EXPT. 5 DETERMINATION OF pk a OF AN INDICATOR USING SPECTROPHOTOMETRY

Transcription

1 EXPT. 5 DETERMITIO OF pk a OF IDICTOR USIG SPECTROPHOTOMETRY Strctre 5.1 Introdction Objectives 5.2 Principle 5.3 Spectrophotometric Determination of pka Vale of Indicator 5.4 Reqirements 5.5 Soltions Provided 5.6 Procedre 5.7 Observations and Calclations 5.8 Reslt 5.1 ITRODUCTIO Yo have so far learnt abot and performed the qantitative determination of inorganic and organic species sing UV-VIS spectrophotometry in this laboratory corse. In this experiment yo wold learn abot an application of spectrophotometry in the determination of a physical constant for an organic compond. Yo wold learn abot and carry ot the determination of the pk a of an acid-base indicator. Yo know that an indicator is sed for the visal detection of the end point of a titration. The indicator sed in acid-base titrations is either a weak acid or weak base which has distinctly different colors in the ionised and nionised form. The end point in an acid-base titration is indicated by a sharp change in the color of the indicator de to a steep change in the ph of the soltion near the eqivalence point of the titration. Spectrophotometry can be sed to determine the concentrations of the ionised (basic) and nionised (acidic) forms of the indicator which in trn is sed for the determination of the acid dissociation constant sing Henderson-Hasselbach eqation. In the next experiment yo wold learn abot the application of IR spectrometry in the detection of the fnctional grop in an organic compond. Objectives fter stdying and performing this experiment yo shold be able to: explain the principle nderlying the spectrophotometric determination of pk a of an acid-base indicator, state and explain Henderson-Hasselbach eqation, prepare a series of bffer soltions and measre the absorbance of the indicator soltion as a fnction of ph, compte the relative concentrations of the ionised and nionised forms of the indicator by simltaneos eqation method and determine the pk a vale of the indicator sing Henderson-Hasselbach eqation, and determine graphically, the pk a of an acid-base indicator sing the ph verss absorbance data. 31

2 Spectroscopic Methods Lab 5.2 PRICIPLE HO O Methyl red 2-(4-dimethylaminophenylazo)benzoic acid s mentioned in the introdction, an acid-base indicator is either a weak acid or a weak base that has distinctly different colors in the ionised and nionised forms. One form of an indicator may be colorless bt the other mst be distinctly colored. Let s take the example of the indicator methyl red. It is a two color indicator; red in its nionised (acidic) and yellow in its ionised (basic) form. Methyl red is a weak organic acid which can be sed as an indicator in the ph range of 4.4 to 6.2. This implies that a soltion of methyl red will be red if the ph is lower than 4.8 and yellow if it is above 6.2. On the other hand, if the ph of the soltion is in this range (4.4< ph > 6.2), the color will be an appropriate mixtre of both the colors. Methyl red is a weak acid and can be represented as say,. The dissociation of the indicator can be expressed as given below. + H + Unionised Ionised represents the ionised or the basic form of the indicator. The acid form () of the indicator is zwitterionic in natre and is a resonance hybrid of two closely related strctres; the basic form on the other hand is an anionic species. The strctres of the acidic and basic forms and the eqilibrim between them are as given below. O O O O + H cid form (RED) + H H + OH O O Base form (YELLOW) Yo have learnt earlier that Henderson-Hasselbach eqation provides the relationship between ph and pk a vale of an indicator. For methyl red we can write the Henderson-Hasselbach eqation as given below. [ ] ph pk a + log (5.1) [] It can be rearranged as following, [ ] K ph log (5.2) [] p a or [ ] log ph pk [] a (5.3) 32

