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Natural Product Isolation - Science method

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We are inviting experts outside India to contribute a book chapter on Phytopharmaceutical for an upcoming publication under the De Gruyter press USA. If you're interested in sharing your expertise in phytopharmaceutical , please contact us for collaboration opportunities at chauhan.nagendra@gmail.com
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Suggestion for oligomers with mass differences m/z 82 daltons. Thank you
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Rapid Commun Mass Spectrom. 2021 Oct 30; 35(20): e9175.
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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
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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?
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Hi Shaimaa Heider is there any chance that you have respiration rates available for cut foliage Fatsia japonica?
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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.
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Andrew K Ottens
Thank you, I will try injecting within 90% ACN and adjust the gradient accordingly so that it starts at 90%. I am using HILIC to further isolate the metabolite of interest after I've already done my best to purify using C18. With C18 I am able to isolate a peak containing the metabolite of interest, but it still contains enough other compounds that would cause issues with NMR. My current workflow is crude extraction with ethyl acetate--> evaporation and resuspension in MeOH (ethyl acetate caused issue with clogging in the HPLC) --> preperatory-hplc with c18 isolating the active peak w/ a time based fractionation collection --> evaporation and resuspension (previously used methanol here but will try 90% ACN) --> HILIC. The goal is to further isolate the metabolite of interest from the other compounds present in the peak so that I can proceed to NMR. If you have any suggestions for alternative routes besides HILIC I would be grateful. The compound appears to be polar/amphipathic and has a MW of <350
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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?
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Did you ever figure out how to do this? I also need to extract nuclei from bark tissue.
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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. 
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The temperature of soxhlet extraction is entirely determined by the selected solvent because it is the boing point of that specific solvent. Any temperature below the B.P. results in a slow extraction because of the limited amount of solvent driven to the extraction chamber and be in contact with the sample. However, if you want to use water for the soxhlet extraction there will a continuous loss of the solvent (incomplete condensation) unless you use chilled water for the cooling condenser... Regards
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Other than sephadex LH 20 and Carbon.
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Before proceeding further for extraction, treat the leaves in boiling water first and then with alcohol (ethanol) as it can remove chlorophyll completely to avoid contamination
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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.
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I proposed that this difference is related to the variation in the active sites' affinities.
Regards
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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?
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Its advisable to keep your samples dry in fume cupboard once you are not running in NMR machine. Keeping them in chloroform for a long time may cause degradation.
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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?
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we've alreday isolated endophytes from diverse spontaneaous plant species, we found very interesting bacterial endophytes from Urtica urens. See (Krimi et al., 2016). Good luck!
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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?
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If you aren't so sure about the Calibration of your machine, try to run same experiments but this time using know verified compounds. Then compare the RTs.
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For a purification of large amount of sample in column chromatography takes long time, so I need some other methods
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Mr Murad Ali Khan can you give more details about the purification of tannins by the solvent extraction method, please?
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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.
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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!
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2-5 g of Retinol 50C (BASF product) are dissolved with vigorous stirring in 10-15 cm3 of n-hexane, using an ultrasonic bath to accelerate dissolution. Remove excess water by adding anhydrous sodium sulfate. The contents of the flask are filtered through a filter paper to separate the undissolved precipitate. The flask is washed twice with 5 cm3 of n-hexane. The filtrates are collected in a volumetric flask with a capacity of 25 cm. The solution is adjusted to the mark with n-hexane. Then an aliquot of the hexane solution is evaporated in a stream of nitrogen and the dry residue is redissolved in the eluent
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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
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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?
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pancreatic alpha shares higher proportion as conmpared to salivary which makes it better. is this the main reason?
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Natural indigo dye produced from different sources contains many impurities in it.What is the best method to purify it ?
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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?
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can anyone tell me the Rf value of standard D-quinovose??? I have searched alot but did not find it.
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I use ethylacetate:hexane solvent system, and is giving me tailing separations. My aim is to isolate flavonoids from the n-butanol fraction. Regards
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We use the following systems for the butanol fraction to identify flavonoids using TLC:
1. Chloroform: methanol: acetic acid: water in a ratio of 7:3:0.5:0.5 (v/v/v/v)
2. n-butanol: acetic acid: water in a ratio of 4: 1: 2 and 4: 1: 5(v/v/v). PS^ after cooking the system is left to saturate, then the non-displaced water must be removed
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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.
