High-Performance Thin Layer Chromatography (HPTLC) Plates

HPTLC is a sophisticated form of thin-layer chromatography (TLC) that provides superior separation efficiency and sensitivity. It adheres to rigorous regulatory standards by integrating validated methodologies for both qualitative and quantitative analysis. 

Section Overview:

• HPTLC Principle
• HPTLC vs. TLC
• Advantages of HPTLC over TLC
• HPTLC Cellulose Plates
• HPTLC Classical Silica Plates
• CN, Diol, NH₂-Modified Silica HPTLC Plates
• Premium Purity HPTLC Plates
• AMD HPTLC Plates
• LiChrospher^®HPTLC Plates
• RP-Modified Silica HPTLC Plates

HPTLC Principle

HPTLC separates components based on their varying affinity for the stationary phase and their differential solubility in the mobile phase. HPTLC utilizes the same basic principle as regular TLC but incorporates sophisticated equipment such as a high-pressure pump, a temperature-controlled chamber, and a scanner or detector to obtain improved resolution and sensitivity. The sample is applied to a thin layer of stationary phase, usually coated on a glass or aluminum plate. This plate is then placed in a developing chamber that contains the mobile phase. Through capillary action, the mobile phase passes through the stationary phase and separates the sample's components depending on their interactions with both phases. The individual constituents can be observed using detection techniques such as UV absorption, fluorescence, or chemiluminescence.

HPTLC vs. TLC

Unlike classical TLC, HPTLC uses optimized silica gel 60 plates containing much smaller particles. This results in a higher packing density and a smoother surface, which further reduces sample diffusion and produces compact bands or spots. This leads to faster analysis times and increased detection sensitivity compared to traditional TLC methods. Consequently, this results in accelerated analysis times and enhanced detection sensitivity compared to conventional TLC techniques.

Comparison of the Particle Size Distribution of TLC and HPTLC Plates

	HPTLC	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 (200 μm)
Typical migration distance	3 - 6 cm	10 - 15 cm
Typical separation time	3 - 20 min	20 - 200 min
Number of samples per plate	< 36 (72)	< 10
Sample volume	0.1 - 0.5 μL	1 - 5 μL
Detection limits UV absorption	100 - 500 pg	1 - 5 ng
Detection limits fluorescence	5 - 10 pg	50 - 100 pg

Advantages of HPTLC over TLC

• Higher Resolution: Superior separation of sample components due to finer particle size in the stationary phase.
• Faster analysis: The use of a high-pressure pump and optimized solvents   accelerates the separations to 3-20 minutes.
• Greater Sensitivity: 5 to 10 times more detection sensitivity than traditional TLC.
• Reduced Solvent Consumption: Requires less solvent for the mobile phase, making it more environmentally friendly and cost-effective.
• Quantitative Analysis: Provides consistent and crisp bands, making it perfect for precise quantification of analytes.
• Wider Range of Detection Methods: Supports advanced detection techniques such as fluorescence, chemiluminescence, and mass spectrometry integration.
• Improved Reproducibility: The automated application, development, and detection processes reduce human error, ensuring consistent results.
• Bioassay Integration: Easily interfaces with bioassays to improve effect-directed analysis.
• Direct MS Analysis: Enables defined zones to be directly analyzed by mass spectrometry (MS) after evaluation, eliminating the need to record every run, including matrix and background.

HPTLC Cellulose plates

Cellulose HPTLC plates are intended to facilitate the separation of hydrophilic substances through partition chromatography. The cellulose layers in the plates are composed of microcrystalline cellulose that is rod-shaped and of high purity. This results in a significantly reduced diffusion of analytes, which is essential for high-performance separations. The plates are available with or without a specialized fluorescent indicator, which is activated to produce intense blue fluorescent emission when exposed to long-wave UV light at 366 nm and short-wave UV light at 254 nm.

Typical applications of the plates include the analysis of nucleic acids, phosphates, carbohydrates, and amino acids. These plates are frequently utilized in metabolic studies and are notably beneficial for two-dimensional separations, such as amino acid "fingerprints."

HPTLC Classical Silica Plates

The classical silica HPTLC plates use an optimized silica gel 60 sorbent with a particle size of only 5 - 6 µm, compared to the 10 - 12 µm used in classical TLC. his results in an increased packing density, a smoother surface, and less band diffusion, resulting in compact sample zones. Using smaller particles and thinner layers (100 µm or 200 µm) improves sensitivity and speeds up processing. The classical silica HPTLC plates are available with either glass or aluminum backing in a variety of formats to meet a wide range of separation requirements. They are equipped with fluorescent indicators, either green fluorescent F254 or blue fluorescent acid-stable F254, both of which fluoresce at 254 nm under UV light.

