# Resolving Polychromatic Radiation Into Different Wavelengths Biology Essay

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To determine the optical density and molar extinction coefficient of both the permanganate and dichromate solutions.

To determine the composition of the dichromate/potassium solution mixture

Experiment B)

To determine the composition of the complex formed when mixing salicylic acid together with ferric ions through visible spectrophotometry.

Introduction (Experiment A and B)

A spectrophotometer or spectrometer is an instrument that resolves polychromatic radiation into different wavelengths. This consists of a source of continuous radiation over the wavelengths of interest, a monochromator for selecting a narrow band of wavelengths from the source spectrum, a sample cell and a detector. The detector is able to convert the radiant energy into electrical energy. The sample present in the curvette absorbs light. The use of a spectrophotometer is of utmost importance since the detection of colour by a human eye is subjective and thus very inaccurate.

In experiment one below the spectra for the standard solutions of 0.0005M potassium permanganate and 0.0005 M potassium dichromate are obtained respectively using the spectrophotometer. These standard solutions were then used to prepare dilutions in which their spectra are obtained and a graph of optical density against concentration is plotted. The same is to be done for the mixtures of the permanganate and the dichromate solutions. The theoretical absorbance of the homologous mixtures produced s then calculated using the Beer-Lambert relationship:

## D= ECrC CCr + EMn CMn

The concentration of the unknown permanganate/dichromate mixture was determined by measures the optical density at the two wavelengths (between 300-400 nm and 500-600 nm) by using the simultaneous linear equation:

D1 = ECr 1C CCr + EMn 1CMn

## D2 = ECr 2C CCr + EMn 2 CMn

In the second experiment Salicylic is added to Ferric ammonium sulfate, in an acidic environment, to form a complex. By measuring the optical absorbance of this formed complex a rough value of the amount of complex formed could be determined. Also, one could also determine the ratio at which most complex forms. This procedure is advantageous since both initial reactants are colourless and thus does not interfere with the absorbance value of the coloured product

## Brand

Potassium permanganate

GPR

BDH

Potassium dichromate

Analar

BDH

## 2.2) Apparatus

Recording Spectrophotometer

Curvette

X 6 50 mL beakers

2 burettes

Funnel

## 2.3) Procedure A

Confirmation of Beer's Law and the Determination of Permanganate and Dichromate in a mixture of the two.

A recording spectrophotometer was used to obtain spectra for a 0.0005M KMnO4 solution and a 0.0005M K2Cr2O7 solution. Particular attention was paid to the 300-400nm and 500-600nm wavelength ranges and the maximum absorption wavelengths were noted.

From the spectra obtained in Step 1, an appropriate wavelength at which Beer's Law could be tested was selected.

Step 2 was repeated for the dichromate solution

Five permanganate solutions of 0.0004, 0.0003, 0.00025, 0.0001 and 0.00005 were prepared.

The optical density of these dilutions was calculated using a non- recording spectrophotometer.

Steps 4 and 5 were repeated for the dichromate solution.

Concentration of both the permanganate and dichromate solution against optical density was plotted for both wavelengths.

Linear regression for both graphs were calculated and noted.

E, extinction coefficient, was calculated by using the equation :

D= EMn CMn

This was done for both the permanganate and dichromate solution.

Five different mixtures of the 0.0005M, 0.0004, 0.0003, 0.00025 and 0.0001 of the original 0.00005M permanganate and 0.0005M dichromate solutions were prepared

The non- recording spectrophotometer was used to measure the absorbance of the mixtures prepared. A graph of optical density against concentration was then plotted

Linear regression analysis of the graph plotted was then used to show that the following equation hold true at both wavelengths

## D= ECrC CCr + EMn CMn

The composition of the unknown permanganate/dichromate mixture was then found by determining the optical density of at 525nm and 351 nm. The simultaneous equation below was then used:

D1 = ECr 1C CCr + EMn 1CMn

## 2.4) Precautions A

The dilutions were prepared using a burette for better accuracy

The curvette was cleaned before measuring the optical density of another sample. This is done to prevent any remaining solution of the previous dilution from affecting the absorbance.

The curvette was placed in such a way that the incident light passes through the clear sides of the curvette, not the opaque sides.

The value on the spectrophotometer was left to settle before taking the final reading due to slight variations.

## 2.5) Sources of error A

Some losses may have been present due to transfers

Contamination of the solution may affect the optical density and thus absorbance of the spectrophotometer.

