SOME BASIC CONCEPTS OF BIOCHEMISTRY (Lesson - 5 : Some Laboratory equipment) FOR DMLT FIRST YEAR STUDENTS (U. P. State Medical Faculty syllabus) in ENGLISH & HINGLISH

Some Laboratory Equipment - Balance, Hot Plate, Magnetic Stirrer, Centrifuge, Vortex Mixer, Incubator, Constant Temperature Water Bath, Colorimeter, Spectrophotometer, Beer-Lambert law, Biochemistry Analyzers, Micropipette types - pipetting techniques - pipette calibration, Water Purification Equipments, Distilled Water, Deionized Water

1. 1
SOMEBASICCONCEPTSOFBIOCHEMISTRYFORDMLTFIRSTYEARSTUDENTS(U.P.
StateMedicalFacultysyllabus)inEnglish&Hinglish
LESSON5
[Somelaboratoryequipment]
INENGLISH
SOMELABORATORYEQUIPMENT
Themostcommonlaboratoryequipmentsusedforbiochemicaltestsare:
BALANCE
HOTPLATE
MAGNETICSTIRRER
CENTRIFUGE
VORTEXMIXER
INCUBATOR
CONSTANTTEMPERATUREWATERBATH
COLORIMETER/SPECTROPHOTOMETER
BIOCHEMISTRYANALYZERS
MICROPIPETTES
WATERPURIFICATIONEQUIPMENTS
BALANCE:
Thebalanceisusedforweighingchemicalsinthelaboratory.
Typesofbalancesusedinlaboratory-
1.Opentwo-panbalance-Itisusedforweighinglargeamountsofsubstances.Itssensitivityis
low(0.5g).Sensitivitymeansthesmallestamountthatcanbeweighedaccuratelybythe
balance.Nowadayselectronicopensinglepanbalancearealsoavailable.
2.Analyticalbalance-Itisusedforweighingsmalleramountsofsubstancesasitssensitivityis
higher(0.1-0.5mg),dependingonthemodel.Analyticalbalancemaybetwopanbalancewith
apointer,singlepanelectricorsinglepanelectronicbalance.

2. 2
TwopanbalancebalanceSinglepanelectricbalanceSinglepanelectronicbalance
Precautionsduringweighing:
●Readthelabelonthebottleofthesubstancetobeweighedthreetimes-beforetakingit
outfromtheshelf,justbeforeweighingitandafterweighingtomakesurethatthe
desiredsubstanceisweighed.
●Thebalanceshouldbeplacedonasturdyvibrationfreeplatform.
●Alwayscheckthatthebalanceisproperlylevelled.Thiscanbecheckedbythelevelling
bubbleatthefloorofthebalanceanditcanbeadjustedbythelevellingscrews.
●Inatwopanbalance,raisethebeamslowlyandnotethatthepointerswingsequal
divisionsonbothsidesofthezeromark.
●Useforcepstopickupthesmallweights.
●Ceilingfanshouldbeeitherataslowspeedorswitchedoffduringweighing.
●Thedoorsoftheanalyticalbalanceshouldbeclosedwhilecheckingthereadingonthe
balance.
●Useacleanpieceofpaper,watchglassoradisposableplasticweighingboattoweigh
chemicals.
Watchglass weighingboats
●Cleanthebalancewithasoftclothorabrushaftercompletionoftheweighing.
HOTPLATE:
Hotplateisusedinthelaboratorytoheatsolutionsinglasscontainers.Thetopofthehotplate
maybeconstructedfromaluminum,ceramicmaterialsorenamel.Somehotplateshave
magneticstirrersalsotostirtheliquidwhileheating.Ithasaknobforon/offandcontroloflow,
mediumandhighheating.

3. 3
Safehandlingofhotplate-
●Donotusehotplateinthepresenceofflammableorcombustiblematerial.
●Setthedesiredheatingoptionviz.Low/High.
●Donotleavethehotplateunattendedwhileusingit.
●Usethermalglovesortongstoremovehotobjectsfromthehotplate.
●Usegoodqualityheatresistantglasswaresuchasborosilicateglassware(notsodalime
glassware).
●Putaplacardshowing"HOT"infrontofthehotplateafterusetopreventanyaccident
becauseitremainshotforsometime.
MAGNETICSTIRRER-Itisusedforstirringormixingasolutionusingastirbarimmersedin
theliquid.Itmaybecoupledwithaheatingsystem.Itemploysarotatingmagneticfield(created
eitherbyarotatingmagnetorasetofstationaryelectromagnets)tocauseastirbarimmersed
inaliquidtospinveryquickly,thusstirringtheliquid.
CENTRIFUGES-
Centrifugeisanessentialequipmentforamedicallaboratoryandisrequiredfortheseparation
ofcells,serum,plasmaandremovingprecipitatesetc.
Principle-Itisbasedontheprincipleofsedimentationandapplicationofcentrifugalforcetoa
heterogeneousmixtureofparticles.Theparticlesareseparatedfromasolutionaccordingto
theirsize,shape,density,theviscosityofthemediumandrotorspeed.Thedenserparticles
settletothebottomofthetesttube,whilelow-densitysubstancesrisetothetop.
rcf=1.118×10
-6
×r×(rpm)
2
Here,'rcf'istherelativecentrifugalforce(relativetotheforceofEarth'sgravity).Relative
centrifugalforcereferstotheamountofforceappliedwhenusingacentrifuge.Itdependson
thespeedofrotationin‘rpm’andthedistanceoftheparticlesfromthecenterofrotation.
'r'isradiusofrotorinmm,
'rpm'isrevolutionsperminute
Unitofrcfisg

4. 4
Sincercfdependsonradius(r)oftherotorthesame'rcf'mayhavedifferentrpmdepending
upontheradiusoftherotor.
Howtomeasuretheradiusoftherotorinaswingoutrotor
HowtomeasuretheradiusofaFixedanglerotor
TypesofCentrifuges-
1.Tabletop/Benchtopcentrifuge-Thesearemoderatespeedcentrifugescommonlyusedin
medicallaboratoriesforseparationofplasma,serum,cells,urinedepositsandprecipitate.The
rotorsofsuchcentrifugesareeitherfixedangletypeorswing-outtype.
2.Microcentrifuge(Microfuge)-Thesearealsousedinmedicallaboratoriesforcentrifuging
smallvolumesofsamples/fluidssuchaspelletingnucleicacidsorproteinsathighspeed.
3.Multipurposehighspeedcentrifuges-Thesearehighspeedrefrigeratedfloorcentrifuges
usedforlargervolumes.Therotorsofsuchcentrifugesareeitherfixedangletypeorswing-out
typeandmaycomewithdifferentadapterstoholdvarioussizesoftesttubes,bottles,or
microtiterplates.

5. 5
Multipurposehighspeedcentrifuge
4.Ultracentrifuge-isasophisticatedandadvancedrefrigeratedcentrifugethatoperatesatan
extremelyhighspeedandseparatessmallermoleculesthatcannotbeseparatedfromthe
traditionalcentrifuges.Theseareusedinmolecularbiologyandbiochemistryresearch.There
aretwotypesofUltracentrifuge-analyticalandpreparative,basedonapplicationandpurpose.
Inthepreparativerunofanultracentrifuge,thecontentsofthetubesareanalyzedafterthe
centrifugationperiod,unliketheanalyticalcentrifugewheretheanalysisisdoneduringthe
centrifugationprocess.
Safetyprecautionsinhandlingcentrifuges-
●Thecentrifugemustbeplacedonrubberpadsoramatonaflatlevelsurface.
●ReadtheInstructionManualtooperatethecentrifuge.
●Neverhandleacentrifugewithwethands.
●Checkthesizeofthetesttubes(neithertoolongnortoosmall)anddonotfillthemmore
than¾withtheliquid.
●Balancethetubesthatareoppositeeachotherbyweighingthemonatwopanopen
balanceandadjusttheirweightbyaddingliquidtothelightertube(ifpermissible)or
watertothebucketcontainingthelightertube.Thegoodqualitytesttubeswithequal
volumeofliquidgenerallydonotrequirebalancingintabletopcentrifuge.Unbalanced
tubemayresultinvibrationsanddamageinthemachineandbreakageoftesttubes.
●Alwayspadthebottomofthebucketswiththerubbercushionsprovidedwiththe
machinetoavoidbreakageoftesttubes.
●Ensurethatthelidisclosedbeforestartingthecentrifuge.
●Increasethespeedgraduallybyturningtheknobslowly,untilthedesiredspeedis
reached.
●Stopthecentrifugegradually(somemodelshaveabrakethatcanbeapplied).Never
trytoslowdowntherotorofthecentrifugedownwithyourhands.
●Neveropenthecentrifugeuntilithascometoacompletestop.
●Cleanthebowlofthecentrifugedailyorafteranyspillageoccurs.Use70%ethanolfor
metalbowlsand1%bleachforplasticones.

6. 6
VORTEXMIXER(CYCLOMIXER):
Itconsistsofanelectricmotorwiththedriveshaftorientedverticallyandattachedtoacupped
rubberpiecemountedslightlyoff-center.Asthemotorrunstherubberpieceoscillatesrapidlyin
acircularmotion.Whenatesttubeorotherappropriatecontainerispressedintotherubbercup
(ortouchedtoitsedge)themotionistransmittedtotheliquidinsideandavortexiscreated
whichresultsingoodmixingoftheliquid.Inabiochemicaloranalyticallaboratorytheymaybe
usedtomixthereagentsofanassayortomixanexperimentalsampleandadiluent.Care
shouldbetakenthat:
●theinstructionmanualshouldbereadbeforeusingitforthefirsttime
●properspeedshouldbeset
●ifvortexingatesttube,itshouldnotbefilledwithliquidmorethanhalfforpropermixing
andavoidingspillage.
INCUBATORS:
Incubatorsaremetallicinsulatedenclosuresthatarethermostaticallyregulatedtomaintaina
constanttemperatureandareusedforbacterialculturebylaboratoriesworkinginmicrobiology.
Hotairiscirculatedoverracksorshelvescontainingthepetridishes,flasks,orotherculture
media.
Thetemperatureintheincubatorsshouldberecordeddailyanditmustbecleanedatregular
intervals(atleasteveryfortnight)andalsoafterspillageofanymaterial,whetherinfectiousor
non-infectious.

