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Experiment 3: Determination of pKa of Bromothymol Blue Purpose : 1.

The purpose of this experiment is to use a spectrophotometry method to evaluate the pKa of bromothymol blue, a commonly used acid base indicator. 2. To learn to use a pH meter 3. To learn doing calculation of pH and pKa 4. To learn using ! visible spectrometer Introduction: " pH meter is an instrument used to measure pH. " special combination electrode that is sensitive to hydrogen ion concentration is dipped into the solution to be tested. "fter the instrument has been calibrated using a solution of #no$n pH the electrode is dipped into the solution to be tested and the pH is read from the meter %or ! and visible spectroscopy , the transitions that result in the absorption of electromagnetic in this region of the spectrum are transitions bet$een electronic energy levels. "s a molecule absorbs energy an electron is promoted from an occupied orbital to an unoccupied orbital of greater potential energy . &enerally the most probable transitions is from the highest occupied molecular orbital 'H()(* to the lo$est unoccupied molecular orbital '+ )(*. The energy differences bet$een electronic levels in most molecules vary from 12, to -,. #/0mole . 1romothymol blue, also called 32,33 4 dibromothymolsulfone 4 phthalein 'molar mass 5 -24.4.g mole61*, is a member of a class of acid base indicators called the 7sulfone 4 phthaleins3. This indicator undergoes three colour changes8 H2ln !ed" pH#1 '1* 9n this experiment $e $ill consider the e:uilibrium bet$een the yello$ and blue form 'Ka2* and ignore other reaction. %or this e:uilibrium, Ka2 * +H+,+ln2 ,-+Hln , Ta#ing the logarithm of both sides yields ;:uation '3* 'remember logxy 5 logx < logy* lo.Ka2 * lo.+H+, + lo.+ln2 ,-+Hln , pon rearrangement, ;:uation '3* becomes, lo.+H+, * /lo. Ka2 + lo.+ln2 ,-+Hln , =ince pH5 6log>H<? and pK 5 6logK, ;:uation '4* simplifies to '4* '3* '2* Ka1 H+ + Hln $ello%"1#pH#& Ka2 2H+ + ln2 !oyal 'lue"pH()

pH * pKa2 + lo.+ln 2 ,-+Hln ,


$hich is the Henderson6Hasselbalch e:uation. This e:uation fits the e:uation for a straight line therefore $e can determine the pKa,2 'y6intercept* by plotting pH versus log>ln26?0>Hln6?. @onse:uently $e need to be able to measure >ln26? and >Hln? at various ph levels. These measurement can be done spectrophotometrically because these t$o species have different absorption spectra. " series of solutions of various pH $ith the same total concentration of indicator $ill be prepared and the absorbance $ill be recorded at these t$o $avelength of maximum absorbance. "n example of the spectrum obtained at the $avelength maximum absorbance for ln26 is sho$n in figure 1, $here "Hln6, is the maximum absorbance of Hln6, "ln26, is the maximum absorbance of ln26, and " is the absorbance of Hln6, "ln6 is the maximum absorbance of ln26, and " is the adsorbance of the mixture at pH A '" is going to vary $ith pH*. %rom these relationship it is clear that +ln2 ,-+HI , * 0 / 0Hln -0ln2 0 '-* sing e:uation - +ln2 ,-+HI , at various ph values can be calculated and these values then substituted into plot in e:uation , to find pKa. 1ethodolo.y: 1. Brepared by laboratory assistant 2, ml of ..1C bromothymol blue '1T1* in 2.C ethanol 'density 5 ..DA.2 g m+61 *, 1.. m+ of ..1. ) a:ueous sodium phosphate, dibasic 'Ea2HB(4*, and 1.. m+ of ..1. ) a:ueous potassium phosphate, monobasic 'KH2B(4* 2. The follo$ing three solutions prepared to obtain absorption spectra. Part 0 pH 18 1..m+ of 1T1 stoc# solution are pipetted into volumetric flas#. F G2 m+ of distilled $ater , are add in to the solution, and then add in drop $ise - ) conncentrated H@l and dilute to the mar# $ith distilled H2(. pH &23: 1.. m+ of 1T1, ,.. m+ ..1. ) Ea2HB(4 and ,.. m+ of ..1. ) KH2B(4 are add a 2,m+ volumetric flas#. Hilute to the mar# $ith distilled $ater. pH13: 1..m+ of 1T1 stoc# solution are pipetted into a 2,m+ volumetric flas#. G2m+ of distilled $ater are add into the solution, and then add drop$ise 12 drops of 4) Ea(H ,then dilute to the mar# $ith distilled $ater.

