Respected Sir,
HPTLC (High Performance Thin Layer Chromatography) is one of the techniques for the qualitative, semi-qualitative and quantitative phytochemical analyses of herbal drugs and formulations. This includes developing TLC finger print profiles and estimation of chemical markers and biomarkers. The major advantage of HPTLC is that several samples can be analyzed simultaneously using a small quantity of mobile phase (Mohana Rao et al., 2005)
Mohana Rao, G.M., Rao, C.V., Pushpangadan, P. and Shirwaikar, A. 2005. Hepatoprotective effects of rubiadin, a major constituent of Rubia cordifolia Linn. J. Ethnopharmacol. 30: 1 - 7.
Alkaloid profile
Samples code
A – Extract of H. leschenaultiana whole plant
NIC – Nicotiine standard as a reference compound
Procedure
Extraction and Test solution preparation
Five gram powdered sample of both the selected plant materials were extracted separately, with ethanol, in Soxhlet apparatus for 3 h. The content was cooled, filtered and concentrated using vacuum flash evaporator. The content was dissolved with 1ml ethanol and centrifuged at 3000 rpm for 5 minutes. These solutions were used as test solution for HPTLC analysis.
Sample application
2 µl of test solutions (two samples) and 4 µl of standard solution were loaded as 6 mm band length in the 4 x 10 Silica gel 60F254 TLC plate using Hamilton syringe and CAMAG LINOMAT 5 instrument.
Spot development
The samples loaded plate was kept in TLC twin trough developing chamber, after saturated with solvent vapour, with respective mobile phase (alkaloids) and the plate was developed in the respective mobile phase up to 90 mm.
Photo-documentation
The developed plate was dried by hot air to evaporate solvents from the plates. The plate was kept in Photo-documentation chamber (CAMAG REPROSTAR 3) and captured the images at white light, UV 254 nm and UV 366 nm.
Derivatization
The developed plate was sprayed with respective spray reagent (alkaloids) and dried at 1200 C in hot air oven. The plate was photo-documented in visible light mode using Photo-documentation (CAMAG REPROSTAR 3) chamber.
Scanning
Before derivatization, the plate was fixed in the scanner stage and scanning was done at 254 nm. The peak table, peak display and peak densitogram were noted.
Analysis details
Mobile phase
Chloroform-methanol (9.9:0.1)
Spray reagent
Dragendorff’s reagent was sprayed and dried at 100C for 10 minutes.
Detection
Orange brown coloured zones at visible light mode were present in the track, it was observed from the chromatogram after derivatization, which confirmed the presence of alkaloids in the given sample.
Flavonoid profile
Samples code
A – Extract of H. leschenaultiana whole plant
RUT – Rutin standard as a reference compound
Procedure
Extraction and Test solution preparation
Five gram powdered sample of both the plant materials were extracted separately, with ethanol, in Soxhlet apparatus for 3 h. The content was cooled, filtered and concentrated using vacuum flash evaporator. The content was dissolved with 1 ml ethanol and centrifuged at 3000 rpm for 5 minutes. These solutions were used as test solution for HPTLC analysis.
Sample application
2µl of test solutions (two samples) and 5µl of standard solution were loaded as 6 mm band length in the 4 10 Silica gel 60F254 TLC plate using Hamilton syringe and CAMAG LINOMAT 5 instrument.
Spot development
The samples loaded plate was kept in TLC twin trough developing chamber, after saturated with solvent vapour, with respective mobile phase (flavonoids) and the plate was developed in the respective mobile phase up to 90 mm.
Photo-documentation
The developed plate was dried by hot air to evaporate solvents from the plate. The plate was kept in Photo-documentation chamber (CAMAG REPROSTAR 3) and captured the images at white light, UV 254 nm and UV 366 nm.
Derivatization
The developed plate was sprayed with respective spray reagent (flavonoids) and dried at 120 C in hot air oven. The plate was photo-documented in UV 366 nm mode using Photo-documentation (CAMAG REPROSTAR 3) chamber.
Scanning
Before derivatization, the plate was fixed in the scanner stage and scanning was done at UV 254 nm. The peak table, peak display and peak densitogram were noted.
