Collaborative trial on the evaluation of HPTLC methods across different laboratories using the Universal HPTLC Mixture as System Suitability Test


As defined by the United States Pharmacopoeia General Chapter <203> [1] and the European Pharmacopoeia General Chapter 2.8.25 [2], HPTLC is a product neutral analytical concept. If the corresponding Standard Operating Procedure (SOP 001) [3] and all relevant analytical parameters are maintained, any lab should be able to obtain comparable results with a previously validated method. During transfer validation of the published method and during routine analysis, a system suitability test (SST) is used to evaluate the actual performance (quality) of the chromatographic system. In HPTLC, a versatile approach was developed to facilitate the system suitability test for a great variety of methods using Silica gel 60 F254: the Universal HPTLC Mixture (UHM) [4].  

The UHM consists of eight substances (guanosine, sulisobenzone, thymidine, paracetamol, phthalimide, 9-hydroxyfluorene, thioxanthen-9-one, and 2-(2H-benzotriazol-2-yl)-4-(1,1,3,3- tetramethylbutyl)phenol) and yields specific patterns of separated zones distributed over the whole RF range for numerous developing solvents. Therefore, the UHM could be used in a generic SST [5]. 

The UHM concept is an ideal candidate for a computer-supported evaluation of the accuracy of a chromatographic result. To collect more data and evaluate the performance of the UHM in various laboratories, an international collaborative trial is proposed. For that, the host institution (HPTLC Association) is currently developing a platform for uploading data.  

Project description: 

The project aims at the evaluation and comparison of the performance of validated HPTLC methods in laboratories across the world. 

Validated HPTLC methods are defined as: any method that is based on USP <203> / Ph. Eur. 2.8.25, uses HPTLC Silica gel 60  F254, and has either been published in a compendium (e.g. pharmacopoeia, HPTLC Association Method Collection) or validated for reproducibility. 

The performance of a method is described by the RF values of the zones obtained for the UHM. 

Participants can use any kind of HPTLC/TLC equipment but must ensure that HPTLC parameters are followed. How HPTLC can be done at various levels of instrumentation is shown here

In addition to the RF values obtained for the major zones of the UHM, information about the chromatographic system (stationary phase with batch number and composition of the developing solvent) and the employed equipment is submitted by the participants. Multiple data on the same chromatographic system are welcome.  

Note: there is no need to disclose any information about the analyte!  

Periodically, the submitted data will be compiled, shared with the participants and published on the project website. 

Project coordination: 

Dr. Tiên Do: Secretary General of the HPTLC Association; 


Preparation of the UHM:

The UHM can be purchased from Merck (article number 91816) [6] or prepared by each collaborator according to SOP 015 [7].  


  1. Apply the 2.0 µL of the UHM on track 8 of each plate.  
  2. After development, record images in shortwave UV (254 nm) and longwave UV (350 nm broadband) and white light after development. 
  3. On the images, align the RF scale with the application position line and the position of the solvent front. 
  4. Generate profiles from the image in UV 254 nm if a scanner is not available in the lab. If the scanner is available scan at 254 nm.
  5. Calculate Rvalues for the major zones obtained for the UHM. If no software is available calculate Rvalues based on measurements with a ruler.
  6. Fill the excel document HPTLC Association - Collaborative trial - UHM excel


[1] General chapter USP <203> 

[2] General chapter Ph. Eur. 2.8.25 

[3] HPTLC Association: SOP 001 General methodology for HPTLC v3

[4] T.K.T. Do, M. Schmid, M. Phanse, A. Charegaonkar, H. Sprecher, M. Obkircher, E. Reich, Development of the first universal mixture for use in system suit- ability tests for high-performance thin layer chromatography, J. Chromatogr. A 1638 (2021) 461830. 

[5] M. Schmid, T.K.T. Do, I. Trettin, E. Reich, Applicability of the Universal Mixture for describing system suitability and quality of analytical data in routine normal phase High Performance Thin Layer Chromatography methods, J. Chromatogr. A. 1666 (2022) 462863.

[6] Universal HPTLC Mix (UHM) for Simplified System Suitability Tests: A Novel Concept for HPTLC Suitability Test (

[7] HPTLC Association: SOP 015 Preparation and quality check of the UHM