The effects of changing solvent composition on the LOD of TLS detection in gradient elution HPLC have been studied from the perspective of thermo-optical properties of the solvent. Hyphenated gradient high-performance liquid chromatography (HPLC)-thermal lens spectrometry (TLS), was used to separate and detect 13 carotenoid compounds and two chlorophylls. Utilization of mixing coils into the system reduces the inhomogeneities during eluent changes and therefore enables the application of thermal lens detection in the gradient HPLC method. For gradient chromatographic conditions in which the thermo-optical properties and related enhancement factor change as much as 50% over 10 min, the LODs for the TLS detector were enhanced by as much as three times in comparison with UV-Vis detection. For the isocratic part of the chromatogram, up to a tenfold improvement of LODs was achieved with TLS detection.