DEPARTMENT OF BIOMEDICAL ENGINEERING FACULTY OF ENGINEERING UNIVERSITY MALAYA

Lab 6 : Spectrophotometer

Objective
The main purpose of this experiment is to expose the usage of spectrophotometer and to determine the absorption spectrum of a substance in solution.

Introduction
Spectrophotometry is mainly based on Quantum energy. Quantum energy states that the molecule needs energy to excite it to a higher level. But when the molecules or ions are in higher levels or excited levels, the ions and molecules need to return back to the original or ground level. This is done by releasing energy. Spectrophotometer are based on the absorption of electromagnetic radiation in visible, ultra-violet(UV) and infra-red ranges. The Beer-Lambert Law or known as Beers Law are the most fundamental and widely applied spectroscopic laws. Beer s Law defined that A=bc, where A is the absorbance of the solution, is the molar absorptivity of the solution, b is the pathlength of the sample cell or simple known as the depth of the well or cuvette, and the c are representing the concentration of the solution. From the formula, we can see that the absorbance is directly proportional to the concentration of the solution. Another spectroscopic formula are A=-logT. A is the absorbance of the solution and T is the transmittance of the solution. Transmittance is the ratio of transmitted light energy to the incident light energy at where T= . Thus we will have the following formula by combining the
Io I

formulas where A=-log I . The main arrangement of the spectrophotometer is as follow.

Io

The source can be a tungsten lamp, a xenon-mercury arc, hydrogen or deuterium discharge lamp. The dispersion device is use to ensure that only the desired light will be emitted and pass through the slit in front of the sample.

Materials
Color dyes(Red, Blue, Green and Yellow) Distilled water

Apparatus
Pipette Cryo-tubes Micro-plate Vortex mixer Spectrophotometer

Procedure
(a) Serial Dilution 1. By using a pipette, 0.10ml of food coloring is place into the cryo-tube A, follow by adding 90ml of distilled water to it. The cryo-tube is labeled as 10% solution and mix well by using a vortex mixer. 2. 0.10ml of distilled water are pipette to all well that marks with B (refer to the diagram 11.1). 3. 0.1ml of the 10% solution in the cryo-tube A are pipette to the well marked X1 according to the color (refer to the diagram 11.1). 4. 0.30ml of the 10% solution are pipette out and place into the cryo-tube B. 0.30ml of distilled water are added to it. This solution is known as the 1st dilution. 5. 0.10ml of the 1st solution, from the cryo-tube B are pipette to the well marked X2 according to the color (refer to the diagram 11.1). 6. 0.30ml of distilled water is added to the cryo-tube B and the solution are mix well. The solution now is known as the 2nd dilution. 7. 0.10ml of the 2nd dilution solution is pipette to the well marked X3 according to the color (refer to the diagram 11.1). 8. 0.30ml of distilled water is then added to the cryo-tube B again as it will produce the 3rd dilution. 9. 0.10ml of the 3rd dilution solution is pipette out and put into the well marked X4. 10. By using the pipette, another 0.30ml of distilled water is added to the cryo-tube B and mix with the 3rd dilution solution. The solution is now known as the 4th dilution. 11. 0.10ml of the 4th dilution is placed to the well marked X5 according to the color (refer to the diagram in 7.77) 12. 30ml of distilled water are added to the 4th solution and mixing is done by using mixer. The solution formed now is known as the 5th solution. 13. 10ml of the 5th solution are place to the well as marked with X6 according to the color (refer to the diagram 11.1). 14. An unknown concentration of food coloring is prepared by the tutor. The unknown solution are place to the well with the marked X7 (refer to the diagram 11.1).

Red 2

Blue 5

Green 8

10

Yellow 11 12

A B C D E F G H
B-Blank X1-10% Solution X2-1st Dilution X3-2nd Dilution X4-3rd Dilution X5-4th Dilution X6-5th Dilution X7-Unknown Concentration

(b) Data Collection 1. With the help of tutors or technicians, the well will be inserted to the machine. 2. The machine will produce data after several minutes. The data should be written down and will be tabulate. 3. Graph will be used to represent the data by using Microsoft Excel 4. The concentration of the unknown solution can be work out by refer to the curve 5. By using Beer s Law, the absorbance of every color dye at 100% can be calculated.