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Use a polar and non-polar solvent. For non-polar compounds, hexane, benzene, petroleum ether, etc. For polar compounds, ethanol 40% and 70% can be used. It depends on what class of compounds you are studying. Methanol and 96% ethanol extracts all secondary and primary metabolites from plants.
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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?
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NMR data is enough to interpret your compound, but you have not specifically written what NMR data you have. It seemed to me that you only have C13 data.
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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
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Butanol:Acetic acid:Water (7:1:2) works well for many polar compounds
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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?
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Organic solvents should be avoided.
But in order to use the appropriate solvent it is necessary to know the target phytocompounds.
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Is fractionation by simply shaking with solvents of increasing polarity successively sufficient or is it needed to always have 2 immiscible solvents?
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1). n- Hexane removes all non polar compounds
2). chloroform removes all pigments such as chlorophyll etc
3). ethyl acetate is where all biological active compounds are excepted such as flavonoids, anthraquinones, anthocyanine ,benzo pyrans etc
4). n-butanol extracts all other hydrocarbons
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am working on determination of total phenolics and flavonoids
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1- First, a Beaker or a crucible should be brought, weighed (Assume that the weight is 100 g)
2- Transfer the extract to the Beaker
3- The solvent used in the extraction process shall be disposed of by evaporation of until the dry matter is obtained as far as possible
4- The beaker is weighed after full evaporation of the solvent (Assume that the weight is 110 gm)
5- The weight of the extract (the weight of the beaker with the extract - the weight of the empty beaker) = 110-100 = 10 g
6- The extract is dissolved in the appropriate solvent Ethyl alcohol is often used and an emulsifier may be added (the amount of the solvent is calculated to obtain the desired concentration(
To prepare different concentrations:
1- The extract is dissolved in the minimum amount of suitable solvent and then a quantitative transfer is done to a volumetric flask with a final size of 100 ml (the appropriate size for the quantity of the extract).
The concentration of the extract is calculated as follows
10gm/100ml = 10×103mg/100ml= 100 mg/ml
Different concentrations could be prepared from that stock solution
To prepare 1 mg / ml with a final volume of 100 ml
V1×C1 (before dilution) = V2×C2 (after dilution)( the concentration which we would to prepare )
? ×100 = 1×100
V1= 1×100/100= 1 ml
Take 1 ml of stock solution (Conc 100mg/ ml) and supplement with appropriate solvent to 100 ml in volumetric flask.
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I am searching for a useful alternative for NIST/EPA/NIH Mass Spectral Library.
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I have the same problem for LC-MS. I would appreciate if any one help me.
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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
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It is possibly a step to eliminate unwanted components from the extract. Formaldehyde act as a precipitate forming agent.
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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.
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Whether you are asking a person or using a program, you need to give some information about the structure, you cannot just put in the numbers and expect to get an answer.
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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.
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Of course you could but you have to select the suitable organic solvents
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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.
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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.
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Do you mean the reference? I used gallic acid for total phenolic content reference
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i want to quantify saponin in Aloe vera genotypes using HPLC. Which steroidal saponin to be used as marker compound?? please suggest.
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Instead of sterols, you can use saponin from other sources such as Solanine (Sigma) as internal standard for quantification (standard), and quality control for HPLC runs and extraction efficiency etc. as well.
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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.
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 hai,,
i have read the procedure u adopted. at the end u used the aqs. Nacl. in fact This step is not necessary. If the saponin u are looking is in glycoside form it will enter into this aqs. NaCl. if only the saponin aglycon it will not enter into water and remain in the organic layer. 
a mistake in procedure is observed. n-butanol addition is not necessary as this is highly polar than ethanol. 
for conformation of saponin availability in water and butanol layers, simply perform liebermann bucchard test.l this is the 1 drop of extract solution in test tube add conc. sulphuric acid without shaking and from the side walls of test tube. at the junction of two layers agreen color appears indicates the presence of saponin.
now the correct extraction procedure is use of alcohol or hydro alcohol solvent. heat at 50 degre C, filter. ensure the removal of solvent from the filtrate. redissolve in water in separatin funnel. add hexane. remove hexane layer repeat 2-3 times. now the water layer evaporate to dryness. this contains saponins will answer the above test. 
another simple test is foam test. take extract put in 25 ml measuring cylinder. add water 10ml. shake well to get foam on above the solution for 1ml ht. and should remain same for 15mins. 
all the best
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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. 
