Welcome to the video question and answer from our webinar The Concept of Fully Automated HPTLC Analysis on the Example of Sugars in Honey. In this video, our HPTLC expert Dr. Melanie Broszat answers questions we didn’t get to during the webinar. Below the video, you will find a transcript.
Answer: There exist different spraying reagents for sugars and for our studies we have used the aniline diphenolamine phosphoric acid reagent because it derivatizes many different types of sugars. You can detect them down to the nanogram range, which means down to the ppm range and also the rapid usability for quantification is quite good, so if you want to have a homogeneous reagent distribution, I would recommend to either do the spraying by Derivatizer or to do the immersion.
Answer: Yes, the initial goal was to quantify the major sugars in honey so they are well separated with a n-butanol isopropanol boric acid developing solvent and also with alternative solvent. What you have to keep in mind is that the sugars are present at different amounts in the honey samples, therefore for evaluation for determination of the sugars in the linear working range it is usually needed to have two different dilutions. Glucose and fructose are present at high amounts in honey and maltose and sucrose in low amounts, therefore usually two different dilutions are needed.
Answer: From an analytical point of view, I would always recommend to do individual standards and to apply of each the same volume. Individual standards will also reduce the chance to have systematic errors. In HPLC or GC you also have individual standards, but no one is asking that there because it's the usual way of working.
Answer: The solvent system with ethyl acetate methanol boric acid better separates trehalose and maltose. Those two are difficult to separate and I would then better try this method. Depending on other sugars you might have also to adjust the composition of the developing solvent.
Answer: Like every analytical method it has limitations and if you talk about HPTLC in general the limitation is the limited separation capacity. In HPTLC you can baseline separate let's say between 10 and 12 analytes, but we can also use selective derivatization to just detect a certain selected class of substances or if we need a higher separation capacity then we can also think about the gradient development.
Answer: We haven't thought about this and I will internally discuss the option what we have done so far is that we included our results of the carbohydrate analysis on the substance database of the HPTLC Association. That includes images of the results, profiles, and UV spectra.
Answer: The Canadian articles have been published in scientific journals and most of them are not open access. We have published a summarized article in our CBS journal, so this you can download from the CAMAG website and in there you will also find the references and the contact to the authors.
Answer: If it's just certain pesticides, HPTLC might be a good technique for analysis, but if it's a multi-target screening of tens or hundreds of pesticides, then I would better recommend multi-target screening methods with MRM mode for doing analysis of let's say 300 pesticides by GC-MS/MS or LC-MS/MS.
Answer: Yes, we have an application note for screening of forensic drugs. Also on the CAMAG website, you can download application notes and an application note about the most relevant street drugs (A-118.1).
Answer: You have to try. There are protocols for plate impregnation and there is also one stationary phase called the chiral plate that has some modification with copper complexes. You will find also protocols in our CCBS literature database via the CAMAG website.
Answer: Regarding bioautography, you will also find a lot of information in the CCBS database, in the CBS journal, and you can also search for courses, so for example the University in Giessen/ Germany. They provide bioautography courses, sometimes in collaboration with the German Chemical Society and some are done via the TransMIT Center.
Answer: On top of the HPTLC PRO Module DEVELOPMENT, you will find three bottles for up to three different developing solvents, but they have to be have to be pre-mixed. Up to now there does not exist a CAMAG add-on for pre-mixing of the developing solvent.
Answer: We don't know yet exactly, but what I can tell you already is that the module derivatization has a nozzle changer that allows to have placed up to three nozzles inside and after spraying the nozzle will be automatically cleaned.
Answer: In a few months we will also launch the HPTLC PRO Module MS-Interface that allows to either directly connect to an MS or to elute selected zones with the integrated pump and with the collected fractions you can then go to the MS site.