Is it possible that we can use TLC as a conformation tool for identify the compounds found in crude extracts?
Dear Sandeep,
TLC run with a crude extract and a pure standard in co-chromatography gives an indication on the presence of the compound in the crude extract based on the Rf and color characteristics (if a derivatisation reagent is used or based on 'quenching' or fluoresence (color) characteristics when viewed under UV light). I do not believe that this is an absolute confirmation tool, it is more or less an indicative tool. The confirmation is to either isolate the compound from the crude extract or run either HPTLC or HPLC of the extract with hyphenated mass spectral detector to confirm the compounds presence. That would be confirmation of the compound's presence in the crude extract.
Dear Sandeep,
TLC run with a crude extract and a pure standard in co-chromatography gives an indication on the presence of the compound in the crude extract based on the Rf and color characteristics (if a derivatisation reagent is used or based on 'quenching' or fluoresence (color) characteristics when viewed under UV light). I do not believe that this is an absolute confirmation tool, it is more or less an indicative tool. The confirmation is to either isolate the compound from the crude extract or run either HPTLC or HPLC of the extract with hyphenated mass spectral detector to confirm the compounds presence. That would be confirmation of the compound's presence in the crude extract.
I'm in accordance with Dr. S.G.
To identify the chemical structure of a compound without any doubt, the substance should be isolated and analysed also by spectroscopic/spectrometric methods as NMR, UV-Vis, IR, MS.
For the correct identification of chiral compounds also the optical activity is a very important datum.
Hi Sandeep
TLC is useful for the preliminary evaluation of crude extract/reaction mixtures and for the preliminary identification of organic compounds. Normally the more polar a compound, the lower it's Rf value, and vice versa. Thus, for a given set of conditions, the Rf values of two spots on a TLC plate may provide some evidence as the identity of a compound. For a given set of conditions, two compounds having different Rf values will be different, while those having identical Rf values may be the same. On the other hand different compounds may have identical Rf values. However, comparison of an unknown compound with an authentic sample(s) can provide some insight as to the identity of that compound. As such, other analytical techniques should be used to confirm the identity of a compound even if it appears to be a single compound by TLC.
Good Luck
TLC is the separation of the compound mixture of solute depending upon there relative solubility to the mobile plate , UPON separation scrap out the seperated portion and go for re doing the sample . Gives the final identification of the compound.
Fully agree with Dr. Suresh and Dr. Alessandro Venditti. TLC can only help to get an indication of the presence of compound and that can be confirmed for identity in the presence of a standard. If you want to establish its presence then you will have to go towards the high end techniques like HPTLC and HPLC and isolate the compound for its structural identification using different analytic and spectroscopic studies. TLC is a very crude method as far as identification of compounds is concerned. Yes it is an unavoidable technique in natural product research and a basic step in almost every chromatographic analysis, so its importance can not be overlooked.
The problem is not so simple even if one have standard compounds. As example oleanolic acid and ursolic acid have the same Rf in many solvent systems. How you can discriminate only by TLC analysis?
As implied in many of the answers, TLC is low resolution in comparison, for example, with HPLC and - even with HPLC - a single peak may often represent more than a single compound. So it's rarely safe to assume that a TLC spot indicates a pure substance. (One can always hope, but that could be scientifically dangerous.)
:)
I wish to add to the points of Dr.Venditti and Dr.Wishnok!
The choice of techniques for deconvolution of the extract matrix is the first and most important step. The choice of 'deconvolution' technique will always be based on the sample matrix, mobile and the stationary phases.
1. There are excellent examples of the utility of HPTLC separations. In my experience, for separation and identification of ginsenosides from Panax ginseng, P.quinquefolium or P. notoginseng, HPTLC coupled with detection after derivatisation is the best bet.
2. HPLC with reverse phase columns are universally accepted; with smaller particle size, smaller bore columns (such as microbore) and use of smaller flow rates (for eg in UPLC) increases the chances of resolution to a very large extent. Chiral columns or ion-pair chromatographic columns are added advantage. In addition to PDA detectors, adding ESI-MS or APCI-MS with tandem mass spectrometry, TOF and Ion-trap systems in series add to generating crucial data on the separated constituents to properly identify.
