vanillabean16
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Postby vanillabean16 » Mon Mar 19, 2007 7:59 am

Here's the reference:

Zhishen, J.; Mengcheng, T.; Jianming, W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 1999, 64, 555-559.

thanks!

Louise
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Postby Louise » Mon Mar 19, 2007 8:29 am

vanillabean16 wrote:Here's the reference:

Zhishen, J.; Mengcheng, T.; Jianming, W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 1999, 64, 555-559.

thanks!


I don't think it matters. I've found looked at 4 more papers (in addition to the two you have) and half use the 0.3 mL amount, and half use 3 mL amount. I don't really understand the chemistry of this reaction yet, and nothing I am reading helps, but I guess the amount of this chemical doesn't matter. I would use the value in the tea, cocoa, wine paper, since it is the closest to what you want to do.

Louise

vanillabean16
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Postby vanillabean16 » Mon Mar 19, 2007 9:01 am

ok. that sound reasonable.
thank you so much!

vanillabean16
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Postby vanillabean16 » Thu Mar 22, 2007 6:14 pm

well i'm doing the experiment in about a week. my biggest issue has been how many samples to do, and of what. This is my preliminary plan, with questions:
1. 3 types of teas, each brewed in 200mL distilled water, to test for the original levels of flavonoids before adding milk. Im thinking 4 samples of each type of tea, each from a different brew. Or 3 samples, or 5 samples?
2. each type of tea with each type of added milk (3 types; whole, skim and 2%) again, probably 4 samples of each. (I'm assuming that i can't take these tea samples from the previously brewed teas; because with an extracted sample (which is 1mL from each) it would be adding milk to 199 mL instead of 200.. not sure if it will really matter? it would certainly save on time..)
3. i was originally planning on testing the milk, but then i realized that we can't test it, because, silly me, milk doesn't have flavonoids in it! and i'm not doing a separate casein-test. So i'm just assuming they're in there.. maybe i'll correlate it to the amount of protein on the labeled package or something.

so total, if each type of tea is individually tested 4 times, and each type of tea is tested with each type of milk 4 times, then thats about 60 samples, 48 of them just testing flavonoid content with added milk. Sufficient?

thanks :)

Louise
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Postby Louise » Thu Mar 22, 2007 7:33 pm

vanillabean16 wrote:well i'm doing the experiment in about a week. my biggest issue has been how many samples to do, and of what. This is my preliminary plan, with questions:
1. 3 types of teas, each brewed in 200mL distilled water, to test for the original levels of flavonoids before adding milk. Im thinking 4 samples of each type of tea, each from a different brew. Or 3 samples, or 5 samples?
2. each type of tea with each type of added milk (3 types; whole, skim and 2%) again, probably 4 samples of each. (I'm assuming that i can't take these tea samples from the previously brewed teas; because with an extracted sample (which is 1mL from each) it would be adding milk to 199 mL instead of 200.. not sure if it will really matter? it would certainly save on time..)
3. i was originally planning on testing the milk, but then i realized that we can't test it, because, silly me, milk doesn't have flavonoids in it! and i'm not doing a separate casein-test. So i'm just assuming they're in there.. maybe i'll correlate it to the amount of protein on the labeled package or something.

so total, if each type of tea is individually tested 4 times, and each type of tea is tested with each type of milk 4 times, then thats about 60 samples, 48 of them just testing flavonoid content with added milk. Sufficient?

thanks :)


I'd test milk anyway, just to make sure you get what you expect.

I'd actually ditch the three types of tea, and do more samples of each type. I have _no_ idea how much variation you are going to get, and having 12 data points to average vs. 4 data points can make a significant difference! For milk, I'm not sure I'd do three types. Maybe whole and skim, and leave out the two percent. Again, I'd do more trials rather than more variables. If your teacher is requiring a certain number of IVs then obviously you have to do that. But, it is better to have a lot of high quality data for a few variables than poor quality data on a lot of variables.

(I'm assuming that i can't take these tea samples from the previously brewed teas; because with an extracted sample (which is 1mL from each) it would be adding milk to 199 mL instead of 200.. not sure if it will really matter?


