Spectrophotometer practical

Name : Joanne Wong
Student ID : 00000012636 (BM1/14)

Title : Spectrophotometer and its function

Introduction Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength.[1] It can measure any of the listed light ranges that usually cover around 200 nm - 2500 nm using different controls and calibrations. [1] There are a few types of spectrophotometer such as calorimeter, UV spectrometer, IR spectrometer, atomic spectrometer and mass spectrometer. [2] It is being used widely in the field of physics, materials science, chemistry, biochemistry, and molecular biology[3] as it is an optical instrument that measure the amount of light absorbed by the solutes in the solution, thus measuring the absorbance of a solution. The mechanism behind this theory is based on the reference on the visible light spectrum. Different compounds absorb different wavelengths of light and appear to be the colour that it’s reflected which can be observed with our naked eyes. The higher the concentration of the absorbing compounds in a solution, the greater the amount of light that is absorbed. The absorbance of a solution can be determined using the methods as follows:
1) Transmittance, T is the amount of light transmitted through a solution and it can be determined by using the formula of
A = log10 I0/I
A = 2 - log10 %T
Where I is the incident light I0 is the energy of light transmitted through the solution A is the absorbance (the amount of light absorbed by a solution)

2) Using the Beer Lambert Law,
Absorbance, A = λbc
Where λ is the molar absorbtivity coefficient in L mg-1 cm-1, a constant for a compound b is the path length of the cuvette in cm c is the concentration of the compound in the sample solution in mg L-1
3) Using Beer Lambert Law to calculate the concentration of different compounds in a mixture,
Atotal = K1C1 + K2C2
Where Atotal is the total absorbance at any specified wavelength of the solution K1 and K2 are the molar absorbtivity coefficient of each compound of the respective wavelength C1 and C2 are the concentrations of each compound in the mixture

Objective
1. To determine the wavelength of maximum absorption, Amax, of bromophenol blue.
2. To construct a standard concentration curve for bromophenol blue.
3. To determine the concentration of the unknown bromophenol blue solutions.
4. To determine the concentration of two different solutes, bromophenol blue and methyl orange, in a mixture.

Materials
Bromophenol blue
Mixture A and B (unknown concentration of bromophenol blue)
Mixture C (mixture of bromophenol blue and methyl orange solutions)
Methyl orange solution
Micropipette
Cuvette
Test tubes
Tips
Dropper
Distilled water
Beaker
Vortex mixer
Spectrophotometer