The point is, one needs to understand the advantages and limitations of each technique. Almost all separation and identification techniques compliment each other and the decision on which one or combination to choose for a given sample (extract) matrix will be a trial and error experimentation.
Hello sandeep,
As all said TLC is only a qualitative test ,we can't assure confirmation of the compound. we can scrap the spot and analyse further with various other chromatograpic techniques .
This is only a separation and identification technique.
Many of them have discussed more, hope u understand
thank u
Of course you can't identify the compound in crude ext. By TLC it may give you a preliminary ideas.
You can do it using analytical HPLC
No, since TLC only tells you that the compound or compounds have similar Rf to the reference compound.
TLC analysis followed by isolation of compound (may be by scraping out or preparative HPTLC), its characterization using techniques like FTIR, HPLC-MS, NMR, XRD will suffice the appropriate confirmation of the compound. Comparing Rf of compound present in the crude extract with known standard, its spectral confirmation, colour of the band usually provides a putative confirmation.
please as you have told that by using HPLC, HPTLC, and by spectroscopy method will help to separate the pure compounds, is there any alternative way to perform isolation with in less time and less money.
I agree with Bhavesh Tiwari that we have to identify the chemical structure of a compound also by sp· ectroscopic/spectrometric methods as NMR, UV-Vis, IR, MS and HPLC with thestandards.
Respected Mr.Deepak,
TLC can be used as qualitative analysis of a compound, to say it may be, but cannot confirm. the confirmation should be followed with spectroscopic methods , FTIR, NMR,MASS,
TLC method will help you to find the compound in that fraction by during the isolation processes.
It will help you to find the end point of the chemical reaction
Dear Sandeep,
As I understand you are trying to identify compounds without those high end expensive methods. The simple practical answer is YES, but (as many highlighted before) indeed you need to be careful with your conclusion.
TLC is indeed relatively low resolution, but using different stationary phase / solvent system combinations you can easily get simple yes/no answers. You need at least three independent methods where your compound in question shows the same chromatographic behaviour as an authentic standard. I'd suggest you do two normal phase (with completely different solvent system components, preferably using one acidic/basic and a neutral, or an acidic and a basic composition; you can do this on one plate with a 2D setup) and a reverse phase TLC. Detectability of the compound (UV absorbing or fluorescent spot, color after using some sprayer, etc.) will give an extra confirmation when compared to that of the reference compound.
If your solvent systems give nice, well-defined spots (without significant tailing) for your compound in between Rf values of let's say 3.5-6.5, this should be enough. In case of certain complicated structures with several stereocenters you can still get false positives with some isomers (that's why I say thrustfulness might depend on the compound of interest), but for example plant poliphenolics can really nicely be identified this way.
To allow results of high reproducibility, I would go for HPTLC for both qualitative and quantitative analyses of samples. If you run the known standards with a concentration series in parallel with your sample on the HPTLC plate, HTPLC can be used for identification. For instance, HPTLC can be successfully used for phospholipid and neutral lipid identification. In addition, you can scrap the band of your interest and then analyze with the mass spectrometry tools.
Theoreticaly, you can use this method for qualitative analysis, but , TLC could be used for preliminary results, because this method has low resolution. You can examine separation of components in different solvents (different mobile phase), and compared your results with reference compound Rf value in used solvents.
If you have HPTLC available in house, use it as a powerful approach! Depending on separation protocols/ solvent systems, the approach gives very reproducible results and has a very good resolving capacity with low detection limits.
Dear, I think you cannot rely on the identification results you get from TLC., specially if you have very similar components in the sample
If you have isolated compounds as standard, the normal compounds could be identified by TLC and special colour reaction. The identification are unreliable, It need to be confirmed by MS or NMR.
Don't lose sight of the original question: Can TLC be used as a final tool for identification. The answer is simply 'No'.
