Unknown Precipitate

To form the iron-curcumin complex, stoichiometric ratios are calculated and volumes of curcumin solution and iron solution are mixed.  Before the iron is added to the curcumin, the pH of the curcumin is adjusted to approximately 7.5 with triethylamine.  It was observed that when this solution is left overnight, a precipitate appears.  When centrifuged, it was discovered that the precipitate was actually a denser fluid and not a solid.  This dense fluid was dark in color and oily in consistency.  It was not soluble in the solvents typically used for FeCl₃ or curcumin.  After testing the oily fluid using HPLC it was discovered that it was not the iron-curcumin complex as had been reported in several papers, but just iron hydroxide.  

In order to further prove that the oily precipitate is not the iron-curcumin complex, different ratios of curcumin and iron(III) were mixed.  If the dense oil is iron hydroxide, then the test tubes containing more iron will have more precipitate.

7/31/18

-Alex Griffith

Trip to NJIT

On Wednesday, July, 25th, we went to NJIT to do mass spectrometry on a few samples of curcuminoids and curcuminoid complex we had prepared the day prior. We were greeted by Dr. Zhang, a research professor, and his post-doctoral assistant. They brought us to a Liquid Chromatography Mass Spectrometry machine to complete the experiments which would identify the masses of fragments of our samples.

We learned that, with the method used, the complex would not appear at its hypothesized molecular weight, and that the mass of one curcumin and one iron appeared to be the most stable fragment. An ongoing topic of interest is the phase, gaseous, or solvated, of the complex.

-Alexander Czechowicz

Microbiology Controls

This week marked an expansion of focus upon plating our hypothetical complex onto various TSA media containing various bacteria. Much of what we analyzed on this side of the project looked like the following:

We saw that the hypothetical complexes did not exhibit larger zones of inhibition across Al3+, Cu2+, Fe3+ and Fe2+ than curcumin alone. Dr. K hypothesized that the formation equilibrium had to be pushed to the right and we increased the mol:mol ratio of cation to curcumin for the next plates. This showed mild evidence supporting Dr. K's hypothesis.

We had more questions about achieving a high concentration of complex in solution and attempted to accomplish that goal by considering the time since mixing our curcumin and the cation. Dr. K had Isaac, who normally runs our HPLC, mix the solution in his syringe which produced results consistent with greater amounts of complex. We decided to try to apply this to the microbiology side of things.

Hypothetically, if we accept that the rate of decomposition of the complex is greater than its rate of formation shortly after mixing, then its concentration should be highest right when mixed. We will check back in next week to see whether or not the plates we streaked today support this hypothesis.

7/19/2018
-Alexander Czechowicz

Crystalization

After crystals were extracted from turmeric solution, it was noted that the crystals were not pure orange in color.  There were several very small flecks of yellow and red.  The lab supplied separated curcuminoids were all slightly different in color, one being yellow, another being orange, and the last being red.  From this information, it was hypothesized that if these crystals were separated by color, they could be used as seed crystals in a super saturated curcumin solution to produce larger single color crystals.  These single colors could possibly be the individual curcuminoids.  If true, this would mean there would be a much easier and cheaper way to separate curcuminoids.

Curcumin Crystals Before Separation

Separating the small crystals by hand had to be accomplished using a dissection microscope, a metal spatula, and tweezers.  Because the extremely low percentage of demethoxycurcumin and bisdemethoxycurcumin in turmeric, the amount of yellow and red crystals in the yield was very small.

Yellow, Orange, and Red Crystals: Seperated

7/18/18
-Alex Griffith

Melting Points

Several solids have been filtered from solutions containing both metal cations and curcumin.  Especially when it comes to iron(III), the identity of the filtered solid has been questionable.  It was possible that the solid was iron oxide and not the curcumin-iron complex.  As a method of identifying the solid, we attempted to find the melting point of the black crystals that were removed from solution.
The melting point of curcumin is 183°C.  The melting point of iron oxide is far beyond the limitations of the melting point apparatus at 1500°C.  The black crystals did not melt at 250°C, although they did begin to change color.  The limitations of the machine made further analysis of the black crystals impossible.

The melting point apparatus was also utilized to confirm the identity of orange crystals extracted from a turmeric solution.  

The melting point of the crystals was 179°C.  Because the melting points of the curcuminoids are all slightly different, the "impurity" caused by having all three curcuminoids together reduced the melting point, meaning that the crystals were confirmed as curcumin by the melting point testing.

7/12/18

-Alex Griffith

Solar Cells and Decomposition

Using solar cells, we tested for current with curcuminoids and the Fe 3+ curcumin complex. This endeavor was inspired by the search for applications of the curcumin complex. We found that there was a significant reduction in potential difference when the curcumin and iron complex was used, which is proof that the Fe 3+ curcumin complex is forming. The change in voltage can be attributed to a loss in conjugation.

