The spectrophotometer can mensurate the strength of light absorbed indirectly by the solutes in solution because each solution has its ain characteristic soaking up movables. The spectrophotometer can be used to mensurate the concentration of compounds in a certain solution. Distinguishing compounds is another utilizations of spectrophotometer.It plants by analysing the form of wavelengths absorbed by the sample.. The optical density can be calculated utilizing:
Beer-Lambert Law, Absorbance, A= I»bc where,
I» is the molar absorbtivity coefficient in L mol-1 cm-1
B is the way length ( in centimeter ) of the cuvette in which the sample is contained
degree Celsius is the concentration of compound in solution, in mol LAA-1
If two compounds are present in a solution, the entire optical density of the solution is the amount of the two separate parts harmonizing to the expression Atotal = K1C1+K2C2 where,
C1 and C2 are the concentrations of bromophenol blue and methyl orange severally in the mixture
K1 and K2 are the molar absorbtivity coefficient of each compound of the several wavelength
Material
Methyl orange solution, bromophenol bluish solution, micropipette, tip droplet, mixture of bromophenol blue and methyl orange, trial tubings.
Method
Part 1: Determination of Amax of bromophenol blue
A cuvette with distilled H2O was placed into the spectrophotometer. The wavelength input is set to 470 nanometer. Auto zero button was pressed to put the optical density into nothing.
The space is removed. Optical density of bromophenol blue is read at different wavelengths.
After a certain wavelength is tested against bromophenol blue, the spectrophotometer is set clean utilizing distilled H2O.
A new wavelength is set to obtain a new optical density value.
A graph of soaking up spectrum was plotted and the wavelength with maximal optical density reading was determined from the graph.
Part 2: The consequence of concentration of bromophenol blue on optical density
The distilled H2O and mixture of distilled H2O with bromophenol blue are prepared harmonizing Table 1.2 and the contents of each tubing are assorted utilizing vortex sociable.
The spectrophotometer is set clean utilizing cuvette with distilled H2O.
Wavelength of the spectrophotometer was set at Amax wavelength of bromophenol blue and the optical density reading is recorded.
The mixture of tubing 1 should be holding a zero optical density.
Concentration of bromophenol blue in tube 1-6 was calculated utilizing information in table 1.2.
Standard concentration curve was plotted and the molar absorbtivity coefficient ( in unit L mg-1 cm-1 ) of the expression from Beer-Lambert Law was calculated utilizing the standard concentration curve.
Part 3: Determination of the concentration of the bromophenol bluish solution of unknown concentration
The optical density reading is set clean utilizing a cuvette of distilled H2O.
The optical densities of the two bromophenol blue of unknown concentrations were measured at the Amaxof bromophenol blue.
The concentration of the two unknown were determined from the graph 2 and besides by utilizing Beer-Lambert Law.
Part 4: Determination of consequence of concentration on optical density of methyl orange solutions
Mixtures of distilled and methyl orange are prepared harmonizing Table 1.2b
The contents of each tubing is assorted utilizing vortex sociable.
The spectrophotometer is set to the Amax wavelength of methyl orange which is 460nm.
The optical density reading of tube 1-6 is measured utilizing the same cuvette you used for tubing 7.
The optical density readings is recorded in Table 1.2b.
The concentration of the methyl orange solutions in tubes 1-6 are calculated.
Graph of standard concentration curve of optical density versus concentration of methyl orange is plotted.
The molar absorbtivity coefficient of methyl orange at 460nm ( in unit L mg-1cm-1 ) is calculated utilizing standard concentration curve from measure 7.
Part 5: Determination of concentrations of two different solutes, bromophenol blue and methyl orange, in mixture C
The spectrophotometer is set to a wavelength of 460nm – Amax of methyl orange.
The spectrophotometer is set clean by pressing the ( auto-zero ) button.
The optical density of the bromophenol bluish solutions in tubes 1-6 in Table 1.2a is measured at the Amax of methyl orange.
The consequences is recorded in table 1.2a
A graph of standard concentration curve of bromophenol blue at Amax of methyl orange.
Molar absorbtivity coefficient of bromophenol blue at 460nm is determined.
The spectrophotometer is set space once more utilizing distilled H2O.
Optical density of methyl orange solutions in tubes 1-6 in Table 1.2b at the Amax of bromophenol blue.
The consequences is recorded in Table 1.2b
A graph of standard concentration curve of methyl orange at Amax of bromophenol blue is plotted.
The molar absorbtivity coefficient of methyl orange at Amax of bromophenol blue is calculated.
The optical density of mixture C incorporating bromophenol blue and methyl orange ( tube C ) is measured at the wavelength of Amax of bromophenol blue and Amax of methyl orange ( 460nm )
The consequences is recorded in Table 1.3
Consequences
Part 1: Determination of Amax of bromophenol blue
Table 1.1: Optical density of bromophenol blue in different wavelengths
Wavelength ( nanometer )
Optical density
470
0.093
500
0.143
530
0.282
560
0.535
590
0.981
620
0.211
650
0.013
680
0.002
From the graph 1, the highest extremum is found to be at the wavelength 590nm. Therefore, the
Amax of bromophenol blue is in wavelength of 590nm.
Part 2: The consequence of concentration of bromophenol blue on optical density
Table 1.2: The optical density values of bromophenol blue in different concentration for the wavelength of 590nm and 460nm.
Tube
1
2
3
4
5
6
A
Bacillus
Distilled H2O ( milliliter )
2.5
2.0
1.5
1.0
0.5
0.0
Bromophenol blue 10 mg/L ( milliliter )
0.0
0.5
1.0
1.5
2.0
2.5
Concentration of Bromophenol blue ( mg/L )
0.0
2.0
4.0
6.0
8.0
10.0
Optical density in 590nm
0.000
0.271
0.459
0.690
0.907
1.091
0.361
0.235
Optical density in 460nm
0.000
0.010
0.026
0.038
0.055
0.079
Beer-Lambert Law,
Optical density, A= I»bc is in the signifier of y=mx ( consecutive line expression ) where,
– B is the way length ( in centimeter ) of the cuvette in which the sample is contained ( 1cm )
– I» is the absorbtivity invariable and degree Celsius is the concentration of the compound in solution, in mol L-1
Therefore, A= I»c ( since B is 1 centimeter )
I»= A/c
Hence, the molar absorbtivity coefficient can be found by ciphering the gradient of the standard concentration curve.
From graph 2, molar absorbtivity coefficient of bromophenol blue in 590nm is
I» =
= 0.113 L mg-1 cm-1
Part 3: Determination of the concentration of the bromophenol bluish solutions of unknown concentration.
The consequence of the optical density of the two unknown concentration bromophenol bluish solutions ( Tube A & A ; B ) is stated in Table1.2
Concentration of the two unknown:
Method 1: By utilizing the standard concentration curve from Part 2 ( chart 2 )
Concentration of bromophenol blue in Solution A = 3.15 mg/L
Concentration of bromophenol blue in Solution B = 2.05 mg/L
Method 2: By utilizing the expression of the Beer-Lambert Law to cipher the concentration os bromophenol blue in solution A and solution B
From the Beer-Lambert Law,
Optical density, A= I»bc
Concentration of solution A:
A= I»bc
0.361= 0.113 ( 1 ) ( degree Celsius )
degree Celsiuss = 3.195 mg/L
Concentration of solution B:
A= I»bc
0.235 = 0.113 ( 1 ) ( degree Celsius )
degree Celsiuss =2.080 mg/L
Part 4: The consequence of concentration on optical density of methyl orange solutions
Table 1.3: The optical density values of methyl orange in different concentration for the wavelength of 590nm and 460nm.
Tube
1
2
3
4
5
6
Distilled H2O ( milliliter )
2.5
2.0
1.5
1.0
0.5
0.0
Methyl orange 10 mg/L ( milliliter )
0.0
0.5
1.0
1.5
2.0
2.5
Concentration of methyl orange ( mg/L )
0.0
2.0
4.0
6.0
8.0
10.0
Optical density in 460nm
0.000
0.150
0.314
0.485
0.606
0.814
Optical density in 590nm
0.000
0.001
0.002
0.003
0.004
0.005
Harmonizing to Beer-Lambert Law,
Optical density, A= I»bc
A= I»c ( since b=1cm )
I» = A/c that is gradient of standard concentration curve
From graph 4, molar absorbtivity coefficient of methyl orange in 460nm is
I» =
= 0.082 L mg-1 cm-1
Part 5: Determination of the concentration of two different solutes, bromophenol blue and methyl orange, in a mixture C
Table 1.3: The optical density of the two mixture solutions of bromophenol blue and methyl orange ( Tube C ) in both the wavelengths of Amax of bromophenol blue ( 590nm ) and methyl orange ( 460nm ) .
Tube
C
Amax of bromophenol blue
0.558
Amax of methyl orange
0.434
From graph 3, molar absorbtivity coefficient of bromophenol blue in 460nm is
I» =
= 0.0069 L mg-1 cm-1
From graph 5, molar absorbtivity coefficient of methyl orange in 590nm is
I» =
= 0.0005 L mg-1 cm-1
Table 1.4: Molar absorbtivity coefficient, ( K ) of BB or MO in Amax of bromophenol blue and methyl orange.
Molar absorbtivity coefficient of BB at Amax
of bromophenol blue ( BB ) / KBB at Amax BB
0.1130
Molar absorbtivity coefficient of MO at
Amax of bromophenol / KMO at Amax BB
0.0005
Molar absorbtivity coefficient of BB at Amax
of methyl orange ( MO ) /KBB at Amax MO
0.0069
Molar absorbtivity coefficient of MO at
Amax of methyl orange ( MO ) ( 460 nanometer ) /
KMO at Amax MO
0.0820
By utilizing the Beer-Lambert Law,
Atotal = K1C1 + K2C2
K1 = Molar absorbtivity coefficient ( in L mol-1 cm-1 ) of bromophenol blue.
K2 = Molar absorbtivity coefficient ( in L mol-1 cm-1 ) of methyl orange.
C1 = Concentration of bromophenol blue in the two mixture solutions of Tube C.
C2 = Concentration of methyl orange in the two mixture solutions of Tube C.
As calculated before, the molar absorbtivity coefficient of methyl orange in Amax of bromophenol blue ( 590nm ) is 0.0005 Fifty mg-1 cm-1 ;
The molar absorbtivity coefficient of bromophenol blue in Amax of bromophenol blue ( 590nm ) is 0.113 Fifty mg-1 cm-1
At Amax of bromophenol blue ( 590nm )
Atotal = K1C1 + K2C2
0.558 = 0.113C1 + ( 0.0005 ) C2
0.113C1= 0.558-0.0005C2
C1= — — — — — — – ( 1 )
As calculated before, the molar absorbtivity coefficient of methyl orange in Amax of methyl orange ( 460nm ) is 0.0820 Fifty mg-1 cm-1
The molar absorbtivity coefficient of bromophenol blue in Amax of methyl orange ( 460nm ) is 0.0064 Fifty mg-1 cm-1
At Amax of methyl orange ( 460nm ) ,
Atotal = K1C1 + K2C2
0.434 = 0.0064C1 + ( 0.0820 ) C2
0.434- 0.0820 C2 = 0.0064 C1
C1= — — — — — — — — ( 2 )
( 1 ) – ( 2 )
=
( 0.558-0.0005 C2 ) x 0.0064 = 0.113 ( 0.434- 0.0820 C2 )
3.843×10-3 – 3.45×10-6 C2 = 0.0490 – 9.266×10-3C2
9.2626×10-3 C2= 0.04516
C2 = 4.876 mg/L
Substitute C2 into ( 2 )
C1=
= 6.841 mg/L
As a consequence, the concentration of bromophenol blue in the two mixture solutions of Tube C is 6.841 mg/L, and the concentration of methyl orange in the two mixture solutions of Tube C is 4.876mg/L.
Discussion
Part 1
The graph of soaking up spectrum, graph of optical density against matching wavelength plotted is a bell form. From the graph, we can analyze that optical density readings increases from 0.093 to 0.981 as the wavelength increases from 470nm to 590nm. The optical density is so lessening when the wavelength applied was 620nm until 680 nanometers. The peak optical density obtained from the graph of soaking up spectrum plotted is in 590nm which is 0.981 optical density. The consequences obtained shows that the maximal soaking up of light bromophenol bluish occur in wavelength of 590nm.
Part 2
Amax of bromophenol blue, which is at 590 nanometer is set in the spectrophotometer to find the optical density readings of different concentration of bromophenol bluish e.g ( 0.0, 2.0, 4.0, 6.0, 8.0, and 10.0 ) mg/L. A standard concentration curve of optical density against concentration of bromophenol blue is plotted. A consecutive line is obtained in the graph. This indicated that the optical density of bromophenol blue is straight relative to the concentration of bromophenol blue. Therefore, the graph is said to obey the Beer-Lambert Law where A= I»bc.
The line of the standard concentration curve of optical density versus concentration of bromophenol bluish base on balls through beginning. However, there is some point that plotted far from the consecutive line.Thus, there are random mistakes might happen during the experiment as all points should be in the consecutive line. The graph shows that distilled H2O shows zero optical density reading. The random mistake that might happen was that hints of chemical were present in the cuvette therefore polluting the distilled H2O which in bend giving the distilled H2O an optical density reading. This random mistake can be reduced by rinsing the cuvette with distilled H2O before usage.
Part 3
The optical density of the two bromophenol bluish solutions ( Tube A & A ; B ) of unknown concentration at the Amax of bromophenol blue ( 590nm ) were measured. The concentrations of the two unknown solutions were determined utilizing two methods. The first method used was extrapolating the graph 2 while the 2nd method used was utilizing the expression of Beer-Lambert Law to cipher the concentrations of the two unknown solutions.
From the first method, the concentration for the Tube A bromophenol blue is 3.15 mg/L while the concentration of Tube B bromophenol blue is 2.05 mg/L. While, from the 2nd method utilizing the Beer-Lambert Law, A = I»bc, the concentration of Tube A bromophenol blue obtained is 3.195 mg/L while the concentration of Tube B bromophenol blue is 2.080 mg/L. Concentration consequence for each unknown obtained utilizing two different methods mentioned above give different consequence. The difference in consequences might due to random mistakes in the experiment. The random mistake that might happen was that the surface of the cuvette was non clear or due some fragment of mixture is non absolutely mixed in the solution. Fingerprints might be printed on the clear surface of the cuvette therefore impacting the supposed visible radiation sum base on balls through.This will impact the optical density reading of bromophenol blue. This random mistake can be reduced by guaranting that the surface of the cuvette was wiped with paper towel before puting it into the spectrophotometer. Furthermore, the orientation of the cuvette may be inserted wrongly into the spectrophotometer when the optical density reading is taken.
Part 4
The peak optical density of methyl orange is at 460nm. The optical density of methyl orange addition with the concentration of methyl orange in the mixtures. The optical density novice from 0.000 til 0.814. A graph of optical density against concentration of methyl orange. A consecutive line go throughing through beginning is obtained. This showed that the concentration of methyl orange increased straight relative to the optical density of light with certain wavelength. Hence, this portion of experiment obeyed the Law of Beer- Lambert.
Somehow, non all point is inside the consecutive line. So, there is some random mistake occur when transporting out this experiment. The random mistake includes, the mixture is non absolutely assorted.Furthermore, there might be fingerprint left on the wall of cuvette.This affect the sum of light go throughing through the solution.There might be caused by hapless pippeting technique that caused inaccuracy in optical density reading of methyl orange. The picks of taking wavelength will impact the sensitiveness and truth of the analysis. Therefore impacting the optical density of solution or Beer- Lambert Law is non obeyed.
The molar absorbtivity coefficient calculated is 0.082 L mg-1cm-1.
Part 5
The experiment shows addition in optical density when concentration bromophenol bluish increased.Bromophenol bluish solutions are tested utilizing Amax of methyl orange- 460nm of visible radiation. The optical density addition from 0.000 to 0.079 of absorbance.A graph of optical density of visible radiation against concentrations of bromophenol blue is plotted.A consecutive line go throughing through the beginning is plotted. This showed that optical density addition straight relative to the concentrations of bromophenol blue. The graph shows scattered point around the consecutive line. This indicated random mistakes occur in the experiment. Some illustrations of random mistake are hapless pippeting technique that may do inaccuracy in mensurating volune of bromophenol blue.
Another portion of the experiment in portion 5 is to change the concentration of methyl orange and trial it utilizing Amax of bromophenol bluish -590 nanometer of visible radiation. The trial shows that there is addition of sum of optical density as the concentration increased. A graph of optical density against concentration of methyl orange is plotted. A consecutive line go throughing through beginning is obtained. This meant that optical density addition proportional to concentration of methyl orange. This shows that it obey the jurisprudence of Beer-Lambert. All points are in the graph. Therefore, there is minimal mistake occurred when the experiment is carried out.
The concentration of bromophenol blue and methyl orange in the two mixture solutions of tubing C was determined utilizing the expression, Atotal = K1C1+K2C2. By work outing coincident equation, the concentration of bromophenol blue in the mixture C was 6.841 mg/L and the concentration for methyl orange in the mixture was found to be 4.876 mg/L.
Decision
Part 1, the Amax of bromophenol blue is at the wavelength of 590nm.
Part 2, the optical density of visible radiation is straight relative to the concentration of bromophenol blue as the standard concentration curve of optical density versus concentration of bromophenol shows a consecutive line go throughing through the beginning. Molar absorbtivity coefficient of bromophenol blue in 590nm is 0.113 L mg-1cm-1.
Part 3, two methods were used to find the concentration of the two terra incognitas ( tube A & A ; B ) . By utilizing insertion of the graph, the concentration of bromophenol blue in Tube A is 3.15 mg/L and the concentration of bromophenol blue in Tube B is 2.05 mg/L. Mean while, utilizing the expression of Beer – Lambert Law, the concentration of bromophenol blue in Tube A is 3.195 mg/L and the concentration of bromophenol blue in Tube B is 2.080 mg/L.
Part 4, the optical density of visible radiation is straight relative to the concentration of methyl orange as the standard concentration curve of optical density against concentration of methyl orange shows a consecutive line go throughing through the beginning. Molar absorbtivity coefficient of methyl orange in 460nm is 0.082 Fifty mg-1 cm-1.
Part 5, the molar absorbtivity coefficient of methyl orange in Amax of bromophenol blue is 0.005L mg-1cm-1.Molar absorbtivity coefficient of bromophenol blue in Amax of methyl orange is 0.0069 Fifty mg-1 cm-1. The concentration of bromophenol blue in Tube C is 6.841 mg/L and the concentration of methyl orange in Tube C is 4.876 mg/L.
