Exercise No. 1 UV- VISIBLE SPECTROPHOTOMETRY
Gerry Mark G!a"#es CHEM 1$%.1 & 'L '"( Se)es#er *Y '+1%-'+1,
Date Performed: February 3, 2018 Date Submitted: February 9, 2018
Mr. e#ro Ma/sa"/kay Laboratory Instructor
*!s#rac# In the e!eriment, the "inear re"ationshi! bet#een absorbance and concentration at a #a$e"en%th and de$iations from the &eer's La# P"ot #ere i""ustrated( )he maimum #a$e"en%ths #ere determined at *12, 39+ and *29 nm for o, -i and ./n +, res!ecti$e"y( )he concentration of an unno#n sam!"ed of o and -i so"utions #ere 0(01* (1030.656 mg/L) and 0(09* 4+*93(8+ m%5L6 /, res!ecti$e"y( Last"y, s!ectro!hotometric ana"ysis of a mu"ti7 com!onent system #as done( It #as used to ca"cu"ate the concentration of benoic acid and caffeine in the miture #hich #ere found to be 1(0310 73 and 1(232010 73 /, res!ecti$e"y(
I.
I"#ro(c#io"
)he "tra$io"et7$isib"e 47is6 s!ectro!hotometry refers to the absor!tion or ref"ectance s!ectrosco!y in the u"tra$io"et7$isib"e s!ectra" re%ion( )his re%ion, moreo$er, is di$ided into three sub7domains termed near 418*;+00 nm6, $isib"e 4+00;00 nm6 and $ery near infrared 400; 1100 nm6( /ost commercia" s!ectro!hotometers co$er the s!ectra" ran%e of 18* to 900 nm 4L6 and e"ectrode"ess dischar%e "am! 4?DL6 !roduce on"y at s!ecific #a$e"en%ths( Furthermore, the sam!"e ho"der is a trans!arent container #hich ho"ds the sam!"e( u$ette, #hich is common"y a$ai"ab"e in 1, *, @ 10 cm !ath "en%ths, is the most #ide"y used sam!"e ho"der( It can be a %"ass, !"astic, Auart or fused si"ica( )he #a$e"en%th se"ector, moreo$er, is a de$ice that iso"ates a restricted re%ion of the s!ectrum for measurement( It can be an o!tica" fi"ter or monochromator( Lie#ise, the detector or transducer con$erts radiant ener%y to a usab"e si%na"( )he common"y used detector are !hoto$o"taic ce""s, !hototubes, !hotomu"ti!"ier tubes 4P/)'s6, !hotoconducti$ity and si"icon !hotodiodes( Last"y, the si%na" !rocessor and readout dis!"ay the transduced si%na"( It can in the form of com!uter dis!"ay, di%ita" or ana"o% readout, stri! chart recorder and inte%rator 4Soo%, et al., 19986( )he 7is s!ectro!hotometer measures the intensity of "i%ht !assin% throu%h a sam!"e 4P 6, and com!ares it to the intensity of "i%ht before it !asses throu%h the sam!"e 4 P o6( =ettin% the
P
ratio
P o
#i"" %i$e the transmittance #hich is usua""y e!ressed as a !ercenta%e 4B)6( )he
absorbance is then re"ated to transmittance %i$en by the eAuation be"o# 4Soo%, et al., 19986( Abs = log (
Po P
1
)= log ( )=−log T =abc T
41(16
)he s!ectrum of the com!ound under ana"ysis #i"" be obtained as a %ra!h re!resentin% the transmittance 4or the absorbance6 as a function of #a$e"en%th a"on% the 7 ais, usua""y %i$en in nanometers( /oreo$er, a"" the absor!tion ana"yses are %o$erned by the &eer7Lambert's "a# 4?Auation 1(16( )he &eer's !"ot can be !re!ared by measurin% the "i%ht absorbed by the so"utions of $aryin% concentration( sin% the re"ationshi! bet#een the t#o, ?Auation 1(1 can be re!resented as 4Soo%, et al., 19986(: Abs = ε bc
41(26
