Spectrophotometry

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Standard Deviation:

[pic 17]

[pic 18]

[pic 19][pic 20]

Experiment 2

[pic 21]

Figure 2. Absorbance spectrum for oxygenated haemoglobin in the visible region (400-700nm).

[pic 22]

Figure 3. Absorbance spectrum for deoxygenated haemoglobin in the visible region (400-700nm).

[pic 23] Figure 4. Absorbance spectrum for protein (BSA) in the UV region (200-400nm).

[pic 24]

Figure 5. Absorbance spectrum for nucleic acid (RNA) in the UV region (200-400nm).

Question and Answer

Experiment 1

[pic 25]

Figure 6. The standard curve of the absorbance at 540nm against the protein concentration (mg/mL)

[pic 26]

- Protein concentration in undiluted sample A = 0.27 ÷ 0.0473

= 5.71mg/mL

- Protein concentration in diluted sample A = 0.03 ÷ 0.0473

= 0.634mg/mL

Protein concentration in undiluted sample A = 0.634 × 10

= 6.34mg/mL

- Protein concentration in undiluted sample B = 1.15 ÷ 0.0473

= 24.31mg/mL

- Protein concentration in diluted sample B = 0.17 ÷ 0.0473

= 3.59mg/mL

Protein concentration in undiluted sample B = 3.59 × 10

= 35.9mg/mL

Two different dilutions of each sample are prepared to get the more accurate results. For the accuracy of the results, the absorbance of protein concentration of the sample solutions must fall between the ranges of standard solutions. Dilution must be done if the absorbances of the sample solutions fall outside the range of standard solutions. For sample A, the undiluted sample has lower protein concentration than the diluted sample which may due to the cloudiness of the protein that caused the light to scatter and cannot measure correctly by the spectrophotometer. For the sample A, both absorbance can be used as both of them fall between the range of the standard solutions and for sample B, the diluted sample should be used as it falls in the range of the absorbance of the standard solutions.

Experiment 2

- The spectrum of haemoglobin change when binding and releasing of the oxygen, which due to the crystal field theory. The binding of oxygen induces a change in the electronic state of Fe2+ that change its absorbance spectrum. Fe2 ion is in the high spin state and is too big to fit into the plane of the porphyrin ring of the heme group when there is the absent of the oxygen. The Fe2 ion will become low spin when binding of the oxygen, which it will shrink and fit into the porphyrin ring of the heme group and eventually caused the spectrum change (Chang, 2000).

- For proteins, amino acids with aromatic rings are the primary reason for the absorbance peak at 280nm. Peptide bond are primarily responsible for the peak at 200 nm (Lee, 1991). For nucleic acid, the purines and pyrimidines cause a strong absorbance peak at 260nm (Mohr & Schopfer, 1995).

- a) The compounds that responsible for the absorption maxima observed in the spectra of bovine serum albumin is due to the aromatic rings of amino acids, such as tryptophan, tyrosine, phenylamine and histidine (Ahmed, 2005). The absorption maxima observed in the spectra of ribonucleic acid (RNA) is due to the purines and pyrimidine bases (Mohr & Schopfer, 1995).

b) The absorbance of RNA at absorption maxima:

[pic 27]

Using Beer-Lambert Law,

A = [pic 28]

0.78 = 25.0 cm-1 (mg/mL)-1 x c x 1cm

c = [pic 29]

= 0.0312 mg/mL

Therefore, the concentration for RNA is 0.0312 mg/mL.

c) The absorbance of BSA at absorption maxima:

[pic 30]

A = [pic 31]

0.67 = x 1 mg/mL x 1cm[pic 32]

= [pic 33][pic 34]

= 0.67 cm-1 (mg/mL)-1

Thus, the extinction coefficient at 280nm for BSA is 0.67 cm-1 (mg/mL)-1.

d) The concentration unit for nucleic acids and proteins often used mg/mL

rather than M (molar). This is because proteins and nucleic acids are made up many subunits and they do not have the exact weight.

Reference

Ahmed, H 2005, Estimation of Protein, Principles and Reactions of Protein Extraction, Purification and Characterization, CRC Press, USA, pp.35-70.

Chang, R 2000, Allosteric Interactions, Physical Chemistry for the Chemical and Biological Sciences, University Science Books, USA, pp. 536-544.

Lee, V 1991, Analysis of Protein Drugs, Peptide and Protein Drug Delivery, Marcel Dekker, Inc., USA, pp. 247-302.

Marczenko, Z & Balcerzak, M 2000, Principles of Spectrophotometry, Separation, Preconcentration and Spectrophotometry in Inorganic Analysis, Elsevier Science, Netherlands, pp. 26-38.

Mohr, H & Schopher, P 1995, Light and UV Stress, Plant Physiology, Springer-Verlag Berlin Heidelberg, New York, pp.551-557.

Parnis, JM & Oldham, KB 2013, Beyond the Beer-Lambert law: The dependence of absorbance on time in photochemistry, Journal of Photochemistry and Photobiology