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Oil of clove is heavier than water , due to its high content of eugenol, as said before, this character of the oil, can be used to differentiate the fresh clove buds from the old or exhausted ones, by placing the buds in water, when sinking down indicate fresh buds. 
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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.
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Have you tried dissolving the crude extract in 100% DMSO at higher concentration, say for e.g. 1 mg/ml and then run serial dilutions to the concentrations that you want to test? The % DMSO will decrease. Normally in my assays, final concentration of DMSO is between 0.2 to 0.5%. 
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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
Method:
  1. Powder (equivalent to 10mg of pure bioactive compound) + 40mL stimulated gastric fluid. Continuous shaking for 2 hours at 37C.
  2. After 2 hour, ph adjusted to ph 6.5. And added with 40mL stimulated intestinal fluid and continue shaking for 2 hours.
  3. Reaction stopped by placing in ice bath.
  4. Bioaccesible fraction obtained by centrifuging at 2000g for 5 minutes.
  5. 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?
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Haha..
Just wondering if my method and calculation is correct for bioaccesibility
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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.
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Collect your blue extract. Filter it. Reduce it to dryness in vacuo under mild conditions to keep it under safer conditions for storage in the refrigerator than in solution.Take a small portion of your blue extract and give it on several TLC plates (but use enough material). Then develop it in small TLC chambers with pure solvents as eluents. These solvents should have different polarities, e.g. hexane, chloroform, ether or MTBE, ethyl acetate, methanol, acetone and others. But do not use mixtures. After the TLC ist developed have a look where the BLUE is. Upstairs, in the middle, downstairs?This will allow you to get an intention on the polarity of your compound and the qualitiy of your extract.
Then, it is possible to find a suitable eluent for column chromatography (CC) by mixing two of the solvents in an appropriate ratio. All of this can be done only by trial and error. But with small amounts it is not difficult to find a solution for the separation. I would recommend to try CC for separation of the blue.
Caution: Real dyestuffs with a high epsilon value may cause a colour already with tiny amounts of material. However, it cannot be seen at the begin if this is the case in your case or not.
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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 ?
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Hm... very interesting. I'd say, unless this is a special case where the peels are very rich in one type of that "phenolic ingredient" it would be only by a strike of luck that one can "identify" the structure. People who do isolation of novel Natural Products would have very little work if things were so simple.
One feature of phenols that can be perhaps useful during initial purification is that they often can be extracted into aqueous NaOH or KOH solutions. Other extractive operations may be necessary, which are normally followed by partitioning on solid phases (Si-gel chromatography, Sephadex, RP HPLC, etc). Analytical HPLC will assist you in assaying the fractions. But even after your component is isolated in a pure form, NMR work (1D-, 2D, nOe-based, etc) alone most probably will not be sufficient (again, unless you are dealing with a relatively simple and well know structure). Typically, HR MS or LCMS data will also be necessary (as Vladimir Khlebnikov indicated to you earlier), and in case of chiral compounds, optical rotation, CD, or chiral chromatography data should also be analyzed.
One perhaps tricky part of your question is that you want to "identify the type" of that compound. I presume that could mean that, for whatever reason, you do not actually need to precisely determine the structure. Perhaps in such case more direct NMR methods may provide some information?
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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
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I would suggest you to perform quick extraction with hydro-alcoholic mixture (start with 80:20 = ethanol: water). Check for TPC and DPPH and compare the results with pure solvents (water and ethanol). 
Additionally, you should calculate the yield of extraction using aforementioned solvent systems.
Best wishes!
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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..
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With phenolics (i.e., flavonoids), I've found a little acetic or formic acid (0.1 - 5%) in the mobile phase helps to tighten up spots.
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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?
