EXPERIMENT 1
USE OF MICROPIPETTOR AND SPECTROPHOTOMETER
Y. A. Azucena, R. M. Baladad, S. C. Baquiran, T. J. Bautista 4BIO5 Departent !" Bi!l!#ical Sciences, C!lle#e !" Science, $ni%ersit& !" Sant! T!as, 'spana A%enue, A%enue, Manila
Keywords:
accurac&, precisi!n, icr!pipett!r, spectr!p(!t!eter
Summary
In the activity, the micropipettor and the spectrophotometer were used to be able to test the accura accuracy cy and preci precisio sion n of studen students ts in using using such such devices. Bromphenol blue and distilled water were used in the experiment as testing agents. In the rst proce procedur dure e a speci specied ed amoun amountt of distil distilled led water water combin combined ed with with dier dierent ent amount amounts s of brom bromphe phenol nol blue were sub!ected for test using a spec pectro trophoto hotom meter eter set at "#$ "#$nm basi asing on absorb absorbanc ance. e. In the second second proced procedur ure e speci specied ed amount amounts s of distil distilled led water were sub ected ected for test
Introducton
T(e icr!pipette !r icr!pipett!r is a precisi!n pup "itted )it( a disp!sa*le tip, desi#ned t! accuratel& trans"er %!lues in t(e icr!liter ran#e. Micr!pipett!rs )!r+ *& air displaceent. T(e !perat!r depresses a plun#er t(at !%es an internal pist!n t! !ne !" t)! di""erent p!siti!ns. T(e "irst st!p is used used t! "ill "ill t(e t(e icr!p icr!pipe ipette tte tip, and and t(e t(e sec!n c!nd st! st!p is used sed t! disp ispens ense t(e t(e c!ntents !" t(e tip. T(e t(ree t(ree t&pe t&pess !" icr!p icr!pipe ipett! tt!rs rs are are dist distin in#u #uis is(e (edd acc! acc!rd rdin in## t! t(e t(e %!lu %!lue e ran#es t(at t(e& eac( can trans"er, eac( rec!#niza*le *& a nu*er at t(e t!p !" t(e plun# plun#er er,, and and *& c!l!r c!l!r.. T(e T(e lar#e lar#e %!lu %!luee icr icr!p !pip ipet ett! t!rr is *lue *lue and and can can assi assist st in !%in# liquids ----- /0, t(e idran#e icr!pipett!r icr!pipett!r is &ell!) and (as a capacit& !" --- /0, and t(e sall %!lue icr!pipett!r is red and capa*le !" !%in# -.5 -.5- /0 !" liqu liquid id.. Di"" Di""er eren entt size sizess !" pipettes tips are !"ten li+e)ise c!l!rc!ded
"!r ease !" rec!#niti!n and are used "!r di"" di""er eren entt size sizess !" icr icr!p !pip ipet ett! t!rs rs.. 1it( 1it( respe respect ct t! %!lue %!lue trans trans"e "erre rred, d, )(ite )(ite tips tips t&picall& trans"er *et)een -.5- 20, &ell!) tips are "!r --- /0, and *lue tips "r! ----- 20. In decidin# )(ic( icr!pipette t! use "!r %!lues t(at can *e easured *& t)! t&pes, it is practical t(at !ne use t(e icr!pipett!r )it( saller %!lue capacit& t! attain accurac& in easureent. A spectr!p(!t!ete spectr!p(!t!eterr is an instruent t(at easures t(e c!ncentrati!n !" s!lutes in s!luti!n *& easurin# t(e a!unt !" t(e li#(t t(at is a*s!r*ed *& t(e s!luti!n in a cu%ette. T(is e3perient in%!l%es t(e use !" a $I IS spectr! ctr!pp(!t!e ete terr )(ic( (ic( !per !perat ates es in t(e t(e ultr ultra% a%i! i!le lett and and %isi %isi*l *lee re#i!ns re#i!ns !" t(e electr! electr!a#n a#netic etic spectru spectru. . T(e T(e spec spectr tr!p !p(! (!t! t!e ete terr e eas asur ures es li#( li#(tt inten intensit& sit& as a "un "unct cti!n i!n !" )a%ele )a%elen# n#t( t( *& di""ractin# t(e li#(t *ea int! a spectru !" )a%elen#t(s, )a%elen#t(s, detectin# t(e intensities intensities )it( a c(ar#ec!upled de%ice, and displa&in# t(e results as a #rap( !n t(e detect!r and t(en 1|Page
t(e displa& de%ice. As t(e c!ncentrati!n !" t(e a*s!r*in# su*stance increases, less li#(t is transitted and !re li#(t is a*s!r*ed *& t(e s!luti!n. A $is spectr!p(!t!eter utilizes t)! li#(t s!urces. T(e "irst is a tun#sten (al!#en lap used as t(e s!urce "!r %isi*le li#(t. T(is lap c!nsists !" a tun#sten "ilaent encl!sed in a #lass en%el!pe, )it( a )a%elen#t( ran#e !" a*!ut 66- t! 7-- n. T(e& are #enerall& use"ul "!r easurin# !deratel& dilute s!luti!ns in )(ic( t(e c(an#e in c!l!r intensit& %aries si#ni"icantl& )it( c(an#es in c!ncentrati!n. T(e sec!nd lap is usuall& a deuteriu arc lap t(at can easure "r! 7-68- n. T(e electrical e3citati!n !" deuteriu at l!) pressure pr!duces a c!ntinu!us $ spectru. T(e ec(anis "!r t(is in%!l%es "!rati!n !" an e3cited !lecular species, )(ic( *rea+s up t! #i%e t)! at!ic species
and an ultra%i!let p(!t!n. T(e 9':'SYS - $is spectr!p(!t!eter, (!)e%er, utilizes a 3en!n "las( lap )(ic( pr!%ides a (i#( ener#& li#(t s!urce )it( a s(!rt )ar up tie and l!n# lap li"e. T(e& easure in *!t( t(e $ and isi*le re#i!ns !" t(e electr!a#netic spectru "r! 7- -n. ;en!n laps t&picall& pr!%ide 8"las(es per sec!nd #i%in# t(e t(eir distincti%e *irdli+e n!ise. A *lan+ is a s!luti!n t(at is identical t! t(e saple s!luti!n *ut d!es n!t c!ntain t(e s!lute t(at a*s!r*s li#(t. T(is easureent re!%es an& a*s!rpti!n !" li#(t and is t(ere"!re used t! set t(e a*s!r*ance t! zer!.
