Measuring PH, Chromatography, ELISA .pdf

PH measuring tools and technique, Chromatography, ELISA test

1. Measuring pH:
Indicators, Paper and Meters
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2. pH indicators
Liquidacid-baseindicatorsareweakorganicacidsorbasesthat
presentasdifferentcolorsintheiracidandbaseforms.An
indicatorhasaspecificpHrangeoverwhichitchangesfromits
acidformtoitsbaseform.
Someofthemostwidely-usedpHtestingtoolsarepHindicators,
includingphenolphthalein(rangepH8.2to10.0;colorlessto
pink),bromthymolblue(rangepH6.0to7.6;yellowtoblue),
andlitmus(rangepH4.5to8.3;redtoblue).
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Dr. Subrata Kundu / [email protected]

3. Acid Alkali low pH high pH
phenolphthalein
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Dr. Subrata Kundu / [email protected]

4. Bromothymolblue
litmus
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Dr. Subrata Kundu / [email protected]

5. Liquidindicatorsareespeciallyusefulinacid-base
titrations,whereanoticeablepHchangeoccursnear
theequivalencepoint.pHindicatorsarealso
commonlyusedtoperformquickchecksonthepHof
watersamples(aquaria,pools,drinkingwater).This
methodofmeasuringpHis quick,inexpensive,and
easy.
TherearedrawbackstousingpHindicators.Thetest
sampleshouldbefairlycolorlesstoclearlyseethe
indicator'scolorchange.Also,theindicators
inherentlymeasurepHatalowaccuracy.
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Dr. Subrata Kundu / [email protected]

6. pH (1-14) Test Paper Strips
Quickandeasytouseandlessexpensive.
SolutioncolorandturbidityarealsoconcernswhenusingpH
stripsandpapers—colorlesssolutionsgivethebestresults
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Dr. Subrata Kundu / [email protected]

7. Digital pH Meter Analog pH Meter
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Dr. Subrata Kundu / [email protected]

8. Pen-type pHTester (Digital Meter)
Portable pH meter
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Dr. Subrata Kundu / [email protected]

9. Thismembraneisfilledwitha
buffersolutionofknownpH
(typicallypH=7).This
electrodedesigncreatesan
environmentwithconstant
bindingofH+ionsonthe
insideoftheglassmembrane,
whiletheoutsideoftheglass
membraneisexposedtothe
samplewhereavariable
amountofH+ionsexist.
ThedifferenceinH+ions
createsapotentialthatisread
versusthestablepotentialof
thereferenceelectrode.
Saturated KCl
solution
pH 7 buffer
Silver wire coated
with AgCl
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Dr. Subrata Kundu / [email protected]

10. WhyisaReferenceNeeded?
Thereferenceelectrodeisdesignedtomaintainaconstant
electricalpotentialthatisindependentofthesample
compositionandtemperature.Incontrast,thehydrogenion
selectiveelectrode(ISE)withglassmembraneprovidesan
electricalpotentialthatisdependentupontheactivityof
hydrogen(H+)ionsinthesamplesolution.Therefore,boththe
referenceelectrodeandthehydrogenISEareneededwhen
determiningpH.
Themostcommontypeofreferenceelectrodeusedtodayisthe
silver/silverchloride(Ag/AgCl)system.Sincesilverisnon-toxic
tohumans,Ag/AgClelectrodescanalsobeusedinmedicineand
foodtechnology.
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Dr. Subrata Kundu / [email protected]

11. ApHmeterwillrequiresingle-,two-,orthree-pointcalibration
foraccurateresults.ThethreemostcommonpHlevelsfor
calibrationare4.0,7.0and10.0.Calibrationintervalsdepend
onthesample,theelectrodeperformance,andtherequired
accuracy.Forhighaccuracymeasurements(≤±0.02pH),the
metershouldbecalibrated immediatelybeforetakinga
measurement.
Theelectrodesmustbehandledwithextremecareastheyare
responsiblefortakingthemeasurements.Theglassbulbofthe
pHelectrodeshouldalwaysbewellhydrated.Neverstorethem
indistilledordeionizedwaterasitdamagesordehydratesthe
sensingglassofthepHelectrodeanddepletesreference
electrolyte.
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Dr. Subrata Kundu / [email protected]