7. 7
CONSTANTTEMPERATUREWATERBATH:
Awaterbathisadoublewalledsteelcontainerfilledwithwaterwhichisheatedbyaheating
elementanditstemperatureiscontrolledbyathermostat.Itisusedtoincubate
samples/reagents/chemicalreactiontubesinwaterataconstanttemperatureoveralong
periodoftime.Somewaterbathshavestirrerstoprovideuniformtemperaturetothewater.
Somewaterbathshaveanextracontrolforconstantshakingwhichmayberequiredin
microbiologicalpractices.
●Thewaterbathshouldbefilledwithdistilledwatertopreventdepositsforminginside.A
crystalofthymolwillhelptopreventalgalgrowth.
●Changethewaterandcleantheinsideofthewater-bathatleastonceamonthor
wheneveritlooksdirty.Ensuretheunitisunpluggedbeforechangingthewater.
●Useathermometertocheckthewatertemperatureeachtimethewaterischangedas
scaleontheheatingelementmaycausethethermostattomalfunction.
●Switchoffthewaterbathafteruseandcoverit.
COLORIMETER/SPECTROPHOTOMETER
PHOTOMETRY:
Photometrydealswiththestudyofthephenomenonoflightabsorptionbymoleculesinsolution.
Whitelightisamixtureofallthecolorsoflight.Whenwhitelightpassesthroughacolored
substance,certaincolorsoflightareabsorbedwhileothercolorspassthroughorarereflected
bythesubstance.Thecolorseenisthevisiblelightthatisnotabsorbedbythesample.For
example,aredsolutionappearsredbecauseitisabsorbingallofthecolorsoflightexceptred.
Differenttypesofphotometricinstrumentsareusedinthelaboratorywhicharebasedonthe
followingprinciple:
Whenmonochromaticlight(lightofonecolour/wavelength)passesthroughasolutionthe
intensityoflighttransmitteddecreasesexponentially-withincreasingpathlength(Lambert's
law)andwithincreasingconcentrationoftheabsorbingsubstance(Beer'slaw).The
combinationofthesetwoiscalledBeer-Lambertlaw.Sinceinphotometricinstruments(suchas
colorimeterandspectrophotometer)themeasurementsareusuallymadeatconstantpath
lengthwithvaryingconcentrationitisusualtorefertoBeer'slaw.Therefore,theabsorptionof
lighttransmittedthroughthecolouredsolutionisdirectlyproportionaltothesolution
concentration.Itmeans,highertheconcentrationoftheabsorbing(coloured)solutionhigherwill
betheabsorptionoflight(absorbance).

8. 8
Theratioofintensityoftheemergentlight(I)andintensityofincidentlight(Io)iscalled
Transmittance(T):
T=I/Io
T×100=%T
-logTorlog(1/T)=extinction(E)oropticaldensity(OD)orabsorbance(A)
Or
absorbance(OD)=log(1/T)=log(100/%T)=2-log%T
Molarextinctioncoefficient-ofanysubstanceisitsextinction(absorbance,OD)ata
concentrationof1mol/latapathlengthof1cm.Forexample-Themolarextinctioncoefficient
ofNADHat340nmis6.22l/mmol/cm(6220l/mole/cm)or0.1mMsolutionofNADHwouldhave
absorbance(OD)of0.622with1cmpathlengthoflight.
TransmittancevsAbsorbance

9. 9
COLORIMETER
AColorimeterconsistsofthefollowingparts:
1.Lightsource(usually6-12Vtungstenlamp)
2.Aperture(Slit)andCondenserlenstoallowaparallelbeamoflighttofallonthefilter.
3.Filters(forselectiveabsorptionofunwantedwavelengths)arrangedonawheel.The
maximumtransmissionofthesefilterisatthefollowingwavelengths:
Deepviolet-420nm
Violet -430nm
Blue -470nm
Bluegreen-490nm
Green -520nm
Yellowgreen-550nm
Yellow -580nm
Orange -600nm
Red -680nm
Deepred-700nm
Thecriteriaforchoosingthecolororwavelengthofthefilterforaparticulartestisthatthe
wavelengthoflightthatistransmittedbythecolorimeterhastobethesameasthatmaximally
absorbedbythesubstancebeingmeasured.Forexample,ifasubstanceabsorbsmaximallyat
520nmthenthegreenfilterwillbeusedforthemeasurementofthatsubstanceusinga
colorimeter.Thiscanbeexplainedasbelow:
Aredsolutionappearsredbecauseitisabsorbingallofthecolorsoflightexceptred.Itwill
havethegreatestabsorbanceinthegreenrangebecausegreenisthecomplementarycolorof
red.Acomplementarycoloristheoppositecoloronthecolorwheel.
4.Cuvettechamber(thetransmittedlightpassesthroughcompartmentwherein
thesolutioncontainingthecoloredsolutioniskeptincuvette,madeofglassordisposable
plastic)
RoundbottomcuvettesSquarecuvettes

10. 10
5.Detector(thisisaphotosensitiveelementthatconvertslightintoelectricalsignals)
6.Galvanometer(measureselectricalsignalquantitatively).
Digitalcolorimeter Analogcolorimeter
Workingofacolorimeter-
Whitelightfromatungstenlamppassesthroughaslit,thenacondenserlens,togiveaparallel
beamwhichfallsonthefilterselectedtoallowmaximumtransmissionofthedesiredwavelength
whichpassesthroughthesolutionunderinvestigationcontainedinacuvette.Thetransmitted
lightthenfallsontoaphotocell,whichgeneratesanelectricalcurrentindirectproportiontothe
intensityoflightfallingonit.Thissmallelectricalsignalisincreasedbytheamplifier,which
passestoagalvanometerofanalogueordigitalreadouttogiveabsorbancereadingdirectly.
Thecolorimeterisfirstsettozeroabsorbance(100%T)withappropriateblanksolution.Then
theabsorbanceofasuitablestandardisreadfollowedbyreadingtheabsorbanceofthetest
(unknown)solution.
Absorbanceofunknown×ConcentrationofStandard
Concentrationofanalyteinunknownsample=--------------------------------------------------------------
AbsorbanceofStandard
SPECTROPHOTOMETER
Spectrophotometersalsomeasuresampleabsorbancetodetermineanalyteconcentrations.
BothcolorimeterandspectrophotometerworkonBeer-LambertLawandoperateinsomewhat
similarwaysasboththeinstrumentsmakeuseofsolutionandbeamoflighttocheckforthe
absorbanceleveloflight.However,somedifferencesexistbetweenthetwo:
●Themaindifferencebetweencolorimeterandspectrophotometeristhatcolorimeterisa
devicewhichmeasuresabsorbanceofspecificcolours,whereasaspectrophotometer
measuresthelight-absorbingortransmittingabilityofsolutions(colouredorcolourless)
whenabeamoflightispassedthroughit.
●Thecolorimeterusescolouredfilterstoobtainmonochromaticlightforvisiblerangeof
spectrumwhereasspectrophotometerusesawiderangeofwavelengthsinUV,visible
andinfraredregionwiththehelpofprismordiffractiongratingstoobtainthedesired
wavelengthoflight.

11. 11
●Thespectrophotometer'slightsourceisatungsten,xenonordeuteriumlamp,whilethe
colorimeterlightsourceisTungsten/LED.
●Thecuvetteofthecolorimeterisusuallycylindricalandmadeofglasswhileinthe
spectrophotometeritisrectangularwithsquarecrosssectionandmadeofglass,silicaor
quartz.
●Thespectrophotometerismorepreciseandaccurateascomparedtoacolorimeter.
●Spectrophotometersareoftwotypes-singlebeamanddoublebeam
spectrophotometers.
SinglebeamSpectrophotometerDoublebeamSpectrophotometer
DigitalSpectrophotometers AnalogSpectrophotometer
BIOCHEMISTRYANALYZERS-Thebasicprincipleoftheseanalyzersisthesameasthatofa
colorimeteroraspectrophotometer.However,theprecisionoftheseanalyzers,especiallythe
fullyautomaticanalyzers,isbetterthanmanualphotometers.TwotypesofBiochemistry
Analyzersareusedindiagnosticlaboratories:
1.Semi-automaticanalyzer-AsthenameisSemi-automatic,thetestisrunmanuallybutthe
finalreadingistakenbyaspiratingthetestsolutionandthemachinegivestheconcentration
valuedirectlybycomparingwiththeabsorbance(OD)andtheconcentrationofthestandard,
hencenoneedforcalculation.Theseanalyzershavesettingsforcuvette/flowcelltemperature
control,programmingforendpoint,fixedtimekineticandkineticreactiontestsandinbuiltquality
controlchartstokeeptrackofqualitycontrol(QC)fordifferenttests.Thesearewellsuitedfor
smallandmediumsizelaboratorieswithlimitednumberofsamplesandtests.

12. 12
2.Fullyautomaticanalyzer-Thesemachinescananalyzealargenumberofsamplesinashort
periodoftimeandwithminimumhumanassistance.AsopposedtoSemi-automaticanalyzer,in
fullyautomaticanalyzeronlythespecimens(blood,serum,plasma,urine,cerebrospinalfluid
etc.)arerequiredtobearrangedintheanalyzeralongwiththerequiredreagentsandthe
machineisprogrammedfortherequiredtestsandthenthemachineperformsthetestsand
givestheprintoutofthefinaltestresults.Theseanalyzersareverymuchsuitableforlarge
laboratorieswithlargenumberoftestsandsamples.
Fullyautomaticanalyzersareoftwotypes-
1.Batchanalyzer-inwhichtheinstrumentsystemsequentiallyperformsasingletestoneachof
agroupofsamples.
2.Randomaccessanalyzer-inwhichthespecimenscanbeaccessedatrandom,i.e.outof
sequencewitheachother.Thusanynumberofselectedtestscanbeprogrammedtobedone
oneachsample.
Inaddition,therearetwotypesofbiochemicalanalyzersbasedontheirreactionprinciple:
1.Wetanalyzer-Inthistypeofanalyzerliquidreagentsareused.Thebloodsampleis
mixedwiththisliquidreagentinareactionvessel(testtube)andthenthereaction
mixture'sabsorbanceisreadonacolorimeter/spectrophotometerandtheconcentration
oftheanalyteisthencalculated.
2.Dryanalyzer-Inthistypeofanalyzerdryreagentisusedinsteadofliquidreagent.This
dryreagentiscoatedonalayerofasupportingmedium(stripsimpregnatedwithdry
reagents).Whenabloodsampleisaddedonittheanalyteofthesamplereactswiththe
dryreagentinpresenceofthemoistureoftheblood.Thereactionproductsoformedis
readbyreflectancespectrophotometryandtheconcentrationoftheanalyteis
calculated.

13. 13
MICROPIPETTES
Micropipettesareakindofpipettesusedinlaboratoryfortransferofsmallvolumesofliquid(as
lowas0.2microliter).
Partsofamicropipette Micropipettesonmicropipettestand
Therearedifferenttypesofmicropipettes:
BasedonthePrinciple/DisplacementMethod-
●Airdisplacementmicropipette-aresuitableforaqueousandnonviscousliquids.These
micropipetteshaveapistoninsideandthereisanaircushionbetweenthepistonand
thesampletohelpdispenseliquids.
●Positivedisplacementmicropipette-Insuchpipettethepistonisnotinsidethepipette
shaftbutinsidethepipettetip.Thereisnoaircushionbetweenthepistonandliquid.The
disposabletipinapositivedisplacementmicropipetteisamicrosyringecomposedofa
capillaryandapiston(movableinnerpart)whichdirectlydisplacestheliquid.Theseare
suitableforviscousandvolatileliquidsalso.
BasedontheNumberofChannels-
●Singlechannel-Onlyashaftispresentinasinglechannelmicropipettethatcantransfer
oneliquidatatime.Thusithasasinglechanneltoaspirateordispensetheliquid.
Theseareavailableinvariouscapacitiesandprovideexcellentaccuracy.Thecapacity
availablerangesfrom0.2to10,000µl.

