DETERMINATION OF ERYTHROSINE CONCENTRATION USING UVVISIBLE

SPECTROPHOTOMETER
INTRODUCTION
A single and double beam spectrophotometer is an instrument which is
designed to measure light by wavelength distribution. There are a wide
variety of different sizes and styles of spectrophotometers both single and
dual beam, with their size and configuration largely dependent on the
specific 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 sample holder for the material being analyzed by means of
spectrophotometry.
Spectrophotometer single beam or double beam models offer the ability to
control the wavelength and intensity of the light source. The results provided
by these instruments are in the form of voltage fluctuations which are the
light energy received by the photo cell into the form of electrical energy,
which is then displayed and/or recorded on a connected computer for further
analysis.
By contrast, spectrophotometers which are designed as double beam
instruments gather data from the difference in light intensity of two beams of
light. One beam's path contains a reference sample with known 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 and the demands of the application. For some purposes, one
instrument is a better choice than the other.
Hence, the absorbance of sample can be determined by using UV-Visible
Spectrophotometer and the concentration can be obtained by using the
principle of Beer Lambert Law.

OBJECTIVES
1. To obtain the absorption spectrum wavelength for the dye solution
2. To determine the wavelength of maximum absorbance (max) from the
spectrum.
3. To produce a standard calibration curve from the series of standard
solutions.
4. To use the standard curve to determine the concentration of an unknown
solution
5. To compare between single-beam and double-beam spectrophotometer.

METHOD
PREPARATION OF STANDARD SOLUTION
1. The concentration ( 200 x 10 ^-4 g/100 mL) of the dye stock solution
( erythrosine) given into a series of different concentration as below :
Blank
5 x 10^-4 g/100 mL
10 x 10^-4 g/100 mL
15 x 10^-4 g/100 mL
20 x 10^-4 g/100 mL
25 x 10^-4 g/100 mL
OBTAINING THE ABSORPTION SPECTRUM
1. The Spectrophotometer (Perkin-Elmer Lambda35) was turned on 30
minutes earlier to allow it to warm up

2. The cuvette was filled about 2/3 full with the blank solution and
another with 1010-4g/100ml concentration of the dye solution.
3. The UV-visible absorbance spectrum was determined for erytrosine.
The range of wavelength to use was 400-700 nm
4. The absorbance maximum wavelength (max) was identified

DETERMINATION OF STANDARD CURVE (BEER-LAMBERTS LAW)

Spectrophotometer type:
Single-beam Spectronic Cole Palmer 1100
Double-beam Perkin Elmer Lambda 35
1. The spectrophotometer wavelength was set using the max obtained
2. The absorbance for each standard solutions and unknown solution was
measured

DISCUSSION
From the experiment, we need to determine the standard curve of
Erythrosine
concentration
using
UV
visible
Spectrophotometer.
Spectrophotometer uses the transmission of light to determine the
concentration of solute within the solution. In this experiment, we used both
single-beam spectrophotometer and double-beam spectrophotometer to
obtain the result where the standard solution needs to be preparing first by
applying dilution method. The sample that has been used is Erythrosine.
Besides, 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 Beer-Lambert law
principle.
From the result, absorbance reading is read at both spectrophotometer
where single-beam spectrophotometer result in 5 10 g/100 mL, 10 10
g/100 mL, 15 10 g/100 mL, 20 10 g/100 mL, 25 10 g/100 mL and
unknown concentration show 0.011, 0.024, 0.039, 0.039, 0.052 and 0.023 of
absorbance respectively The value of absorbance of all concentration show
very higher difference between one concentration to another concentration.
Meanwhile, double-beam spectrophotometer result in 0.04 , 0.05 , 0.07 , 0.10
, 0.13 , 0.058 of absorbance reading in 5 10 g/100 mL, 10 10 g/100
mL, 15 10 g/100 mL , 20 10 g/100 mL , 25 10 g/100 mL and
unknown concentration respectively. The absorbance of double-beam
spectrophotometer
show
very
slightly
differences
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 makes
it difficult to achieve two parallel beam of equal intensity.
The different between both types of spectrophotometer used in this
experiment 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. While, a single beam spectrophotometer measures

the relative light intensity of the beam before and after a test sample is
inserted. In 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 blank is used to set the
instrument to 100%T(0 A),then the samples are read. Besides, a double
beam spectrophotometer has two light paths, both originating from the same
light source. One path is for the sample and other for the blank or reference.
The beam from the source strikes a vibrating or rotating mirror that alternate
directs light through the reference cell and the sample cell. Light passing

through each cell is sent to the detector.

Single-beam spectrophotometry
Double-beam spectrophotometry

Through this experiment, there are some errors that have been done that
may affect the accuracy of the result. For example, 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. Besides, 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 taken,
care during conducting this experiment must be employed besides three
consecutive of reading the absorbance were carried out to eliminate the
errors of the handlers.

CONCLUSION
Through the experiment, both single and double beam spectrophotometry
can read the value of the absorbance and the concentration is obtained by
using Beer Lambert Law where the concentration of Erythrosine is 11.
Hence , the hypothesis is accepted.

REFERENCE
Kokab Khan , Spectrophotometer , ( 2011 ) , Retrieved
http://www.scribd.com/doc/57662820/SPECTROPHOTOMETER

from

Spectrophotometry. (n.d.). Retrieved February 26, 2013 from

http://www.chm.davidson.edu/vce/spectrophotometry/Spectrophoto
metry.html
Double vs. Single Beam Spectrophotometers. (n.d.). Retrieved February 26.
2013 from
http://www.hunterlab.com/appnotes/an11_97.pdf