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This document provides instructions for a lab experiment to analyze the concentrations of caffeine and benzoic acid in a soft drink using UV-Vis spectrophotometry. Students will prepare standard solutions of caffeine and benzoic acid to generate calibration curves. They will then collect absorbance spectra of the standards and an unknown soft drink sample between 240-400nm. Using the calibration curves and Beer's Law, students will analyze the data in Microsoft Excel to calculate the concentrations of caffeine and benzoic acid in the original undiluted soft drink sample.
Descriere originală:
Spectrophotometric Analysis of a
Caffeine and Benzoic Acid in a Soft Drink
This document provides instructions for a lab experiment to analyze the concentrations of caffeine and benzoic acid in a soft drink using UV-Vis spectrophotometry. Students will prepare standard solutions of caffeine and benzoic acid to generate calibration curves. They will then collect absorbance spectra of the standards and an unknown soft drink sample between 240-400nm. Using the calibration curves and Beer's Law, students will analyze the data in Microsoft Excel to calculate the concentrations of caffeine and benzoic acid in the original undiluted soft drink sample.
This document provides instructions for a lab experiment to analyze the concentrations of caffeine and benzoic acid in a soft drink using UV-Vis spectrophotometry. Students will prepare standard solutions of caffeine and benzoic acid to generate calibration curves. They will then collect absorbance spectra of the standards and an unknown soft drink sample between 240-400nm. Using the calibration curves and Beer's Law, students will analyze the data in Microsoft Excel to calculate the concentrations of caffeine and benzoic acid in the original undiluted soft drink sample.
Lab #8 -Spectrophotometric Analysis of a Mixture: Name_________________________
Caffeine and en!oic Acid in a Soft "rin#
(after Harris Exploring Chemical Analysis, $artner%s&_____________________ 2 nd ed, p. 51!51"# $A'( ): )ntroduction In this experiment, we use ultraviolet absorbance to measure two major species in soft drinks. Caffeine is added as a stimulant and sodium benzoate is a preservative. All solutions will contain 0.0 ! "Cl, so the sodium benzoate is protonated to make benzoic acid. Caffeine has no appreciable basicit#, so it is neutral at p" $. %e restrict ourselves to non&diet soft drinks because the su'ar substitute aspartame in diet soda has some ultraviolet absorbance that sli'htl# interferes in the present experiment. %e also avoid darkl# colored drinks because the colorants have ultraviolet absorbance. !ountain (ew, !ello )ello, and, probabl#, other li'htl# colored drinks are suitable for this experiment. *here is undoubtedl# some ul& traviolet absorbance from colorants in these bevera'es that contributes s#stematic error to this experiment. $A'( )): $reparin* Standard Solutions of en!oic Acid and Caffeine %use distilled +ater& . In the data sheet 'o to the section labeled $reparin* Standard Solutions for the Calibration Cur,e. +nter the concentrations of the benzoic acid ,-A. and caffeine ,C. stock solutions in m'/0. Calculate how man# millileters of the stock solutions are needed to produce 00 m0 each of the indicated concentrations of -A and C usin' the dilution e1uation !2 3 !$2$. $. 4repare the standard -A samples b# addin' the calculated volumes of stock solution, 0 m0 of 0.0 ! "Cl, and dilutin' to 00 m0 in the provided volumetric flasks. Caffeine 5! 67.6 8. 4repare the caffeine standards -)(./0( 0.0 ! "Cl. 7. Calculate the molarities of the standard solutions. 