Difference between revisions of "HPA Reactions"
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To see if there is a change in the product formed at different times within a flow run of HPA + Dioxane through an IE HRP Bioreactor, samples where collected at 2 minutes from the run start, 10 min, 20 min, and 30 min. | To see if there is a change in the product formed at different times within a flow run of HPA + Dioxane through an IE HRP Bioreactor, samples where collected at 2 minutes from the run start, 10 min, 20 min, and 30 min. | ||
The rest of the product that was not collected at these specific time intervals was collected together and used as the base comparison, represented by the result labeled 0.25mL/min 20mM H202 (HPA + Dioxane) | The rest of the product that was not collected at these specific time intervals was collected together and used as the base comparison, represented by the result labeled 0.25mL/min 20mM H202 (HPA + Dioxane) | ||
| − | [[File:IE HPA10 02.jpg|500px|thumb| | + | [[File:IE HPA10 02.jpg|500px|thumb|none|IE HRP HPA/H202 (6/27/16)]] |
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| + | Immediately after the IE HRP HPA/H202 (6/27/16) was run, the bioreactor was cleaned with MOPS buffer in preparation for a second run. At a flow rate of 0.25mL/min and 20mM HPA + Dioxide and 20mM H2O2 conditions, the products of the run where expected to behave in the same way as the trial run previously. However it was seen that the products of the second trial have a lower concentration than that of the previous run. [[File:IE HPA10 04.jpg|500px|thumb|none|IE HRP HPA/H202 (6/27/16)-2]] | ||
==Concentration Test== | ==Concentration Test== | ||
Revision as of 17:05, 29 June 2016
4-Hydroxyphenylacetic acid (HPA)
Product Detection Test
Reaction of 50/50 HPA-Dioxane Solution in Buffer with various H202 concentrations and Horse Radish Peroxidase (HRP)
Using the Immobilized Enzyme technique, a reaction of HPA-Dioxane and H202 with Immobilized HRP
Trying out different flow speeds with IE to try and see different products. With the greater absorption of HPA There is only a slight change in the products of the reactions'.
To see if there is a change in the product formed at different times within a flow run of HPA + Dioxane through an IE HRP Bioreactor, samples where collected at 2 minutes from the run start, 10 min, 20 min, and 30 min. The rest of the product that was not collected at these specific time intervals was collected together and used as the base comparison, represented by the result labeled 0.25mL/min 20mM H202 (HPA + Dioxane)
Immediately after the IE HRP HPA/H202 (6/27/16) was run, the bioreactor was cleaned with MOPS buffer in preparation for a second run. At a flow rate of 0.25mL/min and 20mM HPA + Dioxide and 20mM H2O2 conditions, the products of the run where expected to behave in the same way as the trial run previously. However it was seen that the products of the second trial have a lower concentration than that of the previous run.
Concentration Test
To increase the amount of product extractable, the concentration of HPA was increased to 50mM as well as the H202 concentration, however the same amount of products does not appear as well as the formation of polymers that block products at the 0.25mL/Min run of the immobilized enzyme.
Decreasing the Concentration to just 20mM brought the resurgence of the peaks of products. These peaks will be separated in flash chromatography given that they are high enough in yield to be detected by the instrument.
Flash Chromatography Separation
To try to collect a cleaner sample of the products created in the HPA + H2O2 + HRP reaction, Flash chromatography was preformed to separate the peaks.
This is an HPLC of the separation preformed by the Flash Chromatography instrument on the 0.25mL/min 20mM HPA run from 6/23/16. However, the separation of the assumed three product peaks is not clear.