12. The absorption process
How does matter absorb radiation
Whenpolychromaticlight(whitelight),whichcontainsthewhole
spectrumofwavelengthsinvisibleregion,ispassedthoughan
objectwillabsorbcertainofthewavelengths,leavingthe
unabsorbedwavelengthstobetransmitted.Theseresidual
transmittedwavelengthswillbeseenasacolor.Thiscoloris
complementarytotheabsorbedcolors.
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13. Colorimeterisgenerallyanytoolthatcharacterizescolour
samplestoprovideanobjectivemeasureof colour
characteristics.
Thecolorimeterisanapparatusthatallowstheabsorbance
ofasolutionataparticularfrequency(colour)ofvisual
lighttobedetermined.Colorimetershencemakeitpossible
todeterminetheconcentrationofaknownsolute,sinceitis
proportionaltotheabsorbance.
Differentchemicalsubstancesabsorbvaryingfrequenciesof
thevisiblespectrum.
Colorimetersrelyontheprinciplethattheabsorbanceofa
substanceisproportionaltoitsconcentrationi.e.,amore
concentratedsolutiongivesahigherabsorbancereading.
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14. Dr. Subrata Kundu / [email protected]
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15. How colorimeter works?
Whitelightfromatungstenlamppassesthroughaslit,
thenacondenserlens,togiveaparallelbeamwhichfallson
thesolutionunderinvestigationcontainedinanabsorption
cellorcuvette.Thecellismadeofglasswiththesidesfacing
thebeamcutparalleltoeachother.
Light sourceslitcondenser cuvettefilter photocell galvanometer
lens
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16. Single Beam UV-VIS
Spectrophotometer
Double Beam UV-VIS
Spectrophotometer
UV-Visspectrophotometers(ultravioletvisible)measureinthe
UVandvisibleregionsoftheelectromagneticspectrum(190to
380nmand380to760nm,respectively).
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17. Schematicdiagramshowinga
singlebeam(A)andadoublebeam
(B)spectrophotometer
Asinglebeamspectrophotometerhasonlyonebeamoflight,
whileadoublebeamspectrophotometerhastwobeamsoflight,
onepassingthroughareferencesolutionandonepassing
throughthesample.
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18. Typicallyquartzcuvetteareusedwhenthewavelengthofthe
incidentlightisintheUVrangebecausethequartzcuvette
willnotabsorbthelight.Ifyouusethestandardplastic
cuvettes/glasscuvettesintheUVrangetheplasticwillabsorb
someofthelightsoyouwon'tgetacorrectreading.
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19. Likecolorimeters,spectrophotometersareusedtomeasure
thecolorabsorbingpropertiesofasubstance.Thekey
differencebetweenthetwoisthatthespectrophotometer
measuresthetransmittanceandreflectanceasafunctionof
wavelength,whereasthecolorimetermeasurestheabsorbance
ofspecificcolors.
Colorimetersoperateonlyinthe visibleportionofthe
electromagneticspectrumwhereasspectrophotometerswork
withinfraredaswellasvisiblelight.
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20. Dr. Subrata Kundu / [email protected]
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21. Chromatography
Thepurposeofchromatographyistoseparatea
complexmixtureintoindividualcomponent
exploitingthepartitioneffectwhichdistributethe
moleculesintothedifferentphases.
Asdiscussedabove,adistributionofamolecule
betweentwophasesAandBisgivenbya distribution
coefficient,Kd.Inmostofthechromatography
techniques,phaseAisstationaryphaseormatrix
andphaseBismobilephaseorbuffer.
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22. Thin Layer Chromatography
Thethinlayerchromatographytechniqueisananalytical
chromatographytoseparateandanalyzecomplexbiologicalor
non-biologicalsamplesintotheirconstituents.
Itismostpopularformonitoringtheprogressofachemical
reactionorestimationofasubstanceinamixture.Itisalsoone
ofthepopulartechniquefortestingthepurityofasample.
Inthismethod,thesilicaoraluminaasastationeryphaseis
coatedontoaglassor aluminiumfoilasthinlayer and
thenasampleisallowedtoruninthepresenceofa
mobilephase(solvent).Assamplerunsalongwiththemobile
phase,itgetdistributedintothesolventphaseandstationery
phase.Theinteractionofsamplewiththestationeryphase
retardthemovementofthemoleculewhereasmobilephase
impliesaneffectiveforceontothesample.
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23. InTLC,aplastic,glassoraluminumsheetiscoated
withathinlayerofsilicagel.Averysmallamountof
asolutionofthesubstancetobeanalyzedisappliedin
asmallspotwithacapillarytube,~1cmfromthe
bottomoftheTLCplate
TheTLCisdevelopedinachamberwhichcontains
thedevelopingsolvent(themobilephase).A
truncatedfilterpaperplacedinthechamberserves
tosaturatethechamberwithmobilephase.
A B CU D
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Thin Layer Chromatography

