Science method
Natural Product Isolation - Science method
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Questions related to Natural Product Isolation
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Suggestion for oligomers with mass differences m/z 82 daltons. Thank you
i have a dry plant materials leaves, stem and root? if possible tell me to send the plant materials and we will published the paper together. the paper interested with inducing alkaloid production in vinca by some endophytic bacteria and abiotic elicitors
This component is found in Fatsia leaves but I have found no article mentioning the ration or the structure.
Does anyone know anything about this component? The solvents and the color or it's ratio in the plant leaves?
Hello,
I'm designing a generic HILIC gradient to separate a complex mixture containing an unknown metabolite of interest for structure elucidation. The properties of the metabolite are unknown beyond its molecular weight and when it elutes on a C18 reverse-phase column (it is a polar molecule). I'm having difficulty finding online what a good buffer and pH is for a generic HILIC gradient, does anyone have any suggestions? I'll be using ACN and H2O as my solvents. My column is a Poroshell 120.
It is relatively easy to isolate nuclei from leaf tissue. However, when I turn to study the nuclei from the bark, I found it is very hard to obtain intact nuclei from this hard tissue. Is there any solution?
Does anyone here knows whether the temperature of soxhlet extraction can be performed below the boiling point of solvent (in my case is water). I am currently working on the optimization of tannin using soxhlet extraction. If the temperature range use is below than water boiling point, is the concept acceptable? because as far as i know, soxhlet extraction only runs using solvent boiling point.
Other than sephadex LH 20 and Carbon.
Most of the flavonoids that show dual inhibition display good activity against PTP1B as compare to α-glucosidase, mostly prenylated flavoinds showing this behaviour. is related to the insulin sensitivity or absorption of glucose, or it's just an active site binding behaviour.
I was having the issue that a series of compounds degradate by the mild acidity of chloroform when we perform NMR experiments. I think that these compounds could be terpenoids (dollabellane type). And I wonder if other kind of molecules could degradate or are unestable in chloroform?
In general, plants host endophytes (bacteria and fungi) depending on phenological conditions.
Stinging nettle (Urtica dioica) has been widely recognized to be associated with endophytes and especially fungal species.
I am wondering if there are nettle leaves without any endophytic colonization?
I am working on phyto-chemical analysis of Cassia angustifolia extracts. GC-MS conditions were used from published literature. I got GC-MS results in the form of graph and a list of 900 compounds. None of the compounds mentioned in the list can be found in literature. Even the most common compounds like kaempferol etc are not present in my extract. What can be the reason for this?
For a purification of large amount of sample in column chromatography takes long time, so I need some other methods
We have been trying to boost the L- theanine content for a while and we cant get past 10% purity.We have been using cation exchange columns , is there any other methods to do the same.
What are the ways to isolate/extract retinol in its pure form from a mixture of retinol, polysorbate 20, BHT(butyl hydroxy toluene), and BHA(butylated hydroxyanisole)?
Retinol 50C(BASF product) is made up of :
- Retinol (49.5%)
- Polysorbate 20 (48%)
- BHT (3.5%)
- BHA (1%)
I've tried just about every search engine available, however, I am still having trouble finding the relevant information. Your help/guidance would be greatly appreciated!
Hi. I am making a research work in a course on my master degree in biotechnology (not a thesis). The assay is about finding new plants with antimicrobial activity. Plants without any kind of investigation in this area.
I prepared extracts of different plants (10 grams dry plant to 200 mL of solvent) in 99,5% ethanol and I just tested them with the Bauer method (disks).
I now have ethanol extracts of 7 different plants wich already proved to be efficient in some ways against Gram + and Gram - bacteria. But I need to quantify my phenolics.
I never used Folin-C. reagent, neither anything like it so my experience is really zero on this. I read some articles and even the SIngleton et al. paper about this and I can't seem to figure it out how to apply it to my study.
