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Absorption Spectroscopy in the visible region is considered to be one of the
oldest physical methods used for quantitative analysis and structural elucidation.
Spectrometer is mainly used for quantitative analysis and serves as a useful auxiliary
tool for structural elucidation. Analytical application of absorption of radiation by matter
can be either qualitative or quantitative. The qualitative application of absorption
spectrometry depends on the fact that a molecular species absorbs radiation only in
specific regions of the spectrum where the radiation has the energy required to raise
the molecule to some excited state. A display of absorption versus wavelength or
frequency is called an absorption spectrum of that molecular species and serves as a
Fingerprint for identification. The wavelength of visible radiation starts at 8000 and
ends at 4000 . The main types of instruments are use for measuring the emission or
absorption of radiant energy from substance is called by various names such as
Photometers, Colorimeters and spectrophotometers.
Photometer:-
It is an instrument which measures the ratio of some function of two
electromagnetic beams. This is an inexpensive instrument employing a filter to isolate
a narrow wavelength region and photocell or photometer to measure the intensity of
radiation.
Spectrophotometer:-
The instrument measures the ratio of a function of the two of the radiant power
of two electromagnetic beams over large region. In this instrument a monochromatic
radiation is used instead of a filter. A monochromator allows a large wavelength region
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to scan. In addition a spectrophotometer employs most secretive, detectors like
photometer or photomultipliers.
Colorimeters:-
Any instrument used for measuring absorption in the visible region is gradually
called as Colorimeter. In fact some commercial filter photometers are called
Colorimeters.
I.1: Theory of Spectrophotometer:-
When monochromatic or heterogeneous light is incident upon homogeneous
medium a part of incident light is reflected, a part is absorbed by the medium and the
reminder is allowed to transmit. If Io denotes the incident light, Ir the reflected light, I
the absorbed light and It the transmitted light, then we can write,
I0= I + It + Ir -----------------------(1)
If a comparison cell is used, the value of Ir which is very small can be
eliminated for air-glass interfaces, under this condition equation (1) becomes as
I0= I + It ----------------------- (2)
Bouger actually investigated the range of absorption of light with the thickness
of medium. But this credit was enjoyed by Lambert who simply explains the concepts
developed by Bouger. Beer later applied Lamberts concept to solution of different
concentrations and reported his results just power to those of Bernard. However two
laws governing absorption are generally known as Lamberts and Beers law. We will
discus this one by one.
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Lamberts Law:-
This law1,2 can be studied as When a beam of light is allowed to pass through a
transparent medium, the ratio of decrease of intensity with the thickness of the
medium is directly proportional to the intensity of the light.
Mathematically the Lamberts law may be studied as follows
-di / dt I
OR
-di / dt = KI ------------------ (3)
Where I denotes intensity of the incident light of wave length, It denotes the thickness
of medium and K denotes the proportionality factor, on integration equation (3) and
putting I = Io when t = 0 , we get
ln Io / It = Kt
It = Io e-kt --------------------(4)
Where Io denotes the intensity of incident light, Iv denotes the intensity of
transmitted light and K is a constant which depends on the wavelength and absorbing
medium used. On changing equation (4) from natural to common logarithms, we get
It = Io . 10-0.4343kt
= Io . 10-kt
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Where K = k / 2.3026 --------------
(5)
In equation (5) K is absorption coefficient which is defined as The reciprocal of
thickness the light to 1 /10 0f its intensity.
The above definition follows from equation
It / Io = 0.1 = 10-kt
or Kt = 1
or K 1/t ------------- (6)
The ratio It / Io is termed as the transmittance T and log 1/T is termed as
absorbance A of the medium. The ratio Io / It is termed as Optical density.
So that
A = log Io / It -------------- (7)
Beers law:-
Lamberts law shows that there exists a logarithmic relationship between the
transmittance and the length of the optical path through the sample.
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Beer1,2 observed that a similar relationship holds between transmittance and
concentration of the solution, i.e the intensity of a beam of monochromatic light
decreases exponentially with the increase concentration of absorbing substance
arithmetically.
Thus equation (4) becomes as
It = Io e-kc
= It .10-0.4343kt ---------------- (8)
= Io. 10-Kc ----------------(9)
Where k and K are constants and is concentration of the absorbing substance of the
absorbing substance combining equations (5) and (9) we get
It = Io .10-act
Or
log Io / It = act ------------------ (10)
Equation (5) is termed as mathematical statement of Beer- Lamberts law. This is also
a fundamental equation of spectrophotometer.
In equation (10) the value of a depends on the unit of concentration. If C is
expressed in mole dm-3 and in centimeters, then a is replaced by symbol and is
termed as the molar absorption coefficient or molar absorptivity.