9. %arm approx. $0 m0 of soft drink in a beaker on a hot plate to expel C:$. After coolin' to room temperature in ice, pipet 7.00 m0 into a 00&m0 volumetric flask. Add 0.0 m0 of 0.0 ! "Cl and dilute to the mark. $A'( ))): /btainin* the Absorbance Spectra of en!oic Acid and Caffeine 12 5amiliarize #ourself with the basic components of the spectrophotometer b# referrin' to 5i'ure . *he ;$000 is a dual&beam instrument, so the back'round absorbances present are automaticall# ratioed out. 5i'ure 32 )ou will be usin' the "itachi ;$000 dual&beam ;2&2is spectrophotometer. *he ;$000 has a cathode ra# tube ,C<*. for displa# and a ke#board. !ost of the operation of the spectrophotometer is microprocessor controlled. %e can access the instrument controls b# usin' the ke#board ,5i'ure $.. <ead throu'h the ke#board description before proceedin'. 5i'ure $ 42 *he instrument should have the followin' screen displa#ed after we turn it on ,5i'ure 8.. *his is the MA)N M5N0 screen. N/(5: the fi*ure numbers in the illustrations do not match the fi*ure numbers in the instructions2 5i'ure 8 62 4ress the down arrow cursor movement ke# to hi'hli'ht selection number $ or =wavelen'th scan=. After selection press the 5N(5' ke#. *he 7-L SCAN7 menu will appear ,5i'ure 7.. 5i'ure 7 82 !enu option > will be "A(A M/"5. It should alread# be set for "A(A M/"5 9 AS. If not, chan'e it to AS b# selectin' the option displa#ed in the lower left&hand portion of the screen. ?ow move the hi'hli'hted area to choice >$ or (5S( S5(0$. A new screen will appear ,5i'ure 9.. 5i'ure 9 :2 +nter a value of 890 for the S(A'( -L %nm&. 4ress enter. ;2 +nter a value of $0 for the S(/$ -L %nm&. 4ress enter. 82 +nter .$0 for the 0$ SCAL5. A maximum absorbance of .$0 will be displa#ed when we scan the spectrum. <2 +nter 0.00 for the L/ SCAL5. 1=2 @et the SCAN S$55" to $00 nm/min, the )N)( "5LA> to 0 sec, the N0M C>CL5S to , the C>CL5 ()M5 to 0 sec, and the ")S$LA> ?/'MA( to /@5'LA>. 112 4ress the '5(0'N ke# to return to the -L SCAN menu. ?ow move the cursor down to the )NS(' S5(0$ menu and enter. *he screen in 5i'ure A will appear. 5i'ure A 132 +nter the followin' parametersB -aselineB ;ser <esponseB !edium 0amp chan'eB 870 2I@ 0ampB :n ;2 0ampB :n Craph printB :ff *ext printB :ff 0ist intervalB .0 142 <eturn to the -L SCAN menu a'ain and this time select 0S5' AS5L)N5. )ou are 'oin' to record the back'round spectrum, store it in memor#, and this back'round will be subtracted from all subse1uent spectra. 5ill two 1uartz cuvettes with distilled water and place them in the sample and reference slots. 162 4ress the A0(/ A5'/ ke# to start with an absorbance of 0.00 at 890 nm. ?ow press the S(A'( ke# to measure the back'round. 182 %e are now read# to record a spectrum. 4ress the ?/'-A'" ke# on the ke#board. A screen similar to 5i'ure D will appear. 5i'ure D 1:2 <emove the sample cuvette and fill it with the lowest concentration -A sample ,alwa#s take data from lowest to hi'hest concentrations.. 4ress the S(A'( ke# on the ke#board to obtain the spectrum. 1;2 After the spectrum is obtained, select the '5SCAL5 option at the bottom of the screen. A screen similar to 5i'ure E will appear. @elect option . C0'S/' 5N('> and then use the arrow ke#s to move the cross&hair over the spectrum until #ou can read the absorbance at $$6 nm. +nter the absorbance in the data sheet. <epeat at a wavelen'th of $D$ nm. 5i'ure E 182 4ress the return ke# t+ice to return to the screen shown in 5i'ure D. ItFs ver# important that #ou do not lose the data in the window b# pressin' the return ke# too man# timesG *his is because we want all of the spectra to be displa#ed to'ether in the data window. 1<2 <epeat steps A&E for each -A standard. After the last spectrum is obtained, print out the displa# b# selectin' option 7 in 5i'ure D. 3=2 <epeat steps A&6 for the caffeine standards. N/(5: #our spectrum will look different from this & itHs meant to be dia'rammatic. 