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Curcumin Research

2017-05-03T21:21:32Z

Nayala: /* Written Report */

You have reached the page dedicated to the research of curcumin, a secondary plant metabolite and biophenol of interest to the Sturgeon Research Project. This page was created and is maintained by Stephanie Saey and Nadia Ayala.

==Curcumin Research Initiative==

===Preparation of Curcuminoid Standards from Turmeric Plant===
Curcumin is a secondary plant metabolite of the turmeric herb ''Curcuma longa''. The term "curcumin" has been used to refer to the bioactive molecule, but in reality curcumin has three derivatives (I/II/III) of different molecular structures: curcumin (I), demethoxycurcumin (II), and bisdemethoxycurcumin (III). Together, the aforementioned compounds are known as curcuminoids. A review of literature suggests that curcuminoids have chemotherapeutic, antioxidant, and anti-inflammatory activity, among other uses to be discovered. However, difficulty arises when seeking to study these curcuminoids individually. Curcumin I is commercially available (10 mg for $150), while demethoxycurcumin (II) and bisdemethoxycurcumin (III) are not commercially available. Our current research project aims to isolate (methanol in soxhlet), purify (automated Flash/HPLC), and characterize (NMR) the curcuminoids for further investigation.

===The Three Curcuminoids===
Curcumin I (Main Curcuminoid, in picture below), demethoxycurcumin (curcumin II), and bisdemethoxycurcumin (curcumin III) are the three major curcuminoids present in turmeric and of interest to this study. Their respective chemical structures are depicted below:

[[Media:Curcuminoid Analogs with Potent Activity.pdf|Curcuminoid Analogs with Potent Activity (Article)]]

[[File:Curcumin.jpg|400px|thumb|none|Curcumin I, II, and III]]

===Flash Chromatography Data===

We used a 15.5mL C18 Column to run 1mL of our Curcumin extract. This column uses acetyl nitrile and water to run the liquid phase through the column. We ran a gradient for 25 min and got three distinct peaks which we then proceeded to run an HPLC method on.

===Sourcing turmeric===
In order to proceed with the proposed research project, we needed to purchase turmeric from a reliable, science-conscious company. We chose to order 1 lb of organic and 1 lb of non-organic turmeric from Starwest Botanicals [http://www.starwest-botanicals.com/ here].

===Overview of Proposed Methodology===
''Procedure is modeled after the following study:'' Kiuchi, F, Goyto, Y, Sugimoto, N, Akao, N, Kondo, K, Tsuda, Y. Nematocidal activity of turmeric: synergistic action of curcuminoids. Chem Pharm Bull (Tokyo) 1993;41:1640-3.

*Extract curcuminoids with MeOH under soxhlet extraction.
*Partition MeOH supernatant with ethyl acetate (AcOEt) and water for purification purposes.
*Fractionate AcOEt extract by silica gel column chromatography to yield AcOEt curcumin eluents.
*Fractionate AcOEt eluents by silica gel TLC using chloroform: acetone = 8:1 as the solvent system.
*Purify each eluent by removing organic solvent (?) and analyzing under HPLC.

===Extracting Curcuminoids under Soxhlet Distillation===
*Weigh out 150.00g of turmeric powder with an analytical balance.
*Measure out 300.00mL MeOH using a burette.
*Place above compound and solvent in stillpot of soxhlet apparatus.
*Carefully turn on heat source and water source.
*Run extraction for 6-7 hours.

==Written Report==

===1. Descriptive information===
'''Isolation and Purification of Curcuminoids from Tumeric Plant ''Curcuma Longa'''''

Stephanie Saey, Nadia Ayala, and Bradley E. Sturgeon

Special thanks to Michael Prinsell and Broddie Davis

Research work documented in lab notebooks, Stephanie Saey pages 1-13 and Nadia Ayala pages [insert NA page].
===2. Abstract ===
Turmeric, Curcuma longa, is a traditional Indian spice with potential chemotherapeutic, pharmacological, anti-inflammatory, and antioxidative properties. The active component in turmeric, known as curcumin, is what allows the plant to house its well-documented health benefits. Curcumin has three derivatives of different molecular structures: curcumin (I), demethoxycurcumin (II), and bisdemethoxycurcumin (III). Taken together, these structures are referred to as curcuminoids. Due to the safety and availability of turmeric, many studies have reported techniques for isolating and purifying the curcuminoids through methods such as extraction coupled with column chromatography. However, to date, no such methods have been used to prepare large amounts of each curcuminoid individually. Curcumin I is only available in small amounts, while II and III remain unavailable commercially. The current research project aims to successfully isolate (methanol in soxhlet), purify (flash chromatography/HPLC), and characterize (NMR) the curcuminoids (I/II/III) in amounts large enough for further investigation on its radicalisation properties.
===3. Introduction===

Turmeric, ''Curcuma longa,'' is a traditional Indian spice with potential chemotherapeutic, pharmacological, anti-inflammatory, and antioxidative properties (Goel, 2008). The active component in turmeric, known as curcumin, is what allows the plant to house its well-documented health benefits. Curcumin has three derivatives (I/II/III) of different molecular structures: curcumin I, demethoxycurcumin (II), and bisdemethoxycurcumin (III). Taken together, these structures are referred to as curcuminoids. Due to the safety and availability of turmeric, many studies have reported techniques for isolating and purifying the curcuminoids through methods such as extraction coupled with column chromatography (Jayaprakasha). However, to date, no such methods have been used to prepare large amounts of each curcuminoid individually. Curcumin I is only available in small amounts, while II and III remain unavailable commercially. The current research project aims to successfully isolate (methanol in soxhlet), purify (flash chromatography/HPLC), and characterize (NMR) the curcuminoids in amounts large enough for further investigation.

===4. Background from earlier reports===
Stephanie Saey and Nadia Ayala are the first Monmouth College Chemistry students to work on this specific research project involving turmeric.

===5. Experimental===
:'''Sourcing turmeric:'''
In order to proceed with the proposed research project, we needed to purchase turmeric from a reliable, science-conscious company. We chose to order 1 lb of organic and 1 lb of non-organic turmeric from Starwest Botanicals [http://www.starwest-botanicals.com/ here].

:'''Methanol in Soxhlet Extraction:'''
::1. Soxhlet apparatus was set up for extraction, as pictured in Image A.
::2. Approximately 76 grams of organic turmeric from Starwest Botanicals was added to the thimble.
::3. Approximately 350 mL of MeOH was added to the stillpot and a few boiling stones were added.
::4. Heat source and water source were turned on.
::5. Extraction was ran for 6 hours.

[[File:Soxhlet.png|200px|thumb|left|Image A: soxhlet extraction]]

:'''Removal of MeOH and Impurities:'''
::1. MeOH/curcuminoid mixture in the stillpot (from extraction) was transferred to an 1000mL separatory funnel.
::2. 275 mL of ethyl acetate (EtOAc) and 150 mL of water were added to the funnel, along with 150 mL brine solution.
::3. The stopcock was added to the funnel and the mixture was inverted and vented multiple times for 1 minute.
::4. The stopcock was removed, allowing the mixture to separate into two separate phases, one containing EtOAc and curcuminoids, and the other containing MeOH, water, and impurities. Image B displays this separated mixture.
::5. The EtOAc/curcuminioid phase (top layer) was drained into a labeled glassware container.
::6.The bottom layer was readded to the separatory funnel along with 150 mL of EtAOc, 150 mL water, and 100 mL brime.
::7. Again, the stopcock was added to the funnel and the mixture was inverted and vented multiple times for 1 minute.
::8. The stopcock was removed and the mixture was allowed to separate.
::9. The top layer was added to the labeled glassware and the bottom layer was discarded.
[[File:separatoryfunnel.jpg|200px|thumb|left|Image B: Separation of EtOAc and curcuminoids (top layer) from MeOH and impurities (bottom layer)]]

:'''Thin Layer Chromatography'''
::A 5x10cm RediSep silica TLC plate was used to run TLC on the EtOAc/curcuminoid extract. First, 45 mL of the extract was diluted in 25 mL of ethyl acetate. A thin pencil line was drawn horizontally approximately 1.5 cm from the bottom of the plate. Two dots of the diluted extract were placed 4 cm apart on this line in order to conduct two trials on the same plate. An 8:1 chloroform:acetone solvent mixture was used. The fractions were allowed to follow the solvent up the plate and the results were imaged as shown in Figure A. Final Rf values were recorded.

:'''Flash Column Chromatography:'''
::A 15.5mL(13g) gold C18 reverse phase Column was used to run 1mL of the EtOAc/curcuminoid extract. This column uses acetyl nitrile and acidic(TFA 0.1%) water to run the liquid phase through the column.
:: A gradient was ran for 25 min that yielded three distinct peaks. The eluents for each peak were collected and stored in separate viles.

:'''High Pressure Liquid Chromatography (HPLC):'''
::Each 1mL of each Flash eluent was added to a separate HPLC vile and labeled to be compared to the original Curcumin solution extracted through Soxhlet extraction.
[[File:Curcumin HPLC vial image.jpg|400px|thumb|left|Image c: HPLC vials]]

:'''Rotovaporization''':
:: Each fraction of curcumin had its solvent extracted via rotovaportization. The hot bathwater was held at 32 degrees Celsius for about an hour and a half or until completely dry. Spin speed was between 9 and 10, and adjustments in the angle of inundation of the round bottom flask containing the curcumin fractions was adjusted as the solvent was vaporized.

:'''Nuclear Magnetic Resonance (NMR) Testing:'''
::About 1mg of each curcumin was added to separate NMR tubes. 1mL of duderated DMSO was added to resuspend the curcumin samples to be used in the 400Hz NMR at Knox College.

===6. Results===
::'''TLC Results'''
:TLC results from two trials of the diluted EtOAc/curcuminoid mixture (prepared from MeOH in soxhlet extraction) are shown in Figure A. A RediSep silica TLC plate was used with an 8:1 chloroform:acetone solvent front. Three distinct lines are visible for each trial, consistent with the three different curcuminoid structures. Rf values are as follows (same for each trial): .25, .375, .5.
[[File:Example.jpg|200px|thumb|none|Figure A: TLC Results]]

::'''Flash Chromatography Results'''
:With the use of a C18 reverse phase column we successfully separated Curcumin I, II, and III from the original solution. The base line resolution of the curcumin fractions wasadjusted by increasing the run time from the original C18 procedure from 15 min to 25 min.
[[Media:Curcumin Flash graph.pdf|Curcumin Flash Chromatography Image]]

::'''HPLC Results'''
:Having separated the three curcumin through flash chromatography, purity of the sample was tested using flash chromatography using the original solution as a basis of comparison.
[[File:T2 Curcumin HPLC chromatogram.jpg|500px|thumb|none|HPLC chromatogram]]

::'''NMR Results'''
:The concentration of our curcumins where not significant enough to have transmitted a high signal in the NMR for what we precive to be curcumin II and Curcumin III. However, the NMR results for Curcumin II are seen below.
[[File:Curcumin 1 NMR.jpg|500px|thumb|none|NMR Curcumin I Trial 1]]

===7. Discussion===
The flash chromatography and HPLC data have been consistent with the literature regarding the existence of curcumin's three different molecular structures. However, characterization of curcumins through NMR have led to further questions on the structures present within each curcumin sample. Further interpretation and testing are been necessary to have certainty in the purity of our samples as well as specific characterization of each fraction.

===8. Conclusions===
No final conclusions have been reached at this point, as we are still waiting to collect NMR results. The results of the NMR should tell us how successful our method was in isolating and purifying curcuminoids I,II, and III.

===9. Future Directions===
We plan to continue this research project into the second semester of the 2016-17 school year (and beyond). First on our agenda will be to collect NMR data on the three separate curcuminoids that were rotovapped from the separate flash eluents. NMR data will be collected both at Monmouth College and Knox College. Results will indicate the success of our methods, and lead us to either additional method development or mass quantification using the latest method.

===10. Literature references===

Goel A., Kunnumakkara A.B., Aggarwal B.B. (2008). Curcumin as ‘curecumin’: from kitchen to clinic. ''Biochem Pharmacology.'' pp. 787–809, doi:10.1016/j.bcp.2007.08.016

Jayaprakasha, G. K., Gowda, G. A. N., Marquez, S., & Patil, B. S. (2013). Rapid separation and quantitation of curcuminoids combining pseudo two dimensional liquid flash chromatography and NMR spectroscopy. ''Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences'', pp. 937, doi:10.1016/j.jchromb.2013.08.011

Kiuchi, F, Goyto, Y, Sugimoto, N, Akao, N, Kondo, K, Tsuda, Y. Nematocidal activity of turmeric: synergistic action of curcuminoids. (1993). ''Chem Pharm Bull (Tokyo)'' pp. 1640-3.

===10. Signature===
Two copies of the report will be signed and dated and turned in to the Faculty Research Advisor and archived by the Research Coordinator.

File:Curcumin 1 NMR.jpg

2017-05-03T21:20:05Z

Nayala:

File:Curcumin HPLC vial image.jpg

2017-05-03T21:01:30Z

Nayala:

Curcumin Research

2017-05-03T20:48:01Z

Nayala: /* 5. Results */

You have reached the page dedicated to the research of curcumin, a secondary plant metabolite and biophenol of interest to the Sturgeon Research Project. This page was created and is maintained by Stephanie Saey and Nadia Ayala.

==Curcumin Research Initiative==

===Preparation of Curcuminoid Standards from Turmeric Plant===
Curcumin is a secondary plant metabolite of the turmeric herb ''Curcuma longa''. The term "curcumin" has been used to refer to the bioactive molecule, but in reality curcumin has three derivatives (I/II/III) of different molecular structures: curcumin (I), demethoxycurcumin (II), and bisdemethoxycurcumin (III). Together, the aforementioned compounds are known as curcuminoids. A review of literature suggests that curcuminoids have chemotherapeutic, antioxidant, and anti-inflammatory activity, among other uses to be discovered. However, difficulty arises when seeking to study these curcuminoids individually. Curcumin I is commercially available (10 mg for $150), while demethoxycurcumin (II) and bisdemethoxycurcumin (III) are not commercially available. Our current research project aims to isolate (methanol in soxhlet), purify (automated Flash/HPLC), and characterize (NMR) the curcuminoids for further investigation.

===The Three Curcuminoids===
Curcumin I (Main Curcuminoid, in picture below), demethoxycurcumin (curcumin II), and bisdemethoxycurcumin (curcumin III) are the three major curcuminoids present in turmeric and of interest to this study. Their respective chemical structures are depicted below:

[[Media:Curcuminoid Analogs with Potent Activity.pdf|Curcuminoid Analogs with Potent Activity (Article)]]

[[File:Curcumin.jpg|400px|thumb|none|Curcumin I, II, and III]]

===Flash Chromatography Data===

We used a 15.5mL C18 Column to run 1mL of our Curcumin extract. This column uses acetyl nitrile and water to run the liquid phase through the column. We ran a gradient for 25 min and got three distinct peaks which we then proceeded to run an HPLC method on.

===Sourcing turmeric===
In order to proceed with the proposed research project, we needed to purchase turmeric from a reliable, science-conscious company. We chose to order 1 lb of organic and 1 lb of non-organic turmeric from Starwest Botanicals [http://www.starwest-botanicals.com/ here].

