User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/10/09: Difference between revisions
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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. | ||
* | * 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): | ||
<math>\frac{177.16 g}{10}</math> | <math>\frac{177.16 g}{10}</math> = 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) | |||
<math>\frac{53.148 g}{10}</math> = 5.3148 g of luminol in 1000 mL of water (30 mM) | |||
x = <math>\frac{2 mL * 5.3148 g}{1000}</math> = .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== | ==Fluorescence Spectrometer Graph== | ||
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{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'' | | align="center" style="background:#f0f0f0;"|'''' | ||
| 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'' | ||
|- | |- | ||
| 1||30 μL||900 μL||176 μL||1.8 mL | | Molarity of Stock Solutions||2.3 μM||18 mM||10 mM||1.7 mM||neutral | ||
|- | |||
| Volume||30 μL||54 μL||900 μL||176 μL||1.8 mL | |||
|- | |- | ||
Revision as of 06:56, 7 December 2012
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Atomic Absorption Spectroscopy of Au/BSA
Chemiluminescence of Luminol
Image 1. The navy blue color is attributed to a pH of 10 to 11. 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.
Fluorescence Spectrometer GraphNo luminescence was observed for any runs. The concentration of reagents are shown below the table:
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