This document discusses various analytical evaluation and spectrophotometric methods for analyzing plant constituents, including UV-visible spectroscopy, infrared spectroscopy, fluorescence analysis, nuclear magnetic resonance spectroscopy, mass spectroscopy, x-ray diffraction, radioimmunoassay, and various chromatographic techniques such as thin layer chromatography, high performance thin layer chromatography, high performance liquid chromatography, gas chromatography, column chromatography, gel permeation chromatography, and affinity chromatography. Each method is briefly described in terms of its operating principles and applications for identifying compounds.

1. Analytical evaluationof plant constituents
Spectrophptometricmethods:
i) UV- Ultra violet /visible spectroscopy
ii) IR-Infra Red spectroscopy
iii) Fluorescence analysis
iv) NMR-nuclear magnetic resonance spectroscopy
v) MS-Mass spectroscopy
vi) X-ray diffraction
vii) RIA-radio immuno assay
viii) Chromatographic techniques:
a)TLC-Thin layer chromatography
b)HPTLC-High performance thin layer chromatography
c)HPLC-High performance/pressure liquid chromatography
d)GC-Gas chromatography
e)CC-column chromatography
f)Gel permeation chromatography
g)Affinity chromatography
Analytical evaluation
Spectrophptometric methods:
i) UV- Ultra violet/visible spectroscopy Ultra violet–visible absorptiontechniquesencompassanalytical
methodsbaseduponmeasurementof lightabsorptionbysubstancesinwave lengthregionfrom190 to 900
nm 190-380 nm UV region.380-900 nmvisible region.Applications:we cananalyze varietyof pharmaceutical
phytoconstituentslike Lobeline-244nm,Morphine-286 nmAntharaquinone 505nm .
ii)IR-InfraRedspectroscopy
-12,500-400 cm-1, Mid IR-
4000-400 cm-1 Far IR-400-20cm- ometercanbe dividedintosingle anddouble beamand
Fouriertransformspectrophotometer(FTIR) Applications:Identificationof drugs,polymorphsRaw
materials,excipients.
iii) Fluorescence analysis cificrange of wave length,
and manyof themre- -emissionof
absorbedlightlostsonlywhensubstancereceivingexitingrays,andcalledasfluorescence.
iv) NMR-nuclearmagnetic resonance spectroscopy
sample andit occurs at differentfrequenciesfornucleiwithchemicallydifferentenvironment.Applications
NMR is imptool inelucidationof molecularstructure Itisapplicable inidentificationof impurities.Itreveals
positionof protonsina complex
v) MS-Massspectroscopy hthe electronionisation,subsequent
fragmentationof molecules,determinationof the massto charge ratio (m/e).andrelativeabundancesof ions
drug constituents.

2. vi) X-ray diffraction:Many compoundsare capable of crystallisinginmore thanone type of crystal lattice at a
particulartemperature andpressure,sincethe rate of phase transformationof ametastable polymorphtothe
stable one can be quite slow.Polymorphsplaysaveryimprole inpharmaceutical science
vii) RIA-radio immunoassay
labeledformof the same drug.The label maybe particularradio-isotope,active-enzyme oraC14 andiodine
I125 commonlyusedisotopesinRIA.RIA ismethodof choice foridentificationof cardiacglycoside,insulin.
viii) Chromatographic techniques:
a)TLC-Thinlayer chromatography
to analyse Alkaloids,Glycosideslike all bio- y,nature,of
determinationof natural products
Advantages:simpleinoperationandrapid.
Thin layerchromatography(TLC), hasbecome increasinglypopularforbothqualitative andquantitative
evaluationof drugs.•Rf valuesreferstothe rationof distance travelledbythe solute tothe distance moved
by the solventona thinlayeradsorbent
b)HPTLC-Highperformance thin layer chromatography:
-Gel withverysmallparticle size usedas
-HPTLCplatesare producedfrom4-
achievedinabout4 minutes.
c) HPLC-High performance/pressure liquidchromatography
to those methodsinwhichthe separationtakesplace withpackedcolumn.(stationary)A liquidmobilephase
less) flowrate of mobile phase is(100μl /min) Advantages:mostversatile ,safest.
Uses:qualitycontrol of drugslike morhine,emetine,steroids
d) GLC-Gasliquidchromatography
stationaryphase. Principle :GLCworkson partitioningCarriergasusedasmobile phase (Nitrogen,Helium) A
filmof a liquidspreadoveraninertsolid.Actsasstationaryphase.GLC appliedfori.Assayof impurities
ii.Examinationof volatileoilsplantalkaloids.
e)CC-columnchromatography iquidpassesover
chromatographyisoldestandstill practicedtodayfor extractionprocess.
f)Gel permeationchromatography -exclusionchromatography.seperationoccursnotonthe basisof
adsorption/partition,butonthe effective size of solutespresentinsolutionforthe separationpurpose.
g)Affinitychromatography
protiens,enzymes,ant
attachedto a porous stationaryphase andplacedin a column, whenmixture containingthe other
complementof adsorbentpassedthroughstationaryphase.