3 Ths, if we know the concentrations of the ionised and nionised forms of the indicator at a given ph, we can determine the pk a vale of the indicator. s both, the ionised as well as the nionised forms of methyl red are colored, their concentrations can be determined by measring the absorbances at the wavelengths of maximm absorption of the two forms with the help of a spectrophotometer or a colorimeter. These can then be sed to compte the pk a vale of methyl red sing Henderson-Hasselbach eqation. This forms the basis of the spectrophotometric determination of the pk a vale of the indicator. 5.3 SPECTROPHOTOMETRIC DETERMITIO OF pka VLUE OF IDICTOR typical spectrophotometric determination of the pk a vale of the indicator consists of the following steps. It may be noted that the ionised form has small absorption at the wavelength of maximm absorption of the nionised form. Similarly, the nionised form also absorbs to some extent at the wavelength of maximm absorption of the ionised form. Step 1: Obtaining the absorption spectra of the pre nionised and ionised forms of the indicator to determine the wavelengths of their maximm absorption and the corresponding molar absorption coefficients soltion of a known concentration of the indicator is prepared in acidic soltion (low ph) sch that the indicator exists almost exclsively in the nionised form and the spectrm is obtained. Similarly, a spectrm is obtained for a soltion of a known concentration of the indicator in a basic soltion (high ph) sch that the indicator exists almost exclsively in the ionised form. The schematic spectra of the nionised and the ionised forms of the indicator are given in Fig Fig. 5.1: Schematic spectra of ionised (basic) form (ble crve) and nionised (acidic) form (black crve) of methyl red indicator These spectra are then analysed to determine the wavelengths of maximm absorption respectively for the nionised and ionised forms of the indicator. Let these be represented as max,, and respectively. For convenience let s simplify the max, expressions as and ; the sbscript max and the charge on the ionised form being dropped. The molar absorption coefficients of the nionised and ionised forms at the two wavelengths of maximm absorption obtained above are determined sing Beer- 33

4 Spectroscopic Methods Lab Lambert s law. Yo know that the expression for the Beer-Lambert s law can be written as follows. bc (5.4) In the expression, is the absorbance, is the molar absorption coefficient, b is the thickness or the path length (in cm) of the sample and c is the concentration of the absorbing species in moles per litre. For a nit path length at a given concentration the molar absorption coefficient can be written as given below. (5.5) c The for molar absorption coefficients for the nionised and ionised forms of the indicator at the two wavelengths, and can be defined as follows.,,,, (5.6) c, (5.7) c, (5.8) c,, (5.9) c These are determined by sing the absorption vales at the wavelengths of maximm absorption of the nionised and ionised forms in the spectra obtained above. Step 2: Verification of the Beer-Lambert s law for the nionised and ionised forms of the indicator at the wave lengths, and The Beer s law can be verified by measring the absorbances of a series of soltions of varying concentration obtained by dilting the stock soltion of the indicator in the nionised and ionised forms sing the cvettes of path length eqal to 1cm. These absorbance vales are then plotted against relative concentrations of the soltion. The linear plot so obtained establishes the validity of the Beer s law. The slope of the line obtained gives the molar absorption coefficients. Step 3: Obtaining the absorption vales of the indicator at different ph vales small bt fixed amont of the indicator soltion is added to a series of bffer soltions having ph spread over the indicator range (pk a ± 1) sch that the indicator exists in varying proportions of the ionised and nionised form. s the K a vale for acetic acid is in the same range as that for methyl red, we will se acetic acid-acetate bffers to control the ph. The absorption vales of these soltions at and, the wavelengths of maximm absorption of the nionised and ionised form of the indicator respectively are measred with the help of a sitable spectrophotometer or colorimeter. Step 4: Maniplating the data obtained in steps 1-3 to obtain the pk a vale The data obtained in the steps 1-3 can be sed to determine the pk a vale of the indicator. This can be achieved in a nmber of ways. Two of these are described as follows. 34

