In my opinion, we can try following procedure.

First step is literature survey to find out the RF of the target compound.

because RF for a particular compound remains constant in particular Mobile Phase and Stationary Phase System.

Second Step is to collect compound at this RF value by HPTCL and check its Mass.

Compare this mass spectrum with literature, if mass spectrum coincides then we can use it as reference (we can further confirm it by UV and FTIR spectrum or H-NMR (Optional)).

Mass fragmentation is also particular for a specific compound at standard condition (70 eV).

Third step we can use this purified compound as standard for qualitative and quantitative analysis.

I agree with Syed Ali: compare with RF found in the literature.

Additionnaly, you can spray different reagents to get some structural information.

HPTLC is also very reliable for gradient development prior to preparative chromatography (e.g. flash chromatography).

I think any TLC has a number of uses even without the standard. Of course you can use the Rf for a compound if it is known. Other uses include 1) to determine the purity or degree of complexity in the extract (knowing what you are in for in isolation), 2) you can use a number of spray reagents to determine structural type - eg alkaloids often stain well with Dragendorff, and 3) to get some quantitative value of relative concentrations (but this can be misleading as not all spots respond linearly to concentration). I have also taken UV scans of TLC spots to assist in identification, but again this is best if you have the standard.

Dear Alok,

HPTL (and TLC) do not necessarily need of external standards consisting on characteristic compound present in the plant analised (or markers).

If you pay attention to the Pharmacopoea, many qualitative analysis check the identity of the plant drug/ extract using two chemicals not present in the plant or even synthetic. The trick consits in refering your spots to the Rf of these "references" (relative Rfs). Also many pharmacopoeial methods use these references to establih "zones" (i.e. below the lower Rf, in between the two Rfs or above the highest Rf) an explain which kind of bands you should see in each of them.

I am attaching the EU Monograph for Ginkgo for you to see how pharmacopoeial analysis of plant material is conducted in this way.

Best, Jose

If you know the rf value of the compound of interest , you can use preparative plates and load sufficient quantity of extract/semi purified extract and then scrape the bands at the known rf. this can be purified by a short column/crystallization and then run alongside the crude extract. You will get a reasonable qualitative analysis and if you are lucky enough to get pure crystals you can even do quantitative analysis

Dear Jose,

for quantitative analysis you must required an external standard because quantitative analysis is based on comparison. usually in HPTLC, the concentration is check by densitomter, hence you need a standard with known concentration which run side by side with sample and by the difference in densities you get the actual concentration in sample.

for only qualitative analysis you can use RF alone.

Carry out prep TLC or HPLC to isolate your desired compd in small quantity- use it as standard.

In chinese pharmacopeia, a standard medicinal material will be used control model.

For qualitative purposes: You can establish fingerprint of the extract by running it in HPTLC even if you don't have a standard.

For quantitative purpose: As Bapuji sir has told here, isolate a small quantity of the compound of interest, see its purity in HPLC(compare the area obtained for your main compound with the total area obtained in the chromatogram which gives you the purity the of the isolated standard) and compare with the area of the extract which is corresponding with the Rf of the standard.

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Dear Alok, I agree with Jose Prieto

Greetings Dr. Alok,

I Agree with the suggestions given by Mr. Syed Ali :-) Good luck yeah

It depends on yoour problem. Sometimes you do not need such standard (for example, when you need to approximate the amount of a small impurity in a sample, by comparison with the main spot - dilution included).-

@ Dr Kaufmann. What if we want to characterize the extract utilizing this technique. I think without a standard marker it is not possible. Yup for approximation of an unknown impurity we can compare it with the extract. Ok. But for that purpose also we need to know which is the impure spot. How can we say that a particular spot in an extract is an impurity. It may be an active moiety. Might be possible that I can't understand your example. Apologies .......

Best Regards

For any instrumental analysis (qualitative or quantitative) you need a known standard chemical marker.(no exception with HPTLC)...If unknown marker is there (as a purity or impurity moiety ,just same Rf value should not be taken for chemical identity as elution of HPTLC plates depend on various external conditions and mislead ) First it needed to be isolated and its chemical structure be identified (by MS,NMR,IR,UV chemical tests) and use for HPTLC studies of analysis of herbal mixture.

I agree with Dr. Chakravarthy. you need more and more analysis to talk about the certainity of the compound you desired (see sterio structure of the compound). Structure elucidation by means of NMR is a best technique. you should have standard substance. if not, fraction isolation you should do. Semi-prep HPLC you can apply. then follow step by step other instruments as above...

I have drawn the following conclusion from all the suggestions and answers received from all researchers:

We need a standard , may it be a standard marker or may be an isolated compound (from the parent extract) identified with the analytical techniques, for HPTLC. Without markers or standards, this equipment is not useful.

Thanks a lot all of you for your suggestions.

Regards

however, taking into account the improved reproducibility of HPTLC compared to TLC, Rf are much more accurate.

Therefore analysing a large number of standards in the exact same conditions can help building your databases of RF for each compounds you have.

Then you can estimate by RF comparison between your database and the HPTLC profile of your extract some of the compounds.

However, you will need comparison with standards at the end for confirmation.

This strategy can improve the throughput of TLC analysis of herbal extract. You will need at the end to do HPLC, GC of some more accurate analysis. But as a primary approach it should work.

I agree with the answers given. RF value under the same conditions is one of the markers for identifying the compound. In addition to this if you could perform a MS you can authenticate the presence of the compound. HPTLC could be more fused or metabolic fingerprinting and for researches aiming at systemic level information, rather than individual components in the sample.

It can be used. Please use software and compare your peak heights. To certain extant it will help to identify the compound, but not final.

Yes.There is a challenge of getting standard markers.Now one can connect HPTLCMS plates(Merck) to MS interface(CAMAG) and characterise the compound.

Yes it may effective for qualitative purpose or if you want only fingerprint in particular solvent system. Recently in my laboratory one method was developed for quantitative estimation of crude drug present in the formulation by targeting two peaks of extract of crude drug and a calibration plot was obtained vs peak area of different concentrations. The same extract of formulation was prepared and observed the respective peaks area and calculated the amount of crude drug present in the formulation.

Definitely marker / standards are required for any performing HPTLC. However, we can develop the chromatograms with solvent systems optimized for different chemical groups such as coumarins, flavonoids etc. (Ref. Wagner and Bladt, chromatographic drug analysis, Springer-Verlag Berlin Heidelberg New York). These chromatograms (fingerprints) can be used as records for comparing the subsequent extracts. Matching the chromatograms with recorded / published fingerprint is currently done manually. This is a drawback of the method indeed.

One more suggestion: if it is mixture of atleast a few known compounds, note the UV spectrum of these known compounds from the lit; based on Rf values select the spots and run on the plate itself Uv at different wavlengths, If it matches with lit, one can reasonably assume that as the authentic specimen and proceed.