Part B 3. ltra 4 violet !isible =pectrophometer in lab &,1 is use to obtain the absorption spectra for the 3 solutions, and determine the isosbestic point in the spectra. F ;ach absorbance spectra are print out in hardcopies, and then determine the $avelength of maximum absorbance. F Three solutions are save for further analysis. 4. The follo$ing samples are prepared in 2,m+ of volumetric flas#, respectively and dilute to volume, the pH of each of the solutions are measure $ith a pH meter. F 6 26 2 $avelengths $here Hln and ln sho$ a maximum difference in their absorbance are select and the absorbance of each solution are measured.

Eo. 1. 2. 3. 4. ,. -. A.

9ndicator 1T1 0 m+ 1.. 1.. 1.. 1.. 1.. 1.. 1..

KH2B(4 '..1)* 0 m+ ,.. ,.. 1. ,.. 1.. 1.. ...

Ea2HB(4 '..1)* 0 m+ ... 1.. ,.. 1... ,.. 1... ,..

!esult and 4alculation: Bart "8 pH from theory 1.. -.D 13.. pH from experiment Iello$ &reen blue

Bart 18 9ndicator 1T10ml 1.. 1.. 1.. 1.. 1.. 1.. 1..

KH2B(4'..1)*0ml Ea2HB(4'..1)*0ml ,.. ... ,.. 1.. 1... ,.. ,.. 1.. 1.. ... 1... ,.. 1... ,..

pH 4.1 -.. -.4 -.D A.4 A.J.J

9sosbestic point is cutting point for the three spectrum $ith the $ave distance ,.nm. %rom the graph the isosbestic point is e:ual to

9t is given that the center point for every graph give the point $here the concentration of indicator is base is same $ith the concentration of indicator is acid. Therefore, pK 5 pH 4 log>ln6?0>Hln? pK 5 pH 4 log>ln6?0>Hln? it is because that, >ln6? 5 >Hln? so, pKa 5 pH 6 log>ln6?0>Hln? 5 pH 4 log 1 5 pH 5 %rom theory, pKa for bromothymol blue is Therefore C error 5 x 1..C 5 C

9sosbestic point is cutting point for the three spectrum $ith the $ave distance ,.nm. %rom the graph the isosbestic point is e:ual to 'a* To get the value of the percentage for the transmittance "5 log 9 =6log 9 9 9 ThereforeK "5 6log T =o, T5 1.6" x 1..C Eo 1 2 3 4 Lavelength 5,2...nm " 5 ...-J CT 5 1.6...-J x 1..C 5 J,.,.-A " 5 ...,J CT 5 1.6...,J x 1..C 5 JA.4DJ4 " 5 ...D2 CT 5 1.6...D2 x 1..C 5 J..D.D" 5 ..12, CT 5 1.6..12, x 1..C Lavelength 5 4D...nm " =..1A2 CT 5 1.6..1A2 x 1..C 5 -A.2DAA " 5 ..1,. CT 5 1.6..1,. x 1..C 5 A..AD4" 5 ..14D CT 5 1.6..14D x 1..C 5 A..D,AJ " 5 ..12A CT 5 1.6..12A x 1..C ,$here T5 9 9

, A

5 A4.DJD4 " 5 ..1-2 CT 5 1.6...-J x 1..C 5 J,.,.-A " 5 ..1J4 CT 5 1.6..1J4 x 1..C 5 -,.4-3" 5 ..2.. CT 5 1.6..2.. x 1..C 5 -3..D,A

5 A4.-44D " 5 ..121 CT 5 1.6..121 x 1..C 5 A,.-J33 " 5 ..11A CT 5 1.6..11A x 1..C 5 A-.3J3" 5 ..1.3 CT 5 1.6..1.3 x 1..C 5 AJ.JJ-.

