What is the Preparation of Thin-Layer Chromatography?
High-Performance Thin Layer Chromatography (HPTLC) is the modern version of TLC wherein the principles are the same but the practice is fully automated and GLP/USP/EP compliant.
Before starting any method on TLC/HPTLC, one should have prior information about the sample/analyte so that it helps in designing the experiment. Literature review is one of the most important aspects before starting the analysis.
What should I know about my sample before starting TLC/HPTLC analysis?
The aim of the analysis has to be decided before starting the literature review. Refer to common books available on TLC/HPTLC. Please search the following points when doing literature review to analyse a sample by HPTLC.
- Bases for methodology supporting the aim (e.g., Presence of Alkaloids reported in the sample).
- Reported quantities of analyte/marker in botanicals/ formulation.
- Sample processing details are mentioned in literature.
- Related methods on TLC/HPTLC reported for the sample or different sample matrices that can be adopted for the analysis.
- Solubility, polarity, volatility, and stability of standards/ samples used in the analysis.
- Derivatization information on standards and samples (if applicable).
What are the requirements before starting TLC/HPTLC analysis?
Use AR/GR grade solvents for analysis. If you are working on/along with some standards use at least TLC grade standards.
Sample preparation is one of the most important steps for the proper extraction of the analyte of interest. Depending on the sample matrix and aim the analysis extraction procedure should be selected/optimized.
- Selection of suitable stationary phase:
Depending upon the aim of the analysis, a suitable stationary phase should be selected. Generally, Merck TLC plates (Aluminium) Silica gel 60F254 plates work for most of the sample. Still, some specific samples may require usage of different plates/stationary phase e.g., for sugar analysis cellulose plates can be used or for oil samples RP plates are required.
- Selection and optimization of mobile phase:
Depending on the polarity of the molecule to be analyzed, a suitable mobile phase is selected or if you do not have any idea about polarity of the sample to be analysed, dissolve the sample in solvent like methanol and run that sample in mobile phases having different polarities e.g., polar, midpolar and nonpolar.
If a sample shows some response or migration in any of these mobile phase, one can develop a mobile phase having similar polarity or you can optimize the respective mobile phase accordingly.
- Guidelines for analysis of herbal samples:
Follow proper guidelines for HPTLC analysis and suitable instruments e.g. There are a range of CAMAG TLC/HPTLC instruments right from the application to scanning and derivatization for every step of HPTLC analysis. For herbal analysis or for analysis of AYUSH samples USP guidelines should be followed (Chapter 203, 1064, 2254 and 621).
All the Educational institutes, Government Research centres e.g., AYUSH Labs, Herbal drug manufacturing units, commercial testing labs can follow these guidelines for analysis of herbal samples.
Once plate application and development are complete, the next step is to observe and document the image using suitable devices like TLC photo documentation system /Visualizer. Plates should be scanned using suitable scanning parameters using instruments like CAMAG Scanner 4. If the compound is UV active spectra should be recorded for identification and quantification of analyte if required.
Selection of suitable derivatizing reagent if required is necessary especially when the compound is not UV active. Now-a-days TLC/HPTLC can also be coupled with mass spectrometry by means of TLC-MS Interface for identification of compounds.
Anchrom Enterprises Pvt. Ltd is the leader in HPTLC in analysis from last 40 years. Please contact us at lab@anchrom.in for HPTLC analysis of plant extracts, drugs, food, and cosmetic samples.
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