Authors:Eva Kranjc, Alen Albreht, Irena Vovk, Vesna Glavnik, and Damjan Makuc
High-performance thin-layer chromatography (HPTLC) is a powerful separation technique which is often overlooked. In this study, we comprehensively assessed the applicability, ease, and performance of HPTLC in combination with densitometry and mass spectrometry (MS) to characterize physalins — relatively polar secondary metabolites from Physalis alkekengi L. HPTLC silica gel plates were evaluated in combination with 14 developing solvents (13 published in the literature). Bonded stationary phases (HPTLC RP-18, RP-18 W, CN F254S) were also tested. Four detection reagents (sulfuric acid, anisaldehyde, 4-dimethylaminocinnamaldehyde (DMACA), and molybdatophosphoric acid) were compared to ascertain which one is the most suitable. For all chromatographic analyses, a commercial standard physalin L and a P. alkekengi L. crude extract were used. In some cases, physalin L standard appeared as two clearly resolved bands on silica plates, but only after derivatization, where sulfuric acid reagent provided the best selectivity and sensitivity. Physalin L standard impurity was found to belong to the physalin family as confirmed by HPTLC–MS/(MS) and nuclear magnetic resonance (NMR) spectroscopy. Compared to high-performance liquid chromatography (HPLC), our HPTLC method showed extremely high sensitivity for standard impurity (ca. 4% determined by NMR) as it was clearly visible on the plate during image analysis after derivatization. Unlike (ultra)-high-performance liquid chromatography ((U)HPLC), HPTLC was also able to separate physalin L standard from its impurity. We show that (HP)TLC is a suitable chromatographic technique for the analysis of physalins and can even surpass the performance of (U)HPLC, namely, due to a wide array of different developing solvents available.
Authors:Urška Jug, Vesna Glavnik, Eva Kranjc, and Irena Vovk
High-performance thin-layer chromatography (HPTLC) and HPTLC–mass spectrometry (MS)/(MSn) methods for analyses of phenolic acids (chlorogenic acid, rosmarinic acid, protocatechuic acid, gallic acid, syringic acid, ellagic acid, trans-cinnamic acid, o-coumaric acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid) were developed. Separation was performed on HPTLC silica gel plates with and without fluorescent indicator (F254) in a saturated twin-trough chamber using n-hexane–ethyl acetate–formic acid (12:8:2, v/v) as the developing solvent. The developed HPTLC method is also suitable for the preliminary screening of some flavonoids (flavone, apigenin, luteolin, chrysin, quercetin, myricetin, kaempferide, kaempferol, hesperetin, naringenin, pinocembrin), although some interferences of phenolic acids with flavonoids were observed. The effect of pre-development on the HPTLC analysis of phenolic acids on the detection by densitometry and mass spectrometry was also explored. Pre-development of the plates with chloroform–methanol (1:1, v/v) decreased the intensity of secondary front like dark band that appeared at RF 0.7 on unpre-developed plates and enabled densitometric evaluation of phenolic acids at 280 and 330 nm. To eliminate severe spectral background observed during HPTLC–MS analysis, caused by the presence of an acidic modifier in optimized developing solvent, two pre-developments of the plates (1st methanol–formic acid 10:1, v/v and 2nd methanol) were applied. This resulted in a substantial decrease in the intensity of the background signals of sodium formate clusters and considerably improved the analysis of phenolic compounds. The applicability of the developed HPTLC and HPTLC–MS/(MSn) methods was confirmed by analyses of different complex matrix samples, e.g., propolis, roasted coffee, and rose hip crude extracts.
