Science Buddies: "Ask an Expert"

My name is Deo, a student studying in the Philippine Science High School-Western Visayas Campus. We have a Science Research Subject
and my study is all about the anti-fungal activity of solvent fractionated extracts of Indigo plant. I just want to ask you some questions that might
help fill in the knowledge gaps in my methods.

(1)Could anyone please share with me the principles and details concerning the solvent fractionation?
because i can't seem to find a definite procedure on it.

(2)does anyone have a soft copy of an article or a protocol on solvent fractionation?

(3) in a study such as mine, how many fractions of the extract, do you suggest, will be taken for me to measure antifungal activity?

Thank you and more power to you!
I am hoping for your reply!

Hi Deo,

Welcome to the science buddies website. This sounds like a great project! Here's the information you need:

I just found a Wikipedia description of solvent/solvent extraction that is excellent. It explains the general principles behind this technique and the variations that you can use to separate your sample.

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

For this technique, you would use two immiscible solvents and mix them very thoroughly to extract the indigo. Here is a website that shows the chemical structure of indigo:

http://www.chriscooksey.demon.co.uk/indigo/phyto.html

I hope you have had chemistry. You will see from the structure of indigo that it contains 4 ring structures composed mostly of carbon and hydrogen atoms. This makes the molecule very hydrophobic (water-hating). This means that indigo will not dissolve in water, but requires non-polar solvents to dissolve it.

Next, here is a reference that shows the eluotropic series of solvents listed by increasing polarity.

http://www.namedorganicreactions.co.uk/ ... %20tlc.pdf

The solvents at the top (pentane, hexane) are non-polar and will dissolve indigo; the solvents at the bottom (water, methanol, ethanol) and polar and will not dissolve indigo. Acetone is fairly non-polar and should be easy to obtain, but you will have to find out if it's a good solvent for indigo. Please follow safety precautions when working with any solvent, especially the non-polar solvents as these are toxic and flammable. You will need good ventilation!

Next, here is a scientific journal article that describes a method for testing anti-fungal activity. The method is called the minimum inhibitory concentration. You would use different concentrations of indigo and find out if the dye inhibits your test organisms. You would need some test fungi to use for you assay with this method. There are other options for testing for anti-fungal activity; this is just an example of how you might do this and get the measurable result you need for a science fair project. Let us know if you need other suggestions for this part of the experiment.

http://www.sciencedirect.com/science?_o ... 16a14b3e86

The science buddies website has lots of helpful information on microbial techniques that are applicable to growing fungi. You should check out all of the sections under “Microbiology.”
http://sciencebuddies.com/science-fair- ... ndex.shtml

I hope this helps you get started. Do let us know if you need any explanation of the chemistry or microbiology involved in this project.

Donna Hardy

Hi Deo,

If your project is on antifungal properties of indigo on textiles, here is a website that reviews this topic. You will notice that one slide includes a list of the standard methods for measuring antimicrobial activity in textiles.

http://centrum.tul.cz/centrum/itsapt/pr ... #310,1,New Multifunctional Textiles: Antimicrobial Treatments

Here is a website that describes an agar plate method for measuring antimicrobial activity in natural products. You could adapt this method for use with indigo.

http://hillagric.ernet.in/education/cov ... ethods.pdf

Please describe your project and what question you are trying to answer with your experiments if you need more information.

Donna Hardy

Hi Deo,

Here is a website, that is not a scientific reference, but it includes a list of solvents that will dissolve indigo.

http://www.sewanee.edu/Chem/Chem&Art/De ... nts/Indigo

And, here is a suggestion for an alternative to solvent/solvent extraction. You can use thin layer chromatography to isolate the the individual pigments from the sample. If you do use solvent/solvent extraction, you could use the thin layer chromatography technique to verify the purity of your sample.

http://cmuj.chiangmai.ac.th/full/2002/may2002-2f.pdf

Donna Hardy

thank you for providing me with those information. they really helped in conceptualizing my study.
but i have more questions that need answering.

(1) it is said that the indole-derivative isatin has antifungal activity and Isatin was found to be the major degradation product of indigo, so it could be easily
expected that the plant extract has antifungal activity. I plan to isolate the isatin in the plant using fractionation. How can i do this? How can i get the isatin from the extract of indigo? could anyone share with me a lab protocol that answer this question?

(2) how many fractions should be taken in order to isolate isatin?

