Hello Sir, Good day, sir may i ask, let say i want to do the hydrogen peroxide assay for fruit juice/tea, to what concentration should i dilute the sample?
I have one doubt , just assume if my biological system doesn't have any free radicals at present , unknowingly i introduce Anti- oxidants into my bio system . What will happen ?
@@gajalakshmi1915 Thanks for watching. When antioxidNts are added in a bio system, they sequester the free radicals or elevate the antioxidant enzymes such as SOD, Catalase, Glutathione. Also, this will prevent or repair the damage caused by free radicals.
Thank you for your video but i have a question. I did anti oxidant activity on Vernonia amygdalina but it showed low inhibition percentage what could be the problem please.
Thank you. I've experienced multiple reasons that can affect the inhibition 1. Conc. of extract solution 2. Type of extract (aq. Methanol, ethyl acetate etc) 3. Time duration for extraction 4. Method of extraction (soxhlet, microwave, maceration) 5. Part of the plant taken (root, stem, leaves peeled unpeeled etc) It is advised that one should perform quantitative analysis by wither HPTLC or any other methods before DPPH.
Hello, Sir! Thanks for your video, I would like to ask what the reason is for having different concentrations of the standard stock solution? Thank you!
Light induces photo-oxidation in some samples & give false results. Let us take an ex. of an apple when cut & kept for few minutes, it undergoes oxidation & turns red. (light catalyzes the oxidation reaction in some samples, not all) Thanks for watching. Hope, I've tried to clear your doubt.
Just to clarify sir, how do we prepare the blank test tube for H2O2? What volume is needed? And why do we still need to add H2O2 if it is the blank, should it not be just phosphate buffer?
Thank for watching Ollie. Blank test tube has H2O2 alone as it has its own absorbance. Whereas; control has both H2O2 and Phosphate buffer. Before putting in the formula for calculations, the value of blank should be subtracted from both control and standard or sample to have more accurate results.
Thanks for watching. OD decreases with increase in the conc of antioxidants. When a solution of DPPH in methanol is prepared, it has intense purple- violet color. Antioxidants donates [H+] to DPPH and the color fades. If conc of antioxidants is more then more DPPH radicals will receive H+ ions and the color will turn more faint.
Thank you for your response. And what about H2O2 Test, OD should decrease also with the Concentration of antioxidants or it will increase? Many thanks for you
Yes, in H2O2 also there is a decrease in OD with increase in conc of Antioxidants. General rule: oxidation increases color intensity. H2O2 is an oxidizing agent.
Yes, you are right. We just need to substract the abs of blank from every abs before putting in formula Blank = H2O2 Control = H2O2 + Phosp buffer Std= H2O2 + Phosp Buffer + BHT or Rutin Sample = H2O2 + Phosp buffer + Plant extract 1. Abs of control = (Actual absorbance - Absorbance of blank) 2. Abs of Std = (Actual absorbance -Absorbance of blank) 3. Abs of Sample = (Actual absorbance -Absorbance of blank) Now, put value of 1, 2 & 3 in the formula of to cal % Scavenging Activity. Many thanks for watching.
DPPH is soluble in methanol. So methanol is the ideal solvent. Water used for buffer solution may have dissolved oxygen that can create Oxygen free radicals which impact the results.
It depends on the type of "percentage" your stock solution has. The datasheet says "% (w/w)" = weight per weight, which means that the solution contains 30 g H2O2 per 100 g of solution. The density of this solution is 1.11 g/ml, so 100 g have a volume of 90.09 ml. So 1 L contains 333 g of H2O2 (30 g / 90.09 ml x 1000 ml) which is a molar concentration of 9.79 mol/L (333 g/L / 34.01 g/mol). To prepare a 40 mM solution take 4.09 ml of your 30% solution and fill to 1 L with water (40 mM x 1000 ml / 9.79 M). Hope, this will help you.
Buffer prevents the change in color of dpph caused due to pH of sample or standard solutions added. Color change will solely be caused due to antioxidants upon using buffer.
This could be due to some reasons like: 1. DPPH used has little or no free radicals left, all are converted to stable products (DPPH could be expired) 2. Incubation period in dark is not maintained 3. Compounds are not properly soluble in the test-tubes 4. Sample solution itself is more colorful.