Discussion: 1romothymol blue is a member of a class of acid base indicators $hich is $ea# acid. The pKa value for it is A... The pH range is from -.. to A.-. 9n medium acid, it is in yello$ colour. 9n medium al#ali, it is in blue colour. %rom the result of the experiments, for part " $hen the pH change from 1,-.D to 13, the colour change from yello$, green to blue. The changing of the colour sho$s the relative change for the concentration of both acid and base conMugate. 9n this experiment, the pH value $e get is difference from the theory value. The differences maybe is caused by the solution that $ere prepared by the lab assistant is slightly less than the actual volume. 9t also maybe the mass of the chemical used by the assistant is inaccurate, for example is dinatrium hydrogen phosphate ..1), Ea(H 4), and others. 1esides, the pH meter that $e use is very sensitive, so is so tough to get the actual value of the pH. The value of pH al$ays change from time to time, so $e have to use :uite a long time to read the value. 9n the acid condition, the e:uation al$ays move to the left to produce more Hln. 9n base condition, the e:uation move to the right to produce more ln6, HlnN H< < ln6. 9n this experiment originally $e have a spectra absorption from ! visible spectroscopy $hich sho$s three pea#s as 3 solutions $ith the 3 different pH as $ell as isobestic point $here three pea#s Moin. Ho$ever for us the point is not very accurate meaning only t$o pea#s Moin not three graph caused by did not prepare solutions $ith accurate volume. 1esides error occurring during experiment also can cause this to happen.

Le also dra$ t$o graphs of absorption energy versus pH and pH versus log>ln ?0>Hln?. %rom the graph absorption energy versus pH that $e plotted, the value pKa for bromothymol blue that $e use is . the error percentage that $e calculated is C $hen $e compare it $ith the value from theory. The error consider as a minor mista#e, so $e can leave it.

There are some precaution steps $e should ta#e, to get more accurate value for this experiment, $hich are8 1. 2. 3. 4. Lash all instruments $ith distilled $ater cleanly. )a#e sure to use the pH meter in correct $ay. The pH value must be ta#en only after the value become constant. The solution that $e prepared must sha#ed $ell before ta#ing the value from pH meter and ltra 4 violet !isible =pectrophotometer. ,. )a#e sure to choose the instrument that suit $ith it2s sensitivity $ith the :uantity that $e need.

4onclusion: 1. The isosbestic point that $e get is e:ual to 2. The pKa value for bromothymol blue from the experiment is from the theory is A.1.. and the value

3. The error percentage is



@H)34.2 =pectroscopy lab manual upm, page 1161, 9ntroduction to spectroscopy 3rd edition, Bavia Bage 16A0 3,3 @H;)9=TOI matter and its changes ,1rady senese pageA-2 " Text 1oo# of Puantitive 9norganic "nalysis, 3rd edition ".9 !ogel =ilverstein, O.),&.@. 1asster and T.@ )orrill'1DJ1* =pectrometric 9dentification of (rganic @ompounds 4th edition Ee$ Ior#8/ohn Liley Q sons

/ohn ".+andgrebe, 1DD3 Theory and Bractice in the organic laboratory $ith )icroscale and =tandard =cale ;xperiments 4th edition Bacific &rove,@alifornia

Graph Absorption Energy Vs pH

0.30 0.25
Absorption Energy

0.20 0.15 0.10 0.05 0.00 0.00 Series1 Series2