ACID DISSOCIATION CONSTANT OF AN INDICATOR DYE by Clarissa A.

Gomez CHE121L
ABSTRACT
Methyl red is the indicator dye used in this experiment. The maximum absorbance
obtained in its acidic form is 520 nm while 420 nm in its basic form. In the det
ermination of molar absorptivities using 10, 25 and 40 ml aliquots; the absorban
ce increases both in the acidic and basic solution while the volume of aliquots
escalates. Buffer solutions were prepared using the Beers and Henderson-Hasselbal
chs equation. The isosbestic point appears at near 420 nm which means that there
is only one wavelength where two species have the same molar absorptivity. By pl
otting the pH vs. absorbance of the prepared buffer solutions at two maximum abs
orbance (acidic and basic), it was deduced that the spectrum of each solution is
dependent on the pH range. The obtained pka of methyl red using these data shou
ld be compared to the theoretical pKa of methyl red which is 5.05 0.05. However,
this is not achieved due to data deficiency.
INTRODUCTION Indicators are weak organic acids (HIn) that change color when depr
otonated (In-). A few drops of indicator added to the analyte solution before th
e beginning an acid-base titration. When enough base titrant is added to the ana
lyte solution the equilibrium expressed in equation 1 will shift toward products
. HIn(aq) In (aq) + H+(aq) (1) be created by measuring the absorbance of a large
number of solutions of known concentration. An acid is a substance which dissoci
ates to produce hydrogen ions, H , when dissolved in aqueous solution. Once in s
olution, the H ion, which is simply a proton, immediately combines with water to
form the hydronium ion, H O . So The result is the formation of more of the dep
rotonated indicator (In ) and a corresponding color change of the analyte soluti
on (the endpoint). A good indicator for a specific acid-base titration has an en
dpoint with a pH at or near the pH of the equivalence point. In this experiment,
phenolphthalein indicator will be used for each titration. The pH range of the
color change will be observed and compared with the pH of the equivalence point
to determine if the indicator is an appropriate choice for each titration. The a
cid dissociation constant, Ka, is a measure of an acids strength. For weak acids
these values are less than 1 and typically so small that they are expressed with
scientific notation. Taking the negative log of the Ka results in more easily e
xpressed pKa values ranging from 0 to 14 for weak acids. A spectrophotometer sep
arates light into its separate colors. It is able to separate the light into col
ors because each color of light has a different wavelength than the other colors
. The spectrophotometer can shine a narrow band of wavelengths (essentially one
specific color) of light on a sample of substance and then measure how much of t
hat light is absorbed by the sample. Different colored substances absorb varying
amounts of specific wavelengths of light. Therefore, a spectrophotometer can be
Used to measure how much of a substance is present. A calibration curve is a gr
aph of absorbance, how much of a particular wavelength of light is absorbed, ver
sus concentration (Beers law). A calibration curve is specific to a particular su
bstance, and must when aqueous H appears in a chemical equation, it is understoo
d that the actual species exists as H O . An acid is classified as
3 + + 3 + + +
strong or weak depending on the extent to which the molecules of the acid dissoc
iate into H and its anion, A . A strong acid dissociates completely in water (e.
g., HCl dissociates virtually100% into H and its anion, Cl ), while a weak acid
dissociates only partially and forms very little H (e.g., only about 3% of the d
issolved molecules of acetic acid, CH COOH,
3 + + +
dissociate into H and the acetate anion, CH COO ). Weak acid dissociation can be
3
+
represented as a generic reversible reaction: HA(aq) H + A where HA is the weak
acid and A is the anion, or conjugate base, of HA. For this reversible reaction,
equilibrium constant can be defined:
+
