Science Buddies: "Ask an Expert"

Hi Pinkbear,

I'm sorry you have been sick. I hope you are feeling better.

I hope your whole research paper is not due by tomorrow, but if it is, I would recommend explaining why it is important to study anthocyanins for your background information and include some techniques that are used to study anthocyanins.

Here is a link for what should be included in a research paper from the Science Buddies website. I think you have some information on your topic, so if you follow this format, you will include everything that should be included.

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

The Wikipedia article has basic information on anthocyanins and has lots of research papers that you could use in your bibliography:

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

Go back to my post from September 6 and look at the links under 4 and 5. These sources list the reason for studying anthocyanins and would give your project a real world reason for being done. Anthocyanins have a potential benefit for human health, for food coloring, and for studying the relationship between different plants.

http://lpi.oregonstate.edu/ss01/anthocyanin.html

DNA and anthocyanins are very different molecules and your project will not deal with DNA at all. Electrophoresis is a technique that is used for DNA separations, and your project will involve separating anthocyanins, which are the plant pigments that are responsible for red, pink, purple, and blue colors. I have done some additional background reading on this topic, and I don't think that electrophoresis will work well for separating anthocyanins. There are a couple of methods that are probably not suitable for you to use because they require special equipment and are very expensive. These are called high pressure liquid chromatography and capillary zone electrophoresis. However, you should be able to use paper chromatography for your analysis and this is a classic method for separating the anthocyanins.

Here are a couple of links that describe paper electrophoresis. Basically, you extract the dye from the flower, and place a small dot on one in the paper and then develop the paper by letting an organic solvent travel up the paper by capillary action. This is a technique that you would be able to do:

http://www.biochemj.org/bj/083/0637/0830637.pdf

This paper probably has the best protocol that I have found so far:

http://cpr.molsci.ucla.edu/cpr/data/lib ... of_red.pdf

So, don't order the electrophoresis kit. You are going to need other supplies.

Also, please post your project deadlines for the science project and try to work at least one week ahead so you will have time to do a really good job on each assignment. With science projects, communicating the information to others is a really important part of the project, but this takes time to do a really good job.

Good luck on getting your assignment done! I will post more information over the week-end. Let me know what flowers you have available to work with.


Donna Hardy

Hi Pinkbear,

How are you? Did you get your assignment turned in?

As promised, here is some basic information that will be helpful for you to understand your project. You need to read as much as possible and become a subject matter expert on anthocyanins.

This site explains the chemistry of anthocyanins and shows a typical molecular structure. The important points here are to notice that the molecules have three 6-carbon rings with alternating single and double bonds. This is what causes the color of the pigment. Anthocyanins also have a sugar molecule (C6, H11,05) attached.

http://www.demochem.de/p26_anth-e.htm

Here is a recent research paper on anthocyanins in wild bananas. Please note the format of the paper and the techniques used the study the anthocyanins. This is an example of a research report on a topic that is similar to your project, and you can use it as a source of ideas. It includes a procedure for extracting the plant pigments and measuring the stability with different pH’s and temperature.

http://www.resplantbiol.com/getarticle. ... =5&artid=2

pH is a very important concept to understand for your project because anthocyanins will have different colors at different pH levels. Here is an explanation of pH, or concentration of hydrogen ions, from the Science Buddies website:

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

Here is an example of a project, which uses the anthocyanins from purple cabbage to make pH paper:

https://www.sciencebuddies.org/science- ... p041.shtml

You will probably be doing paper chromatography to analyze your samples for this project. Here is an example of a project on paper chromatography. Be sure to read and understand the background section of this project:

https://www.sciencebuddies.org/science- ... background

Unfortunately there is no kit available to purchase for a paper chromatography science project, but here is information for obtaining the supplies you will need. You should check with your teacher and ask if any of the materials are available through your school

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


Now, here are some questions for you to think about.

1. What is your research question? This is going to be the key for designing a really good experiment.

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

2. What type of flowers (or other colored plants) do you have available to use?

3. Do you have any questions about the background reading material? Can you answer the following?

-what causes the color of anthocyanins?
-what is the purpose of the sugar molecule on the anthocyanin molecule?
-what is the effect of changing pH on the color of anthocyanins?
-what is the best way to extract the anthocyanins from flowers?
-what is Rf in paper chromatography?
-using paper chromatography, how will you compare results from different samples?
-what is your independent and dependent variable?
-why can’t you use electrophoresis to separate anthocyanins?