14. 14
●Multichannel-Multipleshaftsarepresentinamultichannelmicropipettethatcantransfer
variousliquidssimultaneously.Thusithasmultiplechannelstoaspirateordispensethe
liquid.Thesemayhave4-18channels.Itisbestfortestsinvolvingmultiplewellslike
ELISA,DNAamplificationtests,etc.Thecapacityofmultichannelmicropipettesranges
from0.2to1200µl.
BasedonthePipettingMechanism-
●Mechanical/Manual-Inthethesemicropipettes,personnelhastoapplypressureto
presstheplungerforaspirationanddispensation.Thepistonhasaspringforthis
purpose.
●Electronic-Anelectronicbuttonreplacesthemechanicalplunger.Itisalsocalledan
automatedpipetteanditreplacesmanuallabor.Thesehavecustomizableprogramsto
adjusttoanyrequirementofthepipetting.
BasedontheVolume-
●Fixedvolumemicropipette-Thesepipettestransferafixedandanequalvolumeof
sampleseverytime.Itisidealfortransferringviscousliquidsinlaboratorieswithalimited
budget.Itdecreasesthechanceoferrorduetomistakenlychangingthevolume.

15. 15
1000µlmicropipette 10µlmicropipette
●Adjustablevolumemicropipette-Thesehavevolumeadjustingknobsbywhichthe
requiredtransfervolumecanbeadjusted.Theseareavailableindifferentvolume
ranges.
0.5-10µlmicropipette10-100µlmicropipette
Differenttypesofpipettingprocedure-
●Forwardpipetting-Thisisthemostfrequentlyusedtechnique.Inforwardpipetting,an
exactlysetvolumeofliquidisaspiredintothetipandthenitisdeliveredtoanewvessel.
Thistechniqueisrecommendedforpipettingofdilutedaqueoussolutions,bufers,diluted
acidsandbases.
●Pipettingsteps-
1.Attachatiptothepipettor
2.Pressthebuttontothefirststop
3.Dipthetipapprox.2-3mmunderthesolutionlevel(dependinguponthe
aspirationvolume)andreleasethebuttonslowly
4.Thesolutionisaspiredtothetip(payattentionnottoaspireairbubblese.g.when
thepistonisreleasedtooquicklyorifthetipisnotattachedproperly).
5.Removethethumbcompletelyfromthebuttonafterithasreachedthereleased
position.

16. 16
6.Takethetipofftheliquidslowly(beforeremovingthetip,waitseveralseconds
especiallywhenworkingwithlargervolumessuchas500–5000μL).
7.Ifnecessary,wipedropletsfromtheexternalsurfaceofthetipwithtissuecloth.
Nevertouchtheorificeofthetipotherwisetheclothwouldabsorbaportionofthe
transferredvolume.
8.Duringdeliveryoftheliquidtoanothervessel(testtube)thetipshouldtouchthe
wallofthevesselatanangle(10–45º).Itshouldbejustabovethelevelofany
liquidthatalreadyisinthevessel.
9.Pushthebuttontothefirststop.Waitforapprox.1secondandpushthebutton
quicklytothesecondstop(astrongerresistancecanbefelt).Nodropletsshould
remaininthetipandnoliquidshouldbesplatteredonthewalls
10.Holdthebuttoninthesecondpositionandremovethetipfromthevesselstill
touchingthewallofthevessel.Nowreleasethebutton.
●Reversepipetting-Thistechniqueissuitableforveryviscousorvolatileliquids,biologic
fluids,foamingsolutionsandformeasuringverysmallvolumes.
Pipettingsteps-
1.Pushthebuttontothesecondstop.
2.Dipthetip2–5mmbelowthelevelofthesolution.Slowlyaspirethesolutionto
thetip.
3.Removethetipslowlyfromthesolution.Ifnecessary,wipedropletsfromthe
externalsurfaceofthetip.
4.Delivertheliquidtoanewvessel,pushingthebuttontothefirststoponly.Thetip
shouldtouchthewallofthevesselsimilarlytotheforwardtechnique.
5.Holdthebuttoninthefirstpositionandremovethetipfromthevessel.
6.Theliquidremaininginthetipcanbereturnedtothestockvesselorthrown
away.

17. 17
●Repetitivepipetting-Thistechniqueisusedwhenthesamevolumeofthesameliquidis
tobemeasuredtoseveraltest-tubesortomanywellsofamicrotitrationplate.Itis
similartoreversepipettingwheresteps2to4arerepeatedseveraltimes.
●Pipettingofheterogeneoussamples-Thistechniqueissuitableforheterogeneous
sampleslikewholeblood.Pre-rinsingofthetipbeforepipettingisnoteasyinthiscase.
Pipettingsteps-
1.Pushthebuttontothefirststopanddipthetipapprox.2–5mmbeneththe
solutionlevel.
2.Slowlyreleasethebutton.Thesampleisaspiredtothetip.
3.Removeslowlythetipfromthesolutionandwipedropletsontheouterwallofthe
tip.
4.Dipthetipintothetargetsolution.
5.Pushthebuttontothefirststopandthenslowlyreleaseback.Thesolutionwillbe
aspired.Donotremovethetipfromthesolutionandrepeatthisstepuntilthe
innerwallofthetipisclean.
6.Touchingthewallofthevessel,placethetipabovethelevelofthesolutionand
pushthebuttontothesecondstop.
7.Holdthebuttonatthesecondstopandremovethetipfromthevessel.
Sometipsforusingmicropipettes-
●Bringtheliquidtoroomtemperature(RT)beforepipettingbecausecoldliquidstendto
beoverdeliveredwhilewarmliquidsmaybeunderdelivered.
●Attachthecorrectsizedisposabletipproperly.
●Pre-rinsethetipatleasttwotimesbyaspiratinganddispensingtheliquid.
●Dipthetipvertically(at90°)approx.2-3mmundertheliquidlevelorlittlemore
dependingupontheaspirationvolume.
●Releasethepistonbuttonslowlytoavoidairbubbles,removethethumbfromthebutton
andwaitforafewsecondsforcompleteaspirationofliquid.
●Takethetipofftheliquidslowlyandifnecessary,wipedropletsfromtheexternalsurface
ofthetipwithtissuepaperwithouttouchingtheorificeofthetip.

18. 18
●Fordeliveringtheliquid,thetipshouldtouchthewallofthevesselatanangle(10–45º)
andjustabovetheleveloftheliquidalreadypresentinthevessel(withouttouchingthe
liquid).
●Pushthepistonbuttontothefirststop,waitforawhileandthenpushthebuttontothe
secondstop.
●Holdthebuttoninthesecondpositionwhileremovingthetipoutofthevesselbutstill
touchingthewallofthevessel.Nowreleasethebutton.
●Discardthetipusingthetipejector.
Calibrationofmicropipette-Themicropipettesshouldbecalibratedfromtimetotime(preferably
everythreemonths)tochecktheiraccuracyandprecision.Thisisdonejustlikewedofor
calibrationofglasspipettesusingasensitiveelectronicbalance:
StepsInvolvedinPipetteCalibration-
●Takedistilledwaterinabeakerandrecorditstemperature.Also,gatheryourpipetteand
thecorrecttipsbasedonboththesmallandlargevolumesthatthepipettecandispense.
●Placeaweighingboatonabalancethatcanaccuratelyweighinthemicrogramrange,
andsetittozeroafterclosingthebalancedoor.
●Pre-rinsethetipbyaspiratinganddispensingthesetvolumethreetimesandpushfully
toremoveanyremainingliquid.
●Aspiratethecalibrationvolumewithoutbubbleformationanddispensetheliquidslowly
intotheweighingboat.Then,recordtheweightonthebalanceandrepeattheprocess
tentimes.
●CalculatethedispensedvolumebyusingtheequationV=WxZwhereWistheweight
ofthewater,ZistheZfactor*,andVisthecalculatedvolumeofdispensedwater.Next,
determinethemeanvaluefromtentrials.Themeanandstandarddeviation(SD)of
thesetenvaluesiscalculated.Theprecisionisdeterminedbycalculatingthecoefficient
ofvariation(CV)usingtheformula%CV=(SD÷Mean)×100.TheCVmustbeless
than1%.
●Finally,calculatetheaccuracybyusingtheequationA=100xVavg/V0,whereAisthe
accuracyofthepipette,VavgistheaveragecalculatedvolumeandV0isthetheoretical
volumeyoutriedtodispense.Iftheaccuracyvalueliesinthe99-101%range,the
pipetteisconsiderednormalandcalibrated.
Z*factor-Theaccuracyofthevolumemeasurementsofwaterisaffectedbyambient
temperature,atmosphericpressureandrelativehumidity.Thesefactorsareusuallycombinedto
givetheZfactor,usedincalculationofvolumeofwater.e.g.
Temperature(°C)Z-factor
15 1.0019
16 1.0020
17 1.0023
18 1.0025
19 1.0027
20 1.0029
21 1.0031
22 1.0033

19. 19
23 1.0035
24 1.0037
25 1.0039
WATERPURIFICATIONEQUIPMENTS
DistilledWater:
Normaltapwatercontainsions/mineralsandothercontaminantswhichmayinterferein
biochemicalestimations,therefore,itisdistilledtoremovethem.
Principleofdistillation-Whenwaterisheatedtoformsteam,theimpuritiesdonotevaporate
withwater.Thenthesteamiscondensedbackintoaseparatecontainerusingcoldwater.
Typesofdistillationequipments-
1.Metallic(steel)Still-wallmountedUnit(Manestytype)-forsingledistilledwater
2.Metallic(steel)Still(Tabletop,barnsteadtype)-forsingledistilledwater

20. 20
3.GlassUnit-fordoubleandtripledistilledwater
4.SolarStill-Forlaboratoriesinremoteareasandwithlimitedresources
Precautions-
●Thewatersupplytothedistillationstill(metallic/glass)shouldbecontinuousthroughout
thedistillationprocess.
●Intheglassdistillationunitthewaterlevelintheflaskshouldbelessthan¾fulland
careshouldbetakenthattheheatingelementshouldalwaysbecompletelydippedin
waterthroughoutthedistillationprocess.
●Thewatersupplyshouldbecontinuedforabout30minutesevenafterswitchingoffthe
unit.
Qualityofdistilledwater-
●pHofdistilledwaterisabout5.5thoughitisabout7.0infreshlydistilledwater.

