Spectrophotometer And Its Function

In fact, light of narrow wavelength ranges can be transmitted through a sample solution by utilizing an optical instrument, spectrophotometer. Normally, it is used in biological and chemical sciences to obtain the measurement of the amount of light absorbed by the solutes in the solution. Hence, concentrations of compounds in solution can be determined by using spectrophotometer because the amount of light absorbed is directly proportional to the concentration of absorbing compounds in the solution. Besides that, spectrophotometer functions as a tool to distinguish compounds because different compounds absorb light at different wavelengths.

There are a few methods which can be used to measure the amount of light absorbed by different solute in a sample solution.

(I)

Transmittance (T) refers to the amount of light transmitted through a sample solution. Its formula is

T = I/I0

%T = 100 x T

I represents the incident light whereas I0 represents the energy of light transmitted through the solution.

Absorbance (A), which is called optical density formerly [2], represents the amount of light absorbed by a solution which is related logarithmically to transmission by the

A = 0 / I

At the end, A = 2 –

(II)

According to Beer Lambert Law,

Absorbance, A = lbc

l, which is called extinction coefficient formerly [2], represents the molar absobtivity coefficient in L mol-1cm-1, a constant for a particular compound, different compound has different molar absorbtivity.

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

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

This shows that there is a relationship between molar absobtivity, path length, molar concentration in Beer Lambert Law [1].

Furthermore, concentration of a solution can be calculated by using Beer-Lambert Law if its molar absobtivity coefficient is known. The equation that has been rearranged can be used to calculate the molar absobtivity. The equation is

l = A/bc

Besides that, concentration of different compounds in a mixture can be determined by using Beer-Lambert Law. If there are presence of two compounds in a solution, the total absorbance at any specified wavelength of the solution is the sum of the two separate contributions as the formula shown below

Atotal = K1C1 + K2C2

C1 and C2 represent the concentrations of each compound in the mixture

K1 and K2 represent the molar absobtivity coefficient of each compound of the respective wavelength.

1.2 Aims

To determine the wavelength of maximum absorption, Amax, of bromophenol blue.

To construct a standard concentration curve for bromophenol blue.

To determine the concentration of the unknown bromophenol blue solutions.

To determine the concentration of two different solutes, bromophenol blue and methyl orange, in a mixture.

In brief, through this experiment, the things that I have learnt are:

the use of spectrophotometer

the use of micropipettes

absorption spectra

absorbance and concentration

absorbance and concentration of different compounds in a mixture

2.0 Materials and Methods

Materials

Bromophenol blue, mixture of bromophenol blue and methyl orange solutions, micropipette, cuvette, test tubes, tips.

Experimental Procedures

The experiments were carried out according the procedures described in the practical manual.

Part 1: Determination of Amax of bromophenol blue

The distilled water was filled into a cuvette (used as blank) and the clear surface of the cuvette was cleaned by using paper towel.

The wavelength of the spectrophotometer was set to 470nm.

The cuvette with distilled water was placed into the spectrophotometer and was set to read zero.

The blank was removed and replaced with another cuvette containing the bromophenol blue.

The absorbance reading was recorded in Table 1.1.

Steps 2 to 5 were repeated with wavelengths 500, 530, 560, 590, 620, 650, and 680nm.

The reading at each wavelength was re-zeroed by using the blank.

The readings obtained were used to plot a graph of absorption spectrum (absorbance readings versus corresponding wavelength).

The wavelength with maximum absorbance reading was determined from the graph.

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

The mixtures of tubes 1-6 were prepared according to the Table 1.2a.

The contents of each tube were mixed by using a vortex mixer.

The spectrophotometer was set to the Amax wavelength for bromophenol blue that had been determined in part 1.

The absorbance reading was zeroed by using the content in tube 1.

The absorbance readings of tubes 2-6 were measured by using the same cuvette used for Tube 1.

The absorbance readings were recorded in Table 1.2a.

The samples were not discarded after taking readings, but were returned to the original test tubes.