24. Dr. Subrata Kundu / [email protected] 39
AsthemobilephaserisesuptheTLCplatebycapillaryaction,the
componentsdissolveinthesolventandmoveuptheTLCplate.Individual
componentsmoveupatdifferentrates,dependingonintermolecular
forcesbetweenthecomponentandthesilicagelstationaryphaseandthe
componentandthemobilephase.
Thin Layer Chromatography
ThestationaryphaseisSiO2andisvery
“polar”.Itiscapableofstrongdipole-
dipoleandH-bonddonatingandaccepting
interactionswiththe“analytes”(the
componentsbeinganalyzed).Morepolar
analytesinteractmorestronglywiththe
stationaryphaseinmoveveryslowlyup
theTLCplate.Morenonpolaranalytes
interactlessstronglywiththepolarsilica
gelandmorestronglywiththelesspolar
mobilephaseandmovehigheruptheTLC
plate.

25. Oncethesolventiswithin~1-2cmof
thetopoftheTLCsheet,theTLCis
removedfromthedevelopingchamber
andthefarthestextentofthesolvent
(thesolventfront)ismarkedwitha
pencil.
Thesolventisallowedtoevaporate
fromtheTLCsheetinthehood.
ThespotsarevisualizedusingaUV
lamp.
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26. X = distance traveled by the solvent front
Y= distance traveled by the substance
R
f
= Y / X
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27. THIN LAYER CHROMATOGRAPHY
Calculation of Rf’s
The R
f
is defined as the distance the centerof the spot moved divided by the
distance the solvent front moved (both measured from the origin)
A B CU
x xx x
Solvent Front
Origen
Distance solvent
migrated = 5.0 cm
Distance A
migrated = 3.0 cm
Distance B
migrated = 2.0 cm
Distance C
migrated = 0.8 cm
0.8 cm
3.0 cm
R
f (A) =
R
f (B) =
R
f (C) =
R
f (U
1) =
R
f (U
2) =
2.0 cm
5.0 cm
= 0.40
= 0.60
= 0.16
= 0.60
= 0.16
3.0 cm
5.0 cm
0.8 cm
5.0 cm
3.0 cm
5.0 cm
0.8 cm
5.0 cm
D
x
R
f (D) = = 0.80
4.0 cm
5.0 cm
4.0 cm
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28. THIN LAYER CHROMATOGRAPHY
Calculation of Rf’s
The R
f
is defined as the distance the centerof the spot moved divided
by the distance the solvent front moved (both measured from the origin)
A B CU
x xx x
Solvent Front
Origen
Distance solvent
migrated = 5.0 cm
Distance A
migrated = 3.0 cm
Distance B
migrated = 2.0 cm
Distance C
migrated = 0.8 cm
0.8 cm
3.0 cm
R
f (A) =
R
f (B) =
R
f (C) =
R
f (U
1) =
R
f (U
2) =
2.0 cm
5.0 cm
= 0.40
= 0.60
= 0.16
= 0.60
= 0.16
3.0 cm
5.0 cm
0.8 cm
5.0 cm
3.0 cm
5.0 cm
0.8 cm
5.0 cm
D
x
R
f (D) = = 0.80
4.0 cm
5.0 cm
4.0 cm
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29. High-PerformanceThin-LayerChromatography
(HPTLC)isthemostadvancedformofTLCand
comprisestheuseofchromatographiclayersofutmost
separationefficiencyandtheemploymentofstate-of-
the-artinstrumentationforallstepsintheprocedure:
precisesampleapplication,standardizedreproducible
chromatogramdevelopmentandsoftwarecontrolled
evaluation.
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30. Dr. Subrata Kundu / [email protected] 45