I can't change my solvent due to time and working limitations on the lab, so is it possible to make the FC quantification test for the TPC with ethanol as a solvent?
Can someone please tell me the steps I need, considering I have the FC reagent avaiable and gallic acid as a standard (and I don't know how to use it).
Thank you so much
There are many research articles exploring alpha glucosidase, salivary and pancreatic alpha amylase inhibition as a therapeutic target for reducing postprandial hyperglycemia (PPHG) in type 2 diabetes. Out of these three, which one is more important/superior and why?
Natural indigo dye produced from different sources contains many impurities in it.What is the best method to purify it ?
My thesis's subject is determination of flavonoids; catechin and quercetin from sea buckthorn leaves with HPLC-DAD. I have extracted the leaves with ethyl acetate 5x12,5ml, and I have now extract about 40ml. I think next step before determination with HPLC is acid hydrolysis, because flavonoids have glucosides and I gues those should dorp out of measurement.
But I have a problem, how should I do the acid hydrolysis? Should I use 4M HCl or weaker, about 2-6M, and should I use reflux 2h at temperature 40C with stirring?
For now I have tried to do this acid hydrolysis like this: 9,5ml ethyl acetate extract + 0,5ml 4M HCl and heated for 5min in waterbath 75°C. The end concentration of sample is 0,6M (I mean extract + HCl). I have found this advice from here page on 47-48
But this doesn't work so well. Am I doing something wrong? I haven't found any good or spefisic advice. So would you guys help me?
I use ethylacetate:hexane solvent system, and is giving me tailing separations. My aim is to isolate flavonoids from the n-butanol fraction. Regards
I am trying to isolate the constituents (polar and non polar organic compounds) from all parts including the edible part of dried herbal plant? Which is the best solvent to use? Or we have to extract polar and non polar organic compounds separately.
Thank you very much for your time and assistance.
I isolated a compound from a plant, sent it to NMR and got some results. Based on its NMR database, I just can guess this compound is a disaccharide and it has a double bond C=C. But I have no idea what compound it is because it has only 12 carbon. Can anybody give me suggestions?
I linked amine functional to Polar compound that contains acid group. I need to purify the compound but it is not soluble in organic solvents and I tried TLC (10,20, 50% Ethylaceate/hexane, 5% methanol/DCM, and chloroform/methanol. but I am not able to get clear spot and while running NMR and Mass so many impurities. Can anyone suggest solvent system for polar compounds?
Thanks
There are so many techniques are available for the extraction of natural product (Phytochemicals) like
1. Classical solvent extraction procedures
2. Ultrasound-assisted extraction
3. Microwave-assisted extraction
4. Extraction with ionic liquids
5. Accelerated (pressurized) solvent extraction
6. Supercritical fluid extraction
7. Extraction on solid phases
8. Distillation methods
All these techniques have their own disadvantages. The design of green and sustainable extraction methods of natural products is currently a hot topic in the multidisciplinary field of applied chemistry, biology and technology. What are the main techniques for green extraction of natural product. This goal can be achieved through optimal consumption of raw materials, improvement and optimization of existing processes, solvents and energy. the green extract obtained in such a way to have the lowest possible impact on the environment. Can anyone suggest best method for the green extraction through easily available and economic equipment?
Is fractionation by simply shaking with solvents of increasing polarity successively sufficient or is it needed to always have 2 immiscible solvents?
am working on determination of total phenolics and flavonoids
I am searching for a useful alternative for NIST/EPA/NIH Mass Spectral Library.
Regards,
I found an isolation protocol for shikimic acid from anise. A abduct from ethanolic extract dried under vacuum is redissolved on distilled water and then formaldehyde is added to the previously obtained aqueous solution. A precipitate is formed and discarded. What is the utility of formaldehyde on this step of the process?
Thank you very much,
Gustavo
Can anyone tell me how to search AIST database with proton/carbon NMR chemical shifts? Before I tried, but in the long run failed to get the suggested chemical structure. Your cooperation would highly be acknowledged.
