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