(3) isatin isn't just the substance that is in indigo. there are still a lot of phytochemicals, substances, metabolites present in the plant. And some of these may possibly have antifungal activity too, right? what are the possible compounds, chemicals, metabolites in indigofera tinctoria that may have antifungal activity?

i am hoping for your reply.
more power to you and thanks

Hi,

Isatin can be synthesized from indigo by oxidizing it with nitric or chromic acids, but it is usually produced synthetically. Here is a reference that explains how to make isatin:

http://www.scielo.br/pdf/%0D/jbchs/v12n3/5590.pdf

Here is another reference that describes how to synthesize different compounds that might have antifungal and other biological activity from isatin:

http://www.sciencedirect.com/science?_o ... 9bbb42f0db

I am having a hard time understanding the purpose of your project. Are you interested in starting with indigo plants and purifying indigo and then converting that to isatin? Or, are you interested in synthesizing isatin and testing that for antifungal activity? Making isatin or purifying indigo from plants would be a complete science fair project. Testing either indigo or isatin for antifungal activity would also be a complete project. Isolating and identifying all of the compounds in indigofera tinctoria that might have antifungal activity would be a good PhD thesis project. For a science project, you need to identify a question that you can answer with an experiment. For example, you could ask the question, "do indigo plants have antifungal activity?" With this question you could make extracts of the plant in different solvents, and then use one of the methods for testing antifungal activity to measure your results.

Also, do you have access to a laboratory where you can work with the organic solvent and grow the microorganisms used for testing? You need to consider what resources you have available and plan an experiment that you will actually be able to do.

I hope this helps you narrow your topic down a little so you can plan a specific project.

Donna Hardy

i am really grateful that you had spent your time in giving me aid in my study.
thank you and more power to you.

just to clarify things,
The aim of this my is to measure the antifungal activity of solvent fractionated extracts of Indigo (Indigofera tinctoria) leaves against Lagenidium sp and determine it’s toxicity on Mud Crab (Scylla serrata) larvae.

i'll try to Isolate isatin or at least reduce unwanted compounds in the methanolic extract from the leaves of indigofera tinctoria and determine it's antifungal activity. after that i will then see if the extract is too toxic for mud crab larvae.
and i won't isolate nor identify all the antifungal compounds present in indigo....

i just thought that it would be beneficial for me if i had a background on chemical components of indigo that may have antifungal properties aside from isatin. i have no plans of including the identification or isolation them in my methods

thanks again!! and more power to you!

Hi Deo,

Isatin is soluble in boiling alcohol and ether.

Measuring the antfungal activity of isatin or indigo plant derivatives would be a complete project. Measuring toxicity on the mud crab larvae would be another complete project. These is a fascinating topic and should make an excellent project, and I'm sure you will learn a lot in doing this. Please check with your teacher to make sure you can get permission to do the experiments with the mud crab larvae; I am not familiar with the local rules for science fair projects in your country.

You have the right approach to study the chemistry of Indigofera tinctoria. It is difficult to find information on the internet for the chemistry of this plant. There is lots of information on indigo and most of the other references are non-scientific references. You might be able to purchase pure isatin, and compare results with the whole plant extracts. That would tell you if there is a difference in the effect of pure isatin and the extract from the plant.

How are you going to measure antifungal activity?

Donna Hardy

hello, thanks again for answering my question. it really means a lot to my research...

me and my adviser decided not to isolate isatin in the plant... we changed my study into determining the antifungal activity of different fractions (obtained through fractionation) of Indigofera tinctoria. could you suggest 1 fraction that would be perfect for my study and it's reason why it is the right one.]. So far, i have already chosen, Dichloromethane fractions and butanol fractions from the plant. I need 1 more.

In relation to this. can anyone tell me how to fractionate Dichloromethane fractions and butanol fractions from Indigofera tinctoria? is there a lab protocol, manual or research article that explains each step in obtaining the fractions?

i don't really have a definite procedure on how to measure antifungal activity. Could you suggest an antifungal assay that would be perfect for a study such as mine? is there a lab protocol, manual or research article that explains each step in measuring antifungal activity.?

thanks! and all answers will be greatly appreciated.

Hi Deo,

It sounds like you are making great progress in defining your project; this sounds like it will be a great project. Methylene chloride is a non-polar solvent and butanol is more polar, so these two solvents will give you a good selection. For your third solvent, you should pick something with different chemical properties. Water is more polar and will not dissolve isatin, so this would not be a good choice. Here’s information from one of the websites I found earlier:

Indigo's chemical properties make it "...difficult to dissolve in hot ethanol, amyl alcohol, acetone, ethyl acetate, and pinene...but readily soluble in boiling aniline, nitrobenzene, naphthalene, phenol and phthalic anhydride.

So I would recommend trying boiling aniline, nitrobenzene, naphthalene, phenol or phthalic anhydride if you have one of these solvents available. Do you understand the significance of the relative polarity of solvents?

All of these solvents are toxic so make sure and work in a hood or other well ventilated area when you work with solvents and avoid direct contact.