5 . Simltaneos Eqations Method Yo wold recall from Experiment 3 of this corse that when the analyte contains a mixtre of two species whose spectra overlap to certain extent then the concentrations of these can be obtained by solving a set of simltaneos eqations. s in the present case also the two species present in the soltions of the indicator at a given ph have overlapping regions in their spectra, we can compte their concentrations in the same way. The relevant eqations can be worked ot as follows. In the mixtres of the acidic and basic forms of methyl red, the total absorbance at the wavelengths of maximm absorptions of the two forms viz., and can be written as follows. [ ], +, [] (5.10) [ ], +, [] (5.11) Solving these simltaneos eqations we get the expressions for the concentrations of the two species as follows. [] [] (5.12),,,,,, (5.13),,,,, These eqations can be sed to obtain an expression for the ratio of the ionised and the nionised form of the indicator in a given mixtre. The expression comes ot to be as follows. [ ],, (5.14) [].,, This can then be sed to obtain the pk a vale of the indicator by sing the Henderson- Hasselbach eqation, viz.,, [ ] pk a ph log (5.2) [] Ths, [. ],, pk a ph log. (5.15) [ ],, In this experiment the concentration of the indicator is to be kept constant in all the absorbance measrements. s we do not need the absolte vales of the concentrations of the nionised and ionised forms of the indicator we jst need their ratio. Therefore, we can do away with the determination of the molar absorption coefficients mentioned above, instead se the absorbance vales for the total concentration of the indicator in the nionised and ionised forms at the and. These can be obtained by 35

6 Spectroscopic Methods Lab extrapolating the linear plots obtained for the verification of Beer s law to relative concentration of 1.0. ccordingly, Eq. (5.10) and (5.11) get modified as follows. [ ] + [], (5.16), [ ] + [], (5.17), Where, the terms containing the sperscript pertain to the absorbance vales at relative concentration of 1.0 or nity. The final expression for the pk a vale then can be written as follows. pk a [.. ],, ph log (5.18) [.. ],, B. Graphical Method Recall that according to the Henderson-Hasselbach eqation, [ ] log ph pk (5.3) a [] This represents an eqation of a straight line of the type, Y mx + C. where, Y eqals [ ] log ; X ph and C pk. Ths, in a plot of [ ] a log verss ph the [] [] slope wold be eqal to 1 and the intercept wold be eqal to pk as shown in a Fig.5.2. Ths, the pk a can be fond by determining the intercept of the plot of [ ] log [] verss ph. lso, the line wold cross the ph axis at ph pk a ( as at this stage the concentrations of the ionised and nionised forms wold be eqal, [ ] [], making the log term eqal to zero. Fig. 5.2: [ ] log verss ph plot for the indicator [] 36

7 The pk a can be obtained either as the point of intersection of the line with the X-axis or from the intercept on the Y-axis. 5.4 REQUIREMETS pparats Spectrophotometer/ Filter photometer Matched cvettes ph meter with glass electrode Volmetric flasks (1 litre) Volmetric flasks (250 cm3) Volmetric flasks (100 cm3) Volmetric flasks (50 cm3) Pipettes (10, 20 cm3) Brette stand with clamp each 1 Chemicals Ethanol Hydrochloric acid Sodim acetate cetic acid Methyl red indicator 5.5 SOLUTIOS PROVIDED i) Sodim acetate (0.04M): It is prepared by accrately weighing 3.28 g of anhydros sodim acetate and transferring to a 1 dm 3 volmetric flask containing abot 100 cm 3 of distilled water. fter dissolving the salt the volme is made p to the mark with distilled water. ii) iii) Sodim acetate soltion (0.01M): It is prepared by dilting 250 cm 3 of the 0.04 M sodim acetate soltion prepared above to 1 dm 3 by distilled water. cetic acid soltion (0.02M): It is prepared by mixing 1.2 cm 3 of glacial acetic acid with 100 cm 3 of distilled water in a 1dm 3 volmetric flask and making p the volme with distilled water. iv) Hydrochloric acid soltion (0.1M): It is prepared by transferring 9 cm 3 of concentrated hydrochloric acid to a 1dm 3 volmetric flask containing 500 cm 3 of distilled water. fter mixing the volme is made p by distilled water. v) Hydrochloric acid soltion (0.01M): It is prepared by dilting 100 cm 3 of the 0.1 M hydrochloric acid soltion prepared above to 1 dm 3 by distilled water. vi) Methyl red indicator (stock) soltion: It is prepared by dissolving 0.1 g of pre crystalline methyl red in 30 cm 3 of 95% ethanol and making p to 100 cm 3 with distilled water. vii) Methyl red in acidic form (Soltion ): It is prepared by mixing 10 cm 3 of the indicator soltion prepared in (vi) above with 10 cm 3 of 0.1 M HCl soltion and dilting to 100 cm 3 with distilled water in a volmetric flask. viii) Methyl red in basic form (Soltion B): It is prepared by dilting 10 cm 3 of the indicator soltion prepared in vi) above with 0.01 M sodim acetate soltion to 100 cm 3 in a volmetric flask. 5.6 PROCEDURE Yo wold recall from section 5.3 that a typical spectrophotometric determination of the pk a vale consists of for steps. These are as follows. a) Determine the wavelengths of maximm absorption for the nionised and ionised forms of the indicator, 37

