User:Floriane Briere/Notebook/CHEM-496/2012/04/04

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Objective

Protocol

  • 166 ratio solution:
  1. 8ml of water
  2. 1ml of BSA (17.7µM)
  3. 1ml of Gold (2.9mM; prepared on the 3/21)
  • 70 ratio solution:
  1. 8.5ml of water
  2. 1ml of BSA (17.7µM)
  3. 0.42ml of Gold (2.9mM; prepared on the 3/21)

Data

One of our objective is to determine the number of Lysine (on the BSA molecule) which are bound to a dye molecule. To do so, we need to evaluate the concentration of dye in our solution and the amount of BSA protein. The amount of dye in each solution can be determined using UV spectrum; the dye in solution has a known-concentration which will allow us to find the molar absorptivity of the dye (at 602nm and 625nm; Gold particles also absorb around 600nm and we want to lower this effect as much as possible).

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

The dye in solution has been prepared on the 21/2 using: m = 1mg of dye 5ml of water + 80µL of DMSO => Total volume = V = 0.0058L MW of the dye = 573.51 g/mol


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


And, C = (1.74* 10^-6)/0.0058 = 3*10^-4 M

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

We are going to use the Absorbance at 602nm (which is the absorption value given by the dye manufacturer); at this wavelength, fluctuation should be the lowest.

Molar absorbtivities (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)

The molar absorptivity of our dye at 602nm is equal to = 3419 L.mol^-1.cm^-1 (It's determined by doing the average of the three different ε values)

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

Molar absorbtivities (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

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Notes

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