TRI Instrument Focus: HPTLC
- TRI Princeton
- 3 hours ago
- 2 min read
HPTLC: A KEY TOOL FOR THE ANALYSIS OF MATERIALS
High-performance thin layer chromatography, also abbreviated to HPTLC, is a specialized technique that is an extension of thin layer chromatography (TLC), an analytical technique used extensively within chemistry. The value of HPTLC is that it is efficient in the quantitative analysis of compounds. The main difference between TLC and HPTLC, is that HPTLC uses TLC plates with finer particle sizes on the stationary phase, resulting in better resolution during separation and lower limits of detection. While visual inspection can sometimes be undertaken, sample bands can also be detected using UV, fluorescence spectroscopy, mass spectrometry, infrared and Raman spectroscopy.
HPTLC: How it works
When running an HPTLC sample, the principle is the same as for a regular TLC.
The sample is loaded on the THPLC plate, which often have silica, modified silica, or cellulose as the stationary phase. Usually, the stationary phase used depends upon the material undergoing separation. At TRI we have an autosampler, where a specified quantity of sample is applied to the plate through spraying as an aerosol in nitrogen gas.
The plate is placed into the developing chamber, which contains an atmosphere saturated with the developing solvent.
The solvent is allowed to run up the HPTLC plate, through capillary action, for a pre-determined height. This causes the components of the mixture being sampled to separate due to differences in their interaction between the solid phase and solvent front, usually due to their solubility in the mobile phase (solvent).
The HPTLC plate is visualized, often under UV light, but sometimes with a chemical agent. This reveals bands of different chemical compounds, allowing a quick visual of the chemical composition of the mixture.
HPTLC at TRI
At TRI, one of the key uses of HPTLC is in the separation of lipids into classes, Figure 1. Differences in lipid composition between a control and treated hair tresses can be readily identified, allowing comparison between different products or formulations. In fact, one of the biggest advantages of HPTLC is the ability to test small hair masses and high throughput. It is also excellent for relative quantification and percent change claims.
Figure 1: HPTLC plate visualised with long-wave UV to show the differences between unknown samples and a sample with known composition. Image adapted from TRI Talks 2025: Scratching the Surface: Lipids, Protection & Sebum - The Microbiome Relationship (Dr Ernesta Malinausktye, TRI Princeton)
The power of HPTLC
HPTLC allows:
Lipid class fingerprinting of hair and skin: HPTLC generates reproducible lipid class profiles from complex biological samples such as hair fibers, nails, and skin. These chromatographic “fingerprints” (e.g., free fatty acids, cholesterol, ceramides, triglycerides, wax esters, and squalene) allow direct comparison between untreated, chemically treated, environmentally exposed, or product-treated samples.
Quantitative evaluation of lipid depletion and restoration: Densitometric analysis enables quantitative or semi-quantitative evaluation of surface and internal lipid loss caused by chemical treatments, washing, UV exposure, or environmental stress, as well as evaluation of lipid replenishment following product application.
Contaminant detection: HPTLC can be used to readily identify if contaminants or adulterants are present in a sample, although this is non-standard at TRI.
