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 acidThe 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.

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.

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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.ConsequencesRiboflavineConcentration of riboflavine ( M )ExtremumOptical density?max ( nanometer )?max ( M-1 cm-1 )1st11.248223.431161521.8662nd12.025267.211241758.







005373.52413188.06584th18.511445.31477883.4294Table 1 – Data of riboflavine obtained from UV spectrophotometer.

ExtremumMean of ?maxSD of ?max1st1187830.71121656.705422nd1253930.90513801.232893rd406919.03524427.7351524th474285.08322725.

587427Table 2 – Mean and standard divergence of the molar soaking up coefficient ( ?max ) for each extremum over the four sample concentrations.Absorbance ratio:Concetration ( M )Mean0.32451550.378255Standard divergence0.0002470.002031Table 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 444nmNo of mole of vitamin B2= 0.1458376= 3.

878 ten 10-4 molConcentration of riboflavine used= 3.878 ten 10-4 ten 10001000= 3.878 ten 10-4 MA = ?bc? = mean of molar soaking up coefficients at 444nmA = 474285.08 ten 1 x ( 3.878 x 10-4 )A = 183.93A ( 1 % ,1cm )183.93 = 323 ten 1 ten degree Celsiusdegree Celsiuss = 0.

569Therefore, 0.569g of riboflavine in 100ml of samplePercentage of vitamin B2 in sample= 0.569 % w/wAscorbic acidNo. of mole of ascorbic acid sample= 0.1200176= 6.81818 ten 10-4 molConcentration of ascorbic acid used= 6.8182 ten 10-4 / ( 100/1000 )= 6.8182 ten 10-3Concentration of ascorbic acid ( M )Optical density?max ( nanometer )?max ( M-1 cm-1 )6.

8182 x 10-60.069012243.9710121.

7Table 4 – Data of ascorbic acid obtained from UV spectrophotometer.Percentage of ascorbic acid in the sample at 243 nanometersA = ?bcA = 10121.7 ten 1 ten ( 6.8182×10-3 )A = 69.01A ( 1 % ,1cm )69.01 = 565 ten 1 ten degree Celsiusdegree Celsiuss = 0.

122Therefore, 0.122g of ascorbic acid contain in 100ml of sample.Percentage of ascorbic acid in sample= 0.

122 % w/wDiscussionAfter 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 acidRiboflavineThe 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 2In 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 MWavelength ( A )Optical density for0.1 M NaOH ( degree Celsius )Optical density for0.

1 M HCL ( degree Celsius )A4850.0520.454A6250.8230.176a. Calculate molar soaking up coefficient for ln- and Hln at 485nm and 625nm.

? = AbcWavelengthMolar soaking up for ln-Molar soaking up for Hln?4850.524.54?6258.231.76b. 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 = 5.39Ka = 4.

0738 ten 10-6A = ?clI ) A485 = ?485 x c x cubic decimeter0.472 = 4.54 ten degree Celsiuss x 1[ acid ] = 0.104 Metertwo ) A625 = ?625 x c x cubic decimeter0.351 = 8.23 x c 1[ base ] = 0.0426 Meterc.

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 = 4.742A = ?clI ) A485 = ?485 x c x cubic decimeter0.

530 = 4.54 ten degree Celsiuss x 1[ acid ] = 0.1167 Metertwo ) A625 = ?625 x c x cubic decimeter0.

216 = 8.23 x c 1[ base ] = 0.02625 MeterDecisionThe 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.


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