21. 21
●SilvernitratetestforChloridecompounds:Take10mldistilledwaterinabeaker,add2
dropsofnitricacidfollowedby1ml1.7%Silvernitratesolution-thedistilledwater
shouldremainperfectlyclear.
Usesofdistilledwater-Freshlypreparedwaterispreferredtousefor:
●preparationofreagents/standardsolutions,
●finalrinsingofglasswaresbeforedrying.
Distilledwatershouldbestoredincappedglassorplasticcontainersbutnotfortoolong.
DeionizedWater:
Deionizedwaterisfreefromions/ionizedsaltbutnotnecessarilyfreefromorganiccompounds.
Themajordifferencebetweendeionizedwateranddistilledwateristhatdistilledwaterusually
haslessorganiccontaminantsanddeionizedwaterisnotfreeofbacteriaandvirus.
Principleofdeionizer-DeionizedwaterisproducedbyanIon-exchangeprocessusingIon
exchangeresinsinwhichthepositiveionsarereplacedbyhydrogenionsandnegativeionsare
replacedbyhydroxideions.Theionexchangeresinismadeoftinypolystyrenebeads(about
thesizeofagrainofsand)composedoforganicpolymerchainsthathavechargedfunctional
groupsbuiltintotheresinbead.Itiscalledamixedbedbecausetwotypesofbeadsaremixed
together.Thesurfaceofthecationbeadscontainhydrogenions(positivecharge).Thesurface
oftheanionbeadscontainhydroxideions(negativecharge).
Precautions-
●Followtheinstructionsgiveninthemanual
●Maintainthewaterflowrateaspertheinstructionmanual
●Checkalltheopticalindicatorsworking
●Checkallthetubingsareleakproof
Qualityofdeionizedwater-
●Onthebasisofelectricalconductivity(10-6S/m;siemens/meter)
●pHofdeionizedwateris6.6-7.0
●Usesilvernitratesolutiontocheckforchloridecompounds.
Usesofdeionizedwater-Itcanbeusedfor:
●rinsingglasswarebeforedrying,
●preparingalmostallthereagents/solutionsusedinmedicallaboratories(especiallyfor
thoseforionicdeterminations),includingstains.

22. 22
INHINGLISH
SOMELABORATORYEQUIPMENT
Biochemicaltestsकेलिएलैबमेंमुख्यतःइस्तेमालमेंआनेवालेequipmentsहैं:
BALANCE
HOTPLATE
MAGNETICSTIRRER
CENTRIFUGE
VORTEXMIXER
INCUBATOR
CONSTANTTEMPERATUREWATERBATH
COLORIMETER/SPECTROPHOTOMETER
BIOCHEMISTRYANALYZERS
MICROPIPETTES
WATERPURIFICATIONEQUIPMENTS
BALANCE:
Balanceकाइस्तेमाललैबमेंchemicalsतौलनेकेलिएहोताहैIलैबमेंकईप्रकारकीbalanceहोतीहैं-
1..Opentwo-panbalance-इसकाउपयोगकिसीsubstancesकीअधिकमात्राकोतौलनेमेंहोताहैIइसकी
sensitivityकमहोतीहै(0.5g)Isensitivityकामतलबहोताहैकिbalanceसेaccuratelyकमसेकम
कितनातौलसकतेहैंIआजकलelectronicopensinglepanbalanceभीमिलतीहैंI
Opentwopanbalance Electronicopensinglepanbalance
2.Analyticalbalance-इसbalanceकाइस्तेमालकिसीsubstanceकीकममात्राकोतौलनेमेंहोताहै
क्योंकिइसकीsensitivityअधिकहोतीहै(0.1-05mg,modelकेअनुसार)Iयेकईप्रकारकीहोतीहैं,जैसे-
Twopanbalancewithapointer,SinglepanelectricऔरSinglepanelectronic(digital)balance.
Twopan Singlepan Singlepanelectronic
Balance Electricbalance balance

23. 23
केमिकल्सकोतौलतेसमयलेनेवालीसावधानियाँ:
●जिसchemicalकोतौलनाहोउसकेलेबलकोतीनबारपढ़नाचाहिए-उसेshelfसेनिकालनेसेपहले,
तौलनेकेपहलेऔरतौलनेकेबादताकियहसुनिश्चितहोसकेकिजोकेमिकलतौलनाथावहीतौला
गयाI
●Balanceकोएकसुदृढ़औरकम्पनरहितplatformपररखनाचाहिएI
●यहसुनिश्चितकरनाआवश्यकहैकिbalanceproperlylevelledहैIयहbalanceकेfloorपरलगे
levellingbubbleसेcheckकरसकतेहैंऔरlevellingscrewsद्वाराआवश्यकतापड़नेपरlevel
adjustकरसकतेहैंI
●Twopanbalanceमेंbeamकोraiseकरकेदेखनाचाहिएकिpointer0markकेदोनोंतरफबराबर
divisionsmoveकरताहैयानहींIअगरनहींतोadjustकरेंI
●छोटेweights(mg)कोचिमटी(forceps)सेउठानाचाहिएI
●तौलतेसमयceilingfanकोयातोबंदकरदेनाचाहिएयाधीमाकरदेनाचाहिएऔरbananceकी
readingपढ़तेसमयउसकेदरवाज़ेबंदरखनेचाहिएI
●Useacleanpieceofpaper,watchglassoradisposableplasticweighingboattoweigh
chemicals.
Watchglass weighingboats
●तौलनासमाप्तकरनेकेबादbalanceकोएकसाफ़कपड़ेयाब्रशसेसाफकरदेनाचाहिएI
HOTPLATE
Hotplateकाइस्तेमालsolutionsकोbeakerयाअन्यglasswareमेंगर्मकरनेमेंहोताहैIHotplateका
topaluminum,ceramicmaterialsयाenamelकाबनाहोताहैIकुछHotplatesमेंएकmagneticstirrer
भीलगाहोताहैजिससेगर्महोरहेsolutionकोstirभीकियाजासकताहैIइसमेंएकON/OFFknobऔरएक
heatcontrolknobलगाहोताहैI
Hotplateकेइस्तेमालमेंसावधानियाँ-
●flammable(ज्वलनशील)याcombustible(दहनशील)materialकीउपस्थितिमेंHotplateका
इस्तेमालनहींकरनाचाहिएI
●Heatinglevel(high/medium/low)आवश्यकतानुसारsetकरनाचाहिएI
●Hotplateकोइस्तेमालकरतेसमयउसेunattendedनहींछोड़नाचाहिएI
●Hotplateसेगर्मglasswareकोहटानेकेलिएtongs(चिमटा)याthermalglovesकाइस्तेमाल
करनाचाहिएI
●Hotplateपरगर्मकरनेकेलिएइस्तेमालहोनेवालाglasswareअच्छीqualityकाऔरheat
resistantहोनाचाहिए,जैसे-borosilicateglass(sodalimeglassनहीं)I

24. 24
●Hotplateइस्तेमालकरनेकेबादउसेOFFकरकेउसकेसामनेएकप्लेकार्डरखदेनाचाहिएजिसपर
"HOT"लिखाहो,जिससेकिसीअन्यलैबकर्मीकेसाथकोईदुर्घटनानहोक्योंकिhotplateOFFकरने
केबादभीकुछसमयकेलिएगर्मरहतीहैI
MAGNETICSTIRRER
इसकाइस्तेमालकिसीsolutionकोएकstirbarकीमददसेmixकरनेमेंहोताहैIइसमेंheatingsystemभी
लगाहोसकताहैIइसमेंrotatingmagneticfield(जोएकrotatingmagnetयाstationary
electromagnetsकाएकsetहोताहै)काइस्तेमालहोताहैजिससेsolutionमेंपड़ाstirbarगोल-गोलतेजीसे
घूमताहै,जिससेsolutionकीmixingअच्छीतरहऔरजल्दीहोजातीहैI
Magneticstirrer
CENTRIFUGES
Centrifugemedicallaboratoryकाएकअतिआवश्यकequipmentहैजिसकाइस्तेमालcells,serum,
plasma,cellorganelles(nuclei,mitochondria,endoplasmicreticulumetc.)औरprecipitatesआदि
केseparationमेंहोताहैI
Principle-Centrifugemachineमेंविजातीयमिश्रण(heterogeneousmixture)केअव्यवों
(components)कोअपकेंद्रीबल(centrifugalforce)औरगुरुत्वाकर्षणबल(gravitationalforce)केउपयोग
सेउनकेआकार(shape),माप(size),घनत्व(density),mediumकीviscosityऔरrotorकीगति(speed
)केआधारपरपृथक्करणकियाजाताहैIइसलिएकिसीheterogeneousmixtureकेsolutionकोcentrifuge
करनेपरअधिकdensityवालेअव्यवनीचेबैठजातेहैंऔरकमdensityवालेऊपररहतेहैंI
Centrifugalforceऔरrpmकेबीचrelationshipकाformulaइसप्रकारहै:
rcf=1.118×10
-6
×r×(rpm)
2
जिसमें-
इसमें'rcf'-relativecentrifugalforce(relativetotheforceofEarth'sgravity)हैIRelative
centrifugalforce(सापेक्षकेन्द्रापसारकबल)centrifugeकरतेसमयsampleपरलगनेवालेबलकीमात्राहै
जोघूर्णनकीगति(rpm)औरsampleकेparticlesकीघूर्णनकेंद्रसेदूरीपरनिर्भरकरताहैI
'r'-radiusofrotorinmm,
'rpm'-revolutionsperminute
Unitofrcf-g
चूंकि'rcf'rotorकेradiusपरनिर्भरकरताहैइसलिएएकही'rcf'का'rpm'rotorकेradiusकेआधारपरअलग
अलगहोसकताहैI
Centrifugeमेंदोप्रकारकेrotorsहोतेहैं-swingoutrotorandfixedanglerotor

25. 25
Howtomeasuretheradiusoftherotorinaswingoutrotor
HowtomeasuretheradiusofaFixedanglerotor
TypesofCentrifuges:
1.Tabletop/Benchtopcentrifuge-इनमध्यमspeedकेcentrifugeकाइस्तेमालसामान्यतःमेडिकल
laboratoriesमेंplasma,serum,cells,urinedepositsऔरprecipitateआदिकेपृथक्करणमेंहोताहैI
इनकेrotorfixedangletypeयाswing-outtypeहोतेहैंI
TabletopCentrifuges
2.Microcentrifuge(Microfuge)-इनकाइस्तेमालभीमेडिकलlaboratoriesमेंहोताहैकिन्तुकमvolume
केsamples/liquidजैसे-nucleicacidयाproteinsकीhighspeedपरpelletingकरनेमेंI