(2) The equilibrium constant is called the acid dissociation constant or acid io
nization constant. If the undissociated acid (HA) is favored and the acid is wea
k, K is measurable
a
and will be much less than one. For strong acids, the dissociated products, (H a
nd A ) are so strongly favored, that [HA] approaches zero and K approaches infin
ity. Thus strong acids, of
a +
which there are very few, do not have values of K associated with them. Mathemat
ically, it is
a
A1 = A1HIn + A1In = 1Hin [Hin] + 1In [In] at 1 (5) The constant does not change with
oncentration, but will change at different wavelengths and/or with a different a
bsorbing species. So, at a second wavelength 2, the following equation for Beers L
aw would be true: A2 = A2HIn + A2In = 2HIn [HIn] + 2In [In] at 2 (6) There are now tw
o equations that can be used to determine the concentrations of the two differen
t colored unknowns that are present in the solution. These equations can be rear
ranged to allow determination of each concentration. [HIn] = ((A1 2In) ( A2 1In)) / (( 2
n 1HIn) ( 1In 2HIn)) (7) [In ] = ((A2 1HIn) ( A1 2HIn)) / (( 2In 1HIn) ( 1
found that the amount of light absorbed by a specific sample depends on three th
ings: 1) the concentration of the solution; 2) the distance the light travels th
rough the sample; and 3) the natural ability of the specific substance to absorb
light. Thus an equation can be written relating these things: A= bc (3) where A =
absorbance = molar absorbtivity how well the material absorbs light b = path
length through which the light passes c = concentration of the solution In gene
ral, the value for b will remain the same and the value for is constant for a sp
ecific chemical at a given wavelength of light. Because the general equation for
a straight line is y = mx + b (4) If A is graphed against c, the result will be
a straight line with a slope of b and a y intercept of zero. A solution that con
tains two colored species can also be analyzed using Beers Law (A= bc). Mathematic
ally, the concentrations of the two species can be determined if two simultaneou
s equations can be developed that relate the two. The two unknown concentrations
are determined by taking readings on each solution twice, using two different w
avelengths of light. The two species that are being studied in this experiment a
re the two colored forms of specific indicators that are weak acids. If there ar
e two species, HIn and In, in a solution with absorbance AHIn and AIn, respectiv
ely, the total absorbance is A = AHIn + AIn . If the sample path length is combi
ned with , then = b and Beers Law for a two component mixture becomes
The best wavelengths for the experiment are selected by measuring the absorbance
vs. wavelength for each of the pure substances. The actual wavelengths are then
chosen such that they will maximize the absorbance for each species. The values
for the four constants can be determined by doing Beers Law plots of absorbance v
s. concentration (of pure samples) at both wavelengths and determining the slope
s of the lines generated. The final set of measurements will be collected by mea
suring the absorbance of solutions of the weak acid at different pHs.
EXPERIMENTAL MEHOD
To obtain the spectra of acid and base forms of the dye, first these two forms w
ere prepared. For the acidic solution of the dye, 0.05M HCl was used. While, 0.0
5M borax was utilized solution for the basic solution. Then the maximum absorban
ces of the solutions were determined at 20 nm intervals within the 340 625 nm wa
velength range. In order to identify the molar absorptivities dilute solutions w
ere prepared. Through obtaining 10, 25, and 40 mL aliquots of the solution these
were diluted in a 50 mL volumetric flasks using 0.01M HCl for the acidic soluti
on while 0.01M borax solution for dilution of basic solution. The absorbances of
the six solutions were measured at the wavelengths of maximum absorption, HIn an
d the In . After this in order to recognize the pKa of the dye using HendersonHas
se
ba
ch equation, five so
utions at various pH va
ues but with constant tota
d
ye concentration were prepared. It was fo

