DETERMINATION OF ERYTHROSINE CONCENTRA C ONCENTRATION TION USING USI NG UVVISIBLE SPECTROPHOTOMETER INTRODUCTION
A singl single e and doubl double e beam beam spect spectro roph photo otomet meter er is an instr instrum ument ent which which is desig designe ned d to measu measure re light light by wavel wavelen ength gth distr distrib ibuti ution on.. Ther There e are are a wide wide variety of dierent sizes and styles of spectrophotometers both single and dual dual beam, beam, with with their their size size and and con congur gurati ation on large largely ly depen dependen dentt on the the specic applications for which they are designed. A single beam instrument is used to measure the intensity of a beam of light before and then after the addition of a sample and uses a light source, a prism and a photocell as well as a sam sample holde lder for for the mater terial being ing anal analy yzed zed by means ans of spectrophotometry. Spectrophotometer single beam or double beam models oer the ability to control the wavelength and intensity of the light source. The results provided by these instruments are in the form of voltage uctuations which are the light energy received by the photo cell into the form of electrical energy, which is then displayed andor recorded on a connected computer for further analysis. !y cont contra rast st,, spec spectr trop opho hoto tome mete ters rs whic which h are are desi design gned ed as doub double le beam beam instruments gather data from the dierence in light intensity of two beams of light. "ne beam#s path contains a reference sample with $nown properties, the other containing the sample being tested. A spectrophotometer, single beam or double suitability for a given application depends on the sample to be tested tested and and the deman demands ds of the the appl applica icatio tion. n. %or some some purpo purposes ses,, one one instrument is a better choice than the other. &ence, the absorbance of sample can be determined by using '()(isible Spectrophotometer and the concentration can be obtained by using the principle of !eer *ambert *aw.
OBJECTIVES
+. To obtain the absorption spectrum wavelength for the dye solution . To determine the wavelength of ma-imum absorbance /ma-0 from the spectrum. 1. To produce a standard calibration curve from the series of standard solutions. 2. To use the standard curve to determine the concentration of an un$nown solution 3. To compare between single)beam and double)beam spectrophotometer.
METHOD
4564A5AT7"8 "% STA89A59 S"*'T7"8 +. The concentration :: - +: ;)2 g+:: m*0 of the dye stoc$ solution erythrosine0 given into a series of dierent concentration as below < !lan$ 3 - +:;)2 g+:: m* +: - +:;)2 g+:: m* +3 - +:;)2 g+:: m* : - +:;)2 g+:: m* 3 - +:;)2 g+:: m* "!TA7878= T&6 A!S"54T7"8 S46>T5'? +. The Spectrophotometer 4er$in)6lmer *ambda130 was turned on 1: minutes earlier to allow it to warm up
. The cuvette was lled about 1 full with the blan$ solution and another with +:@+:)2g+::ml concentration of the dye solution. 1. The '()visible absorbance spectrum was determined for erytrosine. The range of wavelength to use was 2::):: nm 2. The absorbance ma-imum wavelength /ma-0 was identified
96T65?78AT7"8 "% STA89A59 >'5(6 !665)*A?!65TBS *AC0 Spectrophotometer type< Single)beam D Spectronic >ole 4almer ++:: 9ouble)beam D 4er$in 6lmer *ambda 13 +. The spectrophotometer wavelength was set using the /ma- obtained . The absorbance for each standard solutions and un$nown solution was measured
DISCUSSION
%rom the e-periment, we need to determine the standard curve of 6rythrosine concentration using '( visible Spectrophotometer. Spectrophotometer uses the transmission of light to determine the concentration of solute within the solution. 7n this e-periment, we used both single)beam spectrophotometer and double)beam spectrophotometer to obtain the result where the standard solution needs to be preparing rst by applying dilution method. The sample that has been used is 6rythrosine. !esides, the standard that has been prepared is then put at both single and double spectrophotometer and the absorbance was read. The concentration is then obtained by constructing a graph based on !eer)*ambert law principle. %rom the result, absorbance reading is read at both spectrophotometer where single)beam spectrophotometer result in 3 @+:EF g+:: m*, +: @+:EF g+:: m*, +3 @+:EF g+:: m*, : @+:EF g+:: m*, 3 @+:EF g+:: m* and un$nown concentration show :.:++, :.:2, :.:1G, :.:1G, :.:3 and :.:1 of absorbance respectively The value of absorbance of all concentration show very higher dierence between one concentration to another concentration. ?eanwhile, double)beam spectrophotometer result in :.:2 , :.:3 , :.: , :.+: , :.+1 , :.:3H of absorbance reading in 3 @+:EF g+:: m*, +: @+:EF g+:: m*, +3 @+:EF g+:: m* , : @+:EF g+:: m* , 3 @+:EF g+:: m* and un$nown concentration respectively. The absorbance of double)beam spectrophotometer show very slightly dierences between the concentrations that has been prepared. As we can see, single)beam spectrophotometer show better data as it has advantages to has higher signal than double beam. The design of double beam instrument also ma$es it diIcult to achieve two parallel beam of eJual intensity. The dierent between both types of spectrophotometer used in this e-periment is a double beam spectrophotometer compares the light intensity between two light paths, one path containing a reference sample and the
other the test sample. Chile, a single beam spectrophotometer measures the relative light intensity of the beam before and after a test sample is inserted. 7n addition , a single beam spectrophotometer has one light path that passes from the light source through the monochromator system and sample cuvette and then to the detector .A blan$ is used to set the instrument to +::KT: A0,then the samples are read. !esides, a double beam spectrophotometer has two light paths, both originating from the same light source. "ne path is for the sample and other for the blan$ or reference. The beam from the source stri$es a vibrating or rotating mirror that alternate directs light through the reference cell and the sample cell. *ight passing
through each cell is sent to the detector.
Single)beam spectrophotometry 9ouble)beam spectrophotometry
Through this e-periment, there are some errors that have been done that may aect the accuracy of the result. %or e-ample, the cuvette is not placed at the right place, the clear side must face the light source, where the light can emitted into it and reading can obtained. !esides, the standard is also
not prepared properly as the dilution process is not properly conducted by the handler. To minimize these errors various precautions should be ta$en, care during conducting this e-periment must be employed besides three consecutive of reading the absorbance were carried out to eliminate the errors of the handlers.
CONCLUSION
Through the e-periment, both single and double beam spectrophotometry can read the value of the absorbance and the concentration is obtained by using !eer *ambert *aw where the concentration of 6rythrosine is ++. &ence , the hypothesis is accepted.
REFERENCE
Lo$ab Lhan , Spectrophotometer , :++ 0 , 5etrieved http<www.scribd.comdoc3MMH:S46>T5"4&"T"?6T65
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Spectrophotometry. n.d.0. 5etrieved %ebruary M, :+1 from http<www.chm.davidson.eduvcespectrophotometrySpectrophoto metry.html 9ouble vs. Single !eam Spectrophotometers. n.d.0. 5etrieved %ebruary M. :+1 from http<www.hunterlab.comappnotesan++NG.pdf