===Overview of Proposed Methodology===
''Procedure is modeled after the following study:'' Kiuchi, F, Goyto, Y, Sugimoto, N, Akao, N, Kondo, K, Tsuda, Y. Nematocidal activity of turmeric: synergistic action of curcuminoids. Chem Pharm Bull (Tokyo) 1993;41:1640-3.

*Extract curcuminoids with MeOH under soxhlet extraction.
*Partition MeOH supernatant with ethyl acetate (AcOEt) and water for purification purposes.
*Fractionate AcOEt extract by silica gel column chromatography to yield AcOEt curcumin eluents.
*Fractionate AcOEt eluents by silica gel TLC using chloroform: acetone = 8:1 as the solvent system.
*Purify each eluent by removing organic solvent (?) and analyzing under HPLC.

===Extracting Curcuminoids under Soxhlet Distillation===
*Weigh out 150.00g of turmeric powder with an analytical balance.
*Measure out 300.00mL MeOH using a burette.
*Place above compound and solvent in stillpot of soxhlet apparatus.
*Carefully turn on heat source and water source.
*Run extraction for 6-7 hours.

==Written Report==

===1. Descriptive information===
'''Isolation and Purification of Curcuminoids from Tumeric Plant ''Curcuma Longa'''''

Stephanie Saey, Nadia Ayala, and Bradley E. Sturgeon#

Special thanks to Michael Prinsell and Broddie Davis

Research work documented in lab notebooks, Stephanie Saey pages 1-13 and Nadia Ayala pages [insert NA page#].

===2. Introduction===

Turmeric, ''Curcuma longa,'' is a traditional Indian spice with potential chemotherapeutic, pharmacological, anti-inflammatory, and antioxidative properties (Goel, 2008). The active component in turmeric, known as curcumin, is what allows the plant to house its well-documented health benefits. Curcumin has three derivatives (I/II/III) of different molecular structures: curcumin I, demethoxycurcumin (II), and bisdemethoxycurcumin (III). Taken together, these structures are referred to as curcuminoids. Due to the safety and availability of turmeric, many studies have reported techniques for isolating and purifying the curcuminoids through methods such as extraction coupled with column chromatography (Jayaprakasha). However, to date, no such methods have been used to prepare large amounts of each curcuminoid individually. Curcumin I is only available in small amounts, while II and III remain unavailable commercially. The current research project aims to successfully isolate (methanol in soxhlet), purify (flash chromatography/HPLC), and characterize (NMR) the curcuminoids in amounts large enough for further investigation.

===3. Background from earlier reports===
Stephanie Saey and Nadia Ayala are the first Monmouth College Chemistry students to work on this specific research project involving turmeric.

===4. Experimental===
:'''Sourcing turmeric:'''
In order to proceed with the proposed research project, we needed to purchase turmeric from a reliable, science-conscious company. We chose to order 1 lb of organic and 1 lb of non-organic turmeric from Starwest Botanicals [http://www.starwest-botanicals.com/ here].

:'''Methanol in Soxhlet Extraction:'''
::1. Soxhlet apparatus was set up for extraction, as pictured in Image A.
::2. Approximately 76 grams of organic turmeric from Starwest Botanicals was added to the thimble.
::3. Approximately 350 mL of MeOH was added to the stillpot and a few boiling stones were added.
::4. Heat source and water source were turned on.
::5. Extraction was ran for 6 hours.

[[File:Soxhlet.png|200px|thumb|left|Image A: soxhlet extraction]]

:'''Removal of MeOH and Impurities:'''
::1. MeOH/curcuminoid mixture in the stillpot (from extraction) was transferred to an 1000mL separatory funnel.
::2. 275 mL of ethyl acetate (EtOAc) and 150 mL of water were added to the funnel, along with 150 mL brine solution.
::3. The stopcock was added to the funnel and the mixture was inverted and vented multiple times for 1 minute.
::4. The stopcock was removed, allowing the mixture to separate into two separate phases, one containing EtOAc and curcuminoids, and the other containing MeOH, water, and impurities. Image B displays this separated mixture.
::5. The EtOAc/curcuminioid phase (top layer) was drained into a labeled glassware container.
::6.The bottom layer was readded to the separatory funnel along with 150 mL of EtAOc, 150 mL water, and 100 mL brime.
::7. Again, the stopcock was added to the funnel and the mixture was inverted and vented multiple times for 1 minute.
::8. The stopcock was removed and the mixture was allowed to separate.
::9. The top layer was added to the labeled glassware and the bottom layer was discarded.
[[File:separatoryfunnel.jpg|200px|thumb|left|Image B: Separation of EtOAc and curcuminoids (top layer) from MeOH and impurities (bottom layer)]]

:'''Thin Layer Chromatography'''
::A 5x10cm RediSep silica TLC plate was used to run TLC on the EtOAc/curcuminoid extract. First, 45 mL of the extract was diluted in 25 mL of ethyl acetate. A thin pencil line was drawn horizontally approximately 1.5 cm from the bottom of the plate. Two dots of the diluted extract were placed 4 cm apart on this line in order to conduct two trials on the same plate. An 8:1 chloroform:acetone solvent mixture was used. The fractions were allowed to follow the solvent up the plate and the results were imaged as shown in Figure A. Final Rf values were recorded.

:'''Flash Column Chromatography:'''
::A 15.5mL(13g) C18 Column was used to run 1mL of the EtOAc/curcuminoid extract. This column uses acetyl nitrile and water to run the liquid phase through the column.
:: A gradient was ran for 25 min that yielded three distinct peaks. The eluents for each peak were collected and stored in separate viles.

:'''High Pressure Liquid Chromatography (HPLC):'''
::Each Flash eluent was added to a separate HPLC vile. Additionally, a "standard" was made by diluting 1 mL of the original EtOAc/curcuminoid mixture in 4 mL EtOAc. 1mL of the resulting dilution was placed into its on HPLC vile. The HPLC was run for 20 minutes under bes method .... [methods for this portion to be continued when I am able to refer back to the HPLC data...the HPLC is currently under repair].

:'''Preparation for Nuclear Magnetic Resonance (NMR) Testing:'''
::The flash chromatography eluents were each added to their own 50 mL rbf and separately rotovapped for approximately 1 hour and 20 min to remove solvent.

===5. Results===
::'''TLC Results'''
:TLC results from two trials of the diluted EtOAc/curcuminoid mixture (prepared from MeOH in soxhlet extraction) are shown in Figure A. A RediSep silica TLC plate was used with an 8:1 chloroform:acetone solvent front. Three distinct lines are visible for each trial, consistent with the three different curcuminoid structures. Rf values are as follows (same for each trial): .25, .375, .5.
[[File:Example.jpg|200px|thumb|none|Figure A: TLC Results]]

::'''Flash Chromatography Results'''
With the use of a C18 reverse phase column we successfully separated Curcumin I, II, and III from the original solution. The base line resolution of the curcumin fractions wasadjusted by increasing the run time from the original C18 procedure from 15 min to 25 min.
[[Media:Curcumin Flash graph.pdf|Curcumin Flash Chromatography Image]]

::'''HPLC Results'''
having separated the three curcumin through flash chromatography, purity of the sample was tested using flash chromatography using the original solution as a basis of comparison.
[[File:T2 Curcumin HPLC chromatogram.jpg|500px|thumb|none|HPLC chromatogram]]

===6. Discussion===
The flash chromatography and HPLC data have been consistent with the literature regarding the existence of curcumin's three different molecular structures. However, characterization of curcumins through NMR have led to further questions on the structures present within each curcumin sample. Further interpretation and testing are been necessary to have certainty in the purity of our samples as well as specific characterization of each fraction.

===7. Conclusions===
No final conclusions have been reached at this point, as we are still waiting to collect NMR results. The results of the NMR should tell us how successful our method was in isolating and purifying curcuminoids I,II, and III.

===8. Future Directions===
We plan to continue this research project into the second semester of the 2016-17 school year (and beyond). First on our agenda will be to collect NMR data on the three separate curcuminoids that were rotovapped from the separate flash eluents. NMR data will be collected both at Monmouth College and Knox College. Results will indicate the success of our methods, and lead us to either additional method development or mass quantification using the latest method.

===9. Literature references===

Goel A., Kunnumakkara A.B., Aggarwal B.B. (2008). Curcumin as ‘curecumin’: from kitchen to clinic. ''Biochem Pharmacology.'' pp. 787–809, doi:10.1016/j.bcp.2007.08.016

Jayaprakasha, G. K., Gowda, G. A. N., Marquez, S., & Patil, B. S. (2013). Rapid separation and quantitation of curcuminoids combining pseudo two dimensional liquid flash chromatography and NMR spectroscopy. ''Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences'', pp. 937, doi:10.1016/j.jchromb.2013.08.011

Kiuchi, F, Goyto, Y, Sugimoto, N, Akao, N, Kondo, K, Tsuda, Y. Nematocidal activity of turmeric: synergistic action of curcuminoids. (1993). ''Chem Pharm Bull (Tokyo)'' pp. 1640-3.

===10. Signature===
Two copies of the report will be signed and dated and turned in to the Faculty Research Advisor and archived by the Research Coordinator.

Curcumin Research

2017-05-03T20:44:50Z

Nayala: /* 6. Discussion */

You have reached the page dedicated to the research of curcumin, a secondary plant metabolite and biophenol of interest to the Sturgeon Research Project. This page was created and is maintained by Stephanie Saey and Nadia Ayala.

==Curcumin Research Initiative==

===Preparation of Curcuminoid Standards from Turmeric Plant===
Curcumin is a secondary plant metabolite of the turmeric herb ''Curcuma longa''. The term "curcumin" has been used to refer to the bioactive molecule, but in reality curcumin has three derivatives (I/II/III) of different molecular structures: curcumin (I), demethoxycurcumin (II), and bisdemethoxycurcumin (III). Together, the aforementioned compounds are known as curcuminoids. A review of literature suggests that curcuminoids have chemotherapeutic, antioxidant, and anti-inflammatory activity, among other uses to be discovered. However, difficulty arises when seeking to study these curcuminoids individually. Curcumin I is commercially available (10 mg for $150), while demethoxycurcumin (II) and bisdemethoxycurcumin (III) are not commercially available. Our current research project aims to isolate (methanol in soxhlet), purify (automated Flash/HPLC), and characterize (NMR) the curcuminoids for further investigation.

===The Three Curcuminoids===
Curcumin I (Main Curcuminoid, in picture below), demethoxycurcumin (curcumin II), and bisdemethoxycurcumin (curcumin III) are the three major curcuminoids present in turmeric and of interest to this study. Their respective chemical structures are depicted below:

[[Media:Curcuminoid Analogs with Potent Activity.pdf|Curcuminoid Analogs with Potent Activity (Article)]]

[[File:Curcumin.jpg|400px|thumb|none|Curcumin I, II, and III]]

===Flash Chromatography Data===

We used a 15.5mL C18 Column to run 1mL of our Curcumin extract. This column uses acetyl nitrile and water to run the liquid phase through the column. We ran a gradient for 25 min and got three distinct peaks which we then proceeded to run an HPLC method on.

===Sourcing turmeric===
In order to proceed with the proposed research project, we needed to purchase turmeric from a reliable, science-conscious company. We chose to order 1 lb of organic and 1 lb of non-organic turmeric from Starwest Botanicals [http://www.starwest-botanicals.com/ here].

===Overview of Proposed Methodology===
''Procedure is modeled after the following study:'' Kiuchi, F, Goyto, Y, Sugimoto, N, Akao, N, Kondo, K, Tsuda, Y. Nematocidal activity of turmeric: synergistic action of curcuminoids. Chem Pharm Bull (Tokyo) 1993;41:1640-3.

*Extract curcuminoids with MeOH under soxhlet extraction.
*Partition MeOH supernatant with ethyl acetate (AcOEt) and water for purification purposes.
*Fractionate AcOEt extract by silica gel column chromatography to yield AcOEt curcumin eluents.
*Fractionate AcOEt eluents by silica gel TLC using chloroform: acetone = 8:1 as the solvent system.
*Purify each eluent by removing organic solvent (?) and analyzing under HPLC.

===Extracting Curcuminoids under Soxhlet Distillation===
*Weigh out 150.00g of turmeric powder with an analytical balance.
*Measure out 300.00mL MeOH using a burette.
*Place above compound and solvent in stillpot of soxhlet apparatus.
*Carefully turn on heat source and water source.
*Run extraction for 6-7 hours.

==Written Report==

===1. Descriptive information===
'''Isolation and Purification of Curcuminoids from Tumeric Plant ''Curcuma Longa'''''

Stephanie Saey, Nadia Ayala, and Bradley E. Sturgeon#

Special thanks to Michael Prinsell and Broddie Davis

Research work documented in lab notebooks, Stephanie Saey pages 1-13 and Nadia Ayala pages [insert NA page#].

===2. Introduction===

Turmeric, ''Curcuma longa,'' is a traditional Indian spice with potential chemotherapeutic, pharmacological, anti-inflammatory, and antioxidative properties (Goel, 2008). The active component in turmeric, known as curcumin, is what allows the plant to house its well-documented health benefits. Curcumin has three derivatives (I/II/III) of different molecular structures: curcumin I, demethoxycurcumin (II), and bisdemethoxycurcumin (III). Taken together, these structures are referred to as curcuminoids. Due to the safety and availability of turmeric, many studies have reported techniques for isolating and purifying the curcuminoids through methods such as extraction coupled with column chromatography (Jayaprakasha). However, to date, no such methods have been used to prepare large amounts of each curcuminoid individually. Curcumin I is only available in small amounts, while II and III remain unavailable commercially. The current research project aims to successfully isolate (methanol in soxhlet), purify (flash chromatography/HPLC), and characterize (NMR) the curcuminoids in amounts large enough for further investigation.

===3. Background from earlier reports===
Stephanie Saey and Nadia Ayala are the first Monmouth College Chemistry students to work on this specific research project involving turmeric.

===4. Experimental===
:'''Sourcing turmeric:'''
In order to proceed with the proposed research project, we needed to purchase turmeric from a reliable, science-conscious company. We chose to order 1 lb of organic and 1 lb of non-organic turmeric from Starwest Botanicals [http://www.starwest-botanicals.com/ here].

:'''Methanol in Soxhlet Extraction:'''
::1. Soxhlet apparatus was set up for extraction, as pictured in Image A.
::2. Approximately 76 grams of organic turmeric from Starwest Botanicals was added to the thimble.
::3. Approximately 350 mL of MeOH was added to the stillpot and a few boiling stones were added.
::4. Heat source and water source were turned on.
::5. Extraction was ran for 6 hours.

