User:Rebeca Rodriguez/Notebook/Chem 471/2015/09/09

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Objective

Protocol done by via Michael Bible because I'm late to class every Wednesday.

Description

Stock solutions of HAuCl4·H2O and Lysozyme were prepared by Dr. Hartings. The procedure for their preparation can be found here.

The stock solution of HAuCl4·H2O has a concentration of 2.96 mM. The stock solution of Lysozyme has a concentration of 21.4 μM.

Sixteen 1 mL samples and ten 5 mL samples of a 45:1 Au:Lysozyme solution were prepared using the following volumes of stock solutions:

  1. 1 mL samples
    1. Volume of gold stock: 84.4 μL
    2. Volume of protein stock: 259 μL
    3. Volume of water: 656 μL
  2. 5 mL samples
    1. Volume of gold stock: 422 μL
    2. Volume of protein stock: 1,297 μL
    3. Volume of water: 3,281 μL

These solutions were then heated in the oven at 80°C for four hours.

Protocol

A stock solution of lysozyme was created using a 10 mL volumetric flask. The aim was to make a 50 μM stock solution but the actual concentration was 62 μM.

8.88 mg of lysozyme was added to a 10 mL volumetric flask

The following 5 solutions were made using a 10 mL volumetric flask and the volumes of water and other solutions listed below.

  • 15 μM
    • 2.419 mL of 62 μM stock solution
    • 7.581 mL of HPLC water.
  • 12.5 μM
    • 2.016 mL of 62 μM stock solution
    • 7.984 mL of HPLC water.
  • 6.25 μM
    • 5.0 mL of 12.5 μM lysozyme solution
    • 5.0 mL of HPLC water.
  • 3.13 μM
    • 5.0 mL of 6.25 μM lysozyme solution
    • 5.0 mL of HPLC water.
  • 1.56 μM
    • 5.0 mL of 3.13 μM lysozyme solution
    • 5.0 mL of HPLC water.

Calculations

Concentration of Lysozyme stock solution:

(0.00888 g)*[(1 mol lysozyme)/(14307 g lysozyme)] = 6.2*10-7 mols

(6.2*10-7 mols)/(0.010 L) = 62 μM


Standard solutions were made using the following formula C1V1 = C2V2

V2 = [C1V1]/C2

So, in order to make a 15 μM solution the following volume of stock solution was needed:

V62μM = (15 μM * 10 mL)/(62 μM) = 2.419 mL of stock solution

The method described above was used to determine the necessary volumes of previous serial solutions to use in each new dilution.



Data

UV-Vis and Fluorescence spectra were collected for each of the 5 solutions that Michael created. A calibration curve for UV-Vis has been created using the absorbance at λ = 281nm. I made these beautiful graphs.

UV-Vis Lysozyme Calibration Curve MB 9-9-15.png

The Figure above shows the UV-Vis spectra of various concentrations of lysozyme.

Lysozyme Calibration Curve MB 09-09.png

The Figure above shows the calibration curve for the absorbance at λ = 281 as a function of the concentration of lysozyme in solution.

Fluroescence Integration of Lysozyme MB 09-09.png

The Figure above show the calibration curve for the Fluorescence intensity integration as a function of the concentration of lysozyme in solution.

Fluorescence of Lysozyme MB 09-09.png

The Figure above shows the Fluorescence spectra of various concentrations of lysozyme.

Matt Hartings You need a description for how these solutions were made. Also, please list the molar extinction coefficient for lysozyme in M-1 cm-1



BIG NOTE

Michael created all of the solutions on Wednesday as I was commuting back to Georgetown.