Thank you Saurabh for watching. Refer following link www.fda.gov/food/laboratory-methods-food/bam-r61-002-m-phosphate-saline-buffer-ph-73-74 For H2O2: volume strength of H2O2=11.2× molarity. Hence, for 1 M H2O2, volume strength is 11.2. For 0.2 M, vol strength will be 2.24.
Good evening sir.. I need your help for estimating antioxidants like ascorbate, peroxidase, ascorbate peroxidase, gluthionine reductase, superoxide dismutase, catalase etc from the leaf extract of chickpea..it's my genuine request sir..I m ready to pay for your tutorial..how can approach u.. please let me know
@@noreenramzan6727 I am not an expert in Nano-science or Pharmaceutics. Plz let me know if you need any assistance in herbal drug technology, Extraction or isolation of phytoconstituents
You may get several recent articles. I have been following this article bmccomplementmedtherapies.biomedcentral.com/articles/10.1186/1472-6882-8-63 Thank you for watching. All d best
Many thanks Sana for watching the video. Absorbance is the output of UV-Visible spectrophotometer. We need to transfer the solution from test tubes to a cuvette (a square tube) used in UV-visible spectrophotometer. The solution absorbs light wavelength around 517 nm and gives the absorbance o screen. Hope, I've cleared ur query.
Tumchya suggestion baddal dhanyavaad. 🙏🏻🙏🏻 Me nakki try karen. Pan mala he topics English madhe jyast soppe vatale. Next time hinglish vaparen. Thank you.
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Thank you so much Sir to explain in a simple and easy way..very helpful
You are most welcome
Thank You Sir for the wonderful explanation. Could you please give reference for DPPH method any article or links?
Sir i got 157 abs for control (blank) solution. Then i got 195 abs for sample. So scavenging percentage was negative answer. Where is the wrong?
Hello Sir, Good day, sir may i ask, let say i want to do the hydrogen peroxide assay for fruit juice/tea, to what concentration should i dilute the sample?
@@NURATIKAHABDULLAH-f9e Hello, usually the dilution should be done until it is colorless. Else, the color will interfere in absorbance readings.
Very good details. Have similar work concerning phytochemical screening, my doctor. Gratful.
Glad! You like it. Thanks for Watching Sir
I have one doubt , just assume if my biological system doesn't have any free radicals at present , unknowingly i introduce Anti- oxidants into my bio system .
What will happen ?
@@gajalakshmi1915 Thanks for watching. When antioxidNts are added in a bio system, they sequester the free radicals or elevate the antioxidant enzymes such as SOD, Catalase, Glutathione. Also, this will prevent or repair the damage caused by free radicals.
@@bhushansong Thanks for your reply sir....
sir plz tell me the concentration for H202 and phosphate buffer also the ph if needed, because i do not get any colour after performing this reaction
Thank you for your video but i have a question.
I did anti oxidant activity on Vernonia amygdalina but it showed low inhibition percentage what could be the problem please.
Thank you. I've experienced multiple reasons that can affect the inhibition
1. Conc. of extract solution
2. Type of extract (aq. Methanol, ethyl acetate etc)
3. Time duration for extraction
4. Method of extraction (soxhlet, microwave, maceration)
5. Part of the plant taken (root, stem, leaves peeled unpeeled etc)
It is advised that one should perform quantitative analysis by wither HPTLC or any other methods before DPPH.
THANK YOU SIR FOR THIS VIDEO, VERY HELPFUL!
Glad it was helpful! Thanks for Watching
Hello, Sir! Thanks for your video, I would like to ask what the reason is for having different concentrations of the standard stock solution? Thank you!
Extremely sorry for very delayed reply. In order to generte a calibration curve and to determine IC50/effective conc we need it
Sir please answer this question....why dark condition is needed for incubation of Dpph-antioxidant mixture?
Light induces photo-oxidation in some samples & give false results.
Let us take an ex. of an apple when cut & kept for few minutes, it undergoes oxidation & turns red. (light catalyzes the oxidation reaction in some samples, not all)
Thanks for watching. Hope, I've tried to clear your doubt.
Due to photo oxidation, absorbance may fluctuate.
sir if possible give the demo of how to calculate IC 50 value & Antioxident activity Graph
Many thanks for sparing your valuable time to watch the video.