[[File:Soxhlet.png|200px|thumb|left|Image A: soxhlet extraction]]

:'''Removal of MeOH and Impurities:'''
::1. MeOH/curcuminoid mixture in the stillpot (from extraction) was transferred to an 1000mL separatory funnel.
::2. 275 mL of ethyl acetate (EtOAc) and 150 mL of water were added to the funnel, along with 150 mL brine solution.
::3. The stopcock was added to the funnel and the mixture was inverted and vented multiple times for 1 minute.
::4. The stopcock was removed, allowing the mixture to separate into two separate phases, one containing EtOAc and curcuminoids, and the other containing MeOH, water, and impurities. Image B displays this separated mixture.
::5. The EtOAc/curcuminioid phase (top layer) was drained into a labeled glassware container.
::6.The bottom layer was readded to the separatory funnel along with 150 mL of EtAOc, 150 mL water, and 100 mL brime.
::7. Again, the stopcock was added to the funnel and the mixture was inverted and vented multiple times for 1 minute.
::8. The stopcock was removed and the mixture was allowed to separate.
::9. The top layer was added to the labeled glassware and the bottom layer was discarded.
[[File:separatoryfunnel.jpg|200px|thumb|left|Image B: Separation of EtOAc and curcuminoids (top layer) from MeOH and impurities (bottom layer)]]

:'''Thin Layer Chromatography'''
::A 5x10cm RediSep silica TLC plate was used to run TLC on the EtOAc/curcuminoid extract. First, 45 mL of the extract was diluted in 25 mL of ethyl acetate. A thin pencil line was drawn horizontally approximately 1.5 cm from the bottom of the plate. Two dots of the diluted extract were placed 4 cm apart on this line in order to conduct two trials on the same plate. An 8:1 chloroform:acetone solvent mixture was used. The fractions were allowed to follow the solvent up the plate and the results were imaged as shown in Figure A. Final Rf values were recorded.

:'''Flash Column Chromatography:'''
::A 15.5mL(13g) C18 Column was used to run 1mL of the EtOAc/curcuminoid extract. This column uses acetyl nitrile and water to run the liquid phase through the column.
:: A gradient was ran for 25 min that yielded three distinct peaks. The eluents for each peak were collected and stored in separate viles.

:'''High Pressure Liquid Chromatography (HPLC):'''
::Each Flash eluent was added to a separate HPLC vile. Additionally, a "standard" was made by diluting 1 mL of the original EtOAc/curcuminoid mixture in 4 mL EtOAc. 1mL of the resulting dilution was placed into its on HPLC vile. The HPLC was run for 20 minutes under bes method .... [methods for this portion to be continued when I am able to refer back to the HPLC data...the HPLC is currently under repair].

:'''Preparation for Nuclear Magnetic Resonance (NMR) Testing:'''
::The flash chromatography eluents were each added to their own 50 mL rbf and separately rotovapped for approximately 1 hour and 20 min to remove solvent.

===5. Results===
::'''TLC Results'''
:TLC results from two trials of the diluted EtOAc/curcuminoid mixture (prepared from MeOH in soxhlet extraction) are shown in Figure A. A RediSep silica TLC plate was used with an 8:1 chloroform:acetone solvent front. Three distinct lines are visible for each trial, consistent with the three different curcuminoid structures. Rf values are as follows (same for each trial): .25, .375, .5.
[[File:Example.jpg|200px|thumb|none|Figure A: TLC Results]]

::'''Flash Chromatography Results'''
With the use of a C18 reverse phase column we successfully separated Curcumin I, II, and III from the original solution. The base line resolution of the curcumin fractions wasadjusted by increasing the run time from the original C18 procedure from 15 min to 25 min.
[[Media:Curcumin Flash graph.pdf|Curcumin Flash Chromatography Image]]

::'''HPLC Results'''
having separated the three curcumin through flash chromatography, purity of the sample was tested using flash chromatography using the original solution as a basis of comparison.
[[File:T2 Curcumin HPLC chromatogram.jpg|200px|thumb|none|HPLC chromatogram]]

===6. Discussion===
The flash chromatography and HPLC data have been consistent with the literature regarding the existence of curcumin's three different molecular structures. However, characterization of curcumins through NMR have led to further questions on the structures present within each curcumin sample. Further interpretation and testing are been necessary to have certainty in the purity of our samples as well as specific characterization of each fraction.

===7. Conclusions===
No final conclusions have been reached at this point, as we are still waiting to collect NMR results. The results of the NMR should tell us how successful our method was in isolating and purifying curcuminoids I,II, and III.

===8. Future Directions===
We plan to continue this research project into the second semester of the 2016-17 school year (and beyond). First on our agenda will be to collect NMR data on the three separate curcuminoids that were rotovapped from the separate flash eluents. NMR data will be collected both at Monmouth College and Knox College. Results will indicate the success of our methods, and lead us to either additional method development or mass quantification using the latest method.

===9. Literature references===

Goel A., Kunnumakkara A.B., Aggarwal B.B. (2008). Curcumin as ‘curecumin’: from kitchen to clinic. ''Biochem Pharmacology.'' pp. 787–809, doi:10.1016/j.bcp.2007.08.016

Jayaprakasha, G. K., Gowda, G. A. N., Marquez, S., & Patil, B. S. (2013). Rapid separation and quantitation of curcuminoids combining pseudo two dimensional liquid flash chromatography and NMR spectroscopy. ''Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences'', pp. 937, doi:10.1016/j.jchromb.2013.08.011

Kiuchi, F, Goyto, Y, Sugimoto, N, Akao, N, Kondo, K, Tsuda, Y. Nematocidal activity of turmeric: synergistic action of curcuminoids. (1993). ''Chem Pharm Bull (Tokyo)'' pp. 1640-3.

===10. Signature===
Two copies of the report will be signed and dated and turned in to the Faculty Research Advisor and archived by the Research Coordinator.

Curcumin Research

2017-05-03T20:40:44Z

Nayala: /* 5. Results */

You have reached the page dedicated to the research of curcumin, a secondary plant metabolite and biophenol of interest to the Sturgeon Research Project. This page was created and is maintained by Stephanie Saey and Nadia Ayala.

==Curcumin Research Initiative==

===Preparation of Curcuminoid Standards from Turmeric Plant===
Curcumin is a secondary plant metabolite of the turmeric herb ''Curcuma longa''. The term "curcumin" has been used to refer to the bioactive molecule, but in reality curcumin has three derivatives (I/II/III) of different molecular structures: curcumin (I), demethoxycurcumin (II), and bisdemethoxycurcumin (III). Together, the aforementioned compounds are known as curcuminoids. A review of literature suggests that curcuminoids have chemotherapeutic, antioxidant, and anti-inflammatory activity, among other uses to be discovered. However, difficulty arises when seeking to study these curcuminoids individually. Curcumin I is commercially available (10 mg for $150), while demethoxycurcumin (II) and bisdemethoxycurcumin (III) are not commercially available. Our current research project aims to isolate (methanol in soxhlet), purify (automated Flash/HPLC), and characterize (NMR) the curcuminoids for further investigation.

===The Three Curcuminoids===
Curcumin I (Main Curcuminoid, in picture below), demethoxycurcumin (curcumin II), and bisdemethoxycurcumin (curcumin III) are the three major curcuminoids present in turmeric and of interest to this study. Their respective chemical structures are depicted below:

[[Media:Curcuminoid Analogs with Potent Activity.pdf|Curcuminoid Analogs with Potent Activity (Article)]]

[[File:Curcumin.jpg|400px|thumb|none|Curcumin I, II, and III]]

===Flash Chromatography Data===

We used a 15.5mL C18 Column to run 1mL of our Curcumin extract. This column uses acetyl nitrile and water to run the liquid phase through the column. We ran a gradient for 25 min and got three distinct peaks which we then proceeded to run an HPLC method on.

===Sourcing turmeric===
In order to proceed with the proposed research project, we needed to purchase turmeric from a reliable, science-conscious company. We chose to order 1 lb of organic and 1 lb of non-organic turmeric from Starwest Botanicals [http://www.starwest-botanicals.com/ here].

===Overview of Proposed Methodology===
''Procedure is modeled after the following study:'' Kiuchi, F, Goyto, Y, Sugimoto, N, Akao, N, Kondo, K, Tsuda, Y. Nematocidal activity of turmeric: synergistic action of curcuminoids. Chem Pharm Bull (Tokyo) 1993;41:1640-3.

*Extract curcuminoids with MeOH under soxhlet extraction.
*Partition MeOH supernatant with ethyl acetate (AcOEt) and water for purification purposes.
*Fractionate AcOEt extract by silica gel column chromatography to yield AcOEt curcumin eluents.
*Fractionate AcOEt eluents by silica gel TLC using chloroform: acetone = 8:1 as the solvent system.
*Purify each eluent by removing organic solvent (?) and analyzing under HPLC.

===Extracting Curcuminoids under Soxhlet Distillation===
*Weigh out 150.00g of turmeric powder with an analytical balance.
*Measure out 300.00mL MeOH using a burette.
*Place above compound and solvent in stillpot of soxhlet apparatus.
*Carefully turn on heat source and water source.
*Run extraction for 6-7 hours.

==Written Report==

===1. Descriptive information===
'''Isolation and Purification of Curcuminoids from Tumeric Plant ''Curcuma Longa'''''

Stephanie Saey, Nadia Ayala, and Bradley E. Sturgeon#

Special thanks to Michael Prinsell and Broddie Davis

Research work documented in lab notebooks, Stephanie Saey pages 1-13 and Nadia Ayala pages [insert NA page#].

===2. Introduction===

Turmeric, ''Curcuma longa,'' is a traditional Indian spice with potential chemotherapeutic, pharmacological, anti-inflammatory, and antioxidative properties (Goel, 2008). The active component in turmeric, known as curcumin, is what allows the plant to house its well-documented health benefits. Curcumin has three derivatives (I/II/III) of different molecular structures: curcumin I, demethoxycurcumin (II), and bisdemethoxycurcumin (III). Taken together, these structures are referred to as curcuminoids. Due to the safety and availability of turmeric, many studies have reported techniques for isolating and purifying the curcuminoids through methods such as extraction coupled with column chromatography (Jayaprakasha). However, to date, no such methods have been used to prepare large amounts of each curcuminoid individually. Curcumin I is only available in small amounts, while II and III remain unavailable commercially. The current research project aims to successfully isolate (methanol in soxhlet), purify (flash chromatography/HPLC), and characterize (NMR) the curcuminoids in amounts large enough for further investigation.

===3. Background from earlier reports===
Stephanie Saey and Nadia Ayala are the first Monmouth College Chemistry students to work on this specific research project involving turmeric.

===4. Experimental===
:'''Sourcing turmeric:'''
In order to proceed with the proposed research project, we needed to purchase turmeric from a reliable, science-conscious company. We chose to order 1 lb of organic and 1 lb of non-organic turmeric from Starwest Botanicals [http://www.starwest-botanicals.com/ here].

:'''Methanol in Soxhlet Extraction:'''
::1. Soxhlet apparatus was set up for extraction, as pictured in Image A.
::2. Approximately 76 grams of organic turmeric from Starwest Botanicals was added to the thimble.
::3. Approximately 350 mL of MeOH was added to the stillpot and a few boiling stones were added.
::4. Heat source and water source were turned on.
::5. Extraction was ran for 6 hours.

[[File:Soxhlet.png|200px|thumb|left|Image A: soxhlet extraction]]

:'''Removal of MeOH and Impurities:'''
::1. MeOH/curcuminoid mixture in the stillpot (from extraction) was transferred to an 1000mL separatory funnel.
::2. 275 mL of ethyl acetate (EtOAc) and 150 mL of water were added to the funnel, along with 150 mL brine solution.
::3. The stopcock was added to the funnel and the mixture was inverted and vented multiple times for 1 minute.
::4. The stopcock was removed, allowing the mixture to separate into two separate phases, one containing EtOAc and curcuminoids, and the other containing MeOH, water, and impurities. Image B displays this separated mixture.
::5. The EtOAc/curcuminioid phase (top layer) was drained into a labeled glassware container.
::6.The bottom layer was readded to the separatory funnel along with 150 mL of EtAOc, 150 mL water, and 100 mL brime.
::7. Again, the stopcock was added to the funnel and the mixture was inverted and vented multiple times for 1 minute.
::8. The stopcock was removed and the mixture was allowed to separate.
::9. The top layer was added to the labeled glassware and the bottom layer was discarded.
[[File:separatoryfunnel.jpg|200px|thumb|left|Image B: Separation of EtOAc and curcuminoids (top layer) from MeOH and impurities (bottom layer)]]

:'''Thin Layer Chromatography'''
::A 5x10cm RediSep silica TLC plate was used to run TLC on the EtOAc/curcuminoid extract. First, 45 mL of the extract was diluted in 25 mL of ethyl acetate. A thin pencil line was drawn horizontally approximately 1.5 cm from the bottom of the plate. Two dots of the diluted extract were placed 4 cm apart on this line in order to conduct two trials on the same plate. An 8:1 chloroform:acetone solvent mixture was used. The fractions were allowed to follow the solvent up the plate and the results were imaged as shown in Figure A. Final Rf values were recorded.

:'''Flash Column Chromatography:'''
::A 15.5mL(13g) C18 Column was used to run 1mL of the EtOAc/curcuminoid extract. This column uses acetyl nitrile and water to run the liquid phase through the column.
:: A gradient was ran for 25 min that yielded three distinct peaks. The eluents for each peak were collected and stored in separate viles.

:'''High Pressure Liquid Chromatography (HPLC):'''
::Each Flash eluent was added to a separate HPLC vile. Additionally, a "standard" was made by diluting 1 mL of the original EtOAc/curcuminoid mixture in 4 mL EtOAc. 1mL of the resulting dilution was placed into its on HPLC vile. The HPLC was run for 20 minutes under bes method .... [methods for this portion to be continued when I am able to refer back to the HPLC data...the HPLC is currently under repair].

:'''Preparation for Nuclear Magnetic Resonance (NMR) Testing:'''
::The flash chromatography eluents were each added to their own 50 mL rbf and separately rotovapped for approximately 1 hour and 20 min to remove solvent.

===5. Results===
::'''TLC Results'''
:TLC results from two trials of the diluted EtOAc/curcuminoid mixture (prepared from MeOH in soxhlet extraction) are shown in Figure A. A RediSep silica TLC plate was used with an 8:1 chloroform:acetone solvent front. Three distinct lines are visible for each trial, consistent with the three different curcuminoid structures. Rf values are as follows (same for each trial): .25, .375, .5.
[[File:Example.jpg|200px|thumb|none|Figure A: TLC Results]]

::'''Flash Chromatography Results'''
With the use of a C18 reverse phase column we successfully separated Curcumin I, II, and III from the original solution. The base line resolution of the curcumin fractions wasadjusted by increasing the run time from the original C18 procedure from 15 min to 25 min.
[[Media:Curcumin Flash graph.pdf|Curcumin Flash Chromatography Image]]

::'''HPLC Results'''
having separated the three curcumin through flash chromatography, purity of the sample was tested using flash chromatography using the original solution as a basis of comparison.
[[File:T2 Curcumin HPLC chromatogram.jpg|200px|thumb|none|HPLC chromatogram]]

===6. Discussion===
The flash chromatography and HPLC data have been consistent with the literature regarding the existence of curcumin's three different molecular structures.

===7. Conclusions===
No final conclusions have been reached at this point, as we are still waiting to collect NMR results. The results of the NMR should tell us how successful our method was in isolating and purifying curcuminoids I,II, and III.

===8. Future Directions===
We plan to continue this research project into the second semester of the 2016-17 school year (and beyond). First on our agenda will be to collect NMR data on the three separate curcuminoids that were rotovapped from the separate flash eluents. NMR data will be collected both at Monmouth College and Knox College. Results will indicate the success of our methods, and lead us to either additional method development or mass quantification using the latest method.

