Identification of fixed oils by HPTLC


HPTLC is a powerful technique with regard to a high sample throughput capacity, short analysis time, and low running costs. Our case study demonstrates a rapid identification of fixed oils after derivatization with phosphomolybdic acid.


Often chromatographic techniques require a time- and cost-intensive sample preparation step. In many cases HPTLC allows to analyze several samples side by side after only a minimal sample preparation (e.g. dissolving). The identity of samples with unknown composition can be elucidated by comparing the pattern of the sample with that of reference materials. A System Suitability Test (SST) can be applied to check the reproducibility of the chromatographic system during method transfer validation and to qualify the data on each plate.

Description of the procedure

The method is downloaded from the visionCATS Method Library. The Word document (SOP) is opened for reference. In preparation for the analysis a RP-18 HPTLC plate is pre-washed with dichloromethane and then dried in the oven. All samples and references are prepared according to the SOP (in compliance with the USP method <203>, Application note F-39). The visionCATS method file is opened and executed, creating a new analysis file. In this visionCATS analysis file the sequence table is completed (Vial ID, description of samples and references, and selection of the rack position of each vial). An image of the pre-washed clean plate under white light is captured with the TLC Visualizer. The samples and standards are applied band-wise onto the HPTLC plate using the ATS 4, which guarantees precise and reliable application. Then the HPTLC plate is developed using the ADC 2. For visualization of fingerprints of the different fixed oils the developed plate is dipped into phosphomolybdic acid reagent, heated at 120°C for 3 minutes, and documented with the TLC Visualizer under white light. For a successful method transfer validation the SST needs to pass. If the RF values are in the specified range the patterns of the unknown samples and those of the references can be easily compared with the visionCATS Image Comparison.


Track Sample
1 Olive oil, 2 µL
2 Corn oil, 2 µL
3 Almond oil, 2 µL
4 Canola oil, 2 µL
5 Cottonseed oil, 2 µL
6 Evening Primrose oil, 2 µL
7 Flax seed oil, 2 µL
8 Palm oil, 2 µL
9 Peanut oil, 2 µL
10 Sesame oil, 2 µL
11 Soybean oil, 2 µL
12 Chia oil, 2 µL
13 Safflower oil, 2 µL
14 Borage oil, 2 µL
15 Sunflower oil, 2 µL
16 Sunflower oil (Reference), 2 µL
17 Sunflower oil (Sample), 2 µL


Image Comparison of fixed oils and references (USP) after derivatization

SST (Track 1 shows the pattern of olive oil used for the SST)

Benefits of using modern HPTLC for the analysis of food samples

  • HPTLC visualizes at the first glance the similarities and differences between samples and references.
  • HPTLC is also used for determination of purity (adulteration/fraud) and stability studies (shelf life).
  • HPTLC can be used as high throughput and low-cost technique for the analysis of food samples.
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