Spectrophotometric Assay Of Two Vitamins Biology Essay

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Many molecules absorb specific amounts of energy to promote electrons in their bonds from its ground energy state to an excited electronic state. In our surroundings, energy can be obtained through light. Some light energy is absorbed by the molecule as light energy is used to shift electrons from a lower energy state to a higher energy state. Every molecule has different bonds which require a different energy level of light, and therefore absorb different wavelengths of light, as a higher wavelength of light would mean a lower energy value. As the promotion of electrons in a sigma bond to its excited state requires a much higher amount of energy as compared to promoting electrons in a pi bond, molecules with multiple bonds (which have a pi bond) would absorb light of a higher wavelength. In the UV region, absorption only occurs with the presence of pi-electron systems.

Riboflavin Ascorbic acid

The above molecules are the two vitamins investigated in this experiment. By observations, it can be seen that riboflavin has more conjugated pi systems, and therefore it is expected that it would have a higher absorbance value for its specific wavelength of light as compared to ascorbic acid.

For the riboflavin sample, NaOH is used to dissolve the sample but the glacial acetic acid must be added to neutralise the alkali used before the alkali is broken down. To make the reference for ascorbic acid, HCl is used to make the pH similar with the ascorbic acid solution.

As seen in the British Pharmacopoeia 2010, we can see that the absorption maxima of riboflavin are at the wavelength of 223nm, 267nm, 373nm and 444nm; the absorption maximum for ascorbic acid is at the wavelength of 243nm. Using the Beer Lambert law, A=εbc, the absorbance values of the samples can be calculated and compared with its respective theoretical absorbance in the British Pharmacopoeia, which in turn can be used to determine its purity.

Method:

Riboflavin: The experiment is done as per schedule. The mass of riboflavin used is 0.1458 g.

Ascorbic acid: The experiment is done as per schedule. The mass of ascorbic acid used is 0.1200g.

To measure the absorbance, one cuvette is filled with the reference solution while another cuvette is filled with the measured solution, and both cuvettes are placed into the spectrophotometer at the same time. When the cuvette is filled with a solution, it is rinsed with the said solution several times before filling it about three quarters full, and its outer surfaces are cleaned with soft tissues before putting it into the spectrophotometer.

Results

Riboflavine

Concentration of riboflavine (M)

Peak

Absorbance

λmax (nm)

εmax (M-1 cm-1)

1st

11.248

223.43

1161521.866

2nd

12.025

267.21

1241758.573

3rd

3.9062

374.54

403372.7517

4th

4.5696

445.53

471878.5844

1st

22.913

223.45

1183052.565

2nd

24.149

267.09

1246870.178

3rd

7.8301

373.42

404286.6447

4th

9.1512

446.19

472498.1728

1st

34.663

223.44

1193155.299

2nd

36.426

267.03

1253840.549

3rd

11.819

373.50

406828.6786

4th

13.796

445.11

474880.1463

1st

47.009

223.40

1213593.114

2nd

49.320

267.20

1273254.321

3rd

16.005

373.52

413188.0658

4th

18.511

445.31

477883.4294

Table 1 - Data of riboflavine obtained from UV spectrophotometer.

Peak

Mean of εmax

SD of εmax

1st

1187830.711

21656.70542

2nd

1253930.905

13801.23289

3rd

406919.0352

4427.735152

4th

474285.0832

2725.587427

Table 2 - Mean and standard deviation of the molar absorption coefficient (εmax) for each peak over the four sample concentrations.

Absorbance ratio:

Concetration (M)

Mean

0.3245155

0.378255

Standard deviation

0.000247

0.002031

Table 3 - Absorbance ratios for each concentration.

According to the British Pharmacopoeia, the absorbance ratios:

- A373/A267  =  0.31 to 0.33;

- A444/A267  =  0.36 to 0.39.

Since all the absorbance ratios calculated for each of the solution fell within the range of absorbance ratios from British Pharmacopoeia, therefore they comply with the Pharmacopoeial requirements.

