Enriquez and Garcia / Chemistry 26.1/ (2014-2015)
Pa e !1
"uantitati#e $eterminati%n $eterminati%n %& C%''er () C%ncentrati%n y *'ectr%'h%t%metry Giancar+% P%ch%+% ,. Enriquez a uan iue+ Garcia Garcia a
Institute of Biology, University of the Philippines – Diliman, Quezon City, PHL Institute of Biology, University of the Philippines – Diliman, Quezon City, PHL
b
*3C3 *'ectr%'h%t%metry dea+s ith the interacti%n %& radiati%n and matter. t is technique that is uti+ized t% measure the transmittance %r as%rance 'r%'erties %& materia+s as a &uncti%n %& a#e+enth. 3hr%uh s'ectr%'h%t%metry the e'eriment aims t% identi&y the c%ncentrati%n c%ncentrati%n %& an un7n%n s%+uti%n since as%rance as%rance is direct+y direct+y 'r%'%rti%na+ 'r%'%rti%na+ t% c%ncentrati%n. 3he e'eriment as c%m'%sed %& t% 'arts. 3he 8rst 'art aims t% create a ca+irati%n cur#e usin 8#e tria+s ith s%+uti%ns c%ntainin 2.00 4.00 6.00 9.00 and 10.0 10.00m 0m, , %& Cu( Cu()) st%c st%c7 7 s%+u s%+uti ti%n. %n. ean eanhi hi+e +e thre three e tria tria+s +s %& a s%+u s%+uti ti%n %n ith ith an un7n% un7n%n n c%nce c%ncent ntrat rati%n i%n as as he+d he+d under under s'ectr%'h%t%metry &%r the sec%nd 'art. 3he equati%n %tained &r%m the 8rst 'art %& the e'eriment hich is y : 0.001 as used t% acquire the un7n%n c%ncentrati%n %& the s%+uti%n. ;%r the three tria+s the acquired c%ncentrati%n is <60''m. 3hese #a+ues indicate that the data acquired acquired is 'recise. 'recise. ;au+ty ;au+ty hand+in %& the cu#ette instrumenta+ instrumenta+ err%rs and im'r%'er s%+uti%n 're'arati%n 're'arati%n are the '%ssi+e s%urces %& err%r in the e'eriment.
1. Introduction
,iht is a &%rm %& enery transmitted thr%uh s'ace. t is an e+ectr%manetic radiati%n in the #isi+e rei%n %& the e+ectr%manetic s'ectrum &r%m 400nm t% =00 nm. >n the %ther %ther hand hand c%+%r c%+%r is 'erc 'ercei# ei#ed ed +iht +iht ased ased %n emittance emittance %r re?ecti re?ecti%n %n %& +iht +iht rather rather than as%r'ti as%r'ti%n %n (*7 *7%% %% et a+. 2010@ +um P. 1AA= 1AA=). ). ;%r eam'+e i& a sam'+e as%rs a++ a#e+enths in the #isi+e s'ectrum it a''ear a''ears s +ac7 +ac7.. & it as%r as%rs s n%ne n%ne then then it is hite. hite. %re%#er as seen %n ( 3a+e 3a+e 1) 1) ('+ease re&er t% ''endi ) i& a eam %& hite +iht is directed at a sustance that as%rs +ue +iht the c%+%r 'ercei#ed i++ then e ye++% since it has as%red a++ the +ue +iht i#en that ye++% is the c%m'+ementary c%+%r %& +ue ( 3he 3he *tate Bni#ersity %& e D%r7 2005). 2005 ). eanh eanhi+e i+e s'ect s'ectr% r%'h% 'h%t%m t%metr etry y dea+s dea+s ith ith the interacti interacti%n %n %& radiati% radiati%n n and matter. matter. t is an ana+ytic ana+ytica+ a+ tech techni niqu que e hic hich h meas measur ures es the the tran transm smis issi si%n %n %r as%r as%r'ti 'ti%n %n 'r%'e 'r%'erti rties es %& materi materia+s a+s as a &uncti &uncti%n %n %& a#e+ a#e+en enth. th. *ince *ince +iht +iht is a &%rm &%rm %& e+ectr e+ectr%ma %mane netic tic radiati% radiati%n n it can re?ect e as%red as%red %r e as%red as%red then re?ected and transmitted as it &a++s %n a sustance. %e#er %e#er since since s'ectr% s'ectr%'h%t% 'h%t%metr metry y d%es n%t 'rimari+ 'rimari+y y dea+ ith re?ecti%n %n+y as%rance and transmittance %& +iht is c%nsidered ( 3he *tate Bni#ersity %& e D%r7 2005). 