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

Where (5)

K = k / 2.3026

--------------

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.

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 = Io. 10-Kc

---------------- (8)

----------------(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.

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

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:-

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 have been 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. ii.

Instrument should be placed in a clean and dust free environment. Instrument must be installed at a place free from vibration and light. It should be always covered with dust cover when not in use. Only the matched cuvette should be use The sample holder should be cleaned before use to obtained best results.
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iii. iv. v.

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 chapterII 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 Obs No 1 2 3 4 5 6 7 8 9 10 Length (cm) 01 02 03 04 05 06 07 08 09 10 Current (A) 490 470 460 440 430 420 410 400 390 380 Transmittance 0.98 0.94 0.92 0.88 0.86 0.84 0.82 0.80 0.78 0.76 Absorbance 0.0087 0.026 0.036 0.055 0.065 0.075 0.086 0.96 0.107 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 Obs No 1 2 3 4 5 6 7 8 9 10 Length (cm) 01 02 03 04 05 06 07 08 09 10 Current (A) 480 470 450 440 420 400 380 370 360 350 Transmittance 0.96 0.94 0.90 0.88 0.84 0.80 0.76 0.74 0.72 0.70 Absorbance 0.0177 0.026 .045 .055 0.075 0.096 0.119 0.130 0.142 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 Obs No 1 2 3 4 5 6 7 8 9 10 Length (cm) 01 02 03 04 05 06 07 08 09 10 Current (A) 470 460 440 430 410 390 370 360 350 340 Transmittance 0.94 0.92 0.88 0.86 0.82 0.78 0.74 0.72 0.70 0.68 Absorbance 0.0268 0.0362 0.055 0.065 0.086 0.107 0.130 0.142 0.154 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 Obs No 1 2 3 4 5 6 7 8 9 10 Length (cm) 01 02 03 04 05 06 07 08 09 10 Current (A) 450 400 330 300 250 210 180 150 130 100 Transmittance 0.90 0.80 0.66 0.60 0.50 0.42 0.36 0.30 0.26 0.20 Absorbance 0.0457 0.0969 0.180 0.221 0.301 0.376 0.443 0.522 0.585 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 Obs No 1 2 3 4 5 6 7 8 9 10 Length (cm) 01 02 03 04 05 06 07 08 09 10 Current (A) 300 280 250 220 210 190 180 140 120 90 Transmittance 0.60 0.56 0.50 0.44 0.42 0.38 0.36 0.28 0.24 0.18 Absorbance 0.221 0.251 0.301 0.356 0.376 0.420 0.443 0.552 0.619 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 Obs No 1 2 3 4 5 6 7 8 9 10 Length (cm) 01 02 03 04 05 06 07 08 09 10 Current (A) 290 270 240 220 190 180 150 130 110 80 Transmittance 0.58 o.54 0.48 0.44 0.38 0.36 0.30 0.26 0.22 0.16 Absorbance 0.23 0.26 0.31 0.35 0.42 0.44 0.52 0.58 0.65 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 1 2 3 4 5 6 7 8 9 10 Length (cm) 01 02 03 04 05 06 07 08 09 10 Current (A) 280 250 230 210 180 160 140 120 110 95 Transmittance 0.56 0.50 0.46 0.42 0.36 0.32 0.28 0.24 0.22 0.19 Absorbance 0.25 0.30 0.33 0.37 0.44 0.49 0.55 0.61 0.65 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

Obs No 1 2 3 4 5 6 7 8 9 10

Length (cm) 01 02 03 04 05 06 07 08 09 10

Current (A) 260 240 220 200 170 150 130 110 90 80

Transmittance 0.52 0.48 0.44 0.40 0.34 0.30 0.26 0.22 0.18 0.16

Absorbance 0.28 0.31 0.35 0.39 0.46 0.52 0.58 0.65 0.74 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 Obs No 1 2 3 4 5 6 7 8 9 10 Length (cm) 01 02 03 04 05 06 07 08 09 10 Current (A) 240 235 210 190 150 140 120 100 80 70 Transmittance 0.48 0.47 0.42 0.38 0.30 0.28 0.24 0.20 0.16 0.14 Absorbance 0.31 0.32 0.37 0.42 0.52 0.55 0.61 0.69 0.79 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 Obs No 1 2 3 4 5 6 7 8 9 10 Length (cm) 01 02 03 04 05 06 07 08 09 10 Current (A) 210 190 150 115 110 80 70 25 20 10 Transmittance 0.42 0.38 0.30 0.23 0.22 0.16 0.14 0.05 0.04 0.02 Absorbance 0.37 0.42 0.52 0.63 0.65 0.79 0.85 1.30 1.39 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.
Obs No 1 2 3 4 5 6 7 8 9 10 Concentrati on (%) 0.01 0.2 0.5 0.7 1 1.2 1.4 1.6 1.8 2 Density gm/ml 1.7088 1.7112 1.7136 1.7180 1.7201 1.7228 1.7251 1.7284 1.7304 1.7324 Current (A) 490 480 470 450 300 290 280 260 240 210 Absorbance 0.087 0.0177 0.0268 0.0457 0.221 0.23 0.25 0.28 0.31 0.37 Transmittanc e 0.98 0.96 0.94 0.90 0.60 0.58 0.56 0.52 0.48 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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