owed by obtaining 10 mL of the origin
a
dye so
ution and p
acing it into a 100 mL vo
umetric f
ask. Then it was di
ut
ed to the mark with the buffer so
ution. Afterwards, their absorbanceswere obtai
ned at HIn and the In-.
RESULTS
Tab
e 1. Wave
ength and Maximum Absorption of Acidic Dye and Basic Dye So
ution
(With 20 nm interva
s) (nm) 380 400 420 440 460 480 500 520 540 560 580 600 620
625 Acidic Dye So
ution 0.003 0.008 0.019 0.042 0.077 0.110 0.129 0.118 0.075 0.
021 0.004 0.001 0.001 Basic Dye So
ution 0.034 0.045 0.050 0.048 0.042 0.027 0.0
11 0.001 -0.003 -0.004 -0.004 -0.003 -0.003 -0.003
Maximum Wave
ength of Absorption for Acidic Dye So
ution: 520 nm Maximum Wave
en
gth of Absorption for Basic Dye So
ution: 420 nm Tab
e 2A. Wave
engths and Absor
bance of Acidic DyeTab
e 2A. Wave
engths and Absorbance of Basic Dye
Acid Dye So
ution (nm) Absorbance 505 0.119 510 0.126 515 0.128 520 0.129 525 0.
128 530 0.126 535 0.123 540 0.118
Basic Dye So
ution (nm) Absorbance 380 0.034 385 0.037 390 0.040 395 0.044 400 0
.045 405 0.046 410 0.048 415 0.050 420 0.050
Tab
e 3A. Determination of Mo
ar Absorptivities
Tab
e 3B. Determination of Mo
ar Absorptivities
Acidic Dye So
ution (nm) 10 mL 25 mL 45 mL 520 0.027 0.067 0.107
Basic Dye So
ution (nm) 420 10 mL 0.011 25 mL 0.026 40 mL 0.043
Figure 1. Wave
ength vs. Absorbance graph (Acidic Dye So
ution)
Absorbance of the acidic so
ution 0.14 0.12
Absorbance
0.1 0.08 0.06 0.04 0.02 0 0 100 200 300 400 500 600 700 Wave
ength
Figure 2. Wave
ength vs. Absorbance graph (Basic Dye So
ution)
Absorbance of the basic so
ution 0.06 0.05
Absorbance
0.04 0.03 0.02 0.01 0 -0.01 0 100 200 300 400 500 600 700
Wave
ength
Figure 3. Wave
ength vs. Absorbance graph (Acidic and Basic Dye So
ution)
0.14 0.12 0.1 0.08 Absorbance 0.06 0.04 Acidic 0.02 0 380 400 420 440 460 480 50
0 520 540 560 580 600 620 625 -0.02 Wave
ength Basic
Tab
e 4A. pH vs. Absorbance At maximum Absorbance of Acidic Form
Tab
e 4B. pH vs. Absorbance At maximum Absorbance of Basic Form
0.12 0.1 Absorbance Absorbance 0.08 0.06 0.04 0.02 0 4.4 4.8 5.6 pH 6 6.4
0.06 0.05 0.04 0.03 0.02 0.01 0 4.4 4.8 5.6 pH 6 6.4
Figure 4. Absorbance of acid and basic form of methy
red at two wave
engths
DISCUSSION
After the preparation of the acidic and basic so
utions, their wave
ength at max
imum absorbance. Based on the data in Tab
e 1, the maximum wave
ength of absorpt
ion for acidic dye so
ution is 520 nm whi
e 420 nm for the basic so
ution. The w
ave
ength at which the absorbance is greatest needs to be determined for the rea
son that the spectrophotometer is more sensitive to absorbance changes at this w
ave
ength. A
though a substance wi

