Expert Insight: Automate your routine QC with high-performance thin-layer chromatography

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Dr. Melanie Broszat, Scientific Business Development Manager, CAMAG

As an alternative to GC and HPLC, high-performance thin-layer chromatography (HPTLC) is the method of choice for the analysis of complex and variable samples and has proven its value across a wide array of disciplines as a reliable qualitative and quantitative analysis technique for quality assurance.

In this SelectScience^® webinar, Dr. Melanie Broszat, Scientific Business Development Manager at CAMAG, introduces the HPTLC PRO, a fully automated HPTLC sample analysis and evaluation system launched in 2019. Dr. Broszat presents the key benefits of the system for routine quality control applications across multiple industries and provides information on the freely available sources of related methods. Current and future trends in HPTLC are also discussed, as well as the role CAMAG plays in this rapidly accelerating field.

Read on for the highlights of the live Q&A session or register to watch the webinar at a time that suits you.

Q: What is the difference between normal HPTLC and automated HPTLC?

MB: Automated HPTLC will support standardized HPTLC, but it also has further advantages, such as increased inter-laboratory reproducibility and, due to the improved development chamber actively controlling the gas phase, a better separation of compounds. HPTLC requires an individual to move plates from one step to the next but this is not the case with an automated system, meaning you can run more samples compared to classical HPTLC. 

Q: Can you use machine learning for HPTLC?

MB: We have not fully implemented machine learning or artificial intelligence tools, but this will happen together with the new hyperspectral detector. One goal is for the software to automatically evaluate your data and tell you if your sample passes or fails a given criteria. We will also look to implement tools like pattern recognition and hyperspectral imaging which will open up completely new possibilities.

Q: What does high sample throughput in HPTLC mean?

MB: You can easily run 15 plates per day with 15 samples per plate, which results in at least 225 samples a day.

Q: Does automation also mean in-line derivatization without any previous manual intervention?

MB: In the past, manual spraying or immersion was the sole method available, but for our automated system, an automated spraying technique was prerequisite. A few years ago, CAMAG launched the patented, stand-alone Derivatizer, which has a spraying nozzle that uses piezoelectric technology to generate a very fine mist of the reagent that settles on the plate. For the Module DERIVATIZATION in the HPTLC PRO System, we have the same technology, which is now in-line with the automated workflow and is software-controlled. This module allows spraying different reagents and is capable of cleaning and rinsing its nozzles. Additionally, an integrated heating plate allows to heat the plate in an standardized and controlled environment up to 150 °C. 

Q: What about the detection of non-chromophoric compounds, which are in PPM level?

MB: An advantage of HPTLC is that there are many different options for derivatization. We can do a pre-chromatographic or post-chromatographic derivatization, and it can be universal or selective. To reach a very low detection limit, I recommend using a fluorophore instead of a chromophore to enhance the sensitivity of the compound on the plate. This is the most sensitive approach currently available for UV-VIS spectrometry.

Q: Can HPTLC-MS be an economical tool for addressing issues in cases of the non-availability of reference standards or markers?

MB: Yes, it just depends on which type of mass spectrometer you are using. If you use single MS and standardized HPTLC, then you can already guess the type of analyte without a reference. To confirm the structure, I do recommend using a tandem mass spectrometer, if no reference compound is available.

Q: If we are doing a qualitative HPTLC analysis for a phytopharmaceutical formulation that contains three herbal constituents, of which two are non-polar and one is polar, how can we run the plate to prove the presence of these constituents in the formulation?

MB: A mixture of different plant materials is always a difficult task, but the strength of the HPTLC technique based on its market penetration. A lot of methods do already exist in this field. If you want to separate non-polar and polar compounds at the same time on the same plate, several strategies may be followed, e.g., use the first development step to a defined migration distance and then change the polarity of the developing solvent to develop to the final migration distance or e.g. use the gas phase during chromatography of the HPTLC PRO Module DEVELOPMENT.

Q: Do you have to wait for all five plates to run through completely before adding new plates or samples to be run?

MB: Up to five analyses can be submitted in one sequence.  You can interrupt a running sequence, modify and change analysis files, and then the files have to be re-submitted to run the sequence. As an alternative, you can run each plate in a separate analysis file and then you are flexible to stop and redefine your sequence.

Q: Can isomers be identified and quantified with HPTLC?

MB: Yes. You can also separate isomers, using special impregnation of the plates or modified layers on plates specifically made to separate isomers.

Q: Do we need to always run biomarkers together with samples? Does HPTLC PRO have an in-built library?

MB: The unique advantage about HPTLC is that we have the separated compounds in a fixed state on the plate. The Comparison Viewer within the visionCATS software allows comparing reference images generated from different plates and labs. The Method Library of the visionCATS software provides ready-to-use instrumental methods and Comparison Viewer files with reference images. If you use HPTLC as it is defined in the European and American pharmacopeia, you can compare your obtained results with those reference images or create your own Comparison files. I still recommend for each plate a system suitability test to check the reproducibility and quality of your chromatography.

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