2005 ). Bn+i7e Bn+i7e c%+%rime c%+%rimetry try that %n+y uses hite hite +iht +iht that 'asses thr%uh c%+%red 8+ters s'ectr%'h%t%metry s'ectr%'h%t%metry uti+izes u#-#isi+e +iht that is 8+tered y the a#e+enth se+ect%r t% m%r'h int% m%n%chr%matic +iht that 'asses thr%uh the s%+uti%n that causes it t% increase increase %r decrease decrease its intensity intensity. 3he transmitte transmitted d radiati% radiati%n n is then inter're inter'reted ted as the as%ran as%rance ce %& the s%+uti%n s%+uti%n (*7%% et a+. 2010). 2010 ). 3he as%rin as%rin characteristics characteristics %& a sustance %r %r a s%+uti%n can e e'ressed in terms %& as%rance %r transmittance. 3ransmittance is the rati% %& the am%unt %& +iht transmitted t% the am%unt %& +iht radiated i#en y (Equati%n (Equati%n 1). 1). eanhi+e as%rance is de8ned as the C%rres'%ndin auth%r. %i+eH 0AAA92=4<524 0AAA92=4<524 E-mai+ addressH '%chi.enriquezImai+.c%m
neati#e +%arithm %& transmittance de8ned y (Equati%n ( Equati%n 2). I T = I 0
(1) A = log
I 0 I
(2) $ue t% this this it can e in&err in&erred ed that hen i#en i#en t% s%+uti%ns here %ne has a dar7er t%ne than the %ther the dar7er r7er %ne %ne as%r s%rs s m%r m%re +iht. ht. Gi#e i#en a hi hiher her as%rance it can e deduced that it a+s% has a hiher c%ncentrati%n (*7%% (*7%% et a+. 2010@ 3he *tate Bni#ersity %& e D%r7 D%r7 2005@ *in#asan7ar 2009). 2009 ). 3his 'hen%men%n the re+at re+ati% i%nsh nshi' i' etee eteen n as%r as%r't 'ti%n i%n and a s%+uti s%+uti%nF %nFs s c%ncentrati%n is &urther e'+ained y the eerFs ,a %r eer-,amert-%uuer ,a i#en y (Equati%n ( Equati%n <). <). A = abc
(<) 3he eerFs eerFs ,a states states that as%ran as%rance ce is direct+ direct+y y 'r%' 'r%'%r %rti ti%n %na+ a+ t% the the c%nc c%ncen entr trat ati% i%n n %& the the as% as%r rin in s'ecies c and t% the 'ath +enth %& the as%rin medi medium um (*7 *7%% %% et a+ a+. . 20 2010 10). ). %e %e#e #er r there there are are +imitati% +imitati%ns ns t% the eerFs eerFs ,a. ,a. Causes Causes %& n%n-+inear n%n-+inearity ity inc+ud inc+ude e scatte scatterin rin %& +iht +iht due t% 'artic 'articu+a u+ates tes in the sam' sam'+e +e %r smud smude es s and and scra scratc tche hes s in the the cu#e cu#ette tte ?u%rescence %r 'h%s'h%rescence in the sam'+e shi&ts in chemica+ equi+iria %utside +iht and n%nm%n%chr m%n%chr%mati %matic c radiati%n radiati%n (az aze7e e7e#ic #ich h D. 2010 2010 $a# $a#id id 2001). 2001 ). *'ectr *'ectr%'h% %'h%t%metr t%metry y is a''+ied a''+ied in #ari%us #ari%us 8e+ds. 8e+ds. n micr%i%+%y micr%i%+%y.. t is used in %rder t% identi&y the numer %& acteria acteria in a sam'+e sam'+e detect detect im'uriti im'urities. es. ;%r ;%r eam'+e eam'+e enze enzene ne an im'uri im'urity ty &%und &%und in cyc+% cyc+%he heane ane can e
Enriquez and Garcia / Chemistry 26.1/ (2014-2015)
