Spectrophotometry & beer's law Biochemistry Lab experiment

1. 1
Faculty of Medicine
Biochemistry lab
Spectrophotometry & Beer's Law
Prepared By :
Diaa Mohammad Srahin .
Supervisor : Dr. Suheir Jabir .

2. 2
Section 72
SPECTROPHOTOMETRY & BEER'S LAW
Introduction
Every chemical compound absorbs, transmits, or reflects light (electromagnetic radiation) over a
certain range of wavelength . Spectrophotometry is a measurement of how much a chemical
substance absorbs or transmits. Spectrophotometry is widely used for quantitative analysis in
various areas (e.g., chemistry, physics, biology, biochemistry, material and chemical engineering,
clinical applications, industrial applications, etc). Any application that deals with chemical
substances or materials can use this technique. In biochemistry, for example, it is used to
determine enzyme-catalyzed reactions. In clinical applications, it is used to examine blood or
tissues for clinical diagnosis . With the spectrophotometer, the amount of a known chemical
substance (concentrations) can also be determined by measuring the intensity of light detected.
Different Types of Spectrophotometers:
A. Visible Light: useslightat visiblerange (400 - 700 nm) of electromagneticradiationspectrum
, Visiblespectrophotometerscanuse incandescent,halogen,LED,or a combinationof these
sourcesand these spectrophotometersvaryinaccuracy.Plasticandglass cuvettes canbe usedfor
visible lightspectroscopy.
B. UltravioletLight : useslightoverthe ultravioletrange (185 - 400 nm) , UV spectroscopyisusedfor
fluids,andevensolids.Cuvettes,onlymade of quartz,are usedforplacingthe samples.
C. Infrared Light useslightoverthe infraredrange (700 - 15000 nm) of electromagneticradiation
spectrum, IR spectroscopyhelpstostudydifferentstructuresof moleculesandtheirvibrations.
Differentchemical structuresvibrateindifferentwaysdue tovariationof energyassociatedwith
each wave length.
D. Single Beam: In thistype,all the lightpassesthroughthe sample .Tomeasure the intensityof the
incidentlightthe sample mustbe removedsothatall the lightcan pass through.Thistype of
spectrometerisusuallylessexpensive andlesscomplicated.
E. Double Beam: Inthistype,before itreachesthe sample,the lightsource issplitintotwoseparate
beams.Fromthese one passesthroughthe sample andsecondone isusedforreference.Thisgives
an advantage because the referencereadingandsample readingcantake place at the same time .

3. 3
Objectives
1. To identify the parts of a Spectrophotometer and the way it works .
2. To determine Maximum Absorbance (the optimal wavelength for measuring absorbance)
of 4 different colored solution & represent relationship between them .
3. To determine the concentrations of 4 diluted green solutions by the Lambert-Beer Law .
4. To draw the relationship between concentration of a green solution and its absorbance .
Materials
 Spectrophotometer & its components ( Light source , Prism , Detector , Photoelectric cell ) .
 cuvettes & pipettes .
 Distilled water as a Blank solution .
 Four colored solutions ( Red Safranin , Yellow Iodine , crystal Violet , Malachite Green ) .
 Green solution in bottles with different diluted degrees .
Methods & Procedures
Experiment-Part A :
1- five cuvettes were taken from our lab instructor .
2- The first cuvette was filled with distilled water and put in the Spectrophotometer (the blank) .
3- After put the blank, the Spectrophotometer was set at the first wave length which is 380 nm
and reference blank was set at zero ( A = 0 ) .
4- Cuvettes were filled with four colors ( Red , yellow, Violet , and Green ) .
5- Each colored Cuvettes were put in spectrophotometer one by one , and absorbance was
taken & recorded for each one at these wavelengths ( 380,430,480,530,580,630,680,730 ) nm
6- Spectrophotometer was changed to work at other wavelength & it was set "re-zero" at each
changed in the wavelength .
7- Maximum absorbance for each colored was determined & The ( Absorbance – Wavelength )
Relationship was represented .
Experiment-Part B :
1- Four cuvettes were filled with Green solution in different diluted degrees .
2- The bottles and cuvettes were marked from 1-4 starting with the lightest color ( number 1 )
to the darkest color ( number 4 ) .
3- The spectrophotometer was set at the maximum absorbance wavelength of the green
colored which was recorded in Experiment- Part A .
4- Cuvettes were put in spectrophotometer one by one , and absorbance was determined for
each one at the wanted wavelength .
5- The concentrations of four cuvettes were calculated by using Lambert-Beer Law & The
( Absorbance – concentration ) relationship was represented .

