What is HPTLC Fingerprinting ?

High-Performance Thin Layer Chromatography (HPTLC) is a modern version of TLC wherein the principle is the same but practice is different. HPTLC is GLP/USP/EP compliant technique.

USP’s (United States Pharmacopoeia) definition of HPTLC fingerprint is ‘It is the (electronic) image of the visual HPTLC chromatogram. Images are taken at 254nm, 366nm and in white light using suitable TLC photo docsystemstion system like CAMAG visualizer. Fingerprint is evaluated based on Rf, color and intensity of the separated bands.

While developing fingerprint our aim should be to separate maximum number of compound with good resolution.

For fingerprint development, BRM (Botanical Reference Material) and other related or confounding species are used for comparison. The same species of interest from different geographical or climatic conditions are also used to check variation in the samples.

C. rotundus is a traditional herbal medicine i.e. used for the treatment of pain, fever, diarrhea, dysentery, an emmenagogue and other intestinal problems. Mainly it is used to treat stomach ailments, wounds, boils and blisters etc.

HPTLC Method for Cyperus rotundus Rhizome

HPTLC association has developed a method for Cyperus rotundus rhizome. This method also discriminates C. rotundus from other related species. This method is adopted in the International HPTLC Atlas for the identification of herbal drugs and in the British Pharmacopoeia, 2018. Standards isoeugenol and isoeugenyl acetate were used.

USP method of ultrasonic extraction was used for standard and sample preparation with methanol as a diluent.

The plate layout was kept as per the parameters defined in USP chapter 1064. Mobile phase, toluene: ethyl acetate in the ratio 9:1 v/v was used for development.

The development was carried out with controlled humidity of 33%. The plate can be automatically developed under controlled environmental conditions using CAMAG ADC2 (Automatic development chamber). 

After development, the plate was derivatized with anisaldehyde sulfuric acid reagent. After derivatization, standards were identified at Rf 0.43 and 0.50 respectively and α-cyperone was identified after derivatization with anisaldehyde reagent at Rf 0.42.

Other related species like Cyperus articulates, C. corymbosus, C. scariosus, C. alternifolius, Aconitum heterophyllum were used for comparison.

This method has applications in herbal drug manufacturing units, AYUSH labs, Ayurvedic quality control labs and research institutes doing herbal analysis.

For more method details please refer following Link:

https://www.hptlc-association.org/methods/methods_for_identification_of_herbals.cfm

Web reference:

https://www.ijsrp.org/research-paper-0513/ijsrp-p17124.p

Herbal drug testing instrument such as HPTLC is a  powerful tool for identifying and analyzing herbal drugs based on their Rf value, color, and intensity of the separated bands. This technique is especially helpful in distinguishing between related species.

Anchrom Enterprises Pvt. Ltd is one of the leaders in HPTLC analysis and analytical instruments suppliers. Please contact us at lab@anchrom.in for HPTLC analysis of plant extracts, drugs, ingredients in cosmetics, and forensic science.