31. Dr. Subrata Kundu / [email protected] 46

32. Dr. Subrata Kundu / [email protected] 47

33. TLC:
Manualspottingofthesampleleadtoerror.
Whilethesamplehasequivalentproperties,theTLCmethod
givesabadresolution.
TheTLCplatecanbepreparedbytheglass,plastic,orpaper
it'sappliedwithanabsorbentmaterialwhichcontainslarge
particles.
HPTLC:
ItisAutosampler,theaccuracyishigh.
Thisisanadvancedformofthinlayerchromatographythatcan
automatetheanalysisofthesample.
HPTLCmethodgiveshighresolutionascomparedtoTLC.
Thereareverysmallparticlesofadsorbentmaterialusedin
HPTLCplates.
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34. BODIncubator(Bio-OxygenDemand)areusedtomaintain
temperaturefortesttissueculturegrowth,storageofbacterial
culturesandincubationwherehighdegreeofconstanttemperature
accuracyisrequired.BODIncubatorsprovidewithaccurate
conditionsanduniformitythroughoutthechamber.
AdvantagesofBOD(BiologicalOxygenDemand)Incubators
/LowTemperatureIncubators:Forcedconvectiontypei.e.forced
aircirculationofchilledairbydurablecoaxialblowertoachieve
homogeneousheatingandcoolingdissipationthroughoutthe
chamber.
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BOD (Bio-Oxygen Demand)Incubator

35. ELISA(enzyme-linkedimmunosorbentassay)isaplate-based
assaytechniquedesignedfordetectingandquantifyingsubstances
suchaspeptides,proteins,antibodiesandhormones.
InanELISA,anantigenmustbeimmobilizedonasolidsurface
andthencomplexedwithanantibodythatislinkedtoanenzyme.
Detectionisaccomplishedbyassessingtheconjugatedenzyme
activityviaincubationwithasubstratetoproduceameasureable
product.Themostcrucialelementofthedetectionstrategyisa
highlyspecificantibody-antigeninteraction
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Enzyme-linked immunosorbentassay

36. Dr. SubrDrata Kundu / [email protected] 51
DirectELISA
IndirectELISAanantigenisimmobilizedinthewellofanELISAplate.
Theantigenisthendetectedbyanantibodydirectlyconjugatedtoan
enzymesuchashorseradishperoxidase(HRP)oralkalinephosphatase
(AP).
Advantages Disadvantages
Faster than other ELISA –the technique has
fewer steps
Antigen immobilization is not specific -may cause
higher background noise than indirect ELISA. Mainly
because all proteins in the sample, including the target
protein, will bind to the plate
Less flexible -each target protein needs a specific
conjugated primary antibody
No signal amplification -reduces assay sensitivity
Less prone to error –as less reagents and
fewer steps are required

37. Dr. SubrDrata Kundu / [email protected] 52
IndirectELISA
InindirectELISAantigenisadsorbedtoawellinanELISAplate.
Detectionisatwo-stepprocess.First,anunlabeledprimaryantibodybinds
tothespecificantigen.Second,anenzymeconjugatedsecondaryantibody
thatisdirectedagainstthehostspeciesoftheprimaryantibodyisapplied.
Advantages Disadvantages
High sensitivity -more than one labeled
secondary antibody can bind the primary antibody
Possibility of background noise -secondary
antibody may be cross-reactive
Longer procedure than direct ELISA technique -
additional incubation step for secondary antibody
needed
Economical-fewer labeled antibodies are needed
Greater flexibility -different primary antibodies
can be used with a single labeled secondary
antibody

38. Dr. SubrDrata Kundu / [email protected] 53
SandwichELISA
SandwichELISAsrequiretheuseofmatchedantibodypairs(captureand
detectionantibodies).Eachantibodyisthereforespecificforadifferent
andnon-overlappingregionorepitopeoftheantigen.Itisimportantthat
matchedantibodypairsaretestedspecificallyinsandwichELISAto
ensurethattheydetectdifferentepitopes,toachieveaccurateresults.
Advantages Disadvantages
High sensitivity -2-5 times more sensitive than
direct or indirect ELISA
Antibody optimization can be difficult -cross-
reactivity may occur between the capture and
detection antibodies. Needs a standardized ELISA
kit or tested antibody pair.
High specificity -two antibodies are involved in
capture and detection
Flexibility -both direct and indirect detection can
be used