Methods
Part 1 : Determination of Amax of bromophenol blue Distilled water was filled into cuvette to be used as blank. The cuvette was then placed into the spectrophotometer to zero the absorbance. The wavelength of the spectrophotometer was first set to 470nm and the blank earlier was replaced with another cuvette containing the bromophenol blue. The experiment was repeated with wavelengths 500, 530, 560, 590, 620, 650 and 680nm. All the readings of the absorbance were recorded and tabulated in table 1.1 and a graph of absorbance readings versus corresponding wavelength was plotted. The wavelength of maximum absorbance was also determined.
Part 2 : The effect of concentration on absorbance of bromophenol blue solution The mixtures of test tubes 1 to 6 were prepared based on the table 1.2a and the mixtures were made sure to be mixed well using the vortex mixer. The content in test tube 1 was used as the blank to zero the absorbance. The absorbance of all the tubes were measured and recorded at the Amax wavelength determined from part 1. The samples were then kept in the original test tubes without discarding them. All the readings were tabulated in table 1.2a. The concentrations of the bromophenol blue solutions in the 6 test tubes were calculated as well. A standard concentration curve of absorbance versus concentration of bromophenol blue was plotted. By using the standard concentration curve, the molar absorbtivity coefficient of bromophenol blue at Amax of bromophenol blue (in unit L mg-1 cm-1) was determined.
Part 3 : Determination of the concentrations of the bromophenol blue solutions of unknown concentration Distilled water was once again used as the blank. The absorbance of the tubes A and B containing the unknown concentration of bromophenol blue solutions were measured at the Amax of bromophenol blue. The results were recorded in table 1.2a and the concentrations of the two unknown were determined by using two methods which are the standard concentration curve plotted from part 2 and by using the formula of Beer-Lambert Law.
Part 4 : The effect of concentration on absorbance of methyl orange solutions The mixtures of test tubes 1 to 6 were prepared based on the table 1.2b and those mixtures were mixed well by using the vortex mixer. The content in tube 1 was used as the blank to zero the absorbance. The absorbance of all the tubes were measured and recorded at the Amax wavelength of methyl orange which is 460nm. All the readings were tabulated in table 1.2b. The samples were then kept in the original test tubes without discarding them. The same cuvette was used throughout the whole experiment. Then, the concentrations of the methyl orange solutions in the 6 test tubes were calculated and recorded. A standard curve of absorbance versus concentration of methyl orange is plotted and the molar absorbtivity coefficient of methyl orange at 460nm (in unit L mg-1 cm-1) is determined by using the standard concentration curve.
Part 5 : Determination of the concentration of two different solutes, bromophenol blue and methyl orange, in mixture C The absorbance of mixture C containing unknown concentrations of bromophenol blue and methyl orange (Tube C) were measured at both the wavelengths Amax of bromophenol blue and Amax of methyl orange (given as 460nm). All the readings are recorded and tabulated in table 1.3. The table 1.4 regarding the molar absorbtivity coefficient of bromophenol blue and methyl orange with the Amax stated was filled. The values obtained were then substituted into equations 1 and 2 (as shown below) to obtain the concentrations of methyl orange and bromophenol blue in mixture C by solving it simultaneously.
Ac at Amax BB = KBB at Amax BB CBB + KMO at Amax BB CMO………..1
Ac at Amax MO = KBB at Amax MO CBB + KMO at Amax MO CMO………..2
Where Ac at Amax BB is the absorption of mixture C at Amax of bromophenol blue (BB) Ac at Amax MO is the absorption of mixture C at Amax of methyl orange (MO) i.e.460nm KBB at Amax BB is the molar absorptivity coefficient of BB at Amax of BB KMO at Amax BB is the molar absorptivity coefficient of MO at Amax of BB KBB at Amax MO is the molar absorptivity coefficient of BB at Amax of MO KMO at Amax MO is the molar absorptivity coefficient of MO at Amax of MO (560nm) CBB is the concentration of bromophenol blue in the mixture CMO is the concentration of methyl orange in the mixture

Results
Part 1: Determination of Amax of bromophenol blue

Part 2: The effect of concentration on absorbance of bromophenol blue solution

The concentrations of the bromophenol blue solutions in test tubes 1-6 are determined by using the formula of M1V1 = M2V2
Where M1 is the concentration of the bromophenol blue M2 is the concentration of solution in the test tube V1 is the volume of the bromophenol blue V2 is the total volume of the solution in the test tube
Test tube 1: M1V1 = M2V2 (10)(0) = M2 (2.5) M2 = 0.0/2.5 M2 = 0 mg/L
Test tube 2:
M1V1 = M2V2
(10)(0.5) = M2 (2.5)
M2 = 5.0/2.5
M2 = 2.0 mg/L
Test tube 3:
M1V1 = M2V2 (10)(1.0) = M2 (2.5) M2 = 10/2.5 M2 = 4.0 mg/L
Test tube 4:
M1V1 = M2V2 (10)(1.5) = M2 (2.5) M2 = 15/2.5 M2 = 6.0 mg/L
Test tube 5:
M1V1 = M2V2 (10)(2.0) = M2 (2.5) M2 =20/2.5 M2 = 8.0 mg/L
Test tube 6:
M1V1 = M2V2 (10)(2.5) = M2 (2.5) M2 =25/2.5 M2 = 10.0 mg/L
According to Beer-Lambert Law, Absorbance, A = bc Where,  = molar absorbtivity coefficient (L mg-1 cm-1) b = path length of the cuvette in which the sample is contained (cm) c = concentration of the compound in solution (mg L-1) Therefore,  = A/bc
Since b, the path length = 1cm,  = A/c = gradient of curve
From figure 1.2a, molar absorbtivity coefficient = gradient of graph (KBB at Amax BB) = (y2-y1) / (x2-x1) = (1.200 – 0) / (10.0 – 0) = 0.120 L mg-1 cm-1
From the figure 1.2a the molar absorbtivity coefficient of bromophenol blue at Amax of bromophenol blue is 0.120 L mg-1 cm-1.