The plates are well-suited for accurate quantitative studies, such as identifying substances in herbal medicines, performing advanced separations of pharmaceutical medications, conducting quality control and purity testing of complex materials, and detection of trace pollutants in food.

CN, Diol, NH₂-Modified Silica HPTLC Plates

HPTLC plates using modified silica have higher selectivity, making them excellent for complicated separations where unmodified silica falls short. These plates, which include a blue fluorescent, acid-stable UV indicator (F_254s), enable straightforward detection by fluorescence quenching, particularly for substances that absorb UV light at 254 nm.

CN and Diol-Modified Plates

CN and Diol-modified silica plates are moderately polar, making them ideal for both normal and reversed-phase systems. The CN-modified plate is made of silica gel 60 modified with a cyano propyl group, whereas the Diol-modified plate has a silica surface changed with a vicinal diol alkyl ether. The dual feature of the CN plate allows for unique two-dimensional separations by using the normal-phase mechanism in the first direction and the reversed-phase mechanism in the second.

NH₂-Modified Plates

NH₂-modified plates exhibit weakly basic ion-exchange properties and exceptional selectivity for charged molecules. These distinguishing characteristics allow the separation of substances such as nucleotides, purines, pyrimidines, phenols, and sulfonic acids with simple eluent combinations. For many applications, NH₂-modified silica plates provide an alternative to PEI cellulose. Furthermore, they enable the reagent-free identification of certain molecules (e.g., carbohydrates) via thermochemical fluorescence activation.

The separation process is less influenced by atmospheric humidity, making it more reliable under varying conditions. These plates effectively separate both highly non-polar and polar substances using aqueous solvent systems. Additionally, they exhibit no catalytic activity, which prevents the oxidative degradation of unstable compounds, ensuring the integrity of sensitive analytes throughout the separation process.

Premium purity HPTLC plates

Premium purity HPTLC plates are designed for high-performance, contamination-free separations, such as those used in pharmacopeia applications. Premium purity plates are carefully wrapped in plastic-coated aluminum foil to prevent the deposition of plasticizers (such as phthalates), which may emerge as unknown additional zones when employing medium-polar solvent systems like toluene/ethyl acetate (95/5). Furthermore, it prevents staining from derivatization reagents such anis aldehyde.

AMD HPTLC plates

Designed for even more demanding, automated multiple development (AMD) applications, AMD HPTLC plates have an extra-thin layer of 100 μm. The method uses repeated development of the plate in the same direction along with consistent gradient elution. Extremely narrow bands on AMD HPTLC plates enable the whole resolution of up to 40 components over a distance of only 60 mm. For complex investigations like qualitative and quantitative pesticide analysis, the unique qualities of AMD HPTLC plates are quite advantageous.

LiChrospher^® HPTLC plates

The LiChrospher^® HPTLC plates are the first to use spherical silica particles for thin-layer chromatography. These plates provide better performance and faster analysis than regular HPTLC plates, making them ideal for high-throughput examinations of complicated samples. The plates use spherical silica 60 with a 7 µm particle size and a narrow distribution, comparable to that used in HPLC. While LiChrospher^® plates offer a broad selectivity equivalent to that of corresponding HPTLC plates, the plate height, separation numbers, and velocity constants have all been enhanced. This leads to reduced analytical times and higher detection limits.

LiChrospher^® HPTLC plates reduce running times by 20% compared to normal HPTLC, providing highly compact spots or zones that improve detection sensitivity. These plates have low detection limits, making them appropriate for assessing complicated substances with low concentrations. They are particularly suited for applications such as trace analysis of pesticide mixtures and assays of pharmaceutical compounds, where precision and sensitivity are crucial for accurate separations and results.

RP-Modified Silica HPTLC Plates

Reversed-phase (RP) modified silica HPTLC plates provide additional selectivity and significantly broaden thin-layer chromatography applications. As a result, they are ideal for challenging HPTLC separations as well as HPLC pilot experiments. The RP-2, RP-8, and RP-18 plates are made of silica gel 60, which has been treated with aliphatic hydrocarbons. The chain length, together with the degree of modification, determines the plate's ability to tolerate water in the solvent system and has a significant impact on retention. Using the same solvent solution, migration time rises in the following order: RP-2, RP-8, RP-18.

RP-2 HPTLC sorbents have a stronger polarity and affinity for aqueous solutions, with a tolerance of up to 80% water. In contrast, the longer carbon chains, RP-8 and RP-18, may operate with up to 60% water in the solvent solution. The specially constructed RP-18W HPTLC plate has a lower degree of surface modification, thus it may be utilized with 100% water in the solvent solution.

With the RP-modified silica HPTLC plates, the results are less dependent on the atmospheric humidity. They allow the use of aqueous solvent systems and prevent catalytic activity, minimizing the risk of oxidative degradation in unstable compounds.