User:Matthew R Skorski/Notebook/471 - Exp BioChem/2016/02/17

From OpenWetWare

Jump to: navigation, search
Project name Main project page
Previous entry      Next entry

Objective

Today's objective was to use HPLC to determine the concentration of acetylsalicylic acid in the supernatant of the samples we synthesized on February 16. However, we did not get to HPLC today. Instead, we made a calibration curve for the absorption of acetylsalicylic acid using UV-Vis absorbance spectroscopy.


Our second objective for today was to synthesize more acetylsalicylic-doped AuNP fibers for use in measurements next week.

Protocol

  • First, we made a new, 44.72µM lysozyme stock:
    • 0.03199g lysozyme diluted to 50mL in DI water in a volumetric flask


  • We then visually observed our samples synthesized yesterday. We noted whether fibers had formed and how dark the supernatant was. We then added acetylsalicylic acid to the samples that had been designated to receive the acid after incubation in the oven, and we took pictures of all of the samples.


  • We also made a calibration curve for the absorbance of acetylsalicylic acid of 297nm light as a function of concentration. We used UV-Vis absorbance spectroscopy.


  • Next, we made new fiber samples. This time, we decreased the concentration of lysozyme in our samples in order to try to form fibers instead of colloids. (The resulting ratio of gold:lysozyme was 60:1.) We also made 5mL samples instead of 1mL samples, and we made our samples in glass test tubes instead of in plastic Eppindorf tubes. The following table shows the ingredients in each sample.
Table 1: Components of Each Sample

The yellow box indicates that we added 49.88µL of water before putting the sample in the oven (to make up for the extra volume that all the samples containing acetylsalicylic acid had). The green box indicates that we will need to add 49.88µL of water after taking the sample out of the oven (to make up for the extra volume that all the samples containing acetylsalicylic acid had).


  • We put all of the samples in the oven at 80°C for four hours (except for B_200uM_04, B_100uM_04, B_50uM_04, A_200uM_04, A_100uM_04, and A_50uM_04, which we left out on the bench top).

Data, Analysis, and Observations

Samples Synthesized on February 16, 2016

(See the entry for February 16, 2016 for synthesis of these samples.)


First, we observed whether or not fibers had formed from these samples, and we judged the darkness of the color of the supernatant. At first, it appeared as if the only samples to form fibers were the samples that received acetylsalicylic acid before being placed in the oven. The addition of acetylsalicylic acid to the samples that were designated to receive the acid after the oven did not change this observation. However, upon closer observation, these "fibers" actually turned out to be large AuNP colloids because they broke up easily after being shaken.

Table 2: Observations of Samples Synthesized on February 16, 2016
Sample Name Fibers Formed (before adding aspirin, if applicable)? Supernatant (before adding aspirin, if applicable)
B_200uM_01Yes1
B_200uM_02Yes2
B_200uM_03Yes1
B_200uM_04Grey mesh0
B_100uM_01Yes2
B_100uM_02Yes2
B_100uM_03Yes2
B_100uM_04Darker grey mesh0
B_50uM_01Yes2
B_50uM_02Yes2
B_50uM_03Yes1.5
B_50uM_04Even darker grey mesh0
A_200uM_01Weak2.5
A_200uM_02Weak3
A_200uM_03Weak3
A_200uM_04No0
A_100uM_01Weak3
A_100uM_02Weak2.5
A_100uM_03Weak3
A_100uM_04No0
A_50uM_01Weak3
A_50uM_02Weak3
A_50uM_03Weak3
A_50uM_04No0
aspirin200uM_hclNo0
aspirin200uM_hcl_lysNo0
aspirin200uM_auNo0
aspirin100uM_auNo0
aspirin50uM_auNo0
au_lys_beforeWeak2.5
au_lys_afterWeak2.5

The above table shows each sample name in the leftmost column. We made observations about the samples after taking them out of the oven but before adding acetylsalicylic acid to the samples that were designated to receive the acid after the oven. The middle column shows whether or not "fibers" formed. Note that in this case, "fibers" actually meant large AuNP colloids. "Mesh" indicates that a grey mesh suspended in supernatant was formed. The rightmost column describes how dark the supernatant was on a relative scale of 0-5, where 0 was clear and 5 was very dark.