26. 26
Microcentrifuge(Microfuge)
3.Multipurposehighspeedcentrifuges-येhighspeedवालेrefrigeratedfloorcentrifugeहोतेहैं
जिनकाइस्तेमालअधिकvolumeकेsolutionsकोcentrifugeकरनेमेंहोताहैIइनकेrotorfixedangle
typeयाswing-outtypeहोतेहैंऔरइनमेंविभिन्नsizeकीtubes,bottlesऔरmicrotiterplatesको
centrifugeकरनेहेतुअलग-अलगadoptersभीहोतेहैंI
4.Ultracentrifuge-येबहुतहीsophisticatedऔरadvancerefrigeratedcentrifugeहोतेहैंऔरये
अत्यधिकhighspeedपरकामकरतेहैं,इसीलिएजिनparticlesकाtraditionalcentrifugeमेंपृथक्करण
नहींहोपाताहैउन्हेंइसकेद्वारापृथककियाजाताहैIइनकाउपयोगMolecularbiologyऔरBiochemistry
researchlaboratoriesमेंकियाजाताहैIयेदोप्रकारकेहोतेहैं-AnalyticalऔरPreparativeजोइनके
applicationऔरpurposeपरआधारितहैIpreparativeultracentrifugerunमें,tubesकेcontentsकी
analysiscentrifugationसमाप्तहोनेकेबादकीजातीहैIजबकिanalyticalcentrifugeमेंanalysis
centrifugationकेदौरानहीकीजातीहैI
Centrifugeकेइस्तेमालकेसमयलीजानेवालीसावधानियाँ-
●CentrifugeकोrubberpadsयाकिसीmatपरएकflatlevelपररखनाचाहिएI
●Centrifugeoperateकरनेकेपूर्वउसका'InstructionManual'अवश्यपढ़लेनाचाहिएI
●गीलेहाथोंसेcentrifugeनहींछूनाचाहिएI
●Centrifugeमेंनतोबहुतछोटीऔरनबहुतलम्बीtesttubeलगानीचाहिएऔरtesttubeमेंतीन
चौथाईसेअधिकliquidनहींभरनाचाहिएI
●CentrifugerotorमेंलगीआमनेसामनेकीtubesकावजनबराबरहोनाचाहिएIएकtwopanopen
balanceमेंवजनकरकेदेखनाचाहिएऔरहल्कीtubeमेंliquidडालकर(अगरऐसाकरनासंभवहो)
याहल्कीtubeकेbucketमेंdistilledwaterडालकरbalanceकरनाचाहिएIअच्छीqualityकी
समानliquidvolumeवालीtesttubesकोtabletopcentrifugeमेंbalanceनहींकरनापड़ताहैI
बिनाbalanceकीtubesलगाकरcentrifugeचलानेपरvibrationsकेकारणtesttubesटूटसकती
हैंऔरcentrifugeभीख़राबहोसकताहैI

27. 27
●Centrifugeकेtesttubebucketsकेbottomमेंrubbercushionscheckकरलेनाचाहिएताकि
मशीनचलानेपरglasstubesटूटेनहींI
●Centrifugestartकरनेकेपहलेढक्कनअवश्यबंदकरदेंI
●Centrifugespeedकोधीरे-धीरेबढ़ानाचाहिएजबतकवांछितspeedनपहुंचजाएI
●Centrifugeकोबंदकरनेपरspeedकोधीरे-धीरेकमकरनाचाहिएIचलतेहुएrotorकोहाथसे
रोकनेकीकोशिशकभीनहींकरनीचाहिएI
●Centrifugeकाढक्कनखोलनेसेपहलेदेखलेनाचाहिएकिrotorपूरीतरहरुकगयाहोI
●Centrifugeकोरोज़औरcontaminateहोनेपरतुरंत70%अल्कोहल(मैटेलिकpartsकेलिए)या
1%bleach(plasticकेलिए)सेdisinfectकरनाचाहिएI
VORTEXMIXER(CYCLOMIXER)
इसमेंएकcupshapeकाrubberहोताहैजोएकshaftपरथोड़ाoff-centreलगाहोताहैIयहshaftएक
electricmotorकीसहायतासेघूमताहैजिससेrubberpiececircularmotionमेंघूमताहैIजबकोईliquid
सेभरीtesttubeकोइसrubberपरpressकरतेहैंयाrubberकेकिनारेसेtouchकरतेहैंतोtesttubeके
liquidमेंvibrationpassहोतेहैंजिससेliquidमेंभंवर(vortex)साबनजाताहैऔरliquidअच्छीतरहmixहो
जाताहैIइसीलिएइसकाइस्तेमालsolutions,testreagentsआदिकीmixingमेंहोताहैIकिन्तुइसके
इस्तेमालमेंकुछसावधानियाँरखनीचाहिए,जैसे-
●पहलीबारइस्तेमालकरनेसेपहले'InstructionManual'ठीकसेपढ़लेनाचाहिएI
●Properspeedचुनलेनाचाहिए,अन्यथाtesttubeकाliquidछलकसकताहैI
●Testtubeमेंliquidआधेसेअधिकनहींभरनाचाहिएजिससेpropermixingहोऔरliquidछलके
नहींI
INCUBATORS
Incubatorएकinsulatedmetallicenclosureहोताहैजोthermostaticallyregulatedहोताहैऔरइसका
इस्तेमालmicrobiologyमेंbacterialcultureकेलिएconstanttemperatureprovideकरनेहेतुहोताहैI
इसकेअंदरracksऔरshelvesमेंरखीPetridishes,flasksयाअन्यculturemediaकेऊपरगर्महवाका
संचारहोताहैIइसकाtemperatureरोज़recordकियाजानाचाहिए,साथहीहर15दिनमेंऔरकोईspillage
होनेपरतुरंतइसकीसफाईकरनीचाहिएI

28. 28
CONSTANTTEMPERATUREWATERBATH
यहस्टीलकाएकdoublewallवालाcontainerहोताहैजिसमेंपानीभराजाताहैIइसमेंएकheating
elementऔरtemperatureनियंत्रितकरनेहेतुएकthermostatलगाहोताहैIइसकाइस्तेमाल
samples/reagents/chemicalreactiontubesकोएकconstanttemperatureपरलम्बेसमयकेलिए
incubateकरनेमेंहोताहैIकुछwaterbathमेंपानीकोmixकरनेकेलिएएकstirrerलगाहोताहैजिससेपूरे
पानीकाtemperatureएकसारहेIकुछमेंएकshakerभीलगाहोताहैजिसकाइस्तेमालmicrobiological
testsमेंहोताहैI
●WaterbathमेंdistilledwaterहीभरनाचाहिएताकिwaterbathमेंdepositsनजमेंIAlgal
growthकोरोकनेकेलिएthymolcrystalभीपानीमेंडालदेतेहैंI
●इसकेपानीकोमहीनेमेंएकबारयाजबभीपानीगन्दालगेतोपानीनिकालदेतेहैंऔरwaterbathको
साफकरदूसरापानीभरदेतेहैंIपानीबदलतेसमयइसकाelectricplugsocketसेनिकालदेतेहैंI
●पानीबदलनेकेबादtemperaturesettingcheckकरलेनाचाहिएI
●Waterbathइस्तेमालकरनेकेबादswitchoffकरकेइसेढकदेनाचाहिएI

29. 29
COLORIMTER/SPECTROPHOTOMETER
PHOTOMETRY
Photometryमेंकिसीsolutionकेmoleculesद्वाराabsorbed(अवशोषित)lightकाअध्ययनकरतेहैंI
Whitelightएकमिश्रणहोताहैजिसमेंlightकेसभीcolorsहोतेहैंIजबयेwhitelightकिसीपदार्थपरपड़ती
हैतोlightकेकुछcolorsउसमेंabsorbहोजातेहैंऔरकुछcoloursउसपदार्थसेबाहरनिकलजातेहैंया
पदार्थद्वाराreflectहोजातेहैंIतोvisiblelightमेंपदार्थउसcolorकादिखताहैजोपदार्थद्वाराabsorbनहीं
होताIजैसे-एकredsolutionredइसलिएदिखताहैक्योंकियेsolutionvisiblelightकेredcolorकोछोड़
करबाकीसभीcolorsकोabsorbकरलेताहैI
लैबमेंइस्तेमालहोनेवालेविभिन्नphotometricinstrumentsइससिद्धांतपरआधारितहोतेहैं:
जबकोईmonochromaticlight(एकcolourयाएकwavelengthकीlight)किसीsolutionसेpassहोतीहै
तोtransmittedlight(पारेषितप्रकाश)कीतीव्रता(intensity)exponentially(चरघातांकीरूपसे)घटतीहैI
Transmittedlightकीतीव्रतामेंयहकमीpathlength(अवशोषकमाध्यमकीमोटाई)(Lambert'slaw)और
अवशोषकपदार्थकेconcentration(सांद्रता)(Beer'sLaw)केसीधाआनुपातिकहोतीहैIइनदोनोंनियमोंको
मिश्रितरूपसेBeer-LambertLawकहतेहैंI
चूंकिphotometricinstruments(जैसे-colorimeterऔरspectrophotometer)मेंpathlengthconstant
होतीहैकिन्तुsolutionकेविभिन्नconcentrationsकाइस्तेमालहोताहैइसलियेइसेकेवलBeer'sLaw
कहतेहैंIइसप्रकार,किसीरंगीनsolutionद्वाराअवशोषितlightउसsolutionकेअवशोषकपदार्थके
concentrationकेसीधाआनुपातिकहोतीहैIइसकाअर्थहैकिअवशोषकपदार्थकाconcentrationजितना
अधिकहोगाlightकाअवशोषण(absorbance)उतनाहीअधिकहोगाI
Theratioofintensityoftheemergentlight(I)andintensityofincidentlight(Io)iscalled
Transmittance(T):
T=I/Io
T×100=%T
-logTorlog(1/T)=extinction(E)oropticaldensity(OD)orabsorbance(A)
Or
absorbance(OD)=log(1/T)=log(100/%T)=2-log%T
Molarextinctioncoefficient-ofanysubstanceisitsextinction(absorbance,OD)ata
concentrationof1mol/latapathlengthof1cm.Forexample-Themolarextinctioncoefficient
ofNADHat340nmis6.22l/mmol/cm(6220l/mole/cm)or0.1mMsolutionofNADHwouldhave
absorbance(OD)of0.622with1cmpathlengthoflight.

30. 30
TransmittancevsAbsorbance
COLORIMETER
एकcolorimeterकेनिम्नभागहोतेहैं:
1.Lightsource(usually6-12Vtungstenlamp)
2.Aperture(Slit)andCondenserलेंस-जिससेlightकीएकसमानांतरbeamfilterपरपड़ेI
3.Filters(forselectiveabsorptionofunwantedwavelengths)-एकwheelमेंarrangedरहतेहैंIइन
फ़िल्टर्सद्वाराmaximumtransmissionनिम्नwavelengthsपरहोताहै:
Filter Wavelength
Deepviolet-420nm
Violet -430nm
Blue -470nm
Bluegreen-490nm
Green -520nm
Yellowgreen-550nm
Yellow -580nm
Orange -600nm
Red -680nm
Deepred-700nm

31. 31
[Violetfilterसेvioletlightpassहोगी;इसीतरहgreenfilterसेgreenऔरredfilterसेred]
किसीparticulartestकेलिएfilterकाcolourयाwavelengthचुननेकामानदंडयहहोताहैकिcolorimeter
द्वाराtransmittedlightकीwavelengthवहीहोनीचाहिएजोmeasureकियेजारहेपदार्थद्वाराअधिकतम
अवशोषित(absorb)कीजातीहोIउदाहरण-यदिकोईपदार्थ520nmकीlightअधिकतमअवशोषितकरताहै
तोउसकेmeasurementहेतुgreenfilter(520nmwavelength)इस्तेमालकरेंगेIजैसे-एकredsolution
इसलिएredदिखताहैक्योंकिlightकेredcolorकोछोड़करसभीcolorsकोabsorbकरलेताहैIतोयह
solutiongreencolorकीrangeमेंसबसेअधिकabsorbanceदेगाक्योंकिgreencolorredcolorकापूरक
(complementary)colorहै(colorwheelमेंgreenकाcomplementarycolorredहै)I
4.Cuvettechamber-transmittedlightएकcompartmentसेpassहोतीहैजिसमेंएकglassयाplastic
कीगोलयाचौकोरcuvetteमेंcolouredsolutionmeasurementकेलिएरखतेहैंI
RoundbottomcuvettesSquarecuvettes
5.Detector-यहएकphotosensitiveelementहोताहैजोlightकोelectricalsignalsमेंबदलताहैI
6.Galvanometer-electricalsignalकोमात्रात्मकरूपसेमापताहैI
DigitalColorimeter AnalogColorimeter

