EXPERIMENT 5 : THE SIMULT SIMULTANEOUS DETERMINA DETE RMINATION TION OF CAFFEINE AND ACETYLSALICYLIC ACID IN AN ANALGESIC BY ULTRAVIOLET ULTRAVIOLET SPECTROPHOTOMETER. OBJECTIVE : To determine the amount concentration of caffeine and acetylsalicylic acid ac id in an analgesic by ultraviolet spectrophotometer. spectrophotometer. INTRODUCTION
Ultraviolet–visible Ultraviolet–visible spectroscopy or ultraviolet-visi ultraviolet-visible ble spectrophotometry spectrophotometry (UV(UVVis or UV/Vis) refers to absorption spectroscopy spectroscopy or reectance spectroscopy in the ultravioletultraviolet-visible spectral visible spectral region. This means it uses light in the visible and adacent adacent (near (near-UV and near-infrared !"#$%) !"#$%) ranges ranges.. The absorp absorptio tion n or reectance in the visible range directly a&ects the perceived color of the chemicals involved. #n this regio gion of the electro electromagne magnetic tic spectrum spectrum'' molecules molecules under undergo go electronic transitions. transitions . This techniue is complementary to uorescence spectroscopy' spectroscopy' in that uorescence deals uorescence deals ith transitions from the e*cited state to state to the ground state' state' hile absorption measures transitions from the ground state to the e*cited state. UV/Vis spectroscopy is routinely used in anal analyt ytic ical al chem chemis istr try y for the uantitative dete deterrmina minati tion on of di&e di&errent ent anal analyt ytes es'' su such ch astransition astransition metal ions' highly conugated orga organi nic c comp compou ound nds s' a nd biological macromolecules. +pectroscopic analysis is commonly carried out in solutions but solids and gases may also be studied. ,hile interaction ith infrared ligh lightt caus causes es mole molecu cule les s to under undergo go vibr vibrat atio ional nal trans transit itio ions ns'' the the short shorter er avele avelengt ngth' h' higher higher energ energy y radiat radiation ion in the UV ((- nm) and visibl visible e (-0 nm) range of the electromagnetic spectrum causes many organic molecules to undergo electronic transitions. ,hat this means is that hen the energy from UV or visible light is absorbed by a molecule' one of its electrons umps from a loer energy to a higher energy molecular orbital. 1 UV/V UV/Vis is sp spec ectr trop opho hoto tome mete terr may may be us used ed as a dete detect ctor or for for 2345. 2345. The presence of an analyte gives a response assumed to be proportional to the concentration. 6or accurate results' the instrument7s response to the analyte in the un8non should be compared ith the response to a standard9 this is very similar to the use of calibration curves. The response (e.g.' pea8 height) for a particular concentration is 8non as the response factor. factor. The simplest instrument for molecular UV/Vis absorption is a :lter photometer' photometer' hich uses an absorption or interference :lter to isolate a band
of radiation. The :lter is placed beteen the source and the sample to prevent the sample from decomposing hen e*posed to higher energy radiation. 1 :lter photometer has a single optical path beteen the source and detector' and is called a single-beam instrument. The instrument is calibrated to ; T hile using a shutter to bloc8 the source radiation from the detector. 1fter opening the shutter' the instrument is calibrated to <; T using an appropriate blan8. The blan8 is then replaced ith the sample and its transmittance measured. =ecause the source>s incident poer and the sensitivity of the detector vary ith avelength' the photometer must be recalibrated henever the :lter is changed. 3hotometers have the advantage of being relatively ine*pensive' rugged' and easy to maintain. The limitations of :*ed-avelength' single-beam spectrophotometers are minimi?ed by using a double-beam spectrophotometer. 1 chopper controls the radiation>s path' alternating it beteen the sample' the blan8' and a shutter. The signal processor uses the chopper>s 8non speed of rotation to resolve the signal reaching the detector into the transmission of the blan8' P' and the sample' P T. =y including an opaue surface as a shutter' it is possible to continuously adust ; T. The e&ective bandidth of a doublebeam spectrophotometer is controlled by adusting the monochromator>s entrance and e*it slits. @&ective bandidths of .–A. nm are common. 1 scanning monochromator allos for the automated recording of spectra. Bouble-beam instruments are more versatile than single-beam instruments' being useful for both uantitative and ualitative analyses' but also are more e*pensive. Biode 1rray +pectrometer is an instrument ith a single detector can monitor only one avelength at a time. #f a single photomultiplier is replaced ith many photodiodes' the resulting array of detectors can be used to record an entire spectrum simultaneously in as little as .< s. #n a diode array spectrometer the source radiation passes through the sample and is dispersed by a grating. The photodiode array is situated at the grating>s focal plane' ith each diode recording the radiant poer over a narro range of avelengths. 1 full monochromator is replaced ith ust a grating that caused a diode array spectrometer small and compact. +amples for UV/Vis spectrophotometry are most often liuids' although the absorbance of gases and even of solids can also be measured. +amples are typically placed in atransparent cell' 8non as a cuvette. 5uvettes are typically rectangular in shape' commonly ith an internal idth of < cm.
