High Performance Thin Layer Chromatography

High Performance Thin Layer Chromatography (HPTLC) is a sophisticated form of TLC, which provides superior separation efficiency. The HPTLC concept includes validated methods for qualitative and quantitative analysis, and fulfils all quality requirements for use in fully regulated environments. The process steps of HPTLC are identical to classical TLC.

What is HPTLC chromatography

High-Performance Thin Layer Chromatography (HPTLC) is a sophisticated form of TLC, which provides superior separation efficiency. The HPTLC concept includes validated methods for qualitative and quantitative analysis, and fulfils all quality requirements for use in fully regulated environments. The process steps of HPTLC are identical to classical TLC.

The main difference between them is in the characteristics of the separation plate. HPTLC plates are based on optimized silica gel 60 with a significantly smaller particle size than used for classical TLC. This allows a higher packing density and a smoother surface. Hence, sample diffusion is reduced, resulting in compact bands or spots. Furthermore, the smaller particle size and thinner layer significantly increase detection sensitivity and analysis speed.

HPTLC Chromatography Principle

High Performance Thin Layer Chromatography is capable of separating organic compounds. Principle of HPTLC has similar approach and employs the same physical principles of TLC (adsorption chromatography) i.e. the principle of separation is adsorption. 

The mobile phase solvent flows through because of capillary action. The components move according to their affinities towards the adsorbent. The component with more affinity towards the stationary phase travels slower. The component with lesser affinity towards the stationary phase travels faster. Thus, the components are separated on a chromatographic plate.

HPTLC Chromatography Instrumentation

High Performance Thin Layer Chromatography Instrumentation

a) Auto-Sampler

The primary difference between TLC and HPTLC is the sample application part. In TLC, you would manually spot the sample on a silica plate using a capillary tube. An HPTLC auto-sampler however, is a precision instrument that applies a specified amount of sample by spraying it using nitrogen gas. Also, a computer system connected to the auto-sampler is used to feed data about the amount of sample to be applied and the number, width, and position of the bands of the samples.

b) Developing Chamber

Usually, a rectangular glass chamber is used for developing High Performance Thin Layer Chromatography plates, similar to TLC. Here, you saturate the chamber for at least 20-30 minutes by pouring a solvent and covering it.

You can also place a filter paper dipped in the solvent system to allow even distribution of the vapours. After saturation, you must place the plate vertically in the chamber. You must make sure that the sample spots remain above the level of the solvent.

There are two main types of vertical developing chambers viz. flat bottom chamber and a twin trough chamber (this one has a ridge at the base of the chamber dividing it into two troughs). The solvent consumption is less in twin trough chambers as compared to the flat bottom ones.

b) TLC Scanner

When you are scanning your compound for the first time, you can choose a multi wavelength scan (ranging from 190 to 900 nm) to determine the best wavelength for your compound. The best wavelength would show sharper peaks in comparison to others. Once you have found a suitable wavelength for your compound, you can select that particular wavelength every time you scan your plate.

Steps of HPTLC chromatography

a) Selection of plates

Handmade plates: Handmade plates which are made up of cellulose and other materials which are not used much now a day. Pre-coated plates: The plates with different support materials and sorbent layers with different format and thickness are used for qualitative and quantitative analysis. Sorbents used in plates-Silica gel 60F, Aluminium oxide, Cellulose, silica gel chemically modified. Smaller particle size of silica helps in greater resolution and sensitivity.

b) Layer pre-washing

The main purpose of the pre-washing is to remove impurities which include water vapours and other volatile substances from the atmosphere when they get exposed in the lab environment. In case of silica 60F (most widely used sorbent) the major disadvantage of this sorbent is that it contain iron as impurity. This iron is removed by using Methanol: water (9:1), this is the major advantage of the step of pre-washing.

c) Activation

Freshly opened box of High Performance Thin Layer Chromatography plates doesn’t need activation. If plates exposed to high humidity or kept in hand for longer time then activation is required and its activation results by removing moisture. The plates are activated by placing in an oven at 110 – 1200C for 30 min, this step will removes water that has been physically absorbed on surface at solvent layer. Activation at higher temp and for longer time is avoided which leads to very active layer and there is risk of sample being decomposed.