Sure, I'll try about IC50 value in some days.
ua-cam.com/video/ROoMjF4GKcc/v-deo.html
Why don't you give links to some journals.. For further reference
Many thanks for your wonderful suggestion. I'll surely implement it.
Dr. Please answer my question...
I want to do a study using DPPH but in my university don't have. So, where I can to puy DPPH?
Thanks for watching.
You can buy online from www.sigmaaldrich.com/catalog/product/aldrich/d9132?lang=en®ion=IN
All the best!!
@@bhushansong thanks for answer my question, but I live in Africa, it is possible to buy from here?
Yes sure.
Thank you sir
Just to clarify sir, how do we prepare the blank test tube for H2O2? What volume is needed? And why do we still need to add H2O2 if it is the blank, should it not be just phosphate buffer?
Thank for watching Ollie.
Blank test tube has H2O2 alone as it has its own absorbance. Whereas; control has both H2O2 and Phosphate buffer.
Before putting in the formula for calculations, the value of blank should be subtracted from both control and standard or sample to have more accurate results.
Thank you. I have a question about the OD should increase or decrease with increasing concentrations of sample?
Thanks for watching. OD decreases with increase in the conc of antioxidants. When a solution of DPPH in methanol is prepared, it has intense purple- violet color. Antioxidants donates [H+] to DPPH and the color fades. If conc of antioxidants is more then more DPPH radicals will receive H+ ions and the color will turn more faint.
Thank you for your response. And what about H2O2 Test, OD should decrease also with the Concentration of antioxidants or it will increase?
Many thanks for you
Yes, in H2O2 also there is a decrease in OD with increase in conc of Antioxidants.
General rule: oxidation increases color intensity. H2O2 is an oxidizing agent.
Thanks indeed.
Thank you Aliyu.
sir may get the reference of this
AsslamoAlikum Sir! Please share link of video where share graph interpretation of DppH.
Thank you Iqra. Thanks for watching. I'll try to make a clip on graph/result interpretation.
Sir you said that blank will be hydrogen peroxide but in point 6 you have mentioned buffer solution as blank
Yes, you are right.
We just need to substract the abs of blank from every abs before putting in formula
Blank = H2O2
Control = H2O2 + Phosp buffer
Std= H2O2 + Phosp Buffer + BHT or Rutin
Sample = H2O2 + Phosp buffer + Plant extract
1. Abs of control = (Actual absorbance - Absorbance of blank)
2. Abs of Std = (Actual absorbance -Absorbance of blank)
3. Abs of Sample = (Actual absorbance -Absorbance of blank)
Now, put value of 1, 2 & 3 in the formula of to cal % Scavenging Activity.
Many thanks for watching.
Sir please can you give us a reference to this H202 scavenging method
Thanks so much sir
Very Informative and Helpful💜
Glad you think so! Thank you
Sir dpph can be done in buffered aqueous solution also?
DPPH is soluble in methanol. So methanol is the ideal solvent. Water used for buffer solution may have dissolved oxygen that can create Oxygen free radicals which impact the results.
Hats off Sir.
Thank you. Glad you liked it.
Sir, how to make 2 millimolar solution of hydrogen peroxide? Please explain..
It depends on the type of "percentage" your stock solution has.
The datasheet says "% (w/w)" = weight per weight, which means that the solution contains 30 g H2O2 per 100 g of solution.
The density of this solution is 1.11 g/ml, so 100 g have a volume of 90.09 ml. So 1 L contains 333 g of H2O2 (30 g / 90.09 ml x 1000 ml) which is a molar concentration of 9.79 mol/L (333 g/L / 34.01 g/mol).
To prepare a 40 mM solution take 4.09 ml of your 30% solution and fill to 1 L with water (40 mM x 1000 ml / 9.79 M).
Hope, this will help you.
Sir can u give d reference paper of hydrogen peroxide assay
Why you use acetate buffer because most of the articles only referred only methanol as a diluent
Buffer prevents the change in color of dpph caused due to pH of sample or standard solutions added. Color change will solely be caused due to antioxidants upon using buffer.
Sir, can we use phosphate buffer instead of methanolic buffer??
Thanks for watching. Not sure about that.
superb explanation
Thank you so much 🙂
Please explain for super oxide dismutase
Thanks for watching. I will surely try.