(This idth becomes the path length (4) in the =eer-4ambert la.) Test tubes can also be used as cuvettes in some instruments. The type of sample container used must allo radiation to pass over the spectral region of interest. The most idely applicable cuvettes are made of high uality fused silica or uart? glass because these are transparent throughout the UV' visible and near infrared regions. Class and plastic cuvettes are also common' although glass and most plastics absorb in the UV' hich limits their usefulness to visible avelengths Dany molecules absorb ultraviolet or visible light. The absorbance of a solution increases as attenuation of the beam increases. 1bsorbance is directly proportional to the path length (b) and the concentration (c) of the absorbing species. =eer7s 4a states that by the euation E 1 F log (3/3G) F abc ,here a is a proportionality constant called absorptivity and b is the path length of the light beam though the absorbing medium. ,hen c is e*pressed in D (moles per liter) and b in cm. Then a is called the molar absorptivity and is given the special symbol H ith the units 4 cm-< mol-<. Thus' 1 F Hbc Bi&erent molecules absorb radiation of di&erent avelengths. 1n absorption spectrum ill sho a number of absorption bands corresponding to structural groups ithin the molecule. 6or e*ample' the absorption that is observed in the UV region for the carbonyl group in acetone is of the same avelength as the absorption from the carbonyl group in diethyl 8etone. The absorption of UV or visible radiation corresponds to the e*citation of outer electrons. There are three types of electronic transition that can be considered hich are the transition involving p' s' and n electrons' the transitions involving charge-transfer electrons and the transition involving d and f electrons. ,hen an atom or molecule absorbs energy' electrons are promoted from their ground state to an e*cited state. #n a molecule' the atoms can rotate and vibrate ith respect to each other. These vibrations and rotations also have discrete energy levels' hich can be considered as being pac8ed on top of each electronic level. This e*periment is aimed to determine the amount of ca&eine and acetyl salicyclic acid in an analgesic tablet. #t reuire di&erent methods of analysis
for each component. Iften the simultaneous analysis of comple* multicomponent can be done by constructing a matri* of the cross-product of the standard scans ith the sample scan. #n the :nal step it need to calculate the actual sample component concentrations from the 8non concentrations in each standard. The total absorbance of a solution at a given avelength is eual to the sum of the absorbance of individual components present in the solution and is given by the folloing euation E 1
F 1< J 1 F H*bc* J Hybcy
total
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3reparation of standard solution +tanda rd <
5oncentration (D) K*<-L
K*<-L
A
0K*<-L
<*<-L
K
<K*<-L
5a&eine
1cetyl salicylic acid
D
D
+tandard calibration for ca&eine 5oncentration (D) K*<-L K*<-L 0K*<-L
,avelength (mn) < 0P 0
1bsorbance .A .
Transition n M NO n M NO n M NO
<*<-L <K*<-L
0 Q<
<.A< <.Q0
n M NO n M NO
+tandard calibration for acetyl salicylic acid 5oncentration (D) K*<-L K*<-L 0K*<-L <*<-L <K*<-L
,avelength (mn) PQ PQ 0Q 0L
1bsorbance .<0 .<A .PA .<< .P0
Transition n n n n n
M M M M M
NO NO NO NO NO
+tandard 1nalgesic tablet +ample < A
,avelength (mn) P.K K K
Craph of standard calibration ca&eine.
1bsorbance <.0K< <.0Q <.LLP
5oncentration (D) K*<-L K*<-L 0K*<-L <*<-L <K*<-L
1bsorbanc e .A .
Absoba!c" a#a$!s% Co!c"!%a%$o! <.L <. <. < .Q Absoba!c" .L . .
f(*) F
Co!c"!%a%$o!
Craph of standard calibration acetyl salicylic acid.
5oncentration (D) K*<-L K*<-L 0K*<-L <*<-L <K*<-L
1bsorbanc e .<0 .<A .PA .<< .P0
Absoba!c" a#a$!s% Co!c"!%a%$o! .AK .A .K
f(*) F
. Absoba!c"
.
Co!c"!%a%$o! &M'
5oncentration (D) 1nalgesic sample < 1nalgesic sample
5a&eine
1cetylsalicylic acid
1nalgesic sample A