Science Buddies: "Ask an Expert"

hello anybody and everybody.

i am a junior in high school, and i'm doing the science fair. i'd really love to somehow test the theory that adding milk to green tea takes away the healthful benefits. I have found many articles saying that adding milk, which has caseins, to tea, which has catechins, decreases the concentration of catechins, thus taking away its anti-cancer benefits.

So. i basically need a way to test for levels of catechins in a liquid, so i can prove that adding milk decreases their concentrations in tea. i don't know if its at all possible, but it would be incredible if anybody could give me some help!

thank you thank you

vanillabean16 wrote:hello anybody and everybody.

i am a junior in high school, and i'm doing the science fair. i'd really love to somehow test the theory that adding milk to green tea takes away the healthful benefits. I have found many articles saying that adding milk, which has caseins, to tea, which has catechins, decreases the concentration of catechins, thus taking away its anti-cancer benefits.

So. i basically need a way to test for levels of catechins in a liquid, so i can prove that adding milk decreases their concentrations in tea. i don't know if its at all possible, but it would be incredible if anybody could give me some help!

thank you thank you

Have you tried googling for catechin assay or catechin testing? I found tons of papers that listed procedures. Many used an instrument called an HPLC, which I am guessing you don't have access too, but there may be some chemical assays as well.

Catechin levels in green tea are widely measured, so I am sure you will find bleep that will work with what you have.

I have found many articles saying that adding milk, which has caseins, to tea, which has catechins, decreases the concentration of catechins, thus taking away its anti-cancer benefits.

And how did these papers do the testing?

If you want to ask questions about a specific procedure or ask for us to explain the science lingo after you've done some research, feel free to post back

Louise

thanks for writing back!

i looked at a bunch of procedures for catechin testing, and they ALL involved using HPLCs, and you're right, i don't have access to one. However, i may have access to a spectrophotometer. I have seen this option mentioned many places, but i can't find any procedures that actually describe how this can be done.
Do you know of any?

Thank you SO much!!
-Emily.

vanillabean16 wrote:thanks for writing back!

i looked at a bunch of procedures for catechin testing, and they ALL involved using HPLCs, and you're right, i don't have access to one. However, i may have access to a spectrophotometer. I have seen this option mentioned many places, but i can't find any procedures that actually describe how this can be done.
Do you know of any?

Thank you SO much!!
-Emily.

I think it is used with an HPLC. HPLC stands for high pressure (performance?) liquid chromatography, and it is the way you separate out the different components. It is like a tube filled with little beads. Some chemicals stick to the beads and others don't. So, some chemicals go through very quickly (because they don't stick) and others very slowly (because they do stick). So, at each time, you have a different chemical coming out of the tube. You then use a spectrophotometer to tell what the identity of each chemical is.

I will look around a little more, but I was thinking about this last night, and you may be out of luck. HPLC is one of the most widely used modern analytical instruments, and most of the green tea research is pretty recent, so it may be that all the papers use HPLC.


Louise

Louise wrote:

vanillabean16 wrote:thanks for writing back!

i looked at a bunch of procedures for catechin testing, and they ALL involved using HPLCs, and you're right, i don't have access to one. However, i may have access to a spectrophotometer. I have seen this option mentioned many places, but i can't find any procedures that actually describe how this can be done.
Do you know of any?

Thank you SO much!!
-Emily.

I think it is used with an HPLC. HPLC stands for high pressure (performance?) liquid chromatography, and it is the way you separate out the different components. It is like a tube filled with little beads. Some chemicals stick to the beads and others don't. So, some chemicals go through very quickly (because they don't stick) and others very slowly (because they do stick). So, at each time, you have a different chemical coming out of the tube. You then use a spectrophotometer to tell what the identity of each chemical is.

I will look around a little more, but I was thinking about this last night, and you may be out of luck. HPLC is one of the most widely used modern analytical instruments, and most of the green tea research is pretty recent, so it may be that all the papers use HPLC.


Louise

I found a reference that might be useful;
From:http://www.worldnutra.com/Newsletter_Pa ... dants.html

"phenolic compounds such as catechins and the components of Echinacea spp. have been found to have excellent antioxidant properties and are linked to a wide range of health benefits. To determine total polyphenolic levels a spectrophotometric bleep is employed(1). In this assay, samples are extracted with bleep and Folin-Ciocalteu reagent is added. The samples are then placed in a spectrophotometer and the absorbance is compared to a gallic acid reference standard. Results are presented as gallic acid equivalents (GAE)."
[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 will tell you about total polyphenol levels. I have no idea what these chemicals are. I wil look at the original paper at the university tomorrow.

Louise

thanks, that did help. i guess the fact that high performance liquid chromatography is used in conjunction with spectrophotometry accounts for my inability to find any procedures!
i suppose my next step is to find the simplest procedure possible, and to somehow get my hands on those two instruments. Perhaps they could be available for rent commercially, or by local universities?
i wonder why i just HAD to pick the most complicated experiment possible.....

Emily.

i just found this:

http://pubs.acs.org/cgi-bin/abstract.cg ... 0855a.html

entitled: Interactions between Flavonoids and Proteins: effect on the total antioxidant capacity.

it uses a spectrophotometer, and some confusing chemicals.
But this is closer than anything else i have found.
what do you think?

vanillabean16 wrote:thanks, that did help. i guess the fact that high performance liquid chromatography is used in conjunction with spectrophotometry accounts for my inability to find any procedures!
i suppose my next step is to find the simplest procedure possible, and to somehow get my hands on those two instruments. Perhaps they could be available for rent commercially, or by local universities?
i wonder why i just HAD to pick the most complicated experiment possible.....

