Analytical implications of zero-order operation and blazed gratings in chromatographic fluorescence detectors

The use of two grating monochromators in fluorescence detectors coupled to chromatographic systems has been associated with some unexplained optical effects and anomalous results in the literature. The recently introduced zero-order excitation/emission mode, in which the corresponding grating directs the entire lamp spectrum to the flow cell or the emission spectrum to the photomultiplier tube, introduces additional complexities. The consequences of these optical properties in HPLC-FLDs are investigated.

For the tested UV emitters, sensitivity surprisingly dropped up to 10-fold under zero-order diffraction, whereas for the visible fluorophores, sensitivity increased up to 3.6-fold in a nonlinear fashion, depending on the analyte's emission spectrum. This effect is not from a "sag" in photomultiplier gain from scattered light; the rapid increase or decrease in HPLC-FLD signal is caused by (often undisclosed) blazing of holographic diffraction gratings. An optical test based on Rayleigh scattering is developed that predicts the wavelengths at which zero-order diffraction is advantageous versus where it degrades the signal. A one-plus log transform of emission scans revealed a continuous, buried background of higher-order diffraction contributions from monochromators. A robust z-transform tool has been developed in MATLAB®, enabling analysts to view impurities in emission heat maps that would otherwise be invisible.

The proposed optical test for assessing the zero-order advantage applies to all HPLC-FLDs under isocratic or gradient conditions. Zero-order diffraction detection can lower the detection limit for visible-light emitters if the grating is blazed for the UV region. Understanding these optical effects enables more reliable ultra-trace fluorescence detection.