)#o com!ounds in a so"ution, for eam!"e, #hen ana"yed #i"" %i$e different absorbance maima 4Cma6( )his #i"" enab"e their simu"taneous determination in the so"ution( Since the absorbance 4 Abs6 of a so"ution containin% both com!ounds at !articu"ar #a$e"en%th are additi$e, the concentration 4 c 6 of each com!ound can be determined by choosin% t#o #a$e"en%ths 4C 1 and C 26 on #hich to obser$e the Abs( Abs λ 1= ε λ 1 bc 1+ ε λ 1 bc 2 Abs λ 2 =ε λ 2 bc1 +ε λ 2 bc 2
41(36
)he %enera" obecti$e of this e!eriment is to em!"oy 7is s!ectro!hotometry instrumentation( S!ecifica""y, it aims 416 to !erform basic !rocedures in e$a"uatin% condition and !erformance of a 7isib"e s!ectro!hotometer, 426 to determine and com!are the absor!tion s!ectrum of the $arious so"utions, 436 to i""ustrate the "inear re"ationshi! bet#een absorbance and concentration at a !articu"ar #a$e"en%th, 4+6 to i""ustrate de$iations from &eer's La# !"ot, 4*6 to e!erience the use of a doub"e beam s!ectro!hotometer and5or a scannin% 7$isib"e s!ectro!hotometer, 46 to fami"iarie the s!ectro!hotometric ana"ysis of a mu"ti7com!onent system, and 46 to determine the concentrations of unno#n substances(
II.
Me#o(o0o/y
Initia""y, the instrument #as ca"ibrated to chec if it is o!eratin% !ro!er"y( )he instrument used in this e!eriment #as /ini712+0 S!ectro!hotometer, #hich is a sin%"e beam s!ectro!hotometer 4Fi%ure 1(26(
i/re 1.1. UV Mi"i-1'2+ S3ec#ro3o#o)e#er4 a si"/0e !ea) s3ec#ro3o#o)e#er. For the absor!tion s!ectrum and &eer7Lambert's La# of Linearity, the fo""o#in% ste!s #ere fo""o#ed( First, the instrument #as turned on and a""o#ed to #arm u! for 30 minutes( )hen, 2* mL of 0(0* /, 0(10 /, 0(1* /, and 0(20 / so"utions #ere !re!ared usin% the 0(*0 / stoc so"ution of o, -i and ./n +( Efter that, the absorbance of the so"ution #ith hi%hest concentration, 0(20 /, #as read from 00 to 380 nm( )he absorbance a%ainst #a$e"en%th #as !"otted to determine the #a$e"en%th of maimum absor!tion, C ma( )hen, the absorbance of the so"utions and unno#n #as determined at the determined C ma( For the simu"taneous ana"ysis of t#o7com!onent system, benoic acid and caffeine #ere used( Fifty mi""i"iters of each standard so"utions #ere !re!ared( )he concentrations of benoic acid so"utions #ere 2(00 m%5L, +(00 m%5L and 8(00 m%5L( n the other hand, the concentration of caffeine so"utions #ere +(00 m%5L, 8(00 m%5L and 1(00 m%5L( )he reAuired amount of stoc so"ution #as obtained, fo""o#ed by the addition of 0(10/ >" and di"ution #ith disti""ed #ater( E"so, *0(0 mL of a miture of +(00 m%5L benoic acid and 8(00 m%5L caffeine #as !re!ared( )he absor!tion s!ectrum of +(00 m%5L benoic acid so"ution, 8(00 m%5L caffeine so"ution, and the benoic acid7caffeine so"ution #as determined from 3*0 to 210 nm( sin% the obtained s!ectra, t#o a!!ro!riate #a$e"en%ths for a &eer's La# !"ot #as chosen( Last"y, the concentrations of benoic acid and caffeine in three different soft drin sam!"e #ere determined( )he sam!"e used #as S!rite( )#enty mi""i"iters of each sam!"e #ere heated in a beaer to e!e" 2( Eny !artic"es #ere remo$ed $ia fi"tration before it #as coo"ed in room tem!erature( E 0(*0 mL of each sam!"e #ere !"aced in a 2* mL $o"umetric f"as( &efore it #as di"uted #ith d> 2 u! to mar, 0(10 mL >" so"ution #as added( Instead of usin% the sam!"e,
d>2 #as used as method b"an( )he %T or Abs #as read and recorded( sin% the ca"ibration cur$e, the concentrations of benoic acid and caffeine #ere ca"cu"ated(
III.