The concentrations of the bromophenol blue solutions in tubes 1-6 were calculated. The calculations were recorded in Table 1.2a.

A standard concentration curve of absorbance versus concentration of bromophenol blue was plotted.

The molar absorbtivity coefficient of bromophenol blue at Amax of bromophenol blue was determined (in unit L mg-1cm-1, as described in Beer Lambert’s Law ) by using the standard concentration curve from step 9.

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

The absorbance of the two bromophenol bue solutions (Tubes A & B) of unknown concentration were measured at the Amax of bromophenol blue.

The distilled water was used as blank to zero the absorbance reading before the absorbance of the unknown solutions was measured.

The results were recorded in Table 1.2a.

The concentration of the two unknowns was determined by using (1) the standard concentration curve from Part 2 and (2) using the formula of Beer-Lambert Law. The calculations were shown in the report. The results obtained from these two methods were compared.

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

The mixtures of tubes 1-6 were prepared according to Table 1.2b.

The contents of each tube were mixed by using a vortex mixer.

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

The absorbance reading was zeroed by using the content in tube 1.

The absorbance readings of tubes 2-6 were measured by using the same cuvette that used for tube 1.

The absorbance readings were recorded in Table 1.2b.

The samples were not discarded after taking readings, but were returned to the original test tubes.

The concentrations of the methyl orange solutions in tubes 1-6 were calculated. The calculations were recorded in Table 1.2a.

A standard concentration curve of absorbance versus concentration of methyl orange was plotted.

The molar absorbtivity coefficient of methyl orange at 460nm (in unit L mg-1cm-1, as described in Beer Lambert’s Law) was determined by using the standard concentration curve from step 9.

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

Beer-Lambert Law was used in this part.

Atotal = K1 C1 + K2 C2

There are two variables, C1 (concentration of bromophenol blue, BB, i.e. CBB in the mixture) and C2 (concentration of methyl orange, MO, i.e. CMO in the mixture). Hence, two equations are needed to be generated and solved simultaneously. The equations are as below:

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

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. 460 nm

KBB at Amax BB is the molar absorbtivity coefficient of BB at Amax of BB

KMO at Amax BB is the molar absorbtivity coefficient of MO at Amax of BB

KBB at Amax MO is the molar absorbtivity coefficient of BB at Amax of MO

KMO at Amax MO is the molar absorbtivity coefficient of MO at Amax of MO (460 nm)

The Amax of methyl orange is 460nm. The absorbance of the bromophenol blue solutions in Tubes 1-6 in Table 1.2a at the Amax of methyl orange was measured.

The results were recorded in Table 1.2a.

A standard concentration curve of bromophenol blue at Amax of methyl orange was plotted.

The molar absorbtivity coefficient of bromophenol blue at 460nm was determined.

The absorbance of the methyl orange solutions in Tubes 1-6 in Table 1.2b at the Amax of bromophenol blue was measured.

The results were recorded in Table 1.2b

A standard concentration curve of methyl orange at Amax of bromophenol blue was plotted.

The molar absorbtivity coefficient of methyl orange at the Amax of bromophenol blue was determined.

The absorbance of mixture C containing bromophenol blue and methyl orange (Tube C) was measured at both the wavelengths Amax of bromophenol blue and Amax of methyl orange (given as 460nm).

The readings were recorded in Table 1.3.

Table 1.4 was filled in.

The values obtained were substituted into equations 1 and 2. The equations were solved simultaneously to obtain the concentrations of methyl orange and bromophenol blue in mixture C.