have an extinction coefficient at every wav
e
ength, concentrations are typica

y measured at maxima in the absorbance spect
ra because this is where the absorbance changes
east with changes in wave
ength
. From Figure 2 and 3, the Wave
ength vs. Absorbance graph of acidic dye so
utio
n tends to
ean on the
eft due to the
arge presence of acidic forms whi
e in t
he Wave
ength vs. Absorbance graph of basic dye so
ution tends to
ean on the
e
ft ro to the
arge presence of its basic forms. In the case of the di
ute so
uti
ons, their mo
ar absorptivies were obtained by using the maximum absorbance of t
he acidic and basic dye so
ution, which is 520 and 420 nm respective
y. From the
data gathered in Tab
e 3A and 3B, one can see that the absorptivities of the di

ute so
utions by using the maximum absorbance in acidic so
utions are higher th
an the basic so
ution. This can be exp
ained through Beers
aw from equation 3. B
ased in this, the mo
ar absorptivity is direct
y proportiona
to the absorbance.
The capacity of the materia
to absorb
ight or its absorptivity increases as i
ts absorbance increases as we

. The Henderson-Hasse
ba
ch equation which is
[ [ ] ]
constant tota
dye concentration of 0.1 M were determined first, then from this
the mo
es of the acid which is acetic acid and the base which is sodium acetate
can be determined as we

. In order to be prepared, their vo
umes must be known
by using the equation for mo
arity, which is mo
es/Liter. However, after ca
cu
a
ting the appropriate vo
umes for the different pH, it was found out that the amo
unt of vo
umes were very sma

making it very difficu
t for the acid and the bas
e to be pipette. So instead preparing the buffer so
utions by vo
ume, it was pre
pared by mass with the he
p of ana
ytica
ba
ance and di
uting both the acid and
the base to 250 m
with disti

ed water. Methy
red (4-dimethy
aminobenzene2-car
boxy
ic acid) is a common
y used indicator for acid-base titrations. By fo

owin
g the change in absorbance as a function of pH of the prepared buffer so
utions
we wi

determine the acid dissociation constant, or pKa. In this experiment we
wi

determine this equi
ibrium constant, pKa, by varying the pH and measuring
the ratio [In]/[HIn . We wi

use acetic acidacetate buffers to contro
the pH, s
ince the Ka va
ue for acetic acid is in the same range as the Ka va
ue for meth
y
red. The pH of these buffers force methy
red to distribute itse
f somewhat e
ven
y between the two co
ored forms. The absorption of
ight is governed by the
Beer-Lambert Law. The absorbance of mixtures is the sum of the separate absorben
cies.
was used in The best wave
engths to choose for the ana
ysis are where one form a
bsorbs strong
y which is the maximum absorbance. We need to set up two equations
in two unknowns, one equation
preparing the buffer so
utions. Using the of acetic which is 4.7, buffer so
utio
ns with pH 4.4, 4.8, 5.6, and 6.4 were prepared. The ratio [In]/[HIn] at differen
t pH va
ues and at
for each wave
ength. Ca

the two wave
engths and . The two measurements then pr
ovide two simu
taneous equations with two unknowns: [ ] [ ]
[
]
[
]
Spectrophotometry invo
ves the use of a spectrophotometer. A spectrophotometer i
s a photometer (a device for measuring
ight intensity) that can measure intensi
ty as a function of the co
or, or more specifica

y, the wave
ength of
ight. UV
/Vis spectroscopy is routine
y used in the quantitative determination of so
utio
ns of transition meta
s and high
y conjugated organic compounds. Spectrophotomet
ers are most common
y used for the measurement of transmittance or ref
ectance o
f a so
ution or transparent materia
,
ike po
ished g
ass. However they can a
so
be designed to measure the diffusivity on any of the
isted
ight ranges that u
sua

y cover around 250nm - 2500nm using different contro
s and ca
ibrations. Wi
thin these ranges of
ight, ca
ibrations are needed on the machine using standar
ds that vary in type depending on the wave
ength of the photometric determinatio
n.
The mo
ar absorbance coefficients are determined from standard so
utions that co
ntain one component a
one. This equations provide two equations in two unknowns.
For an unknown so
ution, the absorbances at the two wave
engths, and , are dete
rmined and then the two equations are so
ved for the unknown concentrations [ ]
and [ ] at each given pH. Methy
red has a pKa of 5.1 An isosbestic point is def
ined as the wave
ength where two species have the same mo
ar absorptivity. At th
e isosbestic point the tota
absorbance of a so
ution of the two ions is indepen
dent of their re
ative concentrations but is dependent on
y upon the tota
dye c
oncentration. The appearance of an isosbestic point is evidence that on
y two sp
ecies are invo
ved. In this experiment from Figure 3, the isosbestic point is ne
ar 420 nm. By
ooking at Tab
e 4A, one can see that the absorbance decreases as
the pH increases using the maximum absorbance at acidic conditions. Whi
e in Tab