Res0#s a"( 5iscssio"
)he co"ors of coordination com!ounds are com!"ementary in nature( If "i%ht !assed throu%h a substance and absorbs the co"or red, for eam!"e, the co"or that can be obser$ed #ou"d be %reen, since it is the com!"ementary co"or of oran%e( )he co"or #hee" i""ustrates the com!"ementary co"ors 4Soo% et a"(, 19986(
i/re 1.'. Te co0or 6ee0. )he amount of "i%ht absorbed by a substance can be measured in terms of transmittance and absorbance( Ebsor!tion occurs #hen "i%ht !asses throu%h an absorbin% sam!"e, and the absorbin% s!ecies absorb some of the "i%ht #hi"e transmittin% the rest( Incident radiation, Po, enters the sam!"e #hich then absorbs some of the radiation and transmits others this radiation can be "abe"ed as P ( )he transmittance is then measure by tain% the ratio of the difference in amount of "i%ht 4Soo% et a"(, 19986( S!ectro!hotometers are s!ectrometers that a""o# measurement of the ratio of the radiant !o#ers of t#o beams, a reAuirement to measure absorbance( )hey can be c"assified either as doub"e7beam or sin%"e beam instrument(
i/re 1.$.a. B0ock (ia/ra) o7 a si"/0e-!ea) s3ec#ro3o#o)e#er.
i/re 1.$.!. B0ock (ia/ra) o7 a (o!0e-!ea) s3ec#ro3o#o)e#er. In sin%"e7beam instruments, measurin% the transmittance of the sam!"e and of the so"$ent at each #a$e"en%th is reAuired to obtain a s!ectrum( E contro" corres!ondin% to the so"$ent a"one or a so"ution containin% the rea%ents of the measurement is !"aced in the o!tica" !ath( )hen, it is re!"aced by the so"ution !re!ared from the sam!"e of unno#n concentration( It shou"d be noted that it does not contain the com!ound to be measured( Its ad$anta%es inc"ude the !rice and ru%%edness( >o#e$er, sin%"e beam instrument is not !ractica" for recordin% s!ectra since manua""y adustin% the #a$e"en%th and reca"ibratin% the s!ectro!hotometer is tedious and time7consumin%( E"so, the accuracy of a sin%"e7beam s!ectro!hotometer is "imited by the stabi"ity of its source and detector o$er time 4ar$ey, 20006( In doub"e beam instrument, the "i%ht source can be !assed 4simu"taneous"y6 throu%h both a reference and a sam!"e ce""( Ghen the "i%ht !assed throu%h, it #i"" be s!"itted into t#o se!arate beams throu%h the aid of the cho!!er( /oreo$er, the "i%ht out!ut of the instrument is adusted to 100B transmission 40 B absorbance6( )his instrument a""o#s the correction of the sam!"e absorbance si%na" for non7ana"yte absorbance 4
"i%ht at one7ha"f the !ea maimum( It is re"ated to the !hysica" s"it7#idth of the monochromator #hich in turn affects the reso"ution ca!abi"ities of the instrument(
i/re 1.2. Gassia" 5is#ri!#io" o7 Li/# I"#e"si#y.
For the absor!tion s!ectrum and &eer7Lambert's La# of Linearity, 0(0* /, 0(10 /, 0(1* /, and 0(20 / so"utions #ere !re!ared usin% the 0(*0 / stoc so"utions of o, -i and ./n +( )he absorbance of the so"ution #ith hi%hest concentration #as read from 00 to 380 nm( )his #as done to determine #a$e"en%th of maimum absor!tion, C
( Efter obtainin% the C ma, the
ma
absorbances of different so"utions #ere determined(
Ta!0e 1.1. Te (e#er)i"a#io" o7 #e "k"o6"s a"( #e co"s#rc#io" o7 #e s#a"(ar( cr8e.