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It is important to remark here that exist a relationship between the absorbance
A, the transmittance T, and the molar absorption coefficient ,
i.e. A = ct
= log Io / It
= log 1 / T
= - log T --------------------(11)
In spectrophotometers, the scales are calibrated and the absorbance is read directly.
I.2: Deviations from Beers Law:-
From Beers law it follows that if we plot absorbance against concentrations a
straight line passing through the origin should be obtained in figure 1.1. But there is
usually a linear relationship between concentration and absorbance and an apparent
failure of Beers law may ensure. Deviation from the law is reported positive or
negative according to whether the resultant curve upward or concave downwards.
I.3: Deviation from Beers law can arise due to following factors:-
1. Beers law will hold over a wide range of concentrations provided the structure
of coloured ion or of coloured non-electrolyte is the dissolved state does not
change with concentration. If a coloured solution is having foreign substance
whose ions do not react chemically with that coloured components, its small
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concentration (foreign substance) does not affect the light absorption may affect
extinction coefficient.
2. Deviations may also occur if the coloured solute varies, dissociates or
associates in solution.
3. Deviations may also occur due to the presence of impurities that fluoresce or
absorb at absorption wavelength. The interference introduce an error in the
measurement of absorption of radiation penetrating the sample.
4. Deviations may occur if monochromatic light is not used.
5. Deviations may occur if the width of slit is not proper and therefore, it allows
undesirable radiations might be absorbed by impurities present in the sample.
The magnitudes of two deviations becomes appreciable at higher
concentrations.
6. Deviations may occur if the solutions species undergoes polymerization.
7. Beers law can not be applied to suspensions but the lather can be estimated
calorimetrically apply preparing a reference curve with known concentrations.
I.4: The review of work done in absorption measurements:-
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Measurements have been made of visible region absorption spectrum of a
dichloric dye in the chiral nematic host by H.S.Cole, Jr. and S. Aftergut3 . Data are
replaced for a range of conditions of boundary state birefringence and pitch.
Intrinsic polarized ultraviolet absorption of crystalline tetragonal Geo2 are
studied by M.Stapllroek and B.D.Evans4 for in the range 5-103 /cm at room
temperature and below sharpline structure strongly polarized. The optical absorption
spectra from 5000 to 30,000 /cm of single crystals of chromium chloride have be
studied from 300 to 6k by D.R.Rosseinsly and I.R.Dorrily5 . The ultraviolet absorption
spectra of MBBA is the multi stable solids, the nematic and isotropic liquid states havebeen measured by M.Mizuno and T. Shinoda6 . Further more the spectra of dilute
solutions and linear dichroisum spectra of nematic single liquid crystal in
homogeneous orientation have also been observed. The temperature dependence of
absorption of diacetylene chains dispersed in partially polymerized monomer matrices
has been measured by D.Bloor and C.L. Hubble7).Over the range from 2 to 380 k for
number of different monomers. The results are interpreted in terms of the effects of
the lattice environments on the polymer chains. It is shown that in general the polymer
chain length does not have a major effect on the absorption spectrum.
The optical number of organic compounds have been examined by
W.c.mcColgin and etal8, In low temperature glassy solutions. According to the
experimental conditions of excitation a given sample can yield either the usual broad
bands complete with stokes shift or a set of very narrow fluorescence lines,
comparisons of these two distinct type of spectra from the same sample make it
possible to explain such a features of the convention all spectra as their broad band
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width peak positions and stokes shifts. The absorptivity of carefully purified water have
been measured by T.I.Quicken and J.A. Irvin(9) at 1 nm intervals in wavelength range
196 to 320 nm.
2.1: Absorbance and Transmittance Measurements:-
Figure 2.1 shows diagrammatically the measurement of absorbance in
Cuvette(10)
lying vertically. The cuvette is longitudinal to the laser beam. A very
simple apparatus is developed using cuvette detector and laser. The detector
used here is light detecting resistor. Fig 2.2 shows the circuit diagram for
detecting light in terms of current. Through empty cuvette, laser beam is
passed and intensity of laser beam is noted in terms of current Io. Cuvette is
filled with acqueous solution and transmitted intensity of laser beam is noted
in terms of current as It. Noting Io & It for various length of solution in
cuvette ; Absorbance & transmittance have been estimated. Length of liquid
is varied. Concentration dependence of absorbance & transmittance also
found out for various concentration of methyl orange in distilled water. The
absorbance and transmittance have been studied in 0.1%, 0.2%, 0.5%, 0.7%,
1%, 1.2%, 1.4%, 1.6%, 1.8%, and 2% acqueous solution of methyl orange.