Can we use Soxhlet for extracting the secondary metabolites from the Actinobacteria fermented broth? Soxhlet is generally being used for dry/solid materials. If we adopt this to actinobacteria does it work?
or the regular solvent extraction is sufficient? Recommend me some rapid and efficient method to maximize the yield of total metabolites.
I am embarking on an exploratory research project on secondary metabolites of marine organisms particularly on sea stars and brittle stars. What natural products should I consider first? Im looking for a list of natural products and various methods I can use to identify and quantify them. I am planning to do zoochemical analyses, in vitro antioxidant activity, and cytotoxicity.
My 80% MeOH aq extraction of a plant leaves followed by vacuum evaporation and N2 purge resulted thick wax. As most literature express gallic acid equiv (GAE) of total phenolic content (TPC) based on its dry matter, I wonder if there is alternative to express TPC not in terms of dry matter.
i want to quantify saponin in Aloe vera genotypes using HPLC. Which steroidal saponin to be used as marker compound?? please suggest.
10 g of the powder was put into a conical flask and then 50 ml of 20% aqueous ethanol was added. The sample was heated with continuous stirring at 55˚c over a hot waterbath for 4 hours. This mixture was filtered and the remaining residue re extracted with another 100 ml, 20% ethanol. Both the extract combined and reduced up to 40 ml over water bath at 90˚c. The concentrate obtained was transferred into a 250 ml separating funnel and 10 ml of diethyl ether was added and shaken vigorously. In separating funnel ,two separate layers was observed out of which aqueous layer was recovered and the ether layer was discarded. The process of purification was repeated. To the aqueous extract 30 ml of n-butanol was added. The combined extracts were washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation the sample obtained were dried in oven to a constant weight and the saponin percentage was calculated.This is the procedure that I used.
I am extracting clove essential oil with Clavenger hydrodestilation. As can be seen in attachment the oil is accumulating the bottom of bottle. Is it oil or something else? How can I extract it?
Please help. Thank you.
I have tried Tween 80 in distilled water. But it was not helpful. Then a friend suggested DMSO. I used DMSO up to 20% (which is the highest allowed concentration of DMSO for biological activities), but none was helpful. I am using whole plant crude extract of chenopodium for biological activities. Please help me.
According to : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3429087/
For the solubility assay, the intestinal digests need to be centrifuged, to yield a supernatant and precipitate. The nutrients or compounds present in the supernatant represent the soluble components and measured by HPLC etc. Percent solubility is calculated as the amount of soluble compound relative to the total amount of compound in the test sample
* taking idea from: http://www.sciencedirect.com.ezproxy.lib.monash.edu.au/science/article/pii/S0963996906001530
Method:
- Powder (equivalent to 10mg of pure bioactive compound) + 40mL stimulated gastric fluid. Continuous shaking for 2 hours at 37C.
- After 2 hour, ph adjusted to ph 6.5. And added with 40mL stimulated intestinal fluid and continue shaking for 2 hours.
- Reaction stopped by placing in ice bath.
- Bioaccesible fraction obtained by centrifuging at 2000g for 5 minutes.
- 1ml supernatant top up to 20ml with methanol, subjected to hplc
Calculation
HPLC Area: 90.0 muA
Standard curve: Y= 300X
concentration: 90/300= 0.3ug/ml X (20 dilution) = 6ug/ml
6ugml X 80 ml (total volume gastric+intestine)= 480 ug/ml
Initial bioactive compound 10mg ~ 10000 ug
Total bioaccesible content: (480/10000) X100 = 4.8%
= 4.8ug/100ug
So is my method and calculation correct?
I am currently work on endophytic fungi, using rice solid static cultures. One of my fungus shown blue methanol extract. Blue color appear after the molded media immerse using methanol over night and disappear after third day at room temperature. Any idea to isolate this compound? I think it is polar and unstable compound.
