3,5-dinitrobenzoic acid andNaOH gives violet
colour.
Baljet’s test for cardiac glycosides:Sampleand Baljet’s
reagent(picricacid+NaOH)givesorangeorred.
Flavonoids
Shinoda test toan alcoholicsolutionofthesample,add
magnesiumpowder and a few drops of concentrated HCl.Flavones,avonols,thecorresponding2,3-dihydro derivatives,and xanthones produceorange, pink, red topurplecolourswiththistest.Byusingzincinsteadof magnesium,onlyavanonolsgiveadeep-redtomagenta colour;avanonesandavonolswillgive weak pink to
magenta colours or no colour at all.
Lipids
test (Salkowski): Dissolvesample in a little
H
2SO4
chloroformandaddequalvolumeofconc.sulphuricacid. Aplayofcoloursfrombluish-redtocherry-redandpurple isformedinthechloroform,whereastheacidassumesa markedgreenuorescence.
Sterols
1. Liebermann-Burchardtest: Solution—combine 1 ml
acetic anhydride and 1 ml CHCl add one drop concentrated H
,andcoolto0°C,and
3
. Procedure—When
2SO4
thesample is added, either in the solidform or in
solution in CHCl
, blue, green, red, or orange colours
3
thatchangewithtimewillindicateapositivereaction; ablue-greenish colour in particular is observed for sterols, with maximum intensity in 15–30 min. (This testisalsoapplicableforcertainclassesofunsaturated
triterpenoids.)
2. Salkowski reaction: Procedure—dissolve1–2mgofthe
sample in 1 ml CHCl
and add 1 ml concentrated H2SO4,
3
formingtwophases,witharedcolourindicatingthe presenceofsterols.
Saponins
Whenshaken,anaqueoussolutionofasaponincontaining sampleproducesfoam,whichisstablefor15minormore.
Hemolysis test for saponins:Add sample to 1 dropof
horseblood on glass slide, observeunder microscope
hemolytic zone appear.
Other Polyphenols
Vegetabletanninsarelooselydenedbyacombinationof structuralandfunctionalcharacteristicsaspolyphenolic
compounds that precipitate protein.
1. Ferric chloride: Dissolve5% (w/v) FeCl
in water or
3
EtOH.Addition of several drops ofthe solution to anextract produces a blue, blue-black, or blue-green colourreaction in the presence ofpolyphenols. This isnot aspecicreagentfortannins,asotherphenolic compoundswillalsogiveapositiveresult.
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303
2. Gelatin-salt testforthedetectionoftanninsinsolution,
dissolve10mgofanextractin6mlofhot deionised, distilledwater(lteringifnecessary),andthesolution isdivided between three test tubes. To thefirst is addeda1%solutionofNaCl,tothesecond is added
a 1% NaCl and 5% gelatin solution, and to the third is added a FeCl3 solution.Formationofaprecipitateinthe
secondtreatmentsuggeststhepresenceoftannins,and apositiveresponseafteradditionofFeCl3 to the third portionsupportsthisinference.
Chromatography
Theterm chromatography refers to severalrelated techniquesfor analysing, identifying, or separating mixturesofcompounds.
Separationoftwosamplecomponentsinchromatography isbasedontheirdifferentdistributionbetweentwonon-
miscible phases. In each technique, a sample mixture is
placedintoaliquidorgas,calledamobilephase,auid,
is streaming through the chromatographic system.
The mobile phase carries the sample through a solid support, called the stationary phase, which contains an adsorbent or another liquid.
Thedifferentcompoundsinthesamplemixturemove throughthestationaryphaseat different rates, due to differentattractionsforthemobileandstationaryphases.
In gas chromatography, the mobile phase is a gas, in liquid chromatography it is a liquid.
Theory of Chromatography
1. Typical response obtained by chromatography (i.e. a chromatogram):
where: tR =Retentiontime tM =Voidtime Wb=Baselinewidthofthepeakintimeunits Wh=Half-heightwidthofthepeakintimeunits
Theseparationofsolutesinchromatographydependson twofactors:
1. Adifferenceintheretentionofsolutes(i.e.adifference intheirtimeorvolumeofelution.
2. Asufcientlynarrowwidthofthesolutepeaks(i.e.good efciencyfortheseparationsystem).
Solute Retention
Asolute’s retention time or retention volume in chromatographyisdirectlyrelatedtothestrengthofthe solute’sinteractionwiththemobileandstationaryphases.
Retentiononagiven column pertains to theparticulars ofthatsystem:
1. Sizeofthecolumn
2. Flowrateofthemobilephase
Capacityfactor (k’): It is to measure of retention, determinedfromtR or VR. k’=(tR–tM)/tM
or
k’=(VR–VM)/VM
Capacityfactorisusefulforcomparingresultsobtained ondifferent systemssince it isindependent on column lengthandowrate.
