Normal-phase Thin-layer Chromatography Research Articles

Equilibrium Thermodynamics of Macropa Complexes with Selected Metal Isotopes of Radiopharmaceutical Interest.

To pursue the design of in vivo stable chelating systems for radiometals, a concise and straightforward method toolbox was developed combining NMR, isothermal titration calorimetry (ITC), and europium time-resolved laser-induced fluorescence spectroscopy (Eu-TRLFS). For this purpose, the macropa chelator was chosen, and Lu3+, La3+, Pb2+, Ra2+, and Ba2+ were chosen as radiopharmaceutically relevant metal ions. They differ in charge (2+ and 3+) and coordination properties (main group vs lanthanides). 1H NMR was used to determine four pKa values (±0.15; carboxylate functions, 2.40 and 3.13; amino functions, 6.80 and 7.73). Eu-TRLFS was used to validate the exclusive existence of the 1:1 Mn+/ligand complex in the chosen pH range at tracer level concentrations. ITC measurements were accomplished to determine the resulting stability constants of the desired complexes, with log K values ranging from 18.5 for the Pb-mcp complex to 7.3 for the Lu-mcp complex. Density-functional-theory-calculated structures nicely mirror the complexes' order of stabilities by bonding features. Radiolabeling with macropa using ligand concentrations from 10-3 to 10-6 M was accomplished by pointing out the complex formation and stability (212Pb > 133La > 131Ba ≈ 224Ra > 177Lu) by means of normal-phase thin-layer chromatography analyses.

Chromatographic Data in Statistical Analysis of BBB Permeability Indices.

Blood-brain barrier (BBB) permeability is an essential phenomena when considering the treatment of neurological disorders as well as in the case of central nervous system (CNS) adverse effects caused by peripherally acting drugs. The presented work contains statistical analyses and the correlation assessment of the analyzed group of active pharmaceutical ingredients (APIs) with their BBB-permeability data collected from the literature (such as computational log BB; Kp,uu,brain, and CNS+/- groups). A number of regression models were constructed in order to observe the connections between the APIs' physicochemical properties in combination with their retention data from the chromatographic experiments (TLC and HPLC) and the indices of bioavailability in the CNS. Conducted analyses confirm that descriptors significant in BBB permeability modeling are hydrogen bond acceptors and donors, physiological charge, or energy of the lowest unoccupied molecular orbital. These molecular descriptors were the basis, along with the chromatographic data from the TLC in log BB regression analyses. Normal-phase TLC data showed a significant contribution to the creation of the log BB regression model using the multiple linear regression method. The model using them showed a good predictive value at the level of R2 = 0.87. Models for Kp,uu,brain resulted in lower statistics: R2 = 0.56 for the group of 23 APIs with the participation of k IAM.

A Thin Layer Chromatography Prelaboratory Activity Using a 3D-Printed Model to Address Student Misconceptions about Polarity and Intermolecular Forces

Normal phase thin layer chromatography is a widely taught technique in undergraduate organic chemistry laboratory courses. To understand how the technique works, students must have a strong conceptual understanding of polarity and intermolecular forces. Unfortunately, many students have misconceptions regarding how polarity and intermolecular forces affect the separation of compounds via thin layer chromatography. This work aimed to address these misconceptions by designing and implementing a prelaboratory activity using a 3D-printed model of a TLC plate. Data from pre- and postactivity surveys suggest the activity has a positive impact on student understanding, and student perceptions of the activity are overwhelmingly positive.

Normal-Phase TLC and Gradient Reversed-Phase HPLC for the Simultaneous Determination of Enrofloxacin and Bromhexine HCl in Presence of Two of Their Official Impurities.

In this work, two chromatographic methods are developed and validated for the determination of enrofloxacin and bromhexine (BRM) HCl in the presence of two of their specified impurities, ciprofloxacin and BRM impurity C. The suggested chromatographic methods included the use of thin layer chromatography (TLC-densitometry) and high-performance liquid chromatography (HPLC). In case of TLC-densitometry, good separation was achieved by using mobile phase of n.butanol:acetone:water:glacial acetic acid:triethylamine (10:3:1:0.5:0.5, by volume) on silica gel stationary phase at 254-nm detection. The developed HPLC method used BDS HYPERSIL C18 column with a mobile phase of water:acetonitrile:methanol:triflouroacetic acid. A linear gradient elution of 75-10%, 20-50% and 5-40% for water, acetonitrile and methanol, respectively, was applied in 13min at a flow rate of 1.5mLmin-1. These methods were sufficient to separate the four substances simultaneously, and they are validated as per International Conference on Harmonization guidelines.

