Electrophoresis Instrumental method of analysis

Instrumental method of analysis

1. ModernMethodsofPharmaceutical Analysis
Seminar
Ontopieof
ELECTROPHQRESIS
By,
GokulPrasath.A
B.Pharmacy
KMCHCOP

2. ELECTROPHORESIS
Greekwordmeaning“transportbyelectricity"
(Electro-electricity,phoresis-movement)
Electrophoresis isaphysicalanalysiswhichinvolves
theseparationofthecompoundsthatarecapableof
acquiringelectricchargeinconductingelectrodes.
2

3. DEFINITION
Electrophoresismaybedefinedasthemigrationof
thechargedparticlethroughasolutionunderthe
influenceofanexternalelectricalfield.
•Ionsthataresuspended
betweenthetwoelectrodes
Cathode(-)

tendstotraveltowardsthe
electrodesthatbears
oppositecharge.
Anode(+)
3

4. PRINCIPLE
Anychargedionormoleculemigrateswhenplacedinan
electricfield,therateofmigrationdependuponitsnet
charge,size,shapeandtheappliedelectriccurrent.
v=Eq/F
Where,
v=velocityofthemolecule
E=electricfield(Volt/cm)
q=netchargeonmolecule
F=frictionalcoefficient,whichdependsuponma
shapeofthemolecule.
4

5. Therateofmigrationofanioninanelectricalfield
dependson,
1)Netchargeofmolecule
2)Size,massandshapeoftheparticle
3)Strengthofelectricalfield
4)Propertiesofsupportingmedium
5)Temperatureofoperation
5

6. FACTORSAFFECTINGELECTROPHORESIS
1)SAMPLE
a)Charge:rateofmigrationincreaseswithincreasesinnetcharge
b)Size:rateofmigrationdecreasesforlargermolecules. Itisdueto
frictionalandelectrostaticforces.
c)Shape:moleculeshavesimilarchargebutdifferinshapeexhibit
differentmigrationrate
2)ELECTRICFIELD
a)Voltage:increaseinvoltageleadstoincreaseinrateofmigration
b)Current:increaseincurrentleadstoincreaseinvoltage,
migrationalsoincreases
c)Resistance: resistanceincreasesandmigrationdecreases.
6

7. 3)BUFFER
a)Ionicstrength:rateofmigrationincreasesinhighionic
strength
b)pH:ionizationoforganicacidincreasesaspHincreases
4)SUPPORTINGMEDIUM
a)Adsorption:reducesbothrateofmigrationandresolution
ofseparationofmolecule
b)Electro-endosmosis: itisduetothepresenceofcharged
groupsonthesurfaceofthesupportingmedium.
Ex:paper(carboxyl group),agarose(sulphategroup).
Itwillacceleratethemovementofcations,butretardtheanion
movements.
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8. TYPESOFELECTROPHORESIS
a)ZONEELECTROPHORESIS:
1)Paperelectrophoresis
2)Gelelectrophoresis
3)Thinlayerelectrophoresis
4)Celluloseacetateelectrophoresis
b)MOVINGBOUNDARY/FRONTAL ELECTROPHORESIS:
1)Capillaryelectrophoresis
2)Isotachophoresis
3)Isoelectricfocussing
4)Immuno-electrophoresis
8

9. INSTRUMENTATION
(GeneralApparatus)
Itconsistsbasicallyofitems,
1)Powerpack
2)Electrophoresisunit
•Electrodes
Bufferreservoir
•Supportforelectrolysis
medium
•Atransparentinsulating
cover
3)Sampleinjector
4)Detector
Capillarytutbe
Detector
Anode(+) Cathode(-)
Source Destination
reservoir
Sample
reservoir
Powersupply
9

10. GENERALMETHOD OFOPERATION
Saturationofthe
mediumwiththebuffer t=0min
PlasticTray
DWAMnleingdloudegbufe
DNASeparatlon
t=30-45min
DNASample DNA Marker
Loading
Samples DNA
Sampleapplication Power
Supply
60-154V)
Catiodr
ERcbicaf
FRE)
Electrophoretic
separation
Aguosege
Applicattonof(+)Anale
Electrical Field
DNA
Fragments
UV
Detection
WTransilumination
Documentation
10

11. a)ZONEELECTROPHORESIS
•Itinvolvesthemigrationofthechargedparticleonthe
supportingmedia.
•Paper,celluloseacetatemembrane,starchgel,
polyacrylamide areused.
Componentsseparatedaredistributedintodiscretezone
thesupportmedia.
Supportingmediaissaturatedwithbuffersolution,
smallvolumeofthesampleisappliedasnarrow
band.
OnapplicationofPDattheendsofastripcomponents
migratesataratedeterminedbyitselectrophoretic
mobility.
11

