Spectrophotometric Assay Of Two Vitamins Biology Essay

Many molecules absorb specific sums of energy to advance negatrons in their bonds from its land energy province to an aroused electronic province. In our milieus, energy can be obtained through visible radiation. Some light energy is absorbed by the molecule as light energy is used to switch negatrons from a lower energy province to a higher energy province. Every molecule has different bonds which require a different energy degree of visible radiation, and hence absorb different wavelengths of visible radiation, as a higher wavelength of visible radiation would intend a lower energy value. As the publicity of negatrons in a sigma bond to its aroused province requires a much higher sum of energy as compared to advancing negatrons in a pi bond, molecules with multiple bonds ( which have a pi bond ) would absorb visible radiation of a higher wavelength. In the UV part, soaking up merely 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 vitamin B2 has more conjugated pi systems, and therefore it is expected that it would hold a higher optical density value for its specific wavelength of visible radiation as compared to ascorbic acid.

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For the vitamin B2 sample, NaOH is used to fade out the sample but the glacial acetic acid must be added to neutralize the base used before the base is broken down. To do the mention for ascorbic acid, HCl is used to do the pH similar with the ascorbic acerb solution.

As seen in the British Pharmacopoeia 2010, we can see that the soaking up upper limit of vitamin B2 are at the wavelength of 223nm, 267nm, 373nm and 444nm ; the soaking up upper limit for ascorbic acid is at the wavelength of 243nm. Using the Beer Lambert jurisprudence, A=?bc, the optical density values of the samples can be calculated and compared with its several theoretical optical density in the British Pharmacopoeia, which in bend can be used to find its pureness.

Method:

Vitamin b2: The experiment is done as per agenda. The mass of vitamin B2 used is 0.1458 g.

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

To mensurate the optical density, one cuvette is filled with the mention solution while another cuvette is filled with the mensural solution, and both cuvettes are placed into the spectrophotometer at the same clip. When the cuvette is filled with a solution, it is rinsed with the said solution several times before make fulling it about three quarters full, and its outer surfaces are cleaned with soft tissues before seting it into the spectrophotometer.

Consequences

Riboflavine

Concentration of riboflavine ( M )

Extremum

Optical density

?max ( nanometer )

?max ( 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.

Extremum

Mean of ?max

SD of ?max

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 divergence of the molar soaking up coefficient ( ?max ) for each extremum over the four sample concentrations.

Absorbance ratio:

Concetration ( M )

Mean

0.3245155

0.378255

Standard divergence

0.000247

0.002031

Table 3 – Optical density ratios for each concentration.

Harmonizing to the British Pharmacopoeia, the optical density ratios:

– A373/A267 = 0.31 to 0.33 ;

– A444/A267 = 0.36 to 0.39.

Since all the optical density ratios calculated for each of the solution fell within the scope of optical density ratios from British Pharmacopoeia, hence they comply with the Pharmacopoeial demands.

Percentage of riboflavine in the sample at 444nm

No of mole of vitamin B2

= 0.1458

376

= 3.878 ten 10-4 mol

Concentration of riboflavine used

= 3.878 ten 10-4 ten 1000

1000

= 3.878 ten 10-4 M

A = ?bc

? = mean of molar soaking up coefficients at 444nm

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

A = 183.93

A ( 1 % ,1cm )

183.93 = 323 ten 1 ten degree Celsius

degree Celsiuss = 0.569

Therefore, 0.569g of riboflavine in 100ml of sample

Percentage of vitamin B2 in sample

= 0.569 % w/w

Ascorbic acid

No. of mole of ascorbic acid sample

= 0.1200

176

= 6.81818 ten 10-4 mol

Concentration of ascorbic acid used

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

= 6.8182 ten 10-3

Concentration of ascorbic acid ( M )

Optical density

?max ( nanometer )

?max ( 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 nanometers

A = ?bc

A = 10121.7 ten 1 ten ( 6.8182×10-3 )

A = 69.01

A ( 1 % ,1cm )

69.01 = 565 ten 1 ten degree Celsius

degree Celsiuss = 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 optical density ratios of vitamin B2 with values in British Pharmacopeia, we can state here that all the experimental values of optical density ratio lies within the scope stated. As for the variableness of the informations, the ratios is really little since the standard divergence calculated is really little which is 0.000247 for A ( 375nm ) /A ( 267nm ) and 0.002031 for A ( 444nm ) /A ( 267nm ) .

Ascorbic acid

Riboflavine

The diagrams above show constructions of riboflavine and ascorbic acid. Based on the construction, 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 manus, olefine group C=C and carbonyl group are lending chromopheres of ascorbic acid. Riboflavin has greater dual bonds than ascorbic acid. Furthermore, conjugated dual bonds present in riboflavin constructions while it is absent in ascorbic acid because its dual bonds are stray entities which do non interact with each other. Double bonds contain both ?-bond and ?- bond. In riboflavine, P orbitals are able to overlap as junction could convey together the higher and lower unoccupied molecular energy.Therefore, riboflavine requires lower energy in order to excite the negatrons when compared with ascorbic acid. The maximal wavelength absorbed by vitamin B2 is 446.19 while for ascorbic acid is 243.97. This is because the lower the energy the longer the wavelength of the EM moving ridge.

Riboflavine chromophores are different in acid, alkalic solution or impersonal solution, the uncharged molecules. Riboflavine decomposed easy in alkalic solution. The acid/base index Hln undergoes the undermentioned reaction in dilute aqueous solution:

Hln H+ + ln-

Colour 1 Colour 2

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

Table below shows the measuring of optical density at different wavelength and index. Concentration used for each solution = 5.00 ten 10-4 M

Wavelength ( A )

Optical density for

0.1 M NaOH ( degree Celsius )

Optical density for

0.1 M HCL ( degree Celsius )

A485

0.052

0.454

A625

0.823

0.176

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

? = A

bc

Wavelength

Molar soaking up for ln-

Molar soaking up for Hln

?485

0.52

4.54

?625

8.23

1.76

b. cipher the dissociation invariable for index if a pH 5.00 buffer incorporating a little sum of the index exhibits an optical density of 0.472 at 485nm and 0.351 at 625nm.

three ) pH = pKa + log [ base ]

[ acid ]

5 = pKa + log [ 0.0426 ]

[ 0.104 ]

pKa = 5.39

Ka = 4.0738 ten 10-6A = ?cl

I ) A485 = ?485 x c x cubic decimeter

0.472 = 4.54 ten degree Celsiuss x 1

[ acid ] = 0.104 Meter

two ) A625 = ?625 x c x cubic decimeter

0.351 = 8.23 x c 1

[ base ] = 0.0426 Meter

c. What is the pH of solution incorporating a little sum of the index that exhibits an optical density of 0.530 at 485nm and 0.216 at 635nm.

three ) pH = pKa + log [ base ]

[ acid ]

pH = 5.39 + log [ 0.02625 ]

[ 0.1167 ]

pH = 4.742

A = ?cl

I ) A485 = ?485 x c x cubic decimeter

0.530 = 4.54 ten degree Celsiuss x 1

[ acid ] = 0.1167 Meter

two ) A625 = ?625 x c x cubic decimeter

0.216 = 8.23 x c 1

[ base ] = 0.02625 Meter

Decision

The riboflavine solution complies with Pharmacopeial demands as the optical density ratios are between the scopes given in the BP. The concentration of the solution is 0.569 % w/w.

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