Statistical Methods in the Study of Protein Binding and Its Relationship to Drug Bioavailability in Breast Milk.

Protein binding (PB) is indicated as the factor most severely limiting distribution in the organism, reducing the bioavailability of the drug, but also minimizing the penetration of xenobiotics into the fetus or the body of a breastfed child. Therefore, PB is an important aspect to be analyzed and monitored in the design of new drug substances. In this paper, several statistical analyses have been introduced to find the relationship between protein binding and the amount of drug in breast milk and to select molecular descriptors responsible for both pharmacokinetic phenomena. Along with descriptors related to the physicochemical properties of drugs, chromatographic descriptors from TLC and HPLC experiments were also used. Both methods used modification of the stationary phase, using bovine serum albumin (BSA) in TLC and human serum albumin (HSA) in HPLC. The use of the chromatographic data in the protein binding study was found to be positive —the most effective application of normal-phase TLC and HPLCHSA data was found. Statistical analyses also confirmed the prognostic value of affinity chromatography data and protein binding itself as the most important parameters in predicting drug excretion into breast milk.

Novel environment friendly TLC-densitometric method for the determination of anti-coronavirus drugs “Remdesivir and Favipiravir”: Green assessment with application to pharmaceutical formulations and human plasma

A great demand for discovering new therapeutic solutions has been considered all over the world for managing the rapidly progressing COVID-19 pandemic. Remdesivir (REM) and Favipiravir (FAV) are introduced as promising newly developed antiviral agents against the corona virus as evidenced by the clinical findings. Hence, the optimization of an analytical method for their simultaneous determination acquires potential importance in quality control labs and further confirmatory investigations. Herein, a green, sensitive, and selective densitometric method has been proposed and validated for determination of REM and FAV in pharmaceutical formulations and spiked human plasma on normal phase TLC plates. A solvent mixture of ethyl acetate–methanol-ammonia (8:2:0.2 by volume) has been chosen as developing mobile phase system. Well resolved spots have been detected at 235 nm with retardation factors (Rf) of 0.18 and 0.98 for REM and FAV, respectively. A validation study has been carried out in the light of ICH guidelines. Remdesivir and FAV have shown excellent sensitivities with quantitation limits down to 0.12 and 0.07 μg/band, respectively. The developed method has been successfully applied to tablet formulations and spiked plasma with excellent recoveries ranged from 97.21 to 101.31%. The greenness of the method has been evaluated using the standards of greenness profile and Eco-Scale. It has passed the four greenness profile quadrants and achieved 80 score in Eco-Scale.

Normal-Phase TLC and Gradient Reversed-Phase HPLC for the Simultaneous Determination of Enrofloxacin and Bromhexine HCl in Presence of Two of Their Official Impurities

In this work, two chromatographic methods are developed and validated for determination of enrofloxacin (ENR) and bromhexine HCl (BRM) in the presence of two of their specified impurities, ciprofloxacin (CIP) and BRM impurity C. The suggested chromatographic methods included the use of thin layer chromatography (TLC-densitometry) and high performance liquid chromatography (HPLC). In case of TLC- densitometry, good separation was achieved by using mobile phase of n.butanol: acetone: water: glacial acetic acid: triethylamine (10:3:1:0.5:0.5, by volume) on silica gel stationary phase at 254nm detection. The developed HPLC method used BDS HYPERSIL C18 column with a linear gradient elution of 75-10%, 20-50% and 5-40% for water, acetonitrile and methanol, respectively, in 10 min at a flow rate of 1.5 mL min-1.These methods were sufficient to separate the four substances simultaneously and they are validated as per International Conference on Harmonization (ICH) guidelines.