It does matter- so testing fresh samples is best. It may be that the effect is so large that the 1 mL difference wouldn't be super important, but if your flavanoid change is really small, then this difference could make your experiment show the incorrect result.

Here is another thought. You could brew tea in 200 mL. Then divide it in to 2x 100 mL. Test one with and one without milk. Or even divide in to 3 or 4. There is no reason not to do this, as long as you can divide consistently and you always add your milk at the same time (or temperature) of tea.

Louise

vanillabean16
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Postby vanillabean16 » Fri Mar 23, 2007 2:56 pm

im not sure how i would test the milk though.. because the chemicals are for flavonoid testing..and if the milk did for some strange reason turn pink as indicated that would bring about a whole nother area to factor into the results. so what would be the point? again, i'm just trying to concentrate my samples to just tea, and tea with milk.
and i could divide the tea into 100mL samples, and then just add 1 tablespoon of milk to that 100mL instead of 2 tablespoons to 200mL?

then i also was thinking about this: "Absorbances of the mixtures upon the development of pink color were determined at 510 nm relative to a prepared blank." Would the blank just be a plain tea sample, without milk? since i am comparing with milk to without milk? Or should i just use water to be the blank and the zero? since distilled water won't have flavonoids....

Louise
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Postby Louise » Fri Mar 23, 2007 4:02 pm

vanillabean16 wrote:im not sure how i would test the milk though.. because the chemicals are for flavonoid testing..and if the milk did for some strange reason turn pink as indicated that would bring about a whole nother area to factor into the results. so what would be the point.


Because if milk turns the solution pink then you aren't measuring flavanoids. This is a very important control. This test is key to tell if you experiment has any meaning.

again, i'm just trying to concentrate my samples to just tea, and tea with milk.
and i could divide the tea into 100mL samples, and then just add 1 tablespoon of milk to that 100mL instead of 2 tablespoons to 200mL?

Yes.
then i also was thinking about this: "Absorbances of the mixtures upon the development of pink color were determined at 510 nm relative to a prepared blank."

The blank should be all the chemicals but what you are testing.
Louise

vanillabean16
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Postby vanillabean16 » Sun Mar 25, 2007 10:00 am

ok, here is what i plan to be my final procedure.

the tea will be prepared according to instructions, 200mL ddH at 100`C for each 2g tea bag. I decided that splitting them into 100mLs may cause the results to be a little skewed seeing as a lot of the samples would come from the same exact brew of tea. the samples will be centrifuged, and 1mL samples will be taken from the supernatants.
I figured out that the AlCl3 issue i brought up earlier (3mL versus .3mL) was a typo on the original document's part.. it was supposed to add up to 10mL but with 3mL of AlCl3 it added up to be more like 12mL.

The zero will be just ddH, and the blank will be ddH with all the chemicals added.

As far as samples go; I'll prepare 12 samples of each plain tea (hoping to test 10 of them) and a little less of each plain milk (since i should be able to tell pretty quickly whether or not the solution turns pink!).

I'm going to prepare 15 samples of each type of tea with each type of milk (green tea, black tea, skim and whole) in the hopes of doing about 12.. assuming i'll probably mess a couple of them up or run out of time.

Total, about 84 prepared tea samples, although not all of them will actually be tested. I really hope this won't take hours and hours! its worrying me! because i only have day to do it that my teacher is available before break ends.

please tell me how this looks.. and thank you so so so much for helping me get to this point; i honestly could not have done it without your advice!
:)thanks

Louise
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Postby Louise » Sun Mar 25, 2007 10:50 am

vanillabean16 wrote:ok, here is what i plan to be my final procedure.

the tea will be prepared according to instructions, 200mL ddH at 100`C for each 2g tea bag. I decided that splitting them into 100mLs may cause the results to be a little skewed seeing as a lot of the samples would come from the same exact brew of tea. the samples will be centrifuged, and 1mL samples will be taken from the supernatants.
I figured out that the AlCl3 issue i brought up earlier (3mL versus .3mL) was a typo on the original document's part.. it was supposed to add up to 10mL but with 3mL of AlCl3 it added up to be more like 12mL.

The zero will be just ddH, and the blank will be ddH with all the chemicals added.