1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0.04
R² f(x)==1 4.5x
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
0.22
i/re 1.9. Te s#a"(ar( cr8e 7or #e so0#io"s o7 Co!a0#
0.3 0.25 0.2
f(x) = 1.24x # 0 R² = 1
0.15 0.1 0.05 0 0.04 0.06 0.08
0.1
0.12 0.14 0.16 0.18
0.2
0.22
Es sho#n in Fi%ure 1(*(, the standard cur$e sho#s a "inear re"ationshi! bet#een the coba"t and the absorbance( )he ca"cu"ated $a"ues for the < 2 and the s"o!e 4m6 are 0(999+ and +(+98, res!ecti$e"y( )his <
2
$a"ue im!"ies that the re"ationshi! bet#een the %"ucose concentration and the absorbance for the standard is "inear enou%h to be uti"ied as a standard . The equation of the line was obtained as
y = .!"66666#$. Thus% the &al&ulated &on&ent'ation fo' the unnown samle of &obalt is 0.01#56* (1030.656 mg/L) with a e'&ent e''o' of 3.0656+.
i/re 1.:. Te s#a"(ar( cr8e 7or Nicke0. Es sho#n in Fi%ure 1((, the standard cur$e sho#s a "inear re"ationshi! bet#een the nice" concentration and the absorbance( )he ca"cu"ated $a"ues for the < 2, s"o!e 4m6 and the y7 interce!t are 0(9992, 1(2380 and 0(00+*00, res!ecti$e"y( )his < 2 $a"ue im!"ies that the re"ationshi! bet#een the nice" concentration and the absorbance for the standard is "inear enou%h to be uti"ied as a standard( )he eAuation of the "ine #as obtained as y H 1(238000 0(00+*00( )hus, the ca"cu"ated concentration for the unno#n sam!"e of nice" is 0(09*/ 4+*93(8+ m%5L6 #ith a !ercent error of +*,838B(
4.02 4 3.98 3.96 3.94 3.92 3.9 3.88 3.86
f(x) = 3.45x # 3.95 R² = 0.42
0
0.01
0.01
0.02
0.02
0.03
i/re 1.%. Te s#a"(ar( cr8e 7or 3er)a"/a"a#e. /ean#hi"e, for the ./n + so"ution, the standard cur$e sho#s a not "inear re"ationshi! bet#een the !erman%anate concentration and the absorbance( )he ca"cu"ated $a"ues for the < 2, s"o!e 4m6 and the y7interce!t are 0(+22+, 3(++** and 3(9+2, res!ecti$e"y( )his < 2 $a"ue im!"ies that the re"ationshi! bet#een the !erman%anate concentration and the absorbance for the standard is not "inear enou%h to be uti"ied as a standard( )he eAuation of the "ine #as obtained as y H 3(++**+* 3(9+2+0( -o unno#n sam!"e for the ./n +( )he different assum!tions of this "a# are: 416 the incident radiation is monochromatic 426 the absorbin% s!ecies act inde!endent"y of each other in the absor!tion !rocess 436 the absor!tion occurs in a $o"ume of uniform cross7section 4+6 ener%y de%radation is ra!id and 4*6 the refracti$e inde is inde!endent of concentration 4Soo% et a"(, 19986( >o#e$er, &eer's La# has "imitations( First, it is $a"id on"y for "o# concentrations 4J0(01 /6 of ana"yte( Et hi%her concentrations, the e"ectrostatic interactions bet#een !artic"es of ana"yte may chan%e the $a"ue of K( E"so, chemica" de$iations occur #hen the absorbin% s!ecies under%oes association, dissociation, or reaction #ith the so"$ent to %i$e !roducts that absorb different"y from the ana"yte( Edded to these are the instrumenta" de$iations such as !o"ychromatic radiation and stray "i%ht( For eam!"e, a !o"ychromatic "i%ht reaches the sam!"e ce""( )his may resu"t to either a ne%ati$e or a !ositi$e de$iation( )he s!ectra" band #idth of the instrument has therefore a %reat effect on the Aua"ity of the "i%ht that !asses throu%h the sam!"e( E hi%h s!ectra" band #idth #i"" "ead to a "ess accurate absorbance measurement( Stray "i%ht, on the other hand, #i"" "ead to a decrease in the absorbance of the sam!"e since the detector #i"" detect a %reater amount of "i%ht that is transmitted 4>ar$ey, 20006( Furthermore, the s"o!e of the &eer's "a# !"ot can be determined e$en if the on"y a$ai"ab"e data is absor!tion s!ectrum and the no#"ed%e that it obeys &eer's "a#( =i$en that the &eer's "a# the !"ot is "inear, the $a"ue corres!onds to the concentration of the so"ution #hi"e the y $a"ue corres!onds to the absorbance, the s"o!e #i"" be eAua" to y di$ided by ( Since the !ath "en%th is constant, the s"o!e #i"" be eAua" to the absor!ti$ity 4>ar$ey, 20006( )he obtained maimum #a$e"en%th of o, -i and ./n + #ere *12 nm, 39+ nm and *29 nm( )his means that at these #a$e"en%ths, a"" the co"ors of the s!ectrum are absorbed ece!t the co"ors that are emitted in each corres!ondin% so"ution 4red for o, %reen for -i and Dar
io"et for !erman%anate6( )hese corres!ondin% #a$e"en%ths #ou"d be desirab"e for the ana"yses of so"utions #ith concentrations 0(1* o and 0(** / o because there is maimum absor!tion at these #a$e"en%ths( )his is a"so #here the reso"ution and the sensiti$ity are at the hi%hest 4E""en, 2008 >ar$ey, 20006(
)he "ast !art of the e!eriment in$o"$ed the simu"taneous ana"ysis of t#o7com!onent systems( )he absorbance of the !re!ared benoic acid and caffeine standards #ere measured at 23 nm and 230 nm( )hese #a$e"en%ths are the #a$e"en%ths #here the so"utions absorb most(
Ta!0e 1.'. S))ary o7 )easre)e"#s 7or #e ;T a"( *!s o7 !e"
oncentration
benoic acid
, m%5mL 0
affeine
&"an /iture
CH23 nm Ebs
CH230 nm Ebs
0
0
2
0(0*1
0(20
+
0(083
0(+19
8
0(1*2
0(81
0
0
0
+
0(312
0(01
8
0(*2
0(219
1
1(0*2
0(*98
777 777
0(011++10+3 0(**
0(022239* 0(9*
1.2 1
f(x) = 0.06x # 0.02 R² = 1
0.8
Caffene *near (Caffene) +en,oc !cd *near (+en,oc !cd)
0.6 0.4 0.2 0
f(x) = 0.02x # 0.01 R² = 0.99 0 2 4 6 8 10 1 2 1 4 16 18
i/re 1.,. P0o# o7 a!sor!a"ce 8s co"ce"#ra#io" a# '%$") 7or ca77ei"e a"( !e"
1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
f(x) = 0.11x - 0.01 R² = 1 f(x) = 0.04x - 0.05 R² = 0.97
0
2
4
6
8
10 1 2 1 4 16 18
Caffene *near (Caffene) +en,oc !cd *near (+en,oc !cd)
i/re 1.=. P0o# o7 a!sor!a"ce 8s co"ce"#ra#io" a# '$1 ") 7or ca77ei"e a"( !e"
Ta!0e 1.$. 5a#a 7or #e a"a0ysis o7 #e co"ce"#ra#io" o7 !e"
at 230 nm
Benzoic acid
0.0185
0.1078
Caffeine
0.0647
0.0385
From the data %i$en, the t#o7systems of eAuation can no# be constructed as fo""o#s: Abs λ 1= ε λ 1 bc 1+ ε λ 1 bc 2 Abs λ 2 =ε λ 2 bc1 + ε λ 2 bc 2 71
0(29 H 40(018* L m% cm71641cm6c1 40(0+ L m% 71 cm71641cm6c2 0(**3H 40(108 L m% 71 cm71641cm6c1 40(038* L m% 71 cm71641cm6c2 )he concentration of benoic acid 4 16 in the sam!"e is determined as 202(2* m%5L 41(0310 73 /6( /ean#hi"e, the concentration of caffeine 4 26 in the sam!"e is determined as 1*0(++* m%5L 41(232010 73 /6 #ith a !ercent error of 71(3B( )he "iterature $a"ue for caffeine in mountain de# is 1*2(*++* m%5L 4E&E, 201*6( )he !ossib"e sources of errors in this e!eriment are the inc"usion of !ossib"e contaminants, the im!ro!er readin% of the absorbance #hich can cause contaminations and the non7remo$a" of !artic"es in the benoic acid7caffeine sam!"e(
IV.