Dear Researchers
Can I use NMR to identify type of phenolic compound inside the pitaya peel? so i just dry pitaya peel to remove water content by using freeze dryer and then blend the dried sample to get the powder form. after that Can i use NMR directly to identify type of phenolic compound on the powder sample ?
Dear All
My target is to use natural solvent that free toxin such as water in extraction active compound from dragon fruit peel. the active compounds such as phenolic content and mineral profile. These active compound can be used as additive in natural food product. what criteria should I consider if I use water as a solvent into microwave assisted extraction? An addition water gave the highest value TPC and antioxidant activity as description below (Lourith and Kanlayavattanakul, 2013). In this experiment I try to avoid using ethanol because I believe if use water don't need for purification process after extraction such as rotary evaporator.
solvent:Ethanol
TPC (mgGAE/g extract)=1.193+/-0.011
DPPH (ug/ml)=823.580+/-13.250
solvent:Water
TPC (mgGAE/g extract)=1.351+/-0.021
DPPH (ug/ml)=261.520+/-0.980
I'm trying to isolate pure compounds from hexane extract of a plant bark. After VLC, CC, and SEC, I ran 2D-TLC (40% EtOAC in hexane) but the compound assumed to be as a mixture with another compound is still making tails after many separating techniques. Please let me know how to confirm whether the comound tails bcoz of it's phenolic property or is it an another compound runs with same Rf of previous compound or any other degradation scenario?
Thank you..
I have done a gerneral GCMS of plant extracts, results are showing a large number of compounds. I am interested to see whether terpenoids are present or not. What should i do?
Hello,
I was wondering how much sample material can be loaded onto a SPE column until it is saturated and the separation is conducted less efficient. I am working with 500 mg silica gel-NH2 columns. The samples possess a very complex matrix with around 5-15% target molecule.
I think it is also important to note that I am not using SPE for analytical purposes anymore, but for isolation of target compounds. I usually apply 10-30 mg of sample, performing several elution steps and washing steps.
Now, I want to upscale the process and need to know the limitations of SPE columns. I will also upscale the SPE column size and material after or while upscaling the sample mass, but it should be as efficient as possible, of course.
Best regards,
Fabien
My work is on marine sponges and I have isolated few compounds from them .I have performed only NMR and LCMS for the isolated compounds, as the isolated compound quantity was less.
Can I get any idea about my compound from this two analysis alone?
Is it possible to find the compound present in my sample only with NMR results?
Can anyone clear my doubt?
I have read several papers and they said using AR 95% ethanol, but I was told LR grade ethanol will do the trick for rice extraction too and it is cheaper than AR grade. Rice extract will be used in the making of hair tonic for human use, so personally I think using AR grade ethanol is better for safety and health risks reasons. Could anyone help me decide which grade of ethanol I should use? or both can be used in my case? Thank you in advance.
Dear scientists,
i am working on phytochemicals and now i am to analyse that whether a phytochemical Triterpenoid is present in my sample or not, by spectrophotometric method. I did it according to protocol which i found in literature, but now i am confused as how can i know that it is present in my sample or not? kindly guide me for this.
Note: i am not doing color test, i want to know it by spectrophotometric method, about the availability and non- availability of Triterpenoids in my sample.
Thanks
It seems the vapor did not travel up to the condenser, it just dripped at the solvent flask neck. When I wrap the distillation tube, all the vapor gather in the thimble, it was not condensed (just like being steamed). The solvent was ethanol and already boiled, but increasing the heat to 95 celcius didnt resolve the dripping issue. Please help me. Thank you.
process of standardizing herbal formulation
Presently I am trying to isolate native strains of Thraustochytrids from coastal waters of Kerala, India. Pollen bating has been reported as one of the best method, but doesn't know from where to collect pine pollens. Is it available to purchase or we have to collect it from nature?
After the phenolics has been isolated from a crude extract. How do i isolate the bioactive compound from the phenolic extract for in vitro and in vivo studies.