===9. Literature references===

Goel A., Kunnumakkara A.B., Aggarwal B.B. (2008). Curcumin as ‘curecumin’: from kitchen to clinic. ''Biochem Pharmacology.'' pp. 787–809, doi:10.1016/j.bcp.2007.08.016

Jayaprakasha, G. K., Gowda, G. A. N., Marquez, S., & Patil, B. S. (2013). Rapid separation and quantitation of curcuminoids combining pseudo two dimensional liquid flash chromatography and NMR spectroscopy. ''Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences'', pp. 937, doi:10.1016/j.jchromb.2013.08.011

Kiuchi, F, Goyto, Y, Sugimoto, N, Akao, N, Kondo, K, Tsuda, Y. Nematocidal activity of turmeric: synergistic action of curcuminoids. (1993). ''Chem Pharm Bull (Tokyo)'' pp. 1640-3.

===10. Signature===
Two copies of the report will be signed and dated and turned in to the Faculty Research Advisor and archived by the Research Coordinator.

File:T2 Curcumin HPLC chromatogram.jpg

2017-05-03T20:39:30Z

Nayala:

Curcumin Research

2017-05-03T20:33:30Z

Nayala: /* 5. Results */

You have reached the page dedicated to the research of curcumin, a secondary plant metabolite and biophenol of interest to the Sturgeon Research Project. This page was created and is maintained by Stephanie Saey and Nadia Ayala.

==Curcumin Research Initiative==

===Preparation of Curcuminoid Standards from Turmeric Plant===
Curcumin is a secondary plant metabolite of the turmeric herb ''Curcuma longa''. The term "curcumin" has been used to refer to the bioactive molecule, but in reality curcumin has three derivatives (I/II/III) of different molecular structures: curcumin (I), demethoxycurcumin (II), and bisdemethoxycurcumin (III). Together, the aforementioned compounds are known as curcuminoids. A review of literature suggests that curcuminoids have chemotherapeutic, antioxidant, and anti-inflammatory activity, among other uses to be discovered. However, difficulty arises when seeking to study these curcuminoids individually. Curcumin I is commercially available (10 mg for $150), while demethoxycurcumin (II) and bisdemethoxycurcumin (III) are not commercially available. Our current research project aims to isolate (methanol in soxhlet), purify (automated Flash/HPLC), and characterize (NMR) the curcuminoids for further investigation.

===The Three Curcuminoids===
Curcumin I (Main Curcuminoid, in picture below), demethoxycurcumin (curcumin II), and bisdemethoxycurcumin (curcumin III) are the three major curcuminoids present in turmeric and of interest to this study. Their respective chemical structures are depicted below:

[[Media:Curcuminoid Analogs with Potent Activity.pdf|Curcuminoid Analogs with Potent Activity (Article)]]

[[File:Curcumin.jpg|400px|thumb|none|Curcumin I, II, and III]]

===Flash Chromatography Data===

We used a 15.5mL C18 Column to run 1mL of our Curcumin extract. This column uses acetyl nitrile and water to run the liquid phase through the column. We ran a gradient for 25 min and got three distinct peaks which we then proceeded to run an HPLC method on.

===Sourcing turmeric===
In order to proceed with the proposed research project, we needed to purchase turmeric from a reliable, science-conscious company. We chose to order 1 lb of organic and 1 lb of non-organic turmeric from Starwest Botanicals [http://www.starwest-botanicals.com/ here].

===Overview of Proposed Methodology===
''Procedure is modeled after the following study:'' Kiuchi, F, Goyto, Y, Sugimoto, N, Akao, N, Kondo, K, Tsuda, Y. Nematocidal activity of turmeric: synergistic action of curcuminoids. Chem Pharm Bull (Tokyo) 1993;41:1640-3.

*Extract curcuminoids with MeOH under soxhlet extraction.
*Partition MeOH supernatant with ethyl acetate (AcOEt) and water for purification purposes.
*Fractionate AcOEt extract by silica gel column chromatography to yield AcOEt curcumin eluents.
*Fractionate AcOEt eluents by silica gel TLC using chloroform: acetone = 8:1 as the solvent system.
*Purify each eluent by removing organic solvent (?) and analyzing under HPLC.

===Extracting Curcuminoids under Soxhlet Distillation===
*Weigh out 150.00g of turmeric powder with an analytical balance.
*Measure out 300.00mL MeOH using a burette.
*Place above compound and solvent in stillpot of soxhlet apparatus.
*Carefully turn on heat source and water source.
*Run extraction for 6-7 hours.

==Written Report==

===1. Descriptive information===
'''Isolation and Purification of Curcuminoids from Tumeric Plant ''Curcuma Longa'''''

Stephanie Saey, Nadia Ayala, and Bradley E. Sturgeon#

Special thanks to Michael Prinsell and Broddie Davis

Research work documented in lab notebooks, Stephanie Saey pages 1-13 and Nadia Ayala pages [insert NA page#].

===2. Introduction===

Turmeric, ''Curcuma longa,'' is a traditional Indian spice with potential chemotherapeutic, pharmacological, anti-inflammatory, and antioxidative properties (Goel, 2008). The active component in turmeric, known as curcumin, is what allows the plant to house its well-documented health benefits. Curcumin has three derivatives (I/II/III) of different molecular structures: curcumin I, demethoxycurcumin (II), and bisdemethoxycurcumin (III). Taken together, these structures are referred to as curcuminoids. Due to the safety and availability of turmeric, many studies have reported techniques for isolating and purifying the curcuminoids through methods such as extraction coupled with column chromatography (Jayaprakasha). However, to date, no such methods have been used to prepare large amounts of each curcuminoid individually. Curcumin I is only available in small amounts, while II and III remain unavailable commercially. The current research project aims to successfully isolate (methanol in soxhlet), purify (flash chromatography/HPLC), and characterize (NMR) the curcuminoids in amounts large enough for further investigation.

===3. Background from earlier reports===
Stephanie Saey and Nadia Ayala are the first Monmouth College Chemistry students to work on this specific research project involving turmeric.

===4. Experimental===
:'''Sourcing turmeric:'''
In order to proceed with the proposed research project, we needed to purchase turmeric from a reliable, science-conscious company. We chose to order 1 lb of organic and 1 lb of non-organic turmeric from Starwest Botanicals [http://www.starwest-botanicals.com/ here].

:'''Methanol in Soxhlet Extraction:'''
::1. Soxhlet apparatus was set up for extraction, as pictured in Image A.
::2. Approximately 76 grams of organic turmeric from Starwest Botanicals was added to the thimble.
::3. Approximately 350 mL of MeOH was added to the stillpot and a few boiling stones were added.
::4. Heat source and water source were turned on.
::5. Extraction was ran for 6 hours.

[[File:Soxhlet.png|200px|thumb|left|Image A: soxhlet extraction]]

:'''Removal of MeOH and Impurities:'''
::1. MeOH/curcuminoid mixture in the stillpot (from extraction) was transferred to an 1000mL separatory funnel.
::2. 275 mL of ethyl acetate (EtOAc) and 150 mL of water were added to the funnel, along with 150 mL brine solution.
::3. The stopcock was added to the funnel and the mixture was inverted and vented multiple times for 1 minute.
::4. The stopcock was removed, allowing the mixture to separate into two separate phases, one containing EtOAc and curcuminoids, and the other containing MeOH, water, and impurities. Image B displays this separated mixture.
::5. The EtOAc/curcuminioid phase (top layer) was drained into a labeled glassware container.
::6.The bottom layer was readded to the separatory funnel along with 150 mL of EtAOc, 150 mL water, and 100 mL brime.
::7. Again, the stopcock was added to the funnel and the mixture was inverted and vented multiple times for 1 minute.
::8. The stopcock was removed and the mixture was allowed to separate.
::9. The top layer was added to the labeled glassware and the bottom layer was discarded.
[[File:separatoryfunnel.jpg|200px|thumb|left|Image B: Separation of EtOAc and curcuminoids (top layer) from MeOH and impurities (bottom layer)]]

:'''Thin Layer Chromatography'''
::A 5x10cm RediSep silica TLC plate was used to run TLC on the EtOAc/curcuminoid extract. First, 45 mL of the extract was diluted in 25 mL of ethyl acetate. A thin pencil line was drawn horizontally approximately 1.5 cm from the bottom of the plate. Two dots of the diluted extract were placed 4 cm apart on this line in order to conduct two trials on the same plate. An 8:1 chloroform:acetone solvent mixture was used. The fractions were allowed to follow the solvent up the plate and the results were imaged as shown in Figure A. Final Rf values were recorded.

:'''Flash Column Chromatography:'''
::A 15.5mL(13g) C18 Column was used to run 1mL of the EtOAc/curcuminoid extract. This column uses acetyl nitrile and water to run the liquid phase through the column.
:: A gradient was ran for 25 min that yielded three distinct peaks. The eluents for each peak were collected and stored in separate viles.

:'''High Pressure Liquid Chromatography (HPLC):'''
::Each Flash eluent was added to a separate HPLC vile. Additionally, a "standard" was made by diluting 1 mL of the original EtOAc/curcuminoid mixture in 4 mL EtOAc. 1mL of the resulting dilution was placed into its on HPLC vile. The HPLC was run for 20 minutes under bes method .... [methods for this portion to be continued when I am able to refer back to the HPLC data...the HPLC is currently under repair].

:'''Preparation for Nuclear Magnetic Resonance (NMR) Testing:'''
::The flash chromatography eluents were each added to their own 50 mL rbf and separately rotovapped for approximately 1 hour and 20 min to remove solvent.

===5. Results===
::'''TLC Results'''
:TLC results from two trials of the diluted EtOAc/curcuminoid mixture (prepared from MeOH in soxhlet extraction) are shown in Figure A. A RediSep silica TLC plate was used with an 8:1 chloroform:acetone solvent front. Three distinct lines are visible for each trial, consistent with the three different curcuminoid structures. Rf values are as follows (same for each trial): .25, .375, .5.
[[File:Example.jpg|200px|thumb|none|Figure A: TLC Results]]

::'''Flash Chromatography Results'''
With the use of a C18 reverse phase column we successfully separated Curcumin I, II, and III from the original solution. The base line resolution of the curcumin fractions wasadjusted by increasing the run time from the original C18 procedure from 15 min to 25 min.
[[File:Curcumin Flash graph.pdf|200px|thumb|none|Figure A: Flash Results]

::'''HPLC Results'''
[to be added]

===6. Discussion===
The flash chromatography and HPLC data have been consistent with the literature regarding the existence of curcumin's three different molecular structures.

===7. Conclusions===
No final conclusions have been reached at this point, as we are still waiting to collect NMR results. The results of the NMR should tell us how successful our method was in isolating and purifying curcuminoids I,II, and III.

===8. Future Directions===
We plan to continue this research project into the second semester of the 2016-17 school year (and beyond). First on our agenda will be to collect NMR data on the three separate curcuminoids that were rotovapped from the separate flash eluents. NMR data will be collected both at Monmouth College and Knox College. Results will indicate the success of our methods, and lead us to either additional method development or mass quantification using the latest method.

===9. Literature references===

Goel A., Kunnumakkara A.B., Aggarwal B.B. (2008). Curcumin as ‘curecumin’: from kitchen to clinic. ''Biochem Pharmacology.'' pp. 787–809, doi:10.1016/j.bcp.2007.08.016

Jayaprakasha, G. K., Gowda, G. A. N., Marquez, S., & Patil, B. S. (2013). Rapid separation and quantitation of curcuminoids combining pseudo two dimensional liquid flash chromatography and NMR spectroscopy. ''Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences'', pp. 937, doi:10.1016/j.jchromb.2013.08.011

Kiuchi, F, Goyto, Y, Sugimoto, N, Akao, N, Kondo, K, Tsuda, Y. Nematocidal activity of turmeric: synergistic action of curcuminoids. (1993). ''Chem Pharm Bull (Tokyo)'' pp. 1640-3.

===10. Signature===
Two copies of the report will be signed and dated and turned in to the Faculty Research Advisor and archived by the Research Coordinator.

File:Curcumin Flash graph.pdf

2017-05-03T20:32:05Z

Nayala:

Ayala Nadia BIOC430 S17

2017-01-29T22:12:56Z

Nayala: Created page with "Chemistry/Biochemistry Research 430 :Fall 2016 :Nadia Y. Ayala :Senior Biochemistry Major ==Research Times== T/Thur 10am-12pm : section 01 = 0.25 credit = 4 hours per week...."

Chemistry/Biochemistry Research 430
:Fall 2016
:Nadia Y. Ayala
:Senior Biochemistry Major

==Research Times==
T/Thur 10am-12pm
: section 01 = 0.25 credit = 4 hours per week.

==Proposed Research Project==
===Turmeric Curcumin Extraction and Analysis ===

===General Information===
:Advisor: Bradley Sturgeon
:Other research student collaborators: Stephany Saey

===Proposal===
Curcumin is a secondary plant metabolite of the turmeric herb Curcuma Longa. The term "curcumin" has been used to refer to the bioactive moelcule, but in reality curcumin has three different molecular structures: curcumin I, demethoxycurcumin, and bisdemethoxycurcumin. A review of curcumin studies suggest curcumin has chemotherapeutic, antioxidant, and anti-inflammatory activity, among other uses to be discovered. The current research project aims to successfully isolate and purify curcumin and its derivatives from the plant turmeric. With MeOH under reflux, the curcuminoids will be extracted and subsequently subjected to Flash Chromatography for separation. Once the curcuminoids are separated, NMR data collection will take place to compare the chemical properties of each derivative.
[[Curcumin Research|Curcumin Research]]

===Instruments to be used===
Flash Chromatography,
HPLC,
Soxhlet Reflux extraction

===References (2 minimum)===

==Writen Report==
===Enzymatic Oxidation of Biophenols: HPA===
Nadia Ayala, Benjamine Stillwell, Ian Salveson, Dr.Bradley Sturgeon

Summer 2016

===Abstract===
HPA is a model compound of a biologically active pheno, with a OH group that partakes in oxidation naturally. These phenols form radical intermediates during oxidation which are responsible for the resulting stable products. We have investigated various means of oxidation and will present data resulting from using an immobilized enzyme bioreactor. The products have been analyzed by HPLC and separated via flash chromatography.

===Instrumentation===
'''Immobilized Enzyme Bioreactor'''
This technique allowed there to be an oxidation activity that mimics a biological organism by having the Biophenols flow over the enzyme which was attached to agarose beads at different flow rates. The Flow of HPA as well as the concentration of HPA was tested and recorded order to get the most separation of products. To see the progression of the results please see the [[HPA Reactions|HPA Biophenol Reaserch Page]]

'''HPLC'''
High pressure liquid chromatography allowed us to analyze the products formed via the oxidation. The separation is based on polarity of the compound through a column and the separate combination of dimers and trimes where represented by the separate peaks.

'''Flash Chromatography'''
This technique allowed us to separate our products once identified by HPLC, using a C18 Gold column. Once products were separated through flash chromatography they were then ran through HPLC again to confirm the isolated products lined up with the original HPLC data.

'''NMR'''
H-NMR was used in the analysis of the products that where seperated through the flash chromatography.

===Research pledge===
I, Nadia Ayala, have read the Chem/Bioc 430 course syllabus and understand the general structure and expectations of the research program. The above material was prepared after consultation, and in conjunction with my research advisor.

Nadia Y. Ayala

2016-12-06T23:55:01Z

Nayala:

Nadia Y. Ayala

2016-12-06T22:13:32Z

Nayala: /* Instrumentation */

Chemistry/Biochemistry Research 430
:Fall 2016
:Nadia Y. Ayala
:Senior Biochemistry Major

==Research Times==
T/Thur 10am-12pm
: section 01 = 0.25 credit = 4 hours per week.