Emily.

This last article I posted did not use HPLC, but it only measures total polyphenols with a spectrophotometer and two chemicals as indicators and standards.

The HPLC is fairly expensive (I'm guessing $100,000 for a decent system) and is very complicated to use. While the local university probably has many, they probably won't share. A spectrophotometer is also expensive (but much cheaper than an HPLC) and MUCH easier to use (and much harder to screw up if you don't know what you are doing.

These instruments are probably not rentable, and for the HPLC at least, you couldn't operate it even if you had one.

I will try to get the procedure from the Journal of Agricultural Food Chemistry tomorrow. I think that is your best bet... or a new project.

Louise

Louise wrote:

vanillabean16 wrote:thanks, that did help. i guess the fact that high performance liquid chromatography is used in conjunction with spectrophotometry accounts for my inability to find any procedures!
i suppose my next step is to find the simplest procedure possible, and to somehow get my hands on those two instruments. Perhaps they could be available for rent commercially, or by local universities?
i wonder why i just HAD to pick the most complicated experiment possible.....

Emily.

This last article I posted did not use HPLC, but it only measures total polyphenols with a spectrophotometer and two chemicals as indicators and standards.

The HPLC is fairly expensive (I'm guessing $100,000 for a decent system) and is very complicated to use. While the local university probably has many, they probably won't share. A spectrophotometer is also expensive (but much cheaper than an HPLC) and MUCH easier to use (and much harder to screw up if you don't know what you are doing.

These instruments are probably not rentable, and for the HPLC at least, you couldn't operate it even if you had one.

I will try to get the procedure from the Journal of Agricultural Food Chemistry tomorrow. I think that is your best bet... or a new project.

Louise

Looks like "Journal of Agricultural Food Chemistry" likes tea. I posted a citation to this journal (different article) a couple of posts ago. The one you linked to is confusing. Maybe the one I found will be clearer.

would your teacher be able to order chemicals for you?

Louise

i am going to ask her tomorrow if she can order the chemicals, and if the procedure is do-able. Plus, my father is the head of the ER at one of the local hospitals, so he may be able to find connections through the pharmacy. He also thinks that the hospital might have a spectrophotometer. My school has one, but it is from the early 80s, and nobody knows how to work it! But i'm working on trying to find directions through its barely visible serial number

HPLC and Spectography equipment is not bleep you can rent. Long term lease maybe, but not very affordable.

The care of this equipment and the columns isn't bleep you just let anybody do. My wife baby sat this kind of equipment for a pharmacy professor for a couple of years as an undergrad lab assistant.

See if somebody at a local university does similar research and will help you out. If not, then probably need a different project or bleep.

vanillabean16 wrote:i am going to ask her tomorrow if she can order the chemicals, and if the procedure is do-able. Plus, my father is the head of the ER at one of the local hospitals, so he may be able to find connections through the pharmacy. He also thinks that the hospital might have a spectrophotometer. My school has one, but it is from the early 80s, and nobody knows how to work it! But i'm working on trying to find directions through its barely visible serial number

If you can tell me the name and the brand and the model number of the spectrophotometer at school, I might be able to tell you how to use it.

If this is a single wavelength mesurement, then we might be able to construct an instrument with filter glass and a photodiode.

Louise

the spectrophotometer is a Bausch & Lomb Spectronic 20. The only number readable on the back said: CAT NQ. 33-29-95
I typed just its model name and type into google and found it easily. Its pretty old! which means i bet its a sinch to use. that is, if its still working...

vanillabean16 wrote:the spectrophotometer is a Bausch & Lomb Spectronic 20. The only number readable on the back said: CAT NQ. 33-29-95
I typed just its model name and type into google and found it easily. Its pretty old! which means i bet its a sinch to use. that is, if its still working...

Ah, a Spec 20. Those things are pretty durable. This is the "analog" not "digital".

I'm guessing you found a bunch of instructions on the web- they are pretty simple. The first thing to do is see if the lamp works.

You want to turn it on- following the appropriate warmup of 15 minutes, etc. Set the wavelength to 532 nm. Look in the sample comparment. Carefully stick a white piece of paper in the sample cell. You should see a green light. Change to 640 nm, should see red light. If this checks out, you are good to go except for the chemicals, I think. If you see the wrong color light, just write down what color, and we can try to calibrate it.

If no light, then we have a problem... ask if they have an extra tungsten lamp.

Louise

What part of the country do you live in? An organization that I work with has a lab dedicated to high school science fairs, and we have all sorts of things: infared spectrophotomers, UV-VIS spectrophotometers, HPLC, GC-MS, etc. We also have the ability to handle most "nasty" chemicals and materials. Let me know the general area where you live, and I can tell you if using this lab might be a possibility.

phenolic levels a spectrophotometric bleep is employed(1). In this assay, samples are extracted with bleep 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

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.

oh! i just found this. it doesnt require the use of a spectrophotometer, but i assume you need either that or a colorimeter or bleep 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 bleep with vitamin C. But i think i just need flavonoids (?)

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

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 bleep 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 bleep 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

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

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

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

Louise

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.

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?

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.

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.

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

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.

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

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

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