The solar cell project has been testing raspberry solar cells due to favorable conjugation. While raspberries are effective, they are not efficient as they are expensive. Curcumin may yield similar voltages for a fraction of the cost.

We considered that our curcumin samples may be degrading, and occasionally caught scents of vanilla. The following skeletal structure is of vanillin, a vanilla plant derivative that resembles a potential "metabolite."

The following is another candidate:                        

                                                                                                       

Curcumin

                 




Vanillin and the cinnamaldehyde derivative might not be what is actually being produced by natural degradation or leaving our samples is ambient temperature, but we have suspicions that they may be. Further experiments may involve identification of the nature of degradation of curcumin.

"Degradation of curcumin: From mechanism to biological implications" by Schneider, et al, explores this topic. It can be found at this link.

We were having some issues with the IR spec machine. At first in troubleshooting, we tried to re-calibrate the machine to the environment, but this proved ineffective in affording a sound IR graph. We then reset all of the hardware which also proved inconclusive. Using new salt crystals rectified the error. We produced a good overlay of three spectra.

7/3/18
-Alexander Czechowicz

Removing Complexes From Solution

When the complex is dissolved in ethanol, the IR results are changed.  The additional alcohol leads to a greatly exaggerated peak in the IR spectrum around 3500(cm-1).  To combat this, the complex was removed from solution and dried in order to use it in a solvent that did not contain alcohol.

Four complexes were prepared using aluminium, copper, iron (III), and iron (II).  Laboratory supplied curcuminoids (all three were used) were dissolved in methanol.  The cation solutions were then added.  Finally, a drop of triethylamine was added before the solutions were refluxed for an hour.

After refluxing, the solutions were left to evaporate over the weekend.

Curcumin-Aluminum Complex	Curcumin-Copper Complex	With the exception of the iron(III), all complexes were slightly sticky.
Curcumin-Iron(II) Complex	Curcumin-Iron(III) Complex	The complexes were all dissolved in dichloromethane for use with IR spectroscopy.  They were much more soluble in ethanol than dichloromethane.

7/2/18

-Alex Griffith

Iron-Curcumin Complex TLC

Using the curcumin without the curcuminoids, a TLC plate was run.  The spot on the left was a 1:3 ratio of Fe(III):Curcumin.  The spot on the right was just curcumin alone.

The two spots that are circled but not colored were present under UV light. 

The Rf values for the two yellow spots are:
Iron-Curcumin complex=0.589
Curcumin alone=0.759

This shows that the complex is more polar than curcumin alone.  This is consistent with the findings from HPLC.




6/25/18
-Alex Griffith

Iron II or Iron III

Today, we set out to analyze whether using iron II instead of iron III would produce different results with regards to the formation of the iron-curcumin complex.

At the onset, we hypothesized that the proportion of iron II to curcumin would be 1:2, in contrast to the previous iron III 1:1 ratio.

During our observations, we noted that our UV-Vis spectra showed that absorption at the wavelengths corresponding to our iron-curcumin complex decreased after reflux. This likely means that the formation of the complex is exothermic, that is, adding heat to our solutions pushes the reaction in the reverse direction. Dr. K noted that he had been misled by some of the existing literature, and that we should comb our articles to see from where the faulty conclusion about the utility of refluxing our solution came.

6/20/18
-Alexander Czechowicz

Changing the Solvent for Iron

In order to evaluate the iron-curcumin complex, solutions of equal molarity were prepared.  The solution of Fe(III) was prepared using water, and the curcumin solution was prepared using ethanol.  When a UV spectrum was run, the usual peaks for the iron-curcumin complex did not appear.  This problem was rectified when a new solution of the same molarity of Fe(III) was prepared using ethanol instead of water.  The difference was immediately apparent.

Both of these solutions contain the exact same ratio of iron to curcumin, but the solution on the left was made with the iron dissolved in water and the solution on the right was made with the iron dissolved in ethanol.  The color change in the solution on the left indicates the formation of the iron-curcumin complex.





The difference seen visually was supported by the UV spectrum results as well.  The following two runs are the results from the solutions in the previous picture.  The orange line represents the solution made with the iron dissolved in water and the black line represents the solution made with iron dissolved in ethanol.  On the black line, the shift of the tallest peak as well as the appearance of the shoulder around 540nm indicates the formation of the iron-curcumin complex.

These results indicate that iron is complexing with water and once this complex occurs, it cannot be removed for use by the curcumin.  This means that in order to prepare a premade complex for biological use, the iron must not come into contact with anything else it can form complexes with.

6/18/18
-Alex Griffith

Determining the Extinction Coefficient of Curcumin Alone

After making a solution with a known mass of curcumin (alone, no curcuminoids) and a known volume of ethanol, the exact molarity of the solution was known.  Using a known molarity, it was possible to find the extinction coefficient for curcumin without the other curcuminoids.