detected at 255nm. 3he diss%ciati%n c%nstants %& acids and ases can a+s% e determined s'ectr%'h%t%metrica++y &r%m ra'hin as%rance and a#e+enth at diJerent ' #a+ues. $rus can e quantitati#e+y ana+yzed y ma7in a s%+uti%n %& the dru and measurin the as%rance at a s'eci8c a#e+enth. Chemica+ 7inetics can a+s% e studied thr%uh s'ectr%'h%t%metry. 3his is d%ne y measurin as%rance chanes ased %n BK radiati%n 'assin a reacti%n ce++. t is a+s% a''+ied in m%dern at%mic the%ry &%r the determinati%n %& certain m%+ecu+ar structures. t is a+s% used &%r the quantitati#e and qua+itati#e determinati%n %& %th %ranic and in%ranic c%m'%unds (*7%% et a+. 2010@ 'harmatut%r.%r n.d. ). 2. Materials and Methods
*i s%+uti%ns c%ntainin 0.00 2.00 4.00 6.00 9.00 and 10.00 m, %& Cu() st%c7 s%+uti%n ere 're'ared &%r s'ectr%'h%t%metry. 3he sam'+e ith 0.00m, %& Cu() st%c7 s%+uti%n i++ e c%nsidered as reaent +an7 in %rder t% 7ee' the measurements 'recise settin a ase &r%m hich %ther measurements can e 'er&%rmed. mm%nia is a+s% added t% the Cu() s%+uti%ns. 3his is d%ne in %rder t% &%rard the reacti%n eteen c%''er and amm%nia t% &%rm a c%''er-amm%nia c%m'+eH Cu 2L L 4< -M NCu(<)4O2L 3his c%m'+e i#es a m%re intense c%+%r than a standard c%''er s%+uti%n hich a++%s the s'ectr%'h%t%meter t% eamine it m%re eJecti#e+y. 3% determine the ana+ytica+ a#e+enth a BK-Kis s'ectr%'h%t%meter as uti+ized. cu#ette as 8++ed ith the standard s%+uti%n c%ntainin the hihest c%ncentrati%n %r the s%+uti%n c%ntainin 10m, %& Cu() st%c7 s%+uti%n. 3he cu#ette as %n+y he+d at the ti' t% 're#ent smudes that may i#e &a+se +iht as%rance readins. 3he cu#ette must a+s% e rinsed 'r%'er+y ith disti++ed ater and the s%+uti%n e&%re measurement t% a#%id discre'ancies in data r%uht y dirt. &ter rinsin the cu#ette must e i'ed ent+y usin a tissue since ater dr%'+ets may a+s% cause a de#iati%n in the data. >nce the cu#ette is c+ean and dry it is inseted ent+y int% the cu#ette h%+der. 3he machine as then c+%sed t% 're#ent %utside +iht &r%m enterin. 3he s'ectr%'h%t%meter as then set t% *'ectrum %de here the a#e+enth rane as set &r%m <00nm t% =00nm and then scanned. 3he 'ea7 dis'+ayed y the s'ectr%'h%t%meter as then rec%rded as it i++ &uncti%n as the ana+ytica+ a#e+enth. 3% 're'are the ca+irati%n cur#e the as%rance %& the s%+uti%ns c%ntainin 2.00 4.00 6.00 9.00 and 10.00m, Cu() st%c7 s%+uti%n ere measured usin the BK-#is s'ectr%'h%t%meter. reaent +an7 as aain used t% 'recise+y measure the as%rance %& the %r7in standards. >n+y a sin+e cu#ette as used &%r %th the reaent +an7 and %r7in standard t% h%+d it c%nstant since diJerence in materia+ used and thic7ness %& the materia+ may aJect the data (e eic% *tate Bni#ersity 2006). 3he s'ectr%'h%t%meter as set t% G% t% , here the %tained ana+ytica+ a#e+enth as C%rres'%ndin auth%r. %i+eH 0AAA92=4<524 E-mai+ addressH '%chi.enriquezImai+.c%m
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in'utted. *tartin &r%m the standard s%+uti%n ith the +%est c%ncentrati%n the cu#ette as 8++ed ith the s%+uti%n t% e measured. 3he cu#ette as then aain hand+ed %n the ede t% 're#ent smudes and ater dr%'+ets &r%m rinsin ere i'ed t% 're#ent discre'ancies in the data. &ter assurin that the cu#ette is c+ean and dry the s%+uti%n as scanned and the as%rance readin as rec%rded. 3he 'r%cedure as then re'eated unti+ the standard ith the hihest c%ncentrati%n as scanned. 3% determine the Cu() c%ncentrati%n %& the un7n%n s%+uti%n the cu#ette as 8++ed ith the un7n%n s%+uti%n and scanned under the BK-Kis s'ectr%'h%t%meter. 3he as%rance readin as then rec%rded. 3. Results and Discussion