8 Spectroscopic Methods Lab b) Verification of Beer s law for nionised () and ionised ( - ) forms at the wavelengths of their maximm absorption, c) Obtaining the absorption vales of the indicator at different ph vales, d) Maniplating the data obtained in step 1-3 to obtain the pk a vale of the indicator. Follow the instrctions given below in the seqential order to accomplish these tasks. a) Determination of the wavelengths of maximm absorption for the nionised and ionised forms of the indicator 1. Record the absorption spectrm of soltion in the range nm against 0.01 M HCl. 2. In case the instrment is of manal type, measre the absorption vale after every 10 nm over the spectral range and record the readings in colmns 2, 5 and 8 of the Observation Table Similarly, measre the absorption vale for soltion B against 0.01 M sodim acetate after every 10 nm over the spectral range and record the readings in colmns 3, 6 and 9 of Observation Table Draw the spectrm of soltion and soltion B by plotting the absorbance as a fnction of the wavelength in the graph provided in Fig.5.3. Yo may se two different colors to draw the spectra for soltion and soltion B respectively. 5. Select the wavelength which gives maximm absorbance for soltion and soltion B and record the same as and respectively. b) Verification of Beer s law for and at and 1. Pipette ot 40.0 cm 3, 20.0 cm 3 and 10.0 cm 3 of soltion into three separate 50 cm 3 volmetric flasks and make p the volme in each case with 0.01 M HCl soltion. Label these soltions as 1, 2 and 3 respectively. These soltions wold have concentrations eqal to 0.8, 0.4 and 0.2 times the concentration of the stock soltion. 2. Similarly, pipette ot 40.0 cm 3, 20.0 cm 3 and 10.0 cm 3 of soltion B into three separate 50 cm 3 volmetric flasks and make p the volme in each case with 0.01 M sodim acetate soltion. Label these soltions as B1, B2 and B3 respectively. These soltions wold have concentrations eqal to 0.8, 0.4 and 0.2 times the concentration of the stock soltion B. 3. Measre the absorbances of the soltions 1, 2 and 3 at and sing 0.01 M HCl as the reference and record yor observations in Table Similarly, Measre the absorbance vales for the soltions B1, B2 and B3 at and sing 0.01 M sodim acetate as the reference and record yor observations in Observation Table Plot the absorbance vales obtained in step 3 and 4 against the corresponding relative concentrations in the graph provided in Fig The linearity of the plot so obtained establishes the validity of the Beer s law. 7. Extrapolate the linear plots obtained above to compte the absorbance vales of the nionised and ionised forms of the indicator at and and record the same. 38