Sa)30e Ca0c0a#io"s
Determination of the Unknown concentration
Concentration =
absorbance −( y −intercept ) 0.079 = =0.01756 M slope 4.4987
Percent Error%
%error=
10 30.656−1000 actual −theoretical x 100= x 100=3.0656 theoretical 1000
Concentration of benzoic acid and caffeine
sin% ?Auation 1(3, Abs λ 1= ε λ 1 bc 1+ ε λ 1 bc 2 Abs λ 2 =ε λ 2 bc1 + ε λ 2 bc 2 71
0(29 H 40(018* L m% cm71641cm6c1 40(0+ L m% 71 cm71641cm6c2 0(**3H 40(108 L m% 71 cm71641cm6c1 40(038* L m% 71 cm71641cm6c2
(
c 1=[ benzoic acid ] =
c 2=[ caeine ] =
V.
(
3.6454 mg
L 3.0089 mg L
)
(
)(
1g
)
1 mol
1000 mg 122.12 g
1g
)
1 mol
1000 mg 194.19 g
¿ x 50 =1.6603 x 10− M 3
¿ x 50=1.2320 x 10− M 3
Co"c0sio"s In the e!eriment, the absor!tion s!ectrum and &eer7Lambert's La# of "inearity #ere
studied usin% o, -i and ./n + so"utions( )he "inear re"ationshi! bet#een absorbance and concentration at a #a$e"en%th #as i""ustrated and de$iations from &eer's "a# !"ot #ere a"so determined( sin% the absor!tion #a$e"en%th, the maimum #a$e"en%th of o, -i and ./n + is *12, 39+ and *29 nm, res!ecti$e"y( )hese #a$e"en%ths #ere used to determine &eer's La# !"ot for o so"utions that #as further used to determine the concentration of the unno#n so"utions( )he concentration of the unno#n so"utions for o and -i #ere 0(01* and 0(09* /, res!ecti$e"y( Last"y, s!ectro!hotometric ana"ysis of a mu"ti7com!onent system #as done to ca"cu"ate the concentration of benoic acid and caffeine in the miture( )he mo"ar absor!ti$ities of both com!ounds #ere obtained from the &eer's La# !"ot( For benoic acid, K H 0(018* 4at 23 nm6
and 0(108 4at 230 nm6 #hi"e for caffeine K H 0(0+ 4at 23 nm6 and 0(038* 4at 230 nm6( sin% these !arameters, the concentrations #ere ca"cu"ated as 1(0310 73 and 1(232010 73 / for benoic acid and caffeine, res!ecti$e"y(
VI.
Re7ere"ces
E/?E<? D( 2000( /odern Ena"ytica" hemistry( 1 st ed( -e# or: /c=ra#7>i""( <?SSE F, <?SSE E( 200( hemica" Ena"ysis: /odern Instrumentation /ethods and )echniAues( 2 nd ed( Gi"ey( S.= DE, >LL?< FM, -I?/E- )E( 1998( Princi!"es of Instrumenta" Ena"ysis( 2 nd ed(
