The Ultimate Guide to Selecting the Perfect TLC and HPTLC Plates
High Performance Thin layer chromatography is a technique used to separate and identify different components of a mixture. It involves the use of a stationary phase (the TLC/ HPTLC plate) and a mobile phase (the solvent).
The TLC/HPTLC plate is a thin layer of adsorbent material, usually coated on a glass or aluminium foil backing.
When the Plate (Stationary phase) is developed in the mobile phase, the different components of the mixture travels at different rates depending on their interaction with the stationary phase.
The choice of the stationary phase is very crucial, as it determines the selectivity.
The choice should be based on the properties of your sample and your application goals. To choose the suitable TLC/ HPTLC plate, consider the following factors.
Consider the following differences between both the plates before choosing.
|Features of HPTLC versus classical TLC||HPTLC||Classical TLC|
|Mean particle size||5 – 6 µm||10 – 12 µm|
|Particle size distribution||4 – 8 µm||5 – 20 µm|
|Layer thickness||200 µm (100 µm)||250 µm|
|Plate height||12 µm||30 µm|
|Typical migration distance||3 – 6 cm||10 – 15 cm|
|Typical separation time||3 – 20 min||20 – 200 min|
|Sample volume||0.1 – 0.5 µl||1 – 5 µl|
|Detection limits absorption||100 – 500 pg||1 – 5 ng|
|Detection limits: emission (fluorescence)||5 – 10 pg||50 – 100 pg|
Silica gel (unmodified, modified/bonded, chiral and high purity), Aluminum oxide Plates and Cellulose Plates.
- Silica gel is the most common sorbent and is available in different variations. Unmodified silica gel 60 is widely used, providing excellent separation for a wide range of substances. It covers nearly 80% of both adsorption and partition TLC/HPTLC applications.
It enables separation of a large range of diverse substances, such as alkaloids, anabolics, carbohydrates, fatty acids, glycosides, lipids, mycotoxins, nucleotides, peptides, pesticides, steroids, sulfonamides, surfactants, tetracyclines and many others.
- Silica gel 60 G plates utilize gypsum as a binder, making them suitable for customers following older monograph methods.
- Plates with a concentrating zone consist of two silica adsorbents, allowing for efficient sample concentration and clean-up. Clean-up step for analytes in complex matrices, such as oils and cosmetics.
Image 1: concentrating zone plate
- GLP-plates are high-quality silica gel 60 plates with traceability features for easy documentation.
Modified silica TLC/HPTLC plates offer enhanced selectivity, with RP-modified silica plates providing a non-polar stationary phase and amino-modified NH2 silica plates offering weakly basic ion-exchange characteristics for separation of charged moleclues.
CN and Diol-modified silica plates are moderately polar and suitable for both normal phase and reversed phase systems.
- ProteoChromTM HPTLC plates optimized for highly sensitive separations of peptides and protein digests.
- LiChrospher® HPTLC plates based on spherical silica particles for maximum performance and high-throughput analyses of complex samples.
- MS-Grade plates offering exceptional sensitivity and low background for trace analysis in the nanogram range.
- Preparative layer (PLC) plates allow separation and purification of samples in larger quantities, with options for unmodified and modified layers, various thicknesses, and the inclusion of fluorescent indicators.
- Aluminum oxide Plates which exhibits similar selectivity, although slightly different from silica are Excellent Separators of Basic and Neutral Compounds.
- Cellulose Plates: These are available as either microcrystalline or fibrous cellulose. Spots are generally more compact when separated on layers of microcrystalline cellulose than when separated on layers of fibrous cellulose. The best choice for analyzing substances with exchange- active ionic groups, such as amino acids, peptides, nucleotides and nucleosides.
- Kieselguhr Plates is a natural diatomaceous earth that can be used for the separation of inorganic ions, herbicides or steroids.
There are glass and aluminum foil TLC and HPTLC plates are available. Depending on type of method and analysis the backing material of the plate that has to be used can be selected.
The thickness of the stationary phase is another important consideration. Thicker layers offer better separation but slower migration, while thinner layers provide faster migration but lower resolution.
The most commonly used thicknesses are 0.25 mm and 0.5 mm. Preparative TLC plate is 500μm, Glass plates are 250 μm, 200 μm for aluminum and TLC/ HPTLC-MS is of 100 μm.
The particle size of the stationary phase material directly affects the separation efficiency.
Smaller particle sizes provide greater surface area and increased resolution, making them ideal for complex mixtures. However, they also lead to higher back pressure, slower flow rates, and longer development times.
A smaller particle size provides better resolution, but a larger particle size may be necessary for certain applications.
TLC/HPTLC plates come be used in a variety of sizes, it’s important to choose the right size for your application.
If you plan to analyze a small number of samples, a smaller plate may be sufficient. However, if you need to analyze more number of samples, a larger plate may be more appropriate.
The solvent system you plan to use for your analysis is another important factor to consider when choosing a TLC/HPTLC plate. Different solvent systems require different stationary phases to achieve optimal separation.
For example, if you plan to use a non-polar solvent system, you should choose a TLC/HPTLC plate with a non-polar stationary phase like Reverse Phase plates.
Alternatively, if you plan to use a polar solvent system, you should choose a TLC/HPTLC plate with a polar stationary phase like Normal Phase plates.
Finally, you should consider the detection method you plan to use. Different detection methods require different types of TLC/HPTLC plates.
For example, if you plan to use ultraviolet (UV) detection, you should choose a TLC/HPTLC plate with a UV-active stationary phase. Alternatively, if you plan to use fluorescence detection, you should choose a TLC/HPTLC plate with a fluorescent stationary phase.
Selecting the perfect TLC/HPTLC plate is essential for successful chromatographic separations. Understanding these parameters will help you achieve optimal separation and identification of components in your mixture.
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Reference: TLC Brochure by Sigma Aldrich