HPTLC-High performance thin layer chromatography
HPTLC 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 also easy detection and quantification of separated compounds.
The advancement concerning 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 size less than 10μ. It is also uniformly spread and dried.
In TLC chromatography a thick solution of stationary phase is poured on to 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 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, C18 are used for reverse phase chromatography.
In thin layer chromatography mostly silica gel is used.
3. Auto-sampling is present which help minimize handling errors. The techniques like manual spotting and streaking in TLC are time-consuming with possible human error.
4. Greater efficiency in the separation due to the presence of smaller and uniform size particles in the stationary phase. Smaller particle size enhances the surface area of adsorption. While 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 help to chose between isocratic or gradient mode of chromatography. For 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 ratio’s as per the need of the chromatogram.
Detectors and recorders:
Auto-detection of compounds from chromatogram is possible by using built-in UV and fluorescent detectors system.
Recording and storage of data from chromatograms are done by utilizing HPTCL data software in a computer.
Advantages of HPTLC over TLC:
♦ Samples in minute quantities like in nano-gram range can be detected using HPTLC.
♦ Handling and human errors are minimum due to automation.
♦ Better accuracy and sensitivity than TLC.
♣ The system is many folds expensive than TLC.
♣ Bulky instrumentation and large space requirement.
♣ HPTLC Requires stringent condition of operation like 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 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.