Hi, I'd like to concentrate aroma from fruits and flowers at low temperature to avoid degradation and keep the origin fragrances. I read papers, many researchers use liquid extraction like hydro-distillation, steam distillation or with non-polar solvents. Some absorb aroma with porous resin.
Are there more methods or references that can both decrease aroma lose and break polarity selective limitation?
Thank you
I have prepared sequential extracts of a plant (a fern) in hexane, ethyl acetate and methanol in soxhlet. ethyl acetate extract shows promising bioactivity. how can I identify the major phytochemicals in the extract. the plant's data are not reported.
I would like to analyze the ceramide content in cells after addition of NBD-ceramide.
I know that alcohols work, but I can't have that competing with my reagent. I also want to extract water from the organic solvent, so I thought a solvent system would be efficient. I'm just not sure how to go about that.
I am using fungal extracts which are milligrams in quantity after getting a standard curve of gallic acid and quercetin how to get a concentration of phenol and flavonoid from the unknown sample.
Hi all, I want to use a protocol for alkaloid extraction from plants that describes the use of Hyflo Super Cel for filtration. Is there any alternative to Hyflo Super Cel which I can use?
In general, the steroidal alkaloids represent an important class of alkaloids that essentially afford a close structural relationship to sterols i.e., they contain a perhydro-1, 2-cyclopentanophenanthrene nucleus. Interestingly, these group of alkaloids invariably occur in the plant kingdom as glycosidal combination with carbohydrate moieties.
The steroidal alkaloids may be broadly classified into two major groups, namely:
(a) Solanum Alkaloids, and
(b) Veratrum Alkaloids.
how to isolate / crystallize citric acid form water ?
Hi, Currently i am working on isolation of natural products fromMArine actinobacteria, Recently i found out one of my actinobacteria strain which is very strange, when ever i want to do large culture, my all flasks colour is change, from one another.I use Gause medium and same parent sample for all of flasks, The original colour of strain is Pink, after 3 days of culture, it starts to give pink colour then slowly after some days it become dark pink, Some become dark red, Some light yellow, some dark yellow, some dark brown, some green , even every bottle have a strange colour.I even tried dark and light reactions for that, In dark it didnt changed colour but on HPLC-DAD there was no difference, And on hplc this strain didn't show any peak totally blank. I mostly cultured it from 7 -18 days but no peak on HPLC, i don't know what is the reason are they changing because of heat? or what? the temperature is 28.Is someone have this experience?please guide me for that.
possible way to remove stickiness from saponin which i get from crude aqueous extract of albizia lebbeck.
I ran a sephadex column with 50%CHCl3:MeOH solvent system. In first 10 vials, i got a mixture of compounds with tails (streakings) after i run the TLC. But i got 2 compounds in next vials. Should i run this mixture of compounds (present in first 10 vials) again in the sephadex lh-20 with same 50% CHCl3:MeOH or i should better use 100% MeOH solvent for its purification?
cold saponification involves leaving the reaction mixture overnight while hot methods involves reflux for 5 hours.
Dear All,
- i have isolated protein from plants material (leaves or seeds) by Tris-Hcl buffer and precipitate it by 10% TCA, followed by centrifugation to get the pellet.
- i washed the pellet with solvent.
- i want to detect the protein/ Polypeptides by TLC and HPLC.
- please suggest me which buffer OR solvent i can use to dissolve this isolated protein which will not interfere with HPLC and TLC
Thank you.
I get the fragments of Pulegone, Menthofuran, Sabinene hydrate, Dihydro carvone from GC/MS. So, how can I decide the chemical structure of fragments? Is there any reference paper regarding the fragment structure of those terpenoids?
We can use a lot of methods to phytochemical extraction from different sources, Which is the best? Can any one provide me the suitable answer from your experiments?