Part 3 : Determination of the concentration of the bromophenol blue solutions of unknown concentration
To determine the two unknown concentration,
Method 1
From the graph drawn in figure 1.2a in part 2 i. The concentration of bromophenol blue in test tube A is 3.5 mg/ L. ii. The concentration of bromophenol blue in test tube B is 1.8 mg/ L.
Method 2
By using Beer-Lambert Law,  = A/bc Where = 0.120 L mg-1cm-1,
A= absorbance at 590 nm, b = the path length is 1cm.
From test tube A, c = A/ = 0.415/ 0.120 = 3.458 mg/ L
From test tube B, c = A/ = 0.244/0.120 = 2.033 mg /L
Part 4 : The effect of concentration on absorbance of methyl orange solutions

The concentrations of the methyl orange solutions in test tubes 1-6 are determined by using the formula of M1V1 = M2V2
Where M1 is the concentration of the methyl orange M2 is the concentration of solution in the test tube V1 is the volume of the methyl orange V2 is the total volume of the solution in the test tube
Test tube 1: M1V1 = M2V2 (10)(0) = M2 (2.5) M2 = 0.0/2.5 M2 = 0 mg/L
Test tube 2:
M1V1 = M2V2
(10)(0.5) = M2 (2.5)
M2 = 5.0/2.5
M2 = 2.0 mg/L
Test tube 3:
M1V1 = M2V2 (10)(1.0) = M2 (2.5) M2 = 10/2.5 M2 = 4.0 mg/L
Test tube 4:
M1V1 = M2V2 (10)(1.5) = M2 (2.5) M2 = 15/2.5 M2 = 6.0 mg/L
Test tube 5:
M1V1 = M2V2 (10)(2.0) = M2 (2.5) M2 =20/2.5 M2 = 8.0 mg/L
Test tube 6:
M1V1 = M2V2 (10)(2.5) = M2 (2.5) M2 =25/2.5 M2 = 10.0 mg/L
According to Beer-Lambert Law, Absorbance, A = bc Where,  = molar absorbtivity coefficient (L mg-1 cm-1) b = path length of the cuvette in which the sample is contained (cm) c = concentration of the compound in solution (mg L-1) Therefore,  = A/bc
Since b, the path length = 1cm,  = A/c = gradient of curve
From figure 1.2b, molar absorbtivity coefficient = gradient of graph (KMO at Amax MO) = (y2-y1) / (x2-x1) = (0.839 – 0) / (10.0 – 0) = 0.0839 L mg-1 cm-1
From the figure 1.2b, the molar absorbtivity coefficient of methyl orange at Amax of methyl orange (460nm) is 0.0839 L mg-1 cm-1.

