User:Nicole Bonan/Notebook/Chem 571 Lab Notebook/2016/02/24

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Objective

Today's objectives were to:

  • Make a calibration curve for the fluorescence of Rhodamine B as a function of the concentration of AuNP that the Rhodamine is dissolved in
  • Take images of and measure the fluorescence of the supernatants of the samples synthesized on February 17, 2016 that had been incubated in Rhodamine overnight (i.e., the Rhodamine had been added on February 23, 2016)
  • Take images of the samples that were synthesized on February 23, 2016; incubate these samples in Rhodamine for two hours; take images of the samples after the incubation; and then measure the fluorescence of the supernatants of these samples
  • Make new AuNP fiber samples for use next week
  • Use HPLC to measure the impurities in acetylsalicylic acid

Protocol

We used the same Rhodamine and chloroauric acid samples as last week for all samples described today. We made a new lysozyme stock that was 46.79µM.

Incubation of Samples Synthesized on February 23, 2016 in Rhodamine B

Because we had incubated the samples that we synthesized on February 17, 2016 in Rhodamine B for 24 hours, we decided to incubate the samples that we synthesized on February 23, 2016 for 3.5 hours. Then, we could try to determine whether incubating the samples in Rhodamine for longer periods of time helped incorporate more Rhodamine into the fibers.

The following table shows the amount of Rhomdaine B added to each sample.

Table 1: Rhodamine B Added to Samples Synthesized on February 23, 2016

We then covered the tops of the test tubes in parafilm and incubated the samples at room temperature for 3.5 hours.

Fluorescence Measurements

First, we took fluorescence measurements of Rhodamine in varying concentrations of AuNP.


Next, we took fluorescence measurements of the supernatants of each of the samples synthesized on February 17, 2016 after being incubated in Rhodamine for 24 hours. We then took fluorescence measurements of the supernatants of each of the samples synthesized on February 23, 2016 after being incubated in Rhodamine for 3.5 hours. For each measurement, we used 200µL of sample.


We also took one measurement of a 1µM Rhodamine standard (in DI water).


For all the fluorescence measurements, we used the following parameters:

Start 540 nm End 700 nm Ex: 520 nm

Synthesis of AuNP Fiber Samples

We made new AuNP fiber samples according to the following table. (Note that we did not add any Rhodamine. We are going to add the Rhodamine to the samples on March 1, 2016 after the samples have been incubated in the oven.)

Table 2: Components of the AuNP Fiber Samples Synthesized Today

Each of the samples had a total volume of 5mL and were made in glass test tubes. We sealed the test tubes by covering the top with aluminum foil and then taping the foil down with labeling tape so that no vapors could escape.

We then incubated the samples in the oven at 80°C for four hours.

HPLC

MATT PUT YOUR PROTOCOL HERE PLZ THANKSALOT:)

Data, Observations, and Analysis

Calibration Curve for Fluorescence of Rhodamine B as a Function of AuNP Concentration

Figure 1: The figure above plots log(I/I0) against the volume of AuNP added. I is integrated fluorescence intensity and I0 is the integrated fluorescene intensity with no AuNP added.

Fluorescence Data for Supernatants of Samples

Since only measured the fluorescence of one Rhodamine standard, we could not make a calibration curve to determine the change in the concentration of Rhodamine in the supernatant based on our fluorescence measurements of the supernatants. Next week, we will measure Rhodamine standards so that we can determine the change in Rhodamine concentration.


Below is our raw fluorescence data for these samples. See the entry for March 2, 2016 for the analysis of these samples (we're going to take measurements of Rhodamine standards on March 2 so that we can analyze all of the supernatants from this week and in the future.)

  • Note that we forgot to measure the supernatants of all of the samples ending in _04, which were the blanks (they did not receive any Rhodamine).


Figure 1: Raw Fluorescence of Supernatants of Samples Synthesized on February 17, 2016 After 24 Hour Incubation in Rhodamine

The above image shows the raw fluorescence data for each sample we measured. The fluorescence is shown as a function of the wavelength of incident light (nm).