Percentage of riboflavine in the sample at 444nm

No of mole of riboflavin

= 0.1458

376

= 3.878 x 10-4 mol

Concentration of riboflavine used

= 3.878 x 10-4 x 1000

1000

= 3.878 x 10-4 M

A = εbc

ε = mean of molar absorption coefficients at 444nm

A = 474285.08 x 1 x (3.878 x 10-4)

A = 183.93

A(1%,1cm)

183.93 = 323 x 1 x c

c = 0.569

Therefore, 0.569g of riboflavine in 100ml of sample

Percentage of riboflavin in sample

= 0.569% w/w

Ascorbic acid

No. of mole of ascorbic acid sample

= 0.1200

176

= 6.81818 x 10-4 mol

Concentration of ascorbic acid used

= 6.8182 x 10-4 / (100/1000)

= 6.8182 x 10-3

Concentration of ascorbic acid (M)

Absorbance

λmax (nm)

εmax (M-1 cm-1)

6.8182 x 10-6

0.069012

243.97

10121.7

Table 4 - Data of ascorbic acid obtained from UV spectrophotometer.

Percentage of ascorbic acid in the sample at 243 nm

A = εbc

A = 10121.7 x 1 x (6.8182x10-3)

A = 69.01

A(1%,1cm)

69.01 = 565 x 1 x c

c = 0.122

Therefore, 0.122g of ascorbic acid contain in 100ml of sample.

Percentage of ascorbic acid in sample

= 0.122% w/w

Discussion

After comparing the values obtained for the absorbance ratios of riboflavin with values in British Pharmacopeia, we can say here that all the experimental values of absorbance ratio lies within the range stated. As for the variability of the data, the ratios is very small since the standard deviation calculated is very small which is 0.000247 for A(375nm)/A(267nm) and 0.002031 for A(444nm)/A(267nm).

Ascorbic acid

Riboflavine

The diagrams above show structures of riboflavine and ascorbic acid. Based on the structure, we can see that there is two chromophores contribute to riboflavin which is benzene N=C and carbonyl group C=O. While on the other hand, alkene group C=C and carbonyl group are contributing chromopheres of ascorbic acid. Riboflavin has greater double bonds than ascorbic acid. Moreover, conjugated double bonds present in riboflavin structures while it is absent in ascorbic acid because its double bonds are isolated entities which do not interact with each other. Double bonds contain both σ-bond and π- bond. In riboflavine, p orbitals are able to overlap as conjugation could bring together the higher and lower unoccupied molecular energy.Therefore, riboflavine requires lower energy in order to excite the electrons when compared with ascorbic acid. The maximum wavelength absorbed by riboflavin is 446.19 while for ascorbic acid is 243.97. This is because the lower the energy the longer the wavelength of the EM wave.

Riboflavine chromophores are different in acid, alkaline solution or neutral solution, the uncharged molecules. Riboflavine decomposed slowly in alkaline solution. The acid/base indicator Hln undergoes the following reaction in dilute aqueous solution:

Hln H+ + ln-

Colour 1 Colour 2

In NaOH, indicator present as ln- while in HCL it present as Hln.

Table below shows the measurement of absorbance at different wavelength and indicator. Concentration used for each solution = 5.00 x 10-4 M

Wavelength (A)

Absorbance for

0.1 M NaOH (c)

Absorbance for

0.1 M HCL (c)

A485

0.052

0.454

A625

0.823

0.176

a. Calculate molar absorption coefficient for ln- and Hln at 485nm and 625nm.

ε = A

bc

Wavelength

Molar absorption for ln-

Molar absorption for Hln

ε485

0.52

4.54

ε625

8.23

1.76

b. calculate the dissociation constant for indicator if a pH 5.00 buffer containing a small amount of the indicator exhibits an absorbance of 0.472 at 485nm and 0.351 at 625nm.

iii) pH = pKa + log [base]

[acid]

5 = pKa + log [0.0426]

[0.104]

pKa = 5.39

Ka = 4.0738 x 10-6A = εcl

i) A485 = ε485 x c x l

0.472 = 4.54 x c x 1

[ acid ] = 0.104 M

ii) A625 = ε625 x c x l

0.351 = 8.23 x c 1

[ base ] = 0.0426 M

c. What is the pH of solution containing a small amount of the indicator that exhibits an absorbance of 0.530 at 485nm and 0.216 at 635nm.

iii) pH = pKa + log [base]

[acid]

pH = 5.39 + log [0.02625]

[0.1167]

pH = 4.742

A = εcl

i) A485 = ε485 x c x l

0.530 = 4.54 x c x 1

[ acid ] = 0.1167 M

ii) A625 = ε625 x c x l

0.216 = 8.23 x c 1

[ base ] = 0.02625 M

Conclusion

The riboflavine solution complies with Pharmacopeial requirements as the absorbance ratios are between the ranges given in the BP. The concentration of the solution is 0.569% w/w.

The concentration of ascorbic acid solution prepared was 0.122% w/w.

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