Authors:Irena Vovk, Nadja Gerčar, Breda Simonovska, and Mihael Sok
Cholesterol is an essential component of mammalian cells, but its role in cancer is unclear. We have determined the total cholesterol content in the healthy and the cancerous lung tissues of the same patient. Tissues of 13 patients (7 women and 6 men, different histological type, different stages of the disease) were obtained during surgical intervention. The samples (0.056–2.004 g) were hydrolyzed in alkaline medium, and the total cholesterol was extracted into n-hexane and simultaneously determined by thin-layer chromatography (TLC) on silica gel high-performance thin-layer chromatography (HPTLC) plates and nonaqueous reversed-phase high-performance liquid chromatography (HPLC), which both proved to be suitable for the determination of cholesterol in lung tissues and gave comparable results. This is the first report about the comparison of the total cholesterol content in the healthy and the cancerous tissue of the same patient. The difference in the results for the total cholesterol from both types of tissues was remarkable. For 13 patients, the mean contents and standard deviations determined by TLC were 4.01 ± 0.75 mg g−1 in the healthy lung tissue and 7.75 ± 1.93 mg g−1 in the cancerous lung tissue. Comparable results were obtained by HPLC analyses of the same samples: 3.91 ± 0.73 mg g−1 in the healthy and 6.95 ± 1.83 mg g−1 in the cancerous lung tissue. The range of total cholesterol content determined by TLC was between 3.20 mg g−1 and 5.83 mg g−1 in healthy lung tissues, and between 4.64 mg g−1 and 12.01 mg g−1 in lung cancer tissues, while the ranges obtained by HPLC were between 2.80 mg g−1 and 5.49 mg g−1 for healthy lung tissue, and between 4.38 mg g−1 and 11.24 mg g−1 for lung cancer tissue. The cancerous lung tissue of each of the thirteen patients contained higher amounts of total cholesterol compared to their healthy lung tissue. In 8 of the patients, the total cholesterol in the cancer tissue was more than 60% higher than in the healthy tissue; furthermore, in 5 patients, it was more than 100% higher.
Authors:Vesna Glavnik, Breda Simonovska, Alen Albreht, and Irena Vovk
A thin-layer chromatographic (TLC) method for fast screening of trans-resveratrol, pterostilbene, and p-coumaric acid in samples of recombinant bacterial cultures, food supplements, and wine was developed. The separation was performed on high-performance thin-layer chromatography (HPTLC) silica gel 60 plates using n-hexane-ethyl acetate-formic acid (20:19:1, v/v) as developing solvent in tank configuration of horizontal developing chamber, in which better resolution between trans-resveratrol and p-coumaric acid than in sandwich configuration of the same chamber or in automatic developing chamber (ADC) was obtained. Compounds were detected before and after post-chromatographic derivatization (three detection reagents) by image analyzing system (at 366 nm or white light) and by densitometer (absorption-reflectance and fluorescence mode). The lowest densitometric limits of detection (LOD) 2 ng for trans-resveratrol (303 nm), 5 ng for pterostilbene (303 nm), and 15 ng for p-coumaric acid (286 nm) were found before derivatization in absorption-reflectance mode. Post-chromatographic derivatization with anisaldehyde-sulfuric acid detection reagent resulted in higher LOD in the same mode: 13 ng for trans-resveratrol and pterostilbene at 500 nm and 30 ng for p-coumaric acid at 566 nm. Natural fluorescence of both stilbenes was less sensitive than UV absorption and less selective than post-chromatographic derivatization with anisaldehyde reagent at densitometric screening of trans-resveratrol in the samples. A complementary high-performance liquid chromatography (HPLC) method was developed for screening and quantification of the three compounds in recombinant bacterial cultures. Adequate separation of the analytes was performed in 35 min by a gradient elution from a stainless-steel column Hypersil ODS (150 × 4.6 mm I.D., particle size: 5 μm) with the mobile phase consisting of 50 mM sodium acetate buffer pH 5.6 (solvent A) and acetonitrile (solvent B) at the flow rate of 1.5 mLmin−1.
Authors:Katerina Naumoska, Breda Simonovska, Alen Albreht, and Irena Vovk
Separation of three triterpenic acids (ursolic, oleanolic and betulinic acid) was achieved on different thin-layer chromatography (TLC) (silica gel 60) and high-performance thin-layer chromatography (HPTLC) sorbents (silica gel 60, C2 RP and C18 RP) using several developing solvents, based on the non-polar diluent n-hexane, and ester (methyl acetate, ethyl acetate, ethyl propionate) as selector. Anisaldehyde and molybdophosphoric acid detection reagents were used. Finally, a simple method on a C18 RP HPTLC plate was developed using n-hexane-ethyl acetate (5:1 v/v) as a developing solvent in a horizontal developing chamber. The method was used for the screening of ursolic, oleanolic and betulinic acids in different vegetable extracts. Other plant triterpenoids (lupeol, α-amyrin, β-amyrin, cycloartenol, lupenone, friedelin, lupeol acetate, cycloartenol acetate) and phytosterols (β-sitosterol, stigmasterol) did not interfere. TLC-MS was used as a tool for the additional confirmation of the presence of ursolic, oleanolic, and betulinic acids in some of the studied vegetable extracts. Ursolic and oleanolic acids were found in radicchio Leonardo and white-colored radicchio di Castelfranco extracts for the first time, while betulinic acid was not detected in the eggplant extract by MS, although it was suggested at first by TLC analysis. Pre-chromatographic bromination on the HPTLC silica gel 60 plates and subsequent development in toluene-chloroform-diethyl ether-formic acid (20:16:4:0.1, v/v) provided a superior resolution of these compounds.
