This case study demonstrates a rapid identification of fixed oils after derivatization with phosphomolybdic acid.

HPTLC is a powerful technique with regard to a high sample throughput capacity, short analysis time, and low running costs. Our case study demonstrates a rapid identification of fixed oils after derivatization with phosphomolybdic acid.

Often chromatographic techniques require a time- and cost-intensive sample preparation step. In many cases HPTLC allows to analyze several samples side by side after only a minimal sample preparation (e.g. dissolving). The identity of samples with unknown composition can be elucidated by comparing the pattern of the sample with that of reference materials. A System Suitability Test (SST) can be applied to check the reproducibility of the chromatographic system during method transfer validation and to qualify the data on each plate.

The method is downloaded from the visionCATS Method Library. The Word document (SOP) is opened for reference. In preparation for the analysis a RP-18 HPTLC plate is pre-washed with dichloromethane and then dried in the oven. All samples and references are prepared according to the SOP (in compliance with the USP method <203>, Application note F-39). The visionCATS method file is opened and executed, creating a new analysis file. In this visionCATS analysis file the sequence table is completed (Vial ID, description of samples and references, and selection of the rack position of each vial). An image of the pre-washed clean plate under white light is captured with the TLC Visualizer. The samples and standards are applied band-wise onto the HPTLC plate using the ATS 4, which guarantees precise and reliable application. Then the HPTLC plate is developed using the ADC 2. For visualization of fingerprints of the different fixed oils the developed plate is dipped into phosphomolybdic acid reagent, heated at 120°C for 3 minutes, and documented with the TLC Visualizer under white light. For a successful method transfer validation the SST needs to pass. If the RF values are in the specified range the patterns of the unknown samples and those of the references can be easily compared with the visionCATS Image Comparison.

Image Comparison of fixed oils and references (USP) after derivatization

• HPTLC visualizes at the first glance the similarities and differences between samples and references.
• HPTLC is also used for determination of purity (adulteration/fraud) and stability studies (shelf life).
• HPTLC can be used as high throughput and low-cost technique for the analysis of food samples.

In recent years, the need for quality assurance tools to ensure the identity, purity, and quality of botanical material has risen dramatically. HPTLC has emerged as a versatile, high throughput, and cost-effective technology, that is uniquely suited to meet these requirements. According to the regulation of e.g. United States Pharmacopeia (USP 37, Monograph), Ginkgo leaf and Ginkgo extracts are identified by HPTLC. Our case study method demonstrates how Ginkgo biloba extracts can be rapidly identified by HPTLC based on their flavonoid fingerprint.

Most separation techniques do not allow parallel analysis of numerous samples at the same time and they often face problems in separating complex mixtures of substances. However, visual evaluation of HPTLC plates allows for convenient comparison of many samples side by side, where similarities and differences can clearly be seen. Quantification of the separated compounds is possible by densitometry using our TLC Scanner 4.

Ginkgo leaves, Ginkgo extracts and the standards are prepared according to CAMAG’s standardized method (F-16B.1). All parameters (conditions for sample application,chromatogram development, evaluation) are logged into the vision CATS software which controls all major CAMAG instruments needed for the HPTLC analysis. The samples and standards are applied bandwise on a HPTLC plate using the ATS 4 which guarantees for precise and reliable application. Then the HPTLC plate is developed under standardized conditions in a saturated chamber using the ADC 2. The ADC 2 is unsurpassed for reproducibility and universal applicability in HPTLC. The developed HPTLC plate is evaluated and documented by the TLC Visualizer in three modes, white light, UV 254 nm and UV 366 nm. By the use of HPTLC plates containing a fluorescence indicator, all analytes which absorb UV 254 nm can be detected prior to derivatization. Derivatization increases the specificity and selectivity of the method. For the analysis of flavonoids the plate is dipped in “Natural Products” reagent and a solution of PEG 400 and evaluated by the TLC Visualizer under UV 366 nm.

• Several samples can be visually analyzed in parallel. The TLC Visualizer in combination with the visionCATS software features easy comparison and rearrangement of HPTLC fingerprints of samples from the same as well as from different plates. This helps to assess differences in the fingerprint patterns of different samples and over time which is useful for long-term and stability studies of raw materials and finished products. Furthermore adulterated samples are reliably identified.
• The quality of raw materials is rapidly and easily determined by HPTLC. For the identification of herbal drugs and other naturally derived materials standardized HPTLC is the method of choice and recommended by pharmacopoeias worldwide.
• In this case study the identity of different Ginkgo biloba samples were either confirmed, or not, by comparison of their HPTLC fingerprint with those of a “Botanical Reference Material” (BRM) of Ginkgo biloba. With chemical reference standards and measurement by the TLC Scanner cut-off values can be established for limit tests and quantification of single marker compound is possible.

