Isosbestic point of bromothymol blue

determination of isosbestic point of indicator using absorption ratio method

1. Sumitted by :- Vaishnavi . G. Gomase B. Pharmacy 3 rd year VI sem Guide :- Dr. K. B. Gabhane Roll no. 42 “Determination of isosbestic point of indicator using absorption ratio method” Vidyabharati college of pharmacy, Amravati (2018-2019)

2. Introduction In spectroscopy an isosbestic point is a specific wavelength, wavenumber or frequency at which the total absorbance of a sample does not change during a chemical reaction or a physical change of the sample. The word derives from two Greek words: "iso", meaning "equal", and "sbestos", meaning "extinguishable “ When an isosbestic plot is constructed by the superposition of the absorption spectra of two species (whether by using molar absorptivity for the representation, or by using absorbance and keeping the same molar concentration for both species), the  isosbestic point  corresponds to a wavelength at which these spectra cross each other.

3. UV-VISIBLE SPECTROSCOPY Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet and the full, adjacent visible spectral regions. This means it uses light in the visible perceived involved. In this region of the electromagnetic spectrum atoms and molecules undergo electronic transition. Absorption spectroscopy is complementary to fluorescence spectroscopy in that fluorescence deals with transition from the excited state to the ground state while absorption measures transition from the ground state to the excited state.

4. INDICATOR- BROMOTHYMOL BLUE PROFILE Bromothymol blue (also known as bromothymol sulfone phthalein and BTB) is a pH indicator. It is mostly used in applications that require measuring substances that would have a relatively neutral pH (near 7). A common use is for measuring the presence of carbonic acid in a liquid. It is typically sold in solid form as the sodium salt of the acid indicator. IUPAC NAME:- 4,4′-(1,1-Dioxido-3 H -2,1-benzoxathiole-3,3-diyl)bis(2-bromo-6-isopropyl-3-methylphenol) STRUCTURE AND PROPERTIES:- Bromothymol blue acts as a weak acid in solution. It can thus be in protonated or deprotonated form, appearing yellow or blue, respectively.

5. It is bright aquamarine by itself, and greenish-blue in a neutral solution. The deprotonation of the neutral form results in a highly conjugated structure, accounting for the difference in color. An intermediate of the deprotonation mechanism is responsible for the greenish color in neutral solution The protonated form of bromothymol blue has its peak absorption at 427 nm thus transmitting yellow light in acidic solutions, and the deprotonated form has its peak absorption at 602 nm thus transmitting blue light in more basic solutions. [3]  Highly acidic Bromothymol blue is magenta in color.

6. OBJECTIVE AND RATIONAL The main objective of determination of isosbestic point is that this point shows a) There is a process or shift in an equilibrium b) the spectra of the individual compounds are the same in this point. In qualitative assay of two component in mixture can be done by measuring absorbance at two wavelength one being the lambda max of one of the component and other being wavelength of equal absorptivity of two component i.e isobestic or isoabsorptivity component. This method of analysis is reffered as absorbance ratio method or different spectrophotometry.

7. PROCEDURE 1)PREPARATION OF BROMOTHYMOL BLUE:- Dissolve 50mg of bromothymol blue in 4ml of 0.02M sodium hydroxide and 20ml of ethanol (95 percent). After solution is effected , add sufficient water to produce 100ml. 2) PREPARATION OF BUFFERS:- a) Acid phthalate buffer:- Place 50ml of 0.2 M potassium hydrogen phthalate in a 200ml volumetric flask , add the specified volume of 0.2M hydrochloric acid, and then add water to volume. For ph 3.2 add 15.7 ml of 0.2M hydrochloric acid.

8. PH 0.2 M NaOH ml 6 5.6 7 29.1 7.2 34.7 7.4 39.1 8 461 b) Phosphate buffer:- Place 50ml of 0.2M potassium dihydrogen phosphate in a 200ml volumetric flask , add the specified volume of 0.2M sodium hydroxide and then add water to volume.

9. 3) PROCEDURE FOR ISOSBESTIC POINT DETERMINATION:- a) Pipette out 2ml of bromothymol blue solution (0.02%) and transfer to 6 different 50 ml graduated volumetric flask. b) Adjust the volume of each graduated volumetric flask using buffer solution of pH- 3.2, 6, 7, 7.2, 7.4, and 8 respectively. c) Determine the absorption spectrum of each solution over the range 200-800 nm using 1cm cuvette.. d) Record each spectrum on same chart and note the isosbestic point of equal absorbance regardless of pH and position of Tmax.

10. RESULT AND DISCUSSION

11. PH Peak 1 Peak 2 Peak 3 3.2 660 572 461.23 6 641 572 432.87 7 687 572 440.43 7.2 663 572 442.60 7.4 611.18 572 439.44 8 659 572 422.19 The different pH shows 3 peak in which the second peak is constant that mean on that wavelength the buffer solution does not varies. The standard absorptivity wavelength of bromothymol blue is 498nm. And the experimental absorptivity wavelength of bromothymol blue was determine at 480nm which is near about equal to the standard wavelength of bromothymol blue.

12. CONCLUSION The isosbestic point for bromothymol blue indiator solution in various different pH buffer solution is found to be 480 From the given experimental observation it is observed that bromothymol blue indicator shows two distinct peaks in all buffer solution. Among them peak 1 was observed at 572 nm and which didn’t get affected with change in pH of buffer solution. Whereas the second peak show distinct changes with increase in pH of buffer solutions. It is observed that second peak get shifted to shorter wavelength i.e 461.23 nm to 422.19 nm as we change the pH of buffer solution from 3.2 to 7.4. This shift towards shorter wavelength is reffered as blue shift. In respect of change in pH buffer solution at one wavelength i.e at 480 nm the indicator show same absorbance and absorptivity value, this wavelength is reffered as isosbestic point for bromothymol blue indicator solution in various different buffer pH solution.

13. REFERENCE Practical pharmaceutical chemistry part 2 by A.H. Beckett and J.B. Stenlake . Page no. 293-294,330 and 331 Nahhal; et al. (18 July 2012).  "Thin film optical BTB pH sensors using sol–gel method in presence of surfactants"   (PDF) . International Nano Letters. 2 (16): 3 . Retrieved 18 November 2014 . Jump up to: a   b   O'Neil, Maryadele J (2006). The Merck Index. Merck Research Laboratory. p. 1445.  ISBN   978-0-911910-00-1 . Sabnis R. W. (2007). Handbook of Acid-Base Indicators.  CRC Press .  ISBN   978-0-8493-8218-5 . Sabnis R. W. (2010). Handbook of Biological Dyes and Stains: Synthesis and Industrial Applications (1st ed.). Wiley.  ISBN   978-0-470-40753-0 .  IUPAC Gold Book (International Union of Pure and Applied Chemistry)  https://goldbook.iupac.org/html/I/I03310.html

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