I recommend that you test the entire fraction from each solvent to see if there is any antifungal activity. That would be a complete science fair project. You could use techniques called column chromatography or thin layer chromatography to separate individual compounds, but this would make your project much more complicated. Please let me know if you really want to do this part of the project.

For the antifungal test, you will first saturate small filter paper disks, (about the size of a paper punch hole) with the plant extract. You would include a negative control of solvent only, and a positive control that would contain a known antifungal compound. You would then place the paper disks on a plate of agar that contained the Lagenidium. You would incubate the plate and measure the distance between the edge of the paper disk and the start of the fungal growth in millimeters. This gives you the quantitative result (mm of inhibition of growth) that you need for a science fair project. Here is a website that includes a step-by-step protocol for this method:

http://hillagric.ernet.in/education/cov ... ethods.pdf

You would need agar, Petri dishes, and a culture of Lagenidium for this technique. Do you have access to these materials.


Please let me know if you have any questions.

Donna Hardy

thanks ms. donna!, for providing me with the antifungal procedure. i will use it for my study.
i am really glad that you had the time to answer my questions. i am very grateful.

however, 1 part of my methods still isn't complete....(http://panicoco1.multiply.com/photos/album/2/i_dunno#2)the attached link is the concept map of the fractionation process that one author has suggested me with. problem is, he didn't actually explain the methods specifically he only provided me with the overview. so that is why i would to seek aid from you & everyone.
(1) How can i fraction a 500ml methanolic extract of Indigofera tinctoria using dichloromethane as the solvent? what amount of dichloromethane will i use? what materials will be used?
(2) How can i fraction a 250 ml dichloroform fraction from the methanolic extract of Indigofera tinctoria using n-Butanol as the solvent? what amount of N-Butanol will i use? what materials will be used?
(3)How can i acidify a 500ml methanolic extract of Indigofera tinctoria using 92% HCl what amount of HCl will i use? what materials will be used?
(4) in filtering the precipitate? what filter will be used?
(5) How can i basify the precipitate using 10% NH4OH? what amount of NH4OH will i use? what materials will be used?
(6)How can i fraction an acidified an basified extract of Indigofera tinctoria using chloroform as the solvent? what amount of chloroform will i use? what materials will be used?

The answers to these questions are vital to my study...
All logical answers will greatly appreciated.
Thanks and more power!!

Hi Deo,

Please look at the following website, and review the basic information about solvent-solvent extraction carefully.

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

Close to the bottom, you will see a picture of a separatory funnel that is used for this application. For a 500 ml methanol sample, you will need one with a volume of at least 3 liters so you can add an equal volume (500 ml) of butanol and have room in the funnel to shake the solvent mixture and extract the molecules that will partition into the two solvents. You will shake the mixture for several minutes, and then let the solvents separate and collect the two fractions. You will need to use the separatory funnel for each step where an organic solvent is used. If you cannot get a separatory funnel with the correct volume, you will need to scale down the experiment. I don’t know of a substitute for the funnel, so please find out if you can get this equipment. You don’t want to work with nonpolar organic solvents in an open container. The protocol would be the same for each step with two solvents.

Here’s a website that shows the step-by-step details for this technique:

http://orgchem.colorado.edu/hndbksuppor ... edure.html

For the steps where acid and base are added to the sample, there will not be two separate layers of solvent, so you can work in a beaker or flask with a stir bar that has a volume at least twice the sample volume. The outline does not specify a volume, so I would recommend adding acid until no more precipitate forms. Try adding a 5% volume first and stir the sample, then add another 5% volume and check to see if there is any additional precipitate.

Precipitates are collected on laboratory filter paper. The most commonly used filter paper is Whatman filter paper, and it is available in a variety of pore sizes. I would recommend a qualitative filter paper with a 10-15 micron pore size. You will need a funnel that will hold the size of filter paper you plan to use, for example Whatman Grade number 1 with a pore size of 11 microns. This filter paper works for most applications. If you can't get this exact filter paper, then try using a coffee filter and see if it retains all of the particles.

http://www.labdepotinc.com/Category_Details~id~288.aspx

Where did you get this outline? Progress in science is made by taking results of others and planning the next logical experiment. If your outline came from previous research on the purification of antifungal molecules from another type of plant, then it will probably work well for your application and would be the best choice of protocols. Since the outline omits so many details, it doesn’t make complete sense to me either. For example, I can’t tell if you’re supposed to use the precipitate or the supernatant for the base step. Hopefully, the person who supplied this to you can explain it. If the outline was developed for another purpose, then I recommend that you do the first methanol extraction step and then test that for antifungal activity. If you find activity, then do the next step and test each fraction to find out which fraction contains antifungal activity. There’s no reason to do all of the steps unless you verify that the activity you are looking for is present. And, you may have to try the experiment a couple of times to figure out the best way to do the experiment.