Efficiency
Efciencyisrelatedtheoreticallytothevariouskinetic processesthat are involved in solute retention and
transport in the column.
It is needed to determine the width or standard
deviation(s)ofpeaks.
Chromatographicseparations are a result of the
interactions between the analyte and the two phases. In
general,therearevetypesofinteractions.
1. Adsorption chromatography
2. Partition chromatography
 3. Ion-exchangechromatography  4. Afnitychromatography  5. Size-exclusionchromatography.
Section 2 Pharmacognosy
304
Type of chromatography with their description
S. No. Type of chromatography Details Examples
1. Adsorption
chromatography (also known as displacement, liquid/ solid chromatography)
2. Partition
Chromatography
3. Ion-exchange
chromatography
4. Affinity chromatography In affinity chromatography, separations
Adsorption chromatography is an another technique that uses a solid stationary phase, the adsorbent, packed in a glass column, and a solvent, the mobile phase, that moves slowly through the packed column. A solvent used as a mobile phase is called an eluent. It is based on interactions between the solute and fixed active sites on the stationary phase. The active sites of the stationary phase interact with the functional groups of the compounds to be separated by noncovalent bonds, nonpolar interactions, van der Waals forces, and hydrophobic interactions. Stationary phase: A solid adsorbent packed in a column, spread on a plate, or on a porous paper Mobile phase: Usually, a liquid solvent.
It is a very useful technique because it can resolve minute differences in the solubility of the solutes. It is well­suited for separating homologues and isomers. The solute molecules interact between two non-miscible liquid phases according to their relative solubility. This process is also referred to as liquid/liquid chromatography Stationary phase: A film of liquid that is strongly adsorbed to an inert support Mobile phase: A different liquid with a different polarity
The ion-exchange chromatography process allows the separation of ions and polar molecules based on the electrical properties of the molecules Stationary phase: A resin or gel matrix contains covalently bound positive or negative functional groups : Cation exchange column carries negatively charged groups Anion exchange column carries positively charged groups Mobile phase: A buffered aqueous solution carries a counter-ion whose charge is opposite and in equilibrium with the total charge of the resin
are based on specific interactions between interacting pairs of substance such as a macromolecule and its substrate, cofactor, allosteric effector, or inhibitor. Stationary phase: A gel matrix to which a specific ligand is attached Mobile phase: A buffered solution
Separation of alcohols from hydrocarbons using silica gel, silanol groups on the gel interacts with the polar functional groups on the alcohols
Separation of polar compounds such as amino acids, carbohydrates, and water­soluble plant pigments
Contd...
Section 2 Pharmacognosy
305
S. No. Type of chromatography Details Examples
5. Size-exclusion
chromatography (known as gel filtration, gel-permeation chromatography, and molecular sieve chromatography)
In size-exclusion chromatography, no chemical attraction or interaction occurs between the solutes and the stationary phase. The molecules are separated according to their size. High molecular weight molecules ranging between 2000 to 25,000,000 daltons can be separated Stationary phase: A chemically inert material such as a gel or a porous inorganic solid. Common examples are polyacrylamide polymers, porous glass or porous silica beads Mobile phase: Water or an aqueous solution that solely serves as a carrier for the analyte
Adsorption chromatography
Section 2 Pharmacognosy
306
Paper chromatography
1. Ion exchange chromatography begins
2. Adsorption phase
3. Startingofdesorption
4. Endofdesorption
5. Regeneration
Paper Chromatography
Principle
Theprincipleofseparationinpaperchromatographyis
partition chromatography. Analytes are separated based
onthe interactions between two non-miscible liquid phasesaccordingtotheirrelativesolubility.
Themain difference of paper chromatographyis that sheetofpaperisusedfortheinertphase.
The paper usually contains pure cellulose and no lignin,
copper,orotherimpuritiesandisavailableinstripsorin sheets.Low-porositypaperwillproduceaslowrateof movementofthesolventandthickpapershaveincreased
sample capacity.
Stationary phase: Water that is tightly bound to the
cellulosestructure and lls interspaces of the paper bers.
Mobile phase:Anysolvent(alsoknownasadeveloping
solvent)thatispartiallymiscibleinwater.Choiceofthe solventwilldependonthenatureofthesubstancesto beseparated.Often,amixtureoftwoorthreesolvents
is required.
1. Capillary action:Themovementofliquid within the
spacesofaporousmaterialduetotheforcesofadhesion, cohesion,and surface tension. The liquid is ableto moveupthelterpaperbecauseitsattractiontoitself isstrongerthantheforceofgravity.
