Our HPLC specialists in Germering (Germany) recently updated our popular 16-page Fluorescence Method Development Handbook (link to download the PDF) which is proving to be a resource for chromatographers using not just our HPLC systems but also other HPLC systems. The handbook is comprehensive and systematic in explaining the fluorescence theory and the effect on experimental results.
As most of you know, many scientists working with HPLC use optical detection techniques with UV-vis absorption being the most common detection being measured. As selected substances also show fluorescent behavior, they also absorb and emit light with a specific wavelength shift. And, fluorescence detection provides two benefits as compared to UV detection: low limits of detection and high specificity; but, the challenge is the method development as it is much more complex as compared to other optical detectors. For optimum results, it is important to optimize the different parameters in a systematic way.
Our handbook explains the relation between absorbance and excitation of a fluorescent compound and covers how using tools available in our Chromatography Data System (CDS) software (Thermo Scientific Dionex Chromeleon CDS software) and our fluorescence detector (Thermo Scientific Dionex UltiMate 3000 Fluorescence Detector), it is very easy and straight forward to define the best wavelength settings. The tools include 3D synchro, excitation and emission scans, and the zero order mode. Another important setting is the sensitivity of the photomultiplier tube (PMT) that detects the emitted light. A PMT can be regarded as a current source that generates a current proportional to the light intensity to which it is exposed. The sensitivity of the PMT is used to tune the current response to achieve the best signal-to-noise performance of the detector.
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It is industry standard for fluorescence detectors to have a fixed emission light filter between the flow cell and the PMT. The high-end version of our fluorescence detector features a variable emission filter that can dynamically adapt to various wavelength pairs. This makes sure that the instrument always operates under lowest stray light conditions. The handbook further discusses other aspects of detector setting and optimization, such as best suitable data collection rate, data filtering and xenon flash lamp frequency.
Lastly, handbook also includes an easy procedure how to test solvent suitability. This can be used to test whether the mobile phase quality is good enough and whether the quality remains stable between different lots.
Let us know of your challenges with fluorescence detection in the Comments box below; our experts will be pleased to answer your questions!