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How far does your mass spectral library identify the compounds you have?  That is the usual reason for using MS so that you can match the compounds with a library of mass spectra.  Otherwise you have to find published spectra from NIST or in literature to match by eye - very time consuming.  The same is true of trying to match your compounds by using standards.  Terpenoids are such a complex and varied group of compounds you could try for years and never find a match, and many will not be available as standards in any case.
What plant extracts are you looking at?  You could start by looking at other analyses of the same plants (if there are any) which would at least give you a starting point.
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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
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Page 6 and 7 may be of particular applicability to your question. 
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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?
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The answer is certainty comes from the quality of the data and also whether the compound is known or unknown in the literature.  If someone had found the same compound in the same matrix as you have using the same analytical techniques, great!  You just confirmed the other person's results.  It is still possible both of you could be wrong, and if that were true, the person challenging the answer would have to make a compelling case based upon the preponderance of the newly presented evidence.
If you claim you have discovered the same compound in a new matrix,  using the same techniques already published in the literature to determine it would mostly suffice. 
Also excellent if your standard (of a known compound) matches that in your  NMR and LC-MS results.
If there is no standard and/or if you are claiming an entirely new compound has been found, then the criteria  for publication of the results are necessarily significantly higher.  Of course the value of the publication in the peer-review community is greater when in fact a compound detected actually is an entirely new compound or even a new class of compounds.  .     .
Therefore hope you find multiple new compounds very difficult to identify, well worth the level of doubt generated in the analysis,  and that the new compounds are subsequently found to have important biochemical and/or even biomedical properties.   Cannot know until after all the difficult and challenging work has been completed whether successful.  
Our lab identified a-endosulfan correctly after 30 years in the literature incorrectly identified.  The more important the chemical, the bigger the deal if it is properly and correctly identified.
Walter.
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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.
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Hi
 LR, AR, HPLC, Commercial, Rectified, Absolute,  these all grades of ethyl alcohol manufactured for specific use.
if you like to use for research purpose any of above  can be used. it will not interfer the phytochemical assay or extraction efficiency. But Molecular level analysis or biological assay may have some interference.
human health point view, you may use any of the above for extraction but end product, it should pass the limit of residual solvent as per the regulation your are about to follow. USP, ICH, etc,
so you may use any of the grade as above but check the RSL before application and comply the regulation then proceed.
all the best
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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
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Dear Bilal
Spectrophotometer can not confirm you the availability of Triterpenoids in your sample.If you simply run TLC and detect  it by LB Reagent ,you will get your answer. I am also attaching reference article here for your acknowladgement
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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.
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what is the type of condenser that you have used? actually, there are two important things that influence the mobility of solvent in soxhlet extraction process. the first is the phase of solvent, the solvent will travel from the flask to the soxhlet container if the phase of solvent is gas. so, for this matter, the evaporation of the solvent is important, to increase the rate of evaporation can be done by stirring the solvent inside the flask, and by setting the temperature over the boiling point of the solvent. the problem that usually occured for this matter is the vapor of solvent have condensed at the neck of soxhlet before they reach the condenser and drop into the container. this problem occured due to the temperature of the neck is too low, so the temperature of vapor solvent is getting down, condense, and stop travel. you can overcome this problem by adding some heat to the neck, the heat can be delivered from the flask by connecting the flask and the neck using alumunium foil (wrapping the flask and the neck into one pack) and minimize the heat lost by wrapping one more the first pack (the wrapped glass) by using papper/tissue/fabric.
the second thing that influence the mobility of solvent is pressure. simple, the vapor will travel only from higher pressure to the lower. and to make this condition happen in the sochlet system, make sure the air inside the soxhlet container can travel out easilly, so the pressure inside the container is continously lower than the pressure inside the flask and there is any space inside the soxhlet container to be filled by the vapor of solvent. connecting the vacuum system onto the condensser will improve the condition that i have mentioned above. usually the problem of this matter is the rate of condensation is less than the rate of evaporation, and it is combined with the air (any gas phase) inside the soxhlet container can not easily travel out from the container, so there is no space anymore for the vapor inside the container.
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process of standardizing herbal formulation
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Dr Gunawan's suggestion is good,  If one affords or can depend on external support. For most of the simple labs where both teaching and research are to take place the suggested sophisticated equipment may be difficult.
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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?