## 525nm

0.00050 moles

10

0

1.185

0.00040

8

2

0.963

0.00030

6

4

0.767

0.00025

5

5

0.642

0.00010

2

8

0.274

0.00005

1

9

0.158

D = EMnCMn

D/C = Change in y/Change in x = 1374 nm

## Theoretical absorbance 351 nm

0.0005

x

1374

0.687

0.0004

x

1374

0.5496

0.0003

x

1374

0.4122

0.0002

x

1374

0.2748

0.0001

x

1374

0.1374

0.00005

x

1374

0.0687

D = EMnCMn

D/C = Change in y/Change in x = 2292.1 nm

0.0005

x

2292.1

1.14605

0.0004

x

2292.1

0.91684

0.0003

x

2292.1

0.68763

0.0002

x

2292.1

0.45842

0.0001

x

2292.1

0.22921

0.00005

x

2292.1

0.114605

0.00050

1.185

1.14605

0.742

0.00040

0.963

0.91684

0.614

0.00030

0.767

0.68763

0.494

0.00025

0.642

0.45842

0.418

0.00010

0.274

0.22921

0.199

0.00005

0.158

0.114605

0.127

## 525nm

0.00050 moles

10

0

1.160

0.00040

8

2

0.905

0.00030

6

4

0.733

0.00025

5

5

0.552

0.00010

2

8

0.270

0.00005

1

9

0.155

D = ECrCCr

D/C = Change in y/Change in x = 1800 nm

## theoretical absorbance351 nm

0.0005

x

1800

0.9

0.0004

x

1800

0.72

0.0003

x

1800

0.54

0.0002

x

1800

0.36

0.0001

x

1800

0.18

0.00005

x

1800

0.09

D = ECrCCr

D/C = Change in y/Change in x = 2211.1 nm

0.0005

x

2211.1

1.10555

0.0004

x

2211.1

0.88444

0.0003

x

2211.1

0.66333

0.0002

x

2211.1

0.44222

0.0001

x

2211.1

0.22111

0.00005

x

2211.1

0.110555

## 351 nm

0.00050

1.160

1.10555

0.695

0.00040

0.905

0.88444

0.833

0.00030

0.733

0.66333

1.008

0.00025

0.552

0.44222

1.118

0.00010

0.270

0.22111

1.357

0.00005

0.155

0.110555

1.528

Ratio

Wavelength

Permanganate solutions

Permanganate

Dichromate

525nm

0.00050 moles

10

0

0.054

0.00040

8

2

0.053

0.00030

6

4

0.049

0.00025

5

5

0.048

0.00010

2

8

0.042

0.00005

1

9

0.041

## X= dichromate

1.099 = 1810.2x + 1374y (351nm)

0.801 = 2211.1x + 2291.2y (525nm)

Working out using Simultaneous Equations:

1.099 = 1810.2x + 1374y

1374y -1.099 = 1810.2x

0.76- 6.07x 10-4 = x

0.801 = 2211.1x + 2291.2y

0.801 = 2211.1 (0.76- 6.07x 10-4) + 2291.2y

0.801 = 1680.44y - 1.34 + 2291.2y

0.801 + 1.34 = 1680.44y + 2291.2y

2.141=3971.64y

y = 5.39x10-4

1.099 = 1810.2x + 1374y

1.099 = 1810.2x + 1374y (5.39x10-4)

1.099 = 1810.2x + 0.74

1.099 -0.741= 1810.2x

1810.2x = 0.358

x = 1.98x10-4

## Dichromate : Permanganate

CCr = x : CMn = y

1.98x10-4 : 5.39x10-4

2 : 5.4

## 4) Discussion A

The intensity of the colour is directly related to the concentration of the coloured complex, and is measured by recording the absorbance in the visible spectrum. This is done using a spectrophotometer due to the subjectivity of human error.

The permanganate and the dichromate solution are able to absorb light though well separated absorbance. These are seen to be 531 nm and 351 nm. This thus allows for the calculation of two separate ions in an unknown solution mixture. By spectrophotometer analysis the Beer-Lambert Law can be developed and thus two equations may be produced:

## A= ebC

A= absorbance

e= molar absorptivity

b= cell path length

C= molar concentration

In the above practical the cell path length is kept constant at all times. Also the molar absorptivity, e, and path length may be combined and collectively called the molar extinction coefficient, E. Also in this experiment optical density, D, is used rather than absorbance.

By plotting the optical density, of the dilutions against their concentration an analytical calibration curve was obtained. The gradient of the graph is able to give us the experimental value of the molar extinction coefficient, E. The theoretical value was also calculated and compared to the experimental. The differences in the value may be due to contamination errors, together with some inaccuracy in the preparations of the dilutions. From this graph the concentration of iron in the unknown solution was determined.

The theoretical composition of the unknown sample was calculated by use of a simultaneous equation. The values of molar extinction coefficient were used for both the potassium and dichromate solution at absorbance of 525 nm and 351 nm. The ratio obtained was approximately that of 1 dichromate : 2.7 Permanganate solution. This is not seen to be very variant from the theoretical ratio of 1:2. This discrepancy is most likely present due to the loss of mass during transfers and possible contaminations during the procedure.

## Conclusion

From the practical above the Beer-Lambert law was seen to be confirmed by plotting the graphs of optical density against concentration of the solution. The separated absorbance peaks of the permanganate and dichromate solutions at 351nm and 525nm allows the determination of the concentration of the unknown solution. The ratio of moles of the dichromate: Permanganate solutions was found to be 1: 2.7. Thus is close to the theoretical 1:2 and accepted due to the sources of error present in the experiment.