Instead of decreasing the od values are increasing.... What will be reason?
This could be due to some reasons like:
1. DPPH used has little or no free radicals left, all are converted to stable products (DPPH could be expired)
2. Incubation period in dark is not maintained
3. Compounds are not properly soluble in the test-tubes
4. Sample solution itself is more colorful.
@@bhushansong Thank you for the timely reply... I'm doing hydrogen peroxide assay sir... My values are increasing instead of decreasing...
@@maheswari2008 try this it's perfect
cpha.tu.edu.iq/images/%D8%B9%D9%84%D9%8A_%D8%AD%D8%B3%D9%8A%D9%86/2._ASSAY_OF_HYDROGEN_PEROXIDE__SOLUTION.pdf
Nice Explanation, Sir 👍
Many thanks for watching the video.
Very nice explanation sir please make an video regarding graphpad prism
Thanks for watching. Will definitely work on Graphpad.
hi sir do u have any reference about your h2o2 procedure? badly need it 😭
Thanks for watching. Following is one reference. You may get many on net
www.ncbi.nlm.nih.gov/pmc/articles/PMC4492988/
@@bhushansong thank youu
Very informative and interesting.
Thank you for watching
How to prepare 0.2 m PBS ph 7.4?
How to prepare 2 mM H2O2 solution in 0.2 m PBS?
Thank you Saurabh for watching.
Refer following link
www.fda.gov/food/laboratory-methods-food/bam-r61-002-m-phosphate-saline-buffer-ph-73-74
For H2O2:
volume strength of H2O2=11.2× molarity.
Hence, for 1 M H2O2, volume strength is 11.2.
For 0.2 M, vol strength will be 2.24.
Good evening sir..
I need your help for estimating antioxidants like ascorbate, peroxidase, ascorbate peroxidase, gluthionine reductase, superoxide dismutase, catalase etc from the leaf extract of chickpea..it's my genuine request sir..I m ready to pay for your tutorial..how can approach u.. please let me know
Thanks Vishal for watching the video. You may contact at pimplebhushan@yahoo.co.in
lol pay 2 win do ur own research
sir H2O2 solution used is 30%?
Thanks for watching. The conc. of H2O2 solution used is 2mM (video time 12:38)
Sir could you plz provide the reference.
nice explanation
Thanks for sparing your valuable time to watch the video.
@@bhushansong Sir could u make lecture about catalytic degradation (photo and chemo degradation ) of Carbon dots
@@noreenramzan6727 I am not an expert in Nano-science or Pharmaceutics. Plz let me know if you need any assistance in herbal drug technology, Extraction or isolation of phytoconstituents
@@bhushansong thnks for reply Sir .. actually my project is related to nano particles not herbal drug 😊
thank you so much sir
Most welcome. Thanks for watching
Can you please give the refernce of H2O2?
You may get several recent articles. I have been following this article
bmccomplementmedtherapies.biomedcentral.com/articles/10.1186/1472-6882-8-63
Thank you for watching.
All d best
Nice Explanation please share ppt link
Thanks for watching the video. Will upload soon
Well explained 👍🙏
Thank you so much 🙂.
Thx u sir, for informations
Always welcome, Rodoh. Thanks for watching
Thanks.
You're welcome
Make video on DDT ASSAY and AA assay
I'll try. thanks
10/10
Many thanks for sparing your the to watch the video.
what is meant by absorbance at 517nm
Many thanks Sana for watching the video.
Absorbance is the output of UV-Visible spectrophotometer.
We need to transfer the solution from test tubes to a cuvette (a square tube) used in UV-visible spectrophotometer.
The solution absorbs light wavelength around 517 nm and gives the absorbance o screen.
Hope, I've cleared ur query.
@@bhushansong yes sir thanks alot
Try kra ki Hindi madhe thod explain karayala ....mhnje aamha saglyana smjl
Tumchya suggestion baddal dhanyavaad. 🙏🏻🙏🏻 Me nakki try karen. Pan mala he topics English madhe jyast soppe vatale. Next time hinglish vaparen. Thank you.
@@bhushansong ho sir tumhi khup easy way madhe shivkta but kahi terms smjt nhi English madhe clear....and thank you for response
thank you sir
Thanks
Welcome. Thanks for sparing your time for the video.
Thank you sir
Thanks for watching.