Application of uv visible spectroscopy (IMA) (sem viii)

Application of UV visible spectroscopy

1. Application of uv - visible spectroscopy Guided by: Mr. Pratik Terse (Assistant professor ) Prepared by :Ms. Amruta Anil Gawade PRN: 2131861823025 -Amruta gawade

2. contents Spectrophotometric titration Single component analysis method Multi Components analysis method -Amruta gawade 2

3. SPECTROPHOTOMETRIC TITRATION -Amruta gawade 3 In a spectrophotometric titration, the equivalence point is determined with spectrophotometer. In these technique, the titration vessel is kept directly in the light path of the instrument. Then , the absorbance of the solution is determined after adding titrant & a plot of absorbance as a function of volume titrant is prepared. If the titration is complete, the titration curve will consist of two straight line intersecting at the equivalence point, similar to amperometric and conductometric titration. And , if the titration reaction is not complete, there occurs appreciable curvature in equivalence point region but extrapolation of the two linear segments of the titration curve to their intersection gives the equivalence point volume .

4. Following are the some titration curves -Amruta gawade 4 Curve (a) is characteristic of case where only the titrant absorbs. An interesting example of this is the titration of arsenic(III) with bromate –bromide, where the absorbance readings are taken at the wavelength where bromine absorbs. Curve (b) is characteristic of case where only the product of reaction absorbs. An example of this is the titration of Cu(II) with EDTA, carried out at745 nm wavelength. This wavelength is selected because at this position the EDTA-copper complex possess a much greater absorbance compared to copper solution alone .

5. -Amruta gawade 5 Curve (c) is characteristic of case where only the substance being titrated absorbs, while the titrant and the product do not absorb. An example is the titration of p- toludine in butanol with perchloric acid at 290nm wavelength Curve (d) is obtained when a colored analyte is converted into a colorless product by a colored titrant. When titrant is added, the analyte color starts fading due to the formation of colorless product. But after the equivalence point, absorbance again rises due to color of the titrant alone .

6. CHOICE OF WAVELENGTH : -Amruta gawade 6 A number of criteria are considered in the choice of wavelength. It may be necessary to balance one against another. 1) The highest absorption peak will yield the maximum sensitivity. 2) A chosen wavelength should yield a minimum interference from other species that may be in the system. 3) Because of the errors that arise from sharply rising or sharply falling portions of the absorption curves, these sections should be avoided. 4) Care should be taken to avoid a sharp band if the slit width exceeds the bandwidth .

7. ADVANTAGES : -Amruta gawade 7 Spectrophotometric titrations have several advantages over direct spectrophotometric analysis : It can be applied to a large number of non-absorbing constituents as only one absorber is to be present among the reactant, the titrant, or the reaction products. Presence of other absorbing species at the analytical wavelength does not cause interference because only the change in absorbance is significant. It can be applied to highly colored solutions that could not be determined by the visual indicator. It can be applied to such reactions that tends to be appreciably incomplete at the equivalence point. More dilute solutions may be employed than in other types of titrations. A variety of equipment may be employed.

8. SINGLE COMPONENT ANALYSIS METHODS -Amruta gawade 8 The methods involved in the calculation of single component analysis are : Direct Analysis : Essentially all compounds containing conjugated double bond or aromatic rings , and mainly inorganic species absorbs light in the UV-Visible regions. In these techniques, the substance to be determined is dissolved in a suitable solvent and diluted to the required concentration by appropriate dilutions, and absorbance is measured. Indirect Analysis : This method involves analysis after addition of some addition of some reagent. These methods are based on the conversions of analyte by a chemical reagent having different spectral properties.

9. Methods for Calculating Concentration in Single Component Analysis:- -Amruta gawade 9 By using the relationship A = abc . By using the formula Cu = (A u /A s )*C s . By using the equations y = mx + c . By using the Beer’s curve.

10. MULTI-COMPONENT ANALYSIS METHOD -Amruta gawade 10 UV Spectrophotometric techniques are mainly use for multi component analysis, thus minimizing the cumbersome task of separating interferents and allowing the determination of an increasing number of analytes, consequently reducing analysis time and cost. Multicomponent UV Spectrophotometric methods are based on recording and mathematically processing absorption spectra. The multicomponent analysis methods offer the following advantages : Avoiding prior separation techniques , eg : extraction, concentration of constituents and clean-up steps that might be required. Spectral data are readily acquired with ease. The process is fast, accurate and simple. Wide applicability to both inorganic and organic systems. Typical detection limits of 10 -4 and 10 -5 M and moderate to high sensitivity.

11. Different UV spectrophotometric multicomponent analysis methods include : -Amruta gawade 11 Simultaneous equation method, Difference spectrophotometry, Derivative spectrophotometry, Absorbance ratio spectra method, Derivative ratio spectra method, Double divisor ratio spectra derivative method, Successive ratio-derivative spectra method, Q-absorbance ratio method, Isobestic “ isoabsorptive ” point method, Absorptivity factor method, Dual wavelength method Ratio subtraction method(RSM), Mean centering of the ratio spectra, Absorption Factor Method(AFM), and Multivariate chemometric method .

12. 1. SIMULTANEOUS EQUATION METHOD ( Vierordt’s Method) : -Amruta gawade 12 If a sample contains two absorbing drugs (x & y) each of which absorbs at the λ max of the other, both the drugs can be determined by this method. The information required is : The absorptivities of x at λ 1 and λ 2 (ax 1 and ax 2 respectively). The absorptivities of y at λ 1 and λ 2 (ay 1 and ay 2 respectively). The absorbance of diluted samples at λ 1 and λ 2 (A 1 and A 2 respectively). let C x and C y be the concentration of x and y respectively in the diluted samples. Two equations are constructed based upon the fact that at λ 1 , the absorbance of the mixture is the sum of the individual absorbance of x and y : A 1 =a x1 bC x + a y1 b C y ..................(1) A 2 =a x2 bC x + a y2 b C y ………………..(2) For measurements in 1cm cells, b= 1cm. On rearranging equation (2) : C y =(A 2 – a x2 C x )/a y2 …………………(3) On substituting equation (3) in equation (1) and rearranging : C x = (A 2 a y1 -A 1 a y2 )/(a x2 a y1 -a x1 a y2 ) ……….(4) C y = (A 1 a x2 -A 2 a x1 )/(a x2 a y1 -a x1 a y2 ) …………(5)

13. 2. DIFFERENCE SPECTROPHOTOMETRY -Amruta gawade 13 The selectivity and accuracy of spectrophotometric analysis of samples containing absorbing interferents may be markedly improved by this ttechnique . The essential feature of tis method is that the measured value is the absorbance difference between two equimolar solutions of the analyte in different chemical forms exhibiting different spectral characteristics. The criteria for applying difference spectrophotometry to the assay of the substance in the presence of other absorbing substances are that : Reproducible changes may be introduced in the spectrum of the analyte by adding one or more reagents. The absorbance of the interfering substances is not altered by the reagent. The simplest and the most commonly employed techniques for altering the spectral properties of the analyte is the adjustment of pH by means of aqueous solutions of acids , alkalis or buffers.

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