New dihydroxycucurbitacin D's from the Namib desert endemic plant Acanthosicyos horridus (!nara)

Acanthosicyos horridus Welw. ex Hook.f. (!nara) is a leafless, thorny, melon-producing plant endemic to the hyper-arid Namib Desert. The methanol crude extract prepared from the ripe fruits of !nara afforded the known dihydroxycucurbitacin 7β-hydroxy-23,24-dihydrocucurbitacin D (1), along with four new congeners 7β,15β-dihydroxy-23,24-dihydrocucurbitacin D (2), 25-O-β-glucopyranosyl-7β-hydroxy-23,24-dihydrocucurbitacin D (3), 25-O-β-glucopyranosyl-7β-hydroxy-23,24-dihydroisocucurbitacin D (4) and 25-O-β-glucopyranosyl-7β-hydroxy-23,24-dihydro-3-epi-isocucurbitacin D (5). These compounds were isolated through a combination of preparative normal phase thin-layer chromatography (TLC) and semi-preparative reversed phase high performance liquid chromatography (HPLC). Their structures were established by comprehensive analysis of HR-ESI-MS data, 1D and 2D NMR spectroscopic data and by comparison with literature values of similar cucurbitacins. The five isolated compounds exhibited poor cytotoxic activity against the MDA-MB-231 breast cancer cell line. To the best of our knowledge, this is the first report of glycosylated cucurbitacins in Acanthosicyos horridus.

Normal phase thin layer chromatography for aromatic derivatives of 3-chloro-1,4-naphtochinone

The chromatographic characteristics were investigated for eight aromatic derivatives of 3-chloro-1,4-naphthoquinone under conditions of normal-phase thin-layer chromatography for benzene-based binary mobile phase and such polar solvents as chloroform, acetone, acetonitrile, methanol and propan-2-ol. The slope of linear retention dependencies for the investigated compounds on the concentration of the polar component in the mobile phase satisfactorily correlates with the area occupied by the adsorbed analyte molecule in the stationary phase. The intercept in the Soczewinski equation depends on the polar component of the mobile phase.

HPTLC METHOD FOR ESTIMATION AND QUANTIFICATION OF β-SITOSTEROL FROM IN-VIVO AND IN-VITRO SAMPLES OF MERREMIA AEGYPTIA AND MERREMIA DISSECTA

Objective: A normal-phase high-performance thin-layer chromatography (HPTLC) method has been developed and validated for estimation and quantitation of beta-sitosterol from the methanolic fraction of different plant parts of two medicinally important plants viz. Merremia aegyptia and Merremia dissecta. These plants have been reported to possess antimicrobial, antioxidant, and anti-inflammatory activities.
 Methods: Chromatographic separation of beta-sitosterol from the methanolic extracts of plant parts of M. aegyptia and M. dissecta was performed on TLC aluminum plates pre-coated with silica gel 60F254 using a suitable mobile phase. The densitometric scanning was done after derivatization at ????-580 nm for ????-sitosterol.
 Result: Only M. dissecta leaf sample was reported to contain ????-sitosterol (4.6 ng/μl), whereas other samples such as seed, stem, and callus extracts of M. aegyptia and M. dissecta did not showed its presence.
 Conclusion: The developed HPTLC method is simple, rapid, and precise and can be used for routine analysis and quantification of ????-sitosterol and other useful plant bioactives that are phytopharmaceutically important.

Low-Temperature Plasma Probe Mass Spectrometry for Analytes Separated on Thin-Layer Chromatography Plates: Direct vs Laser Assisted Desorption.

Thin-layer chromatography (TLC) is a widespread technique because it allows fast, simple, and inexpensive analyte separations. In addition, direct analysis of the compounds separated on TLC plates via mass spectrometry (MS) has been shown to provide high sensitivity and selectivity while avoiding time-consuming sample extraction protocols. Here, direct desorption low-temperature plasma-mass spectrometry (LTP-MS) as well as diode laser assisted desorption (LD) LTP-MS are studied for direct spatially resolved analysis of compounds from TLC plates. Qualitative and quantitative characterization of amino acids, pharmaceuticals, and structural isomers were performed. The nature of the TLC plate stationary phase was found to have a significant influence, together with the analyte's characteristics, on the desorption efficiency. Tandem MS is shown to greatly improve the limits of detection (LODs). Direct desorption LTP-MS, without external thermal assisted desorption, demonstrates its best performance with cellulose TLC plates (LODs, 0.01 ng/mm2 to 2.55 ng/mm2) and restricted performance with normal-phase (NP) TLC plates (several analytes without observable signal). LD LTP-MS, with systematic optimization of irradiance and focal point diameter, is shown to overcome the direct-desorption limitations and reach significantly improved LODs with NP TLC plates (up to ×1000 better). In addition, a wide-ranging characterization of amino acid analytical figures of merit with LD LTP-MS shows that LODs from 84 pg/mm2 down to 0.3 pg/mm2 are achieved on NP TLC plates.