312 !easure the spectrum of the prepared soda and enter the absorbances at $$6 and $D$ nm in the data sheet. 332 OPTIONAL: If #our instructor chooses, measure the spectrum of a s#nthetic, unknown mixture of benzoic acid and caffeine prepared b# the instructor. $A'( )@: Analy!in* the "ata 0sin* Microsoft 5xcel A2 (heory In lecture an e1uation will be derived that allows #ou to calculate the concentration of each component in a binar# mixture usin' -eerFs 0aw ,A 3 bc.. *he two e1uations areB IJK 3 ,A ) & A ). / ( use this e1uation for benzoic acid I)K 3 ,A J & A J. / ( use this e1uation for caffeine where ( 3 b,J ) & ) J. A 3 total absorbance at wavelen'th ,$$6 nm. A 3 total absorbance at wavelen'th $ ,$D$ nm. J 3 molar absorptivit# of molecule J ,benzoic acid. at wavelen'th ,$$6 nm. J 3 molar absorptivit# of molecule J ,benzoic acid. at wavelen'th $ ,$D$ nm. ) 3 molar absorptivit# of molecule ) ,caffeine. at wavelen'th ,$$6 nm. ) 3 molar absorptivit# of molecule ) ,caffeine. at wavelen'th $ ,$D$ nm. 2 $rocedure 12 (ownload the +xcel anal#sis file from the class webpa'e b# ri'ht&clickin' on the link and savin' to a flopp# disk ,the file name is 0ab>A_;2&2is.xls.. 32 @tart !icrosoft +xcel and enter the file. *he spreadsheet will look similar to thisB 42 5rom the 0@-@is "ata section of the data sheet, enter the benzoic acid molarities and absorbances fom the data sheet into the top half of the spreadsheet. (o the same for the caffeine data in the bottom half of the spreadsheet. Add a trendline to each set of data b# ri'ht clickin' on a data point and selectin' the Add *rendline option. *he resultin' linear trends in absorbance will be plotted in the 'raphs alon' with the linear least s1uares best fits. 62 *he slopes of the calibration curves are e1ual to the molar absorptivities ,.. ?otice that the e1uations are ke#ed to the wavelen'ths ,#H and #= refer to $$6 and $D$ nm respectivel#.. +nter the molar absorptivities in the spaces belowB J for benzoic acid at $$6 nm 3 ___________________ cm & ! & J for benzoic acid at $D$ nm 3 ___________________ cm & ! & ) for caffeine at $$6 nm 3 ___________________ cm & ! & ) for caffeine at $D$ nm 3 ___________________ cm & ! & 82 Click on the tab on the bottom of the spreadsheet labeled =calculations= and a new sheet appears. In the upper left&hand corner of the sheet enter the molar absorptivities from step 7 ,in the sheet the# are labeled with an =e= rather than a .. :2 In the space below the molar absorptivities enter the absorbance of the soda sample at $$6 and $D$ nm ,AH and A= respectivel#.. ;2 *he spreadheet will calculate the concentrations of -A and C in the diluted sample. +nter the values for the molarities of -A and C in the soda sampleB I-AK 3 ______________ ! ICK 3 ______________ ! 82 Calculate the molarit# of -A and C in the soda before dilution: calculationsB I-AK 3 ______________ ! ICK 3 ______________ ! <2 ?ow calculate the 'rams/liter of -A and C in the sodaB calculationB benzoic acid !% 3 $$.$, caffeine !% 3 67.6 I-AK 3 ______________ '/0 ICK 3 ______________ '/0 1=2 A $0 fl. oz. bottle is 96$ m0. Calculate the 'rams/bottle for each componentB calculationB benzoic acid !% 3 $$.$, caffeine !% 3 67.6 I-AK 3 ______________ '/bottle ICK 3 ______________ '/bottle Ch 413: Lab #8 - Spectrophotometric Analysis of a Mixture: Caffeine and en!oic Acid in a Soft "rin# "A(A S.55( $reparin* Standard Solutions for the Calibration Cur,e Concentration of -A stock solution 3 ______________ m'/0 Concentration of C stock solution 3 ______________ m'/0 BACD ppm @olume A Stoc# BACD molEL BCCD ppm @olume CA Stoc# BCCD molEL Solution Solution $ 7 7 E A $ E A 0 $0 0@-@is "ata BACD M Abs F 33< nm Abs F 3;3 nm BCCD M Abs F 33< nm Abs F 3;3 nm Soda Abs F 33< nm Abs F 3;3 nm 7B00 dilution s#nthetic sample