The slope of the following graph gives the extinction coefficient of curcumin alone:

The Extinction Coefficient of Curcumin Alone: 57000

6/14/18

-Alex Griffith

Egg Albumin and Curcumin-Iron Complexes

Using a UV spectrum analysis, the effects of egg albumin were tested on the formation of curcumin-iron complexes.  It was discovered that the iron bonded quickly to the albumin, leaving nothing for the curcumin to complex with.  In addition to leaving no excess iron, it was discovered that curcumin cannot remove iron when it is attached to the protein.  Although this result does not indicate that the curcumin-iron complex can form from iron that is being used biologically, it could indicate that curcumin may not worsen anemia.
The next step will be adding a pre-made curcumin-iron complex to the albumin mixture.


6/11/18
-Alex Griffith

Using Commercially Produced Curcuminoids

To ensure the purity of the samples that we extracted from the turmeric powder directly, we used commercially available samples from Sigma-Aldrich.  The first sample tested was a mixture of all three curcuminoids.  5mg of the commercially available powder was added to ethanol to make 100ml of solution that was tested by UV and HPLC against the solution extracted from the turmeric powder.  This confirmed that our original sample extracted from the turmeric was just as pure as the commercially available powders.  The benefit of using the powder was that the exact molarity of the solution was known.

6/5/18
-Alex Griffith

Column Chromatography

Column chromatography was used in an attempt to separate the curcuminoids.  The silica gel used was a 200-425 mesh.  The solvent used for the gel was the same as the solvent used for TLC, a 95:5 mixture of CH₂Cl₂:CH₃OH.

Initially, one darker layer appeared, it was expected to split into three separate layers, one for each of the curcuminoids.  Instead, the gel began to crack, possibly because of the interaction between the solvent used for the gel and the solvent used for the extraction of the curcumin, which was ethanol.  Whatever the reason, it ruined the run.  The crack in the gel was at the top of the column so the experiment continued to run.  The layer initially seen did not split into separate layers, in fact, it disappeared completely. 



Overall, this method was not effective, at least with the solvents used for this run.  The next attempt to separate the curcuminoids will be done with solid phase extraction.

6/4/18
-Alex Griffith

Initial Hand Cream

A basic hand cream was made using polar and non-polar ingredients.

In addition to the standard hand cream ingredients, 2ml of curcumin extract and 0.5g of synthesized aspirin were added to the hand cream.  Essential oils were also added for scent.

The cream was very yellow despite the small amount of curcumin that was added.  However, the cream did not stain skin like the curcumin extract did.




6/4/18
-Alex Griffith

Curcumin-Iron Complex

It was visually apparent that the combination of iron and curcumin resulted in a chemical reaction.

The vial on the left contains the filtered curcumin solution.
The vial on the right contains 0.1M Fe (3+).

The color of the solution in this vial is the result of adding 50µl of the iron solution to the curcumin solution.









5/24/18
-Alex Griffith

Curcumin TLC with Metals

The first TLC plate run showed very promising results, meaning there was complete separation of the curcuminoids.  Tests using UV showed that adding metals, especially iron and aluminium, to the curcumin solution changed the molecule, meaning that the curcuminoids were bonding to the metals as predicted.  TLC was then used on the curcumin-metal complexes to see if the different curcuminoids reacted differently with the metals.  Unfortunately, the results from the TLC were inconclusive.  In order to check each curcuminoid's reaction to the metal, the better option will likely be an HPLC analysis.

The TLC plate on the left had 4 identical spots of filtered curcumin extract.
The TLC plate on the right had, in order from left to right, (1) filtered curcumin extract,  (2) 100µl Fe (0.1M) in 2ml curcumin solution, (3) 20µl Fe (0.1M) in 2ml curcumin solution, (4) 100µl Al (0.1M) in 2ml curcumin solution.


The valley on the second plate originally indicated that the addition of iron to the curcumin was producing a complex with each curcuminoid that had slightly less affinity for the solvent.  However, the plate on the left that had the same sample in each spot showed the exact same valley so no conclusions could be made about the curcumin-iron complex based on the TLC.


The fourth spot on the second plate which contained aluminum was interesting because it did not show three spots like the other seven spots shown.  It produced a few spots along a smear.  This means the aluminum was forming complexes with curcumin and changing the molecule.  For more detailed results about the curcumin-aluminum complex, testing will need to be done with HPLC, UV, and IR spectroscopy. 


5/24/18
-Alex Griffith

Curcumin HPLC

High performance liquid chromotography, or HPLC, is a technique used in chemistry to separate, identify, and quantify each component in a mixture. The pumps switch between solvents A and B. Solvent A was composed of 2% acetic acid and 98% water, while solvent B was composed of 2% acetic acid and 98% acetonitrile. The LC Time Progress was set for 50% of solvent B at 1:00 minute, 53% of solvent B at 3:00 minutes, and 56% of solvent B at 5:00 minutes; at fifteen minutes the run was stopped. 

