User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/09

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(Chemiluminescence of Luminol)
Current revision (09:56, 7 December 2012) (view source)
(Fluorescence Spectrometer Graph)
 
(11 intermediate revisions not shown.)
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* The reaction of 3-aminophthalhydrazide (luminol) and hydrogen peroxide is catalyzed by the addition of horseradish peroxidase (HRP) producing 3-aminophthalate.  
* The reaction of 3-aminophthalhydrazide (luminol) and hydrogen peroxide is catalyzed by the addition of horseradish peroxidase (HRP) producing 3-aminophthalate.  
* The reagents originated from the HRP Assay of aminoantipyrine (AAP) laboratory period [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/02|10/02/2012.]] For this procedure, 4-iodophenol, hydrogen peroxide, HRP, and water will be used. The AAP will be replaced in the reaction with luminol.
* The reagents originated from the HRP Assay of aminoantipyrine (AAP) laboratory period [[User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/02|10/02/2012.]] For this procedure, 4-iodophenol, hydrogen peroxide, HRP, and water will be used. The AAP will be replaced in the reaction with luminol.
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* It was decided to prepare 10 mM of luminol since this will be further diluted down to 3 mM upon the addition of water in the cuvette.  
+
* A concentration of 10 mM of luminol was used. This will be further diluted down to 3 mM upon the addition of water in the cuvette.  
* The following calculations were made for luminol (FW 177.16):
* The following calculations were made for luminol (FW 177.16):
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* The actual of the yellow pastel powder of luminol transferred in 2 mL of water was 0.0108 g. Luminol was insoluble in water. Hence, 0.0729 g of the white granular solid of sodium carbonate was added into solution. The solution of luminol, sodium carbonate in 2 mL water was vortexed for faster dissolution.
+
* The actual amount of the yellow pastel powder of luminol transferred in 2 mL of water was 0.0108 g. Luminol was insoluble in water. Hence, 0.0729 g of the white granular solid of sodium carbonate was added into solution. The solution of luminol and sodium carbonate in 2 mL water was vortexed for faster dissolution.
 +
* To buffer the alkalinity of sodium carbonate, the amount of 0.4373 g of sodium bicarbonate was added.
 +
* Sodium bicarbonate precipitated in the solution. Addition of 4 mL of water diluted luminol from 30 mM to 10 mM.
 +
* A pH paper was used to estimate the pH level of the solution since the electrode of the pH meter did not fit into the falcon tube of the solution.
 +
* The images of the pH paper during qualitative analysis of the solution are embedded below:
 +
 
 +
 
 +
[[Image:2012-10-09 14.17.45.jpg]]
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Image 1. The navy blue color is attributed to a pH of 10 to 11.
 +
 
 +
 
 +
[[Image:2012-10-09 14.59.55.jpg]]
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 +
Image 2. Below each pH paper is shown the number of drops of 1 M HCl into the solution of luminol. The light green color corresponds to the pH level of 7 to 8.
 +
 
 +
 
 +
* The lamp of LS 55 Perkin Elmer fluorescence spectrometer was turned off. The signal was set to a function of time. The wavelength was set at 425 nm. The excitation slit was placed at 15 nm while the emission slit was placed at 20 nm. The duration of data collection was settled at 300 sec.
 +
* The reagents were transferred into the cuvette using automatic delivery micropipets. The HRP was added last for this initiates the reaction through catalysis of hydrogen peroxide.
 +
* The collected data are shown in the next section.
==Fluorescence Spectrometer Graph==
==Fluorescence Spectrometer Graph==
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{| {{table}}
{| {{table}}
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| align="center" style="background:#f0f0f0;"|''sample''
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| align="center" style="background:#f0f0f0;"|''''
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| align="center" style="background:#f0f0f0;"|''HRP''
| align="center" style="background:#f0f0f0;"|''4-iodophenol''
| align="center" style="background:#f0f0f0;"|''4-iodophenol''
| align="center" style="background:#f0f0f0;"|''luminol''
| align="center" style="background:#f0f0f0;"|''luminol''
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| align="center" style="background:#f0f0f0;"|''H<sub>2</sub>O''
| align="center" style="background:#f0f0f0;"|''H<sub>2</sub>O''
|-
|-
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| 1||30 μL||900 μL||176 μL||1.8 mL
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| Molarity of Stock Solutions||2.3 μM||18 mM||10 mM||1.7 mM||neutral
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|-
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| Volume||30 μL||54 μL||900 μL||176 μL||1.8 mL
|-
|-
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Atomic Absorption Spectroscopy of Au/BSA