==Proposed Research Project==
===Turmeric Curcumin Extraction and Analysis ===

===General Information===
:Advisor: Bradley Sturgeon
:Other research student collaborators: Stephany Saey

===Proposal===
Curcumin is a secondary plant metabolite of the turmeric herb Curcuma Longa. The term "curcumin" has been used to refer to the bioactive moelcule, but in reality curcumin has three different molecular structures: curcumin I, demethoxycurcumin, and bisdemethoxycurcumin. A review of curcumin studies suggest curcumin has chemotherapeutic, antioxidant, and anti-inflammatory activity, among other uses to be discovered. The current research project aims to successfully isolate and purify curcumin and its derivatives from the plant turmeric. With MeOH under reflux, the curcuminoids will be extracted and subsequently subjected to Flash Chromatography for separation. Once the curcuminoids are separated, NMR data collection will take place to compare the chemical properties of each derivative.
[[Curcumin Research|Curcumin Research]]

===Instruments to be used===
Flash Chromatography,
HPLC,
Soxhlet Reflux extraction

===References (2 minimum)===

==Writen Report==
===Enzymatic Oxidation of Biophenols: HPA===
Nadia Ayala, Benjamine Stillwell, Ian Salveson, Dr.Bradley Sturgeon

Summer 2016

===Abstract===
HPA is a model compound of a biologically active pheno, with a OH group that partakes in oxidation naturally. These phenols form radical intermediates during oxidation which are responsible for the resulting stable products. We have investigated various means of oxidation and will present data resulting from using an immobilized enzyme bioreactor. The products have been analyzed by HPLC and separated via flash chromatography.

===Instrumentation===
'''Immobilized Enzyme Bioreactor'''
This technique allowed there to be an oxidation activity that mimics a biological organism by having the Biophenols flow over the enzyme which was attached to agarose beads at different flow rates. The Flow of HPA as well as the concentration of HPA was tested and recorded order to get the most separation of products. To see the progression of the results please see the [[HPA Reactions|HPA Biophenol Reaserch Page]]

===Research pledge===
I, Nadia Ayala, have read the Chem/Bioc 430 course syllabus and understand the general structure and expectations of the research program. The above material was prepared after consultation, and in conjunction with my research advisor.

Nadia Y. Ayala

2016-12-06T22:12:47Z

Nayala:

Chemistry/Biochemistry Research 430
:Fall 2016
:Nadia Y. Ayala
:Senior Biochemistry Major

==Research Times==
T/Thur 10am-12pm
: section 01 = 0.25 credit = 4 hours per week.

==Proposed Research Project==
===Turmeric Curcumin Extraction and Analysis ===

===General Information===
:Advisor: Bradley Sturgeon
:Other research student collaborators: Stephany Saey

===Proposal===
Curcumin is a secondary plant metabolite of the turmeric herb Curcuma Longa. The term "curcumin" has been used to refer to the bioactive moelcule, but in reality curcumin has three different molecular structures: curcumin I, demethoxycurcumin, and bisdemethoxycurcumin. A review of curcumin studies suggest curcumin has chemotherapeutic, antioxidant, and anti-inflammatory activity, among other uses to be discovered. The current research project aims to successfully isolate and purify curcumin and its derivatives from the plant turmeric. With MeOH under reflux, the curcuminoids will be extracted and subsequently subjected to Flash Chromatography for separation. Once the curcuminoids are separated, NMR data collection will take place to compare the chemical properties of each derivative.
[[Curcumin Research|Curcumin Research]]

===Instruments to be used===
Flash Chromatography,
HPLC,
Soxhlet Reflux extraction

===References (2 minimum)===

==Writen Report==
===Enzymatic Oxidation of Biophenols: HPA===
Nadia Ayala, Benjamine Stillwell, Ian Salveson, Dr.Bradley Sturgeon

Summer 2016

===Abstract===
HPA is a model compound of a biologically active pheno, with a OH group that partakes in oxidation naturally. These phenols form radical intermediates during oxidation which are responsible for the resulting stable products. We have investigated various means of oxidation and will present data resulting from using an immobilized enzyme bioreactor. The products have been analyzed by HPLC and separated via flash chromatography.

===Instrumentation===
'''Immobilized Enzyme Bioreactor'''
This technique allowed there to be an oxidation activity that mimics a biological organism by having the Biophenols flow over the enzyme which was attached to agarose beads at different flow rates. The Flow of HPA as well as the concentration of HPA was tested and recorded order to get the most separation of products. To see the progression of the results please see the [[HPA Biophenol Reaserch Page|HPA Reactions]]

===Research pledge===
I, Nadia Ayala, have read the Chem/Bioc 430 course syllabus and understand the general structure and expectations of the research program. The above material was prepared after consultation, and in conjunction with my research advisor.

Nadia Y. Ayala

2016-12-06T22:00:34Z

Nayala: /* Instruments to be used */

HPA Reactions

2016-11-18T02:44:21Z

Nayala:

4-Hydroxyphenylacetic acid (HPA)
[[File:4-Hydroxyphenylacetic acid.svg.png|200px|thumb|none|HPA]]

==Enzyme Oxidation of HPA==

The oxidation of HPA forms radicals which form resonance structures and can be combined with each other to form dimers, trimes, and other polymers as a result of oxidation and reduction through an enzyme mechanism.
[[File:HPA radicals.png|500px|thumb|none|HPA Radicals]]

===Radical-Radical Recombinations===
The combination of these radicals are the expected products of the reaction and can begin as a combination of the following dimers.
An A-A Dimer would create an Organic Peroxide, in which the oxygen radicals create a bond.
[[File:HPA A-A Dimer .png|thumb|none|A-A Dimer]]

A B-B Dimer creates a Carbon-Carbon bond between the 3rd carbon of each HPA monomers. This is also commonly refereed to as a 3-3' bond and is the same as a D-D dimer in that the D radical is on the mirror opposite of the the C radical.
[[File:HPA B-B Dimer 01.png|thumb|none|B-B Dimer]]

A C-C Dimer would form a bond between the carbon which is attached to the acetic acid group on the HPA phenol. The position of this R group would cause there to be steric hindrance on the bond thus is less likely to form. This is also applicable to other C combination between the radicals.
[[File:HPA C-C Dimer 02.png|thumb|none|C-C Dimer]]

An A-B Dimer forms an ether bond between the HPA monomers.
[[File:HPA A-B Dimer 01.png|thumb|none|A-B Dimer]]

==Product Identification==

Reaction of 50/50 HPA-Dioxane Solution in Buffer with various H2O2 concentrations and Horse Radish Peroxidase (HRP)
[[File:Layout HPA 02.jpg|500px|thumb|Left|HPA/H2O2/HPA (6/3/16)]]
[[File:Layout HPA2 01.jpg|500px|thumb|none|HPA/H2O2/HPA (6/3/16)]]

Using the [[Enzyme Immobilization|Immobilized Enzyme]] technique, a reaction of HPA-Dioxane and H202 with Immobilized HRP

[[File:IE HPA3 T1 1.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/8/16)]]
[[File:IE HPA3 T1.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/8/16)]]

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'.
[[File:IE HPA5 03.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/15/16)]]
[[File:IE HPA5 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/15/16)]]

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 H2O2 (HPA + Dioxane)
[[File:IE HPA10 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/27/16)]]

Immediately after the IE HRP HPA/H2O2 (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|center|IE HRP HPA/H2O2 (6/27/16)-2]]
This change bring into question the change in the conditions within the bioreactor when used continually.

==Concentration Test==

To increase the amount of product extractable, the concentration of HPA was increased to 50mM as well as the H2O2 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.
[[File:IE HPA7 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/20/16)]]
[[File:IE HPA7 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/20/16)]]

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.
[[File:IE HPA9 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/23/16)]]
[[File:IE HPA9 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/23/16)]]

==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 as can be seen on result labeled Flash Peak 3.
[[File:IE HPA11 04.jpg|500px|thumb|Left|IE HPA Flash#2 Results (6/27/16)]]
[[File:IE HPA11 03.jpg|500px|thumb|none|IE HPA Flash#2 Results (6/27/16)]]

This separation was preformed on a 0.5mL/min 20mM HPA from 6/23/16.
[[File:IE HPA13 04.jpg|500px|thumb|Left|IE HPA Flash#4 Results (6/29/16)]]
[[File:IE HPA13 03.jpg|500px|thumb|none|IE HPA Flash#4 Results (6/29/16)]]

This separation was performed on a 0.25mL/min 20mM HPA from 6/27/16.
[[File:IE HPA13 02.jpg|500px|thumb|Left|IE HPA Flash#3 Results (6/29/16)-2]]
[[File:IE HPA13 01.jpg|500px|thumb|none|IE HPA Flash#3 Results (6/29/16)-2]]

This seperation was preformed on a 0.25mL/min 50mM HPA + Dioxane + 50mM H2O2 from 6/30/2016.
[[File:HPA17 02.jpg|500px|thumb|Left|IE HPA Flash#7 Results (7/8/16)]]
[[File:HPA17 01.jpg|500px|thumb|none|IE HPA Flash#7 Results (7/8/16)]]

This separation was preformed on a 0.5mL/min 50mM HPA from 7/6/16.
[[File:HPA18 02.jpg|500px|thumb|Left|IE HPA Flash#9 Results (7/6/16)]]
[[File:HPA18 01.jpg|500px|thumb|none|IE HPA Flash#9 Results (7/6/16)]]

==IE Without Dioxane Tests==

Without adding dioxane to the original solution that is to be flown through the IR, Different flow times and concentration of HPA will be tested.

To see the efficiency of the bioreactor, an test of 20mM HPA with 20mM H2O2 was preformed ad various flow speeds.
[[File:IE HPA12 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/28/16)]]
[[File:IE HPA12 01.jpg|500px|thumb|none|IE HPA HPA/H2O2 (6/28/16)]]

With the deduction of Dioxane from the reaction, a 50mM concentration was tested using the Immobilized Enzyme technique and the results are shown below. Because the concentration of the HPA is higher than previous, there seems to be too much for the HPLC to detect fully thus the first standard peak is shown with a rounded tip.
[[File:IE HPA15 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (7/05/16)]]
[[File:IE HPA15 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (7/05/16)]]
These results suggest that degradation of the HRP on the beads did not occur as it had with the reaction that included dioxane. this is shown by the continual reaction of the beads with the HPA and H2O2 evn after the 0.24mL/min run was completed, and the speed reduced to 0.5 and 1.0mL/min.

==NMR Results==

After collecting samples through Flash Chromatography, we are able to identify the proton structure of a product using NMR.
[[File:NMR full.png|700px|thumb|none|NMR of Flash #3 peak 3 (7/13/16)]]
[[File:NMR product 1.png|700px|thumb|none|NMR of Flash #3 peak 3 (7/13/16)]]

File:NMR full.png

2016-11-18T02:42:58Z

Nayala:

Curcumin Research

2016-11-18T02:11:33Z

Nayala: /* The Three Curcuminoids */

Curcumin Research

2016-11-18T02:11:08Z

Nayala:

Curcumin Research

2016-11-18T02:08:21Z

Nayala:

Nadia Y. Ayala

2016-09-28T21:53:38Z

Nayala: /* Instruments to be used */

Nadia Y. Ayala

2016-09-28T21:51:48Z

Nayala: /* Proposal */

Nadia Y. Ayala

2016-09-28T21:49:12Z

Nayala: /* Proposal */

Ice box

2016-09-22T22:11:53Z

Nayala:

Ice box

2016-09-22T22:11:40Z

Nayala:

Ice box

2016-09-22T22:11:25Z

Nayala:

Ice box

2016-09-22T22:10:56Z

Nayala:

File:Freezerfuz.png

2016-09-22T22:10:38Z

Nayala:

Ice box

2016-09-22T22:08:17Z

Nayala:

[[File:Freeer.png|thumb|none|Ice box]]

The inside of the icebox was measured at -4.1 degrees Celsius by the thermal imaging camera, while the rest of the box was measured at room temperature.

File:Freeer.png

2016-09-22T22:07:54Z

Nayala:

Ice box

2016-09-22T22:03:34Z

Nayala:

[[File:Freeer.bmp|thumb|none|Ice box]]

The inside of the icebox was measured at -4.1 degrees Celsius by the thermal imaging camera, while the rest of the box was measured at room temperature.

Ice box

2016-09-22T22:02:01Z

Nayala:

[[File:Freeer.bmp]]

The inside of the icebox was measured at -4.1 degrees Celsius by the thermal imaging camera, while the rest of the box was measured at room temperature.

File:Freeer.bmp

2016-09-22T22:01:30Z

Nayala:

Ice box

2016-09-22T21:59:55Z

Nayala: Created page with "The inside of the icebox was measured at -4.1 degrees Celsius by the thermal imaging camera, while the rest of the box was measured at room temperature."

The inside of the icebox was measured at -4.1 degrees Celsius by the thermal imaging camera, while the rest of the box was measured at room temperature.

Nadia Y. Ayala

2016-09-22T21:35:33Z

Chem 430 Fall 2016

2016-09-22T21:29:51Z

Nayala:

[[Styles_Bitchly_Chem430_F16|Styles Bitchly]] <-- use this an an example.

[[David_Thul_Chem430_F16|David Thul]]

[[Mohanad_Ahmad_Chem430_F16|Mohanad Ahmad]]

[[Mirna_Alhanash_Chem430_F16|Mirna Alhanash]]

[[Samer_Aljundi_Chem430_F16|Samer Aljundi]]

[[Brandon_Allen_Chem430_F16|Brandon Allen]]

[[Antonetta_Axup_Chem430_F16|Antonetta Axup]]

[[Nadia Y. Ayala]]

[[Louis_Badang_Chem430_F16|Louis Badang]]

[[Luis_Badang_Chem430_F16|Luis Badang]]

[[Tyler_Bailey_Chem430_F16|Tyler Bailey]]

[[Brittney_Book_Chem430_F16|Brittney Book]]

[[Rachel_Book_Chem430_F16|Rachel Book]]

[[Jasmine_Davila_Chem430_F16|Jasmine Davila]]

[[Broddie_Davis_Chem430_F16|Broddie Davis]]

[[Bradley_Dulee_Chem430_F16|Bradley Dulee]]

[[Raquel_Emeterio_Chem430_F16|Raquel Emeterio]]

[[Carley_Folluo_Chem430_F16|Carley Folluo]]

[[Morgan_Gulley_Chem430_F16|Morgan Gulley]]

[[Alan_Kuhlemier_Chem430_F16|Alan Kuhlemier]]

[[Sarah_Lang_Chem430_F16|Sarah Lang]]

[[YeJun_Park_Chem430_F16|YeJun Park]]

[[Austin_Reeder_Chem430_F16|Austin Reeder]]

[[Derek_Rineberg_Chem430_F16|Derek Rineberg]]

[[Stephanie_Saey_Chem430_F16|Stephanie Saey]]

[[Ian_Salveson_Chem430_F16|Ian Salveson]]

[[Kathryn_Saulcy_Chem430_F16|Kathryn Saulcy]]

[[Benjamin_Stillwell_Chem430_F16|Benjamin Stillwell]]

[[Taylor_Welch_Chem430_F16|Taylor Welch]]

[[Brandi_Yoder_Chem430_F16|Brandi Yoder]]

Chem 430 Fall 2016

2016-09-06T15:56:54Z

Nayala:

[[Styles_Bitchly_Chem430_F16|Styles Bitchly]] <-- use this an an example.