First, an HPLC was run for a 6.78 x 10^-3M sample of Curcumin. 

Second, an HPLC was run for a sample of curcumin one hundred times more concentrated than the first run.

Next, an HPLC was run for a 1:5-Iron:Curcumin ratio at 420nm.

Finally, an HPLC was run for a 5:1-Iron:Curcumin ratio at 420nm.

5/30/2018
-Kaitlyn Jordan

Second Extraction

After the first week, our original filtered extract was almost gone and the diluted mixtures that were made from it were showing that the curcumin left out at room temperature was oxidizing and therefore no longer usable.  The new extract is going to be kept in refrigeration when not in use and extractions will be more frequent, most likely weekly.

A larger volume of extract was filtered this time and will be used to attempt to separate the curcuminoids by column chromatography. 

5/29/18
-Alex Griffith

UV results

The curcumin extract was run through the UV machine again using the dilution that was found to be optimal before, 1:500 curcumin:ethanol.  This time, trials were run adding 20µl of a solution containing metal.  Results showed that both aluminium and iron bonded to the curcumin and changed its shape, however they changed the shape in different ways because the resulting curves were very different.  Calcium did not change the shape of the original curcumin UV curve, it did change the absorbance but that was only due to the addition of the calcium solution dilution the curcumin solution further.

There was a problem with getting the data from the machine.  The results could not be exported digitally because the software was so out of date.  The computer reading the results was also not connected to the internet and could not be connected, and in addition, it could not be connected to a printer.  Further analysis of the results will have to wait until the data can be exported.

5/24/18
-Alex Griffith

100uL Fe-Curcumin Complex IR Results

Today, an infrared (IR) spectra was run for the filtered curcumin extract.

Next, an infrared (IR) spectra was run for the 100uL Fe (3+)-curcumin complex.

















Analysis of both IR spectra revealed the loss of the carbon-oxygen double bonds of a curcuminoid of curcumin. The IR spectra of the Iron (Fe 3+)-Curcumin complex does not contain that extra spike slightly above 1500ccm^-1, indicating that iron has replaced the original carbon-oxygen double bond.


5/24/2018
-Kaitlyn Jordan

Initial UV Preliminary Testing

Today the preliminary scans of curcumin and the curcumin with metal were run to check for trends.
Using the program UV Probe- (Spectrum), the initial scans showed a large difference between the UV curves of the curcumin solutions with and without metals.

The optimal dilution for the machine was found to be 1:500 curcumin solution to ethanol.

-Alex Griffith

TLC- Thin Layer Chromatography

Once the solution was purified, a TLC was run to see if:
1. The sample was pure.
2. Three distinct spots were visible, meaning all three curcuminoids were present in solution.

The solvent used in the TLC chamber was a 95:5 mixture of CH₂Cl₂:CH₃OH.
Two spots of the filtered curcumin sample were used initially.
The resulting plate looked as follows:

The result produced three distinct spots for each original run.  This means that all three curcuminoids were in solution and the solvent in the chamber was appropriate for the test.

The spots were assigned designations A, B, and C, from top to bottom, meaning that the spot that traveled 4.10cm was A.

The Rf values were:
Rf A- 0.788
Rf B- 0.654
Rf C- 0.558





-Alex Griffith

Initial Extraction

The powdered turmeric root was used as the source of the curcumin.
In a round bottomed flask, 3.0026g of the powder was combined with 60ml of pure ethanol.
The mixture was heated (with boiling chips) for approximately two hours.
The instrument set up was as pictured:

After heating the apparatus was turned off.  The flask was left to cool and settle overnight (5-21-18).

The next day the solution was filtered using a syringe and a syringe filter into a clean bottle:

Due to the concentrated color of curcumin, staining of skin, surfaces, and glassware was extremely common.

-Alex Griffith 

Important Initial Information

Meeting times: Mon-Thurs 10-3
Mentor: Dr. Kahyaoglu, Dr. Scala
Location: SA116
Team Members: Alex Griffith, Kaitlyn Jordan, Isaac Kim, Alexander Czechowicz, Liliana Torres, Andrew Markov, Clara Kim
Dr. Kahyaoglu Availability: Monday and Wednesday from 10-3 in SA116

Goals and Objectives:
1. Extraction of curcumin from ground turmeric.
2. Use of TLC/HPLC/IR/UV to test for curcuminoids and to test our extracted curcumin against the commercially available curcuminoids.
3. Testing the reactions with metals of each curcuminoid, comparing UV results with the computational results.
4. Creating an extract based arthritis cream.  Application and effectiveness will be recorded.

-Alex Griffith