How to identify whether a reaction has been complete using TLC?
Reaction monitoring is a process that can be carried out during research and manufacturing process. During R & D, reaction monitoring can be used to optimize process conditions. In the manufacturing process, reaction monitoring can help to make sure that the reaction is progressing as expected.
Thin Layer Chromatography for Reaction Monitoring:
There are number of instrumental techniques used to monitor reactions. Thin Layer Chromatography is one of the most popular technique used in pharma industries for reaction monitoring. As stated above, it can be used to monitor progress of a reaction, identify compounds present in a given reaction mixture and determine the purity of a substance.
Three spots/bands are made on the TLC, one with the Starting material (SM), another one with the reaction mixture (RM), and another one in the middle (co-spot or cross-spot) in which you put both a solution of the SM and the reaction mixture. This way you can clearly visualize, after elution, that your SM actually reacted to form a new product. This is particularly important if both SM and product have very similar Rf, and it is difficult to see if you actually have a new product or just SM in the reaction mixture.
TLC and HPTLC difference:
Although, TLC is useful for reaction monitoring it has many drawbacks or limitations like it is only qualitative technique, non GLP compliant and prone to manual errors. These limitations can be overcome by subjecting to HPTLC analysis. HPTLC is modern version of TLC wherein the principles are same but practice is fully automated and GLP/USP/EP compliant. HPTLC has latest generation equipment with USP compliant software. It is 21CFR Rule 11 compliant designed for rapid analysis, more output at reduced cost.
CAMAG HPTLC in pharmaceutical analysis:
CAMAG HPTLC system has many applications in pharmaceutical analysis like identification, quantification and validation studies of API, identification of known/unknown impurities, content uniformity test (CUT studies), cleaning validation and reaction monitoring etc.
Advantages of HPTLC for drug analysis -In reaction monitoring:
A number of enhancements can be made to the original method to automate the different steps, to increase the resolution achieved with TLC and to allow more accurate quantitative analysis. This can be achieved by performing High performance Thin Layer Chromatography (HPTLC).
Unique features of HPTLC are that it is the fastest, simplest, most economical and flexible, “visible” technique which can analyse in parallel more than 100 samples. It is risk free and multiple detections can be made without repeating chromatogram.
While doing reaction monitoring, samples from many batches can be applied simultaneously on the same plate, solvent requirement and analysis time is significantly less as 20x20cm plate requires only 20ml mobile phase and plate development time is roughly 20-30 minutes.
HPTLC Chromatography instrumentation:
CAMAG, the world’s pioneer and leader in HPTLC instrumentation has introduced range of instruments/modules for each step in HPTLC i.e., application(CAMAG Linomat 5/ATS4), development(CAMAG ADC2), photo documentation(CAMAG Visualizer 1 / 2) and derivatization (CAMAG Derivatizer).
One can get very clear picture about reaction progress as known/unknown intermediates and products can be visualized and identified by Rf values, spectral evaluation and if required by using suitable derivatizing reagent. Even we can go for quantitative analysis of the respective components and if unknown intermediates are observed, that can be further subjected to MS analysis by means of HPTLC-MS interface or FTIR for further identification and characterisation of unknown compound.
Thus, HPTLC has many advantages in the reaction monitoring process whether it is research or manufacturing process.
- Application of thin layer chromatography for qualitative analysis of gunpowder in purpose of life prediction of ammunition, Namir H. et al., International Journal of Biosensors and Bioelectronics, 5(1):4-12, 2019.
Dr. Manjusha Phanse
Anchrom Enterprises Pvt. Ltd is one of the leaders in HPTLC in food analysis. Please contact us at email@example.com for HPTLC analysis of plant extracts, drugs, ingredients in cosmetics, and forensic science.