Authors:Mirko Prosek, Alenka Golc-Wondra, Irena Vovk, and Janko Zmitek
A wide spread of measurements is typical in quantitative TLC. Improved reproducibility and speed can be achieved by automatic and controlled sample application, chromatographic development, and data acquisition and processing. Secondary chromatography is the main reason for poor precision in TLC. With
up to 10% this is by far the largest source of uncertainty. During the drying process mobile phase evaporates from the upper surface of the plate, and molecules of separated components inside the layer move up or down. Our experimental results show the strong dependence of the intensity of reflected diffuse light on the position of the spots inside the layer. Experimental results gave us an idea how to construct a device for drying and derivatization of TLC plates and a device which reduces uncontrolled propagation and non-homogeneous vertical in-depth distribution of spots during drying and derivatization was constructed. In addition the device designed is safer to use than a hair dryer. A laminar flow of air or inert gas constantly removes solvent vapor from the upper layer of the adsorbent and accelerates drying. Temperature is controlled and varies in a predetermined manner at predetermined intervals. Temperature gradient, which cannot be avoided in flow systems is controlled and is oriented in the direction of chromatographic development. The construction of the device results in identical drying conditions for substances with the same
. Diffusion of the analyte is controlled and standardized and inhomogeneous in-depth distribution of compounds inside the adsorbent is minimized. Heating grade, heating intervals, pulses, switching, and other conditions are preset or programmable. The TLC dryer constructed reduces uncontrolled propagation and non-homogeneous vertical in-depth distribution of spots during drying and derivatization, which results in significantly improved reproducibility and precision. This is very important because in quantitative TLC most measurements are performed in reflectance mode.
Authors:Tero Wennberg, Irena Vovk, Pia Vuorela, Breda Simonovska, and Heikki Vuorela
The computer-assisted simulation program DryLab has been used to simulate TLC separations. The simulations were based on data from preliminary TLC separations. For DryLab data entry
values from TLC were converted to retention times, the development distance on the plate was used as column length, and the plate thickness was used as the column diameter. To achieve reasonably accurate simulations it was found necessary to run three preliminary runs in which differences between organic modifier concentration in two adjacent runs was more than 5%. The possibility of predicting HPLC separation on the basis of TLC separations was also studied. It was found that the method can be transferred from TLC to HPLC and that DryLab can be used to predict HPLC separation on the basis of the information obtained from TLC experiments. To produce a reasonably accurate HPLC simulation on the basis of TLC data, however, a relatively large number of preliminary experiments is required.
Authors:Irena Vovk, Breda Simonovska, Pia Vuorela, and Heikki Vuorela
The separation of (+)-catechin and (−)-epicatechin on cellulose TLC plates with 1-butanol-water-acetic acid, 4 + 2 + 1 (
), has been studied under different development conditions on prewashed and untreated TLC plates. Plates were developed in horizontal chambers (tank and sandwich configuration) and in twin trough (unsaturated and saturated) developing chambers. Tank configurations were used with and without preconditioning.Prewashing of the TLC plates, preconditioning, and the type of development chamber used had a large effect on the chromatographic results. Without prewashing of the TLC plate unsatisfactory or misleading results were obtained.Anisaldehyde-sulfuric acid dipping reagent proved to be more selective than vanillin-phosphoric acid as detection reagent for the detection of flavan-3-ols in oak bark extract; the latter was, however, more sensitive.
Authors:Irena Vovk, Breda Simonovska, Lidija Kompan, and Mirko Prošek
The ratio of lactulose/mannitol excretion in urine after their administration is of great importance for evaluation of malabsorption and intestinal permeability disruption in some diseases. An analytical method has been developed for determination of lactulose and mannitol in urine on the same amino HPTLC plate. The method enables densitometric quantification of lactulose by use of fluorescence mode, and mannitol by use of absorption mode after detection with AgNO