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Monday, September 6, 2021

Principle and Procedure of HPTLC Chromatography

Learn the principle, types, applications and how HPTLC works.

What is High-Performance Thin Layer Chromatography?

The HPTLC technique is an automated and sophisticated form of thin layer chromatography with superior and advanced separation efficiency and detection limits and is often an exceptional alternative to high-performance liquid chromatography (HPLC) and gas chromatography (GC). The high-performance thin-layer chromatography is also known as flat-bed chromatography or as planar chromatography.

HPTLC Principle:

The HPTLC works on the same principles as TLC such as the principle of separation is adsorption. The mobile phase or solvent flows through the capillary action. The analytes move according to their affinities towards the stationary phase (adsorbent). The higher affinity component travels slower towards the stationary phase. A low-affinity component travels rapidly toward the stationary phase. On a chromatographic plate, then, the components are separated.

Experimental Procedure of HPTLC:

Before beginning an experiment, we must recognize the various components essential to perform the process.

1. A tool suitable for sampling as bands to monitor the size and position of the test, as well as the sample volume applied.
2. An appropriate chromatographic chamber that provides developing distance and control of saturation.
3. A device appropriate for controlling stationary phase behavior through relative humidity.
4. A tool appropriate for reproducible drying of the developed plate.
5. Appropriate equipment for reagent transfer and heating.
6. A tool for electronic documentation of chromatograms.

Stepwise procedure:

1. Sample Preparation:
This requires a highly concentrated solution since much less sample quantity needs to be applied. The plate’s solvents must be non-polar of the volatile type. Polar solvents are commonly used to dissolve samples for reversed-phase chromatography.
2. Selection of Chromatographic Layers:
The layer of HPTLC is available in the form of very fine particle size silica gel pre-coats which are widely used as adsorbents.
3. Pre Washing:
To water vapor or volatile impurities, the plates must be cleaned. It may be clean with a suitable solvent such as methanol.
4. Conditioning:
Plates are placed in an oven at 120° C for 15 to 20 minutes to perform conditioning.
5. Sample Application:
The size of the sample spot is not greater than 1 mm in diameter. There are various methods for spotting samples. One is a self-loading capillary in which small quantities of samples can apply to the HPTLC plate.
6. Pre-Conditioning:
Saturation is necessary for highly polar mobile phases although there is no need for saturation for low polarity mobile phases.
7. Mobile Phase of HPTLC:
Through trial and error, the mobile phase of the suitable solvents is to be selective.
8. Chromatographic Development:
The linear development method in high-performance thin-layer chromatography is the most common technique here the plate is positioned vertically in an appropriate container with a solvent or mobile phase. The mobile phase is generally fed by capillary action and both sides may produce chromatograms.
9. Detection of spot and Scanning:
The instrument has attached to computer and data recording devices. The development of spots is viewed as peaks at wavelengths of selected UV regions. The height and the area of the peaks are determined by the instrument and recorded as a percentage.

HPTLC Applications:

  • High-performance thin-layer chromatography is used to analysis of molecules in both qualitative and quantitative terms.
  • It can estimate the concentration of components although TLC can only separate components.
  • It can analyze a complex structure or a very small amount of compounds.
  • This method is used in the food industry to evaluate nutrients, beverages, vitamins, and pesticides in fruit, vegetables, and other foodstuffs.
  • It is useful in the forensic detection of substances, including adulteration, overdose, counterfeit drugs, and drug misuse.
  • To identify the substances including drug abuse, overdose, adulteration, counterfeit drugs it is used forensic dept.
  • HPTLC is used in pharmaceuticals for quality control.
  • It is used for the analysis of forced degradation studies, stability testing, and to check the presence of impurities in the drug.

Advantages of HPTLC:

  • More than one analyst works on the system simultaneously.
  • It can be sharable, as it is not devoted to any sample.
  • The pre-coated plates of HPTLC are available at low prices.
  • There is less maintenance cost as compared to other equipment.
  • It has a wide range of stationary phases.
  • It has no risk of contamination, since the use of the freshly prepared mobile phase and stationary phase.
  • Mobile phases are not required for filtration and degassing such as HPLC.
  • It is highly sensitive, reproducible, and precise as compared with a thin layer chromatography.

Disadvantages of HPTLC:

  • Short separation bed is a major disadvantage of HPTLC.
  • A limited number of samples per plate can be tested.
  • Sometimes silica gel is present during detection.

Commonly asked questions on HPTLC chromatography are as follows.

What is the principle of HPTLC?
HPTLC has similar approaches and follows the same physical principles as TLC, i.e. adsorption chromatography.

What type of chromatography is HPTLC?
HPTLC is a type of planar chromatography and is the most advanced instrumental method of TLC.

What is the HPTLC plate made of?
Silica gel is widely used for plate preparation. HPTLC plates are available on a glass or aluminum support with various sorbent materials.

What is the major advantage of high-performance thin-layer chromatography?
Its main advantage is that it is capable of simultaneously analyzing multiple samples.


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