d) Sample preparation

It’s important to prepare proper sample for successful separation. Sample and reference substances should be dissolved in the same solvent to ensure comparable distribution at starting zones. It needs a high concentrated solution, as very less amount of sample need to be applied. After that dry the plates and store in dust free atmosphere.

e) Sample application

Usual concentration range is 0.1-1µg/µl, above this causes poor separation and volume recommended for High Performance Thin Layer Chromatography-0.5-5μl. The size of sample spot applied must not exceed 1mm in diameter. Problem from overloading can be overcome by applying the sample as band.

f) Mobile phase selection

Chemical properties of analytes and sorbent layer factors should be considered while selection of mobile phase. Various components of Mobile Phase should be measured separately and then placed in mixing vessel. The less amount of mobile phase is required then TLC. This prevents contamination of solvents and also error arising from volumes expansion or contraction on mixing. Multi component mobile phase once used not recommended for further use due to different evaporation and adsorption by layer.

g) Pre-conditioning

Un-saturated chamber causes high Rf values. Saturated chamber by lining with filter paper for 30min prior to development-uniform distribution of solvent vapours-less solvent for the sample to travel-lower RF values. For low polarity mobile phase there is no need of saturation. However saturation is needed for highly polar mobile phase.

h) Development

Plates are spotted with sample and air dried and placed in the developing chambers. Automatic multiple development, Circular, anti-circular device and multiple developments are some other methods. After development, remove the plate and mobile phase is removed from the plate to avoid contamination of lab atmosphere. Dry in vacuum desiccators with protection from heat and light.

i ) Detection

Detection under UV light is first choice. Non-destructive and spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length). Spots of non-fluorescent compounds can be seen fluorescent stationary phase is used – silica gel Gf.  Non-UV absorbing compounds like ethambutol, dicylomine dipping the plates in 0.1% iodine solution.

j) Scanning

The scanner converts band into peak and peak height or area is related to the concentration of substance on spot/band. The peak height and area under spot are measured by instrument and recorded.

Applications of HPTLC chromatography

High Performance Thin Layer Chromatography is one of the most widely applied methods for the analysis in pharmaceutical industries, clinical chemistry, forensic chemistry, biochemistry, cosmetology, food and drug analysis, environmental analysis, and other areas.

a) Pharma

High Performance Thin Layer Chromatography is also used in analysing the purity and efficacy of many pharmaceutical preparations and dosage forms.

b) Testing

The High Performance Thin Layer Chromatography technique is rapid, comparatively simple, robust, and extremely versatile. HPTLC not only confirm but also establish its identity. It is also an ideal screening tool for adulterations and is highly suitable for evaluation and monitoring of cultivation, harvesting, and extraction processes and testing of stability.

c) Other Fields

In recent years, High Performance Thin Layer Chromatography is a globally accepted practical solution to characterize small molecules in quality assessment throughout the developing world. HPTLC is used for purity control of chemicals, pesticides, steroids, and water analysis. HPTLC is also widely used for analysis of vitamins, water-soluble food dyes, pesticides in fruits, vegetables, and other food stuffs.

Advantages of HPTLC chromatography

1. More than one analyst works on the system simultaneously.

2. HPTLC can be sharable, as it is not devoted to any sample.

3. The pre-coated plates of HPTLC are available at low prices.

4. There is less maintenance cost as compared to other equipment.

 5. HPTLC has a wide range of stationary phases.

6. HPTLC has no risk of contamination, since the use of the freshly prepared mobile phase and stationary phase.

7. Mobile phases are not required for filtration and degassing such as HPLC.

8. It is highly sensitive, reproducible, and precise as compared with a thin layer chromatography.

Limitations of HPTLC chromatography

1. Limitations of HPTLC include short separation bed (62 mm effective length), limited number of samples per plate, presence of silica gel during detection (unless MS is the detector).

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