2.2: materials & preparation of solution:-
In problem methyl present orange is selected, for the preparation
of acqueous solution methyl orange is of A R grade. Supplied by Fishery
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Industries. Acqueous solution of methyl orange is prepared for0.1% , 0.2% ,
0.5% , 0.7% , 1% , 1.2%, 1.4% , 1.6% , 1.8% , and 2% acqueous solution of
methyl orange in distilled water. The absorption and transmittance of all
concentration have been studied at room temperature 30.2 C0 using the
procedure described in article 2.1.
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2.3: density measurement:-
Density measurement are carried out using specific gravity bottle,
electronic balanced with accuracy of 0.01 gm has been used for weighing
empty and filled specific gravity bottle with various concentration of
acqueous solutions of methyl orange. Density measurements are carried out
at room temperature 30.2 0C
2.4: precautions taken during measurements:-
Following precautions must be observed using the absorption and
transmittance measurement.
i. Instrument should be placed in a clean and dust free environment.
ii. Instrument must be installed at a place free from vibration and light.
iii. It should be always covered with dust cover when not in use.
iv. Only the matched cuvette should be use
v. The sample holder should be cleaned before use to obtained best
results.
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vi. The instrument should not be used in presence of inflammable gasses.
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The absorption measurement technique has been already discussed in chapter-
II and all the results obtained during this study are presented in table 3.1 to 3.12 and
figure 3.11 to 3.12. table shows the experimental values obtained during the
absorption and transmission study in aqueous solutions of methyl orange. Figure
shows variation of absorbance and transmission of laser beam through the aqueous
solutions of methyl orange.
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Table - 3.1:
Variation of Transmittance & Absorbance with length for
0.01 % methyl orange aqueous solution
The intensity of incident light Io = 500 A
Density = 1.7088 gm/ml
ObsNo
Length (cm) Current (A) Transmittance Absorbance
1 01 490 0.98 0.0087
2 02 470 0.94 0.026
3 03 460 0.92 0.036
4 04 440 0.88 0.055
5 05 430 0.86 0.065
6 06 420 0.84 0.075
7 07 410 0.82 0.086
8 08 400 0.80 0.96
9 09 390 0.78 0.107
10 10 380 0.76 0.119
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Table - 3.2:
Variation of Transmittance & Absorbance with length for
0.2 % methyl orange aqueous solution
The intensity of incident light Io = 500 A
Density = 1.7112 gm/ml
ObsNo
Length (cm) Current (A) Transmittance Absorbance
1 01 480 0.96 0.0177
2 02 470 0.94 0.026
3 03 450 0.90 .045
4 04 440 0.88 .055
5 05 420 0.84 0.075
6 06 400 0.80 0.096
7 07 380 0.76 0.119
8 08 370 0.74 0.130
9 09 360 0.72 0.142
10 10 350 0.70 0.154
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Table - 3.3:
Variation of Transmittance & Absorbance with length for
0.5 % methyl orange aqueous solution
The intensity of incident light Io = 500 A
Density = 1.7136 gm/ml
ObsNo
Length (cm) Current (A) Transmittance Absorbance
1 01 470 0.94 0.0268
2 02 460 0.92 0.0362
3 03 440 0.88 0.055
4 04 430 0.86 0.0655 05 410 0.82 0.086
6 06 390 0.78 0.107
7 07 370 0.74 0.130
8 08 360 0.72 0.142
9 09 350 0.70 0.154
10 10 340 0.68 0.167
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Table - 3.4:
Variation of Transmittance & Absorbance with length for
0.7 % methyl orange aqueous solution
The intensity of incident light Io = 500 A
Density = 1.7180 gm/ml
ObsNo
Length (cm) Current (A) Transmittance Absorbance
1 01 450 0.90 0.0457
2 02 400 0.80 0.0969
3 03 330 0.66 0.180
4 04 300 0.60 0.221
5 05 250 0.50 0.301
6 06 210 0.42 0.3767 07 180 0.36 0.443
8 08 150 0.30 0.522
9 09 130 0.26 0.585
10 10 100 0.20 0.698
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Table - 3.5:
Variation of Transmittance & Absorbance with length for
1 % methyl orange aqueous solution
The intensity of incident light Io = 500 A
Density = 1.7201 gm/ml
ObsNo
Length (cm) Current (A) Transmittance Absorbance
1 01 300 0.60 0.221
2 02 280 0.56 0.251
3 03 250 0.50 0.301
4 04 220 0.44 0.3565 05 210 0.42 0.376
6 06 190 0.38 0.420
7 07 180 0.36 0.443
