Extra exercises Spectroscopy Course 2014-2015

1. A beam of light with a wavelength of 256 nm is passed through a solution 0.05 M from

C6H6 (1.0 mm-thick cell). In this case, the intensity of light decreases to 6% of the initial

value. Calculate the absorption and molar Absorption coefficients of the sample.

Calculate the transmittance when a cell of 2.0 mm thick is used.

R/ A= 0.795, =159.2 L.mol-1.cm-1 T=2.56%

2. Potassium thiocyanate is a detection agent for Fe3+ ions. The detection effect is due to a

reaction in the Fe3+ ions with the thiocyanate in a aqueous solution forming the

complex (Fe (SCN) (H2O)5)+2 .The complex shows a maximum peak absorption at =

580nm, and the = 7, 00.103 M-1 . cm-1.

a) Calculate the absorbance and the transmittance of the complex in a solution

2.22.10-4 M, which was measured in a cell of 1.25 cm at = 580 nm.

b) A 50 mL aliquot of well water is treated with an excess of KSCN and diluted to 1000

mL. Calculate the concentration of iron (III) in the sample in ppm when the diluted

solution has an A= 0.506 at =580 nm and a cell d = 1:50 cm.

R/ a)A= 1.94, T=1.15%

b) c=4.82. 10 -5M, 53.4 ppm

3. A solution 0:10 M of CuSO4 was measured at =600 nm in a 5.0 mm thick cell and the

value observed was 0:30. Calculate the molar absorption coefficient of Cu2 +(aq) at a

wavelength of =600 and the absorbance of this solution. Calculate the transmittance

when 1.0 mm cell is used?

R/ =10.5 L.mol-1.cm-1 , A=0.523, T= 78.5%

4 Determining the concentration of a protein in aqueous solution by Lowry assay (Lowry

assay, Lowry et al., J. Biol. Chem 193, 1951, pp 265-275) commonly used "Bovine

Serum albumin "(BSA) as a standard for the calibration. The assay is performed

through a colorful reaction between the proteins and the reagents, which are

subsequently measured at 750 nm by UV spectroscopy.

To prepare the calibration, different solutions with different concentration of BSA were used.

All the samples were measured at 750 nm and the absorbance values are shown below:

mg BSA in 3 mL solution Abs

0 0 5 0.033

10 0.068 15 0.069

20 0.12 40 0.229 60 0.327 80 0.41

a) Determine graphically the slope and intercept of the linear function.

b) For 5, 10, 15, 20 and 40 microliters of a solution of the protein with the desired X

concentration mg / mL were these volumes of water each to 3 ml replenished. There

were for the absorptions 0.065, 0.1281, 0.2092, 0.257, 0.4064 measured. Determine

the average of the desired protein concentration in mg / ml.

c) For the protein was determined by means of SDS gel electrophoresis, a molecular

weight of 35000 g / mol found. What is the molar concentration of the protein?

R/b) mean=2.3 g/L. c=65 mol/L