uant|tat|ve

App||cat|on of
UV-v|s|b|e
Spectroscopy



Prepored by :
Kadri Uvvah Kadri Uvvah Kadri Uvvah Kadri Uvvah
(Department of ua||ty Assurance)
S]09]07
uant|tat|ve App||cat|on of UV-v|s|b|e Spectroscopy
Introduct|on
1

CuallLaLlve analysls esLabllshes Lhe chemlcal ldenLlLy of Lhe specles ln Lhe sample.
CuallLaLlve analysls deLermlnes or lndlcaLes Lhe amounL of each subsLance or analyLe ln
a sample. AnalyLes are Lhe componenLs of a sample LhaL are Lo be deLermlned.
1he resulLs of a Lyplcal quallLaLlve analysls are compuLed from Lwo measuremenLs :
a) 1he mass or Lhe volume of sample belng analysed
(AbsorpLlon/Lmlsslon/1ransmlslon).
b) 1he second ls Lhe measuremenL of some quanLlLy LhaL ls proporLlonal Lo Lhe
amounL of Lhe analyLe ln Lhe sample such as mass, volume, lnLenslLy of llghL, eLc.
SpecLroscoplc meLhods are based on measuremenL of Lhe lnLeracLlon beLween
elecLromagneLlc radlaLlons and analyLe aLoms or molecules or on Lhe producLlon of such
radlaLlon by analyLes.
1he general Lerm for chemlcal analysls Lhrough measuremenL of radlaLlon ls
absorpLlomeLry.
SpecLrophoLomeLry ls a dlvlslon of absorpLlomeLry LhaL ls concerned speclflcaLlon Lo Lhe
use of Lhe specLrophoLomeLer.
SpecLroscopy ls Lhe sLudy of maLLer by lnvesLlgaLlng llghL, sound or parLlcles LhaL ls
emlLLed, absorbed or scaLLered by Lhe maLLer under lnvesLlgaLlon. SpecLroscopy may
also be deflned as Lhe sLudy of Lhe lnLeracLlon beLween llghL and maLLer.

uant|tat|ve ana|ys|s
3

1hls alms Lo deLermlne Lhe concenLraLlon and amounL of drug ln sample soluLlon and Lhus Lhe
percenLage purlLy can be deLermlned.
lor Lhe quanLlLaLlve analysls of drugs by colorlmeLry, Lhe followlng characLerlsLlc musL be
known.
1) LlghL absorpLlon characLerlsLlc - l.e \max Lo be known. 1hls wavelengLh has Lo be used
because of hlgh senslLlvlLy, speclflcaLlon and accuracy.
2) valldlLy of 8eer's law - l.e ConcenLraLlon range ln whlch Lhe sysLem obeys llnearlLy (eg
10-30g/ml, 2-10g/ml, eLc.)
3) SolvenL, reagenL, oLher condlLlon, L(1/1cm) - lf avallable from llLeraLure eLc.

1he severa| quant|tat|ve methods are,
A. MeLhods for Slngle ComponenLs.
8. MeLhods for MulLl-componenL sysLem.



A. Methods for S|ng|e Components
2,3
:

a) Us|ng L(1]1cm) va|ues : 1hls meLhod can be used for esLlmaLlons from formulaLlon or
raw maLerlal, when reference sLandard ls noL avallable. L(1/1cm) means Lhe
absorbance of 1 w/v soluLlon, uslng a paLhlengLh of 1cm. L(1/1cm) aL a wavelengLh ls
a consLanL value for each drug and can be seen ln harmacopoela and sLandard books
on Lhe sub[ecL.

Lg :- stimotion of Porocetomo/ - Pere L(1/1cm) for araceLamol aL 237nm ls 713.

1he concenLraLlon can be calculaLed by Lhe formula : C = A/b
Where , C - ConcenLraLlon of sample
A - Absorbance of sample (0.370)
- LxLlncLlon value (713)
b - aLhlengLh (1cm)

C = 0.370/713 x 1 = 0.000317 gm/ml.