  • A rack of Au/BSA solutions was recovered and used for the assigned Atomic Absorption Spectroscopy (AAS). AAS was used to strip the molecule to its elemental state.
  • The entire solution set were observed to lack fibers even at high mole ratios where formation of fibers is typical. The date of which the solutions were made is unknown.
  • The equipment used for AAS is by the make of Shimadzu AA-6200 Atomic Absorption Flame Emission.
  • The sample cell used for this procedure is the ignited flame.
  • A gold hollow cathode tube was selected for the procedure. The wavelength of the lamp was 242.8 nm.
  • Two blanks were used; water and HCl. Water was chosen since it is part of the ternary mixture. The standards provided by Dr. Miller were made with HCl. In addition, HCl ionizes better and keeps the metal in solution. The standards were 5, 8, 10, 15, 20, 25, 30, 40 ppm Au which appeared as clear and colorless liquid solutions.
  • Measurement was done by inserting a transparent, plastic tube attached to the equipment into the solution.
  • Each blank, water and HCl, was measured and followed by autozero. Next, standards 1, 2, and 3 were measured.
  • The flame is blue green in its normal state. Having run the blanks, the flame remained blue green during the re-measurement of each blank.
  • During the measurement of the standards, the flame was observed to turn orange. The color orange is indicative of the ions present in the solution.
  • The other standards and samples were run. The calibration curve is on Puja's notebook.

Chemiluminescence of Luminol

  • The reaction of 3-aminophthalhydrazide (luminol) and hydrogen peroxide is catalyzed by the addition of horseradish peroxidase (HRP) producing 3-aminophthalate.
  • The reagents originated from the HRP Assay of aminoantipyrine (AAP) laboratory period 10/02/2012. For this procedure, 4-iodophenol, hydrogen peroxide, HRP, and water will be used. The AAP will be replaced in the reaction with luminol.
  • A concentration of 10 mM of luminol was used. This will be further diluted down to 3 mM upon the addition of water in the cuvette.
  • The following calculations were made for luminol (FW 177.16):


\frac{177.16 g}{10} = 17. 716 g of luminol in 1000 mL of water (.1M)


17.716 g × 3 = 53.148 g of luminol in 1000 mL of water (.3 M = 300 mM)


\frac{53.148 g}{10} = 5.3148 g of luminol in 1000 mL of water (30 mM)


x = \frac{2 mL * 5.3148 g}{1000} = .0106296 g of luminol in 2 mL of water (30 mM)


  • The actual amount of the yellow pastel powder of luminol transferred in 2 mL of water was 0.0108 g. Luminol was insoluble in water. Hence, 0.0729 g of the white granular solid of sodium carbonate was added into solution. The solution of luminol and sodium carbonate in 2 mL water was vortexed for faster dissolution.
  • To buffer the alkalinity of sodium carbonate, the amount of 0.4373 g of sodium bicarbonate was added.
  • Sodium bicarbonate precipitated in the solution. Addition of 4 mL of water diluted luminol from 30 mM to 10 mM.
  • A pH paper was used to estimate the pH level of the solution since the electrode of the pH meter did not fit into the falcon tube of the solution.
  • The images of the pH paper during qualitative analysis of the solution are embedded below:


Image:2012-10-09 14.17.45.jpg

Image 1. The navy blue color is attributed to a pH of 10 to 11.


Image:2012-10-09 14.59.55.jpg

Image 2. Below each pH paper is shown the number of drops of 1 M HCl into the solution of luminol. The light green color corresponds to the pH level of 7 to 8.


  • The lamp of LS 55 Perkin Elmer fluorescence spectrometer was turned off. The signal was set to a function of time. The wavelength was set at 425 nm. The excitation slit was placed at 15 nm while the emission slit was placed at 20 nm. The duration of data collection was settled at 300 sec.
  • The reagents were transferred into the cuvette using automatic delivery micropipets. The HRP was added last for this initiates the reaction through catalysis of hydrogen peroxide.
  • The collected data are shown in the next section.

Fluorescence Spectrometer Graph

Image:Fluor10.09.12.png

No luminescence was observed for any runs.

The concentration of reagents are shown below the table:

' HRP 4-iodophenol luminol H2O2 H2O
Molarity of Stock Solutions2.3 μM18 mM10 mM1.7 mMneutral
Volume30 μL54 μL900 μL176 μL1.8 mL


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