Please let me know if you have questions. I know that there’s lots of chemistry and new information for you here, but I need to make sure that you understand the basics before you continue. It’s very important to understand the science behind your project because this will help you design a better experiment.

When is your next deadline?

Donna Hardy

yikes actually thats kinda a HUGE problemo... Is it 100% impossible to test on an aragose gel chamber, because my teacher gave me an extenstion until monday. I already got the kit, and half my paper is on the process of gel electrophoeris. haha ohhh snap, thats a problem... thanks!

Hi Pinkbear,

Don’t worry. This is not a problem. This is just a challenging moment in your science fair project and you need to decide what to do.

Since you have been researching electrophoresis, you know that it is used to separate charged molecules like proteins, DNA, and artificial dyes. Anthocyanins are neutral molecules and are usually separated by organic solvents based on their relative polarity. I don’t think electrophoresis will work well for separating anthocyanins.

Here are some possible solutions:

1. You just need to change molecules. So do an internet search and look for a project that involves separation of molecules that are charged. DNA and proteins are colorless, so you will need a stain to detect them in the agarose gel, but that’s a solvable problem.
Since you have already invested in the electrophoresis kit, you really should go ahead and use it, but you need a meaningful project to work on.

2. You could write up your paper with the background information on electrophoresis and plan to do the anthocyanin separation. However, knowing that this method is not optimum for separating these molecules, you would have to start a backup plan. Please do an internet search and look for an electrophoresis method for anthocyanins. You will find a method called capillary zone electrophoresis, but this requires a special instrument and the capillaries are very expensive. However a search of the scientific literature will certainly help clarify the matter and perhaps give you more information to include in your paper.

Probably, the most important thing to do at this point is focus on finishing your paper. Depending on what you decide to do for your experiment, you may need to make extensive revisions in the future based on your pilot experiments.

Donna

what flowers do you suggest i test on?
thaaaaaaank u

Hi Pinkbear,

You will need a steady supply of flowers to test and you will want to pick flowers that are readily available. Anthocyanins deteriorate with age, so you need fresh flowers to test, so you will have to let me know what might be available. Here are some possibilities.

What is blooming in your geographic area now? I live in a mild climate and we will have roses that will be in bloom until January and many other flowers blooming all winter. There are public gardens and botanical parks that might be able to supply unusual flowers with permission. Camellias will start blooming in November and different varieties will be in bloom all winter.

Do you know a local florist? I think it would be expensive to purchase lots of flowers, so do you know a florist who could provide short dated flowers at a nominal or no cost?

Do you have any neighbors that grow flowers? Usually gardeners who grow flowers are willing to share if you ask in advance. What varieties of flowers would be available?

I would suggest analyzing flowers that have a similar color, for example, all red, all blue, or all purple flowers. This would make an interesting and colorful project.

Or, do you live in an area where the trees have good fall colors? Perhaps you could compare the anthocyanin content of leaves of different varieties of trees that change colors during the fall.

Or, maybe you could do a fruit and vegetable project. Perhaps compare the anthocyanins in purple cabbage, plums and eggplant, or strawberries, red apples, and pomegranates.

I recommend that you do a pilot project as quickly as possible to find out how many flowers you will need for your project. One of the papers I saw noted that results were better if a large quantity of concentrated flower extract is used. This might require more than one flower.

Also doing a pilot experiment will help confirm if the electrophoresis method is going to work, or if it needs to be optimized. Be sure to run the control sample with food coloring as described first to make sure your electrophoresis unit is working and that you are operating it properly. If this works, then try one flower sample, perhaps a red carnation.


Donna Hardy

Hi Pinkbear,

How is everything going with your science project? Have you done any experiments yet? Please post if you have questions.

Donna Hardy

ok its experiment time! ive had to focus on a lot of other school stuff , and now that its over w/ time to focus on my sfp. based on what the kit comes with, and how it co-insides w/ my project, what other materials do i need? thaaaaaaanks!

Hi Pinkbear,

I understand; it’s hard to get all of your school work done, but I’m glad you are going to focus on your sfp again. Before you continue, you need to write down the question that you are going to answer by doing your project. What is your question? This will help you decide how to design the experiment.

I assume that you will be trying the electrophoresis gel box for a flower pigment project. But you need to decide what to focus on. Read back at earlier posts, and decide what specific topic you want to investigate. This is what will make your project unique.

Have you received your kit yet? When you do, I would recommend doing a trial run with food coloring as described in the project outline. This will give you experiment in working with the unit and verify that your unit is working properly. Let me know what happens.