9 c) Obtaining the absorption vales of the indicator at different ph vales 1. Prepare for soltions of the indicator in bffer soltion of different ph vales by mixing sodim acetate, acetic acid, methyl red and water as detailed in colmn 2 to 5 of the Observation Table 5.3. Use 100 cm 3 volmetric flasks labelled as 1, 2, 3 and 4 for this prpose. 2. Measre the ph vales of these soltions with the help of a sitably calibrated ph meter. Record these vales in the colmn 6 of Table Measre the absorbance vales of these soltions at and against water as a blank. Record the same nder colmns 7 and 8 of Observation Table 5.3. d) Calclation of pk a vale for the indicator from the data obtained 1. Calclate the vales of [], [], and [ ] respectively from the [] observed absorbance vales at different ph vales sing eqations given nder step D of Section 5.6. Record the same in Observation Table Use these to calclate pk a vale with the help of Henderson-Hasselbach eqation and record in Observation Table Find average vale and report the reslt. 4. Plot a graph between ph (x-axis) and [ ] log (y-axis) in Fig.5.5. [] Determine the pk a vale from the point of intersection of the line and the ph axis and also in terms of the intercept on the y-axis and report the reslt. 5.7 OBSERVTIOS D CLCULTIOS. Determination of the wavelengths of maximm absorption for the nionised and ionised forms of the indicator Observation Table 5.1: bsorbance vales of the soltion and soltion B at different wavelengths Wavelength (nm) Colmn bsorbance Wavelength bsorbance Wavelength bsorbance (nm) (nm) Soltion Soltion B Soltion Soltion B Soltion Soltion B 39

10 Spectroscopic Methods Lab B. Spectra for nionised form of the indicator (soltion ) and ionised form of the indicator (soltion B) sing the data recorded in Table 5.1 bsorbance Wavelength (nm) Fig. 5.3: Visible spectra for methyl red in the nionised and ionised forms From the spectra obtained above, the wavelengths of maximm absorption for the nionised and ionised forms of the indicator methyl red are as follows. For nionised form, nm For nionised form,.nm C. Verification of Beer s law for and - at the and Observation Table 5.2: bsorbance vales of the soltion and soltion B at different wavelengths For Unionised form, Soltion Volme of Soltion Volme of 0.01 M HCl Relative concentration For ionised form, - Soltion Volme of Soltion B Volme of 0.01 M CH 3 COOa B B B Relative concentration bsorbance bsorbance 40

11 bsorbance Relative concentration Fig. 5.4: bsorbance vales (at and ) verss relative concentration plot for methyl red in the nionised and ionised forms The absorbance vales at and for nionised and ionised forms of methyl red at relative concentration of 1.0 are fond to be as given below.,,,, D. Obtaining the absorption vales at and for the indicator at different ph vales. Observation Table 5.3: bsorbance vales of the indicator soltion in bffer soltions of different ph vales Colmn S.o. Volme of 0.04 M 0.02 M Indicator Water CH 3 COOa CH 3 COOH Stock (cm 3 ) (cm 3 ) (cm 3 ) (cm 3 ) To make p to the mark To make p to the mark To make p to the mark To make p to the mark ph bsorbance 41

12 Spectroscopic Methods Lab E. Calclation of pk a vale for the indicator from the data obtained a) Simltaneos Eqation Method [ ] The vales of [], [],, can be calclated from the observed [] absorbance vales at different ph vales sing the following eqations. [].,.,,,..,, [].,,.,.,.,, [ - ] []..,,,..,,, pk a [ ph log [..,,..,, ] Observation Table 5.4: Comptation of the pka vales of methyl red indicator sing Handerson-Hesselbalch eqation. ] S.o. ph [] [] [ ] [] [ ] log [] [ ] pk a ph log [] verage vale of pk a The average vale of pk a from the simltaneos eqation method is fond to be b) Graphical method Graph between ph and [ ] log [ ] 42

13 ph Fig. 5.5: Plot of [ ] log [] verss ph to determine the pka of methyl red. The vale of pk a from the graphical eqation method is fond to be 5.8 RESULT The pk a of the indicator (methyl red) sing simltaneos eqation method is fond to be The pk a of the indicator (methyl red) sing graphical method is fond to be