[[David_Thul_Chem430_F16|David Thul]]

[[Mohanad_Ahmad_Chem430_F16|Mohanad Ahmad]]

[[Mirna_Alhanash_Chem430_F16|Mirna Alhanash]]

[[Samer_Aljundi_Chem430_F16|Samer Aljundi]]

[[Brandon_Allen_Chem430_F16|Brandon Allen]]

[[Antonetta_Axup_Chem430_F16|Antonetta Axup]]

[[Nadia_Ayala|Nadia Y. Ayala]]

[[Louis_Badang_Chem430_F16|Louis Badang]]

[[Luis_Badang_Chem430_F16|Luis Badang]]

[[Tyler_Bailey_Chem430_F16|Tyler Bailey]]

[[Brittney_Book_Chem430_F16|Brittney Book]]

[[Rachel_Book_Chem430_F16|Rachel Book]]

[[Jasmine_Davila_Chem430_F16|Jasmine Davila]]

[[Broddie_Davis_Chem430_F16|Broddie Davis]]

[[Bradley_Dulee_Chem430_F16|Bradley Dulee]]

[[Raquel_Emeterio_Chem430_F16|Raquel Emeterio]]

[[Carley_Folluo_Chem430_F16|Carley Folluo]]

[[Morgan_Gulley_Chem430_F16|Morgan Gulley]]

[[Alan_Kuhlemier_Chem430_F16|Alan Kuhlemier]]

[[Sarah_Lang_Chem430_F16|Sarah Lang]]

[[YeJun_Park_Chem430_F16|YeJun Park]]

[[Austin_Reeder_Chem430_F16|Austin Reeder]]

[[Derek_Rineberg_Chem430_F16|Derek Rineberg]]

[[Stephanie_Saey_Chem430_F16|Stephanie Saey]]

[[Ian_Salveson_Chem430_F16|Ian Salveson]]

[[Kathryn_Saulcy_Chem430_F16|Kathryn Saulcy]]

[[Benjamin_Stillwell_Chem430_F16|Benjamin Stillwell]]

[[Taylor_Welch_Chem430_F16|Taylor Welch]]

[[Amy_Wollenberg_Chem430_F16|Amy Wollenberg]]

[[Brandi_Yoder_Chem430_F16|Brandi Yoder]]

Nadia Ayala

2016-07-21T18:47:14Z

Nayala: /* Presentations */

==Personal Information==
Senior Biochemistry Major.

Hometown: Chicago IL

Contact Info: nayala@monmouthcollege.edu

==Undergraduate Research Activities==

Aug 2013: SOfIA (2013) Delayed Gratification related to Personality with Marsha Dopheide, Monmouth IL

Summer 2014: Summer Medical and Dental Education Program (SMDEP) at Case Western Reserve University, Cleveland OH

Fall 2015: BIOC 430 with B. Sturgeon

[[SPME|SPME]]

Summer 2016: Kieft Summer Research with Bradley Sturgeon

[[HPA_Reactions|HPA Oxidation Reactions]]

==Presentations==
Aug 2013: '''SOfIA Presentation'''

Title: "Delayed Gratification"

July 2016: '''Keift Presentation'''

Title: "Oxidation of Biophenols"

==Interests==

Music

Languages

Pokemon

==Career Plans==

I plan to seek a higher education in either Medical School or immunological research.

HPA Reactions

2016-07-21T18:27:27Z

Nayala: /* The combination of these radicals are the expected products of the reaction and can begin as a combination of the following dimers */

4-Hydroxyphenylacetic acid (HPA)
[[File:4-Hydroxyphenylacetic acid.svg.png|200px|thumb|none|HPA]]

==Enzyme Oxidation of HPA==

The oxidation of HPA forms radicals which form resonance structures and can be combined with each other to form dimers, trimes, and other polymers as a result of oxidation and reduction through an enzyme mechanism.
[[File:HPA radicals.png|500px|thumb|none|HPA Radicals]]

===Combinations===
The combination of these radicals are the expected products of the reaction and can begin as a combination of the following dimers.
An A-A Dimer would create an Organic Peroxide, in which the oxygen radicals create a bond.
[[File:HPA A-A Dimer .png|thumb|none|A-A Dimer]]

A B-B Dimer creates a Carbon-Carbon bond between the 3rd carbon of each HPA monomers. This is also commonly refereed to as a 3-3' bond and is the same as a D-D dimer in that the D radical is on the mirror opposite of the the C radical.
[[File:HPA B-B Dimer 01.png|thumb|none|B-B Dimer]]

A C-C Dimer would form a bond between the carbon which is attached to the acetic acid group on the HPA phenol. The position of this R group would cause there to be steric hindrance on the bond thus is less likely to form. This is also applicable to other C combination between the radicals.
[[File:HPA C-C Dimer 02.png|thumb|none|C-C Dimer]]

An A-B Dimer forms an ether bond between the HPA monomers.
[[File:HPA A-B Dimer 01.png|thumb|none|A-B Dimer]]

==Product Detection Test==

Reaction of 50/50 HPA-Dioxane Solution in Buffer with various H2O2 concentrations and Horse Radish Peroxidase (HRP)
[[File:Layout HPA 02.jpg|500px|thumb|Left|HPA/H2O2/HPA (6/3/16)]]
[[File:Layout HPA2 01.jpg|500px|thumb|none|HPA/H2O2/HPA (6/3/16)]]

Using the [[Enzyme Immobilization|Immobilized Enzyme]] technique, a reaction of HPA-Dioxane and H202 with Immobilized HRP

[[File:IE HPA3 T1 1.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/8/16)]]
[[File:IE HPA3 T1.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/8/16)]]

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'.
[[File:IE HPA5 03.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/15/16)]]
[[File:IE HPA5 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/15/16)]]

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 H2O2 (HPA + Dioxane)
[[File:IE HPA10 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/27/16)]]

Immediately after the IE HRP HPA/H2O2 (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|center|IE HRP HPA/H2O2 (6/27/16)-2]]
This change bring into question the change in the conditions within the bioreactor when used continually.

==Concentration Test==

To increase the amount of product extractable, the concentration of HPA was increased to 50mM as well as the H2O2 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.
[[File:IE HPA7 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/20/16)]]
[[File:IE HPA7 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/20/16)]]

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.
[[File:IE HPA9 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/23/16)]]
[[File:IE HPA9 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/23/16)]]

==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 as can be seen on result labeled Flash Peak 3.
[[File:IE HPA11 04.jpg|500px|thumb|Left|IE HPA Flash#2 Results (6/27/16)]]
[[File:IE HPA11 03.jpg|500px|thumb|none|IE HPA Flash#2 Results (6/27/16)]]

This separation was preformed on a 0.5mL/min 20mM HPA from 6/23/16.
[[File:IE HPA13 04.jpg|500px|thumb|Left|IE HPA Flash#4 Results (6/29/16)]]
[[File:IE HPA13 03.jpg|500px|thumb|none|IE HPA Flash#4 Results (6/29/16)]]

This separation was performed on a 0.25mL/min 20mM HPA from 6/27/16.
[[File:IE HPA13 02.jpg|500px|thumb|Left|IE HPA Flash#3 Results (6/29/16)-2]]
[[File:IE HPA13 01.jpg|500px|thumb|none|IE HPA Flash#3 Results (6/29/16)-2]]

This seperation was preformed on a 0.25mL/min 50mM HPA + Dioxane + 50mM H2O2 from 6/30/2016.
[[File:HPA17 02.jpg|500px|thumb|Left|IE HPA Flash#7 Results (7/8/16)]]
[[File:HPA17 01.jpg|500px|thumb|none|IE HPA Flash#7 Results (7/8/16)]]

This separation was preformed on a 0.5mL/min 50mM HPA from 7/6/16.
[[File:HPA18 02.jpg|500px|thumb|Left|IE HPA Flash#9 Results (7/6/16)]]
[[File:HPA18 01.jpg|500px|thumb|none|IE HPA Flash#9 Results (7/6/16)]]

==IE Without Dioxane Tests==

Without adding dioxane to the original solution that is to be flown through the IR, Different flow times and concentration of HPA will be tested.

To see the efficiency of the bioreactor, an test of 20mM HPA with 20mM H2O2 was preformed ad various flow speeds.
[[File:IE HPA12 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/28/16)]]
[[File:IE HPA12 01.jpg|500px|thumb|none|IE HPA HPA/H2O2 (6/28/16)]]

With the deduction of Dioxane from the reaction, a 50mM concentration was tested using the Immobilized Enzyme technique and the results are shown below. Because the concentration of the HPA is higher than previous, there seems to be too much for the HPLC to detect fully thus the first standard peak is shown with a rounded tip.
[[File:IE HPA15 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (7/05/16)]]
[[File:IE HPA15 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (7/05/16)]]
These results suggest that degradation of the HRP on the beads did not occur as it had with the reaction that included dioxane. this is shown by the continual reaction of the beads with the HPA and H2O2 evn after the 0.24mL/min run was completed, and the speed reduced to 0.5 and 1.0mL/min.

==NMR Results==

After collecting samples through Flash Chromatography, we are able to identify the proton structure of a product using NMR.
[[File:NMR product 1.png|700px|thumb|none|NMR of Flash #3 peak 3 (7/13/16)]]

HPA Reactions

2016-07-21T18:26:13Z

Nayala: /* The combination of these radicals are the expected products of the reaction and can begin as a combination of the following dimers */

4-Hydroxyphenylacetic acid (HPA)
[[File:4-Hydroxyphenylacetic acid.svg.png|200px|thumb|none|HPA]]

==Enzyme Oxidation of HPA==

The oxidation of HPA forms radicals which form resonance structures and can be combined with each other to form dimers, trimes, and other polymers as a result of oxidation and reduction through an enzyme mechanism.
[[File:HPA radicals.png|500px|thumb|none|HPA Radicals]]

===The combination of these radicals are the expected products of the reaction and can begin as a combination of the following dimers===

An A-A Dimer would create an Organic Peroxide, in which the oxygen radicals create a bond.
[[File:HPA A-A Dimer .png|thumb|none|A-A Dimer]]

A B-B Dimer creates a Carbon-Carbon bond between the 3rd carbon of each HPA monomers. This is also commonly refereed to as a 3-3' bond and is the same as a D-D dimer in that the D radical is on the mirror opposite of the the C radical.
[[File:HPA B-B Dimer 01.png|thumb|none|B-B Dimer]]

A C-C Dimer would form a bond between the carbon which is attached to the acetic acid group on the HPA phenol. The position of this R group would cause there to be steric hindrance on the bond thus is less likely to form. This is also applicable to other C combination between the radicals.
[[File:HPA C-C Dimer 02.png|thumb|none|C-C Dimer]]

An A-B Dimer forms an ether bond between the HPA monomers.
[[File:HPA A-B Dimer 01.png|thumb|none|A-B Dimer]]

==Product Detection Test==

Reaction of 50/50 HPA-Dioxane Solution in Buffer with various H2O2 concentrations and Horse Radish Peroxidase (HRP)
[[File:Layout HPA 02.jpg|500px|thumb|Left|HPA/H2O2/HPA (6/3/16)]]
[[File:Layout HPA2 01.jpg|500px|thumb|none|HPA/H2O2/HPA (6/3/16)]]

Using the [[Enzyme Immobilization|Immobilized Enzyme]] technique, a reaction of HPA-Dioxane and H202 with Immobilized HRP

[[File:IE HPA3 T1 1.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/8/16)]]
[[File:IE HPA3 T1.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/8/16)]]

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'.
[[File:IE HPA5 03.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/15/16)]]
[[File:IE HPA5 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/15/16)]]

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 H2O2 (HPA + Dioxane)
[[File:IE HPA10 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/27/16)]]

Immediately after the IE HRP HPA/H2O2 (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|center|IE HRP HPA/H2O2 (6/27/16)-2]]
This change bring into question the change in the conditions within the bioreactor when used continually.

==Concentration Test==

To increase the amount of product extractable, the concentration of HPA was increased to 50mM as well as the H2O2 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.
[[File:IE HPA7 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/20/16)]]
[[File:IE HPA7 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/20/16)]]

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.
[[File:IE HPA9 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/23/16)]]
[[File:IE HPA9 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/23/16)]]

==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 as can be seen on result labeled Flash Peak 3.
[[File:IE HPA11 04.jpg|500px|thumb|Left|IE HPA Flash#2 Results (6/27/16)]]
[[File:IE HPA11 03.jpg|500px|thumb|none|IE HPA Flash#2 Results (6/27/16)]]

This separation was preformed on a 0.5mL/min 20mM HPA from 6/23/16.
[[File:IE HPA13 04.jpg|500px|thumb|Left|IE HPA Flash#4 Results (6/29/16)]]
[[File:IE HPA13 03.jpg|500px|thumb|none|IE HPA Flash#4 Results (6/29/16)]]

This separation was performed on a 0.25mL/min 20mM HPA from 6/27/16.
[[File:IE HPA13 02.jpg|500px|thumb|Left|IE HPA Flash#3 Results (6/29/16)-2]]
[[File:IE HPA13 01.jpg|500px|thumb|none|IE HPA Flash#3 Results (6/29/16)-2]]

This seperation was preformed on a 0.25mL/min 50mM HPA + Dioxane + 50mM H2O2 from 6/30/2016.
[[File:HPA17 02.jpg|500px|thumb|Left|IE HPA Flash#7 Results (7/8/16)]]
[[File:HPA17 01.jpg|500px|thumb|none|IE HPA Flash#7 Results (7/8/16)]]

This separation was preformed on a 0.5mL/min 50mM HPA from 7/6/16.
[[File:HPA18 02.jpg|500px|thumb|Left|IE HPA Flash#9 Results (7/6/16)]]
[[File:HPA18 01.jpg|500px|thumb|none|IE HPA Flash#9 Results (7/6/16)]]

==IE Without Dioxane Tests==

Without adding dioxane to the original solution that is to be flown through the IR, Different flow times and concentration of HPA will be tested.

To see the efficiency of the bioreactor, an test of 20mM HPA with 20mM H2O2 was preformed ad various flow speeds.
[[File:IE HPA12 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/28/16)]]
[[File:IE HPA12 01.jpg|500px|thumb|none|IE HPA HPA/H2O2 (6/28/16)]]

With the deduction of Dioxane from the reaction, a 50mM concentration was tested using the Immobilized Enzyme technique and the results are shown below. Because the concentration of the HPA is higher than previous, there seems to be too much for the HPLC to detect fully thus the first standard peak is shown with a rounded tip.
[[File:IE HPA15 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (7/05/16)]]
[[File:IE HPA15 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (7/05/16)]]
These results suggest that degradation of the HRP on the beads did not occur as it had with the reaction that included dioxane. this is shown by the continual reaction of the beads with the HPA and H2O2 evn after the 0.24mL/min run was completed, and the speed reduced to 0.5 and 1.0mL/min.