Table 1.2b: Absorbance of different concentration of methyl orange at 590nm and 460 nm

Tube

C

Absorption of mixture C at Amaxof bromophenol blue (BB),Ac at Amax BB

0.202

Absorption of mixture C at Amaxof methyl orange (MO), Acat Amax MO (i.e. 460nm)

0.241

Table 1.3: Absorbance of Mixture C at Amax

Molar absorbtivity coefficient of BB at Amax of bromophenol blue (BB) i.e. KBB at Amax BB

3.7 0x 10-2L mg-1cm-1

Molar absorbtivity coefficient of MO at Amax of bromophenol i.e. KMO at Amax BB

2.80 x 10-4L mg-1cm-1

Molar absorbtivity coefficient of BB at Amax of methyl orange (MO)i.e. KBB at Amax MO

1.30 x 10-3L mg-1cm-1

Molar absorbtivity coefficient of MO at Amax of methyl orange (MO) (460nm) i.e. KMO at Amax MO

7.56 x 10-2L mg-1cm-1

Table 1.4: Molar absobtivity coefficient of bromophenol blue and methyl orange at Amax of bromophenol blue and methyl orange

3.2 Figures

Figure 1

4.6 mg/ L

y1-y2: 0.37 -0 = 0.37

x1-x2:

10-0 = 10

Figure 2

y1-y2:

0.013 – 0 = 0.013

x1-x2:

10-0=10

Figure 3

x1-x2:

10-0 = 10

y1-y2:

0.756-0 = 0.756

Figure 4

Figure 5

3.3 Calculations

Wavelength with Amax of bromophenol blue: 590nm

Calculations of concentrations [M1V1 = M2V2]

Tube 1

10 ( 0 ) = M2 (2.5)

M2 = 10 ( 0 ) / 2.5

= 0 mg / L

Tube 2

10 ( 0.5 ) = M2 (2.5)

M2 = 10 ( 0.5 ) / 2.5

= 2 mg / L

Tube 3

10 ( 1.0 ) = M2 (2.5)

M2 = 10 ( 1.0 ) / 2.5

= 4 mg / L

Tube 4

10 ( 1.5 ) = M2 (2.5)

M2 = 10 ( 1.5 ) / 2.5

= 6 mg / L

Tube 5

10 ( 2 ) = M2 (2.5)

M2 = 10 ( 2 ) / 2.5

= 8 mg / L

Tube 6

10 ( 2.5 ) = M2 (2.5)

M2 = 10 ( 2.5 ) / 2.5

= 10 mg / L

Calculation of molar absobtivity coefficient,l of

bromophenol blue at Amax of bromophenol blue (590nm):

(0.37-0)/ (10 – 0) = 3.70 x 10-2 L mg-1 cm-1

bromophenol blue at Amax of methyl orange (460nm):

(0.015-0)/ (10-2.4) = 1.30 x 10-3 L mg-1 cm-1

methyl orange at Amax of methyl orange (460nm):

(0.756 – 0)/ (10-0) = 7.56 x 10-2 L mg-1 cm-1

methyl orange at Amax of bromophenol blue (590nm):

(0.0025-0.0008)/ (10-0) = 2.80 x 10-4 L mg-1 cm-1

Calculation of the concentrations of the bromophenol blue solutions of unknown concentration (Tube A and Tube B)

Using the standard concentration curve from part 2 (Figure 2)

Concentration of solution A = 6.3 mg/L

Concentration of solution B = 4.6 mg/L

Using the formula of Beer-Lambert Law

A = lbc c= A/lb

Concentration of solution A =

c = 0.234/3.70 x 10-2 (1)

= 6.32 mg/ L

Concentration of solution B =

c = 0.169/3.70 x 10-2 (1)

= 4.57 mg/L

Calculation of the concentrations of two different solutes, bromophenol blue and methyl orange, in mixture C

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

0.202 = (3.70 x 10-2) CBB + (2.80 x 10-4) CMO…………. 1

0.241 = (1.30 x 10-3) CBB + (7.56 x 10-2) CMO………….2

From equation 1, CBB = (0.202 – 2.80 x 10-4CMO) / 3.70 x 10-2

Substitute CBB = (0.202 – 2.80 x 10-4CMO) / 3.70 x 10-2 into equation 2:

0.241=(1.30 x 10-3)[ (0.202 – 2.80 x 10-4CMO) / 3.70 x 10-2]+ (7.56 x 10-2)CMO………….3

Multiply equation 3 with 3.70 x 10-2:

