Need Help using Chromatography paper

[tigergirl1009]

I'm doing the project which compares the pigments which make-up different red flowers. I'm using a red rose and a red carnation and chromatography paper ordered from science kit. My research indicates that the paper should separate the red color into different pigments, but after removing it from the alcohol and water solution-the paper still show only red-no separated pigments. Please help!

[donnahardy2]

Hi,

I think you are doing this project:

https://www.sciencebuddies.org/science- ... p035.shtml

The separation of the pigments depends on the solubility of the pigments in the solvent and the migration of the pigments through the paper. Did the red pigment move through the paper strip? What does the paper strip look like now? Did you put a small concentrated dot of pigment on the origin? Was the red pigment soluble in the alcohol/water mixture? Here are some possible solutions:

1. The paper strip was not long enough to allow separation of the pigments.
2. You didn't use enough sample at the origin
3. What concentration of isopropanol did you use? Perhaps you should increase the concentration of isopropanol?
4. Perhaps using a different solvent, such as acetone (fingernail polish remover) would give a different result.

Please try your experiment again and let us know what happens. I'm sure there is a solution.

Donna Hardy

[tigergirl1009]

Yes I am doing the Reveal the Red project. I tried my experiment with 91% and 70% isopropyl alcohol but the different concentrations didn't seem to make a difference. I put plenty of the sample on the origin, so I don't believe that's my problem. The red sample of both of my flowers moves up the paper, but the pigments don't separate out like they are supposed to. My results from my red rose and red carnation are the same, and I'm pretty sure that's not right. I would like to to my experiment well, but I have done it multiple times with the same result. Any ideas?

[donnahardy2]

Hi,

Did you run the chromatogram in an enclosed glass jar? How did you extract the flower pigments?

I think you need to run some more chromatograms. Look at the next to the last slide in the slide show in this website:

http://peer.tamu.edu/podium_poster_pres ... lternative Experiments

Notice that there is a difference in separation of the plant pigments with different proportions of water and isopropanol.

What grade are you in? How much chemistry have you had? Understanding the chemistry of the plant pigments will help a lot on this project.

The red pigments found in flowers are organic molecules known as anthocyanins and carotenes. For each class of pigment, the red color is due to alternating double and single carbon bonds in the base structure of the pigment molecule. However, there’s an important difference in the chemistry of the pigments.

Anthocyanins are water soluble because they have a relatively high concentration of hydrogen and oxygen atoms attached to the carbon backbone of the pigments. Look at the chemical structure in this website:

http://en.wikipedia.org/wiki/Anthocyanin

Carotenes also have the alternating single and double carbon bonds, but do not have many oxygen atoms in the structure. This means that they will be soluble only in organic solvents such as isopropanol, acetone, or petroleum ether. You can see the chemical structure of carotene in this website:

http://en.wikipedia.org/wiki/File:Alpha-carotene.svg

Now, look at the structure of the solvents you are using. Here is isopropanol; notice that there is carbon and hydrogen atoms on one end of the molecule and a single oxygen atom with a hydrogen atom (called a hydroxyl group) on the other end. This solvent will interact primarily with molecules that contain carbon and hydrogen atoms. However, because of the hydroxyl group, it can also be mixed with water.

http://en.wikipedia.org/wiki/Isopropyl_alcohol

Now, here’s the structure of water.

http://en.wikipedia.org/wiki/Water

As you can see, water is composed of only oxygen and hydrogen, so will interact with similar structures such as the hydroxyl groups on anthocyanins. It will not dissolve carotene pigments very well because of the difference in chemical structure.

You have used 70 and 91% isopropyl alcohol, which would dissolve carotene pigments better than anthocyanins. If you try again with a higher percentage of water, you will get different results.

Please note that the photographs of paper chromatograms are usually fairly diffuse. You will get the best results if you start with a very concentrated dot of red dye that is as small as possible.