==NMR Results==

After collecting samples through Flash Chromatography, we are able to identify the proton structure of a product using NMR.
[[File:NMR product 1.png|700px|thumb|none|NMR of Flash #3 peak 3 (7/13/16)]]

HPA Reactions

2016-07-21T18:12:50Z

Nayala: /* Enzyme Oxidation of HPA */

4-Hydroxyphenylacetic acid (HPA)
[[File:4-Hydroxyphenylacetic acid.svg.png|200px|thumb|none|HPA]]

==Enzyme Oxidation of HPA==

The oxidation of HPA forms radicals which form resonance structures and can be combined with each other to form dimers, trimes, and other polymers as a result of oxidation and reduction through an enzyme mechanism.
[[File:HPA radicals.png|500px|thumb|none|HPA Radicals]]

===The combination of these radicals are the expected products of the reaction and can begin as a combination of the following dimers===

An A-A Dimer would create an Organic Peroxide, in which the oxygen radicals create a bond.
[[File:HPA A-A Dimer .png|thumb|none|A-A Dimer]]

A B-B Dimer creates a Carbon-Carbon bond between the 3rd carbon of each HPA monomers. This is also commonly refereed to as a 3-3' bond and is the same as a D-D dimer in that the D radical is on the mirror opposite of the the C radical.
[[File:HPA B-B Dimer 01.png|thumb|none|B-B Dimer]]

==Product Detection Test==

Reaction of 50/50 HPA-Dioxane Solution in Buffer with various H2O2 concentrations and Horse Radish Peroxidase (HRP)
[[File:Layout HPA 02.jpg|500px|thumb|Left|HPA/H2O2/HPA (6/3/16)]]
[[File:Layout HPA2 01.jpg|500px|thumb|none|HPA/H2O2/HPA (6/3/16)]]

Using the [[Enzyme Immobilization|Immobilized Enzyme]] technique, a reaction of HPA-Dioxane and H202 with Immobilized HRP

[[File:IE HPA3 T1 1.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/8/16)]]
[[File:IE HPA3 T1.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/8/16)]]

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'.
[[File:IE HPA5 03.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/15/16)]]
[[File:IE HPA5 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/15/16)]]

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 H2O2 (HPA + Dioxane)
[[File:IE HPA10 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/27/16)]]

Immediately after the IE HRP HPA/H2O2 (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|center|IE HRP HPA/H2O2 (6/27/16)-2]]
This change bring into question the change in the conditions within the bioreactor when used continually.

==Concentration Test==

To increase the amount of product extractable, the concentration of HPA was increased to 50mM as well as the H2O2 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.
[[File:IE HPA7 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/20/16)]]
[[File:IE HPA7 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/20/16)]]

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.
[[File:IE HPA9 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/23/16)]]
[[File:IE HPA9 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/23/16)]]

==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 as can be seen on result labeled Flash Peak 3.
[[File:IE HPA11 04.jpg|500px|thumb|Left|IE HPA Flash#2 Results (6/27/16)]]
[[File:IE HPA11 03.jpg|500px|thumb|none|IE HPA Flash#2 Results (6/27/16)]]

This separation was preformed on a 0.5mL/min 20mM HPA from 6/23/16.
[[File:IE HPA13 04.jpg|500px|thumb|Left|IE HPA Flash#4 Results (6/29/16)]]
[[File:IE HPA13 03.jpg|500px|thumb|none|IE HPA Flash#4 Results (6/29/16)]]

This separation was performed on a 0.25mL/min 20mM HPA from 6/27/16.
[[File:IE HPA13 02.jpg|500px|thumb|Left|IE HPA Flash#3 Results (6/29/16)-2]]
[[File:IE HPA13 01.jpg|500px|thumb|none|IE HPA Flash#3 Results (6/29/16)-2]]

This seperation was preformed on a 0.25mL/min 50mM HPA + Dioxane + 50mM H2O2 from 6/30/2016.
[[File:HPA17 02.jpg|500px|thumb|Left|IE HPA Flash#7 Results (7/8/16)]]
[[File:HPA17 01.jpg|500px|thumb|none|IE HPA Flash#7 Results (7/8/16)]]

This separation was preformed on a 0.5mL/min 50mM HPA from 7/6/16.
[[File:HPA18 02.jpg|500px|thumb|Left|IE HPA Flash#9 Results (7/6/16)]]
[[File:HPA18 01.jpg|500px|thumb|none|IE HPA Flash#9 Results (7/6/16)]]

==IE Without Dioxane Tests==

Without adding dioxane to the original solution that is to be flown through the IR, Different flow times and concentration of HPA will be tested.

To see the efficiency of the bioreactor, an test of 20mM HPA with 20mM H2O2 was preformed ad various flow speeds.
[[File:IE HPA12 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/28/16)]]
[[File:IE HPA12 01.jpg|500px|thumb|none|IE HPA HPA/H2O2 (6/28/16)]]

With the deduction of Dioxane from the reaction, a 50mM concentration was tested using the Immobilized Enzyme technique and the results are shown below. Because the concentration of the HPA is higher than previous, there seems to be too much for the HPLC to detect fully thus the first standard peak is shown with a rounded tip.
[[File:IE HPA15 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (7/05/16)]]
[[File:IE HPA15 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (7/05/16)]]
These results suggest that degradation of the HRP on the beads did not occur as it had with the reaction that included dioxane. this is shown by the continual reaction of the beads with the HPA and H2O2 evn after the 0.24mL/min run was completed, and the speed reduced to 0.5 and 1.0mL/min.

==NMR Results==

After collecting samples through Flash Chromatography, we are able to identify the proton structure of a product using NMR.
[[File:NMR product 1.png|700px|thumb|none|NMR of Flash #3 peak 3 (7/13/16)]]

HPA Reactions

2016-07-21T18:12:17Z

Nayala: /* Enzyme Oxidation of HPA */

4-Hydroxyphenylacetic acid (HPA)
[[File:4-Hydroxyphenylacetic acid.svg.png|200px|thumb|none|HPA]]

==Enzyme Oxidation of HPA==

The oxidation of HPA forms radicals which form resonance structures and can be combined with each other to form dimers, trimes, and other polymers as a result of oxidation and reduction through an enzyme mechanism.
[[File:HPA radicals.png|500px|thumb|none|HPA Radicals]]

The combination of these radicals are the expected products of the reaction and can begin as a combination of the following dimers:

An A-A Dimer would create an Organic Peroxide, in which the oxygen radicals create a bond.
[[File:HPA A-A Dimer .png|thumb|none|A-A Dimer]]

A B-B Dimer creates a Carbon-Carbon bond between the 3rd carbon of each HPA monomers. This is also commonly refereed to as a 3-3' bond and is the same as a D-D dimer in that the D radical is on the mirror opposite of the the C radical.
[[File:HPA B-B Dimer 01.png|thumb|none|B-B Dimer]]

==Product Detection Test==

Reaction of 50/50 HPA-Dioxane Solution in Buffer with various H2O2 concentrations and Horse Radish Peroxidase (HRP)
[[File:Layout HPA 02.jpg|500px|thumb|Left|HPA/H2O2/HPA (6/3/16)]]
[[File:Layout HPA2 01.jpg|500px|thumb|none|HPA/H2O2/HPA (6/3/16)]]

Using the [[Enzyme Immobilization|Immobilized Enzyme]] technique, a reaction of HPA-Dioxane and H202 with Immobilized HRP

[[File:IE HPA3 T1 1.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/8/16)]]
[[File:IE HPA3 T1.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/8/16)]]

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'.
[[File:IE HPA5 03.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/15/16)]]
[[File:IE HPA5 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/15/16)]]

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 H2O2 (HPA + Dioxane)
[[File:IE HPA10 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/27/16)]]

Immediately after the IE HRP HPA/H2O2 (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|center|IE HRP HPA/H2O2 (6/27/16)-2]]
This change bring into question the change in the conditions within the bioreactor when used continually.

==Concentration Test==

To increase the amount of product extractable, the concentration of HPA was increased to 50mM as well as the H2O2 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.
[[File:IE HPA7 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/20/16)]]
[[File:IE HPA7 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/20/16)]]

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.
[[File:IE HPA9 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/23/16)]]
[[File:IE HPA9 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/23/16)]]

==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 as can be seen on result labeled Flash Peak 3.
[[File:IE HPA11 04.jpg|500px|thumb|Left|IE HPA Flash#2 Results (6/27/16)]]
[[File:IE HPA11 03.jpg|500px|thumb|none|IE HPA Flash#2 Results (6/27/16)]]

This separation was preformed on a 0.5mL/min 20mM HPA from 6/23/16.
[[File:IE HPA13 04.jpg|500px|thumb|Left|IE HPA Flash#4 Results (6/29/16)]]
[[File:IE HPA13 03.jpg|500px|thumb|none|IE HPA Flash#4 Results (6/29/16)]]

This separation was performed on a 0.25mL/min 20mM HPA from 6/27/16.
[[File:IE HPA13 02.jpg|500px|thumb|Left|IE HPA Flash#3 Results (6/29/16)-2]]
[[File:IE HPA13 01.jpg|500px|thumb|none|IE HPA Flash#3 Results (6/29/16)-2]]

This seperation was preformed on a 0.25mL/min 50mM HPA + Dioxane + 50mM H2O2 from 6/30/2016.
[[File:HPA17 02.jpg|500px|thumb|Left|IE HPA Flash#7 Results (7/8/16)]]
[[File:HPA17 01.jpg|500px|thumb|none|IE HPA Flash#7 Results (7/8/16)]]

This separation was preformed on a 0.5mL/min 50mM HPA from 7/6/16.
[[File:HPA18 02.jpg|500px|thumb|Left|IE HPA Flash#9 Results (7/6/16)]]
[[File:HPA18 01.jpg|500px|thumb|none|IE HPA Flash#9 Results (7/6/16)]]

==IE Without Dioxane Tests==

Without adding dioxane to the original solution that is to be flown through the IR, Different flow times and concentration of HPA will be tested.

To see the efficiency of the bioreactor, an test of 20mM HPA with 20mM H2O2 was preformed ad various flow speeds.
[[File:IE HPA12 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/28/16)]]
[[File:IE HPA12 01.jpg|500px|thumb|none|IE HPA HPA/H2O2 (6/28/16)]]

With the deduction of Dioxane from the reaction, a 50mM concentration was tested using the Immobilized Enzyme technique and the results are shown below. Because the concentration of the HPA is higher than previous, there seems to be too much for the HPLC to detect fully thus the first standard peak is shown with a rounded tip.
[[File:IE HPA15 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (7/05/16)]]
[[File:IE HPA15 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (7/05/16)]]
These results suggest that degradation of the HRP on the beads did not occur as it had with the reaction that included dioxane. this is shown by the continual reaction of the beads with the HPA and H2O2 evn after the 0.24mL/min run was completed, and the speed reduced to 0.5 and 1.0mL/min.

==NMR Results==

After collecting samples through Flash Chromatography, we are able to identify the proton structure of a product using NMR.
[[File:NMR product 1.png|700px|thumb|none|NMR of Flash #3 peak 3 (7/13/16)]]

HPA Reactions

2016-07-21T18:11:21Z

Nayala: /* Enzyme Oxidation of HPA */

4-Hydroxyphenylacetic acid (HPA)
[[File:4-Hydroxyphenylacetic acid.svg.png|200px|thumb|none|HPA]]

==Enzyme Oxidation of HPA==

The oxidation of HPA forms radicals which form resonance structures and can be combined with each other to form dimers, trimes, and other polymers as a result of oxidation and reduction through an enzyme mechanism.
[[File:HPA radicals.png|500px|thumb|none|HPA Radicals]]

The combination of these radicals are the expected products of the reaction and can begin as a combination of the following dimers.

An A-A Dimer would create an Organic Peroxide, in which the oxygen radicals create a bond.
[[File:HPA A-A Dimer .png|thumb|none|A-A Dimer]]

A B-B Dimer creates a Carbon-Carbon bond between the 3rd carbon of each HPA monomers. This is also commonly refereed to as a 3-3' bond and is the same as a D-D dimer in that the D radical is on the mirror opposite of the the C radical.
[[File:HPA B-B Dimer 01.png|thumb|none|B-B Dimer]]

==Product Detection Test==

Reaction of 50/50 HPA-Dioxane Solution in Buffer with various H2O2 concentrations and Horse Radish Peroxidase (HRP)
[[File:Layout HPA 02.jpg|500px|thumb|Left|HPA/H2O2/HPA (6/3/16)]]
[[File:Layout HPA2 01.jpg|500px|thumb|none|HPA/H2O2/HPA (6/3/16)]]

Using the [[Enzyme Immobilization|Immobilized Enzyme]] technique, a reaction of HPA-Dioxane and H202 with Immobilized HRP

[[File:IE HPA3 T1 1.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/8/16)]]
[[File:IE HPA3 T1.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/8/16)]]

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'.
[[File:IE HPA5 03.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/15/16)]]
[[File:IE HPA5 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/15/16)]]

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 H2O2 (HPA + Dioxane)
[[File:IE HPA10 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/27/16)]]

Immediately after the IE HRP HPA/H2O2 (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|center|IE HRP HPA/H2O2 (6/27/16)-2]]
This change bring into question the change in the conditions within the bioreactor when used continually.

==Concentration Test==

To increase the amount of product extractable, the concentration of HPA was increased to 50mM as well as the H2O2 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.
[[File:IE HPA7 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/20/16)]]
[[File:IE HPA7 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/20/16)]]

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.
[[File:IE HPA9 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/23/16)]]
[[File:IE HPA9 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/23/16)]]

==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 as can be seen on result labeled Flash Peak 3.
[[File:IE HPA11 04.jpg|500px|thumb|Left|IE HPA Flash#2 Results (6/27/16)]]
[[File:IE HPA11 03.jpg|500px|thumb|none|IE HPA Flash#2 Results (6/27/16)]]

This separation was preformed on a 0.5mL/min 20mM HPA from 6/23/16.
[[File:IE HPA13 04.jpg|500px|thumb|Left|IE HPA Flash#4 Results (6/29/16)]]
[[File:IE HPA13 03.jpg|500px|thumb|none|IE HPA Flash#4 Results (6/29/16)]]

This separation was performed on a 0.25mL/min 20mM HPA from 6/27/16.
[[File:IE HPA13 02.jpg|500px|thumb|Left|IE HPA Flash#3 Results (6/29/16)-2]]
[[File:IE HPA13 01.jpg|500px|thumb|none|IE HPA Flash#3 Results (6/29/16)-2]]

This seperation was preformed on a 0.25mL/min 50mM HPA + Dioxane + 50mM H2O2 from 6/30/2016.
[[File:HPA17 02.jpg|500px|thumb|Left|IE HPA Flash#7 Results (7/8/16)]]
[[File:HPA17 01.jpg|500px|thumb|none|IE HPA Flash#7 Results (7/8/16)]]

This separation was preformed on a 0.5mL/min 50mM HPA from 7/6/16.
[[File:HPA18 02.jpg|500px|thumb|Left|IE HPA Flash#9 Results (7/6/16)]]
[[File:HPA18 01.jpg|500px|thumb|none|IE HPA Flash#9 Results (7/6/16)]]

==IE Without Dioxane Tests==

Without adding dioxane to the original solution that is to be flown through the IR, Different flow times and concentration of HPA will be tested.