Part 5 : Determination of the concentrations of two different solutes, bromophenol blue and methyl orange, in mixture C

According to Beer Lambert law,

Atotal = K1C1 + K2C2
Where,
C1 and C2 = concentrations of each compound in mixture
K1 and K2 = molar absorbtivity coefficient of each compound of the respective wavelength

Values obtained from Table 1.3 and Table 1.4 is substituted into equation 1 and 2 to be solved simultaneously. Thus the concentrations of methyl orange and bromophenol blue in mixture C is calculated as follows:
Ac at Amax BB = KBB at Amax BB CBB + KMO at Amax BB CMO ------- Equation 1
Ac at Amax MO = KBB at Amax MO CBB + KMO at Amax MO CMO ------- Equation 2
Where Ac at Amax BB is the absorption of mixture C at Amax of bromophenol blue (BB)
Where Ac at Amax MO is the absorption if mixture C at Amax of methyl orange (MO)
i.e. 460nm
Where KBB at Amax BB is the molar absorbtivity coefficient of BB at Amax of BB
Where KMO at Amax BB is the molar absorbtivity coefficient of MO at Amax of BB
Where KBB at Amax MO is the molar absorbtivity coefficient of BB at Amax of MO
Where KMO at Amax MO is the molar absorbtivity coefficient of MO at Amax of MO
(460nm)

For Equation 1, 0.675 = (0.1221) CBB + (0) CMO CBB = (0.675 – 0) / (0.1221) = 5.528 mg L-1
For Equation 2, 0.448 = (0) CBB + (0.0839) CMO CMO = (0.448– 0) / (0.0839) = 5.340 mg L-1

The concentration of bromophenol blue in mixture C is 5.528 mg L-1.
The concentration of methyl orange in mixture C is 5.340 mg L-1.

Discussion In part 1 of the experiment, the trend of the graph plotted is a dumb-bell shape with a peak showing a maximum absorbance reading. As the absorbance increases, the wavelength increases until it reaches a maximum point which is at the wavelength of 590nm, it then decreases tremendously after the peak from 620nm to 680nm. This shows that the bromophenol blue absorbs the maximum transmittance of light at the wavelength of 590nm. The reason why it decreases is due to the properties of the bromophenol blue having the ability to give different readings of absorbance at different wavelength. Thus, the optimum wavelength for the maximum absorbance, Amax of bromophenol blue which is 1.085 is st 590nm.
In part 2 of the experiment, the trend of the graph plotted is a straight line. As the concentration of bromophenol blue increases, the absorbance also increases. Thus, the concentration of bromophenol blue is said to be directly proportional to the absorbance at 590nm which is the Amax of the bromophenol blue. As the concentration of bromophenol blue increases, more solutes are present in the solutions absorbing more light, giving a rise in the absorbance. The R-squared value of the trend line is 0.9951 which is near to 1, portraying an almost best fit graph. Thus, the graph is said to obey Beer-Lambert’s Law. However, some random errors occurred as there are points plotted far from the straight line, this might due to contamination of the chemicals in the cuvette as well as the presence of fingerprints on the cuvette that might affect the readings of absorbance. These random errors can be reduced by rinsing the cuvette well with the solution filled in the cuvette before using and cleaning it properly before putting into the spectrophotometer. In part 3 of the experiment, the concentration of the bromophenol blue in both mixture A and B are determined by using two methods, determining from the graph plotted and through calculation using Beer-Lambert Law. The concentration of bromophenol blue in mixture A obtained from the graph is 3.5 mgL-1 whereas it’s 3.458 mgL-1 from calculation. On the other hand, the concentration of the bromophenol blue in mixture B obtained from the graph is 1.8 mgL-1 whereas its reading obtained from calculation is 2.033 mgL-1. There is small difference in the reading obtained which can be due to random errors such as incomplete mixing of the solution or fingerprints and water droplet present on the clear surface of the cuvette which can affect the amount of light passing through the cuvette. This can be avoided by mixing the solution well as well as cleaning the cuvette properly with paper towel. In part 4 of the experiment, the trend of the graph plotted is a straight line. As the concentration of the methyl orange increases, the absorbance also increases. Thus, the concentration of methyl orange is said to be directly proportional to the absorbance at 460nm which is the Amax of the methyl orange. As the concentration of methyl orange increases, more solutes are present in the solutions absorbing more light, thus increasing the absorbance. The R-squared value of the trend line is 0.9998 which is near to 1, portraying an almost best fit graph. Thus, the graph is said to obey Beer-Lambert’s Law. However, there is a slight deviation in one of the reading from its straight line indicating the presence of error. This might be due to poor pipetting technique which can be avoided by making sure that there is no air bubbles present in the tips as well as pipetting vertically. Another reason might be due to the presence of fingerprints and water droplets on the clear surface of the cuvette which can be avoided by wiping it using paper tower before inserting it into the spectrophotometer. In part 5 of the experiment, the concentrations of bromophenol blue and methyl orange in mixture C are determined by using the Beer-Lambert Law. The molar absorbtivity coefficient of MO at Amax of bromophenol and the molar absorbtivity coefficient of BB at Amax of methyl orange is assumed to be zero due to the time constriction in conducting the experiment. Besides that, if we were to take the Amax of both the bromophenol blue and methyl orange from the graphs, both of them has very small intersection point indicating there’s minimal activity between those two solutions, and the graph will appear as a straight line if graphs were to be plotted. Also, their molar absorbtivity coefficients are taken from the gradient calculated by the Microsoft Excel software as it is more accurate. Thus, the concentration of bromophenol blue in mixture C is 5.528 mgL-1 and the concentration of methyl orange in mixture C is 5.340 mg L-1.

Conclusion
For part 1, the wavelength of maximum absorption, Amax of bromophenol blue is 590nm.
For part 2, the absorbance of bromophenol blue is directly proportional to the concentration of bromophenol blue. The molar absorbtivity coefficient of bromophenol blue at Amax of bromophenol blue is 0.120 L mg-1 cm-1.
For part 3, the concentration of bromophenol blue in tube A and tube B obtained from the graph plotted is 3.5 mg L-1 and 1.8 mg L-1 respectively. The concentration of bromophenol blue in tube A and tube B obtained is 3.458 mg L-1 and 2.033 mg L-1 respectively by using Beer Lambert Law. Therefore, the result obtained from formula is more accurate as random and systematic errors might occur during experiment.
For part 4, the absorbance of methyl orange is directly proportional to the concentration of methyl orange. The molar absorbtivity coefficient of methyl orange at Amax of methyl orange (460nm) is 0.0839 L mg-1 cm-1.
For part 5, the molar absorbtivity coefficient of methyl orange at Amax of bromophenol blue and the molar absorbtivity coefficient of bromophenol blue at Amax of methyl orange are both assumed to be zero. The concentration of bromophenol blue in mixture C is 5.528 mg L-1 whereas the concentration of methyl orange in mixture C is 5.340 mg L-1.

References
1. Allen, D., Cooksey, C., & Tsai, B. (2010, October 5). Spectrophotometry. Retrieved from http://www.nist.gov/pml/div685/grp03/spectrophotometry.cfm (accessed 8 October 2014)
2. Fundamentals of Analytical Chemistry; Douglas A. Skoog, Donald M. West, and F. James Holler; 1988. Retrieved from http://www.ehow.com/about_5444167_types-spectrometers.html (accessed 8 October 2014)
3. Rendina, George. Experimental Methods in Modern Biochemistry W. B. Saunders Company: Philadelphia, PA. 1976. pp. 46-555

References: 1. Allen, D., Cooksey, C., & Tsai, B. (2010, October 5). Spectrophotometry. Retrieved from http://www.nist.gov/pml/div685/grp03/spectrophotometry.cfm (accessed 8 October 2014) 2. Fundamentals of Analytical Chemistry; Douglas A. Skoog, Donald M. West, and F. James Holler; 1988. Retrieved from http://www.ehow.com/about_5444167_types-spectrometers.html (accessed 8 October 2014) 3. Rendina, George. Experimental Methods in Modern Biochemistry W. B. Saunders Company: Philadelphia, PA. 1976. pp. 46-555