As far as samples go; I'll prepare 12 samples of each plain tea (hoping to test 10 of them) and a little less of each plain milk (since i should be able to tell pretty quickly whether or not the solution turns pink!).

I'm going to prepare 15 samples of each type of tea with each type of milk (green tea, black tea, skim and whole) in the hopes of doing about 12.. assuming i'll probably mess a couple of them up or run out of time.

Total, about 84 prepared tea samples, although not all of them will actually be tested. I really hope this won't take hours and hours! its worrying me! because i only have day to do it that my teacher is available before break ends.

please tell me how this looks.. and thank you so so so much for helping me get to this point; i honestly could not have done it without your advice!
:)thanks


I think this is fine. I agree you don't have to test a lot of milk samples, just a few to get an idea if milk gives a reading in the spectrometer.

I think that you can get all this done, but don't rush... you are more likely to make mistakes if you rush through this. It probably will be take a while. I wouldn't do all the samples at once, in case you run out of time. So, I would do maybe:

Half of the controls and half of the experimental groups.

Then, if you still have time, do the other half. This way, if you don't have time to complete everything, you can still have a full data set.

The originial procedure had pretty specific timing requirements, so again, doing all at once will be difficult.

Louise

vanillabean16
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Postby vanillabean16 » Sun Mar 25, 2007 2:20 pm

i will most likely split the samples into 2 parts. thats a good idea.
i also just found this:
http://209.85.165.104/search?q=cache:w0 ... cd=5&gl=us

an experiment in which the concentration of catechins in black tea with and without milk were determined with an HPLC. These samples were centrifuged for 20 mins instead of 5 mins. I'm wondering if maybe longer would be better, since this experiment uses milk, whereas my procedure does not?

Louise
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Postby Louise » Sun Mar 25, 2007 2:55 pm

vanillabean16 wrote:i will most likely split the samples into 2 parts. thats a good idea.
i also just found this:
http://209.85.165.104/search?q=cache:w0 ... cd=5&gl=us

an experiment in which the concentration of catechins in black tea with and without milk were determined with an HPLC. These samples were centrifuged for 20 mins instead of 5 mins. I'm wondering if maybe longer would be better, since this experiment uses milk, whereas my procedure does not?


I think the first procedure was at 20000g, this one is at 13000g. This is how "fast" the centrifuge spins. Longer cannot hurt, but I don't know how many samples you can run at one time, so you may have to do shorter than 20 min just to get them all spun.


Louise


Louise

vanillabean16
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Postby vanillabean16 » Fri Mar 30, 2007 7:34 am

hello again!
i was wondering if anybody could explain to me or suggest a website or something that could help me understand what exactly is going on in my experiment when the NaOH, NaNO2, and AlCl3 are added to the tea and tea with milk to form a pink color.
I've heard that AlCl3 can turn pink when exposed to other metal ions (like Na?) to form color, i've heard that NaNO2 is used to manufacture dyes and colored things.. there are so many reactions that could be taking place between these chemicals and the stuff in the tea that i just don't know which one is happening!
and ofcourse the introduction to the procedure i'm following and its major reference doesn't help me out at all. i'd just like to know exactly what i'm doing...
thanks!

Louise
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Joined: Mon Jan 16, 2006 2:17 pm

Postby Louise » Fri Mar 30, 2007 8:06 am

vanillabean16 wrote:hello again!
i was wondering if anybody could explain to me or suggest a website or something that could help me understand what exactly is going on in my experiment when the NaOH, NaNO2, and AlCl3 are added to the tea and tea with milk to form a pink color.
I've heard that AlCl3 can turn pink when exposed to other metal ions (like Na?) to form color, i've heard that NaNO2 is used to manufacture dyes and colored things.. there are so many reactions that could be taking place between these chemicals and the stuff in the tea that i just don't know which one is happening!
and ofcourse the introduction to the procedure i'm following and its major reference doesn't help me out at all. i'd just like to know exactly what i'm doing...
thanks!


I'm not exactly sure what the chemistry is either... I read a bunch of the papers and didn't find anything useful. I'll look around some more.