M!ter!"s !nd Met#odo"o$y
T(e aterials and equipents used "!r t(e "irst pr!cedure !" testin# accurac& and precisi!n in usin# a icr!pipett!r )ere Br!p(en!l *lue, icr!centri"u#e tu*es, icr!pipett!rs, icr!pipett!r tips, 9':'SYS - u%%is spcetr!p(!t!eter, %!rte3 i3er, seiicr! cu%ettes and distilled )ater.
T(e spectr!p(!t!eter )as )ared up and )as set at 54-n. 0 !" distilled )ater )as placed in eac( !" "i%e icr!centri"u#e tu*es.
until t(e d&e )as in s!luti!n. T(e spectr!p(!t!eter )as zer!ed )it( distilled )ater. T(e c!ntents !" t(e icr!centri"u#e tu*es )ere trans"erred t! t(e cu%ettes. A"ter )(ic( t(e a*s!r*ance !" t(e d&e s!luti!ns )ere read and )ere rec!rded.
0 !" distilled )ater )as placed eac( !" "i%e icr!centri"u#e tu*es. 1it( t(e icr!pipett!rs, =.5 /0 !" *r!p(en!l *lue )as added *& eac( e*er in eac( icr!centri"u#e tu*e. T(e tu*e )as ten %!rte3ed until t(e d&e )as in s!luti!n. T(e spectr!p(!t!eter )as zer!ed )it( distilled )ater. T(e c!ntents !" t(e icr!centri"u#e tu*es )ere trans"erred t! t(e cu%ettes. A"ter )(ic( t(e a*s!r*ance !" t(e d&e s!luti!ns )ere read and )ere rec!rded. T(e standard de%iati!n and t(e c!e""icient !" %ariati!n )ere calculated, )(ere> SD ? @:3= E 3F= G::F F F C ? SDMean
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Testn$ !ccur!cy !nd %recson n usn$ ! &cro%%ettor Testn$ !ccur!cy o' o%er!tors
In t(e "irst part !" t(e e3perient a spectr!p(!t!eter )as utilized t! c!rrelate a*s!r*ance )it( t(e a!unt d&e.
Hara"il !r aluiniu "!il )as placed in a an anal&tical *alance. Tare )as pressed a"ter it )as )ei#(ed. $sin# a icr!pipett!r, 5- /0 !" distilled )ater )as added int! t(e para"il. T(e )ei#(t )as rec!rded up t! t(e "!urt( decial place. Tare )as pressed a#ain and t(e pr!cedure )as repeated until all t(e e*ers !" t(e #r!up "inis(ed per"!rin# t(e pr!cedure. T(e a%era#e )ei#(t and standard de%iati!n )ere c!puted.
Hara"il !r aluiniu "!il )as placed in a anal&tical *alance. Tare )as pressed a"ter it )as )ei#(ed. $sin# a icr!pipett!r, 5- /0 !" distilled )ater )as added int! t(e para"il. T(e )ei#(t )as rec!rded up t! t(e "!urt( decial place. Tare )as pressed a#ain and t(e pr!cedure )as repeated until all t(e e*ers !" t(e #r!up "inis(ed per"!rin# t(e pr!cedure. T(e a%era#e )ei#(t, percent err!r err!rF, standard de%iati!n S.D.F and standard err!r !" t(e ean S.'.F )ere calculated, )(ere> err!r?standard %aluee3periental %alueFstandard %alueF-S'? SD@:F Resu"ts ( Dscusson
T(e "!ll!)in# "!rula )ere used t! calculate "!r t(e standard de%iati!n and c!e""icient !" %ariati!n
√
2
2
[ N ∑ x – ( ∑ x ) ] SD = N −( N −1)
C ?