## Brand

0.001 Salicylic acid

GPR

Aldrich

0.001 Ferric ammonium sulfate

GPR

BDH

0.002 M Hydrochloric acid

GPR

BDH

## 2.2) Apparatus

X2 500 mL round bottomed flasks x2 burettes

Measuring cylinder x11 50 mL beakers

Analytical balance Stirring rod

Spatula Spectrophotometer

Weighing boat

## 2.3) Procedure

0.069 g of Salicylic acid was weighed and placed in volumetric flask filled with 500 mL of 0.002 hydrochloric acid in order to make 0.0001 M solution of salicylic acid.

0.0239 g of ferric ammonium sulfate solution was weighed and placed in volumetric flask filled with 500 mL of 0.002 hydrochloric acid in order to make 0.0001 M solution of ferric ammonium sulfate

Two burettes wre then used to prepare 11 mixtures of the two solutions prepared above. These solutions being:

Vol. (A)/ mL

0

1

2

3

4

5

6

7

8

9

10

Vol. (B)/ mL

10

9

8

7

6

5

4

3

2

1

0

4) The solutions were then mixed at intervals of two minutes and allowed to stand for 5 minutes.

5) Their optical density was then measured by placing some of the solutions in a glass cell of 1 cm, against a water blank, at a wavelength of 520 nm

6) a graph of optical density against composition of the solution was then plotted

7) Using the graph the composition of the blue complex was then found.

## 2.4) Precautions

The dilutions were prepared using a burette for better accuracy

The weighing boat was rinsed with the respective solution in order to transfer as much solid as possible into the volumetric flask

The curvette was cleaned before measuring the optical density of another sample. This is done to prevent any remaining solution of the previous dilution from affecting the absorbance.

The curvette was placed in such a way that the incident light passes through the clear sides of the curvette, not the opaque sides.

The value on the spectrophotometer was left to settle before taking the final reading due to slight variations.

## 2.5) Sources of error

Some mass of the ferric ammonium sulfate and the salicylic acid may have been lost due to the incomplete transfer of the solid into the volumetric flask

Not all the solid may have dissolved, thus this would not result in a 0.001M solution.

0.137

0.277

0.425

0.568

0.652

0.821

0.910

1.069

1.182

1.182

## 10

0.086

Molar composition at maximum wavelength and thus the most likely composition of the complex is seen to be 9:1. Salicylic acid : ferric ions

## Thus number of moles of ferric ions:

0.001 moles ferric ammonium sulfate = 1000 mL

? = 1 mL

(1*0.001)/1000 = 1x 10-6 moles

## Thus number of moles of Salicylic acid:

0.001 moles ferric ammonium sulfate = 1000 mL

? = 9 mL

(9*0.001)/1000 = 9x 10-6 moles of salicylic acid

## 4) Discussion

Salicylic acid is a weak acid also known as 2-hydroxybenzoic acid. Salicylic acid is not seen to absorb light in the visible region of the spectrum; however, it does form a coloured complex with Fe3+ as shown by the reaction below:

The complex is seen to form different coloured compounds depending on the pH of the solution. In the experiment below hydrochloric acid was used making the solution acidic. This thus forms a blue-violet complex. At neutral pH, however, a dark-red complex forms, and in basic solution an orange complex.

The intensity of the coloured complex is measured using a spectrophotometer at a wavelength of 520 nm. It was noted that the darker the colour of the solution the higher the intensity thus the more concentrated the complex. Thus one could conclude that colour intensity is directly proportional to concentration.

In the experiment the mixture of the salicylic acid,A, with the Ferric ammonium sulfate, B, react with each other to form a complex that balance out each other at equilibrium.:

K

mA + nB AmBn

Where AmBn is seen to be the empirical formula of the complex formed between the salicylic acid and the Fe3+ ions. If one of the two reagents are not present in their exact amounts, then the colour intensity is seen to be less and thus the absorbance is seen to decrease. K is highest when the both the salicylic acid and Fe3+ ions are present in the stoichiometric ratio, as seen from the equation below:

The intensity was determined using a UV-Light spectrophotometer. The liquid solutions prepared were placed into a plastic cell and an incandescence bulb supplied the sample with the incident light ray. The sample then absorbs this incident light depending on the amount of complex formed. In the experiment one can deduce that the complex that contained the highest absorbance of complex formation at a ratio of 9 salicylic acid: 1 ferric ammonium sulfate.

Theoretically, however, this is seen to be incorrect, since it is actually most abundant at a ratio of 5 salicylic acid: 5 ferric ammonium sulfate. Thus the actual number of moles that should be present should be at a ratio of 5 x 10-6: 5 x 10-6. This could have been due to the loss of sample during the transfers and may be due to some solid that had not dissolved.

## Conclusion

From Experiment B it was concluded that the mixture of salicylic acid with ferrous ammonium sulfate formed a complex of salicylic acid: ferric ions. This blue complex is seen to form at different concentrations when different proportions of the two chemicals are used. The experimental results concluded that the highest absorbance was seen to be at the molar ration of 9 salicylic acid:1ferric ions. However, the theoretical ratio of this complex is seen to be 5:5. Thus would thus give a high concentration of complex and also high optical density.