12. ADVANTAGES:
1)Usefulinbiochemical investigations.
2)Costislow
3)Smallquantityofsamplecanbeanalysed.
4)Easymaintenance
DISADVANTAGES:
1)Unsuitableforaccuratemobilityandisoelectricpoint
determination.
2)Duetothepresenceofsupportingmedium,technical
complicationssuchascapillaryflow,electroosmosis,
adsorptionandmolecularsievingareintroduced.
12

13. 1)PAPERELECTROPHORESIS
Itistheformofelectrophoresis thatiscarriedoutonfilter
paper.Thistechniqueisusefulforseparationofsmall
chargedmoleculessuchasaminoacidsandsmall
proteins.
+Migratingbands Filterpaper
BufferSolution
Anodechamber
Cathodethamber
13

14. FILTERPAPER
Itisthestabilisingmedium.Whatmanfilterpaper,cellulose
acetatefilterpaperorchromatographicpaperscanbe
used.
APPARATUS
Powerback,electrophoreticcellthatcontainselectrodes,buffer
reservoirs,supportforpaper,transparentinsulatingcover.
SAMPLEAPPLICATION
Thesamplemaybeappliedasaspot(about0.5cmindiameter)
orasauniformstreak.
14

15. ELECTROPHORETICRUN
Thecurrentisswitchedonafterthesamplehasbeenappliedto
thepaperandthepaperhasbeenequilibrated withthebuffer.
Thetypesofbufferuseddependsuponthetypeofseparation.
Onceremoved,thepaperisdriedinvacuum
DETECTIONANDQUANTITATIVE ASSAY
Toidentifyunknowncomponentsintheresolvedmixturethe
electrophoretogrammaybecomparedwithanother
electrophoretogram onwhichstandardcomponentshavebeen
electrophorescedunderidenticalconditions.
Physicalpropertieslikefluorescence,UVabsorptionor
radioactivityareexploitedfordetection.
15

16. WORKING
1)Inthissampleisappliedas:
circularsupportonastripof
whatmanfilterpapermoistened
withthebuffersolution.
2)Theendsofthepaperare
immersedinseparate
eetaolyte
eservoirscontainingbufferand
inwhichtheelectrodesarefitted.
3)Uponpassingelectriccurrent,the
ionsinthesamplemigratetowards
oppositelychargedelectrodes.
4)Thismethodissuitableforlow
molecularweightcompounds
suchasaminoacidsand
nucleotides.
pnnl
pperfiter
16

17. 2)GELELECTROPHORESIS
ItisatechniqueusedfortheseparationofDNA,RNAorprotein
moleculesaccordingtotheirsizeandelectricalchargeusingan
electriccurrentappliedtoagelmatrix.
GEL:Gelisaerosslinkedpolymerwhosecompositionandporosity
ischosenbasedonthespecificweightandporosityofthetarget
molecules.
TYPESOFGEL
1)Agarosegel
2)Polyacrylamide gel
3)Sephadexgel
Clothwick
Powersupply
Gelslab
Samplewells
ingel
Electrolyte
solution
17

18. AGAROSEGELELECTROPHORESIS
Ahighlypurifiedunchargedpolysaccharidederivedformagar.
Usedtoseparatemacromolecules suchasnucleicacid,larger
proteinsandproteincomplexes.
ItispreparedbydissolvingO.5%agaroseinboilingwaterand
allowingittocoolto40°C.
Itisfragilebecauseoftheformationofweakhydrogenbonds
andhydrophobicbonds.
Sideview: Sampleloaded
intowell Gel Plasticgelbox
buffer
Electricfeldand
direction ofmigration
Negative()Electrode
Positive()Electrode
18

19. 3.Coolthemixture
to65°Candpour
intomold.
4Gelsolidifiedat
roomtemperature.
Combinsertedinto
Combremoved;
moldtomakewells.
vellsremain.
2.Boilmixture
inmicrovave.
1.Mixagarose
andbuffer.
Finishedgel
Anoverviewof
gel
electrophoresis
|oetainprepared
DNASamples Inte
gelles
6esd
Separate fragment
Dyelectreptoresis
repareastandardcurve
etermtnefragentaizes
100,00o
StatnDNAFragments
andmesuredistances
10,000
DutaneeCmen)
19

