2.
CoLORIIM67RV AND
SP6CTROPHoToM ETRY
Colooimety It is the most tommon
analytica theehniue used tn biochunvial
estimaton tn chemical la bootafouy I
oh (olaur.
tntev es quati tatie examinahontolouumutnal,
A substane to be estinatd
must be Colowed o it s hould be able
to foum chomoqens Ctoleuxcd Complxes),?
tisough the additon of teagen b.
CoLORIMETER
’ TE s an cnstoument that meau uore thu
amount of lght absosbed by Colowted
Solvedh ts fomed by subrtan es
ond analyu's.
Pxinuplus - lohen a monochemahic ight
peses thotoug h a colouted solution,
Some specie oavelength o ight ate
absorbed which s ste lated to the
Co lour tntináity
())Beer's law:- When a mono chomaht
pusses thotoug h a toloured solutos
i¡ht
amount of u¡ he toans mited decreaste,
Cx ponen tially with inrase io
Contentra tion o Colowted Solution
i.e., the a mount of Wght abtoUte d b
a solu Hion is dionetly pioportonal
to Conuen tration of slbshute in
Colowted soluton
Aac
i) Lambertt's lay- When a ay of
Monochomahic i'ght passes thoto ugh. an.
medam, its in tensify cdecreauet
absorbing
Cxponuntialy asthe length o 'the path.
thätoug h th lht absorbing mafeoral
Ln xease
by tombinin g obove equaions ,oe get
Kcl
K’ lonstant o< to loured sebsfanta /tola
fo tests,
AK x C,xL
for standoxd
As.
As Cs
G- Ar
A
Conertaahon
Test Soluhon
Absosbant
Test e
Concenhation
X. D, shandaxd
Absosbanxe
Stún davd
Conien tahon g Test Absoxbcun e d toncenboho
Iest
Soluhon pes l00 me' Absoxbante o
Transmlane I
Lo
Asbo:bane lA)
1lo-A
log 1-( abe)
-log 7 - - f-aby
fet - Op T - oD 98
Conenhaton
pes looml
vl00
(ompornunó o Colouindes
’ Soute!- ungafent lamp whuih emb
Continuow Spetn
Wavekung fh.
’ ilter;- Mecle o colowed gla filtea
au wsel fot selething length
Uaveng ths. Fltes sed is alw ald auys
Copli mun tany h to lour to colsun
Ssluhon
Supl Cell (Cavette)-: Tt can be tound (or)
Squece mad o leus e) plaatie. Tt holdu
Soluton wohose absoothane is to be masued.
Thu light path must be Kept constant in
oxtdos to have Been-Lambert ao oleyed
'e., odth ed cuveta, apotesent path f
ight a kepi consfant.
’ Pbotodeetox :- The puypose o photod etceton.
which s stmple semicon ductor dv'e
is to convett tans mited ocliant
enenqy tn to cn egivaent cUnount o
eletn enqy, measwud in a
gavanometex.
Ruad outi:- These aoe braadly eleinhied
as mefes, seceoxdrs, or duqi bal
sianal foms, The de tectoy galuano meter
18
Scale:
d) Absorbnle bcau - Giadaated from O-)
ui) Transmtance Scalu. Gatated Fom 0
loo7.
Comporunts o Colonineler
L
LPotoells balaromtes
Tungln gut Cardusi Cuveite
lamp Filte
Dev iation s from Be er's kao !=
)If an analytt ox Solu hon s
hgh tonentahoo, Beer's
present in
lauo is not obeyed.
i) Tf inciclurnt tadiant enorgy ia nob
nonochiomatic
ci ) rt soluent absonp hon is Signihiant
Compay ed to solute aboapfion
Civ) I?I absorbant» a flous(ent
Soluhion is being heasubel
In co lovum et estmuhon.it t's
neces cony to prepase 8 soluhons
6) Blank CB) - Some ot tnuclunt
enegy is otellected back bythe
Swfate of the luvettu . Thse
fa ctos must be elimincaud. I, one
i's to tonsicbn only absoxphon o
this
the tompound i.e, solute', blunk/
0Can. be don e by using a
ate fetente soluhon"tontuning
the to luhon to
cvenyhing cxrpt It Can be sumpl
be mecs ured.
oaagent blonk oroY dustilad
OF
watr
Exampl: Alkaire picarat is usel as Juagent
blaunK in creahinune es timaton
ci) Standad Cs:- suhos to a soleutionbut
Same Substane as of a test
with known oncentration
(o) stoch Sfandoaud- It r's contentaud
solution Khown Contenta tion
(6) Wotting stundaacl:- It is prepured
fom sfock 3tandad atby dilaton
() Test (TJ: Test solution is made by
treting a spuiftcvolume of test
sampl& with' Oteoqcn ta.
Applitatiors
I: It is widely used in hospital and
tabo atoiy for estimation biothemita!
fluids ike plauma, Csf, uhñe and sem
d It is also usecl to quntitative estimation
: of the sexunm lomponent
qleuose, psotein, et
Buaclventage
’ Can' t be
Compounds
used fox Coloualess
-’ Doesn't ooK in UVor IR oruglon,
’’ Less sensitive | Limited ounge
tilters availabe
Simila coloun
intervenen9
Substantes can proclue colou
SPECTRD PHO10 M ETER
otefers to a
Pinciple: Specbophoto mutex se7ers
tech niguu that meas uustes
absorbane o the colowt f Soluton
fhs ohich is
at specibt'e oavelengconcentration
Propovfion al to the
of solution or compound : The
tnshument s used to neasuste
aborp tion af bo th UV s well an
Viss ible o1eqton.
Corgonenls of Spechophotondry
Detectr
0:20
Light Monuchonato Digtil
lo linato Sut
Source (ens)hhbee
Sample Meler
0Solulion
A spestophofamater haa all Co mpo
a basic photodetecbre toloxi metou oith
More sop his hicton
" Light is sepeatd intt a Continos
specfsum of ave lng ths anc pasel
though solution.
" In co lo ou'meln , wovengths one
:Colou cte goouptud togth et
" UV
uV iyht is pazhially absonbed by thu
and hinte specbophotome tur
Lvetles wte sed foy 0V jtange.
’’ Clasikied in to d type :
)Single beam specoopho to meter - lo mesus
Hhein tensity of in cicd en t iqht,the sumpl
must be Semoved 8o that the efene
can be. plated each time .Thi's type of
spechophoométor is usually lew expensive
and less complicated.
2) Ooublu- beam Speetropho tome her - Tp ths
typé, before it eaches the sam ple lig ht
Sowste , it splits into two sepeta tod beams. bom
thee,ome passes thotough the Sample and
Second one is used foY ote7erente This gives
an advauntage becawe the otcfeune at.
ot eadlking aample taoabing Can take plate
Same ftme.
D;ffesences
CoLDRIMETER SPELTRoPH070 ME7ER
’ Limitd fos v isible ’UV,IR Y Vibible vtegio
tegionoE:340 mn
’ Cheapa Vety o
’ Tuco digit oeading fou diqit te ading
after deimal point affey deimal point
’ o r e sensitive
’less sensitive
’ lesS flters ase Pisns ase ul ed.
used
u t is usec ohich
’ Gula test tbes
Cuvcttes ase used
quaty
dloesno t absorb 340 mm
whth absorbs 34o mm
light lampt ate
au welHalogen
lngsan lamp
>Tungstin use
’ lan't se suufl specij'c
fltes
flter
Kineh'c
’ Can't do Kinetie ’ lan do
hethod.dolotl muthod
