The idea of developing a substance database for natural products arose from a previous collaboration on HPTLC of flavonoids and phenolic acids, between CAMAG and the ZHAW(Zurich University of Applied Sciences, Wädenswil). The project investigated the chromatographic behavior of about 72 substances. In a pilot phase in collaboration with the company Extrasynthese we worked on two smaller projects on iridoids and coumarins to practically implement the database concept.
The HPTLC Association Substance Database is a systematic collection of data, easily accessible electronically from the website of the HPTLC Association . It contains retention and spectral data of representative substances from different classes, analyzed with different developing solvents and derivatization reagents.
With the substance database, HPTLC laboratories have a free and convenient tool to help with identification of zones in a chromatogram. The user can compare RF values, and colors prior to and after derivatization, and UV spectra of unknown zones with those of the references. As the collection will be regularly expanded with substances not limited to constituents of herbal drugs, different fields of application can benefit from the HPTLC Association substance database. Iridoids and coumarins are described in this article.
Iridoids and coumarins were dissolved in methanol at a concentration of 1.0 mg/mL, and concentrations were adjusted if needed.
HPTLC plates silica gel 60 F254 (Merck), 20 x 10 cm are used.
2.0 μL of solutions were applied as bands with the Automatic TLC Sampler (ATS 4), 15 tracks, band length 8.0 mm, distance from left edge 20.0 mm, distance from lower edge 8.0 mm.
Plates were developed to 70 mm (from the lower edge) in the ADC 2 with chamber saturation (20 min, with filter paper) and after activation at 33% relative humidity for 10 min using a saturated aqueous solution of magnesium chloride. Different mobile phases were investigated (see table), followed by drying for 5 min.
Iridoids were derivatized with anisaldehyde sulfuric acid (AS) and vanillin sulfuric acid (VS) reagents. Coumarins were derivatized with AS, potassium hydroxide (KOH), and natural product (NP) / polyethylene glycol (PEG) reagents. AS reagent: slowly and carefully mix 170.0 mL of ice-cooled methanol with 20.0 mL of acetic acid and 10.0 mL of sulfuric acid; allow the mixture to cool to room temperature, then add 1.0 mL of anisaldehyde (p-methoxy benzaldehyde); VS: dissolve 1 g of vanillin in 100 mL of ethanol 96% and carefully add 2 mL of concentrated sulfuric acid; KOH: dissolve 5 g potassium hydroxide in 100 mL of methanol (96%); NP: dissolve 1 g of 2-aminoethyl diphenylborinate in 200 mL of ethyl acetate; PEG: mix 10 g of polyethylene glycol 400 (macrogol) with 200 mL of dichloromethane.
Images of the plates were captured with the TLC Visualizer 2 in UV 254 nm, UV 366 nm and white light, and in UV 366 nm and white light after derivatization.
Absorbance measurements at 254 nm with the deuterium lamp for iridoids, and at 313 nm with the mercury lamp for coumarins were performed with TLC Scanner 4 and visionCATS, slit dimension 5.00 mm x 0.20 mm, scanning speed 20 mm/s. Spectra of the corresponding zones were recorded in the absorbance-reflectance mode from λ=190–450 nm using a combination of deuterium and tungsten lamps with a slit size of 5.00 mm x 0.20 mm.
Results and discussion
Analysts who want to identify unknown zones in the HPTLC chromatogram are limited to the availability of reference standards in their laboratory. To help with selecting appropriate standards, the substance database can be a useful screening tool. For each class of compounds, six steps were taken: evaluation of the existing HPTLC methods, selection of three developing solvents of diverse properties, evaluation of the derivatization reagents, analysis of all substances of each class, and creation of the documentation.
The HPTLC Association provides method documents (PDF format) for each class and all evaluated developing solvents for a convenient check of available substances.
The documentation of the database also includes PDF files for each individual substance including peak profiles from image, UV spectra and images of chromatograms prior to and after derivatization.
Users of visionCATS can download all the data as comparison files and import their own data for comparison.
The following instruments and devices were used in this work (discontinued products are replaced with current versions)