1o flnd Lhe purlLy of paraceLamol LableLs, Lhe followlng formula can be used.

urlLy = Cbserved Absorbance 100
'L' value
x
ConcenLraLlon

ln Lhls example, Lhe absorbance of 0.00092 w/v soluLlon aL 237nm was 0.63, Pence

urlLy = 0.63 x 100 = 98.81 w/w
713 0.00092


b) L(1]1cm) |s not ava||ab|e, but keference standard ava||ab|e : ln Lhls case L(1/1cm)
value can be deLermlned by observlng Lhe absorbance aL dlfferenL sLandard soluLlon and
Lhe average L value ls Laken. AfLer calculaLlng Lhe L value, Lhe meLhod as ln (a) can be
followed. AlLernaLlvely we can use any of Lhe followlng meLhods :


(l) S|ng|e Standard or D|rect Compar|son method : ln Lhls meLhod, Lhe absorbance of a
sLandard soluLlon of known concenLraLlon and a sample soluLlon ls measured. 1he
concenLraLlon of unknown can be calculaLed uslng Lhe formula :

A
1
= C
1
L

A
2
= C
2
L


Where, A
1
& A
2
- Absorbance of sLandard & sample.
C
1
& C
2
- ConcenLraLlon of sLandard & sample.
- Molecular exLlncLlon coefflclenL.
L - aLhlengLh (1cm)

Cn dlvldlng, we geL

A
1
/ A
2
= C
1
L / C
2
L

A
1
/ A
2
= C
1
/ C
2

C
2
= C
1
x A
2
/ A
1

Slnce C
1
, A
1
and A
2
are known, C
2
can be calculaLed. lrom Lhe concenLraLlon of Lhe
sample, Lhe amounL as well as purlLy of Lhe drug can be calculaLed.

Lg : stimotion of uic/ofenoc 5odium
Sample absorbance A
2
- 0.329
SLandard absorbance A
1
- 0.413
ConcenLraLlon of SLandard C
1
- 10 g/ml
ConcenLraLlon of Sample C
2
- ?

C
2
= C
1
x A
2
/ A
1
= 10 x 0.329 / 0.413
= 7.96 g/ml


(ll) Ca||brat|on curve method or Mu|t|p|e standard method : ln Lhe slngle sLandard
meLhod, when error ls lnLroduced ln preparlng Lhe soluLlon or measuremenL of
absorbance, Lhe error ln resulLs would be greaLer. 1o ellmlnaLe or Lo mlnlmlze Lhls
error, we can use callbraLlon curve meLhod. CallbraLlon deLermlnes Lhe relaLlonshlp
beLween Lhe analyLlcal response and Lhe analyLe concenLraLlon. 1hls ls accompllshed
by Lhe use of chemlcal sLandards.

Methodo|ogy : ln callbraLlon curve meLhod, a callbraLlon curve ls ploLLed uslng
ConcenLraLlon vs Absorbance value of 3 or more sLandard soluLlons. A sLralghL llne ls
drawn elLher Lhrough maxlmum no. of polnLs or ln such a way LhaL Lhere ls equal
magnlLude of poslLlve and negaLlve errors. 1hls meLhod ls called as Llne of besL flL.
1he llne may or may noL be pass Lhrough orlgln.





8. Methods for Mu|t|component system
2,3
:

1. S|mu|taneous mu|t|component method :
lf a mlxLure of Lwo componenLs a and b are presenL ln x and y w/v respecLlvely, by
measurlng Lhe absorbance of mlxLure aL Lwo wavelengLh \
1
and \
2
, Lhe concenLraLlon or
amounL of componenLs a and b can be esLlmaLed. lf a, and a, and b, and b, are
L(1/1cm) values deLermlned aL \, and \, for componenL a and b respecLlvely.
k
1
k
2
x w]v of comp. a a
1
a
1
y w]v of comp. y b
1
b
2
M|xture S
1
S
2

Slnce 100S
1
= a
1
x + b
1
y
100S
2
= a
2
x + b
2
y
x w/v = 100 [(b
1
S
2
- b
2
S
1
) / (b
1
a
2
- b
2
a
1
)]

y w/v = 100 [(a
1
S
2
- a
2
S
1
) / (a
1
b
2
- a
2
b
1
)]

Note
4
1he lnLenslLy of Lhe absorpLlon ls proporLlonal Lo Lhe number, Lype and locaLlon of
colour absorblng sLrucLure ln Lhe molecule.
A meLhod based on measuremenL aL wavelengLhs.
1wo dlsslmllar chromophores musL necessarlly have dlfferenL powers of llghL absorpLlon
aL some polnL or polnLs ln Lhelr absorpLlon specLra.
lf measuremenLs are made on each soluLlon aL such Lwo polnLs, a palr of slmulLaneous
equaLlon may be obLalned form whlch Lhe Lwo unknown concenLraLlons may be
obLalned.
llrsL, lL ls necessary Lo selecL Lwo polnLs on Lhe wavelengLh scale where Lhe raLlo of Lhe
molar absorpLlvlLles ls maxlmal.
nexL, lL ls necessary Lo calculaLe Lhe molar absorpLlvlLy for each componenL aL each
wavelengLh selecLed.
1hus, Lwo slmulLaneous equaLlons may be wrlLLen :

C
1
(
1
) \
1
+ C
2
(
2
) \
1
= A \
1

C
1
(
1
) \, + C
2
(
2
) \
2
= A \
2

1he equaLlon are solved for Lhe concenLraLlon of each componenLs.
SlmulLaneous deLermlnaLlons resL on Lhe assumpLlon LhaL Lhe subsLances concerned
conLrlbuLe addlLlvely Lo Lhe LoLal absorbance aL an analyLlcal wavelengLh.