HPTLC is the only single test that allows for 100% botanical ingredient identification. HPTLC is the primary chemical test for identification of all pharmacopoeia monographs on botanicals. According corresponding monographs of USP, PhEur and AHP on Ginkgo leaf and Ginkgo leaf extract are identified by HPTLC based on fingerprints of flavonoids. In addition to the identification, our case study method demonstrates how ginkgolides and bilobalide in Ginkgo biloba extracts can be quantified by HPTLC.

Most techniques can address identification only in part. They either are limited to plant parts (microscopy), have difficulties with natural variability of botanical materials (IR techniques), or focus on quantitative comparison of separated markers (HPLC). All other techniques lack the versatility and flexibility of HPTLC for the analysis of botanicals. HPTLC allows for convenient comparison of many samples side by side, where similarities and differences can be clearly seen. Quantification of the separated compounds is possible by densitometry using our TLC Scanner 4.

• HPTLC shows visually the similarities and differences between samples and references.
• For the identification of herbal drugs and other naturally derived materials standardized HPTLC is the method of choice and recommended by pharmacopoeias worldwide.
• By using internal or external chemical reference standards and the TLC Scanner 4 marker compounds can be quantified precisely.
• HPTLC can also be used for assays (potency), purity (adulteration), and stability studies (shelf life).
• The whole sample is detectable on the plate and allows to judge all components of the sample even if components remain at the application zone.
• Following separation and localization of target zones by non-destructive detection analytes can be directly eluted from the plates with the TLC-MS Interface and identified by hyphenated techniques (e.g. MS, NIR, NMR).

This HPTLC case study demonstrates a rapid way of in-process control during chemical synthesis.

HPTLC is a powerful technique enabling high sample throughput capacity, short analysis time, and low operating costs. Our case study demonstrates a rapid way of in-process control during chemical synthesis.

Commonly used chromatographic techniques require a time- and cost-intensive sample preparation step. In many cases HPTLC allows one to analyze several samples side-by-side with little to no sample preparation. The progress of a reaction during a chemical synthesis and of purification can easily be monitored by comparing samples on the same plate side-by-side.

All samples are directly applied from each step of the chemical synthesis without sample preparation on an HPTLC plate (silica gel 60 F₂₅₄). The visionCATS method file is opened and executed, creating a new analysis file. In this visionCATS analysis file the sequence table is completed (Vial ID, description of samples and references, and selection of the rack position of each vial). An image of the clean plate under UV 254 nm is captured with the TLC Visualizer. The samples and standards are applied bandwise onto the HPTLC plate using the Automatic TLC Sampler 4 (ATS 4), which guarantees precise and reliable application. Then the HPTLC plate is developed using the Automatic Developing Chamber 2 (ADC 2) allowing for reproducibility between plates. For visualization the plate is documented with the TLC Visualizer under UV 254 nm and UV 366 nm. Spectra from 200 to 500 nm are recorded with the TLC Scanner 4 and visionCATS. The purified product zone of interest is eluted with the TLC-MS Interface 2 and confirmed by HPTLC-MS.

• HPTLC is suitable as a high throughput and low cost technique for the analysis of drugs.
• HPTLC is used for determination of purity and for stability studies (shelf life).
• HPTLC allows the separation of several samples in parallel on one plate and is therefore excellently suited for process monitoring.
• HPTLC visualizes the similarities and differences between samples at a glance on a plate (e.g. to monitor up- and downscaling processes) and by using the software visionCATS to even compare between plates created years apart.
• HPTLC-MS is a fast way for substance confirmation.

This case study demonstrates a reliable characterization of impurities in biodiesel (according to UNE EN 14214:2013) and additionally presents the concept for identity confirmation of monoacylglycerides by mass spectrometry, which allows for origin identification.

HPTLC is a powerful technique with high sample throughput capacity, short analysis time, and low running costs. This case study demonstrates a reliable characterization of impurities in biodiesel (according to UNE EN 14214:2013) and additionally presents the concept for identity confirmation of monoacylglycerides by mass spectrometry, which allows for origin identification (e.g. vegetable, animal, waste cooking oil).