Louise

vanillabean16
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Postby vanillabean16 » Fri Apr 06, 2007 2:48 pm

and here comes yet ANOTHER dilemma! what a surprise :)
well everything was going quite smoothly. we mixed the solutions, and even tried one of the samples to see if it actually turned pink, which it did. And then.............. the spectrophotometer DIED. Literally minutes before we were going to take the first reading.
We had let it warm up for about 2 hours.. we had it on while we were making the solutions.. i don't know if thats why it wasn't working, because when we first turned it on it was working just fine! but after 2 hrs it didn't produce those green and red lights when a white piece of paper was put inside when it was set at 532 and 640 nm. When we put the blank into it, it didn't read 100% transmittance like it had before, it read like 5, and the needle wouldn't move above about 10 no matter what knobs we turned.
Now that i have EVERYTHING ready to go, and if the spec 20 doesn't recover overnight, is there a qualititative way that i can measure this? Technically i could line all the samples up next to one another and say hmm, this one looks more pink than this other one.. which i may have to do, because i really don't want to just throw all of my work out the window.
This is very discouraging.

Louise
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Joined: Mon Jan 16, 2006 2:17 pm

Postby Louise » Fri Apr 06, 2007 5:56 pm

vanillabean16 wrote:and here comes yet ANOTHER dilemma! what a surprise :)
well everything was going quite smoothly. we mixed the solutions, and even tried one of the samples to see if it actually turned pink, which it did. And then.............. the spectrophotometer DIED. Literally minutes before we were going to take the first reading.
We had let it warm up for about 2 hours.. we had it on while we were making the solutions.. i don't know if thats why it wasn't working, because when we first turned it on it was working just fine! but after 2 hrs it didn't produce those green and red lights when a white piece of paper was put inside when it was set at 532 and 640 nm. When we put the blank into it, it didn't read 100% transmittance like it had before, it read like 5, and the needle wouldn't move above about 10 no matter what knobs we turned.
Now that i have EVERYTHING ready to go, and if the spec 20 doesn't recover overnight, is there a qualititative way that i can measure this? Technically i could line all the samples up next to one another and say hmm, this one looks more pink than this other one.. which i may have to do, because i really don't want to just throw all of my work out the window.
This is very discouraging.


The lamp is probably burned out if there is no red and green and the transmittance is low. Can you turn the wavelength to 0? This should produce white light (if you can do this, do not let the white light go to the detector. Keep the paper in, change the wavelength to 530 or something, and then take the paper out. If there is no light at 0, it is dead for sure. Also, this will be very bright, so be careful looking at it. If there is light, but it is very dim, then the lamp is dying... You can try to open the slits all the way open.


You can rank the samples if that is all you can do. Here are some other ideas:

1) see if they have one at the hospital you could use. It might be called a "UV-VIS" and common brands are Cary, Varian, and Shimadzu.

2) Try to fix the spec 20 with your teacher. See if there is a replacement lamp. The lamp is a fancy light bulb, so you cannot just run out and buy them. There might be one lying around. I couldn't find instructions. But, I can tell you this-
Wear gloves and goggles.
Turn off the system. Unplug it. Wait 10 minutes before starting. Make sure the old lamp is cold before you take it out.
Don't touch anything with your bare hands. Wear gloves. Oil from your hands can cause the lamp to explode when you turn the system on.
Wipe off the new lamp with a chimwipe or optical paper to remove any finger prints before you put it in. Be careful with the lamp. The inside is under vacuum, if you break it, it implodes and glass goes every where.
Close the system. Then turn everything back on. Don't turn the system on with it open, because if something goes wrong (lamp exploding) you want that contained inside the case.

If you don't have the replacement lamp, you can still open it up and see if any connections are loose. You can take out the lamp and put it back in. Follow the safety precaustions above.

3) You had mentioned a colorimeter... will that not work?

4) If the lamp is dead, and you have a very small very bright flashlight, you can try to put it in. You don't want to take the lamp out, because when you turn the power on it wil be dangerous not to have a part there, but try to fit the flashlight in. I have no idea how much space there is inside. Follow all the safety instructions from part 2. (Gloves, goggles, no power)

Here is a diagram:
Image

You will want to point the flashlight toward the slits. Turn it on, and check for light at 530, 640. If you have light, and it is the right color, try your blank. Obviously, the lamp knobs will do nothing with this setup, so you should turn them to the lowest setting.


Louise


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