32. 32
Workingofacolorimeter-
Tungstenलैंपसेनिकलीwhitelightएकslitऔरएकlensसेpassहोतीहुईएकसमानांतरbeamकेरूपमें
चुनेहुएfilterपरपड़तीहैऔरfilterसेनिकलीवांछितwavelengthकीmonochromaticlightcuvetteके
solutionसेpassहोतीहैऔरउसsolutionकेconcentrationकेआधारपरabsorbहोनेकेबादयहlightएक
photocellपरपड़तीहैजिससेlightकीतीव्रताकेसीधेअनुपातमेंएकelectriccurrentपैदाहोतीहैIयह
electricsignalएकgalvanometerद्वाराabsorbance(OD)readingमेंपढ़ीजातीहैI
सर्वप्रथमblanksolution(plainreagent/distilledwater)कोcuvetteमेंडालकरcolorimeterकोzero
absorbance(100%T)परsetकरतेहैंIफिरउपयुक्तstandardreactiontubeकीabsorbance(OD)
readकरतेहैंऔरउसकेबादtest(unknown)reactiontubeकीabsorbance(opticaldensity,OD)read
करतेहैंI
Absorbanceofunknown×ConcentrationofStandard
Concentrationofanalyteinunknownsample=--------------------------------------------------------------
AbsorbanceofStandard
SPECTROPHOTOMETER
Spectrophotometerसेभीsampleद्वाराlightabsorbancemeasureकरकेanalyteकाsampleमें
concentrationमापतेहैंIColorimeterऔरspectrophotometerदोनोंहीBeer-LambertLawपरआधारित
होतेहैंऔरलगभगएकहीतरीकेसेकामकरतेहैंIकिन्तुदोनोंकेबीचकुछअंतरहोताहै:
●मुख्यअंतरयहहोताहैकिcolorimeterमेंविशेषcolourद्वाराlightकाabsorbanceदेखतेहैंऔर
spectrophotometerमेंcolouredयाcolourlesssolutionकीlightabsorbingयाlight
transmittingability(क्षमता)मापतेहैंI
●Colorimeterमेंlightspectrumकेvisiblerangeकीmonochromaticlightप्राप्तकरनेकेलिए
colouredfiltersकाइस्तेमालकरतेहैंजबकिspectrophotometerमेंprismयाdiffraction
gratingsद्वाराUV,visibleऔरinfraredregionकीwavelengthsकीएकwiderangeका
इस्तेमालहोसकताहैI
●Colorimeterमेंlightsourceकेलिएtungsten/LEDbulbहोताहैजबकिspectrophotometerमें
यहtungsten,xenonयाdeuteriumलैंपहोताहैI

33. 33
●Colorimeterकीcuvettecylindricalshapeकीऔरglassकीबनीहोतीहैजबकि
spectrophotometerमेंयहglass,silicaयाquartzकीrectangularऔरsquarecrosssection
वालीहोतीहैI
●Spectrophotometercolorimeterकीअपेक्षाअधिकpreciseऔरaccurateहोताहैI
●Spectrophotometerदोप्रकारकेहोतेहैं-singlebeamऔरdoublebeamspectrophotometer
Singlebeamspectrophotometer Doublebeamspectrophotometer
DigitalSpectrophotometer AnalogSpectrophotometer
BIOCHEMISTRYANALYZERS-इनanalyzersकाbasicprincipleवहीहोताहैजोcolorimeterया
spectrophotometerकाहोताहैIहालांकि,इनanalyzersकाprecisionअधिकहोताहैIdiagnostic
laboratoriesमेंdegreeofautomationकेआधारपरदोप्रकारकेanalyzersप्रयोगहोतेहैं:
1.Semiautomaticanalyzer-इसमेंtestतोmanuallyलगानापड़ताहैऔरfinallytestsolutionकी
readingलेनेकेलिएsolutionकोanalyzerमेंaspirateकरादेतेहैंऔरanalyzerइसकेabsorbanceको
standardकेabsorbanceएवंconcentrationसेतुलनाकरकेसीधेtestsolutionकाconcentrationबता
देताहैIइसतरहहमेंकोईcalculationनहींकरनापड़ताIइसमेंcuvette/flowcellकेtemperaturecontrol,
endpoint,fixedtimekineticandkineticreactiontestsकीprogrammingकाप्रावधानहोताहैतथा
inbuiltqualitycontrolchartsकाभीप्रावधानहोताहैजिससेहमdifferenttestsकेqualitycontrol(QC)
परनियंत्रणरखसकतेहैंIइनanalyzersकाइस्तेमालछोटीऔरमध्यमश्रेणीकीलैबमेंहोताहैजहाँएक
सीमितसंख्यामेंsamplesऔरtestsहोतेहैंI
2.Fullyautomatedanalyzer-इनanalyzersद्वाराकमसेकममानवसहायताकेकमसमयमेंबड़ीसंख्या
मेंsamplesकेtestsकियेजासकतेहैंIsemiautoanalyzerकेविपरीतइनमेंकेवलsamples(blood,
serum,plasma,urine,cerebrospinalfluidआदि)कोreagentsकेसाथmachineमेंarrangeकरदेतेहैं
औरmachineकोtestsकेअनुसारprogramकरदेतेहैंImachineसारेtestsकरकेfinalresultsका
printoutदेदेतीहैIइनकाइस्तेमालबड़ीlaboratoriesमेंहोताहैजहाँबड़ीसंख्यामेंsamplesऔरtestsहोते
हैंI

34. 34
Fullyautomatedanalyzersमुख्यतःदोप्रकारकेहोतेहैं:
1.Batchanalyzer-इसमेंinstrumentsystemsamplesकेप्रत्येकgroupमेंएकtestकोक्रमिकरूपसे
(sequentially)analyzeकरताहैI
2.Randomaccessanalyzer-इसsystemमेंकोईभीsampleकोक्रमसेबाहर(outofsequence)
बेतरतीबअभिगम(randomlyaccess)करकेकोईभीtestकरसकतेहैंIइसप्रकारएकहीsampleमेंकई
testsएकहीबारमेंprogramsetकरकेकरसकतेहैंI
इसकेअतिरिक्तanalyzerकेreactionprincipleकेआधारपरदोप्रकारकेbiochemicalanalyzersहोतेहैं:
1.Wetanalyzer-इसमेंreagentsकोliquidformमेंइस्तेमालकरतेहैंऔरbloodsampleकोइस
reagentकेसाथएकreactionvessel(testtube)मेंreactionकरातेहैंऔरफिरreactionmixture
काएकcolorimeter/spectrophotometerपरabsorbanceकीमददसेanalyteकाconcentration
calculateकरतेहैंI
2.Dryanalyzer-इसमेंliquidreagentकेस्थानपरdryreagentकाइस्तेमालहोताहैजिसेएक
supportingmediumकीlayerपरcoatकरदियाजाताहै(dryreagentsसेसंसेचितstrips)Iजब
bloodsampleइसपरडालतेहैंतोsampleकाanalytebloodकीmoistureकेकारणdryreagent
सेreactकरकेproductबनाताहैजिसेreflectancespectrophotometryद्वाराreadकरकेanalyte
काconcentrationमापतेहैंI
MICROPIPETTES
Micropipettesareakindofpipettesusedinlaboratoryfortransferofsmallvolumesofliquid(as
lowas0.2microliter).
Partsofamicropipette Micropipettesonmicropipettestand

35. 35
Micropipettesकेविभिन्नप्रकार:
BasedonthePrinciple/DisplacementMethod-
●Airdisplacementmicropipette-येaqueousऔरnonviscousliquidsकेलिएउपयुक्तहोतेहैंI
इनmicropipettesमेंअदंरएकpistonहोताहैऔरpistonएवंsampleकेबीचएकaircushion
होताहैजोliquidsकोdispenseकरनेमेंमददकरताहैI
●Positivedisplacementmicropipette-इनpipetteमेंpistonpipetteshaftकेअदंरहोनेकेबजाए
pipettetipकेअदंरहोताहैIpistonऔरliquidकेबीचकोईaircushionनहींहोताIइसमें
disposabletipएकmicrosyringeकेरुपमेंहोतीहैजोएकcapillaryऔरएकpiston(movable
innerpart)सेबनीहोतीहैऔरयहliquidकोसीधेdisplaceकरतीहैIयेmicropipettesviscous
औरvolatileliquidsकेलिएभीउपयुक्तहोतेहैंI
BasedontheNumberofChannels-
●Singlechannel-इसमेंकेवलएकshaftहोताहैजोएकबारमेंकेवलएकliquidकोट्रांसफरकर
सकताहैIइसप्रकारइसमेंliquidकोaspirateयाdispenseकरनेहेतुकेवलएकchannelहोताहैIये
विभिन्नcapacities(from0.2से10,000μl)मेंउपलधहोतेहैंऔरइनकीaccuracyexcellentहोती
हैI
●Multichannel-इनmicropipettesमेंकईshaftsहोतेहैंजोकईliquidsएकसाथट्रांसफरकरसकते
हैंIइसतरहइसमेंliquidकोaspirateऔरdispenseकरनेकेलिएकईchannels(4-18)होतेहैंIये
pipettesउनtestsकेलिएसर्वोत्तमहोतेहैंजिनमेएकाधिक(multiple)wellsमेंliquidsडालनेहोतेहैं,
जैसे-ELISA,DNAamplificationtestsआदिIइनmultichannelmicropipettesकीcapacity
0.2से1200μlतकहोसकतीहैI

36. 36
BasedonthePipettingMechanism-.
●Mechanical/Manual-इनmicropipettesमेंकार्मिक(personnel)कोaspirationऔर
dispensationहेतुplungerकोpressure
केसाथदबानापड़ताहैऔरइसकेलिएpiston
मेंएकस्प्रिंगहोतीहैI
●Electronic-इसमेंmechanicalplungerकेस्थानपरएकelectronicbuttonहोताहैIइसे
automatedpipetteभीकहतेहैंक्योंकिइसमेंelectronicbuttonकेकारणmanuallabourबहुत
कमहोजाताहैIइसमेंpipettingकीअवश्यक्तानुसारcustomizableprogramsकीसुविधाभीहोती
हैI
BasedontheVolume-
●Fixedvolumemicropipette-येpipettesप्रत्येकबारsampleकेएकfixedऔरequalvolume
काट्रांसफरकरतेहैंIयेसीमितबजटकीलबैमेंviscousliquidsकेलिएउपयुक्तहोतेहैंIइसमें
pipettingकरतेसमयगल्तीसेvolumeमेंपरिवर्तनकाखतराकमहोताहैI
1000μlmicropipette10μlmicropipette
●Adjustablevolumemicropipette-इसमेंआवश्यक्तानुसारvolumeपरिवर्तनहेतुvolume
adjustableknobहोताहैंIयेविभिन्नvolumerangeमेंउपलब्धहोतेहैंI