:)
TLC can, with the aid of chromogenic reagents in cases of non- coloured components, be used to give clue to the chemical composition of crude extracts. it can also for identification of predicted components present by comparison of Rf values with a known standard.
TLC with a crude extract and a pure standard in co-chromatography gives an indication on the presence of the compound in the crude extract based on the Rf ., so TLC is a preliminary test not a confirmatory. The confirmation must be done by isolate the active compound from the crude extract and then identified by HPLC .
TLC of crude extract or Co-TLC with standard is not the final answer so far identification of pure compounds in concerned. You have to go for precise separation by HPLC and then characterization of unknown compounds by spectral analysis. However, known compounds can be identified by HPTLC and/or superimposable IR using standard (authentic sample) of the known compounds.
If you have standard compounds which can be presented in the plant extract, you can confirm the presence of those compounds. Also spray reagents for type of compounds as alkaloids (Dragendorff), triterpenes and steroids (Liebermann-Buchard) etc. could be used for more confirmation.
TLC cannot be used as a final tool for identification because of its "low resolution". It is usually used "qualitatively" to indicate the presence of an analyte of interest by comparison with Rf values of a known standard. However, crude extracts may contain compounds with relatively similar Rf values. Thus, confirmation of the presence of the analyte of interest using more robust analytical tools viz., HP-TLC, HPLC, LC-MS, LC-MS-MS is imperative.
To identify a compound present in an extract you have either to isolate the compound prior to its characterization using the proper analytical tool (MNR, MS, ...) or to use a separation technique combined to an analytical tool (GC-MS, LC-MS,...). Definitely TLC is not an identification tool.
If you compare with a standard compound and they have the same Rf you would have, in principle, the same compounds and you have a preliminary results, but you necessarily should run many others TLC chromatography's using many others solvents system to check them. Finally, to confirm you should follow Micaela Nali indications.
TLC cannot be used as confirmation tool, it is only indicative, you have to isolate the compound and give for MS and NMR
TLC comparison with a standard compound is not enough to confirm the chemical structure, you need more information as a HPLC fingerprint or a NMR spectroscopic data for example.
By preparative thin layer chromatography you can isolate main compounds of plant extracts and then proceed with espectroscopic analysis to determine structure.
Agree with views of Micaela, Selene Maia, Kelvin, Ifeoluwa. TLC can't be the final tool for identification of compound in a crude extract. Spots appeared on the TLC plate after proper development may provide preliminary indication about the class of compounds present in the compounds when visualized by using various detecting reagents.
TLC is basic tool that is used in basic lab. To have more precise information go for gc and LC
Problem is being a final here. If you have only TLC and HPLC only. ıt is better to use TLC first, then run the HPLC for quantitave analysis. TLC is best tool if you consider only absence or presence of compounds, since it is cheaper, or paper chromatography is much cheaper. If your aim is to isolate new compound etc, and then publication in a good journal, it is just a start
I believe you can use TLC or better HPTLC as fingerprint techinque to aid in plant identification. As a tool for definitive identification of a marker within an extract has limitations, but it can provide unique patterns composed of multiple bands with different colors that provide an easy recognition of a particular plat species even with plants within the same genus. You may also use an orthogonal HPTLC procedure for fingerprinting using a different development reagent or/and development solvent to identify a complementary pattern of a different class of compounds. For examples for a given plant you may want to fingerprint a flavonoid profile and a saponin profile using complementary HPTLC profiles.
Tlc can only be used for as preliminary phytochemical screening tool
TLC can not used as a confirmatory tool for identification of compounds from crude extract. Spectra comparison in HPTLC and HPTLC-MS may be used for this purpose.
As already was pointed out, TLC is an indication only. But also HPLC or HPTLC is generally an indication although being a more sound argument. You should be aware that all chromatographic methods are comparative methods; the result taken critically is: "compound in question has the same tR or Rf as compound X." Color detection (TLC) or UV spectrum (HPLC) may help to get closer to the compound X, but again it does not make the compound in question and X identical. This is especially true when it comes to optically active compounds (even if you work with chiral stationary phases). HPLC/MS may even be a better method, but when it comes to an end, the compound has to be isolated and identified with absolutely working methods (NMR, optical rotation).
