Hello Devesh:

First of all, quantification by TLC even HPTLC is problematic.  The best research I have read uses an automatic spotter and a high quality densitometer.  Internal standards also help.  You can use a secondary standard but your response factors will have to be close.  You need to find a phytochemical with a structure similar to your extract; this would serve as a suitable internal standard.  You would prepare your samples by keeping the concentration of the internal standard the same, and comparing the concentration of the extract to the internal standard.  This will give you a calibration curve from which you can make measurements.  If your extract absorbs UV light, the densitometer should be able to scan the small spot uniformly.  If you have to use a derivatizing agent, things get more complicated.  Hope this helps or at least points you in a positive direction.  Good luck!

Hi Devesh

If you have a standard for comparison, then HPTLC is not a bad option. You can find the exact quantity using this technique. You can confirm the presence of standard in your sample by using the UV overlain spectral function of the instrument which is very accurate and confirms your compound. Later you can match the Rf with the standard and find out the AUC of that particular spot. From the amount of sample applied by the applicator which you set yourself, you can find how much of the compound is present in the sample. 

For other queries, you can follow the attached link which constitutes the input of many researchers on the same question asked by me before. 

Regards 

https://www.researchgate.net/post/What_is_the_utility_of_HPTLC_analysis_if_you_dont_have_a_standard_marker_for_comparison_with_the_mixture_extract1?_tpcectx=profile_questions

 Attached is the calibration and quantitation using an internal standard (ex. for GC, can be used for other analytical techniques, as principle is the same)

Hello Davesh,

according to what Alok has written, taking the UV spectra of the compound, which are you going to quantify, is very important step, since in HPTLC of plant extracts, one spot often contains more than one chemical compound (usually with the similar structure). Therefore you have to be sure that you quantify the band of the single metabolite. If you do not have the standard, but you predict/know that type of compound is present in the band, sometimes it is also possible to perform the hydrolysis and quantify the products of the reaction (e.g. sugars and aglycones after the hydrolysis of glycosides). However, this makes the procedure more complicated and is used basically if you are interested directly in aglycone quantification...

Hope this would help

Best regards