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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
sebach equation, five soutions at various pH vaues but with constant tota d
ye concentration were prepared. It was foowed by obtaining 10 mL of the origin
a dye soution and pacing it into a 100 mL voumetric fask. Then it was diut
ed to the mark with the buffer soution. Afterwards, their absorbanceswere obtai
ned at HIn and the In-.
RESULTS
Tabe 1. Waveength and Maximum Absorption of Acidic Dye and Basic Dye Soution
(With 20 nm intervas) (nm) 380 400 420 440 460 480 500 520 540 560 580 600 620
625 Acidic Dye Soution 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 Soution 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 Waveength of Absorption for Acidic Dye Soution: 520 nm Maximum Waveen
gth of Absorption for Basic Dye Soution: 420 nm Tabe 2A. Waveengths and Absor
bance of Acidic DyeTabe 2A. Waveengths and Absorbance of Basic Dye
Acid Dye Soution (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 Soution (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
Tabe 3A. Determination of Moar Absorptivities
Tabe 3B. Determination of Moar Absorptivities
Acidic Dye Soution (nm) 10 mL 25 mL 45 mL 520 0.027 0.067 0.107
Basic Dye Soution (nm) 420 10 mL 0.011 25 mL 0.026 40 mL 0.043
Figure 1. Waveength vs. Absorbance graph (Acidic Dye Soution)
Absorbance of the acidic soution 0.14 0.12
Absorbance
0.1 0.08 0.06 0.04 0.02 0 0 100 200 300 400 500 600 700 Waveength
Figure 2. Waveength vs. Absorbance graph (Basic Dye Soution)
Absorbance of the basic soution 0.06 0.05
Absorbance
0.04 0.03 0.02 0.01 0 -0.01 0 100 200 300 400 500 600 700
Waveength
Figure 3. Waveength vs. Absorbance graph (Acidic and Basic Dye Soution)
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 Waveength Basic
Tabe 4A. pH vs. Absorbance At maximum Absorbance of Acidic Form
Tabe 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 waveengths
DISCUSSION
After the preparation of the acidic and basic soutions, their waveength at max
imum absorbance. Based on the data in Tabe 1, the maximum waveength of absorpt
ion for acidic dye soution is 520 nm whie 420 nm for the basic soution. The w
aveength 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
aveength. Athough a substance wi have an extinction coefficient at every wav
eength, concentrations are typicay measured at maxima in the absorbance spect
ra because this is where the absorbance changes east with changes in waveength
. From Figure 2 and 3, the Waveength vs. Absorbance graph of acidic dye soutio
n tends to ean on the eft due to the arge presence of acidic forms whie in t
he Waveength vs. Absorbance graph of basic dye soution tends to ean on the e
ft ro to the arge presence of its basic forms. In the case of the diute souti
ons, their moar absorptivies were obtained by using the maximum absorbance of t
he acidic and basic dye soution, which is 520 and 420 nm respectivey. From the
data gathered in Tabe 3A and 3B, one can see that the absorptivities of the di
ute soutions by using the maximum absorbance in acidic soutions are higher th
an the basic soution. This can be expained through Beers aw from equation 3. B
ased in this, the moar absorptivity is directy 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-Hassebach equation which is
[ [ ] ]
constant tota dye concentration of 0.1 M were determined first, then from this
the moes 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 voumes must be known
by using the equation for moarity, which is moes/Liter. However, after cacua
ting the appropriate voumes for the different pH, it was found out that the amo
unt of voumes were very sma making it very difficut for the acid and the bas
e to be pipette. So instead preparing the buffer soutions by voume, it was pre
pared by mass with the hep of anaytica baance and diuting both the acid and
the base to 250 m with distied water. Methy red (4-dimethyaminobenzene2-car
boxyic acid) is a commony used indicator for acid-base titrations. By foowin
g the change in absorbance as a function of pH of the prepared buffer soutions
we wi determine the acid dissociation constant, or pKa. In this experiment we
wi determine this equiibrium 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 vaue for acetic acid is in the same range as the Ka vaue for meth
y red. The pH of these buffers force methy red to distribute itsef somewhat e