4. 4
Results
 1st
Experiment :
380 430 480 530 580 630 680 730
Red 0.095 0.124 0.218 0.317 0.101 0.068 0.073 0.077
Yellow 0.450 0.185 0.090 0.082 0.085 0.078 0.076 0.072
Violet 0.164 0.083 0.126 0.190 0.232 0.137 0.074 0.077
Green 0.128 0.173 0.153 0.146 0.196 0.214 0.090 0.081
Table 1 : The absorbance of different colored solutions at different wavelengths .
Maximum Absorbance for green solutionAt 630 nm .
Figure 1 : The Relationship between Absorbance & wavelength for different colored solutions .

5. 5
 2nd Experiment :
Sample Number Absorbance Concentration
1 0.122 1.018*10-6
2 0.194 1.618*10-6
3 0.251 2.093*10-6
4 0.317 2.644*10-6
Table 2 : The absorbance & concentrations of 4 green solutions at wave length of 630 nm .
 We used Lambert Beer Law to calculate the unknown concentration of 4 green solutions as
it shown below :
A= Є * L * C
A : Absorbance . Є : Molar Extinction Coefficient in (M . cm)-1 .
L : The sample path length in cm ( the width of the cuvette = 1 cm ) .
C : The molar concentration of the solution .
Є = 1.19897 * 105 (M . cm)-1 for Malachite green in water at 630 nm .
C1 =
0.122
1∗1.19897∗105 = 1.018*10-6 M C2 =
0.194
1∗1.19897∗105 = 1.618*10-6 M
C3 =
0.251
1∗1.19897∗105 = 2.093*10-6 M C4 =
0.317
1∗1.19897∗105 = 2.644*10-6 M
Figure 2 : The relationship between Absorbance & Concentration at 630 nm for Malachite green .

6. 6
Discussion
We noticed in first lab experiment , that If a substance absorbs all wavelengths in the visible
range, none of the light is reflected back to our eyes and the substance appears black. If the
substance absorbs none of the incident visible light, it appears white (all light reflected) or
colorless (all light transmitted). And we noticed that the maximum absorbance wavelength differs
in colors , It’s because of the “Complementary Color”.
Complementary colors are pairs of colors which, when combined in the right proportions,
produce white or black . If a substance absorbs blue and red light, but not green light, we will see it
as green since that is the only light that reaches us from that substance. Therefore, when you look
at the absorption spectrum of a green solution, it should show low absorbance of wavelengths in
the green part of the spectrum but high absorption of light in the red and blue parts of the visible
spectrum .
The complementary colors for the basic 3 colors are given by mixing the two others (for instance,
Complementary color of Green is Blue + Yellow = Red ) .
If we return to the results of our experiment, we can notice that the maximum absorbance of
green solution was at 630 nm, which is near the wavelength of red color, which is the
complementary color of green solution ! That will be right for the other 3 solution or any solution .
In the second experiment , we noticed that if we have a solution in different concentrations
and we know maximum absorbance wavelength , we can use the spectrophotometer & calculate
the unknown concentration by Lambert-Beer Law ( A= Є * L * C ) .
We observed that the concentration of solution is directly proportional with the amount of
light that absorbed , As solution concentration increases, the absorbance increases . It’s because
Absorption is reversely proportion to Transmittance, also Transmittance is reversely proportion to
Concentration .
Biologist measure absorption rather than transmission by this relationship so , the Beer's Law
is very useful to calculate the concentration of a solution if the extinction coefficient is known .
References
http://amrita.vlab.co.in/?sub=2&brch=190&sim=338&cnt=1 .
http://chemwiki.ucdavis.edu/Physical_Chemistry/Kinetics/Reaction_Rates/Experimental_Determinatio
n_of_Kinetcs/Spectrophotometry .
http://omlc.org/spectra/PhotochemCAD/html/030.html .
http://www.columbia.edu/itc/barnard/biology/biobc2004/edit/experiments/Experiment1-Spec.pdf .
http://mason.gmu.edu/~jschorni/chem211lab/Chem%20211-212%20Absorption.pdf .