To see the efficiency of the bioreactor, an test of 20mM HPA with 20mM H2O2 was preformed ad various flow speeds.
[[File:IE HPA12 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/28/16)]]
[[File:IE HPA12 01.jpg|500px|thumb|none|IE HPA HPA/H2O2 (6/28/16)]]

With the deduction of Dioxane from the reaction, a 50mM concentration was tested using the Immobilized Enzyme technique and the results are shown below. Because the concentration of the HPA is higher than previous, there seems to be too much for the HPLC to detect fully thus the first standard peak is shown with a rounded tip.
[[File:IE HPA15 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (7/05/16)]]
[[File:IE HPA15 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (7/05/16)]]
These results suggest that degradation of the HRP on the beads did not occur as it had with the reaction that included dioxane. this is shown by the continual reaction of the beads with the HPA and H2O2 evn after the 0.24mL/min run was completed, and the speed reduced to 0.5 and 1.0mL/min.

==NMR Results==

After collecting samples through Flash Chromatography, we are able to identify the proton structure of a product using NMR.
[[File:NMR product 1.png|700px|thumb|none|NMR of Flash #3 peak 3 (7/13/16)]]

HPA Reactions

2016-07-21T17:59:10Z

Nayala:

4-Hydroxyphenylacetic acid (HPA)
[[File:4-Hydroxyphenylacetic acid.svg.png|200px|thumb|none|HPA]]

==Enzyme Oxidation of HPA==

The oxidation of HPA forms radicals which form resonance structures and can be combined with each other to form dimers, trimes, and other polymers as a result of oxidation and reduction through an enzyme mechanism.
[[File:HPA radicals.png|500px|thumb|none|HPA Radicals]]

==Product Detection Test==

Reaction of 50/50 HPA-Dioxane Solution in Buffer with various H2O2 concentrations and Horse Radish Peroxidase (HRP)
[[File:Layout HPA 02.jpg|500px|thumb|Left|HPA/H2O2/HPA (6/3/16)]]
[[File:Layout HPA2 01.jpg|500px|thumb|none|HPA/H2O2/HPA (6/3/16)]]

Using the [[Enzyme Immobilization|Immobilized Enzyme]] technique, a reaction of HPA-Dioxane and H202 with Immobilized HRP

[[File:IE HPA3 T1 1.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/8/16)]]
[[File:IE HPA3 T1.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/8/16)]]

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'.
[[File:IE HPA5 03.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/15/16)]]
[[File:IE HPA5 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/15/16)]]

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 H2O2 (HPA + Dioxane)
[[File:IE HPA10 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/27/16)]]

Immediately after the IE HRP HPA/H2O2 (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|center|IE HRP HPA/H2O2 (6/27/16)-2]]
This change bring into question the change in the conditions within the bioreactor when used continually.

==Concentration Test==

To increase the amount of product extractable, the concentration of HPA was increased to 50mM as well as the H2O2 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.
[[File:IE HPA7 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/20/16)]]
[[File:IE HPA7 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/20/16)]]

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.
[[File:IE HPA9 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/23/16)]]
[[File:IE HPA9 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/23/16)]]

==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 as can be seen on result labeled Flash Peak 3.
[[File:IE HPA11 04.jpg|500px|thumb|Left|IE HPA Flash#2 Results (6/27/16)]]
[[File:IE HPA11 03.jpg|500px|thumb|none|IE HPA Flash#2 Results (6/27/16)]]

This separation was preformed on a 0.5mL/min 20mM HPA from 6/23/16.
[[File:IE HPA13 04.jpg|500px|thumb|Left|IE HPA Flash#4 Results (6/29/16)]]
[[File:IE HPA13 03.jpg|500px|thumb|none|IE HPA Flash#4 Results (6/29/16)]]

This separation was performed on a 0.25mL/min 20mM HPA from 6/27/16.
[[File:IE HPA13 02.jpg|500px|thumb|Left|IE HPA Flash#3 Results (6/29/16)-2]]
[[File:IE HPA13 01.jpg|500px|thumb|none|IE HPA Flash#3 Results (6/29/16)-2]]

This seperation was preformed on a 0.25mL/min 50mM HPA + Dioxane + 50mM H2O2 from 6/30/2016.
[[File:HPA17 02.jpg|500px|thumb|Left|IE HPA Flash#7 Results (7/8/16)]]
[[File:HPA17 01.jpg|500px|thumb|none|IE HPA Flash#7 Results (7/8/16)]]

This separation was preformed on a 0.5mL/min 50mM HPA from 7/6/16.
[[File:HPA18 02.jpg|500px|thumb|Left|IE HPA Flash#9 Results (7/6/16)]]
[[File:HPA18 01.jpg|500px|thumb|none|IE HPA Flash#9 Results (7/6/16)]]

==IE Without Dioxane Tests==

Without adding dioxane to the original solution that is to be flown through the IR, Different flow times and concentration of HPA will be tested.

To see the efficiency of the bioreactor, an test of 20mM HPA with 20mM H2O2 was preformed ad various flow speeds.
[[File:IE HPA12 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/28/16)]]
[[File:IE HPA12 01.jpg|500px|thumb|none|IE HPA HPA/H2O2 (6/28/16)]]

With the deduction of Dioxane from the reaction, a 50mM concentration was tested using the Immobilized Enzyme technique and the results are shown below. Because the concentration of the HPA is higher than previous, there seems to be too much for the HPLC to detect fully thus the first standard peak is shown with a rounded tip.
[[File:IE HPA15 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (7/05/16)]]
[[File:IE HPA15 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (7/05/16)]]
These results suggest that degradation of the HRP on the beads did not occur as it had with the reaction that included dioxane. this is shown by the continual reaction of the beads with the HPA and H2O2 evn after the 0.24mL/min run was completed, and the speed reduced to 0.5 and 1.0mL/min.

==NMR Results==

After collecting samples through Flash Chromatography, we are able to identify the proton structure of a product using NMR.
[[File:NMR product 1.png|700px|thumb|none|NMR of Flash #3 peak 3 (7/13/16)]]

File:HPA C-C Dimer 02.png

2016-07-21T17:55:54Z

Nayala:

File:HPA B-B Dimer 01.png

2016-07-21T17:54:43Z

Nayala:

File:HPA A-B Dimer 01.png

2016-07-21T17:54:22Z

Nayala:

File:HPA A-A Dimer .png

2016-07-21T17:53:56Z

Nayala:

File:HPA radicals.png

2016-07-21T17:53:32Z

Nayala:

HPA Reactions

2016-07-14T19:17:45Z

Nayala: /* NMR Results */

4-Hydroxyphenylacetic acid (HPA)
[[File:4-Hydroxyphenylacetic acid.svg.png|200px|thumb|none|HPA]]

==Product Detection Test==
Reaction of 50/50 HPA-Dioxane Solution in Buffer with various H2O2 concentrations and Horse Radish Peroxidase (HRP)
[[File:Layout HPA 02.jpg|500px|thumb|Left|HPA/H2O2/HPA (6/3/16)]]
[[File:Layout HPA2 01.jpg|500px|thumb|none|HPA/H2O2/HPA (6/3/16)]]

Using the [[Enzyme Immobilization|Immobilized Enzyme]] technique, a reaction of HPA-Dioxane and H202 with Immobilized HRP

[[File:IE HPA3 T1 1.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/8/16)]]
[[File:IE HPA3 T1.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/8/16)]]

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'.
[[File:IE HPA5 03.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/15/16)]]
[[File:IE HPA5 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/15/16)]]

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 H2O2 (HPA + Dioxane)
[[File:IE HPA10 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/27/16)]]

Immediately after the IE HRP HPA/H2O2 (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|center|IE HRP HPA/H2O2 (6/27/16)-2]]
This change bring into question the change in the conditions within the bioreactor when used continually.

==Concentration Test==

To increase the amount of product extractable, the concentration of HPA was increased to 50mM as well as the H2O2 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.
[[File:IE HPA7 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/20/16)]]
[[File:IE HPA7 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/20/16)]]

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.
[[File:IE HPA9 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/23/16)]]
[[File:IE HPA9 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/23/16)]]

==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 as can be seen on result labeled Flash Peak 3.
[[File:IE HPA11 04.jpg|500px|thumb|Left|IE HPA Flash#2 Results (6/27/16)]]
[[File:IE HPA11 03.jpg|500px|thumb|none|IE HPA Flash#2 Results (6/27/16)]]

This separation was preformed on a 0.5mL/min 20mM HPA from 6/23/16.
[[File:IE HPA13 04.jpg|500px|thumb|Left|IE HPA Flash#4 Results (6/29/16)]]
[[File:IE HPA13 03.jpg|500px|thumb|none|IE HPA Flash#4 Results (6/29/16)]]

This separation was performed on a 0.25mL/min 20mM HPA from 6/27/16.
[[File:IE HPA13 02.jpg|500px|thumb|Left|IE HPA Flash#3 Results (6/29/16)-2]]
[[File:IE HPA13 01.jpg|500px|thumb|none|IE HPA Flash#3 Results (6/29/16)-2]]

This seperation was preformed on a 0.25mL/min 50mM HPA + Dioxane + 50mM H2O2 from 6/30/2016.
[[File:HPA17 02.jpg|500px|thumb|Left|IE HPA Flash#7 Results (7/8/16)]]
[[File:HPA17 01.jpg|500px|thumb|none|IE HPA Flash#7 Results (7/8/16)]]

This separation was preformed on a 0.5mL/min 50mM HPA from 7/6/16.
[[File:HPA18 02.jpg|500px|thumb|Left|IE HPA Flash#9 Results (7/6/16)]]
[[File:HPA18 01.jpg|500px|thumb|none|IE HPA Flash#9 Results (7/6/16)]]

==IE Without Dioxane Tests==

Without adding dioxane to the original solution that is to be flown through the IR, Different flow times and concentration of HPA will be tested.

To see the efficiency of the bioreactor, an test of 20mM HPA with 20mM H2O2 was preformed ad various flow speeds.
[[File:IE HPA12 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/28/16)]]
[[File:IE HPA12 01.jpg|500px|thumb|none|IE HPA HPA/H2O2 (6/28/16)]]

With the deduction of Dioxane from the reaction, a 50mM concentration was tested using the Immobilized Enzyme technique and the results are shown below. Because the concentration of the HPA is higher than previous, there seems to be too much for the HPLC to detect fully thus the first standard peak is shown with a rounded tip.
[[File:IE HPA15 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (7/05/16)]]
[[File:IE HPA15 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (7/05/16)]]
These results suggest that degradation of the HRP on the beads did not occur as it had with the reaction that included dioxane. this is shown by the continual reaction of the beads with the HPA and H2O2 evn after the 0.24mL/min run was completed, and the speed reduced to 0.5 and 1.0mL/min.

==NMR Results==

After collecting samples through Flash Chromatography, we are able to identify the proton structure of a product using NMR.
[[File:NMR product 1.png|700px|thumb|none|NMR of Flash #3 peak 3 (7/13/16)]]

HPA Reactions

2016-07-14T19:15:32Z

Nayala: /* NMR Results */

4-Hydroxyphenylacetic acid (HPA)
[[File:4-Hydroxyphenylacetic acid.svg.png|200px|thumb|none|HPA]]

==Product Detection Test==
Reaction of 50/50 HPA-Dioxane Solution in Buffer with various H2O2 concentrations and Horse Radish Peroxidase (HRP)
[[File:Layout HPA 02.jpg|500px|thumb|Left|HPA/H2O2/HPA (6/3/16)]]
[[File:Layout HPA2 01.jpg|500px|thumb|none|HPA/H2O2/HPA (6/3/16)]]

Using the [[Enzyme Immobilization|Immobilized Enzyme]] technique, a reaction of HPA-Dioxane and H202 with Immobilized HRP

[[File:IE HPA3 T1 1.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/8/16)]]
[[File:IE HPA3 T1.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/8/16)]]

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'.
[[File:IE HPA5 03.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/15/16)]]
[[File:IE HPA5 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/15/16)]]

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 H2O2 (HPA + Dioxane)
[[File:IE HPA10 02.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/27/16)]]

Immediately after the IE HRP HPA/H2O2 (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|center|IE HRP HPA/H2O2 (6/27/16)-2]]
This change bring into question the change in the conditions within the bioreactor when used continually.

==Concentration Test==

To increase the amount of product extractable, the concentration of HPA was increased to 50mM as well as the H2O2 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.
[[File:IE HPA7 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/20/16)]]
[[File:IE HPA7 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/20/16)]]

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.
[[File:IE HPA9 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/23/16)]]
[[File:IE HPA9 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (6/23/16)]]

==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 as can be seen on result labeled Flash Peak 3.
[[File:IE HPA11 04.jpg|500px|thumb|Left|IE HPA Flash#2 Results (6/27/16)]]
[[File:IE HPA11 03.jpg|500px|thumb|none|IE HPA Flash#2 Results (6/27/16)]]

This separation was preformed on a 0.5mL/min 20mM HPA from 6/23/16.
[[File:IE HPA13 04.jpg|500px|thumb|Left|IE HPA Flash#4 Results (6/29/16)]]
[[File:IE HPA13 03.jpg|500px|thumb|none|IE HPA Flash#4 Results (6/29/16)]]

This separation was performed on a 0.25mL/min 20mM HPA from 6/27/16.
[[File:IE HPA13 02.jpg|500px|thumb|Left|IE HPA Flash#3 Results (6/29/16)-2]]
[[File:IE HPA13 01.jpg|500px|thumb|none|IE HPA Flash#3 Results (6/29/16)-2]]

This seperation was preformed on a 0.25mL/min 50mM HPA + Dioxane + 50mM H2O2 from 6/30/2016.
[[File:HPA17 02.jpg|500px|thumb|Left|IE HPA Flash#7 Results (7/8/16)]]
[[File:HPA17 01.jpg|500px|thumb|none|IE HPA Flash#7 Results (7/8/16)]]

This separation was preformed on a 0.5mL/min 50mM HPA from 7/6/16.
[[File:HPA18 02.jpg|500px|thumb|Left|IE HPA Flash#9 Results (7/6/16)]]
[[File:HPA18 01.jpg|500px|thumb|none|IE HPA Flash#9 Results (7/6/16)]]

==IE Without Dioxane Tests==

Without adding dioxane to the original solution that is to be flown through the IR, Different flow times and concentration of HPA will be tested.

To see the efficiency of the bioreactor, an test of 20mM HPA with 20mM H2O2 was preformed ad various flow speeds.
[[File:IE HPA12 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (6/28/16)]]
[[File:IE HPA12 01.jpg|500px|thumb|none|IE HPA HPA/H2O2 (6/28/16)]]

With the deduction of Dioxane from the reaction, a 50mM concentration was tested using the Immobilized Enzyme technique and the results are shown below. Because the concentration of the HPA is higher than previous, there seems to be too much for the HPLC to detect fully thus the first standard peak is shown with a rounded tip.
[[File:IE HPA15 02.jpg|500px|thumb|Left|IE HRP HPA/H2O2 (7/05/16)]]
[[File:IE HPA15 01.jpg|500px|thumb|none|IE HRP HPA/H2O2 (7/05/16)]]
These results suggest that degradation of the HRP on the beads did not occur as it had with the reaction that included dioxane. this is shown by the continual reaction of the beads with the HPA and H2O2 evn after the 0.24mL/min run was completed, and the speed reduced to 0.5 and 1.0mL/min.

==NMR Results==

[[File:NMR product 1.png|500px|thumb|none|NMR of Flash #3 peak 3 (7/13/16)]]