The New Test Procedure for Group-Type Composition of Base Oils of Lubricating Oils, Especially Emitted into the Environment

Mineral base oil, a product of multistep fine refining of the relevant fractions from vacuum distillation of crude oil, is a main component of so-called mineral lubricating oils containing aliphatic and alicyclic substituted aromatic hydrocarbons, i.e., derivatives of benzene and biphenyl. Mineral lubricating oil is composed mostly of mineral base oil and a low amount of enriching additives, most often products of advanced organic chemical technology. The application of mineral lubricating oils in open cutting systems has a very negative impact on environment and on the operator’s health. This work presents a simple, cheap and fast methodology allows identification of the group-type composition of base oil in lubricating oil and to estimate the content or total absence of base oil of mineral and vegetable origin in lubricating oil. The first step of the test is an in-situ screening for fluorescence of petroleum fraction under the 365 nm light. The next is the performance of infrared spectra with Fourier transformation (FT-MIR) to identify and estimate the content of vegetable oil and its derivatives and the performance of UV-Vis spectra to identify and determine the content of aromatic hydrocarbons, as well as dyes present in the lubricating oil. The last stage is normal phase thin layer chromatography (NP-TLC) using different visualization methods to evaluate the group-type composition of lubricating oil. Effectivity of the developed procedure has been confirmed during control of group-type composition evaluation of lubricating oils in cutting systems. The procedure can be also applied with respect to different oil matrices.

Thin-layer chromatographic separation of a number of bile acids with mobile phases based on surfactants

The aim of this study was the separation of cholic, ursodeoxycholic, chenodeoxycholic, deoxycholic, and lithocholic bile acids by thin-layer chromatography (TLC) with mobile phases based on surfactants. The effect of surfactant type and concentration, the pH of the mobile phase, the type and amount of the mobile phase modifier, and the effect of temperature and type of material backed silica gel TLC layers on the investigated bile acid separation were studied. The separation was carried out on normal-phase TLC plates Sorbfil PTLC-P-A-UV-254 and Sorbfil PTLC-AF-A-UV-254. The acid zones after separation were visualized by using sulfuric acid solutions followed by heating the TLC plate and acid spots observing under 365-nm ultraviolet (UV) light. Mobile phases based on surfactants, namely, cationic cetylpyridinium chloride, anionic sodium dodecyl sulfate, and nonionic Tween-80, were used. Aliphatic alcohols 1-propanol, 1-butanol, and 1-pentanol as well as the nonionic surfactant Tween-80 were used as modifiers of the mobile phase. The optimal separation of the studied acids was achieved using the two-dimensional TLC method with sequential elution with mobile phases based on cetylpyridinium chloride, first with the addition of 1-butanol and then, perpendicular to the initial direction, with the addition of 1-pentanol. The proposed method for the bile acid separation can be used in the analysis of human biological fluids and drugs.

Bioactive compounds from the leaf extract of Bauhinia galpinii (Fabaceae) used as antidiarrhoeal therapy in southern Africa