So, please try again, and let me know what happens. Also, let me know if you need more explanation about the chemistry of this project.

Donna Hardy

[donnahardy2]

Hi,

I just noticed that your project is due tomorrow. In that case, you should not do any more experiments. You should concentrate on writing up your board and presenting your results, including the fact that your results were reproducible. You have a very good and complete project as it is. Here is the very helpful information from the Science Buddies website on preparing your board. Be sure to include all of the sections. Don’t worry that your results did not turn out as expected. You will be able to explain what happened in your project and what you would do if you had more time to do another experiment.

https://www.sciencebuddies.org/science- ... oard.shtml

Here is some more helpful background information.

Here is an abstract from the scientific literature reporting the identification of pigments from roses and carnations. Both references describe the presence of anthocyanins and don’t mention carotenes. I hope you can open these abstracts:

http://www.sciencedirect.com/science?_o ... archtype=a

http://www.sciencedirect.com/science?_o ... archtype=a

Here is a color chart with a list of pigments found in roses. Notice that most of the pink and red pigments are anthocyanins; the carotenes are more orange and yellow.

http://holeman.org/Dons%20Handy%20Rose% ... 0Chart.pdf

I hope this helps.

Donna Hardy

[tigergirl1009]

Thank you so much for your help. I didn't know until last night but I have an extra week to work on my project. I'll redo my experiment with more water and try to put a small dot of the sample on the origin. I'm in 8th grade, and I haven't done much chemistry. Some of this stuff is a little over my head, but I think I'll have more success on my next experiment. I will post as soon as I have my new results.

-Savannah

[donnahardy2]

Hi Savannah,

The extra time is really goods news for this project! Try a range of solvent concentrations if you can. In the reference photograph, the red pigments separated best at 40 and 60% isopropanol. It would be excellent can run chromatograms with a range of solvent concentrations from 0 to 100%, However, if you can only do one more concentration, try using 50%.

I’m sorry the chemistry was confusing. Since the chemistry is key for understanding this project, it would be worthwhile to spend some time looking though the information I sent and see if you can make some sense from it. The key concept that you need to understand is the polarity of molecules. Since you haven’t had chemistry yet, I know it’s difficult.

Organic molecules: Anthocyanins and carotenes are organic molecules because they contain carbon.

http://www.google.com/search?hl=en&defl ... d=0CBMQkAE


Polarity: This website explains the difference between polar and non-polar molecules. See how much you can understand:

http://en.wikipedia.org/wiki/Chemical_polarity

If you look at the chemical structure of anthocyanins and carotenes, you will notice that carotenes have more carbon and hydrogen and fewer oxygen atoms. This means than anthocyanins are more polar and more hydrophilic compared to carotenes and carotenes are less polar and more hydrophobic compared to anthocyanins. Anthocyanins will be more soluble in higher concentrations of water; carotenes will be more soluble in organic solvents such as isopropanol.

Another example might help. Another hydrophobic molecule that you are familiar with is olive oil; this is a very large hydrophobic molecule that only mostly carbon and hydrogen atoms and floats on water. An example of a hydrophilic molecule is the red dye that is used in food coloring and is commonly used in water-based products like sports drinks. Look at the chemical structure of these two molecules:

http://en.wikipedia.org/wiki/Allura_Red_AC

http://www.cardiactherapy.org/images/OliveOil.gif

Can you “see” the relationship between the chemical structure (relative number of carbons, hydrogens, and oxygen atoms) of the molecules and their physical characteristics?

You will see better separation of compounds with paper chromatography if the molecules are more soluble in the solvent you are using.

For your science board, you should include background information to explain how molecules separate on the paper and as much of the chemistry of anthocyanins and carotenes as you can understand. You should also include a discussion about your initial results and what you are doing to improve the resolution on the chromatogram. The science fair judges will be impressed by your chemistry knowledge and by what you learned in the course of doing the project.

Let me know if you have any questions, and do let me know about your results.

Donna Hardy