I have posted details of this isolate before also, but after getting suggestions from esteemed researchers, i have got the NMR, IR, Mass Spectra, UV spectrum done again on my sample. I have isolated probably a phenolic compound from ethanolic extract of a bark by using column chromatography technique. I used Hexane and Butanol as mobile phase , with linearly increasing butanol in the mobile phase. I have done super isolation by running the isolate obtained onto column chromatography again. Absorption maximum was found to be 280 nm and around 370-375 nm using UV spectrophotometer. I have attached all the spectrums
For NMR, sample was dissolved in DMSO
For Mass spectrum, Sample was dissolved in DMSO
IR was carried out by making pellet of KBr
For UV spectrum, sample was dissolved in Methanol
Isolate was dried using lyophilizer before carrying out any analysis.
My previous question is here


I want to extract total alkaloid from plants. I have tried several methods and still not get the alkaloid. The method that I used is:
1. 500 gram sample is boiled with water for 30 minutes and add lime juice 50 mL (pH 3), then squeezed (3X). Results of the filter boiled again until the solution becomes viscous. Once cooled, add caustic soda to pH 11. The solution was fractionated using petroleum and petroleum fractions evaporated.
2. 100 gram samples soxhleted using methanol. The extract was concentrated fractionated using n-hexane to remove chlorophyll and fat. The remaining methanol extract was added 50 mL of acetic acid and homogenized for 30 minutes. The extract was then added with 50 mL of 10% caustic soda and fractionated with ethyl acetate. Ethyl acetate fraction evaporated.
Whether the method used is correct? or is there any other method is better?
Best regards,
Ayu Muthia
for tlc suitable solvent system as saponins doesnot have any double bonds in the structure
Hello all,
I want to determine the amount of terpene compounds in different plant parts. Can anyone suggest a method for extracting all terpenes? Is methanolic extraction of powdered samples good enough for this purpose?
We are standardizing the technique of Hydrodistillation. We have no problem with plants such as eucalyptus, but other species the oil is obtained but does not come out to the Clevenger, but that it stays trapped in the balloon of distillation along with the water (the two phases is seen). Some idea of how to make the oil flows out of the balloon of distillation toward the apparatus of Clevenger? Thank you
Hi.
I am trying to extract flavonoids from plants and (if present) from animal tissues. I will then proceed to the estimation of the overall antioxidant capacity of the extract (FRAP, TEAC, ORAC, etc...).
Now, the issue is the following:
The different samples to analyze have different concentrations of tocopherols (already measured by HPLC). Therefore, I would like to selectively remove them from the sample, before extraction of other compounds (e.g.: phenolics). In this way, I'd like to avoid interferences from the tocopherols which actively react in the antioxidant capacity assays.
I cannot use polar solvents (methanol, ethanol or acetone) for this, because they would also extract the compounds I am interested in.
Therefore, i am thinking of using hexane or petroleum ether, as phenolics are poorly soluble in these solvents. Do you this these solvents will do a satisfactory job in selectively removing tocopherols? Which one would be the best candidate?
How can I isolate Trigonelline from fenugreek seeds (Methi seeds)?
I have isolated a compound from bark of a plant. I have HNMR , CNMR and IR Spectra of this isolate . HNMR and CNMR was done by dissolving my compound in DMSO.
I have done a very simple method of washing and rinsing 3 flower heads with 1 ml of distilled water in a 1 ml centrifuge tube. The total time for washing rinsing was 1 hour. Then I centrifuged the rinsate at 2540 rpm for 10 minutes.
I did not get a clear separation of the liquids but when I collected a small amount of liquid at the top and measured the sugar content (glucose), the amount was very small, some nectar was still contained in the flowers, they didn't all wash out.
Why do we used 70% of methanol for extraction of total phenolics but not Hexane or petroleum ether?
I'm conducting a research about the phytochemical properties of essential oils extracted from Cupressus sempervirens and I would kindly want to know whether it is necessary to de-fat the plant materials with petroleum ether prior to methanolic extraction.