SD Mean
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Ta*le .= Accurac& !" !perat!r Br!p(en!l *lue %!lue 20F -.5 ..5 =.=.5
Standard De%iati!n
A*s!r*ance 54-nF
Trial Trial = Trial 6 A%era#e -.=5 -.=5 -.=5 -.=5 -.=8 -.=8 -.=8 -.=8 -.64= -.64= -.64= -.64= Average Absorbance versus Volume of BPB -.65K -.65K -.65K -.65K -.46 -.46 -.46 -.46
-
C!e""icient ariati!n -
Average absorbance (540 (nm)
$."11."%%."& Bromphenol blue volume (μL)
Ta*le .6 Testin# precisi!n !" !perat!rs A*s!r*ance
Student = 6 4
Trial -.487 -.5=-.45L -.554
Trial = -.488 -.5=-.45L -.554
T(e results s(!) t(e a!unt !" d&e in t(e s!luti!n is directl& pr!p!rti!nal t! t(e a!unt !" li#(t a*s!r*ed. T(e increasin# a!unt !" Br!p(en!l *lue in t(e s!luti!n resulted t! a (i#(er a*s!r*ance readin#.
Trial 6 -.487 -.5=-.45L -.555
A%era#e -.487 -.5=-.45L -.554
Standard De%iati!n
C!e""icient ariati!n
5.LL- 4 5.LL- 4
.8-6 .-4-6
In t(e sec!nd part !" t(e e3perient, icr!pipett!rs )ere used t! trans"er t(e )ater saple and )ei#( t(e in nuer!us trials. T(e icr!pipett!rs )ere used t! test t(eir accurac& and precisi!n.
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Ta*le .4 1ei#(t !" di""erent )ater saples *& e*er !" t(e #r!up !lue !" )ater 20F 5-5--
Student
Standard De%iati!n
1ei#(t !" saple #F Trial -.47 -.-777 -.5-
Trial = -.475 -.-77= -.478-
Trial 6 -.487 -.--8 -.47L7
Trial 4 -.47L -.--4 -.47LK
Trial 5 -.47K -.-77L -.477L
A%era#e -.-474 -. -.4787
Ta*le .5 1ei#(t !" di""erent )ater saples *& e*ers !" t(e #r!up 5-20 --20 1ei#(t 1ei#(t 1ei#(t De%iati!n 'rr!r De%iati!n 'rr!r #F #F #F
-.-475
6-4
=
-.-475
6-4
6
-.-484
.4-6
4
-.-54K
4.8-6
A%era#e Standard De%iati!n
6.44- 4 K.=-- 4 .478- 6
5--20 De%iati!n
'rr!r
-.K-
-.--5
4 8-
-.8-
-.5--=
6 .L-
-.64
-.K-
-.--6
4 K-
-.K-
-.4786
4 =-
-.-4
=.8
-.--6
4 K-
-.K-
-.4788
4 6-
-.-K
7.K4
4 8-
-8-
-.477L -.4776
6 .=-
-.=4
-.-5-5
-.--5 -.--4
=.8-6
.=-4
Accurac& is de"ined as t(e cl!seness !" t(e easured %alue t! t(e true %alue )(ile precisi!n is de"ined as t(e cl!seness !" !ne easured %alue t! !t(er easured %alues. T(e l!)er err!r eans t(at t(e %alue !*tained is !re accurate. T(e l!)er standard de%iati!n and c!e""icient %ariati!n eans t(at t(e %alue !*tained is !re precise. T(e %alues !*tained )ere cl!se t! eac( !t(er *ecause !" t(e pr!per trans"er !" )ater usin# t(e appr!priate icr!pipett!r. T(e in!r di""erence !" t(e di""erent %alues a& *e due t! t(e ad(erence !" )ater !n
8.K-4
t(e pipette tip !r *ecause t(e sae pipette tip )it( )ater residue )as used. Conc"uson
T(e results !*tained "r! usin# icr!pipett!rs in )ei#(in# t(e )ater saple pr!duced accurate and precise %alues. In t(e utilizati!n !" a spectr!p(!t!eter, an increase in t(e a*s!r*ance !" a su*stance results t! a decrease in t(e transittance. M!re!%er, t(e increase in t(e c!ncentrati!n !" t(e BHB cuases an in increase in t(e a*s!r*ance.
Re'erences
9aines, H. =-4F. Accurac&, Hrecisi!n, Mean and Standard De%iati!n. Retrie%ed "r!
(ttp>))).in!r#anic%entures.c!accurac& precisi!neanandstandardde%iati!n.
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Reusc(, 1. =-6F. isi*le and ultra%i!let spectr!sc!p&. Retrie%ed =6 Septe*er =-5, "r! (ttp>)))=.c(eistr&.su.edu"acult&reus c(%irtt3tlspectrp&u%%isspectru.(t
Cap*ell, M. =--=F. N!) t! $se a Micr!pipett!r. Retrie%ed =6 Septe*er =-5, "r! (ttp>))).*i!.da%ids!n.edu!lecularHr!t !c!lspipette.(tl
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