Louise
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Postby Louise » Tue Mar 13, 2007 6:17 am

phenolic levels a spectrophotometric method is employed(1). In this assay, samples are extracted with methanol and Folin-Ciocalteu reagent


This reagent (Folin-Ciocalteu) appears to be a phosphomolybdic-phosphotungstic acid reagent. The good news is the experiment was done in 1965, so it the Spec 20 would probably be a super advanced machine compared to what they used.

You can buy this chemical from sigma aldrich. The product is:
F9252
Sigma-Aldrich Folin & Ciocalteu’s phenol reagent

However it is also used as an assay for total protein, so milk would probably make it "go" too.


[1] Sakakibara, H., Honda, Y., Nakagawa, S., Ashida, H., and Kanazawa, K. "Simultaneous Determination of all Polyphenols in Vegetables, Fruits, and Teas," J. Agric. Food Chem. 51, 572-580 (2003).


This reference seems to be wrong/ does not exist/ etc.

Louise

vanillabean16
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Postby vanillabean16 » Tue Mar 13, 2007 12:48 pm

I will try seeing if the spectrophotometer works tomorrow at school. i know it atleast turns on, because a red light came on when we plugged it in.

oh, and i live in new england.

vanillabean16
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Postby vanillabean16 » Tue Mar 13, 2007 5:21 pm

oh! i just found this. it doesnt require the use of a spectrophotometer, but i assume you need either that or a colorimeter or something of the sort, because it requires read wavelength.
http://72.14.209.104/search?q=cache:xZ4 ... cd=2&gl=us

i dont know if the link will work but it tested: "the phenolic and flavonoid contents and totalantioxidant capacities of cocoa, black tea, green tea, and redwine."
obviously i'm only doing tea, and not phenols. But this is the procedure for flavonoid content determination:

" Sample Preparation. The serving size of each beverage was defined as follows: commercial cocoa powder manufactured using a nonalka-lized process from Ghanaian cacao beans (7.3 g, 2 tablespoons in accordance with the manufacturer’s instructions) was dissolved in 200mL of distilled water (ddH2O) at 100 °C; commercial black tea (2 gbag) and green tea (2 g bag) were each extracted with 200 mL of ddH2Oat 100 °C for 2 min (according to the manufacturer’s instructions);and 140 mL of red wine (Merlot, California) as one serving size (14).The samples then were centrifuged in a Sorvall RC-5B refrigerated superspeed centrifuge (DuPont, Biomedical Products Department,Wilmington, DE) at 12000g using a GSA rotor for 5 min, and theresulting supernatants were used as the final samples. "
" Total Flavonoid Content. The total flavonoid concentration wasmeasured using a colorimetric assay developed by Zhishen et al. (8,9). Briefly, 1 mL of appropriately diluted sample was added to a 10mL volumetric flask containing 4 mL of ddH2O. At time zero, 0.3 mLof 5% NaNO2was added to each volumetric flask; at 5 min, 0.3 mL of10% AlCl3was added; at 6 min, 2 mL of 1 M NaOH was added. Eachreaction flask was then immediately diluted with 2.4 mL of ddH2Oand mixed. Absorbances of the mixtures upon the development of pinkcolor were determined at 510 nm relative to a prepared blank. The total flavonoid contents of the samples are expressed in milligrams per serving of epicatechin equivalents (ECE). All samples were prepared in five replications. "

it also measures antioxidant capacity with a procedure that is very complicated and involves the ABTS assay and something with vitamin C. But i think i just need flavonoids (?)

this just seems so much more simple. I dont know... second opinion please?

Louise
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Postby Louise » Tue Mar 13, 2007 5:38 pm

vanillabean16 wrote:oh! i just found this. it doesnt require the use of a spectrophotometer, but i assume you need either that or a colorimeter or something of the sort, because it requires read wavelength.
http://72.14.209.104/search?q=cache:xZ4 ... cd=2&gl=us

i dont know if the link will work but it tested: "the phenolic and flavonoid contents and totalantioxidant capacities of cocoa, black tea, green tea, and redwine."
obviously i'm only doing tea, and not phenols. But this is the procedure for flavonoid content determination:

" Sample Preparation. The serving size of each beverage was defined as follows: commercial cocoa powder manufactured using a nonalka-lized process from Ghanaian cacao beans (7.3 g, 2 tablespoons in accordance with the manufacturer’s instructions) was dissolved in 200mL of distilled water (ddH2O) at 100 °C; commercial black tea (2 gbag) and green tea (2 g bag) were each extracted with 200 mL of ddH2Oat 100 °C for 2 min (according to the manufacturer’s instructions);and 140 mL of red wine (Merlot, California) as one serving size (14).The samples then were centrifuged in a Sorvall RC-5B refrigerated superspeed centrifuge (DuPont, Biomedical Products Department,Wilmington, DE) at 12000g using a GSA rotor for 5 min, and theresulting supernatants were used as the final samples. "
" Total Flavonoid Content. The total flavonoid concentration wasmeasured using a colorimetric assay developed by Zhishen et al. (8,9). Briefly, 1 mL of appropriately diluted sample was added to a 10mL volumetric flask containing 4 mL of ddH2O. At time zero, 0.3 mLof 5% NaNO2was added to each volumetric flask; at 5 min, 0.3 mL of10% AlCl3was added; at 6 min, 2 mL of 1 M NaOH was added. Eachreaction flask was then immediately diluted with 2.4 mL of ddH2Oand mixed. Absorbances of the mixtures upon the development of pinkcolor were determined at 510 nm relative to a prepared blank. The total flavonoid contents of the samples are expressed in milligrams per serving of epicatechin equivalents (ECE). All samples were prepared in five replications. "

it also measures antioxidant capacity with a procedure that is very complicated and involves the ABTS assay and something with vitamin C. But i think i just need flavonoids (?)

this just seems so much more simple. I dont know... second opinion please?



I think this is fine. Do you have access to a centrifuge- that woulld be really useful. I am worried that the milk proteins will scatter light and ruin your reading. You could use the spectrophotometer for that, or, I suppose you might be able to do it by eye.

Look up references 8,9 and see if they have more detail. They may also list hazards and sources for the chemicals.

Good job finding this!

Louise

Craig_Bridge
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Postby Craig_Bridge » Tue Mar 13, 2007 5:40 pm

510 nm is in the infrared spectrum so you are going to need something like a spectrograph to measure it. Check out the Spec 20 using Louise's method.
-Craig

Louise
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Postby Louise » Tue Mar 13, 2007 5:41 pm

Craig_Bridge wrote:510 nm is in the infrared spectrum so you are going to need something like a spectrograph to measure it. Check out the Spec 20 using Louise's method.


510 nm is green-blue light. Maybe you are thinking microns and not nm?

Louise

vanillabean16
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Postby vanillabean16 » Tue Mar 13, 2007 5:53 pm

i do have access to a centrifuge. i'm sure they have some at my dad's hospital!
maybe i could dilute the milk... i hope it doesnt really skew the measurements... or explosively react with any of the chemicals. that would be my luck :)
i'll see what my chem teacher has to say.

vanillabean16
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Postby vanillabean16 » Tue Mar 13, 2007 6:00 pm

but you know, i just thought... they did the same process with red wine, and it didn't skew the results... so maybe it will work?

Louise
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Postby Louise » Tue Mar 13, 2007 6:03 pm

vanillabean16 wrote:i do have access to a centrifuge. i'm sure they have some at my dad's hospital!
maybe i could dilute the milk... i hope it doesnt really skew the measurements... or explosively react with any of the chemicals. that would be my luck :)
i'll see what my chem teacher has to say.



I forgot about the hospital... that is great.

So, do the procedure like this:
1) make the tea
2) add milk
3) centrifuge (all the protein and tea bits and anything else will turn in to a solid ball on the bottom). Test the liquid. If the flavanoids have reacted with the casein, then they will be stuck in the ball of milk protein.
4) do the colormetric test. The solutions will be pink-ish, they will absorb the blue green light at 510 nm.

If you cannot remove the milk protein, it isn't dangerous. You cannot see through milk, right? Well, neither can a spectrophotometer. So milky tea is hard to measure. It is the protein that causes this problem. Centrifuging will remove this.

tdaly
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Postby tdaly » Tue Mar 13, 2007 11:11 pm

vanillabean16,

The facility I work with is on the west coast, so it won't be of much help for you. :(

Good luck working with the Spec20 and centrifuge; your project sounds really interesting.
All the best,
Terik

Louise
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Postby Louise » Wed Mar 14, 2007 7:26 am

vanillabean16 wrote:but you know, i just thought... they did the same process with red wine, and it didn't skew the results... so maybe it will work?


Red wine is clear. It is a dark color, but it is transparent. Milk isn't. It is cloudy and opaque. It might be that you could get it to work, the chances are much better if you can centrifuge it.