8.917 x 10-3 = (1.30 x 10-3)[ (0.202 – 2.80 x 10-4CMO) + 2.7972 x 10-3 CMO

8.917 x 10-3 = 2.626 x 10-4 – 3.64 x 10-7 CMO + 2.7972 x 10-3 CMO

Then,

8.917 x 10-3 – 2.626 x 10-4 = – 3.64 x 10-7 CMO + 2.7972 x 10-3 CMO

CMO =[ 8.917 x 10-3 – 2.626 x 10-4]/ – 3.64 x 10-7 + 2.7972 x 10-3]

= 3.0944 mg/ L

= 3.09 mg/ L

Substitute CMO = 3.0944 mg/ L into equation 2

0.241 = (1.30 x 10-3) CBB + (7.56 x 10-2) (3.0944)

CBB = [0.241 – (7.56 x 10-2) (3.0944)] / (1.30 x 10-3)

=5.4334 mg/ L

=5.43 mg/ L

4.0 Discussion

Determination of the wavelength with maximum absorbance of bromophenol blue

From the bell-shaped graph in Figure 2, the wavelength with maximum absorbance of bromophenol blue is 590nm. This value is taken from the peak of the graph. It means that bromophenol blue absorbs maximum light intensity at 590nm.

Calculations of concentrations

The concentrations of bromophenol blue and methyl orange are calculated by using the formula M1V1 = M2V2. M represents the concentration of the solution. V represents the volume of the solution.

(iii) Calculation of molar absobtivity coefficient,l

By using the concentrations of bromophenol blue and methyl orange, standard concentration curve of absorbance versus concentration of bromophenol blue and methyl orange can be plotted. Those graphs are drawn for the wavelength of 460nm and 590nm respectively. As a result, there are four graphs (Figure 2 to Figure 4) which have been plotted. Those graphs show that absorbance is directly proportional to the concentration of the solution. The molar absobtivity coefficient is the gradient of the graph because

A= lbc

Since b = 1,

A = lc

Y = mX

l = m (gradient).

Since some of the points in Figure 2 to Figure 5 cannot pass through the straight line in the graph, a best fit line has been drawn by using Microsoft Office. There are a few reasons that result some of the points in the graph cannot pass through the best fit line which are:

The surface of cuvette is not wiped cleanly.

The solution of bromophenol blue or methyl orange is not mixed well with the distilled water.

Calculation of the concentrations of the bromophenol blue solutions of unknown concentration (Tube A and Tube B)

There are two methods to determine the concentrations of the bromophenol blue solutions in Tube A and Tube B which are by using the standard concentration curve from Figure 2 and by using the Beer-Lambert law.

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The results from these two methods are close to each other. However, since the results from using calculation of Beer-Lambert law ( A-6.32 mg/L, B-4.57 mg/L) contain two decimal places whereas the results from the standard concentration curve (A-6.3 mg/ L, B-4.6 mg/L) contain only one decimal place, the results from using calculation of Beer-Lambert law are more precise than the results from the standard concentration curve.

Calculation of the concentrations of two different solutes, bromophenol blue and methyl orange, in mixture C

For this part of calculation, two equations formed from Beer-Lambert Law can help to get the concentrations of bromophenol blue and methyl orange in mixture C. The equations are:

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

Concentrations of bromophenol blue and methyl orange in mixture C can be obtained by substituting the results obtained into the equations and solving them simultaneously.

5.0 Conclusion

In conclusion, molar absobtivity coefficient,l of bromophenol blue at maximum absorbance of bromophenol blue, bromophenol blue at maximum absorbance of methyl orange, methyl orange at maximum absorbance of methyl orange and methyl orange at maximum absorbance of bromophenol blue are 3.7 x 10-2 L mg-1 cm-1 ,1.3 x 10-3 L mg-1 cm-1, 7.56 x 10-2 L mg-1 cm-1 and 2.8 x 10-4 L mg-1 cm-1 respectively. Moreover, concentrations of bromophenol blue and methyl orange in mixture C are 5.43 mg/ L and 3.09 mg/ L respectively.