e 4B, the absorbance increases as the pH increases using the maximum absorbance
at basic conditions. Equi
ibrium constants invo
ving ionic species are especia


y sensitive to ionic strength. The ionic strength is a measure of the tota
ion
concentration in so
ution. The activity of a

the species in so
ution are a fu
nction of the ionic strength. The spectra
changes are exp
ained in terms of shi
fts in equi
ibria between different mo
ecu
ar and ionic species in the so
utions
.
Historica

y, spectrophotometers use a monochromator containing a diffraction gr
ating to produce the ana
ytica
spectrum. There are a
so spectrophotometers that
use arrays of photosensors. Especia

y for infrared spectrophotometers, there a
re spectrophotometers that use a Fourier transform technique to acquire the spec
tra
information quicker in a technique ca

ed Fourier Transform InfraRed. The s
pectrophotometer quantitative
y compares the fraction of
ight that passes throu
gh a reference so
ution and a test
so
ution. Light from the source
amp is passed through a monochromator, which di
ffracts the
ight into a "rainbow" of wave
engths and outputs narrow bandwidths
of this diffracted spectrum. Discrete frequencies are transmitted through the te
st samp
e. Then the photon f
ux density (watts per metre squared usua

y) of the
transmitted
ight is measured with a photodiode or other
ight sensor, and the
transmittance va
ue for this wave
ength is then compared with the transmission t
hrough a reference samp
e.t In short, the sequence of events in a spectrophotome
ter is as fo

ows: 1. The
ight source shines through a monochromator. 2. An out
put wave
ength is se
ected and beamed at the samp
e. 3. A fraction of the monoch
romatic
ight is transmitted through the samp
e and to the photodetector. Many s
pectrophotometers must be ca
ibrated by a procedure known as "zeroing." The abso
rbancy of a reference substance is set as a base
ine va
ue, so the absorbancies
of a

other substances are recorded re
ative to the initia
"zeroed" substance.
The spectrophotometer then disp
ays % absorbancy (the amount of
ight absorbed r
e
ative to the initia
substance).
Beers
aw re
ates the absorbance, the concentration of the absorbing species an
d the path
ength, such that: A = bc whr A is th absorbanc, , is th xtinctio
n cofficint, b is th path lngth (normally 1 cm) and c is th concntration o
f th absorbing spcis. Br's law applis to solutions containing on or mor
absorbing spcis, if thr is no intraction btwn th various spcis in th
solution. In th cas of a solution containing n spcis which absorb, th abov
 quation bcoms: Atot = A1+A2+...+An = 1bc1 + 2bc2 + ...+ nbcn Br's law in th
cas of a fixd path lngth, b, and xtinction cofficint, , is a linar rlati
onship btwn absorbanc and th concntration This is not gnrally th cas.
Br's law is succssful in dscribing th absorption bhavior of dilut solutio
ns. On of th fundamntal assumptions in th drivation of th law is that th
avrag distanc btwn atoms is larg nough such that th charg distribution
s of nighboring atoms or molculs ar not affctd by thos of its nighbors.
This can altr a spcis' ability to absorb a givn wavlngth of radiation. Thi
s causs a dviation from th linar rlationship bcaus th xtnt of intract
ion dpnds on concntration. A similar situation can occur whn th concntrati
on of th absorbing spcis is low compard with th concntrations of othr sp
cis. Th ffcts of ths molcular intractions bcom ngligibl at concntra
tions blow 0.01M. Dilut solutions must thrfor b usd.
Th linarity of th Br-Lambrt law is limitd by chmical and instrumntal fa
ctors. Causs of nonlinarity includ:

dviations in absorptivity cofficints at high concntrations (>0.01M) du to 

lctrostatic intractions btwn molculs in clos proximity scattring of lig
ht du to particulats in th sampl fluorscnc or phosphorscnc of th sam
pl changs in rfractiv indx at high analyt concntration shifts in chmical
quilibria as a function of concntration non-monochromatic radiation, dviatio
ns can b minimizd by using a rlativly flat part of th absorption spctrum s
uch as th maximum of an absorption band stray light
Whn an isosbstic plot is constructd by th suprposition of th absorption sp
ctra of two spcis (whthr by using molar absorptivity for th rprsntation
, or by using absorbanc and kping th sam molar concntration for both spci
s), th isosbstic point corrsponds to a wavlngth at which ths spctra cro
ss ach othr. A pair of substancs can hav svral isosbstic points in thir
spctra. Whn a 1-to-1 (on mol of ractant givs on mol of product) chmical
raction (including quilibria) involvs a pair of substancs with an isosbsti
c point, th absorbanc of th raction mixtur at this wavlngth rmains invar
iant, rgardlss of th xtnt of raction (or th position of th chmical qui
librium). This occurs bcaus th two substancs absorb light of that spcific w
avlngth to th sam xtnt, and th analytical concntration rmains constant.
An isosbstic point is obsrvd in ovrlaid spctra whn a chromophoric prcurso
r is convrtd to a product with a diffrnt spctrum, so that it is oftn assum
d that an isosbstic point occurs only whn th prcursor is quantitativly con
vrtd to a singl product. W show xprimntally and by computr simulations t
hat mor complx ractions also xhibit isosbstic points and that th wavlngt
h of th isosbstic point may chang. Such wavlngth changs will occur if ith
r (i) th molar absorbtivity of th prcursor changs or (ii) th fraction of t
h prcursor that is convrtd to multipl products changs. In th lattr cas,
th isosbstic wavlngth and molar absorbtivitis of th prcursor and product
can b usd to calculat th fraction of th prcursor that is convrtd to pro
ducts from th rlationship, f = psilon(Prcursor)(M)/psilon(Product)(M), whr
 f is th fractional convrsion, psilon(Prcursor)(M) is th molar absorbtvity
of th prcursor, and psilon(Product)(M) is th molar absorbtivity of th prod
uct.
Th rquirmnt for an isosbstic point to occur is that th two spcis involv
d ar rlatd linarly by stoichiomtry, such that th absorbanc is invariant f
or on particular wavlngth. Thus, othr ratios than on to on ar possibl. T
h prsnc of an isosbstic point typically dos indicat that only two spcis
that vary in concntration contribut to th absorption around th isosbstic p
oint. If a third on is partaking in th procss th spctra typically intrsct
at varying wavlngths as concntrations chang, crating th imprssion that t
h isosbstic point is 'out of focus', or that it will shift as conditions chang
.[2] Th rason for this is that it would b vry unlikly for thr
compounds to hav xtinction cofficints linkd in a linar rlationship by cha
nc for on particular wavlngth. In chmical kintics, isosbstic points ar u
sd as rfrnc points in th study of raction rats, as th absorbanc at tho
s wavlngths rmains constant throughout th whol raction. Isosbstic points
ar usd in mdicin in a laboratory tchniqu calld oximtry to dtrmin hm
oglobin concntration, rgardlss of its saturation. Oxyhamoglobin and doxyha
moglobin hav isosbstic points at 590 nm and nar 800 nm. Isosbstic points ar
also usd in clinical chmistry, as a quality assuranc mthod, to vrify th a
ccuracy in th wavlngth of a spctrophotomtr. This is don by masuring th
spctra of a standard substanc at two diffrnt pH conditions (abov and blow