Analysis details
Mobile phase
Ethyl acetate-butanone-formic acid-water (5:3:1:1)
Spray reagent
1% ethanolic aluminum chloride reagent and dried at 100°C for 3 minutes.
Detection
Blue coloured and yellow coloured fluorescent zones at UV 366 nm mode were present in the track, it was observed from the chromatogram after derivatization, which confirmed the presence of flavonoids in the given sample.
Glycoside profile
Samples code
A – Extract of H. leschenaultiana whole plant
STE – Stevioside standard as a reference compound
Procedure
Extraction and Test solution preparation
Five gram powdered sample of both the plant materials were extracted separately, with ethanol, in Soxhlet apparatus for 3 h. The content was cooled, filtered and concentrated using vacuum flash evaporator. The content was dissolved with 1ml ethanol and centrifuged at 3000 rpm for 5 minutes. These solutions were used as test solution for HPTLC analysis.
Sample application
2µl of test solutions (two samples) and 5µl of standard solution were loaded at 6 mm band length in the 4 x 10 Silica gel 60F254 TLC plate using Hamilton syringe and CAMAG LINOMAT 5 instrument.
Spot development
The samples loaded plate was kept in TLC twin trough developing chamber, after saturated with solvent vapour, with respective mobile phase (glycosides) and the plate was developed in the respective mobile phase up to 90 mm.
Photo-documentation
The developed plate was dried by hot air to evaporate solvents from the plate. The plate was kept in Photo-documentation chamber (CAMAG REPROSTAR 3) and captured the images at white light, UV 254 nm and UV 366 nm.
Derivatization
The developed plate was sprayed with respective spray reagent (glycosides) and dried at 120ºC in hot air oven. The plate was photo-documented in visible light mode using Photo-documentation (CAMAG REPROSTAR 3) chamber.
Scanning
After derivatization, the plate was fixed in the scanner stage and scanning was done at 366 nm. The peak table, peak display and peak densitogram were noted.
Analysis details
Mobile phase
Ethyl acetate-methanol-ethanol-water (8.1:1.1:0.4:0.8)
Spray reagent
Antimony III chloride reagent was sprayed and dried at 100ºC for 10 minutes.
Detection
Blue, green and red coloured zones at UV 366 nm mode were present in the track, it was observed from the chromatogram after derivatization, which confirmed the presence of glycosides in the given sample.
Saponin profile
Samples code
A – Extract of H. leschenaultiana whole plant
SAP – Saponin standard as a reference compound
Procedure
Extraction and Test solution preparation
Five gram powdered sample of both the plant materials were extracted separately, with ethanol, in Soxhlet apparatus for 3 h. The content was cooled, filtered and concentrated using vacuum flash evaporator. The content was dissolved with 1 ml ethanol and centrifuged at 3000 rpm for 5 minutes. These solutions were used as test solution for HPTLC analysis.
Sample application
2 µl of test solutions (two samples) and 5µl of standard solution were loaded at 6 mm band length in the 4 10 silica gel 60F254 TLC plate using Hamilton syringe and CAMAG LINOMAT 5 instrument.
Spot development
The samples loaded plate was kept in TLC twin trough developing chamber, after saturated with solvent vapour, with respective mobile phase (saponins) and the plate was developed in the respective mobile phase up to 90 mm.
Photo-documentation
The developed plate was dried by hot air to evaporate solvents from the plate. The plate was kept in Photo-documentation chamber (CAMAG REPROSTAR 3) and captured the images at white light, UV 254 nm and UV 366 nm.
Derivatization
The developed plate was sprayed with respective spray reagent (saponins) and dried at 120ºC in hot air oven. The plate was photo-documented in visible light mode using Photo-documentation (CAMAG REPROSTAR 3) chamber.
Scanning
After derivatization, the plate was fixed in the scanner stage and scanning was done at 500 nm. The peak table, peak display and peak densitogram were noted.
Analysis details
Mobile phase
Chloroform-methanol-water (9:1:0.1)
Spray reagent
Anisaldehyde sulphuric acid reagent was sprayed and dried at 100ºC for 10 minutes.
Detection
Blue coloured zones at visible light mode were present in the track, it was observed from the chromatogram after derivatization, which confirmed the presence of saponins in the given sample.