3he #a+ues used &%r the data treatment are sh%n in ( 3a+e 2). 3he #a+ues ere used t% create a ca+irati%n cur#e that i++ 8nd the equati%n %& the est 8t +ine. Bsin this +ine the c%ncentrati%n %& the un7n%n s%+uti%n can e determined y measurin its as%rance. 3a+e 2H Ca+irati%n Cur#e Ka+ues K%+ume %& C%ncentrati%n %& %r7in *tandard Cu() *tandard ''m *%+uti%n 2.00 100 4.00 200 6.00 <00 9.00 400 10.00 500
s%rance .014 .029 .041 .054 .0=0
Bsin the equati%n y : m L as%rance c%u+d e determined y equatin it t% y. 3his is determined y the s'ectr%'h%t%meter. eanhi+e is equi#a+ent t% the c%ncentrati%n %& the s%+uti%n m is equa+ t% the as%r'ti#ity and %u+d e the y-interce't. Bsin icr%s%&t Ece+ the ca+irati%n cur#e is sh%n as seen in (;iure 1) (re&er t% ''endi 2). n the equati%n y : 0.0001 thus the as%rance is equa+ t% 0.0001. %re%#er since there is n% y-interce't in the data acquired it sini8es that there is n% de#iati%n &r%m the the%retica+ #a+ue. n the y-interce't there is n% c%ncentrati%n %& Cu() ecause : zer%. 3he%retica++y the as%rance at this '%int %u+d e zer% ecause there is n% c%+%red s%+uti%n due t% the +ac7 %& c%''er hich means that n% +iht sh%u+d e as%red due t% the s%+uti%n ein c%+%r+ess. 3he 'resence %& a y-interce't e#en a&ter neatin the eJectin %& the ce++ and the amm%nia s%+uti%n %n the #a+ues %& as%rance sina+s that s%methin e+se is as%rin +iht due t% the 'resence %& an as%rance #a+ue e#en at zer% ''m c%ncentrati%n. n this case a y-interce't %u+d de#iate &r%m the the%retica+ #a+ue y increasin the #a+ues %& as%rance. 3he data acquired ere d%ne under the a#e+enth %& maimum as%r'ti%n %& 541nm. 3a+e
Enriquez and Garcia / Chemistry 26.1/ (2014-2015)
3ria+
s%rance
C%ncentrati%n %& *t%c7 *am'+e Cu () ''m
1 2 < #erae
0.0<6 0.0<6 0.0<6 0.0<6
<60 <60 <60 <60
++ %& the three tria+s &%r the s%+uti%n c%ntainin the un7n%n c%ncentrati%n had an as%rance %& 0.0<6. e'+acin y ith 0.0<6 in the equati%n y:0.0001 the c%ncentrati%n %& the un7n%n sam'+e %u+d e <60''m. *ince a++ %& the tria+s had the same as%rance #a+ues it can e in&erred that the data acquired is 'recise. P%ssi+es s%urces %& err%rs &%r this e'eriment %u+d e rand%m systematic and r%ss. and%m err%rs are %&ten una#%ida+e. 3hey resu+t &r%m the usae %& +assare and may aJect the 'recisi%n %& the resu+ts. *ystematic err%rs c%u+d %ccur &r%m meth%d %r instrument. 3he as%rance #a+ues %tained &r%m the s'ectr%'h%t%meter may increase i& the reacti%n ce++ as he+d im'r%'er+y. 3his is ecause %& the 8ner'rints may e +e&t in the ce++. 