Often chromatographic techniques require time- and cost-intensive sample preparation steps. However, in many cases HPTLC allows to analyze samples after a minimal sample preparation (e.g. dissolving) in combination with the separation of several samples side by side on the same plate. Quantification of the separated compounds is possible by densitometry using the CAMAG TLC Scanner. In addition, the identity can be proven by eluting the spot of interest using the CAMAG TLC-MS Interface and coupling it to any commercially available mass spectrometer.

Sample and standard preparation are performed according to Cebolla et al. (CBS 114, p. 5-7). All parameters (conditions for sample application, chromatogram development, and evaluation) are logged into the winCATS software, which controls all major CAMAG instruments required for the HPTLC analysis. The samples and standards are applied band-wise onto the HPTLC plate using the ATS 4, which guarantees precise and reliable application. Then the HPTLC plate is developed using the AMD 2. For the analysis of monoacylglycerides the developed plate is dipped into primuline reagent. For quantification each track is scanned in fluorescence mode at 366/>400 nm with the TLC Scanner and evaluated with winCATS software. If further investigations of separated sample components are of interest, their eluted zones can be analyzed with other techniques like MS, NIR, and NMR (off-line hyphenation).

• HPTLC can be used as high throughput and low-cost technique for a broad variety of analytical tasks in industrial applications settings.
• By using internal or external chemical reference standards compounds can be quantified precisely with the TLC Scanner.
• The entire sample is detectable on the plate and allows evaluation of all components even if some of them remain at the application zone or migrate in the solvent front.
• HPTLC can analyze samples with high matrix content due to the disposable stationary phase.
• Thanks to the non-destructive nature of the chromatographic method, analytes can be eluted after separation from the plates using the CAMAG TLC-MS Interface and analyzed by hyphenated techniques (e.g. MS, NIR, NMR).

This work was done at the CSIC, Instituto de Carbochímiqa, in Zaragoza, Spain. We are grateful to Prof. Dr. Vicente Cebolla and his team for the excellent collaboration!

HPTLC bioautography links an effect to a separated compound from a complex mixture (non-target screening). By the use of RP-18 W stationary phases (wettable with water) large volumes of aqueous samples can be applied resulting in sharply-bounded zones. HPTLC bioautography offers a great option with regard to a high sample throughput capacity at low running costs. This case study demonstrates a new way of non-target screening for estrogen active compounds in food samples or water.

Endocrine active compounds (EACs) are ubiquitous in food. They have impact on human health by controlling and regulating essential functions of metabolism, growth and development. Endocrine active compounds include natural estrogens, phytoestrogens as well as food contaminants like plasticizers, pesticides and biocides. To detect all EACs specific non-target screening methods are needed. The planar Yeast Estrogen Screen (pYES) offers a very sensitive way to analyze known and unknown EACs in various sample matrices.

The yeast is cultivated according to Klingelhoefer and Morlock (2015). For the HPTLC analysis the samples (in the case shown e.g. beer) are degassed by sonication and 750 µL of each beer sample is mixed with 750 µL of methanol. After centrifugation for 5 min the supernatant is transferred to vials. In the first step an image of the clean RP-18 W HPTLC plate is captured under white light and UV 254 nm with the CAMAG TLC Visualizer. 300 µL of each sample are applied in form of a rectangle (10 x 30 mm) onto the HPTLC plate using the CAMAG Automatic TLC Sampler 4 (ATS 4) to spread the huge beer matrix over a large adsorbent area. Then the HPTLC plate is developed in 3 steps using the Automatic Developing Chamber 2 (ADC 2). The first two steps are for focusing the applied sample (front elution of the analytes) with isopropyl acetate up to 35 mm, followed by the development with n-hexane – toluene – ethyl acetate 6:3:4 up to 70 mm. An image of the developed plate is captured under UV 254 nm, UV 366 nm and white light. In the next step, the developed plate is dipped with the CAMAG Chromatogram Immersion Device 3 into the yeast culture. The plate is kept humid and incubated at 30°C for 3 h. After that, the plate is dipped into the substrate solution and incubated at 37°C for 1 h. The evaluation is done under UV 366 nm. The active zones are further analyzed by HPTLC-MS. Zones are directly eluted with CAMAG TLC-MS Interface to an ESI-MS for confirmation.

• HPTLC bioautography couples effect-directed analysis with chromatography which allows non-target screening
• HPTLC bioautography can be used for screening several samples side by side
• pYES detects EACs down to the very low µg/kg range (for some even down to the ng/kg range)
• pYES is a specific detection method for the substance class of EACs
• With HPTLC-MS the presence of known estrogens can easily be confirmed