Thin-layer chromatography (TLC) is a special case of planar chromatography. TLC is being used since the mid-20th century for diverse separation purposes, and until now, it is generally done either manually or with a simple instrument set-up. It is a simple, flexible, and rapid tool for qualitative investigations. As there is no agreement on a standardized TLC methodology, it is challenging to get reproducible results between laboratories (if that is the method’s aim).
The term High-Performance Thin-Layer Chromatography (HPTLC) was introduced when plates with smaller particle sizes and dimensions were brought to the market. HPTLC is considered the evolution of classical TLC. With time, HPTLC was clearly defined by Compendia and became a concept that prioritizes the quality and reproducibility of the results. The HPTLC concept includes:
- HPTLC glass plates of fixed dimensions (20×10 cm) with layers of Silica gel (or other adsorbents) with particle sizes between 2 and 10 µm and a fluorescence indicator;
- Simple to sophisticated instruments, available for all chromatography steps;
- HPTLC software;
- Well-defined methods with optimized and standardized parameters;
- Qualified data through method-specific system suitability test (SST);
- Validated methods, compliant with cGxP;
- Traceable digital data.
A standardized and optimized TLC method on a 20×20 cm plate can be scaled down to run on a 20×10 cm HPTLC plate. Thus, HPTLC can be considered as miniaturized TLC.
The main differences between TLC and HPTLC are shown in the table below:
| TLC | HPTLC | |
| Principle | Planar chromatography | Planar chromatography |
| Primary focus | Simplicity, low cost | Reproducibility, separation power |
| Process | Flexible, no rules | Highly standardized methodology/ optimized parameters |
| Methods | Only a few parameters defined | Well defined and validated |
| Flexibility | Very high | None for validated methods High between methods |
| Target | Rapid, preliminary results | Reliable analytical results |
| Data structure | Simple chromatograms/ photographs | Traceable digital images/ scan data qualified by system suitability test (SST) on each plate cGMP compliant reporting |
| Samples/ references | Side by side on the plate | On the same or on different plates |
| Plate | TLC any format | HPTLC 20×10 cm |
| Instrumentation | None to simple | Simple to sophisticated |
| Cost | Very low | Medium to high compared to TLC Low per sample compared to HPLC |
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