Thin Layer Chromatography (TLC) - Principle, procedure

 

 

Thin Layer Chromatography TLC Principle Procedure


   What is Chromatography?

Chromatography means the separation of mixture of compounds. The components travel through a stationary phase at different speeds and get separated. The mixture is dissolved in the mobile phase and is carried through the stationary phase.

Thin Layer chromatography is a form of planar chromatography. The stationary phase is a TLC plate (silica gel) or alumina. The plate is run through a solvent which is known as the mobile phase.

The principle of TLC is where mixture components are separated between a fixed stationary phase and a liquid mobile phase by differential affinities between the two phases. The separation is through adsorption.

Thin-layer chromatography can be used to monitor the progress of a reaction, identify compounds present in a given mixture, and determine the purity of a substance.

 

What is High-Performance Thin Layer Chromatography?

HPTLC (High-Performance Thin Layer Chromatography) is an advanced version of TLC. TLC is qualitative whereas HPTLC is qualitative and quantitative. HPTLC works on similar principles as TLC but the practice is fully automated and GLP/USP/EP compliant. As a liquid chromatography technique, HPTLC can analyze most non-volatile organic samples with ease.

 

TLC Procedure

TLC is performed manually. Samples are applied on TLC plates. The plate (stationary phase) is silica gel backed by Alumina or glass. The plate is run through a solvent (Mobile phase) system for the separation of the samples.

Steps:

 1. Preparation of sample and plate for the experiment. Cut the TLC plate in 10x10, 20x10       dimensions. Mark the plate with a pencil at 70mm. 

2. Application of sample on TLC plate. TLC involves manual spotting whereas HPTLC has an automatic sampler for e.g., CAMAG ATS 4. You can spot the samples on the plate. Mark the sample application area with a pencil and start the spotting process.

3. Development of plate. The plate is kept in a solvent system such that the samples are not immersed in the mobile phase. For this purpose, use CAMAG TTC (Twin through chambers). Observe the plate while it is developed and remove the plate.

4. After development is finished. Air-dry the plate and observe the separated compounds. Only the compounds observed in white light can be seen by the naked eye. For UV active and fluorescent compounds, we need a UV chamber or CAMAG Visualizer.

5. Analysis of results requires interaction with detected compounds. CAMAG TLC-MS coupling uses Mass Spectrometry for the verification of the chemical structure of analytes. Analytes can be directly sent to an MS or the results can be collected for further analysis offline.

 

We offer free HPTLC training for using CAMAG HPTLC machines in Mumbai. Call us today on 9833830898 today!

 

Comments

Post a Comment

Popular posts from this blog

Factors Affecting Resolution in Chromatography

Image
  As we all know, Chromatography is a science of separation of mixtures into its components. There are several types of chromatography, each differing in the kind of stationary phase and mobile phase they use. There are many chromatographic techniques like HPLC, HPTLC and GC. Let us understand what is the resolution in Thin Layer Chromatography? Many times we use the term ‘Resolution’ but what exactly does it mean in TLC/HPTLC?      In Chromatography resolution is ability (of the method) to separate two peaks. In Thin Layer Chromatography or  High-Performance Thin Layer Chromatography , as it is a visual technique, we can also say it is the ability of the method to separate two bands or two peaks. Resolution is termed good when there is a difference of Rf 0.05 between two bands. Ideal resolution is considered when there is base to base separation between adjacent bands. What are the factors affecting resolution in high performance thin layer chromatography? HPTLC is modern version of T
Read more

How do You Test for Colorless Substances in Chromatography?

Image
  How Do You Test For Colorless Substances In Chromatography? by  Anchrom Lab   Thin Layer Chromatography/High Performance Thin Layer Chromatography is suitable for detection of organic and nonvolatile compounds. There are many compounds which do not have chromophoric group therefore we can not see them with naked eyes. Sometimes these compounds have UV/fluorescence. Such compounds can be detected by using suitable instrument like UV cabinet or CAMAG visualizer which can detect compounds in short UV i.e., 254nm, long UV i.e., 366nm and white light or if any compound is not UV active, a technique of derivatization can be used to make them visible. UV detection, spectra and derivatization are powerful tools which helps in detection of colorless substances in  High Performance Thin Layer Chromatography technique. How colorless substances are analyzed by HPTLC? HPTLC is a stepwise technique. One has to follow certain sequence while doing analysis. First step is application of sample on sui
Read more

Automate Your Analytical Processes with the Continuous Flow Analyzer

Image
  Automatic Continuous Flow Wet Chemistry Analyzer In the world of analytical chemistry, efficiency and reliability are paramount. Laboratories dealing with various sample matrices, such as sea water, drinking water, wastewater, environmental samples, soil, food, beverages, tobacco, and more, face the challenge of testing different parameters accurately and efficiently. Manual sample preparation methods can be complex and time-consuming. However, a revolutionary solution has emerged— the AMS Alliance FUTURA 3 Continuous Flow Analyzer. This cutting-edge instrument automates the testing process, providing a complete and reliable solution for laboratories seeking streamlined operations. Each FUTURA 3 console integrates a high precision 3-speed peristaltic pump (medium, fast (startup and washout more speed) and slow (for low consumption of reagents)), a removable drawer for reagents, an analytical manifold, a heater, a detector, and a built-in display for the presentation of essential diag
Read more