No, we cannot use TLC for final confirmation, however it can be use as preliminary tool for identification. For final confirmation other technique such as mass, NMR, HPLC, GC can be use depending on the nature of compound and study.
Absolutely No, I agree with Kannan, 1D, 2D NMR and MS are the only best and final ways for the identification of pure compounds, after TLC from the crude extract.
It pays to be thorough in isolation process to avoid doubt about your work, follow this pattern,:TLC,HPLC,NMR,MS,IR(if necessary)
I second the views of almost all the respondents that TLC cannot be used as a tool for identification compounds in plant extract. However it can be indicative.
With advancement of MS interface deeveloped by CAMAG now it is quite possible to identify the plant extract composition by using TLC-MS/HPTLC-MS plates developed by MerckMillipore.Pl. refer the applications on the brochure.
Yes.Hyphenation of technique leads to techniques like TLC-MS/HPTLC-MS which can help for identification easily.
TLC gives only indications how ever with tlc scanner and appropriate digistore documentation can be easily detect and analyse specific retention zone, which can be extracted and additionaly analysed by ftir ms and nmr .
TLC gives only indication of compounds under a specific methodology. This may vary according to the solvent systems used. TLC is not the final step for identification
The limitation of TLC in general is in terms of sensitivity and separating power, but unlike many analytical methods can identify a wide range of analytes without any risk that components are lost during the chromatographic process.
No, TLC can't be used a final tool for identification or separation of compounds from crude extract. if you want to check the presence of desired compounds of your interset, then you have to load standards of paticular compounds which you want to seek in your crude extract. By that way you can get an idea of prsence and absence of your desired compounds in crude extract and then simply go for separation of compounds by applying various chromatographic techniques like VLC, Flash chromatography and finally go for NMR andGC-MS spectroscopic analysis.
TLC analysis followed by isolation of compound (may be by scraping out or preparative HPTLC), its characterization using techniques like FTIR, HPLC-MS, NMR, XRD will suffice the appropriate confirmation of the compound. Comparing Rf of compound present in the crude extract with known standard, its spectral confirmation, colour of the band usually provides a putative confirmation. Hence hyphanated techniques like mass tlc-ms/lc-ms/gc-ms are generally preferred.
The statement of Mr Bhavesh is also in agreement with the fact that TLC alone cann't be used as final tool for identification of phytochemical constituent in a plant extract
Dear Dr. Ashok K Shakya, I am not agreed with your answer. Every bioactive constituenets have been identified by the help of 1D and 2D NMR, GC-MS spctroscopic analysis. You would'nt be assure for the characterization of compounds either from plant/bcterial or from Fungal crude extract untill you go for chromatographic techniques (VLC,TLC,CC) for getting pure single compound from your crude extract.
No, for conclusive identification of a unknown compound by TLC as John S Wishnok's simple answer.
It still works if a known compound contained in the sample tested by adding the related chemical reference substance (standard) for comparison thier Rf value, the derivated color by sparaying special reagent or its original visual color, or its fluorescence image in parallel, etc.
@Seema Dharni, FC, TLC CC and other chromatographic techniques are required for the separation of the compounds not for the identification of the unknown substances. I think the question is related to identification rather than purification. We are having better techniques for identification Like LC-NMR, LC-MS/MSn, High resolution mass, rather than identification using TLC.
TLC finds importance in preparative analysis for separation of components given a mixture but not for the final analysis and identification of the component substance. Crude fractions obtained from TLC bands are to be analyzed further either by GC or LC-MS with the use of high purity standards and comparison with the ones reported in the libraries. TLC can not, definitely, serve as final tool to identify component substance of a given mixture.