Bauhinia galpinii N. E. Br is a shrub or small tree that grows 3–9 m tall throughout South Africa in nature and as ornamental plant. Various parts of the plant are used to cure gastrointestinal tract (GIT) disorders, epilepsy, convulsion, infertility, and amenorrhoea. Despite popular use, the phytochemicals responsible for the medicinal efficacy of the plant has not been exhaustively investigated.Bioautography-guided fractionation was used to isolate the antimicrobial phytochemicals of the acidified 70% acetone leaf extract. A combination of open column chromatography using silica gel 60, Sephadex LH-20, normal phase thin-layer chromatography (TLC) on precoated Kieselgel 60F254 plates and the spots visualized under UV366 and UV254 light, and spray reagents (vanillin/sulphuric acid) were used to isolate compounds. The compounds were identified by UV spectra, extensive 1D and 2D NMR spectroscopy, mass spectrometry and comparison of the data with those reported in the literature. The antimicrobial activity of isolated compounds was determined against Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), Candida albicans (ATCC 10231), clinical isolates of Cryptococcus neoformans, Aspergillus fumigatus, C. albicans (M0824), C. albicans (M0825), C. albicans (1051604),and C. albicans (1051608) using a serial dilution microplate method.Result: Six compounds including two novel (24-Isopropylcholest-5-en-3,8-diol(1) and 5, 7, 4′ 5′ tetrahydroxy-2′-methoxyflavone (isoetin 2′ methyl ether) (4)) and four known (3, 5, 7, 3′, 4′-pentahydroxyflavone (quercetin) (2); 3, 5, 7, 3′, 4′, 5′-hexahydroxyflavone (myricetin) (3); quercetin-3-O-β-galactopyranoside (5) and myricetin-3-O-β-galactopyranoside (6)) were isolated and characterised. The isolated compounds inhibited antimicrobial growth with MICs ranging between 3.9 and 125 μg/.The results may provide a rational support for the traditional use of Bauhinia galpinii leaf in the treatment of infectious gastrointestinal disorders, since antimicrobial compounds especially the flavonoids isolated from the 70% acetone extract may also present in the traditional water extract. They could be responsible for the broad spectrum of antimicrobial activity of B. galpinii extract.

Identification of Apigenin and Luteolin in Artemisia annua L. for the Quality Control

Objective: To identify active compounds and establish the chemical fingerprint of Artemisia annua L. for the quality control. Methods: Thin-layer chromatography (TLC) conditions were developed to screen for 2 common flavonoids (apigenin and luteolin). Three mobile phases were used to isolate these flavonoids in 80% ethanolic extract of A. annua .Hexane : ethyl acetate : acetic acid (31:14:5, v/v) and toluene : 1,4-dioxane : acetic acid (90:25:4, v/v) were used in normal phase TLC (NP-TLC), and 5.5% formic acid in water : methanol (50:50, v/v) were used in reverse phase TLC (RP-TLC). Chromatograms were visualized under visible light after spraying with Fast Blue B Salt. Apigenin and luteolin bands were checked by comparing their Rf values and UV-Vis absorption spectra with reference markers. Results: Apigenin and luteolin were simultaneously detected with good specificity in RP-TLC condition, while only apigenin was detected in NP-TLC condition. Apigenin band intensity was higher than luteolin band intensity in both conditions. Conclusion: This knowledge can be applied to the development of quality control assessments to ensure product efficacy and consistency.

Normal-phase versus reversed-phase thin-layer chromatography (TLC) to monitor oxidized phosphatidylcholines by TLC/mass spectrometry.

Normal-phase thin-layer chromatography (NP-TLC) is an established method for the separation of all major phospholipid classes according to the different polarities of the head groups. In contrast, reversed-phase (RP)-TLC is much less frequently used for this purpose. This study aimed to compare the NP and the RP approach regarding their separation potential of phospholipid oxidation products. Commercially available 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC) (PC 16:0/18:1) and 1-palmitoyl-2-linoleoyl-sn-phosphatidylcholine (PLPC) (PC 16:0/18:2) were oxidized by NaMnO4 . Oxidation products were subsequently separated by NP- and RP-TLC and analyzed by electrospray ionization mass spectrometry. In comparison with NP-TLC, RP-TLC was clearly superior regarding the separation of oxidation products of phospholipids. RP-TLC enabled the separation not only of primary oxidation products of POPC such as alcohols and ketones but also of secondary oxidation products. Furthermore some oxidation products, such as aldehydes, were only detectable by ESI after RP-TLC but not after NP-TLC. RP-TLC is the method of choice to characterize oxidized PL such as oxidized phosphatidylcholines.

Rapid autographic method for detection of enzymatic browning inhibitors based on enzyme immobilization

This work describes a TLC-coupled bioautographic assay suitable for the separation and detection of apple polyphenol oxidase (PPO) inhibitors from natural extracts. PPO was immobilised in agar containing l-DOPA as substrate and 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) to enhance colour development. The inhibition was detected as white spots on reddish background. Minimum amount of PPO inhibitors detected was 0.0125 µg of 4-hexylresorcinol, 0.025 µg of ascorbic acid, 0.5 µg of cysteine and 1 µg of kojic acid. The assay was compatible with normal and reverse phase TLC systems and allows detecting compounds that directly had action on the enzyme as well as agents that could convert quinones back to their reduced form. The chromatographic run evidenced the different nature of enzymatic browning inhibitory compounds from garlic and onion extracts. Using natural enzymes will provide a fast and cheap alternative for target specific exploration of natural enzymatic inhibitors.