veny between the two coored 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 waveengths to choose for the anaysis are where one form a
bsorbs strongy which is the maximum absorbance. We need to set up two equations
in two unknowns, one equation
preparing the buffer soutions. Using the of acetic which is 4.7, buffer soutio
ns with pH 4.4, 4.8, 5.6, and 6.4 were prepared. The ratio [In]/[HIn] at differen
t pH vaues and at
for each waveength. Ca the two waveengths and . The two measurements then pr
ovide two simutaneous equations with two unknowns: [ ] [ ]
[
]
[
]
Spectrophotometry invoves 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 coor, or more specificay, the waveength of ight. UV
/Vis spectroscopy is routiney used in the quantitative determination of soutio
ns of transition metas and highy conjugated organic compounds. Spectrophotomet
ers are most commony used for the measurement of transmittance or refectance o
f a soution or transparent materia, ike poished gass. However they can aso
be designed to measure the diffusivity on any of the isted ight ranges that u
suay cover around 250nm - 2500nm using different contros and caibrations. Wi
thin these ranges of ight, caibrations are needed on the machine using standar
ds that vary in type depending on the waveength of the photometric determinatio
n.
The moar absorbance coefficients are determined from standard soutions that co
ntain one component aone. This equations provide two equations in two unknowns.
For an unknown soution, the absorbances at the two waveengths, and , are dete
rmined and then the two equations are soved 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 waveength where two species have the same moar absorptivity. At th
e isosbestic point the tota absorbance of a soution of the two ions is indepen
dent of their reative concentrations but is dependent ony upon the tota dye c
oncentration. The appearance of an isosbestic point is evidence that ony two sp
ecies are invoved. In this experiment from Figure 3, the isosbestic point is ne
ar 420 nm. By ooking at Tabe 4A, one can see that the absorbance decreases as
the pH increases using the maximum absorbance at acidic conditions. Whie in Tab
e 4B, the absorbance increases as the pH increases using the maximum absorbance
at basic conditions. Equiibrium constants invoving ionic species are especia
y sensitive to ionic strength. The ionic strength is a measure of the tota ion
concentration in soution. The activity of a the species in soution are a fu
nction of the ionic strength. The spectra changes are expained in terms of shi
fts in equiibria between different moecuar and ionic species in the soutions
.
Historicay, spectrophotometers use a monochromator containing a diffraction gr
ating to produce the anaytica spectrum. There are aso spectrophotometers that
use arrays of photosensors. Especiay for infrared spectrophotometers, there a
re spectrophotometers that use a Fourier transform technique to acquire the spec
tra information quicker in a technique caed Fourier Transform InfraRed. The s
pectrophotometer quantitativey compares the fraction of ight that passes throu
gh a reference soution and a test
soution. Light from the source amp is passed through a monochromator, which di
ffracts the ight into a "rainbow" of waveengths and outputs narrow bandwidths
of this diffracted spectrum. Discrete frequencies are transmitted through the te
st sampe. Then the photon fux density (watts per metre squared usuay) of the
transmitted ight is measured with a photodiode or other ight sensor, and the
transmittance vaue for this waveength is then compared with the transmission t
hrough a reference sampe.t In short, the sequence of events in a spectrophotome
ter is as foows: 1. The ight source shines through a monochromator. 2. An out
put waveength is seected and beamed at the sampe. 3. A fraction of the monoch
romatic ight is transmitted through the sampe and to the photodetector. Many s
pectrophotometers must be caibrated by a procedure known as "zeroing." The abso
rbancy of a reference substance is set as a baseine vaue, so the absorbancies
of a other substances are recorded reative to the initia "zeroed" substance.
The spectrophotometer then dispays % absorbancy (the amount of ight absorbed r
eative to the initia substance).
Beers aw reates 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: