Using Spectrophotometer to measure the concentration of compounds

The spectrophotometer can measure the intensity of light absorbed indirectly by the solutes in solution because each solution has its own characteristic absorption chattels. The spectrophotometer can be used to measure the concentration of compounds in a certain solution. Differentiating compounds is another uses of spectrophotometer .It works by analyzing the pattern of wavelengths absorbed by the sample.. The absorbance can be calculated using:

Beer-Lambert Law, Absorbance, A= λbc where,

λ is the molar absorbtivity coefficient in L mol-1 cm-1

b is the path length (in cm) of the cuvette in which the sample is contained

c is the concentration of compound in solution, in mol L­­-1

If two compounds are present in a solution, the total absorbance of the solution is the sum of the two separate contributions according to the formula Atotal = K1C1+K2C2 where,

C1 and C2 are the concentrations of bromophenol blue and methyl orange respectively in the mixture

K1 and K2 are the molar absorbtivity coefficient of each compound of the respective wavelength

Material

Methyl orange solution , bromophenol blue solution , micropipette , tip droplet , mixture of bromophenol blue and methyl orange, test tubes .

Method

Part 1: Determination of Amax of bromophenol blue

A cuvette with distilled water was placed into the spectrophotometer. The wavelength input is set to 470 nm. Auto zero button was pressed to set the absorbance into zero.

The blank is removed. Absorbance of bromophenol blue is read at different wavelengths.

After a certain wavelength is tested against bromophenol blue, the spectrophotometer is set blank using distilled water.

A new wavelength is set to obtain a new absorbance value.

A graph of absorption spectrum was plotted and the wavelength with maximum absorbance reading was determined from the graph.

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

The distilled water and mixture of distilled water with bromophenol blue are prepared according Table 1.2 and the contents of each tube are mixed using vortex mixer.

The spectrophotometer is set blank using cuvette with distilled water.

Wavelength of the spectrophotometer was set at Amax wavelength of bromophenol blue and the absorbance reading is recorded.

The mixture of tube 1 should be having a zero absorbance.

Concentration of bromophenol blue in tube 1-6 was calculated using information in table 1.2.

Standard concentration curve was plotted and the molar absorbtivity coefficient (in unit L mg-1 cm-1) of the formula from Beer-Lambert Law was calculated using the standard concentration curve.

Part 3: Determination of the concentration of the bromophenol blue solution of unknown concentration

The absorbance reading is set blank using a cuvette of distilled water.

The absorbances 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 also by using Beer-Lambert Law.

Part 4: Determination of effect of concentration on absorbance of methyl orange solutions

Mixtures of distilled and methyl orange are prepared according Table 1.2b

The contents of each tube is mixed using vortex mixer.

The spectrophotometer is set to the Amax wavelength of methyl orange which is 460nm.

The absorbance reading of tube 1-6 is measured using the same cuvette you used for tube 7.

The absorbance 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 absorbance versus concentration of methyl orange is plotted.

The molar absorbtivity coefficient of methyl orange at 460nm (in unit L mg-1cm-1) is calculated using standard concentration curve from step 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 blank by pressing the (auto-zero) button.

The absorbance of the bromophenol blue solutions in tubes 1-6 in Table 1.2a is measured at the Amax of methyl orange.

The results 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 blank again using distilled water.

Absorbance of methyl orange solutions in tubes 1-6 in Table 1.2b at the Amax of bromophenol blue.

The results 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 absorbance of mixture C containing bromophenol blue and methyl orange (tube C) is measured at the wavelength of Amax of bromophenol blue and Amax of methyl orange (460nm)

The results is recorded in Table 1.3

Results

Part 1: Determination of Amax of bromophenol blue

Table 1.1: Absorbance of bromophenol blue in different wavelengths

Wavelength (nm)

Absorbance

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 peak is found to be at the wavelength 590nm. Therefore, the

Amax of bromophenol blue is in wavelength of 590nm.

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

Table 1.2: The absorbance values of bromophenol blue in different concentration for the wavelength of 590nm and 460nm.

Tube

1

2

3

4

5

6

A

B

Distilled water (ml)

2.5

2.0

1.5

1.0

0.5

0.0

Bromophenol blue 10 mg/L (ml)

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

Absorbance in 590nm

0.000

0.271

0.459

0.690

0.907

1.091

0.361

0.235

Absorbance in 460nm

0.000

0.010

0.026

0.038

0.055

0.079

Beer-Lambert Law,

Absorbance, A= λbc is in the form of y=mx (straight line formula) where,

– b is the path length (in cm) of the cuvette in which the sample is contained (1cm)

– λ is the absorbtivity constant and c is the concentration of the compound in solution, in mol L-1

Therefore, A= λc (since b is 1 cm)

λ= A/c

Hence, the molar absorbtivity coefficient can be found by calculating the gradient of the standard concentration curve.

From graph 2, molar absorbtivity coefficient of bromophenol blue in 590nm is

λ =

= 0.113 L mg-1 cm-1

Part 3: Determination of the concentration of the bromophenol blue solutions of unknown concentration.

The result of the absorbance of the two unknown concentration bromophenol blue solutions (Tube A&B) is stated in Table1.2

Concentration of the two unknown:

Method 1: By using the standard concentration curve from Part 2 (graph 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 using the formula of the Beer-Lambert Law to calculate the concentration os bromophenol blue in solution A and solution B

From the Beer-Lambert Law,

Absorbance, A= λbc

Concentration of solution A:

A= λbc

0.361= 0.113(1)(c)

c = 3.195 mg/L

Concentration of solution B:

A= λbc

0.235 = 0.113(1)(c)

c =2.080 mg/L

Part 4: The effect of concentration on absorbance of methyl orange solutions

Table 1.3: The absorbance values of methyl orange in different concentration for the wavelength of 590nm and 460nm.

Tube

1

2

3

4

5

6

Distilled water (ml)

2.5

2.0

1.5

1.0

0.5

0.0

Methyl orange 10 mg/L (ml)

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

Absorbance in 460nm

0.000

0.150

0.314

0.485

0.606

0.814

Absorbance in 590nm

0.000

0.001

0.002

0.003

0.004

0.005

According to Beer-Lambert Law,

Absorbance, A= λbc

A= λc (since b=1cm)

λ = A/c that is gradient of standard concentration curve

From graph 4, molar absorbtivity coefficient of methyl orange in 460nm is

λ =

= 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 absorbance 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

λ =

= 0.0069 L mg-1 cm-1

From graph 5, molar absorbtivity coefficient of methyl orange in 590nm is

λ =

= 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 nm)/

KMO at Amax MO

0.0820

By using 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 L mg-1 cm-1;

The molar absorbtivity coefficient of bromophenol blue in Amax of bromophenol blue (590nm) is 0.113 L 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 L mg-1 cm-1

The molar absorbtivity coefficient of bromophenol blue in Amax of methyl orange (460nm) is 0.0064 L 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 result, 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 absorption spectrum , graph of absorbance against corresponding wavelength plotted is a bell shape. From the graph, we can analyse that absorbance readings increases from 0.093 to 0.981 as the wavelength increases from 470nm to 590nm. The absorbance is then decrease when the wavelength applied was 620nm until 680 nm. The peak absorbance obtained from the graph of absorption spectrum plotted is in 590nm which is 0.981 absorbance. The results obtained shows that the maximum absorption of light bromophenol blue occur in wavelength of 590nm.

Part 2

Amax of bromophenol blue, which is at 590 nm is set in the spectrophotometer to determined the absorbance readings of different concentration of bromophenol blue e.g (0.0 , 2.0 , 4.0 , 6.0 , 8.0 , and 10.0) mg/L. A standard concentration curve of absorbance against concentration of bromophenol blue is plotted. A straight line is obtained in the graph. This indicated that the absorbance of bromophenol blue is directly proportional to the concentration of bromophenol blue. Therefore, the graph is said to obey the Beer-Lambert Law where A= λbc.

The line of the standard concentration curve of absorbance versus concentration of bromophenol blue pass through origin. However, there is some point that plotted far from the straight line.Thus, there are random errors might occur during the experiment as all points should be in the straight line. The graph shows that distilled water shows zero absorbance reading. The random error that might occur was that traces of chemical were present in the cuvette thus contaminating the distilled water which in turn giving the distilled water an absorbance reading. This random error can be reduced by rinsing the cuvette with distilled water before use.

Part 3

The absorbance of the two bromophenol blue solutions (Tube A & B) of unknown concentration at the Amax of bromophenol blue (590nm) were measured. The concentrations of the two unknown solutions were determined using two methods. The first method used was interpolating the graph 2 while the second method used was using the formula of Beer-Lambert Law to calculate 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 second method using the Beer-Lambert Law, A = λbc, 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 result for each unknown obtained using two different methods mentioned above give different result. The difference in results might due to random errors in the experiment. The random error that might occur was that the surface of the cuvette was not clear or due some fragment of mixture is not perfectly mixed in the solution. Fingerprints might be printed on the clear surface of the cuvette thus affecting the supposed light amount pass through.This will affect the absorbance reading of bromophenol blue. This random error can be reduced by ensuring that the surface of the cuvette was wiped with paper towel before placing it into the spectrophotometer. Furthermore, the orientation of the cuvette may be inserted wrongly into the spectrophotometer when the absorbance reading is taken.

Part 4

The peak absorbance of methyl orange is at 460nm. The absorbance of methyl orange increase with the concentration of methyl orange in the mixtures. The absorbance initiate from 0.000 til 0.814. A graph of absorbance against concentration of methyl orange . A straight line passing through origin is obtained. This showed that the concentration of methyl orange increased directly proportional to the absorbance of light with certain wavelength. Hence , this part of experiment obeyed the Law of Beer- Lambert.

Somehow, not all point is inside the straight line. So ,there is some random error occur when carrying out this experiment. The random error includes , the mixture is not perfectly mixed .Furthermore , there might be fingerprint left on the wall of cuvette.This affect the amount of light passing through the solution.There might be caused by poor pippeting technique that caused inaccuracy in absorbance reading of methyl orange. The choices of choosing wavelength will affect the sensitivity and accuracy of the analysis. Thus affecting the absorbance of solution or Beer- Lambert Law is not obeyed.

The molar absorbtivity coefficient calculated is 0.082 L mg-1cm-1 .

Part 5

The experiment shows increase in absorbance when concentration bromophenol blue increased .Bromophenol blue solutions are tested using Amax of methyl orange- 460nm of light. The absorbance increase from 0.000 to 0.079 of absorbance.A graph of absorbance of light against concentrations of bromophenol blue is plotted.A straight line passing through the origin is plotted. This showed that absorbance increase directly proportional to the concentrations of bromophenol blue. The graph shows scattered point around the straight line. This indicated random errors occur in the experiment. Some examples of random error are poor pippeting technique that may cause inaccuracy in measuring volune of bromophenol blue.

Another part of the experiment in part 5 is to vary the concentration of methyl orange and test it using Amax of bromophenol blue -590 nm of light . The test shows that there is increase of amount of absorbance as the concentration increased. A graph of absorbance against concentration of methyl orange is plotted. A straight line passing through origin is obtained. This meant that absorbance increase proportional to concentration of methyl orange. This shows that it obey the law of Beer-Lambert . All points are in the graph. Thus , there is minimum error occurred when the experiment is carried out.

The concentration of bromophenol blue and methyl orange in the two mixture solutions of tube C was determined using the formula, Atotal = K1C1+K2C2. By solving simultaneous 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 .

Conclusion

Part 1, the Amax of bromophenol blue is at the wavelength of 590nm.

Part 2, the absorbance of light is directly proportional to the concentration of bromophenol blue as the standard concentration curve of absorbance versus concentration of bromophenol shows a straight line passing through the origin. Molar absorbtivity coefficient of bromophenol blue in 590nm is 0.113 L mg-1cm-1.

Part 3, two methods were used to determined the concentration of the two unknowns (tube A & B). By using interpolation 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, using the formula 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 absorbance of light is directly proportional to the concentration of methyl orange as the standard concentration curve of absorbance against concentration of methyl orange shows a straight line passing through the origin. Molar absorbtivity coefficient of methyl orange in 460nm is 0.082 L 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 L 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.