Development of the first universal mixture for use in system suitability tests for High-Performance Thin Layer Chromatography

•New HPTLC-based approach for qualification of the chromatographic system•Development of a universal mixture of substances covering the entire RF range•Usefulness of Universal HPTLC mix in a wide range of System Suitability Tests The use of chromatographic methods in routine analysis includes System Suitability Tests (SST). This paper presents a novel approach to SST in HPTLC, which allows qualification of the entire RF range of an HPTLC plate independently of the samples. It is based on the Universal HPTLC mix (UHM), a pre-defined mixture of eight reference substances: guanosine, sulisobenzone, thymidine, paracetamol, phthalimide, 9-hydroxyfluorene, thioxanthen-9-one, and 2-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol, selected to cover a broad range of polarities and functional groups. The chromatographic behavior of the UHM was evaluated for 20 different mobile phases on Silica gel 60 F254. At least three constituents were baseline separated. In a collaborative trial with four laboratories the reproducibility of RF values for three representative mobile phases, was found to be within a confidence interval of 0.040 RF units. The response characteristics of the UHM were assessed with respect to changes in chromatographic conditions, such as variation of the relative humidity, improperly employed saturation, or mistakes in the preparation of the mobile phase. Based on the RF values of the individual constituents significant responses were found for most changes. This qualifies the UHM for use in SST.