Louise

vanillabean16
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Postby vanillabean16 » Wed Mar 14, 2007 2:48 pm

ok that procedure sounds good. i was thinking that i would centrifuge JUST the tea first, so i can compare the concentration of flavonoids before adding milk to after adding milk.
i didn't get a chance to test the spectrophotometer because my teacher was super busy today. but i definitely will tomorrow.
also, are the chemicals mentioned in the procedure relatively easy to get? that is, compared to the other really confusing experiment i was going to do... just because my spring vacation starts after tomorrow, and so i won't be seeing my teacher for a couple of weeks, so i don't know if she can order them for me. and i would like to do the experiment over break.
just a thought.
also... just to clarify.. do i really need a spectrophotometer? or can i use a colorimeter?
as usual, thank you so much. :)

Louise
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Postby Louise » Wed Mar 14, 2007 3:31 pm

vanillabean16 wrote:ok that procedure sounds good. i was thinking that i would centrifuge JUST the tea first, so i can compare the concentration of flavonoids before adding milk to after adding milk.
i didn't get a chance to test the spectrophotometer because my teacher was super busy today. but i definitely will tomorrow.
also, are the chemicals mentioned in the procedure relatively easy to get? that is, compared to the other really confusing experiment i was going to do... just because my spring vacation starts after tomorrow, and so i won't be seeing my teacher for a couple of weeks, so i don't know if she can order them for me. and i would like to do the experiment over break.
just a thought.
also... just to clarify.. do i really need a spectrophotometer? or can i use a colorimeter?
as usual, thank you so much. :)



You want to centrifuge all samples, with and without milk. You want to make your solution (tea, tea with milk, milk, water... (the last two might be good controls)). Then centrifuge, then do the chemical part.

You cannot test the tea, then use it for the milk experiment, because you will destroy part of the sample when you add the chemicals. Best to do several trials of plain tea, tea with milk, etc. First make sure you can do the experiment. Then, once you know how to do it, you can do several trials.

You will probably have to have your teacher order the chemicals. He or she may already have them. All look common though.

Do you have access to a colorimeter? A colorimeter is basically a simpler version of a spectrometer. The spec 20 is pretty close to a colorimeter. Some colorimeters only test certain colors, so you would need to make sure it works for 510 nm.

Louise

vanillabean16
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Postby vanillabean16 » Wed Mar 14, 2007 4:32 pm

ok. i understand what i'm supposed to do.
i do have access to a colorimeter, i believe... im not sure if it reads 510 nm. Just incase, if the spec 20 does work, i'm gonna bring it home with me over break so i can use either that or a colorimeter.
and i will ask my teacher about the chemicals.. hopefully she will have some of them..
also, do the concentrations of the chemicals matter? i'm trying to find the reference where it describes the original colorimetric assay.... which may have it that information...
and i will have to get the samples centrifuged at the hospital, so there will be some time between when they are spun and when i get to test them. will that make a significant difference?

thanks :)

Louise
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Postby Louise » Wed Mar 14, 2007 5:41 pm

vanillabean16 wrote:ok. i understand what i'm supposed to do.
i do have access to a colorimeter, i believe... im not sure if it reads 510 nm. Just incase, if the spec 20 does work, i'm gonna bring it home with me over break so i can use either that or a colorimeter.
and i will ask my teacher about the chemicals.. hopefully she will have some of them..
also, do the concentrations of the chemicals matter? i'm trying to find the reference where it describes the original colorimetric assay.... which may have it that information...
and i will have to get the samples centrifuged at the hospital, so there will be some time between when they are spun and when i get to test them. will that make a significant difference?

thanks :)


I don't the the wait after centrifuging will matter beause you haven't added any chemicals yet.

You should try to use the concentrations and volumes the same as the paper. If you cannot find the papers, let me know and I will try to find them. If your dad works at a teaching hospital, he may be able to access the papers on line through the med school library.

I will be out of town until Sunday, so I may not answer your questions for a few days. I don't know how my email access will be.

Your teacher probably has the NaOH. Maybe the NaNO2. I think AlCl3 may be the problem... as a solution it should be fine, but as a solid it is problematic. Please don't do anything with it (alcl3) until I can look up more safety information. In some forms it reacts explosively with water. You should definitely pull the other papers and see which form they used. If you can, talk to your teacher about it. If he or she has it, he or she probably knows how to work with it, and can give you safety information.

Louise


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