3hese 8ner'rints may as%r +iht &r%m the s'ectr%'h%t%meter hich increases as%rance #a+ue. *ince the same ce++ as used &%r a++ tests in this e'eriment it is im'%rtant that it is ashed ith the s%+uti%n a%ut t% e tested thr%uh+y. 3his is d%ne t% ensure that the c%rrect c%ncentrati%n %& the Cu() is ein ana+yzed %therise the as%rance #a+ue may increase %r decrease de'endin %n the c%ntaminated c%ncentrati%n. 3he reaent +an7 test must e 'er&%rmed in %rder t% neate the eJects %& the ce++ and the amm%nia s%+uti%n %n as%rance %therise the as%rance #a+ues i++ increase. nstrumenta+ err%rs may %ccur in this e'eriment &r%m the s'ectr%'h%t%meter. ny stray +iht in the s'ectr%'h%t%meter i++ decrease the as%rance #a+ue. 3his is due t% the additi%na+ 'resence %& unas%red +iht. 3he ce++ used &%r ana+ysis must e standardized ecause a ider ce++ resu+ts in a +%ner 'ath +enth. 3he +%ner 'ath +enth i++ increase as%rance #a+ues and the ra'h may de#iate &r%m its +inear &%rm. 3he s'ectr%'h%t%meter must a+s% e ins'ected e&%re use t% ensure it is sti++ eJecti#e &%r ana+ysis. Gr%ss err%rs can resu+t &r%m c%ntaminated +assare es'ecia++y durin s%+uti%n 're'arati%n. Bnanted reaents may enter the 're'ared st%c7 s%+uti%ns and aJect the c%ncentrati%n %& c%''er hich i++ aJect the as%rance #a+ue. 3his i++ reat+y aJect the c%nstructi%n %& the ca+irati%n cur#e hich i++ aJect the ca+cu+ati%ns &%r the c%ncentrati%n %& the un7n%n sam'+e. ++ c%ncentrati%ns must e unc%ntaminated e&%re testin t% 're#ent such err%rs &r%m %ccurrin. 4. Conclusion and Recommendations
3he quantitati#e ana+ysis %& the c%''er s%+uti%n #ia s'ectr%'h%t%metry as success&u+. n accurate ca+irati%n cur#e as c%nstructed ith the a#e+enth at maimum as%r'ti%n at 541 nm. 3he ra'h as near+y c%m'+ete+y +inear hich a++%s &%r an accurate equati%n %& the +ine hich as &%und at y : 0.0001. ased %n the as%rance #a+ue %& 0.0<6 the ca+cu+ated c%ncentrati%n C%rres'%ndin auth%r. %i+eH 0AAA92=4<524 E-mai+ addressH '%chi.enriquezImai+.c%m
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%& the c%''er s%+uti%n as &%und at <60 ''m hich is true &%r a++ tria+s. 3his a+s% ensures a++ #a+ues are 'recise t% %ne an%ther &%r the sam'+e ana+ysis. %e#er the ca+cu+ated c%ncentrati%n %& the c%''er s%+uti%n may s+iht+y de#iate &r%m the the%retica+ #a+ue due t% the ca+irati%n cur#e n%t ein c%m'+ete+y +inear hich may ha#e resu+ted in s%me min%r err%rs. 3% increase the accuracy %& the resu+ts it is rec%mmended t% address the systematic err%rs. $urin the testin ce++ must e hand+ed 'r%'er+y and ith utm%st care t% 're#ent unanted err%rs. 3he ce++ must a+s% e thr%uh+y ashed t% 're#ent c%ntaminati%n and di+uti%n &r%m aJectin the c%ncentrati%n %& the sam'+e. 3he cur#e must e c%nstructed as +inear as '%ssi+e. hen testin standardized s%+uti%ns &%r the c%nstructi%n %& the ca+irati%n cur#e s%me tests may e re'eated t% 8nd the m%st accurate resu+t t% reduce any de#iati%ns in the +inear ra'h. ny instrument err%r may e addressed thr%uh 'r%'er ins'ecti%n %& the s'ectr%'h%t%meter e&%re the 'er&%rmance %& the e'eriment. n additi%n it is im'%rtant that a++ s%+uti%ns used &%r the c%nstructi%n %& the ca+irati%n cur#e are 'r%'er+y 're'ared and unc%ntaminated durin s%+uti%n 're'arati%n t% ensure that the %tained as%rance #a+ues are accurate. 5. References
Q''+icati%ns %& s%r'ti%n Pharmatut%r.%r. n.d. e. = ay 2015.