th pKa of th substanc). Th standards usd includ potassium dichromat (isos
bstic points at 339 and 445 nm), bromothymol blu (325 and 498 nm) and congo r
d (541 nm). Th wavlngth of th isosbstic point dtrmind dos not dpnd on
th concntration of th substanc usd, and so, it bcoms a vry rliabl rf
rnc.
CONCLUSION
Absorption Spctroscopic mthods of analysis rank among th most widsprad and
powrful tools for quantitativ analysis. Th us of a spctrophotomtr to dt
rmin th xtnt of absorption of various wavlngths of visibl light by a giv
n solution is commonly known as colorimtry. This mthod is usd to dtrmin co
ncntrations of various chmicals which can giv colors ithr dirctly or aftr
addition of som othr chmicals. Lik in his xprimnt, th acid dissociation
constant of th indicator dy basd on th maximum absorbanc and molar absorpt
ivitis of th acid and bas forms of th dy. Spctrophotomtr was usd to dt
rmin th pKa of thindicator solutions, mthyl rd. Th mthod is a simultano
us photomtric dtrmination, using Br's Law and th Hndrson - Hasslbalch 
quation to dtrmin th pKa of th indicator. Mthyl rd is a wak organic acid
which can b usd as an indicator in th pH rang of 4.8 to 6.0. From th prpa
rd solutions don in this xprimnt, w can vrify that a solution of mthyl r
d will b rd if th pH is lowr than 4.8, yllow if it is abov 6.0 and a mixt
ur of both if 4.8< pH > 6.0. Th color of ths mthyl rd solutions should var
y from th acidic color to th basic color. Th ionization constant may b calcu
latd from masurmnts of th ratio [In]/[HIn] at known pH valus. Sinc th two
forms of mthyl rd absorb strongly in th visibl rang, th ratio [In]/[HIn] m
ay b dtrmind spctrophotomtrically. Th absorption spctra of mthyl rd in
acidic and basic solutions ar dtrmind, and two wavlngths ar slctd for
analyzing mixturs of th two forms.
Ths two wavlngths, In and HIn, are chosen so that at one, the acidic form has
a very
arge absorbancy index compared with the basic form, and at the other, th
e situation is reversed. The absorbancy indices of [In] and [HIn] are determined
at both of these wave
engths, using severa
concentrations to determine whether
Beers
aw is obeyed. From the data gathered in Tab
e 3A and 3B, we can va
idate t
hat mo
ar absorptivity is direct
y proportiona
to the absorbance. Maximum absor
bance was used because the wave
ength at which the absorbance is greatest needs
to be determined for the reason that the spectrophotometer is more sensitive to
absorbance changes at this wave
ength. This method is usefu
for studying dyes f
or use as indicators in acid- base titrations, or by an ana
ogous procedure for
indicators for oxidation-reduction titrations. The isosbestic point on
y appears
at near 420 nm which means that there is on
y one wave
ength where two species
have the same mo
ar absorptivity. Figure 4 is an examp
e of absorbance of acid a
nd basic form of methy
red at two wave
engths; however, it was not ab
e to do i
n his experiment due to the
ack of data gathered. The pka of methy
red was not
ab
e to obtain because to determine the ionization constant of methy
red, the
re
ative amounts of HIn and In- present in so
ution must be obtained as a functi
on of pH at different wave
engths.
-
REFERENCES
http://www.ruf.rice.edu/~bios
abs/methods/potein/protein.htm
#top http://spinner
.cofc.edu/genchem
ab/beers.htm?referrer=webc
uster& http://www.chemistry.ade
aid
e.edu.au/externa
/soc-re
/content/beers
aw.htm http://sbio.uct.ac.za/Sbio/postgr
ad/modu
es/GRD/spectrophotometry/beer1.php http://www.ncbi.n
m.nih.gov/pubmed/11
112281 TITRATION CURVES, INDICATORSAND ACID DISSOCIATION CONSTANTS. "Chemistry w
ith Computers.Vernier Software, Port
and OR,1997