A Spectroscopy Guide for PAT

By Emil W. Ciurczak, Contributing Editor In fact, when HPLC is used for analysis of dosage forms, the spectrum of the analyte, combined with the retention time, may be used as the “specificity” portion of the analysis. HPLC has also been incorporated (since the 1980s) as a process technique in bioprocessing. Eli Lilly was a pioneer in the field, using LC on-line to monitor the formation and purification of insulin. The earliest process equipment was designed and built by Lilly for this work. While LC is not instantaneous, several minutes is, in effect, real-time when a process (especially a biological-type) takes days. In the 20+ years since its inception, process HPLC has come a long way. While fluorescence spectra resemble vibrational spectra, they are considered electronic because of the mode of their mechanism. Energy (in the form of photons) is absorbed, causing the electrons to become excited and move to a higher level. The electron excitation depends on the incident light (higher light flux causes higher peaks) while the emitted light can be strongly influenced by the matrix and temperature. Emitted photons may be reabsorbed or the excited electrons may interact with solvent at higher temperatures and not emit a photon at all. However, unlike the very simple UV and visible spectra, fluorescence spectra are “rich.” That is, they contain many more peaks and give more structural information than are seen in either visible or ultraviolet spectra. Since the mechanism of fluorescing is “multidirectional,” the detectors are placed perpendicular to the incident light. Thus, unlike UV/Vis, small absorbances (followed by emission) and not the total ouput of the light source are seen against a “dark” background. This allows for detection of trace amounts of analyte. Where applicable, these monitors (grating or filter) can give molecular information with little interference from the