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