Figure 2: Raw Fluorescence of Supernatants of Samples Synthesized on February 23, 2016 After 2 Hour Incubation in Rhodamine

The above image shows the raw fluorescence data for each sample we measured. The fluorescence is shown as a function of the wavelength of incident light (nm).

Images of Samples Synthesized on February 17, 2016 After 24 Hour Incubation in Rhodamine

Figure 1: Samples Synthesized on February 17, 2016 After 24 Hour Incubation in Rhodamine

This image was taken after the samples had been incubating in Rhodamine for 24 hours. From left to right, the samples are:

  • A_200uM_01
  • A_200uM_02
  • A_200uM_03
  • A_100uM_01
  • A_100uM_02
  • A_100uM_03
  • A_50uM_01
  • A_50uM_02
  • A_50uM_03
  • au_lys_after

The concentration of Rhodamine added to the A_200uM samples was 1µM. The concentration of Rhodamine added to the A_100uM samples was 0.1µM. The concentration of Rhodamine added to the A_50uM samples was 0.01µM. No Rhodamine was added to the au_lys_after sample; this sample was a blank.


The pink hue in the samples, especially the A_200uM samples, is due to the Rhodamine and not the presence of AuNP.

Images of Samples Synthesized on February 23, 2016 Before Addition in Rhodamine

Figure 2a-c: Samples Synthesized on February 23, 2016 Before Addition in Rhodamine
Figure 2a: From Left to Right: Samples A_1uM_01, A_1uM_02, A_1uM_03, A_1uM_04 These samples were synthesized on February 23, 2016. They were going to be incubated in 1µM Rhodamine for 3.5 hours. This image was taken before the incubation in Rhodamine.
Figure 2a: From Left to Right: Samples A_1uM_01, A_1uM_02, A_1uM_03, A_1uM_04 These samples were synthesized on February 23, 2016. They were going to be incubated in 1µM Rhodamine for 3.5 hours. This image was taken before the incubation in Rhodamine.
Figure 2b: From Left to Right: Samples A_0.1uM_01, A_0.1uM_02, A_0.1uM_03, A_0.1uM_04 These samples were synthesized on February 23, 2016. They were going to be incubated in 0.1µM Rhodamine for 3.5 hours. This image was taken before the incubation in Rhodamine.
Figure 2b: From Left to Right: Samples A_0.1uM_01, A_0.1uM_02, A_0.1uM_03, A_0.1uM_04 These samples were synthesized on February 23, 2016. They were going to be incubated in 0.1µM Rhodamine for 3.5 hours. This image was taken before the incubation in Rhodamine.
Figure 2c: From Left to Right: Samples A_0.01uM_01, A_0.01uM_02, A_0.01uM_03, A_0.01uM_04 These samples were synthesized on February 23, 2016. They were going to be incubated in 0.01µM Rhodamine for 3.5 hours. This image was taken before the incubation in Rhodamine.
Figure 2c: From Left to Right: Samples A_0.01uM_01, A_0.01uM_02, A_0.01uM_03, A_0.01uM_04 These samples were synthesized on February 23, 2016. They were going to be incubated in 0.01µM Rhodamine for 3.5 hours. This image was taken before the incubation in Rhodamine.

Images of Samples Synthesized on February 23, 2016 After 3.5 Hour Incubation in Rhodamine

Figure 3: Samples Synthesized on February 23, 2016 After 3.5 Hour Incubation in Rhodamine

From left to right, the samples in this image are:

  • A_0.01uM_02
  • A_0.01uM_01
  • A_0.01uM_03
  • A_0.01uM_04
  • A_0.1uM_03
  • A_0.1uM_02
  • A_0.1uM_01
  • A_0.1uM_04
  • A_1uM_03
  • A_1uM_02
  • A_1uM_01
  • A_1uM_04


This image was taken after the samples had been incubated in Rhodamine for 2 hours. We did not measure the fluorescence of the supernatants of these samples until they had incubated in Rhodamine for 3.5 hours.


All of the samples ending in _04 did not receive any Rhodamine. These were blanks.

HPLC Data for Acetylsalicylic Acid

MATT ALSO PLZ PUT UR DATA HERE THAT WOULD BE COOL


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