Donna

ok, so im thinking of doing my project today or tomorrow, im thinking of teting on some brightly colored geraniums
http://www.bing.com/images/search?q=gge ... C&first=36

and getting other plants of the same genus. im thinking of testing 4 flowers, each three times. i purchased the kit, and im worried i dont have enough agarose and buffer to do three experiments. since i can remove the gel, i was thinking of preserving the gel and using it as a model in front of my sfp. Where should i go from here?

thank You, and happy holidays

Hi Pinkbear,

Welcome back. Happy holidays to you also! This is great weekend to work on your science fair project.

You have an excellent idea. I agree that you can probably save the agarose and reuse it. Since you are doing an original project with unknown results, I suspect that you will need to do a few runs to obtain good results with the electrophoresis cell, so preserving the agarose will be helpful.

After you have run a gel, carefully transfer it to an open container and add deionized water. In time, the water soluble pigments should diffuse out of the agarose. It will probably be helpful to mix the water and gel gently occasionally to expedite the diffusion process. And, you may need to change the water 3-4 times. Once the gel is clear, you can let it dry briefly and remelt it to make a new gel.

If this does not work, you can buy more agarose. Agar is an ingredient sold in Oriental grocery stores and it is available from Carolina Biologicals. The grocery store product will not have the same purity as the laboratory grade material, however, the lab agarose is a fairly expensive item.

http://www.carolina.com/catalog/search- ... SearchForm

Now, before you set up too many gels, please look at the structure of the plant pigments found in geraniums. Look at the charge of peonidins, cyanidins, and anthocyanins. Do these molecules have a negative or positive charge?

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

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

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

Now, look at the structure for a Red dye # 40, common food dye. What is the charge (negative or positive) of this molecule? What is the charge of the other food coloring molecules you are planning to try?

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

In electrophoresis, molecules will migrate towards the oppositely charged electrode. Negative molecules are attracted to the cathode and positive molecules to the anode. Do you expect the food coloring molecules to migrate in the same direction as the plant pigments? You need to run at least one food dye to verify the electrophoresis box is working properly . What are you going to do to ensure that your geranium samples migrate through the gel?

Donna Hardy

Awesome! i checked how much agarose i have (4 grams) and it seems to be suffecient for my 3 (and tester) trials! i got some diffrent colored geraniums together in that bright pigmented pink, a peach, a red, and a light pink. Will the specimens be visible in the gel? Thanks!

Hi Pinkbear,

The plant pigments will be visible in the gel. The samples will become diluted as they diffuse and migrate in the gel, so you need to start with very concentrated samples. How are you going to extract the pigments from the petals?

Also, how are you going to measure your results?

Donna

i added some alchohol with petals from the flower, and grinded them up. i tried the chamber out, and it works, with dye. i did my samples and am waiting for the alchohol to evaporate, but i dont know how much time to give them, and what its suppose to look like when the alchohol evaporates? Ill be running my 3 trials, measuring how long the band for each sample in cms, and finding and recording the averages. Thaaaaaanks!


also, since im testing the same exact species of geranium, differing in color, what exactly would be my problem? "Does the pigment of a flower affect the size of dna fragments?"

Hi Pinkbear,

That’s great news. I’m glad your electrophoresis chamber works. You want to load a sample that’s as concentrated as possible, so look for an intense color similar to the food dye.

You will not be separating DNA at all, just plant pigments. So your problem will be "what pigments are found in different colored flowers," or "are the same plant pigments found in different colored flowers." or something similar. If you were compared different species of red flowers, your question would be, "do red flowers contain the same plant pigment.."

Donna

Sorry, but isnt measuring the size of dna fragments measured in gel elctrophoresis? In my reasearch, it said the size of the dna fragments are what varied the bands of colors length from the origin. Im a little confused...?

Hi Pinkbear,

Sorry I did not include more detail. DNA is the molecule found in a cell nucleus that can vary in size and always has a negative charge. The traditional method to separate DNA fragments is by agarose gel electrophoresis. The gel module you are using was designed for this application.

http://en.wikipedia.org/wiki/Gel_electr ... leic_acids

However, electrophoresis is a general technique that can be used to separate any type of charged molecule.

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

You are purifying anthocyanins. Look at the structure of the anthocyanins in the link below. The ring structures are composed of carbon and hydrogen, the R groups are usually sugar molecules, which are composed of carbon, hydrogen, and oxygen and have a neutral charge. Notice that there is an oxygen with a positive charge in the molecule. The molecular weight is about 450 Daltons.