8 08 140 0.28 0.552
9 09 120 0.24 0.619
10 10 90 0.18 0.654
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Table - 3.6:
Variation of Transmittance & Absorbance with length for
1.2 % methyl orange aqueous solution
The intensity of incident light Io = 500 A
Density = 1.7228 gm/ml
ObsNo
Length (cm) Current (A) Transmittance Absorbance
1 01 290 0.58 0.23
2 02 270 o.54 0.26
3 03 240 0.48 0.31
4 04 220 0.44 0.35
5 05 190 0.38 0.42
6 06 180 0.36 0.44
7 07 150 0.30 0.52
8 08 130 0.26 0.58
9 09 110 0.22 0.65
10 10 80 0.16 0.79
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Table - 3.7:
Variation of Transmittance & Absorbance with length for
1.4 % methyl orange aqueous solution
The intensity of incident light Io = 500 A
Density = 1.7251 gm/ml
Obs No Length (cm) Current (A) Transmittance Absorbance
1 01 280 0.56 0.25
2 02 250 0.50 0.30
3 03 230 0.46 0.33
4 04 210 0.42 0.37
5 05 180 0.36 0.44
6 06 160 0.32 0.497 07 140 0.28 0.55
8 08 120 0.24 0.61
9 09 110 0.22 0.65
10 10 95 0.19 0.73
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Table - 3.8:
Variation of Transmittance & Absorbance with length for
1.6 % methyl orange aqueous solution
The intensity of incident light Io = 500 A
Density = 1.7284 gm/ml
ObsNo
Length (cm) Current (A) Transmittance Absorbance
1 01 260 0.52 0.28
2 02 240 0.48 0.31
3 03 220 0.44 0.35
4 04 200 0.40 0.395 05 170 0.34 0.46
6 06 150 0.30 0.52
7 07 130 0.26 0.58
8 08 110 0.22 0.65
9 09 90 0.18 0.74
10 10 80 0.16 0.79
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Table - 3.9:
Variation of Transmittance & Absorbance with length for
1.8 % methyl orange aqueous solution
The intensity of incident light Io = 500 A
Density = 1.7304 gm/ml
ObsNo
Length (cm) Current (A) Transmittance Absorbance
1 01 240 0.48 0.31
2 02 235 0.47 0.32
3 03 210 0.42 0.37
4 04 190 0.38 0.42
5 05 150 0.30 0.52
6 06 140 0.28 0.55
7 07 120 0.24 0.61
8 08 100 0.20 0.69
9 09 80 0.16 0.79
10 10 70 0.14 0.85
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Table - 3.10:
Variation of Transmittance & Absorbance with length for
2 % methyl orange aqueous solution
The intensity of incident light Io = 500 A
Density = 1.7324 gm/ml
ObsNo
Length (cm) Current (A) Transmittance Absorbance
1 01 210 0.42 0.37
2 02 190 0.38 0.42
3 03 150 0.30 0.52
4 04 115 0.23 0.63
5 05 110 0.22 0.65
6 06 80 0.16 0.797 07 70 0.14 0.85
8 08 25 0.05 1.30
9 09 20 0.04 1.39
10 10 10 0.02 1.69
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Table - 3.11:
Variation of Transmittance & Absorbance with length for
all concentrations of methyl orange aqueous solutions
Length =1 cm.
ObsNo
Concentration (%)
Densitygm/ml
Current(A)
Absorbance Transmittance
1 0.01 1.7088 490 0.087 0.98
2 0.2 1.7112 480 0.0177 0.96
3 0.5 1.7136 470 0.0268 0.94
4 0.7 1.7180 450 0.0457 0.90
5 1 1.7201 300 0.221 0.60
6 1.2 1.7228 290 0.23 0.58
7 1.4 1.7251 280 0.25 0.56
8 1.6 1.7284 260 0.28 0.52
9 1.8 1.7304 240 0.31 0.48
10 2 1.7324 210 0.37 0.42
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Study of tables & figures shows that as the length of the liquid medium is
increased in cuvette the absorbance is increased, at same time the variation of
transmittance decreasing as the length of the medium is in the cuvette increased.
Figure show that there is linear increase and decrease of absorbance and
transmittance of laser light in acqueous solution of methyl orange.
Figure 3.11 shows variation of absorbance with concentration of acqueous
solution of methyl orange. According to Beers it seems that this dependence of
absorbance should linear with concentration but this solution does not seems to verify
the Beers law. Study of fig 3.11 shows that in the region 0 - 0.6% of acqueous solution
of methyl orange verifies Beers law but after that from 0.8 2% concentration Beers
law does not verified. That is only at low concentration Beers law is verified.
The over all study of the results shows that for each concentration of acqueous
solution of methyl orange, the length of the medium increases absorbance increases
and transmittance decreases. That is absorbance of light is directly proportional to
length and concentration of liquid in cuvette, and transmittance is inversely
proportional to the concentration and length of liquid in the cuvette. One can not verify
Beers and Lamberts law in the high concentration region.
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