2. Der|vat|ve spectrophotometr|c method :
ln Lhls meLhod, speclal lsolaLlon ls achleved raLher Lhan chromaLographlc lsolaLlon. ln a
derlvaLlve specLrum Lhe changes ln absorbance wlLh respecL Lo wavelengLh vs
wavelengLh ls recorded. 1
sL
or 2
nd
derlvaLlve specLrum ls recorded and Lhe characLerlsLlc
peak for Lhe lndlvldual componenL can be ldenLlfled and quanLlfled, uslng a callbraLlon
curve of pure subsLances.

Note
4
uerlvaLlve specLrophoLomeLrlc ls of use ln quanLlLaLlve analysls Lo measure Lhe
concenLraLlon of analyLe whose peak ls obscured by a larger overlapplng peak due Lo
someLhlng else ln a sample.
1hls meLhod can be used Lo clarlfy absorpLlon band ln mosL complex uv specLra.
uerlvaLlve specLra are llLerally Lhe derlvaLlon of Lhe normal specLra.
ln a derlvaLlve specLrum Lhe ablllLy Lo deLecL and Lo measure mlnor specLrum feaLure ls
conslderably enhanced.
A shlfL ln absorpLlon specLrum of Lhe deslred analyLe can be accompllshed by converLlng
lL lnLo a derlvaLlves.
1he sLrucLure change wlll resulL ln a specLra change. Lg : ueLermlnaLlon of Lhe amounL
of pseudoephedrlne ln an ellxler conLalnlng pseudoephedrlne dexLromeLhorphane and
Lrlprolldlne (13mg, 10mg and 1.23mg respecLlvely).


3. D|fference spectrophotometr|c method :
1hls meLhod ls especlally useful Lo quanLlfy a subsLance when lnLerferlng specles are
presenL. 1he prlnclple ls LhaL Lhe absorbance dlfference beLween Lwo forms of Lhe same
drug ls measured. 1he absorbance dlfference ls achleved by uslng pP manlpulaLlon uslng
a palr of buffers. 1hls meLhod ls used Lo quanLlfy drugs ln blologlcal flulds.

Note
4
ln dlfference specLrophoLomeLrlc a componenL ln a mlxLure ls analysls by carrylng ouL a
reacLlon whlch ls selecLlve for Lhe analyLe.
ulfference specLrophoLomeLrlc lnvolvlng Lhe chemlcal modlflcaLlon of Lhe
specLrophoLomeLrlc proflle of Lhe analyLe alone ln Lhe presence of lnLerference.
1he analyLe may be modlfled by alLeraLlon of lLs or Lhrough chemlcal reacLlon such as
oxldaLlon or reducLlon.
1hls brlng abouL a shlfL ln Lhe wavelengLh. Lg : 1he selecLlve alkallne shlfL of asplrln ls
used Lo deLermlne lL ln a preparaLlon also conLalnlng dexLropropoxyphene,
naphLhalene, naphLhalene sulphonlc acld and caffelne.
Caffelne, dexLropropoxyphene and naphLhalene sulphonlc acld wlll donoL undergo
appllcable alkallne shlfL where as asplrln does.
SpecLra of Lhe capable exLracLs ln 0.1M PCl & 0.1M naoP ln reference & sample cells
respecLlvely where absorbed.
1he absorbance aL 299nm ln Lhe sample cell was only due Lo asplrln.

keference

1. Skoog, West, no||er, Crouch : " Iundamenta| of Ana|yt|ca| Chem|stry" 8
th

ed|t|on - page no. ( 3-4, 192, 784, 78S, 789-790 )
2. 8.k Sharma : "Instrumenta| Method of Chem|ca| Ana|ys|s" 13
th
ed|t|on -
page no. ( 40, 41, S1, 88 )
3. kav|shankar : "1extbook of harmaceut|ca| Ana|ys|s" 3
rd
ed|t|on - page
no. ( 29-30 )
4. W|||ard, Mer|t : "Instrumenta| Method of An|ays|s" page no. ( 77, 78, 82 )