20. 3)THINLAYERELECTROPHORESIS
Electrophoreticstudiescanbecarriedoutinthinlayerofsilica,
keisulghur,alumina.
ADVANTAGES:
1)Lesstimeconsuming
2)Goodresolution
APPLICATION:
Usedincombinedelectrophoretic-chromatography studiesin
twodimensionalstudyofproteinsandnucleicacid
hydrolysates.
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21. 4)CELLULOSEACETATEELECTROPHORESIS
tcontains2-3acetylgroupsperglucoseunitanditsadsorption
capacityislessthanthatofpaper.
Itgivessharperbands.
Providesagoodbackgroundforstainingglycoproteins.
ADVANTAGES:
1)Notailingofproteins
2)Givessharpbandsandgoodresolution.
DISADVANTAGES:
1)Expensive
2)Presenceofcarboxylicresiduecausesinducedelectro-osmosis
duringprocess.
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22. PRINCIPLE
InanalkalinepH(8.2-8.6)
Haemoglobin isa
negativelycharged(-)
moleculeandwillmigrate
towardtheanode(+).
ThevariousHaemoglobin
movesatdifferentrates
dependingontheirnet(-)
charge,whichinturnis
controlledbythe
compositionofthe
Haemoglobin molecule.
Cathode
Sample
spot
Bufter
Bands
Celuloseacetate
fiterpaper
Anode
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23. b)MOVINGBOUNDARYELECTROPHORESIS
PRINCIPLE:
Themovingboundarymethodallowsthechargedspeciestomigrate
freemovingsolutionwithoutthesupportingmedium.
ADVANTAGES:
1)
denaturingagents.
Biologicallyactivefractionscanberecoveredwithouttheuseof
2)Minuteconcentrationsofthesamplecanbedetected.
DISADVANTAGES:
1)Costlier
2)Elaborateopticalsystemarerequired.
APPLICATIONS:
1)Tostudyhomogenecityofamacromolecularsystem.
2)Analysisofcomplexbiologicalmixture.
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24. INSTRUMENTATION
ConsistsofaUshapedglass
cellofrectangularcross
sectionwithelectrodesplaced
ontheoneeachofthelimbsof
thecell.
Samplesolution
isintroduced
atthebottomorthroughthe
sidearmandtheapparatusis
placedinaconstanttemp.
bathat4o°C.
Detectionisdoneby
measuringrefractiveindex
throughoutthesolution
(Schilerenopticalsystem).
+
Before
Platinum
Electrode
So
After
-Deionized
Water
26

25. 1)CAPILLARYELECTROPHORESIS
Capillarytubeisplacedbetweenthetwobufferreservoirand
anelectricfieldisapplied,separationdependson
electrophoretic mobilityandelectro-osmosis.
Definedvolumeofanalyteisintroducedintothecapillaryby
replacingonebufferreservoirwithsamplevial.
Electrophoreticseparationismeasuredbydetector.
Usingnarrowboretubes,capillaryelectrophoresisremoves
thejouleheatingeffectwhichdecreasesbandbroadening,
givingfasterseparationsthangel.
CEusestubes20-100umdiameterand20-100cm
length.
CEisusedwith/without gel.
Longitudinaldiffusionisthemainsourceofband-broadening.
27

26. Higherelectricfieldsresult
inhighefficiencyand
narrowpeaks(analyte
migratesfaster).
Allanalytestravelthesame
distance,butthe
migrationtime(tm)for
thatdistanceismeasured.
Relatetimetoidentity.
Relatepeakareaorheight
toamount.
CaplarywindowN
Gelreno
Detector
Electrolyte
Cahode(
Hghvoltage
Pouersupply
Anode(t)
Eectopherogam
Computer
Cation
Deteetor
response
Neptral
Anion
Migrationtime
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27. APPLICATIONS OFELECTROPHORESIS
1)DNAsequencing
2)Medicalresearch
3)Proteinresearch/purification
4)Agricultural testing
5)Separationoforganicacid,alkaloids,carbohydrates,amino
acids,alcohols,phenols,nucleicacids,insulin.
6)Infoodindustry
7)Itisemployedinbiochemicalandclinicalfieldsie.,inthe
studyofproteinmixturessuchasbloodserum,haemoglobin
andinthestudyofantigen-antibody interactions.
8)Electrophoresisincombinationwithauto-radiograhy isused
tostudythebindingofirontoserumproteins.
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28. 9)Usedforanalysisofterpenoids,steroidsandantibiotics.
10)Fortestingpurityofthyroidhormonesbyzone
electrophoresis.
11)Paperchromato-electrophoresis isusedtoseparatefree
insulinformplasmaproteins.
12)Itisusedfordiagnosisofvariousdiseaseofkidney,liverand
CVS.
13)Itisalsousedforseparationofscopolamineandephedrine
usingbufferatpH4.2
14)Itisalsousedforseparationofcarbohydratesandvitamins.
15)Quantitativeseparationofallfractionsofcellularentities,
antibiotics,RBC,enzymes,etc.,arepossible.
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29. Thankyou.
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