HPTLC is a sophisticated form of Thin-Layer chromatography (TLC).
In full form, it is High-Performance Thin Layer Chromatography.
This is so-called because of the features like the greater efficiency and higher resolution.
It has added advantages like auto-sampling, faster development of spots, and easy detection and quantification of separated compounds.
The advancement in better efficiency and resolution is due to the
1) Use of precoated HPTLC plates with an optimized stationary phase. The stationary phase has smaller particles with a size less than 10μ. It is also uniformly spread and dried.
In TLC chromatography, a thick solution of the stationary phase is poured onto the plates.
The solution is spread and allowed to dry.
This is a time-consuming process.
Further, there are chances of non-uniform distribution of the stationary phase on the plate, which can lead to errors in the chromatogram.
2) A choice to use a wide variety of stationary phases is possible in HPTLC. Silica gel is used for normal-phase chromatography.
While materials like C8 and C18 are used for reverse-phase chromatography.
In thin-layer chromatography, mostly silica gel is used.
3. Auto-sampling is present, which helps minimize handling errors.
Techniques like manual spotting and streaking in TLC are time-consuming and prone to human error.
4. Greater efficiency in the separation due to the presence of smaller and uniform-sized particles in the stationary phase.
Smaller particle size enhances the surface area of adsorption.
The uniform size enabled better flow and movement of sample particles.
5. Better and efficient solvent usage:
a) There is a minimal requirement of solvents due to the presence of advanced development chambers, which require less amount of solvents for developing the chromatograms.
b) Online mixing of solvents helps to choose between the isocratic or gradient mode of chromatography.
For the isocratic mode, a single solvent is required during chromatogram development.
But for gradient mode, a mixture of solvents in a predefined ratio is required.
In HPTLC, the mixing of solvents helps to change the solvent ratios as per the needs of the chromatogram.
Detectors and recorders:
Auto-detection of compounds from a chromatogram is possible by using the built-in UV and fluorescent detector system.
Recording and storage of data from chromatograms are done by utilizing HPTCL data software on a computer.
Advantages of HPTLC over TLC:
♦ Samples in minute quantities, like in the nano-gram range, can be detected using HPTLC.
♦ Handling and human errors are minimized due to automation.
♦ Better accuracy and sensitivity than TLC.
Disadvantages:
♣ The system is manyfold more expensive than TLC.
♣ Bulky instrumentation and large space requirement.
♣ HPTLC requires stringent conditions of operation, such as a dust-free environment and temperature-controlled conditions.
♣ A technically skilled person with the knowledge to run the system is needed.
Application of HPTLC:
1) For the detection and analysis of components of phytochemicals obtained from plants. For analysis of compounds of medicinal chemistry & organic chemistry.
2) In TLC, one can identify the elements of an extract. Whereas using this method, one can even estimate its concentration.
2) Compounds having a complicated structure or those available in very scarce quantities can be analyzed.