*'ectr%sc%'y.R
+um P. 1AA=. e!e"tan"e #pe"trophotometry an$ Colorimetry% PP and%%7. r%n 3.. 2012. Chemistry 3he Centra+ *cience. Pears%n Educati%n nc. Cr%uch *. %++er ;. . *7%% $. est $. 2014. ;undamenta+s %& na+ytica+ Chemistry. Cenae ,earnin. Ath editi%n. Cr%uch * %++er ;. ;undamenta+s %& na+ytica+ Chemistry. n.'H Cenae ,earnin 201<. Print. $a#id G. na+ytica+ Chemistry. ndiaH Bni#ersities Press (ndia) Pri#ate ,imited 2001. Print. aze7e#ich D. 2010. na+ytica+ Chemistry. *et%n a++ Bni#ersity. Petrucci . 2010. Genera+ Chemistry S Princi'+es and %dern ''+icati%ns. 10 th Editi%n. Pears%n Canada nc. *c%tt . 200=. ChemistryH 3he Cenae ,earnin 10th Editi%n.
Practica+
*cience.
*in#asan7ar . Enineerin Chemistry. e $e+hi 3ata cGra-i++ Pu+ishin C%m'any ,imited 2009. Print. *+%ins7i E. %+sey . and %ssi 2011. Chemica+ Princi'+es n 3he ,a%rat%ry. r%%7s/C%+e. 10 th editi%n.
Enriquez and Garcia / Chemistry 26.1/ (2014-2015)
*i+erer . Princi'+es %& Genera+ Chemistry. 200=. e D%r7H cGra-i++. #pe"trophotometry% *tate Bni#ersity %& e D%r7 2006. e D%r7.
C%rres'%ndin auth%r. %i+eH 0AAA92=4<524 E-mai+ addressH '%chi.enriquezImai+.c%m
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Appendix A: ist of !a"les !a"le 1: Pairs %& c%m'+imentary c%+%rs at their c%rres'%ndin a#e+enth ranes
a#e+enth 400-4<5 4<5-490 490-4A0 4A0-500 500-560 560-590 590-5A5 5A5-650 650-=50
C%+%r s%red Ki%+et +ue +ue-reen Green-+ue Green De++%-reen De++% >rane ed
C%+%r >ser#ed De++%-reen De++% >rane ed Pur'+e Ki%+et +ue +ue-reen Green-+ue
!a"le 2: Ca+irati%n Cur#e Ka+ues C%ncentrati%n %& K%+ume %& *tandard Cu() %r7in ''m *tandard
*%+uti%n 2.00 4.00 6.00 9.00 10.00
s%rance
100 200 <00 400 500
.014 .029 .041 .054 .0=0
!a"le 3: s%rance &%r the Bn7n%n na+ysis
3ria+
s%rance
C%ncentrati%n %& *t%c7 *am'+e Cu () ''m
1 2 < #erae
0.0<6 0.0<6 0.0<6 0.0<6
<60 <60 <60 <60
Appendix #: ist of $i%ures $i%ure 1: Cali"ration Cur&e
s%rance 0.09 0.0= 0.06
&() : 0 - 0 T : 1
0.05 0.04 0.0< 0.02 0.01 0 50 100 150 200 250 <00 <50 400 450 500 550 Appendix C: Calculations
C%ncentrati%n %& *t%c7 *am'+e Cu()H Equati%n %& the ,ine (;r%m $ata 3reatment)H y : 0.0001 y : : as%rance %& the s%+uti%n
: c : c%ncentrati%n %& the s%+uti%n
1st 3ria+H y : 0.0<6 0.0<6 : 0.0001 : 0.0<6/0.0001 : <60 ''m 2nd 3ria+H y : 0.0<6 0.0<6 : 0.0001 : 0.0<6/0.0001 : <60 ''m