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

Here is the structure of DNA. Notice all of the phosphorus atoms on the molecule; these all have a negative charge and the molecule is much larger, up to several million Daltons.

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

As I recall, you did not want to do the DNA project because DNA is colorless and you would have to stain the DNA molecules with a DNA specific stain. Since you wanted to separate molecules that were already colored, you chose flower pigments. So you are using the same technique that is used for DNA separations, however, your basic application is different.

I see that your project deadline is coming soon, so you will need to revise whatever background information you have written and make sure your project board refers to plant pigments/anthocyanins and not to DNA. With your extraction protocol, I would not expect there would be any DNA at all in the samples. Since the anthocyanins are positively charged, not negatively charged like the DNA and food coloring, you will need to run the gel in the opposite direction compared to the food colors.

Does this help? If not, then post more questions. I want to make sure you understand all of the details before you put your project board together. You need to be able to explain the science behind your project to the science fair judges.

Donna Hardy

So far since im basing my sfp on this http://www.usc.edu/CSSF/History/2009/Projects/J0403.pdf, this contains basically everything that im doing. For the reversing and putting the pigments opposite to were it was, im slighty confuzzled. When i ran the project i mixed a couple flower petals into the dye, and they still migrated to the negative end, and how is their a specefic distinction between the positive & negative ends? thanks

Hi Pinkbear,

This is helpful. Pansies get their purple color from anthocyanins, so this experiment should work for you also. There is no description of the polarity of the electrodes in the experiments described in the summary. If the pigments migrated towards the anode in your pilot experiment, then do go ahead and set up your next experiment the same way. I was basing my suggestion on my experience with protein electrophoresis, and I have never done the experiment that you are doing.

With proteins, if they have a net positive charge, then it is necessary to reverse the polarity of the electrophoresis module. However, it could be that the plant pigments have enough polarity that they will migrate towards the anode. You will verify what will happen with your results.

Go ahead and set up the experiment with the different colored geraniums petal extracts and let me know what happens. Will you be able to take a photograph of the gel as the colored bands migrate through the gel? If not, then measure the distance of each colored band from the origin every 15-20 minutes, if you can, in centimeters.


Donna Hardy

ok so went through one trial, but the steel wire at the negative end (or whatever the pigments migrate to) got like, rusty! Um, what thhe heck happened,lol! anyway, since NOW im all out of steel wire were would you suggest i find some? also, since im trying to set up my board, i want to get the title out of the way. Would "The Molecular Migration of Plant Pigments" work? Thaaaaaaanks!

Hi Pinkbear,

Your electrode corroded, and this can happen with inexpensive electrodes. Electrodes on regular laboratory equipment are made of platinum, but using this metal would have made the kit prohibitively expensive.

The electrode is probably made from a paper clip, so try to find some more paper clips that are the same size as the ones included in the kit. If you can find galvanized paper clips, they will be more resistant to corrosion. I recommend changing the electrodes for every run.

Are you using the bicarbonate buffer that is recommended? If there are any chloride ions in your sample or buffer, this would accelerate the corrosion.

It's good that you are working on your board. "Molecular Migration of Plant Pigments," is a nice title. I like the alliteration. You can add a subtitle with the research questions in smaller type, such as "how many plant pigments do geraniums contain?" of "how is geranium color related to plant pigments?"

Donna Hardy

Hi Pinkbear.

How are your doing? Have you had a chance to repeat your experiment with new paper clips?

One of the good things about having unexpected results is that it gives you an opportunity to include more scientific information in your discussion and conclusion section. Here is a Science Buddies project on corrosion. There is helpful information in the discussion and references that explain the rusting process that you observed with your electrodes.

https://www.sciencebuddies.org/science- ... background

The paper clip contains elemental iron, Fe. The Wikipedia article includes chemical equations for the oxidation of Fe to FeIII, or iron oxide.

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

The question is why did the electrode rust so quickly?

So, you have actually done two science experiments Let me know if you have any questions.


Donna Hardy

hi, im wondering is my project including gel electrophoresis or molecular migration? Im just going to include my findings about the plant pigments. Thanks!

Hi Pinkbear,

In your project you used gel electrophoresis as a technique to separate the plant pigments, which are molecules. In gel electrophoresis, molecules migrate in an electrical field, based on charge and molecular weight.

Does this help?

Donna