37. 37
0.5-10μlmicropipette10-100μlmicropipette
Differenttypesofpipettingprocedures-
●Forwardpipetting-यहसर्वाधिकउपयोगहोनेवालीतकनीकहैIForwardpipettingमेंliquidका
एकexactlysetकियाvolumetipमेंaspirate(चषूण)कियाजाताहैऔरफिरइसेदूसरेcontainer
मेंdeliverकियाजाताहैIयहतकनीकdilutedaqueoussolutions,bufers,dilutedacidsऔर
basesकीpipettingहेतुउचितहोतीहैI
Pipettingsteps-
1.सर्वप्रथमpipetteसेएकउचितdisposabletipकोठीकसेattachकरतेहैंI
2.PlungerbuttonकोअंगूठेसेपहलेstopतकpressकरतेहैंI
3.Tipकोliquidमेंलगभग2-3mmनीचेतकdipकरतेहैं(यहनिर्भरकरताहैकिकितना
volumeliquidलेनाहै)औरफिरbuttonकोधीरेसेछोड़तेहैंI
4.Buttonछोड़तेहीliquidtipमेंaspirateहोनेलगताहै(ध्यानरखतेहैंकिकोईairbubble
नहींआये)IBubbleकीसम्भावनातबहोतीहैजबplungerbuttonकोएकदमसेछोड़ाजाए
याtipठीकसेpipetteसेattachनहींहोती,याbuttonपूराछोड़नेसेपूर्वहीtipliquidकी
सतहसेबाहरआजाएI
5.जबbuttonपूरीतरहreleasepositionपरआजाएतोअंगूठाहटालेतेहैंI
6.Liquidtipमेंaspirateहोनेकेबादकुछसेकंडकेलिएरूकतेहैंताकिliquidपूरीतरह
aspirateहोजाए,खासतौरपरअधिकvolumeकेलिए,जैसे-500–5000μl,औरअबtipको
liquidसेधीरेसेबाहरनकालतेहैंI
7.यदिआवश्यकहोतोtipकेबाहरीसतहसेलगीliquiddropletsकोएकमुलायमtissue
paperयाclothसेसावधानीसेपोछदेतेहैंIलेकिनtipकेछेदकोpaperयाclothसेनहींछुएं
वरनाpaper/clothमेंtipकेअंदरकाकुछliquidabsorbहोसकताहैI
8.दूसरेvessel(जैसे-testtube)मेंpipetteमेंलिएहुएliquidकोdeliverकरतेसमयtipको
testtubeकीअंदरूनीwallसे10-45°केangleसेtouchकरनाचाहिएऔरयदिtesttube
मेंपहलेसेहीकोईliquidहोतोउसकेस्तरसेथोड़ाऊपरtipकाछोरहोनाचाहएI
9.अबplungerbuttonकोपहलेfirststopतकदबातेहैं,फिरलगभग1सेकंडरूककरbutton
कोजल्दीसेsecondstopतकदबातेहैंILiquiddeliverकरनेपरtipमेंकोईdropletsनहीं
रहनीचाहिएऔरtesttubeकीwallsपरliquidकाकोईछितरावनहींहोनाचाहिएI
10.अबbuttonकोsecondstopपरदबायेहुएहीtipकोtesttubeकीwallसेtouchकरातेहुए
बाहरनिकालतेहैंऔरतबbuttonकोreleaseकरतेहैंI

38. 38
11.Accuracyबढ़ानेकेलिएयहअच्छारहताहैकिpipettetipकोliquidकाfinalvolume
pipetteकरनेसेपूर्वउसliquidसे2-3बारpre-wet(pre-rinse)करलियाजाए,खासतौरपर
बहुतकमvolumeकीpipettingकेलिएI
●Reversepipetting-इसतकनीककाउपयोगबहुतviscousयाvolatileliquids,biologicfluids,
foamingsolutionsऔरबहुतकमvolumeकेमापनमेंउपयुक्तरहताहैI
Pipettingsteps-
1.Plungerbuttonकोसर्वप्रथमsecondstopतकदबातेहैं,
2.Pipettetipकोliquidकेस्तरसे2–5mmनीचेdipकरतेहैंऔरbuttonकोधीरेसेrelease
करतेहैंजिससेliquidtipमेंaspirateहोजाएI
3.अबtipकोliquidसेधीरेसेनिकालतेहैं,यदिआवश्यकहोतोtipकीबाहरीsurfaceपरलगे
dropletsकोtissuepaperसेसाफ़करतेहैंI
4.दूसरेvesselयाtesttubeमेंliquiddeliverकरनेकेलिएtipकोforwardतकनीककीतरह
हीtesttubeकीinnerwallकोtouchकरातेहुएplungerbuttonकोकेवलfirststopतक
दबाकरliquiddeliverकरतेहैंI
5.अबbuttonकोfirststoppositionपरदबायेहुएहीtipकोtesttubeसेबाहरनकालतेहैंI
6.DeliverकरनेकेबादTipमेंबचेहुएliquidकोयातोफेकसकतेहैंयाफिरवापसliquid
containerमेंडालसकतेहैंI
1.
●Repetitivepipetting-इसतकनीककाउपयोगतबकरतेहैंजबएकहीliquidकाएकहीvolumeकई
testtubesयाmicrotiterplateकेकईwellsमेंdispenseकरनाहोताहैIयहreversepipettingके
समानहीहोताहैकिन्तुइसमेंreversepipettingकेsteps2से4कोकईबारदोहरातेहI

39. 39
●PipettingofHeterogeneousSamples-यहतकनीकbloodजैसेheterogeneoussamplesके
लिएउपयुक्तहोतीहैक्योंकिऐसेsamplesमेंpipettingकेपूर्वtipकीpre-rinsingआसाननहींहोती
हैI
Pipettingsteps-
1.Plungerbuttonकोfirststopतकदबाकरtipकोliquidsampleकेस्तरके2-5mmनीचे
तकdipकरतेहैं,
2.फिरधीरेसेbuttonकोreleaseकरतेहैंजिससेsampletipमेंaspirateहोजाताहैI
3.Tipकोधीरेसेबाहरनिकालतेहैंऔरtipकीबाहरीसतहपरलगीdropletsकोसावधानीसे
tissuepaperसेपोछदेतेहैंI
4.अबtipकोउसsolutionमेंथोड़ाdipकरतेहैंजिसमेंtipकेheterogeneousliquidकोडालना
होताहैI
5.Buttonकोfirststopतकदबातेहैंऔरधीरेसेbuttonकोवापसreleaseकरतेहैंजिससेtip
मेंsolutionaspirateहोजाताहैIअबइसstepकोबिनाtipकोsolutionसेबाहरनिकालेतब
तकदोहरातेहैंजबतकtipकीinnerwallपरलगाheterogeneousliquidपूरीतरहसाफ़
नहींहोजाताI
6.अबtesttubeकीinnerwallकोtouchकरतेहुएtipकोsolutionकेस्तरसेथोड़ाऊपरकरके
buttonकोsecondstopतकदबातेहैंI
7.SecondstopतकbuttonकोदबायेहुएहीtipकोtesttubeसेबाहरनकाललेतेहैंI
Sometipsforusingmicropipettes-
●Pipettingकेपूर्वliquidकोroomtemperature(RT)परलेआनाचाहिएक्योंकिठंडाliquidसही
volumeसेअधिकऔरगर्मliquidसहीvolumeसेकमdeliverहोसकताहैI
●PipetteमेंसहीsizeकीtipलगानाचाहिएI
●AdjustablevolumemicropipetteमेंवांछितvolumeठीकसेsetकरलेनाचाहएI
●Tipकोliquidसेकमसेकमदोबारpre-rinseकरनेसेpipettingकीaccuracyबढ़जातीहैI
●Liquidaspirateकरतेसमयtipकोliquidमेंvertically(at90°)औरliquidकेस्तरसेलगभग2-3
mm(अधिकvolumeaspirateकरनेकेलएथोड़ाअधिकनीचे)नीचेतकdipकरनाचाहिएI
●Liquidaspirateकरतेसमयplungerbuttonकोधीरेसेreleaseकरनाचाहिए(ताकिairbubbles
नहींआएं)औरकुछsecondsकेलिएtipकोdipकियेरखनाचाहिए(ताकिपूराvolumeaspirateहो
जाए)IउसकेबादtipकोबाहरनिकलनाचाहिएI
●Pipetteकीusedtipकोtipejectorद्वाराहीनिकालनाचाहिएI
Calibrationofmicropipette-Micropipettesकोसमय-समयपर(प्रत्येकतीनमहीनेपर)calibrateकरते
रहनाचाहिएजिससेउनकीaccuracyऔरprecisionकोबराबरcheckकियाजासकेIयहcalibrationएक
sensitiveelectronicbalanceपरवैसेहीकियाजाताहैजिसप्रकारएकglasspipetteकाcalibrationकरते
हैंI

40. 40
StepsInvolvedinPipetteCalibration-
●एकbeakerमेंdistilledwaterलेतेहैंऔरwaterकाtemperaturenoteकरलेतेहैंIसाथहीजिस
pipetteकाcalibrationकरनाहोऔरउचितtipsएकत्रकरलेतेहैंI
●एकsensitiveelectronicbalanceकेpanपरएकweighingboatरखतेहैंऔरbalanceकेdoors
बंदकरकेbalanceकोzeroपरsetकरतेहैंI
●Micropipetteकाvolumesetकरकेउचितtipलगाकरtipकोbeakerकेwaterसेतीनबार
aspirateऔरdispenseकरकेpre-rinseकरतेहैंऔरफिरtipकाwaterपूरीतरहनिकालदेतेहैंI
●अबबिनाकोईairbubbleकेbeakerसेtipमेंwateraspirateकरतेहैंऔरधीरेसेweighingboat
मेंdispenseकरतेहैंIफिरbalanceकेdisplayपरआयेweightकोnoteकरलेतेहैंIइसप्रक्रियाको
दसबारदोहरातेहैंऔरदसreadingsnoteकरलेतेहैंI
●अबdispenseकिएwaterकेvolumeकोइसequationद्वाराcalculateकरतेहैं:V=WxZ
जिसमेंWwaterकाweightहै,Zएकfactor(Z-factor)*औरVdispenseकिएगएwaterका
calculatedvolumeहैIफिरइसप्रकारप्राप्तदसcalculatedvolumesकीmeanvalueऔर
standarddeviation(SD)calculateकरतेहैंImeanऔरSDvalueसेहम%coefficientof
variation(%CV)calculateकरतेहैं[%CV=(SD÷Mean)×100]औरयदिCV1%सेकमहैतो
इसकाअर्थहैकिpipetteकाprecisionअच्छाहैI
●अतंमेंpipetteकीaccuracyनिकालतेहैं,जिसकाformulaहै:A=100xVavg/V0,जिसमेंA
pipetteकीaccuracy,VavgcalculatedmeanvolumeऔरV0pipetteकादियाहुआvolumeहैI
यदिaccuracyvalue99-101%होतोpipetteकोaccurateऔरcalibratedमानतेहैंI
*[Zfactor]-waterकेvolumeमापनकीaccuracyपरिवेशीतापमान(ambienttemperature),
atmosphericpressureऔरसापेक्षआर्द्रता(relativehumidity)परनिर्भरकरतीहैIइनfactorsकोमिलाकर
ZfactorनिकालतेहैंजिसकाउपयोगwaterकेvolumecalculationमेंकरतेहैंI
Temperature(°C)Z-factor
15 1.0019
16 1.0020
17 1.0023
18 1.0025
19 1.0027
20 1.0029
21 1.0031
22 1.0033
23 1.0035
24 1.0037
25 1.0039
WATERPURIFICATIONEQUIPMENTS
आसुतजल(DistilledWater)-
साधारणtapwaterमेंकईअशुद्धियाँहोसकतीहैं,जैसे-ions/mineralsऔरअन्यcontaminants,जो
biochemicalestimationsमेंहस्तक्षेप(interfere)करसकतेहैं,इसलियेइन्हेंदूरकरनेकेलिएwaterको
distillकियाजाताहैI
आसवनकासिद्धांत(Principleofdistillation)-जबपानीकोगर्मकरतेहैंतोभापबनतीहैलेकिनपानीमें
उपस्थितअशुद्धियाँevaporateनहींहोतीहैंIजबभापकोठन्डेपानीद्वाराठंडाकरतेहैंतोभापcondense
होकरपुनःपानीबनजातीहैजोअशुद्धिरहितहोताहैIइसेहीdistilledwaterकहतेहैंI

