Planar Chromatography in Practice Separation of common plant triterpenoids by HPTLC

Standard solutions
Standard solutions and mixtures were prepared in n-propanol (0.1 mg/mL).

Sample preparation
Fresh tomato fruit and fresh leaves of cabbage, rosemary and sage were immersed into dichloromethane. Pulverized oak bark was extracted with the same solvent. After ltration and evaporation of the solvent, nal test solutions were prepared in n-propanol with concentrations of 5 mg/mL (cabbage and rosemary leaves), 30 mg/mL (sage leaves), 10 mg/mL (oak bark) and 2 mg/mL (tomato fruit) [2].

Left to right: Dr. Irena Vovk , Dr. Breda Simonovska and Mitja Martelanc
*

The Laboratory for Food Chemistry at the National Institute of Chemistry in Ljubljana (www.ki.si) is the leading research group in the eld of planar chromatography in Slovenia, providing consulting for industry and other institutions. Research and development activities are primarily focused on the eld of nutraceuticals and the development of analytical methods based on chromatographic techniques.

Chromatogram layer
HPTLC plates silica gel 60 (Merck), 20x10 cm, prewashed by developing in chloroform methanol 1:1 and HPTLC plates RP18 (Merck), 20x10 cm, prewashed by developing in acetone, followed by drying on the TLC plate heater at 110 C for 10 minutes.

Sample application
Bandwise with Linomat, band length 6 mm, distance from lower plate edge 5 mm, from left edge 12 mm, track distance 10 mm, application volumes as follows: 6 L (cabbage leaves), 7 L (rosemary leaves), 5 L (sage leaves), 4 L (oak bark), and 6 L (tomato), for standard solutions 2-8 L.

Introduction
Triterpenoids represent a large class of secondary metabolites commonly present in plants, especially in leaf and fruit epicuticular waxes. Some of them exhibit benecial activities, such as anti-inammatory ones. Therefore, they are of emerging importance as constituents of food supplements and functional foods. In general HPLC is used for determination of triterpenoids, but due to their lack of chromophores and numerous possible isomeric structures, the choice of detector and mobile phases has limitations. On the other hand, HPTLC is indispensible in the investigation of triterpenoids, because it provides unique separations with a wealth of potential mobile phases and with possibilities for in situ derivatization, which results in characteristic specic coloration and uorescence of the separated bands [1, 2]. In the investigation of triterpenoids in plant extracts, the proposed HPTLC methods enable easy, fast and inexpensive screening. Depending on the sample characteristics, quantication is possible.

Chromatography
In the Horizontal Developing Chamber with n-hexane ethyl acetate 5:1 on silica gel plate (developing time 15 min), whereby the conditioning tray was covered with the developing solvent, and with ethyl acetate acetonitrile 3:2 or acetone acetonitrile 5:1 on RP18 plate (developing time 17 min), running distance 8 cm

Post-chromatographic derivatization
Dried plates were dipped for 2 s in anisaldehyde sulfuric acid reagent (16 mL sulfuric acid and 1 mL p-methoxybenzaldehyde were added to 20 mL acetic acid and 170 mL methanol during cooling with ice water) using the Chromatogram Immersion Device (vertical speed 3.5 cm/s), dried in a stream of warm air and heated at 110 C with a TLC Plate Heater for

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2 min (silica gel plate) or 30 s (RP18 plate).

Note (editor):In case of quantication the plate has to be placed on the cold TLC Plate Heater, being heated to 110 C. By doing so, homogeneous heating across the whole plate is assured, and thus a good precision guaranteed.

acetonitrile 5:1 separated the mentioned isomeric esters, but cycloartenol was not fully separated from -amyrin.

Documentation
With DigiStore 2 under UV 366 nm and under white light illumination

Results and discussion

The separation of triterpenoids with different functional groups (alcohols, acids, ketones and esters) has been generally achieved on silica gel plates, although the use of RP18 plates was crucial for the separation of four isomeric triterpenols and two triterpenol esters.

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Characteristic uorescence of bands under UV 366 nm after derivatization with anisaldehyde sulfuric acid reagent; separation on RP18 plates with ethyl acetate acetonitrile 3:2 (left) and acetone acetonitrile 5:1 (right)

Moreover, both reversed phase methods enabled to some extent the separation of triterpenoids with different functional groups, yet with no interference from the structurally related sterols. For identication of compounds, the characteristic colors and uorescence of bands obtained after derivatization with anisaldehyde sulfuric acid reagent were helpful. Through this selective, postchromatographic derivatization for all samples in parallel, HPTLC provides easy, fast and inexpensive screening of triterpenoids.

Separation of triterpenoids on silica gel (A) and RP18 phases (B: ethyl acetate acetonitril 3:2, C: acetone acetonitrile 5:1). Tracks: 1 -amyrin; 2 - amyrin; 3 - amyrin; 4 lupeol; 5 lupeol acetate; 6 cycloartenol; 7 cycloartenol acetate; 8 lupenon; 9 ursolic acid; 10 oleanolic acid; 11 -amyrin, -amyrin and lupeol; 12 cabbage; 13 rosemary; 14 sage; 15 oak bark; 16 tomato

The separation of isomeric triterpenols (-amyrin, amyrin, lupeol and cycloartenol) was achieved using the solvent system ethyl acetate - acetonitril 3:2, but with this system the separation of isomeric esters failed. On the other hand, solvent system acetone

Further information is available on request from the author(s).

[1] M. Martelanc, I. Vovk, B. Simonovska, J. Chromatogr. A 1164 (2007) 145 and [2] dito 1216 (2009) 6662
* Dr. Irena Vovk, National Institute of Chemistry, Laboratory for Food Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia, irena.vovk@ki.si

CAMAG CH-4253 Muttenz (Switzerland) Tel. + 41 61 467 34 34 info@camag.com www.camag.com

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