It is very important for you to understand how solvent-solvent extraction works, and what types of molecules are going to separate into the various solvents. Please read the background information and let me know if you have any questions about the chemistry. Please let me know if you have had high school chemistry. I need to make sure you understand both the theory and how to do the techniques for this very ambitious project.

Donna Hardy

how do you transfer filtered-off precipitates to erlenmeyer flasks.

I will filter off precipitates from a solution of methanol extracts of plants and hydrochloric acid.
what are the right and safe procedures for transferring precipitates from the filter paper to a erlenmeyer flask?

how do you dispose methanol extracts?

how do you dispose of it safely?

where will you put the waste?

Hi,

The answer to your question depends on what you will do to the precipitate. If the next step is to redissolve the precipitate, my old chemistry text (Blaedel & Melochie,”Elementary Quantitative Analysis, Theory and Practice”) recommends pouring the dissolving solution over the washed precipitate while still on the filter paper in the funnel and letting the solution and dissolved precipitate drain through the funnel into the flask. You continue to add dissolving reagent to the filter in the funnel until no precipitate is left on the filter paper.

If the redissolution is too slow to do in place on the funnel, the recommendation is to transfer the paper and precipitate to the beaker/flask and do the redissolution there. The problem with this is that the filter paper tends to disintegrate in the beaker/flask, leaving a hard to separate mess.

If you intend to dry and weigh the precipitate, they recommend transferring the filter paper and precipitate to a weighed ashing vessel, then heating the vessel until the paper is burned off and reaches constant weight.

What really does not work is to try to scrape the precipitate off the wet filter paper and into the flask.

You will have to devise your own proceedure based on your specific situation. You want to avoid getting your fingers on the wet filter paper if it is wet with acid and or organic solvents (use tongs or tweasers or gloves if you must).

I hope this helps. If not, please talk to a chemistry teacher in your school for advice - they should be able to demonstrate good techniques for you.

Best wishes for a great project.

Barrett Tomlinson

Hi,

The answer of how you dispose of methanol extracts may depend on where you are working. In an industrial laboratory all chemical wastes are normally collected in 55 gallon drums, and sent off to an authorized hazardous waste disposal company. This is a legal requirement, and companies get into huge trouble with the government if they fail to do this.

If you are working in a school lab, talk to the person in charge of the laboratory and follow their instructions.

If you are working at home, I cannot give you advice. Strictly speaking, it is not legal to pour any chemical waste down the drain - in most jurisdictions you must send the waste to an authorized hazardous waste disposal company (for an expensive fee) to comply with the environmental laws. If you know someone working in a chemical lab, perhaps they can help you with the disposal. Alternatively, contact your city government - some cities now sponsor hazardous waste disposal events where you can drop off your chemical wastes for safe disposal.

Good luck with the project!


Barrett Tomlinson

Hi,

I have merged all of your topics back under your original topic--please keep all your questions in this topic in the future; it will help the experts better help you.

Hi Deo,

It's very important to always reply on the same thread so everyone is notified of your response. If you do, everyone who is helping you will be notified when you post a question. I look for new inquiries on Sundays only, but I usually respond on other days if I see a notification in my e-mail.

For the methanol disposal, you need to follow the local laws for disposal. In the US, we collect methanol and other solvents in a special container in the hood and the container is taken a hazardous waste collection site when the container is full. I imagine that the requirements for organic solvent disposal are similar in the Philipines, but you need to find out what the legal requirements are for your city, and follow the law. It is very important for scientists to follow are laws and safety regulations when doing research projects. Methanol is toxic, so must be handled accordingly.

It sounds like you may have started doing your experiment already. It would be very helpful if you could post your experiment and explain what you are planning to do. We can do a better job of answering your specific questions if we know what the general project is. It’s hard to give a good answer to an isolated question if we don’t understand what is happening. Perhaps next time you post a question, you could explain what is happening that made you think of asking the question. Do you have a teacher or someone who can show you the specific techniques involved with this project, or are you trying to do this on your own?

Have you had a chance to read the articles and references I have previously posted? Please let me know if you could not access any of the information and I will print it out, scan it and upload it so you can see it. I want to make sure you understand the chemistry of what you are doing. The reason I am suggesting this, is that it sounds like you are following a protocol for indigo extraction from the plants. Methanol will extract the indigo precursor, but might not extract all possible antifungal compounds. If you look at the structure of antifungal compounds, you will see that most are hydrophobic and some are more polar than others. This means that some of these compounds will dissolve in polar solvents like methanol, and others will not dissolve in methanol, but might dissolve in a less polar solvent like methylene chloride or chloroform. If you use just methanol extraction, you will miss antifungal compounds that are soluble in non-polar solvents. Can you tell me if you think any of the known antifungal compounds are soluble in water?

.
Donna Hardy