It was interesting to note that the samples that were left on the bench top instead of being placed in the oven formed a sort of grey mesh suspended in supernatant. A possible explanation for this formation is that the acetylsalicylic acid was acting as a reducing agent that aided in the formation of AuNP. Since the samples were left on the bench instead of being heated, the AuNP formation was much slower. Thus, larger nanoparticles were formed, creating a mesh that was grayish in color (since smaller AuNP are purple while larger AuNP are grey).


Figure 1a-h: Images of Samples Synthesized on February 16, 2016 After Incubation in Oven and Addition of Acetylsalicylic Acid to Designated Samples
Figure1a: Samples B_200uM_01 through B_200uM_04 These samples received 200µM acetylsalicylic acid before being incubated in the oven. Sample 04 was left on the bench top overnight.
Figure1a: Samples B_200uM_01 through B_200uM_04 These samples received 200µM acetylsalicylic acid before being incubated in the oven. Sample 04 was left on the bench top overnight.
Figure1b: Samples B_100uM_01 through B_100uM_04 These samples received 100µM acetylsalicylic acid before being incubated in the oven. Sample 04 was left on the bench top overnight.
Figure1b: Samples B_100uM_01 through B_100uM_04 These samples received 100µM acetylsalicylic acid before being incubated in the oven. Sample 04 was left on the bench top overnight.
Figure1c: Samples B_50uM_01 through B_50uM_04 These samples received 50µM acetylsalicylic acid before being incubated in the oven. Sample 04 was left on the bench top overnight.
Figure1c: Samples B_50uM_01 through B_50uM_04 These samples received 50µM acetylsalicylic acid before being incubated in the oven. Sample 04 was left on the bench top overnight.
Figure1d: Samples A_200uM_01 through A_200uM_04 These samples received 200µM acetylsalicylic acid after being incubated in the oven. Sample 04 was left on the bench top overnight.
Figure1d: Samples A_200uM_01 through A_200uM_04 These samples received 200µM acetylsalicylic acid after being incubated in the oven. Sample 04 was left on the bench top overnight.
Figure1e: Samples A_100uM_01 through A_100uM_04 These samples received 100µM acetylsalicylic acid after being incubated in the oven. Sample 04 was left on the bench top overnight.
Figure1e: Samples A_100uM_01 through A_100uM_04 These samples received 100µM acetylsalicylic acid after being incubated in the oven. Sample 04 was left on the bench top overnight.
Figure1f: Samples A_50uM_01 through A_50uM_04 These samples received 50µM acetylsalicylic acid after being incubated in the oven. Sample 04 was left on the bench top overnight.
Figure1f: Samples A_50uM_01 through A_50uM_04 These samples received 50µM acetylsalicylic acid after being incubated in the oven. Sample 04 was left on the bench top overnight.
Figure1g: aspirin200uM_hcl; aspirin200uM_hcl_lys; aspirin200uM_au; aspirin100uM_au; aspirin50uM_au These samples are blanks. aspirin200uM_hcl consisted of 200µM acetylsalicylic acid and HCl (to adjust the pH) only. aspirin200uM_hcl_lys consisted of 200µM acetylsalicylic acid, lysozyme, and HCl. aspirin200uM_au, aspirin100uM_au, and aspirin50uM_au consisted of chloroauric acid and either 200, 100, or 50µM acetylsalicylic acid, respectively.
Figure1g: aspirin200uM_hcl; aspirin200uM_hcl_lys; aspirin200uM_au; aspirin100uM_au; aspirin50uM_au These samples are blanks. aspirin200uM_hcl consisted of 200µM acetylsalicylic acid and HCl (to adjust the pH) only. aspirin200uM_hcl_lys consisted of 200µM acetylsalicylic acid, lysozyme, and HCl. aspirin200uM_au, aspirin100uM_au, and aspirin50uM_au consisted of chloroauric acid and either 200, 100, or 50µM acetylsalicylic acid, respectively.
Figure 1h: au_lys_before; au_lys_after These samples are blanks that did not receive any acetylsalicylic acid.
Figure 1h: au_lys_before; au_lys_after These samples are blanks that did not receive any acetylsalicylic acid.

Calibration Curve for UV-Vis Absorbance of Acetylsalicylic Acid

Figure 2: Calibration Curve for UV-Vis Absorbance of Acetylsalicylic Acid

The above calibration curve shows the absorbance of acetylsalicylic acid of light of wavelength 297nm as a function of the concentration of acetylsalicylic acid (µM).



Personal tools