Results
(a) Chromatic 1, which is blue color wavelength Concentration (%) X1 Average Raw Data 1.163 3.020 0.909 0.725 0.912 3.020 0.650 0.593 1.001 3.020 0.407 0.323 0.949 2.440 0.296 0.175 0.290 0.968 0.211 0.157 0.276 0.435 0.186 0.241 1.155 0.360 0.166 0.260 Blank (distilled water) 0.211 0.260 0.213 0.150 0.211 0.260 0.213 0.150 0.211 0.260 0.213 0.150 0.211 0.260 0.213 0.150 0.211 0.260 0.213 0.150 0.211 0.260 0.213 0.150 0.211 0.260 0.213 0.150 Average - Blank (Absorbance, A) 0.952 2.760 0.696 0.575 0.701 2.760 0.437 0.443 0.790 2.760 0.194 0.173 0.738 2.180 0.083 0.025 0.079 0.708 -0.002 0.007 0.065 0.175 -0.027 0.091 0.944 0.100 -0.160 0.110 Transmittance, T (%) 11.17 0.174 20.14 26.61 19.91 0.174 36.56 36.06 16.22 0.174 63.97 67.14 18.28 0.66 82.60 94.41 83.36 19.59 99.54 98.40 86.10 66.83 93.97 81.10 11.38 79.43 144.54 77.62

X2

X3

X4

X5

X6

X7

Chromatic 2, which is red color wavelength Concentration (%) X1 Average Raw Data 3.562 2.890 2.895 3.971 4.348 2.066 2.763 3.375 2.057 1.159 1.886 2.192 1.161 0.712 1.088 0.939 0.484 0.404 0.653 0.281 0.238 0.252 0.394 0.153 3.075 0.207 0.774 3.440 Blank (distilled water) 0.151 0.131 0.114 0.096 0.151 0.131 0.114 0.096 0.151 0.131 0.114 0.096 0.151 0.131 0.114 0.096 0.151 0.131 0.114 0.096 0.151 0.131 0.114 0.096 0.151 0.131 0.114 0.096 Average - Blank (Absorbance, A) 3.411 2.759 2.781 3.875 4.197 1.935 2.649 3.279 1.906 1.028 1.772 2.096 1.010 0.581 0.974 0.843 0.333 0.273 0.539 0.185 0.087 0.121 0.280 0.057 2.924 0.076 0.660 3.344 Transmittance, T (%) 0.039 0.17 0.17 0.013 0.64 1.16 0.22 0.053 1.24 9.38 1.69 0.80 9.77 26.24 10.62 14.35 46.45 52.55 28.91 65.13 81.85 75.68 52.48 87.70 0.12 83.95 21.88 0.045

X2

X3

X4

X5

X6

X7

Micro-plate with various dyes.

(b) To find concentrations of the solution By using the formula of M1V1=M2V2 [X2], (0.10)(300)=[X2](600) [X2]=0.05=5% [X3], (0.05)(300)=[X3](600) [X3]=0.025=2.5% [X4], (0.025)(300)=[X4](600) [X2]=0.0125=1.25% [X5], (0.0125)(300)=[X5](600) [X5]=0.00625=0.625% [X6], (0.00625)(300)=[X6](600) [X6]=0.003125=0.3125
4.5 4 3.5 3 Absorbance, A 2.5 2 1.5 1 0.5 0 0% -0.5 2% 4% 6% Concentration(%) 8% 10% 12% Red Blue Green Yellow Linear (Red) Linear (Blue) Linear (Green) Linear (Yellow) y = 11.997x y = 38.651x y = 7.2636x y = 6.3477x

Absorbance against Concentration (Chromatic 1, Blue)

From the graph, we can determine the equation of each line. Red, y=11.99x Green, y=7.263x Blue, y=38.65x Yellow, y=6.347x Since we can get the linear equation from the graph, we can calculate the unknown concentration instead of finding the values in the curve. Thus, the unknown concentrations of each color dye are Red, y=11.99x Green, y=7.263x 0.944=11.99x -0.160=7.263x X=0.0788=7.79% x=-0.0220=-2.20%(*) Blue, y=38.65x Yellow, y=6.347x 0.100=38.65x 0.110=6.347x -3 X=-2.87x10 =0.26% x=0.01726=17.26% If the concentrations of the color dyes are 100%, then the absorbance will be as follows.

This is based on Beer-Lambert Law, Red, y=11.99x Green, y=5.625x y=11.99(1.00) y=5.625(1.00) y=11.99 y=5.625 Blue, y=38.65x Yellow, y=6.347x y=38.65(1.00) y=6.347(1.00) y=38.65 y=6.347 (*)-This data is not correct. May be due to the errors and also human errors.
4.5

Absorbance against Concentration (Chromatic 2, Red) 4

3.5 3 2.5 2 1.5 1 0.5 0 0% 2% 4%

y = 30.51x

y = 35.354x

Red Blue Green Yellow Linear (Red) Linear (Blue) Linear (Green) Linear (Yellow)

6%

8%

10%

12%

From the graph, we can determine the equation of each line. Red, y=46.03x Green, y=35.35x Blue, y=30.51x Yellow, y=46.19x Since we can get the linear equation from the graph, we can calculate the unknown concentration instead of finding the values in the curve. Thus, the unknown concentrations of each color dye are Red, y=46.03x Green, y=35.35x 2.924=11.99x 0.660=5.625x X=0.2439=24.39% x=0.1173=11.73% Blue, y=30.51x Yellow, y=46.19x 0.076=38.65x 3.344=6.347x -3 X=1.966x10 =0.20% x=0.5269=52.69% If the concentrations of the color dyes are 100%, then the absorbance will be as follows.