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Thank you Reynold
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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. 
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When it is possible, an online detection as a kind of postcolumn derivatization would be also a good possibility. There, you can directly get informations after the separation. This is not possible for every bioactivity, but for antioixdant activity, enzyme intactions/binding, this works really well. With this approach one can quickly find out which compound is the most responsible one in an extract....
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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 
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I agree that the advantage of using supercritical fluid CO2 extraction is that you can make a very efficient extraction of components and eliminate subsequent separation steps.  The challenge is how close is this going to be to the right "natural" composition and ratio of chemicals of what you want. You have to find the happy medium. Depending on the source of your plant material, and plant part used, you may have to adjust temperature, solvent-to-material ratio and extraction time. There are many examples in the literature that can guide you.
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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.
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Yes. However, comparison of the retention times, mass spectral data and fragmentation pattern of the phytoconstituents with those in a standard library must be meticulously guided.
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I would like to analyze the ceramide content in cells after addition of NBD-ceramide.
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Hi Maiku. Do you mean you want to see the NBD-ceramide by TLC or unlabelled endogenous ceramide?  For either, collect the cells in PBS, add 5 volumes of chloroform:methanol 2:1 and vortex vigorously several times. You should have 2 phases with cell debris at the interphase. Centrifuge to quickly clarify the 2 phases or allow to sit several hours.  Both ceramide and NBD-ceramide are in the lower phase- dry this down and run a portion on silica TLC plates in the solvent chloroform:methanol:water, 90:15:1.  
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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.
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Try using a Dean-Stork assembly to get water out! You may use non-alcoholic and ionic liquids as solvents!
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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.
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Use FC reagent and Construct a standard linear curve with quercetin. In the same way perform the reaction with unknown sample also, calculate how flavonoid concentration how we do in protein estimation by using BSA standard curve
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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?
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I'd try the following two methods- both take advantage of the basic nature of alkaloids
The first method is extraction. First, partition the extract between dichloromethane and acidic water (I like to use acetic or formic acid). Set the dichloromethane layer aside for other compounds if desired. Make the aqueous solution basic with ammonium hydroxide, pH 10 is good. Extract again with dichloromethane. The dichloromethane extract will contain your alkaloids, often clean enough for a single-column purification.
The other way of capturing alkaloids is with a strong cation exchange (SCX) column. Details in the following links:
I use the double gradient because it allows me to resolve different classes of alkaloids. Step gradients can also be used if one doesn't have an automatic system.
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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.
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Dear Rasoul,
Please note that C18 is a reverse phase column whereas silica is a normal phase chromatographic column. 
C18 columns are broadly used in a wide variety of applications including pharmaceuticals, steroids, fatty acids, phthalates, environmental and more. They are the most retentive of the alkyl-bonded phases. Therefore, C18 may accomplish the task you are going to conduct.
Good luck,
Rafik
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how to isolate / crystallize citric acid form water ?
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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.
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pH is the main factor behind different pigment production. Actinobacterial colour tends to change after sporulation and with increase and decrease of pH. My suggestion is to check its colour in ISP-2 and ISP-4 medium.
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possible way to remove stickiness from saponin which i get from crude aqueous extract of albizia lebbeck.
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One way I have dealt with sticky sample is by dissolving them in the minimum amount of ethanol and adding, drop wise at first, into a large volume (3-4 liters) of petroleum ether with vigorous stirring. Your compound will precipitate. Adding the sample too fast (or too much) will result in a sticky  mass again.
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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?
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Dear Sir
Please, find in attch, an Instructions 56-1190-97 AD Gel Filtration for Sephadex LH-20. It might be useful for you.
Best wishes
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cold saponification involves leaving the reaction mixture overnight while hot methods involves reflux for 5 hours.
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yes Prof. that's what i want to know. what is the difference in yield?
thanks for your concern
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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.
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Dear Sir
Beside the intersting answers provided to you.Please, find in attach a Chapter on: Peptide and Amino Acids Separation and Identification from Natural Products Ion Neda, Paulina Vlazan, Raluca Oana Pop,Paula Sfarloaga, Ioan Grozescu and Adina-Elena Segneanu.
Good luck
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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?