TLc is a very useful tool , but with hptlc you can identify , quantify and confirm analites by its UV spectra in case that you only use a TLc you must confirm your results with another technique such as GCMS
No, TLC cannot be used to identify the compound in the crude extract. It can only be used to indicate the number of possible compounds that may likely be present in the crude extract.
It is clear that TLC (HPTLC) needs confirmation by more informative methods like NMR or MS, but for known substances preliminary conclusions could be made using combination of TLC with in-situ densitometry. You can get either UV-Vis spectra of the spot or use absorption ratios at different wavelengths. As the method is non-destructive (in most cases) you can later combine it with other methods for final confirmation.
Only TLC can not be used for Identification because it is not highly specific like IR. TLC along with other test like UV can be used for identification.
TLC alone cannot be used as a final tool for identification. It is simple a prior analytical technique. It only gives a rough idea that there are compounds present in the sample. GC-MS is one of the best methods for identification provided your compounds are volatile.
TLC can be used as a final tool for identification if and only if you have a pure compound which you want to confirm with its standard. Then running TLCs of the two in different solvent systems and comparing theirs Rfs could confirm whether the isolate is similar to the standard nothing more nothing less!!
TLC is a simple technique that can be used for example to distinguish the compounds of different polarity or preliminary identification of individuals after the classical column chromatography. Unfortunately, many compounds have similar polarity, so only in the case of separation of a mixture of known composition, using the standards of pure substances we can talk about some identification (but certainly not the final identification). Most next step is to confirm the structure with a set of a NMR and IR analysis and the final proof - the elemental analysis or MS analysis (when elemental analysis is not possible).
Hallo!
I do not agree with Mr. Wishnock. In the fifties and sixties thousands of substances have been identified by TLC, often using several different solvents. These methods have also been used by forensic medical institutes. I would rather soggest "you can't identify a substance as fast as with GC-MS or TLC-MS".
In addition, using MS, you only get a fit (suggetsion of identity) and under normal conditions, I never have obtained a 100% fit.
Sighart Walter Golf
Put the question in another form:Can TLC be used to identify a named compound in a mixture of related compounds? The answer may not be emphatically yes. There may be synergy among the compounds of the mixture, which may not allow the specified compound display its chemical properties. If a known standard is used to monitor the components of the mixture, the Rf value of the named compound may be higher or lower than the Rf value of the standard even if both sample are the same compound, because of the synergy.
The best method is to use TLC as analytical tool and GC
Hi, Walter -
You make an interesting point, but remember - in that era TLC was important, but still not sufficient. In order to report (publish) an identification, a compound had to be purified to a constant melting or boiling point, and enough isolated for combustion analysis to verify the empirical formula.
I agree with Dr.Wishnok fully. Prior to the TLC era, a compound was considered pure, when one would repeatedly recrystallize a compound for a constant melting point. They also used degradative techniques to get parts of molecules to reconstruct. a structure. Introduction of TLC changed this approach and a number of structures were then revisited and revised based on the use of silica both for prep column separations and TLC. With the introduction of HPLC, purity checks of number of elucidated compounds with TLCs were again revisited. With the most modern separation techniques utilising much smaller particle sizes and micro bores, chiral separations etc, it is now possible to resolve even very closely moving chiral substances. With the advancement of knowledge in separation technologies, we now know resolution limitations of TLC, which can at best be an indicative technique and definitely not a final tool. Again, it is a good complementary technique and personally I would definitely not use it as a confirmatory technque to establish purity.
TLC can not be a final tool in the identification of a compound from a given mixture sample. TLC is one preparative chromatography mainly useful in the separation of the different non-volatie components of a given mixture subject for further purification. High performance TLC results in better resolution and allows quantification where identification of the compound may be made possible by running standards along with sample at the same time in the same plate and then Rf values were compared. Two substances with the same Rf values are likely, but not necessarily, one and the same compound but if they have different Rf values, they are definitely different compounds. Liquid chromatography combined with mass spectrometry (LC-MS) can serve as a better final tool in the identification of the compound separated by TLC.
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