This is based on Beer-Lambert Law, Red, y=46.03x y=46.03(1.00) y=46.03 Blue, y=30.51x y=30.51(1.00) y=30.51
120

Green, y=35.35x y=35.35(1.00) y=35.35 Yellow, y=46.19x y=46.19(1.00) y=46.19

100

Transmittance against Concentration (Chromatic 1, Blue)

80 Tranmittance,T

60

Green Yellow

40

Log. (Red) Log. (Blue)

20

Log. (Green) Log. (Yellow)

0 0% -20 2% 4% 6% Concentration(%) 8% 10%

y = -23.3ln(x) - 55.15 12% y = -16.1ln(x) - 50.60 y = -23.77ln(x) - 29.802 y = -17.9ln(x) - 7.928

100

Transmittance against Concentration (Chromatic 2, Red)

80

60 Transmittance, T

Red Blue Green

40

Yellow Log. (Red) Log. (Blue)

20

Log. (Green) Log. (Yellow) y = -22.8ln(x) - 69.00 0% 2% 4% 6% 8% 10% 12% y = -22.6ln(x) - 63.68 y = -14.7ln(x) - 43.63

-20

Concentration(%)

y = -26.6ln(x) - 79.59

Discussion
By assuming the depth of the solutions in each well are 5mm, we are able to calculate the molar absorptivity of Green Dye (X3, Chromatic 1) based on the standard curve and the Beer-Lambert Law. The Beer-Lambert Law states that, A=bc. Since the absorbance of the green dye (X3, Chromatic 1) is 0.194, and the depth of the well is 0.5cm, and the concentration of the dye is 0.025mol/L. Thus, the of the Green Dye (X3, Chromatic 1) are 6.48 L/mol cm. Calculation step will be as follow, A=bc (0.194)=(0.5)(0.025) =15.52 L/mol cm From the graph, we can see the relationship between the concentrations of the solution and the absorbance of the solution. The relationship is the concentration of the solution is proportional to the absorbance of the solution. As the concentration decrease, the absorbance will decrease and the absorbance will increase with the increase of concentration. Based on the data, we can see that there are some values having the same value of absorbance. This might be because of the errors of the data. The presence of foreign particle at the microplate will affect the results obtained. The foreign particles include fingerprint and dust. Spectrophotometer are widely used in medical field, for example, spectrometer are used to evaluated and measure the color of a bleached tooth. Besides that, it also can used to determine

the manganese(ll) present in the human blood serum with the aid of oxidation of sulphonilic acid by potassium periodate with an activator which is 1,10-phenanthroline. If given 3 solutions of Tang, one were dissolved 2,0g in 20ml of water(Solution A), one were dissolved 0.2g in 1ml of water(Solution B), and one where dissolved 0.2g in 15ml water(Solution C). The solution with highest absorbance will be solution B and the solution with lowest absorbance will be solution C. This can be seen by Beer-Lambert Law. Since the law state that the absorbance of a solution is proportional to the concentration of the solution. Therefore, the more concentrate the solution, the higher absorbance the solution will be. A solution is made by placing one drop of blue dye in water. The absorbance value recorded is 0.522 with the aid of spectrophotometer. The solution is placed into a 1-cm cuvette and is being measured using 630nm. The molar absorptivity of the dye is 1.38x105 L/(mole cm). By using Beer-Lambert Law, we can calculate the concentration of the blue dye in moles per litre. Beer-Lambert Law are defined as A=bc (0.522)=(1.38x105)(1.0)c C=3.6x10-3 mole/L Thus the concentration of the blue dye is 3.6x10-3 mole/L. In practical, introduction of the solvent decrease the amount of light received by the light intensity measuring system. Thus, by adding the distilled water is used in the 1st column in the microplate, it can eliminate this problem. Another reason of putting the distilled water in the 1st column of the microplate is because we need to adjust the system to read zero absorbance or 100% transmittance. As we can do comparison and can determine the concentration of the solution.

Conclusion
The usage of spectrophotometer are learnt and the absorption spectrum of various color dyes(red, blue, green and yellow) are determined. We also proved that Beer-Lambert Law which it state that the absorbance of the solution is increase with the increase in concentration of the solution is true.

References
1. L.A. Geddes, L.E. Baker, Principles of Applied Instrumentation 3rd Edition, Wiley Interscience Publication, Canada, 1989. 2. R. S. Khandpur, Biomedical Instrumentation Technology & Applications, McGraw-Hill, 2005. 3. http://www.gmi-inc.com/Categories/cary5000.htm