Science Buddies: "Ask an Expert"

I am trying to separate chlorophyll a and chlorophyll b from a main batch of chlorophll using gel electrophoresis. The problem is that I need information about the differences between chlorophyll a and chlorophyll b. Is there a charge difference? Is there a size or weight difference? Is there any difference that can help me perform gel electrophoresis successfully? Thank You for the help!

Hi,

This is a very interesting topic and you will learn a lot of chemistry with this project. Here is a website that shows the structure of chlorophyll a and b.

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

Please note that chlorophyll is a large hydrophobic molecule and that the difference between a and b is only one aldehyde group (COH). This means there is very little difference in molecular weight no difference in charge; just a slight difference in polarity. This means that gel electrophoresis would not be suitable for separating these two molecules.

One method to separate chlorophyll a and b is with a reversed phase C8 or C18 column using an HPLC chromatography system.

http://pubs.acs.org/doi/abs/10.1021/jf00006a020

Do you have access to an HPLC system? Do you know anyone who works in a chemistry or biochemistry lab who could help you with this analysis?

Another classic method is to use paper chromatography or cellulose chromatography. You won't be able to read this article, but the abstract describes the separation that you want to do.

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

The paper chromatography requires Whatman filter paper, paraffin oil, methanol, and petroleum ether. The science buddies website is a good reference for this technique:

http://www.sciencebuddies.com/science-f ... rces.shtml

What experiment are you doing? Please let me know if you need more information about either of the chromatography techniques for separating chlorophyll a and b.

Donna Hardy

donnahardy2 wrote:Hi,

This is a very interesting topic and you will learn a lot of chemistry with this project. Here is a website that shows the structure of chlorophyll a and b.

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

Please note that chlorophyll is a large hydrophobic molecule and that the difference between a and b is only one aldehyde group (COH). This means there is very little difference in molecular weight no difference in charge; just a slight difference in polarity. This means that gel electrophoresis would not be suitable for separating these two molecules.

One method to separate chlorophyll a and b is with a reversed phase C8 or C18 column using an HPLC chromatography system.

http://pubs.acs.org/doi/abs/10.1021/jf00006a020

Do you have access to an HPLC system? Do you know anyone who works in a chemistry or biochemistry lab who could help you with this analysis?

Another classic method is to use paper chromatography or cellulose chromatography. You won't be able to read this article, but the abstract describes the separation that you want to do.

http://www.sciencedirect.com/science?_o ... earchtype=

The paper chromatography requires Whatman filter paper, paraffin oil, methanol, and petroleum ether. The science buddies website is a good reference for this technique:

http://www.sciencebuddies.com/science-f ... rces.shtml

What experiment are you doing? Please let me know if you need more information about either of the chromatography techniques for separating chlorophyll a and b.

Donna Hardy

I was wondering will the other methods of extracting chlorophyll a and chlorophyll b, be able to give me a significant amount to test the extractions using a spectrophotometer. Thank You for you help, this has already enlightened me greatly!

Hi,

Here is a website that includes a procedure for extracting chlorophyll using acetone, which is the solvent found in fingernail polish removal products. This lab protocol also includes a procedure for separating the pigments using thin layer chromatography.

http://facstaff.bloomu.edu/mpugh/Experiment2.pdf

Chlorophyll is a very hydrophobic molecule, so dissolves best in non-polar solvents.

Here's a website with an explanation of polarity. With science projects, for best results, it's important to understand the scientific principles behind your project.

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

Here's a table showing the relative polarity of organic solvents. Notice the position of acetone on the chart. Chlorophyll will be more soluble in all solvents above acetone and less soluble in the solvents below acetone. Benzene would be perfect to use, but it's a very toxic solvent, so I don't recommend using it for a science project. Acetone is much less toxic, and it's readily available, so would be a good choice. But you should still be very careful when working with any type of solvent.

http://www.chemical-ecology.net/java/solvents.htm

If you extract chlorophyll from a plant, you will have a mixture of pigments in solution. What were you planning to do with the spectrophotometer? What is the objective of your project? Please let me know more about what you are doing, and I'm sure I will have more information to share.

Donna Hardy

donnahardy2 wrote:Hi,

Here is a website that includes a procedure for extracting chlorophyll using acetone, which is the solvent found in fingernail polish removal products. This lab protocol also includes a procedure for separating the pigments using thin layer chromatography.

http://facstaff.bloomu.edu/mpugh/Experiment2.pdf

Chlorophyll is a very hydrophobic molecule, so dissolves best in non-polar solvents.

Here's a website with an explanation of polarity. With science projects, for best results, it's important to understand the scientific principles behind your project.

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

Here's a table showing the relative polarity of organic solvents. Notice the position of acetone on the chart. Chlorophyll will be more soluble in all solvents above acetone and less soluble in the solvents below acetone. Benzene would be perfect to use, but it's a very toxic solvent, so I don't recommend using it for a science project. Acetone is much less toxic, and it's readily available, so would be a good choice. But you should still be very careful when working with any type of solvent.

http://www.chemical-ecology.net/java/solvents.htm

If you extract chlorophyll from a plant, you will have a mixture of pigments in solution. What were you planning to do with the spectrophotometer? What is the objective of your project? Please let me know more about what you are doing, and I'm sure I will have more information to share.

Donna Hardy

I was planning to use the chlorophyll a and chlorophyll b extract in a spectrophotometer to determine exactly what wavelengths they absorb best. Also, I am subjecting the chlorophyll a and chlorophyll b to different pH concentrations because my main objective is to see how pH effects the absorption and reflection spectrum of chlorophyll a and chlorophyll b. Thank You again for everythin!

Hi,

Are you going to separate the chlorophyll a and b and then do a pH study using the individual pigments? Chlorophyll is susceptible to degradation by lower pH values. If you use a plant extract, chlorophyllases can be present that will degrade the chlorophyll. Temperature and oxidation can also affect the integrity of the chlorophyll molecule.

Have you tried to do a literature search on your topic to find out what research has been done in the past? That would give you a foundation that will allow you to design a unique experiment for your science project.

Please let me know if you need more information on doing literature searches.

Donna Hardy