User:Omar Choudary/Notebook/Omar Choudary CHEM-571 AU-2011/2012/2012/04/04

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Objective

  • Determine the number of Lysine residues on BSA that are bound to a dye molecule. To do so, we need to calculate the concentration of dye in our dye solutions and the concentration of BSA. The amount of dye in each solution can be determined using the UV-Vis spectra; the dye in solution has a known-concentration which will allow for calculation of molar absorptivity of the dye (at 602nm and 625nm;

Description and Data

  • Determination of the molar absorptivity of the dye (at 602nm) using a known-concentration solution.

1. Determining concentration of the dye in solution.

The dye in solution was prepared on the 2/21 using: mass = 1 mg of dye in 5 ml of water + 80µL of DMSO = Total volume (V) = 0.0058L

MW of dye = 573.51 g/mol

So, n = 10^-3/573.51 = 1.74* 10^-6 moles

And, concentration of dye in solution (c) = (1.74* 10^-6)/0.0058 = 3*10^-4 M

2. Determination of the molar absorptivity of the dye using Beer's law.

Use the Absorbance at 602nm (which is the absorption value given by the dye manufacturer); at this wavelength, fluctuations should be the lowest.

Molar absorptivity (ε, L/mol/cm) were determined using Beer's law: A = ε*l*c

With A = absorbance at 602nm l = width of the cuvette (1cm) c = dye concentration (3*10^-4 M)

Image:4-4-2012-1.jpg

The molar absorptivity of the dye at 602nm = 3419 L.mol^-1.cm^-1 (Average of the three different ε values)

Determination of the molar absorptivity of the dye (at 625nm) using a known-concentration solution.

Molar absorptivity (ε, L/mol/cm) were determined using Beer's law: A = ε*l*c

With A = absorbance at 625nm l = width of the cuvette (1cm) c = 3*10^-4 M


Image:4-4-2012-2.jpg

The molar absorptivity of our dye at 625nm is equal to = 2204 L.mol^-1.cm^-1 ( Average of the three different ε values)

Determination of the dye concentration in 166, 70 and BSA/HCl solutions (using 3/27 UV spectrum).

Image:4-4-2012-3.jpg

In this table are corrected value for Absorbance (Abs with dye - Abs without dye)

According to the data, we can calculate the dye concentration using Beer's law.

We know that: ε at 625nm = 2204 L.mol^-1.cm^-1

ε at 602nm = 3419 L.mol^-1.cm^-1

Notes

  • Dye Molecule / BSA Ratio:

Image:4-4-2012-4.jpg


  • Determining Dye Concentration:

Using the UV-VIS of a known dye standard, the molar absorptivity of the dye was determined using Beers Law

The molar absorptivity value calculated from the UV spectra of the dye was too low, so used the value obtained in literature

Beer’s Law and the calculated molar absorptivity were used to determine the concentration of dye in all of the dye reactions

To account for the presence of gold nanoparticles and BSA solutions, corrected values for absorbance were used.

The corrected values were acquired by subtracting the absorbance of the control solutions (166, 70, and BSA/HCl without dye) from the absorbance values obtained from the spectra for each trial

Used the initial concentrations of BSA and assumed that no BSA had been lost during the experiment

  • BSA is composed of approximately 60 Lys residues:

Hypothetically 50% of Lys are bound with a dye molecule in 2nd trial

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