High performance Thin layer chromatography(HPTLC) is a sophisticated form of Thin layer chromatography. It provides superior separation efficiency. The concept of HPTLC includes validated methods for qualitative and quantitative analysis and fulfills all requirements to be used in the fully-fledged environment. It is also known as planar or flatbed chromatography.
A particular advantage of this technique is that it can be combined with mass spectrometric approaches, thus allowing detailed structural characterization of carbohydrate and ceramide moieties. Furthermore, HPTLC can be easily used in the context of so-called overlay techniques
Principle of HPTLC
HPTLC possesses a similar principle to that of Thin-layer chromatography(TLC) which is principle of adsorption chromatography. The solvent used in the mobile phase flows due to capillary action and the component moves according to their affinity towards adsorbent and eluent.
The component with more affinity towards the stationary phase travels faster and the component with less affinity towards the stationary phase travels slower.
Usually, HPTLC plates are made using alumina rather than silica because of more conventional separation. Thus the stationary phase used is alumina. For the mobile phase usually, polar solvents are used.
Process and Performance of HPTLC
Unlike Thin layer chromatography, High Performance Thin Layer Chromatography requires a more sophisticated presentation and assemblage of instruments. The usual chromatographic techniques are amplified with the help of attached electronic detectors. Therefore the procedure of HPTLC includes varied steps.
1. Sample Preparation
The silica or alumina pre-coated chromatography plates are used for the sample dropping. The solvents used could be polar. Sample and standard substances used should be dissolved in the same solvent.
The selection of a chromatographic plate or layer is the most important part of sample preparation. The plates with different support materials and sorbent layers with different formats and thickness are used as a base plate or chromatographic base.
The sorbent thickness can vary around 100-250 micrometers. As HPTLC involves visualization under an external source of light like UV, binders like gypsum and starch are used as fluorescent indicators. Other than alumina and silica, cellulose microcrystals are used which sometimes contain amino acids, peptides, sugars, etc.
The chromatographic plate needs to be pre-washed to remove water vapors or volatile temperatures. It is done usually using methanol.
To prevent the glitches in retardation factor values(Rf values), plates are pre-conditioned in chamber saturation. To complete the saturation, plates are kept for twenty minutes in a clean drying oven at 120 degrees celsius. It is placed after lining the oven with filter paper for twenty minutes before the saturation procedure to uniformly distribute the solvent vapors.
2. Sample Application
The size of the sample spot applied must not exceed 1 mm in diameter. The usual concentration of the sample could vary around 0.1-1 micrograms per microlitre for errorless qualitative analysis. The application can be of varied methods, Manual, Semi-automatic, and Automatic application.
Manual: It is usually done by a Nanomat. It serves for easy application in the form of spots and this can also be used in TLC.
Semi-automatic: It is done using an instrument called Linomat and the samples are sprayed onto the chromatographic layers.
Automatic: Samples are either applied as spots through contact transfer or as bands or rectangles using spray techniques. Rectangular application is preferred due to the accuracy it portrays.
3. Chromatographic Development
They are developed in flat bottom chambers or twin chambers. After the separation, the mobile phase is removed to avoid contamination of the laboratory. It can be developed vertically, horizontally, in multiple methods per Automated multiple developments (AMD).
After removal of the mobile phase from the developed plate by heating, zones are detected on the layer by their natural color, natural fluorescence, quenching of fluorescence, or as colored UV-absorbing, or fluorescent zones after reaction with a reagent (post chromatographic derivatization).
Zones with fluorescence or quench fluorescence are viewed in cabinets that incorporate short-wave (254 nm) and long-wave (366 nm) UV lamps. Detection under UV light is the first choice as it is nondestructive. An important advantage of the off-line operation of TLC is the flexibility afforded by the use of multiple methods for zone detection and identification.
For example, the layer can be viewed under long and short-wave UV light, followed by one or more chromogenic, fluorogenic, or biological detection methods. Many hundreds of reagents and detection methods have been described in various literature sources.
In TLC, derivatization is used to enable the detection of separated compounds that are colorless and cannot be visualized with UV radiation or fluorescence. A suitable reagent is applied to the TLC plate, which reacts with the sample compounds and transforms them into detectable derivatives.
Depending on the sample and application goals, the detection reagent may be applied to the TLC plate by spraying, dipping (immersion), or as part of the solvent system.
The procedure is further detected using high volt detectors and read through graphs. Most modern HPTLC quantitative analyses are performed in situ by measuring the zones of samples and standards using a chromatogram spectrophotometer usually called a densitometer or scanner with a fixed sample light beam in the form of a rectangular slit.
Generally, quantitative evaluation is performed with the TLC Scanner 3 using winCATS software. It can scan the chromatogram in reflectance or transmittance mode by absorbance or by fluorescent mode.
Scanning speed is selectable up to 100 mm/s. Spectra recording is fast. Calibration of single and multiple levels with linear or non-linear regressions is possible.
Applications of High Performance Thin Layer Chromatography
- In the pharmaceutical industry it is used for quality control, content uniformity, identity/purity check.
- In food industry it is used for Quality control, additives, pesticides, stability testing, and analysis of submicron levels of aflatoxins, etc.
- It can also be used for clinical Applications in metabolism studies such as drug screening, stability testing, etc.
- Industrial Applications of HPTLC include, process development and optimization, in-process Q.C. check, validation, etc.
- In Forensic science, it is generally used for the investigation of Poisoning cases or any such case where separation of components is highly necessary.