Steroid profile
Samples code
A – Extract of H. leschenaultiana whole plant
SGL – Stigmasterol standard as a reference compound
Procedure
Extraction and Test solution preparation
Five gram powdered sample of both the plant materials were extracted separately, with ethanol, in Soxhlet apparatus for 3 h. The content was cooled, filtered and concentrated using vacuum flash evaporator. The content was dissolved with 1ml ethanol and centrifuged at 3000 rpm for 5 minutes. These solutions were used as test solution for HPTLC analysis.
Sample application
2µl of test solutions (two samples) and 4µl of standard solution were loaded at 6 mm band length in the 4 10 Silica gel 60F254 TLC plate using Hamilton syringe and CAMAG LINOMAT 5 instrument.
Spot development
The samples loaded plate was kept in TLC twin trough developing chamber, after saturated with solvent vapour, with respective mobile phase (steroids) and the plate was developed in the respective mobile phase up to 90 mm.
Photo-documentation
The developed plate was dried by hot air to evaporate solvents from the plates. The plate was kept in Photo-documentation chamber (CAMAG REPROSTAR 3) and captured the images at white light, UV 254 nm and UV 366 nm.
Derivatization
The developed plate was sprayed with respective spray reagent (steroids) and dried at 120ºC in hot air oven. The plate was photo-documented in visible light mode using Photo-documentation (CAMAG REPROSTAR 3) chamber.
Scanning
Finally, the plate was fixed in the scanner stage and scanning was done at 500 nm. The peak table, peak display and peak densitogram were noted.
Analysis details
Mobile phase
Ethyl acetate-methanol-acetic acid-water (10:2.2:1.1:2.6)
Spray reagent
Libermann-Burchard reagent was sprayed and dried at 100ºC for 10 minutes.
Detection
Blue coloured zones at visible light mode were present in the track, it was observed from the chromatogram after derivatization, which confirmed the presence of steroids in the given sample.
Terpenoid profile
Samples code
A – Extract of H. leschenaultiana whole plant
SOL – Solanesol standard as a reference compound
Procedure
Extraction & Test solution preparation
The given dried plant materials (5gm) were extracted with methanol in soxhlet apparatus for 3hrs. Cooled, filtered the content and concentrated using vacuum flash evaporator. Dissolved the content with 1 ml methanol and centrifuged at 3000rpm for 5min. These solutions were used as test solution for HPTLC analysis.
Sample application
2 µl of test solutions (two samples) and 5 µl of standard solution were loaded as 6 mm band length in the 4 x 10 silica gel 60F254 TLC plate using Hamilton syringe and CAMAG LINOMAT 5 instrument.
Spot development
The samples loaded plate was kept in TLC twin trough developing chamber (after saturated with solvent vapour) with respective mobile phase (terpenoids) and the plate was developed in the respective mobile phase up to 90mm.
Photo-documentation
The developed plate was dried by hot air to evaporate solvents from the plate. The plate was kept in Photo-documentation chamber (CAMAG REPROSTAR 3) and captured the images at White light, UV 254nm and UV366nm.
Derivatization
The developed plate was sprayed with respective spray reagent (terpenoids) and dried at 120 C in Hot air oven. The plate was photo-documented in UV 366nm mode using Photo-documentation (CAMAG REPROSTAR 3) chamber.
Scanning
Finally, the plate was fixed in scanner stage and scanning was done at UV 366nm. The Peak table, Peak display and Peak densitogram were noted.
Analysis details
Mobile phase
n-hexane-ethyl acetate (1:1)
Spray reagent
Sprayed Libermann-Burchard reagent and dried at 100 C 10min.
Detection
Blue coloured fluorescent zones at UV 366nm mode were present in the track, it was observed from the chromatogram after derivatization, which confirmed the presence of terpenoids in the given sample.
Both HPLC and HPTLC is a standardization technique used for plant drug analysis. The fingerprinting means the standard of drug must be carbon copy of reference standard already reported in official compendium. Which fix the phytochemical content in order to standardize the extract of particular plants. In case of Saffron, try to find out the reported method in HPLC and perform accordingly to match your results with previous ones.