Reversed-phase and normal-phase thin-layer chromatography and their application to the lipophilicity prediction of synthetic pyrethroids based on quantitative structure–retention relationships

Synthetic pyrethroids are pesticides derived from naturally occurring pyrethrins. Nowadays, they are one of the most commonly used insecticides. Synthetic pyrethroids are applied in many areas including agriculture, veterinary medicine, households, and public health. The chromatographic properties of 7 synthetic pyrethroids with known lipophilicity have been studied by use of planar chromatography. Reversed-phase and normal-phase thin-layer chromatography approaches were applied. The main goal of this study was to evaluate retention parameters as the possible measure of the lipophilicity of synthetic pyrethroids. Upon the obtained results, it can be concluded that the best chromatographic system to predict log P is reversed-phase thin-layer chromatography and a mixture of tetrahydrofuran and water (r = 0.972). This conclusion was also confirmed by the performed principal component analysis. Furthermore, the utilization of computational descriptors, calculated by Dragon software, showed which physicochemic...

Centrifugal partition chromatography enables selective enrichment of trimeric and tetrameric proanthocyanidins for biomaterial development

Proanthocyanidins (PACs) find wide applications for human use including food, cosmetics, dietary supplements, and pharmaceuticals. The chemical complexity associated with PACs has triggered the development of various chromatographic techniques, with countercurrent separation (CCS) gaining in popularity. This study applied the recently developed DESIGNER (Depletion and Enrichment of Select Ingredients Generating Normalized Extract Resources) approach for the selective enrichment of trimeric and tetrameric PACs using centrifugal partition chromatography (CPC). This CPC method aims at developing PAC based biomaterials, particularly for their application in restoring and repairing dental hard tissue. A general separation scheme beginning with the depletion of polymeric PACs, followed by the removal of monomeric flavan-3-ols and a final enrichment step produced PAC trimer and tetramer enriched fractions. A successful application of this separation scheme is demonstrated for four polyphenol rich plant sources: grape seeds, pine bark, cinnamon bark, and cocoa seeds. Minor modifications to the generic DESIGNER CCS method were sufficient to accommodate the varying chemical complexities of the individual source materials. The step-wise enrichment of PAC trimers and tetramers was monitored using normal phase TLC and Diol-HPLC-UV analyses. CPC proved to be a reliable tool for the selective enrichment of medium size oligomeric PACs (OPACs). This method plays a key role in the development of dental biomaterials considering its reliability and reproducibility, as well as its scale-up capabilities for possible larger-scale manufacturing.

Unprecedented sensitivity of the planar yeast estrogen screen by using a spray-on technology

The planar yeast estrogen screen (p-YES) can serve as a highly valuable and sensitive screening tool for the detection of estrogenic compounds in various sample matrices such as water and wastewater, personal care products and foodstuff. The method combines the separation of sample constituents by thin layer chromatography with the direct detection of estrogenic compounds on the surface of the HPTLC-plate. The previous protocol using the immersion of a normal phase silica HPTLC-plate in a cell suspension for bio-autography resulted in blurred signals due to the accelerated diffusion of compounds on the wet surface of the HPTLC-plate. Here, the application of the yeast cells by spraying on the surface of the HPTLC-plate is described as an alternative approach. The presented method for the hyphenation of normal phase thin layer chromatography with a yeast estrogen screen results in much sharper signals compared to reports in previous publications. Satisfying results were achieved using cultures with cell densities of 1000 FAU. Due to the reduced signal broadening, lower limits of quantification for estrogenic compounds were achieved (Estrone (E1)=2pg/zone, 17β-estradiol (E2)=0.5pg/zone, 17α-ethinylestradiol (EE2)=0.5pg/zone and Estriol (E3)=20pg/zone). As demonstrated, it is possible to characterize profiles of estrogenic activity of wastewater samples with high quality and reproducibility. The improved sensitivity opens the stage for applications using native samples from waste- or even surface water directly applied on HPTLC-plates without the need for prior sample treatment by e.g. solid phase extraction.