2. Solubility: The degree to which a material (solute)
dissolvesintoasolvent.Solutesdissolveintosolvents thathave similar properties. This allowsdifferent
solutesto be separatedby different combinationsof solvents.Separationof components depends onboth theirsolubilityinthemobilephaseandtheirdifferential afnitytothemobilephaseandthestationaryphase.
Techniques Used in Paper Chromatography
Ascending technique: In a chromatographic chamber,
thesolventrisesupthepaper by capillary action and allowsaseparationofthecomponentsasitascends.
Descending technique: In a chromatographic chamber,
apaperstripishangedverticallywiththespottedend beup.Thesolventrisesupthewickanddescendsacross
the spot and down the paper. This technique permits a
separationoveralongerdistanceandallowsanincrease
in resolution.
Applications
1. Itisusedfordiagnosisofpresenceofaminoacidsin
urine.
2. Infoodindustry,paperchromatographyisusuallyused forhighlypolarcompoundssuchassugars,aminoacids,
and natural pigments.
Theanalytes thatare highly water-solubleor have the greaterhydrogen-bondingcapacitycanmovesloweralong thepaper,while lesspolarcompoundswilltravelfaster withthesolvent. Thedegree of retention of acomponent is called the retardationfactor(Rf).
Distancemigratedbyananalyte(Da)
(Rf)=
Distancemigratedbythesolvent(Ds)
Thin layer chromatography
Thestationaryphaseisathin layer of a solid such as
alumina or silica supported on an inert base such as
glass,aluminiumfoilorinsolubleplastic.
Thin layer chromatography
Section 2 Pharmacognosy
307
Themixtureis‘spotted’atthebottomoftheTLCplate
and allowed to dry.
Theplateisplacedinaclosedvesselcontainingsolvent (themobilephase)sothattheliquidlevelisbelowthe
spot.
Thesolvent ascends the plate by capillary action,the liquidllingthespacesbetweenthesolidparticles.
Thistechniqueisusuallydoneinaclosedvesseltoensure thattheatmosphereissaturatedwithsolventvapourand thatevaporationfromtheplateisminimisedbeforethe
run is complete.
Theplateisremovedwhenthesolventfrontapproaches thetopoftheplateandthepositionofthesolventfront recordedbeforeitisdried.
Advantages
1. More reproducible.
2. Separations arevery efficient because of the much smallerparticlesizeofthestationaryphase.
High-performance Liquid Chromatography (HPLC)
HPLCisaseparationtechniquethatinvolvesinjection ofasmallvolumeofliquidsampleintoatubepacked
with tiny particles [3 to 5 micron (µm) in diameter called
thestationaryphase]whereindividualcomponentsof thesamplearemoveddownthepackedtube(column) withaliquid(mobilephase)forcedthroughthecolumn byhighpressuredeliveredbyapump.
Thesecomponentsareseparatedfromoneanotherbythe columnpackingthatinvolvesvariouschemicaland/or
physical interactions between their molecules and the packing particles.
Theseseparatedcomponentsaredetectedattheexitof thistube(column)byaow-throughdevice(detector) thatmeasurestheiramount.Anoutputfromthisdetector
is called a “liquid chromatogram”
High-performance thin layer Chromatography (HPTLC)
HPTLCis an advanced and automatedform of TLC thatprovides superior separationpower, suitable for qualitativeandquantitativeanalyticaltasks.
Itis a flexible, versatile andeconomical process in which thevarious stages are carried out independently.Itpromotesforsamplepreparation requirementsare often minimal, simultaneous processing of sample and standard, no prior treatmentforsolventslikefiltrationanddegassing, higherseparationefficiencies,shorteranalysistime, lessconsumptionofmobilephase,visualdetection possible,efficientdataacquisitionandprocessing, lowcostperanalysisandlowmaintenancecostof
the instrument.
Itis a valuable tool forreliable identification, as it provideschromatographic fingerprints thatcan be visualisedandstoredaselectronicimage.
High-performance liquid chromatography (HPLC)
Application of chromatography in evaluation of herbal drugs
S. No. Herbal drug Chromatography Solvent system
1. St John’s wort TLC Ethyl acetate : dichloromethane : acetic acid : formic acid : water (100 : 25 : 10 : 10 : 11)
2. Ginseng TLC Chloroform : Methanol : ethyl acetate : water (15 : 22 : 40 : 1)
3. Rhubarb TLC Acetone : water : formic acid (18 : 1 : 1)
4. Valerinic acid TLC Methanol : water (7 : 3)
5. Cinchona alkaloids TLC Chloroform : diethylamine (90 : 10)
6. Rhubarb TLC Acetone : water : formic acid (18 : 1 : 1)
7. Atropine TLC Methanol : acetone : diethylamine (25 : 24 : 1)
Section 2 Pharmacognosy
308
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