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Dear Somnath,
Since, you already have mass spectrum of each of your compound of interest. You may directly match your recorded spectra with available libraries if installed in your mass spectrometer.  I am sure that the  resultant hits (library) will be very much correct as these compounds are already known.
If you are willing to understand in detail about mass spectral fragmentation technique specific to type of functional groups, you should consult textbooks like Spectrometric Identification of Organic Compounds by Robert M. Silverstein, Francis X. Webster, David J. Kiemle, David L. Bryce etc.
Otherwise, attachment named terpene by Mr. Raj is also useful in order to understand this concept.
Good Luck
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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? 
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Dear Ibrahem Khalifa,
Please find the rest of the method attached.
A.R. Abdel-Moemin, To investigate the antioxidant properties of dietary flavonoids in human metabolism (Ph.D. Thesis, The Queen’s University of Belfast, United Kingdom, 2004)
All the best
Aly
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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
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Hi, you have ESI HR MS data,  let me share our experiences at our laboratory for determining the structure of isolate (for known compound).
Usually  at first we measured our isolate using (LC) ESI HR MS, by this we can predict its chemical structure by applying some  free online MS data base (Metfrag, Metlin, mzCloud, MassBank or others), if the compounds are known compound, it will listed in the data base;  the predicted  structure can be confirmed by NMR (H-NMR, C-NMR, 2D NMR), or by measured (HRMS) of authentic standard; you can get the link of the free data base by searching in Google. Did you have check the purity of your isolate (e.g. by using 2DTLC or HPLC-DAD or DSC)? If it is not pure, it will nice you can run LC-ESI HRMS, best regards
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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
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You can follow this procedure:
For the purpose of phytochemical analysis of the selected plants,
0.2 g of the selected plant samples were added in each test tube and
3 ml of hexane were mixed in it, shaken well and filtered. Then took
5 ml of 2% HCl and poured in a test tube having the mixture of plant
extract and hexane. Heated the test tube having the mixture, filtered it
and poured few drops of picric acid in a mixture. Formation of yellow
color precipitate indicates the presence of alkaloids
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for tlc suitable solvent system as saponins doesnot have any double bonds in the structure
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Hello Tanvi,
I worked with saponin purification and the best technique is to perform column chromatography (CC). First is recomended to use CC with reverse phase silica gel (RP-8 or RP-18) uson mixtures MeOH/H2O as solvents to eliminate small polar molecules from the crude. Then, apply CC with normal phase column cromatography on silica gel using solvent mixtures like CHCl3/MeOH/H2O (70/30/5).
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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?
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use apolar solvent such as hexane, chloroform, petrol then concentrate the extract and inject in gaz chromatography!
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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
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you can try stream distillation to remove volatils compounds, good luck
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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?
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Hexane or petroleum ether should do what you wish, for the reasons you stated. Tocopherols are soluble in either one. Diethyl ether is another candidate solvent for this purpose. I've used hexanes to dissolve tocopherols. Also consider dichloromethane, though that is getting just polar enough that it may start affecting your other compounds.
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How can I isolate Trigonelline from fenugreek seeds (Methi seeds)?
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Thanks, Shiv Gupta.
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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.
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To start, we need to know the method used to make the product isolation. What types of solvents were used in the extraction process? The NMR spectra has a  showed two types of deuterated solvents CDCl3 and DMSO (see 13C). What didn´t you make a APT or DEPT spectra? Your 1H spectrum seems to be not calibrated to DMSO, but CDCl3. In my opinion you have it is a mixture of solvents used in the extraction process. Look improve the quality of NMR spectra. Search the literature the kind of already isolated substances from its plant, can be of great help. Good lucky
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Medicinal value of Bael fruits
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pinene, pheladrene and myrecene is  the chemical composition of Bael or Aegle marmeoles.
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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.
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Hi Martrin,
No I haven't tried extraction with calibrated micro pipette but , I will do that.
Thank you for the suggestion. 
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Why do we used 70% of methanol for extraction of total phenolics but not Hexane or petroleum ether?
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Dear Zahoor
Considering that phenolics contain hydroxyl groups they are more soluble in polar solvents (methanol) than in nonpolar ones (hexane, petroleum ether)
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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.
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ThanQ all for your contributions
Best regards