41. 41
Typesofdistillationequipments-
1.Metallic(steel)Still-यहदीवारपरfixहोतीहै(Manestytype)-इसकाइस्तेमालsingledistilled
waterबनानेमेंहोताहैI
2.Metallic(steel)Still(Tabletop,barnsteadtype)-इसकाइस्तेमालभीsingledistilledwater
बनानेमेंहोताहैI
3.GlassUnit-इसकाइस्तेमालdoubleऔरtripledistilledwaterबनानेमेंहोताहैI

42. 42
4.SolarStill-इसकाइस्तेमालदूरदराज़केक्षेत्रोंमेंऔरजहाँसंसाधनसीमितहो,वहाँहोताहैI
सावधानियाँ(Precautions)-
●Distillationprocessकेदौरानdistillationstill(metallic/glass)मेंwatersupplyलगातारबनी
रहनीचाहिएI
●Glassdistillationunitकेflaskमेंwaterlevel¾सेथोड़ाकमरहनाचाहिएऔरयहध्यानरखना
चाहिएकिheatingelementपूरेdistillationप्रक्रियाकेदौरानपानीमेंपूराडूबाहोनाचाहिएI
●Distillationunitकोswitchoffकरनेकेबादभीलगभग30मिनटतकwatersupplyबनीरहनी
चाहिएI
Qualityofdistilledwater-
●distilledwaterकाpHलगभग5.5होताहैहालांकिfreshlydistilledwaterकाpHलगभग7.0होता
हैI
●SilvernitratetestforChloridecompounds:एकbeakerमें10mldistilledwaterलेकरउसमें
nitricacidकी2dropsऔरफिर1.7%Silvernitratesolutionका1mlडालतेहैं-सहीdistilled
waterकोperfectlyclearरहनाचाहिएI
Usesofdistilledwater-
●reagents/standardsolutionsकोबनानेमेंfreshlydistilledwaterइस्तेमालकरतेहैंI
●धुलेGlasswaresकोसुखानेसेपूर्वdistilledwaterसेrinseकरतेहैंI
Distilledwaterकोcappedglassयाplasticcontainersमेंरखतेहैं,किन्तुबहुतलम्बेसमयतकनहींI
विआयनितयाअनायनितजल(DeionizedWater)-
DeionizedwaterकामतलबवोwaterजिसमेंसेionsऔरionizedsaltsremoveकरदिएगयेहोंI
Deionizedwater,ions/ionizedsaltसेमुक्तहोताहैकिन्तुयहआवश्यकनहींहैकिउसमेंसेorganic
compoundsभीremoveहोगएहोंIdeionizedwaterऔरdistilledwaterमेंमुख्यअंतरयहहैकि
distilledwaterमेंorganiccontaminantsकमहोतेहैंकिन्तुionsकाcontaminationहोसकताहैऔर
deionizedwaterमेंionsremoveहोजातेहैंकिन्तुइसमेंbacteriaऔरवायरसहोसकतेहैंIDeionized
waterबनानेहेतुउपयोगकीजानेवालीmachineकोdeionizerकहतेहैंI
Principleofdeionizer-DeionizedwaterकोएकIon-exchangeprocess,जिसमेंIonexchange
resinsकाइस्तेमालहोताहै,द्वाराबनायाजाताहैIइसप्रक्रियामेंpositiveionsकोhydrogenionsद्वारा
औरnegativeionsकोhydroxideionsद्वाराप्रतिस्थापित(replace)कियाजाताहैIIonexchangeresin
tinypolystyrenebeads(जोबालूकेकणोंकेsizeकेहोतेहैं)सेबनाहोताहैजिसमेंresinbeadsपर

43. 43
chargedfunctionalgroupsयुक्तorganicpolymerchainsहोतीहैंIइसेmixedbedकहतेहैंक्योंकिइसमें
दोप्रकारकीbeadsकामिश्रणहोताहैIcationbeadsकीसतहपरhydrogenions(positivecharge)और
anionbeadsकीसतहपरhydroxideions(negativecharge)होतेहैंIइसतरहपानीकेउपस्थितcations
औरanionsकाhydrogenऔरhydroxideionsद्वाराexchangeहोनेसेdeionizerसेनिकलापानी
ion-मुक्तहोताहैI
सावधानियाँ-
●'InstructionManual'मेंदिएनिर्देशोंकापालनकरेंI
●ManualकेअनुसारwaterflowmaintainरखेंI
●इसकाध्यानरखेंकिसभीopticalindicatorsकामकररहेहोंI
●सभीtubingsleakproofहोनीचाहिएI
deionizedwaterकीqualitycheckकरना-
●electricalconductivity10-6S/m(siemens/meter)होनीचाहिएI
●deionizedwaterकाpH6.6-7.0होनाचाहिएI
●silvernitratesolutionद्वाराchloridecompoundsकीउपस्थितिकीजाँच(जैसेdistilledwaterमें
करतेहैं)करनीचाहिएI
Usesofdeionizedwater-deionizedwaterकाइस्तेमालहोताहै:
●glasswaresकोसुखानेसेपूर्वउन्हेंrinseकरनेमेंI
●Medicalलैबमेंसभीप्रकारकेreagents/solutions(विशेषतौरपरजिनकाइस्तेमालions
determinationमेंहोताहै)औरstainsबनानेमेंI
Somepracticequestions
Q.1.Fillintheblanks-
1.Extremelyhighspeedवालेcentrifugeको______centrifugeकहतेहैंI
2.एकcentrifugeको_____हाथसेकभीनहींछूनाचाहिएI
3.एकcolorimeter______________lawपरआधारतहोताहैI
4._____________lawकेअनुसारजबकोईmonochromaticlightकिसीsolutionसेpassहोतीहैतो
transmittedlight(पारेषितप्रकाश)कीतीव्रता(intensity)exponentially(चरघातीरुपसे)घटतीहैऔर

44. 44
Transmittedlightकीतीव्रतामेंयहकमीpathlength(अवशोषकमायमकीमोटाई)केसीधाआनुपातिक
होतीहैI
5.____________lawकेअनुसारजबकोईmonochromaticlightकसीsolutionसेpassहोतीहैतो
transmittedlight(पारेषितप्रकाश)कीतीव्रता(intensity)exponentially(चरघातीरूपसे)घटतीहैऔर
Transmittedlightकीतीव्रतामेंयहकमीअवशोषकपदार्थकेconcentration(सांन्द्रता)केसीधाआनुपातिक
होतीहैI
6.Theratioofintensityofthetransmittedlight(I)andintensityofincidentlight(Io)iscalled
___________.
7.-logTorlog(1/T)=__________
8.Thewavelengthofvisiblelightrangesfrom______nmto______nm.
9.Thecriteriaforchoosingthecolororwavelengthofthefilterforaparticulartestisthatthe
wavelengthoflightthatistransmittedbythecolorimeterhastobethesameasthat
maximally________bythesubstancebeingmeasured.
10.एकcolorimeterमेंmonochromaticlightप्राप्तकरनेकेलिएcoloredfiltersकाउपयोगकिया
जाताहैजबकिspectrophotometerमेंइसकेलिए_________या_____________काउपयोग
होताहैI
11.Thespectrophotometerismore______andaccurateascomparedtoacolorimeter.
12.Thetwotypesoffullyautomaticbiochemistryanalyzersare__________analyzersand
_______analyzers.
13.Colorimeterमेंlightsourceकेलिए_____/____bulbहोताहैजबकिspectrophotometerमेंयह
_______,______या______लैंपहोताहैI
14.rcf=1.118×10-6×r×______
Ans.1-ultra,2-गीले,3-Beer-Lambert,4-Lambert's,5-Beer's,6-transmittance,7-absorbance
(OD),8-400,700,9-absorbed,10-gratings,prism,11-precise,12-randomaccess,batch,13-
tungsten/LED,tungsten,xenon,deuterium,14-(rpm)
2
Q.2.Matchthefollowing:
(i)Desiccator (a)hearingsolutions
(ii)vortex/cyclomixer(b)heatingchemicalreactiontubesataconstanttemperature
(iii)waterbath (c)bacterialculture
(iv)incubator (d)stirringthesolution
(v)magneticstirrer(e)mixingliquidinatesttube
(vi)hotplate (f)Preservingmoisture-sensitiveitems/hygroscopicchemicals
Ans.
(i)-(f),(ii)-(e),(iii)-(b),(iv)-(c),(v)-(d),(vi)-(a)
Q.3.Identifytrue/falsestatements:
A.Tapwatercanbeusedtomakethereagents-
B.Thedoorsoftheanalyticalbalanceshouldbeclosedwhilecheckingthereadingonthe
balance-
C.Forcepsshouldbeusedtopickupthesmallweightsduringweighing-
D.Hotplatecanbeusedinpresenceofflammableandcombustiblematerial-
E.Itisnotnecessarytobalancethetubesbeforecentrifugingthem-
F.Thewaterbathshouldbefilledwithtapwater-
G.Deionizedwatercontainsions-
Ans.A-false,B-true,C-true,D-false,E-false,F-false,G-false

45. 45
REFERENCES
1.PracticalClinicalBiochemistry.VarleyH,GowenlockAH,BellM.Fifthedition,1991.
2.Manualofbasictechniquesforahealthlaboratory,WorldHealthOrganization,Geneva,
Secondedition,2003.
3.Pipettinghttps://www.wikilectures.eu/index.php?title=Pipetting&oldid=20889
Disclaimer:ThepicturesgiveninthetexthavebeendownloadedfromGoogleimagesandIam
thankfultothepersonswhohaveuploadedthesepictures.
Dr.P.K.Nigam
Ph.D.(RetiredBiochemist)